This study aimed to investigate the dose-effect of iron on growth performance,antioxidant function.intestinal morphology,and mRNA expression of jejunal tight junction protein in 1-to21-d-old yellow-feathered broilers....This study aimed to investigate the dose-effect of iron on growth performance,antioxidant function.intestinal morphology,and mRNA expression of jejunal tight junction protein in 1-to21-d-old yellow-feathered broilers.A total of 7201-d-old yellow-feathered maleb roilers were allocated to 9 treatments with 8 replicate cages of 10 birds per cage.The dietary treatments were consisted of a basal diet(contained 79.6 mg Fe kg^(-1))supplemented with 0,20,40,60,80,160,320,640,and 1,280 mg Fe kg^(-1)in the form of FeSO_(4)·7H_(2)O.Compared with the birds in the control group,birds supplemented with 20mg Fe kg^(-1)had higher average daily gain(ADG)(P<0.0001).Adding 640 and 1,280 mg Fe kg^(-1)significantly decreased ADG(P<0.0001)and average daily feed intake(ADFI)(P<0.0001)compared with supplementation of 20mg Fe kg^(-1).Malondialdehyde(MDA)concentration in plasma and duodenum increased linearly(P<0.0001),but MDA concentration in liver and jejunum increased linearly(P<0.05)or quadratically(P<0.05)with increased dietary Fe concentration.The villus height(VH)in duodenum and jejunum,and the ratio of villus height to crypt depth(V/C)in duodenum decreased linearly(P?0.05)as dietary Feincreased.As dietary Fe increased,the jejunal relative mRNA abundance of claudin-1 decreased linearly(P=0.001),but the jejunal relative mRNA abundance of zona occludens-1(ZO-1)and occludin decreased linearly(P?0.05)or quadratically(P?0.05).Compared with the supplementation of 20 mg Fe kg^(-1),the supplementation of640 mg Fe kg^(-1)or higher increased(P?0.05)MDA concentrations in plasma,duodenum,and jejunum,decreased VH in the duodenum and jejunum,and the addition of 1,280 mg Fe kg^(-1)reduced(P?0.05)the jejunal tight junction protein(claudin-1,ZO-1,occludin)mRNA abundance.In summary,640 mg of supplemental Fe kg^(-1)or greater was associated with decreased growth performance,increased oxidative stress,disrupted intestinal morphology,and reduced mRNA expression of jejunal tight junction protein.展开更多
Background:Bacillus cereus is an important pathogen that causes human food poisoning,specifically diarrhea and vomiting.B.cereus can also induce mastitis in dairy cows and has a strong survival ability in milk,as it c...Background:Bacillus cereus is an important pathogen that causes human food poisoning,specifically diarrhea and vomiting.B.cereus can also induce mastitis in dairy cows and has a strong survival ability in milk,as it cannot be inactivated by high-temperature short-time pasteurization.Therefore,B.cereus can enter the market through pasteurized milk and other dairy products,imposing enormous hidden dangers on food safety and human health.Results:In this study,B.cereus 2101(BC)was isolated from milk samples of cows with mastitis.BC grew rapidly with strong hemolysis,making it difficult to prevent mastitis and ensure food security.MAC-T cells were treated with BC and/or Lactobacillus rhamnosus GR-1(LGR-1).Pretreatment with LGR-1 protected the integrity of tight junctions and the expression of zonula occludens-1(ZO-1)and occludin destroyed by BC.Furthermore,LGR-1 pretreatment reduced the expression of NOD-like receptor family member pyrin domain-containing protein 3(NLRP3),caspase recruitment and activation domain(ASC),Caspase-1 p20,gasdermin D(GSDMD)p30,inflammatory factors(interleukin(IL)-1βand IL-18),and cell death induced by BC.Moreover,LGR-1 pretreatment reduced NLRP3 inflammasome activity and increased expressions of ZO-1 and occludin induced by lipopolysaccharides(LPS)+ATP stimulation.MAC-T cells were transfected with NLRP3 si RNA or MCC950 and/or treated with BC and/or LGR-1.NLRP3-si RNA transfection and MCC950 attenuated BC-induced NLRP3 inflammasome activity.Expression of inflammatory cytokines and cell death suggested that the inflammatory pathway might play an important role in the induction of the NLRP3 inflammasome by BC and the protection of LGR-1.Conclusions:These results suggest that LGR-1 might be a probiotic alternative to antibiotics and could be administered to prevent mastitis in dairy cows,thus ensuring food security.展开更多
The chronic complications of diabetes mellitus constitute a major public health problem.For example,diabetic eye diseases are the most important cause of blindness,and diabetic nephropathy is the most frequent cause o...The chronic complications of diabetes mellitus constitute a major public health problem.For example,diabetic eye diseases are the most important cause of blindness,and diabetic nephropathy is the most frequent cause of chronic kidney disease worldwide.The cellular and molecular mechanisms of these chronic complications are still poorly understood,preventing the development of effective treatment strategies.Tight junctions(TJs)are epithelial intercellular junctions located at the most apical region of cell-cell contacts,and their main function is to restrict the passage of molecules through the paracellular space.The TJs consist of over 40 proteins,and the most important are occludin,claudins and the zonula occludens.Accumulating evidence suggests that TJ disruption in different organs,such as the brain,nerves,retina and kidneys,plays a fundamental pathophysiological role in the development of chronic complications.Increased permeability of the blood-brain barrier and the blood-retinal barrier has been demonstrated in diabetic neuropathy,brain injury and diabetic retinopathy.The consequences of TJ disruption on kidney function or progression of kidney disease are currently unknown.In the present review,we highlighted the molecular events that lead to barrier dysfunction in diabetes.Further investigation of the mechanisms underlying TJ disruption is expected to provide new insights into therapeutic approaches to ameliorate the chronic complications of diabetes mellitus.展开更多
The placenta plays an important role in nutrient transport to maintain the growth and development of the embryo.Gestational diabetes mellitus(GDM),the most common complication during pregnancy,highly affects placental...The placenta plays an important role in nutrient transport to maintain the growth and development of the embryo.Gestational diabetes mellitus(GDM),the most common complication during pregnancy,highly affects placental function in late gestation.Advanced glycation end-products(AGEs),a complex and heterogeneous group of compounds engaged by the receptor for AGEs(RAGE),are closely associated with diabetes-related complications.In this study,AGEs induced a decrease in the expression of tight junction(TJ)proteins in BeWo cells and increased the paracellular permeability of trophoblast cells by regulating RAGE/NF-κB.Sprague-Dawley(SD)rats injected with 100 mg/kg AGEs-rat serum albumin(RSA)via the tail vein from embryo day 2 were set as the placental barrier dysfunction model group(n=10).The effect of AGEs on placental permeability was determined using the Evans-Blue dye extravasation method.The ultrastructure of the placenta samples was observed by transmission electron microscopy.The effects of AGEs on the placenta were confirmed by treating rats with RAGE antagonist FPS-ZM1 and soluble forms of RAGE(sRAGE).AGEs treatment increased placental permeability and disrupted the tight junctions in pregnant rat placenta,but has no effect on blood glucose.The expression of TJ-related proteins,including ZO-1,Occludin,and Claudin 5,were downregulated after AGEs treatment.Further,AGEs treatment increased the expression of RAGE and nuclear factor-κB in the placenta of rats and upregulated the levels of vascular endothelial growth factor.The effects of AGEs on the placenta were blocked by RAGE antagonist FPS-ZM1 and sRAGE.This study demonstrates the mechanism underlying AGEs-induced disturbance in placental function in pregnant rats and highlights the potential of AGEs in the treatment of GDM.展开更多
AIM To examine the effects of Acanthopanax senticosus polysaccharides(ASPS) on intestinal tight junction(TJ) disruption and nuclear factor-kappa B(NF-κB)/myosin light chain kinase(MLCK) activation in endotoxemia.METH...AIM To examine the effects of Acanthopanax senticosus polysaccharides(ASPS) on intestinal tight junction(TJ) disruption and nuclear factor-kappa B(NF-κB)/myosin light chain kinase(MLCK) activation in endotoxemia.METHODS BALB/C mice(6-8-weeks-old) received continuous intragastric gavage of ASPS for 7 d before injection of lipopolysaccharide(LPS), or received ASPS once after LPS injection. Blood and intestinal mucosal samples were collected 6 h after LPS challenge. Clinical symptoms, histological injury, intestinal permeability,TJ ultrastructure, and TJ protein expression were determined.RESULTS Compared with mice in the LPS group, pretreatment with ASPS improved clinical and histological scores by 390.9%(P < 0.05) and 57.89%(P < 0.05), respectively, and gut permeability change in endotoxemic mice was shown by a 61.93% reduction in reduced leakage of fluorescein isothiocyanate-dextran 6 h after LPS injection(P < 0.05). ASPS pretreatment also prevented LPS-induced TJ ultrastructure breakdown supported by increased electron dense materials between adjoining cells, sustained redistribution and expression of occludin(0.597 ± 0.027 vs 0.103 ± 0.009, P < 0.05) and zonula occludens-1(0.507 ± 0.032 vs 0.125 ± 0.019, P < 0.05), and suppressed activation of the NF-κB/MLCK pathway indicated by reduced expression of NF-κB, phospho-inhibitor kappa B-alpha, MLCK and phospho-myosin light-chain-2 by 16.06%(P < 0.05), 54.31%(P < 0.05), 66.10%(P < 0.05) and 64.82%(P < 0.05), respectively. CONCLUSION ASPS pretreatment may be associated with inhibition of the NF-κB/MLCK pathway and concomitant amelioration of LPS-induced TJ dysfunction of intestinal epithelium in endotoxemia.展开更多
Background: Weaning is one of the major factors that cause stress and intestinal disease in piglets. Protocatechuic acid(PCA) is an active plant phenolic acid which exists in Chinese herb, Duzhong(Eucommia ulmoides Ol...Background: Weaning is one of the major factors that cause stress and intestinal disease in piglets. Protocatechuic acid(PCA) is an active plant phenolic acid which exists in Chinese herb, Duzhong(Eucommia ulmoides Oliver), and is also considered as the main bioactive metabolite of polyphenol against oxidative stress and inflammation. This study aimed to investigate the effect of PCA on growth performance, intestinal barrier function, and gut microbiota in a weaned piglet model challenged with lipopolysaccharide(LPS).Methods: Thirty-six piglets(Pig Improvement Company line 337 × C48, 28 d of age, 8.87 kg ± 0.11 kg BW) were randomly allocated into 3 treatments and fed with a basal diet(CTL), a diet added 50 mg/kg of aureomycin(AUR), or a diet supplemented with 4000 mg/kg of PCA, respectively. The piglets were challenged with LPS(10 μg/kg BW) on d 14 and d 21 by intraperitoneal injection during the 21-d experiment. Animals(n = 6 from each group) were sacrificed after being anesthetized by sodium pentobarbital at 2 h after the last injection of LPS. The serum was collected for antioxidant indices and inflammatory cytokines analysis, the ileum was harvested for detecting mRNA and protein levels of tight junction proteins by PCR and immunohistochemical staining, and the cecum chyme was collected for intestinal flora analysis using 16 S rRNA gene sequencing.Results: Dietary supplementation of PCA or AUR significantly increased the expression of tight junction proteins including ZO-1 and claudin-1 in intestinal mucosa, and decreased the serum levels of thiobarbituric acid reactive substances(TBARS) and IL-6, as compared with CTL group. In addition, PCA also decreased the serum levels of IL-2 and TNF-α(P < 0.05). Analysis of gut microbiota indicated that PCA increased the Firmicutes/Bacteroidetes ratio(P < 0.05). Spearman's correlation analysis at the genus level revealed that PCA reduced the relative abundance of Prevotella 9, Prevotella 2, Holdemanella, and Ruminococcus torques group(P < 0.05), and increased the relative abundance of Roseburia and Desulfovibrio(P < 0.05), whereas AUR had no significant effect on these bacteria.Conclusions: These results demonstrated that both PCA and AUR had protective effect on oxidative stress, inflammation and intestinal barrier function in piglets challenged with LPS, and PCA potentially exerted the protective function by modulating intestinal flora in a way different from AUR.展开更多
The tight junction (TJ) is a critical cellular component for maintenance of tissue integrity, cellular interactions and cell-cell communications, and physiologically functions as the "great wall" against ext...The tight junction (TJ) is a critical cellular component for maintenance of tissue integrity, cellular interactions and cell-cell communications, and physiologically functions as the "great wall" against external agents and the surrounding hostile environment. During the host-pathogen evolution, viruses somehow found the key to unlock the gate for their entry into cells and to exploit and exhaust the host cells. In the liver, an array of TJ molecules is localized along the bile canaliculi forming the blood-biliary barrier, where they play pivotal roles in paracellular permeability, bile secretion, and cell polarity. In pathology, certain hepatic TJ molecules mediate virus entry causing hepatitis infection; deregulation and functional abnormality of the TJ have also been implicated in triggering liver cancer development and metastasis. All these findings shed new insights on the understanding of hepatic TJs in the development of liver disease and provide new clues for potential intervention.展开更多
BACKGROUND Progressive familial intrahepatic cholestasis(PFIC)encompasses a group of autosomal recessive disorders with high morbidity and mortality.Variants in the gene encoding tight junction protein-2(TJP2)have bee...BACKGROUND Progressive familial intrahepatic cholestasis(PFIC)encompasses a group of autosomal recessive disorders with high morbidity and mortality.Variants in the gene encoding tight junction protein-2(TJP2)have been linked to PFIC type 4(PFIC4),which predominantly presents in childhood.However,there are only limited data from adults with TJP2-related PFIC4.We report a family with an autosomal recessive disorder with a novel variant in the TJP2 gene in adults with very variable expression of PFIC4.CASE SUMMARY The index patient presented at 19 years old with liver cirrhosis and variceal bleeding and was treated with endoscopic banding and beta-blockers.In 2018,he developed primary liver cancer that was treated with radiofrequency ablation followed by liver transplantation in 2019.Genetic testing revealed a novel homozygous TJP2 variant causing PFIC4(TJP2([NM_004817.3]:c.[3334C>T];[3334C>T])).The consanguineous family consists of the father and mother(both heterozygous)and their 12 children,of which five carry the variant in a homozygous state;however,these five siblings have highly variable expression of PFIC4.Two homozygous brothers had cirrhosis and portal hypertension at diagnosis at the ages of 19 and 36.Two other homozygous brothers,age 23 and 19,and the homozygous sister,age 21,have elevated liver enzymes but presently no cirrhosis,which may suggest an age-dependent penetrance.In addition,five sisters had severe and mild intrahepatic cholestasis of pregnancy and carry the TJP2 variant in a homozygous and heterozygous state,respectively.CONCLUSION This novel TJP2 variant is associated with PFIC4 causing severe liver disease with cirrhosis and primary liver cancer in adolescents/adults.展开更多
Tight junctions(TJs) are the most apical intercellular junctions of epithelial cells formed by occludin, claudins, junctional adhesion molecules(JAMs), and zonula occludens(ZO). Tight junction proteins can sense the p...Tight junctions(TJs) are the most apical intercellular junctions of epithelial cells formed by occludin, claudins, junctional adhesion molecules(JAMs), and zonula occludens(ZO). Tight junction proteins can sense the presence of bacteria and regulate the transcription of target genes that encode effectors and regulators of the immune response. The aim of this study was to determine the impact of TJ proteins in response to Porphyromonas gingivalis(P. gingivalis), P. gingivalis lipopolysaccharide(P. gingivalis LPS), and extracellular adenosine triphosphate(ATP) in the oral epithelial cell culture model. Quantified real timepolymerase chain reaction(RT-PCR), immunoblots, and immunostaining were performed to assess the gene and protein expression in TJs. It was found that P. gingivalis infection led to transient upregulation of the genes encoding occludin, claudin-1, and claudin-4 but not JAM-A, claudin-15, or ZO-1, while P. gingivalis LPS increased claudin-1, claudin-15, and ZO-1 and decreased occludin, JAM-A, and claudin-4. Tight junction proteins showed significant upregulation in the above two groups when cells were pretreated with ATP for 3 h. The findings indicated that P. gingivalis induced the host defence responses at an early stage. P. gingivalis LPS exerted a more powerful stimulatory effect on the disruption of the epithelial barrier than P. gingivalis.ATP stimulation enhanced the reaction of TJ proteins to P. gingivalis invasion and LPS destruction of the epithelium.展开更多
Objective:In this study,the influence of puerarin,paeoniflorin,and menthol on the structure and barrier function of tight junctions(TJs)in MadineDarby canine kidney epithelial(MDCK)and MDCK-multi-drug resistance 1(MDR...Objective:In this study,the influence of puerarin,paeoniflorin,and menthol on the structure and barrier function of tight junctions(TJs)in MadineDarby canine kidney epithelial(MDCK)and MDCK-multi-drug resistance 1(MDR1)cells was evaluated to determine the mechanisms by which the drugs cross the bloodebrain barrier(BBB).Method:Cells were treated with puerarin,paeoniflorin,and menthol followed by immunohistochemical staining with occludin,claudin-1,and F-actin.The cells were then observed using laser-scanning confocal microscopy.Average optical density(AOD)of the immunofluorescence images of the proteins were analyzed using ImageJ software while Transepithelial electrical resistance(TEER)was measured using an epithelial voltohmmeter.Results:Confocal microscopy revealed that puerarin-and paeoniflorin-treated tight junction proteins were conspicuous while menthol suppressed their expression.Correspondingly,AOD values of cells treated with puerarin or paeoniflorin,or both showed no difference compared to the control group(P>.05)while the menthol group value was downregulated.In 3 h,TEER of cells not treated with menthol were similar to the control group,while treatment with menthol significantly decreased TEER value(P<.05).In addition,application of menthol decreased TEER in MDCK cells earlier than in MDCK-MDR1 cells.Conclusion:Menthol but not puerarin and paeoniflorin may enhance paracellular transport and improve drug penetration of the BBB by disrupting the structure and,thereby,weakening the barrier function of TJs.展开更多
AIM:To investigate the mechanism of the tight junction(TJ) disruption and the association between tumor necrosis factor(TNF)-α and matrix metalloproteinase(MMPs) under hyperosmotic condition in primary human corneal ...AIM:To investigate the mechanism of the tight junction(TJ) disruption and the association between tumor necrosis factor(TNF)-α and matrix metalloproteinase(MMPs) under hyperosmotic condition in primary human corneal epithelial cells(HCECs).METHODS:The cultured HCECs were exposed to media which adding sodium chloride(Na Cl) for hyperosmolar stress or adding rh-TNF-α(10 ng/m L). NF-κB inhibitor(5 μmol/L) or GM-6001(potent and broad spectrum MMP inhibitor, 20 μmol/L)was added 1 h before that treatment. The integrity of TJ proteins was determined by immunofluorescent(IF) staining. The m RNA levels of TNF-α and MMPs were evaluated by quantitative reverse transcription polymerase chain reaction(RT-q PCR) and the protein expression by enzyme-linked immunosorbent assay(ELISA).RESULTS:TJ proteins ZO-1 and Occludin were disrupted in primary HCECs exposed to hyperosmotic medium. The m RNA expression and protein production of TNF-α increased significantly in hyperosmotic media at 500 m Os M. TNF-α mediated the expression and production of MMP-1, MMP-13, MMP-9, and MMP-3 stimulated by hyperosmotic stress. The production of MMPs in hyperosmolar media were increased through the increase of TNF-α. GM-6001 prevent the destruction of ZO-1 and Occludin in hyperosmolar stress and rh-TNF-α treated medium. TNF-α induced activation of MMPs was involved in the TJ disruption by hyperosmolarity.CONCLUSION:TJ proteins ZO-1 and Occludin are disrupted by hyperosmolar stress and TNF-α, but protected by MMP inhibitor(GM-6001). It suggests that TNF-α/MMP pathway mediates the TJ disruption in primary HCECs exposed to hyperosmotic stress.展开更多
The differentiation of cells composing mature human hairs produces layers with different corneous characteristics that would tend to flake away one from another,as in the corneous layer of the epidermis,without anchor...The differentiation of cells composing mature human hairs produces layers with different corneous characteristics that would tend to flake away one from another,as in the corneous layer of the epidermis,without anchoring junctions.It is likely that cell junctions established in the forming cells of the hair bulb are not completely degraded like in the corneous layer of the epidermis but instead remain in the hair shaft to bind mature cuticle,cortex,and medulla cells into a compact hair shaft.During cell differentiation in hairs,cell junctions seem to disappear,and little is known about the fate of junctional proteins present in the mature human hair shaft.The present ultrastructural immunogold study has detected some marker proteins of adhesion junction(cadherin and beta-catenin)and tight junctions(occludin and cingulin)that are still present in cornified hairs where numerous isopeptide bonds are detected,especially in the medulla.This qualitative ultrastructural study indicates that aside from the cell membrane complex,a long corneo-desmosome bonding cortex and cuticle cells,also sparse adherens and tight junction remnants are present.It is suggested that the cornification of these junctions with the incorporation of their proteins within the mature corneous material of the hair shaft likely contributes to maintaining the integrity of the mature hair.This information will also allow us to evaluate the effects of different chemical components present in hair formulations and stains on these junctional proteins and the consequent integrity of the hair shaft.展开更多
BACKGROUND Altered tight junction(TJ)proteins are correlated with carcinogenesis and tumor development.Nimbolide is a tetranotriterpenoid that has been shown to have antioxidant and anti-proliferative properties;howev...BACKGROUND Altered tight junction(TJ)proteins are correlated with carcinogenesis and tumor development.Nimbolide is a tetranotriterpenoid that has been shown to have antioxidant and anti-proliferative properties;however,its anticancer effects and molecular mechanism in hepatocellular carcinoma(HCC)remains obscure.AIM To investigate the effect of nimbolide on TJ proteins,cell cycle progression,and hepatic inflammation in a mouse model of HCC.METHODS HCC was induced in male Swiss albino mice(CD-1 strain)by a single intraperitoneal injection of 100 mg/kg diethylnitrosamine(DEN)followed by 80 ppm N-nitrosomorpholine(NMOR)in drinking water for 28 wk.After 28 wk,nimbolide(6 mg/kg)was given orally for four consecutive weeks in DEN/NMOR induced HCC mice.At the end of the 32nd week,all the mice were sacrificed and blood and liver samples were collected for various analyses.Macroscopic examinations of hepatic nodules were assessed.Liver histology and HCC tumor markers such as alpha-fetoprotein(AFP)and glypican-3 were measured.Expression of TJ proteins,cell proliferation,and cell cycle markers,inflammatory markers,and oxidative stress markers were analyzed.In silico analysis was performed to confirm the binding and modulatory effect of nimbolide on zonula occludens 1(ZO-1),nuclear factor of kappa light polypeptide gene enhancer in B-cells(NF-κB),and tumor necrosis factor alpha(TNF-α).RESULTS We found nimbolide treatment at a concentration of 6 mg/kg to HCC mice reduced hepatic tumor size by 52.08%and tumor volume(P<0.01),and delayed tumor growth in HCC mice with a concomitant reduction in tumor markers such as AFP levels(P<0.01)and glypican-3 expression(P<0.05).Furthermore,nimbolide treatment increased tight junction proteins such as ZO-1 and occludin expression(P<0.05,respectively)and reduced ZO-1 associated nucleic acid binding protein expression(P<0.001)in HCC mice liver.Nimbolide treatment to HCC mice also inhibited cell proliferation and suppressed cell cycle progression by attenuating proliferating cell nuclear antigen(P<0.01),cyclin dependent kinase(P<0.05),and CyclinD1(P<0.05)expression.In addition,nimbolide treatment to HCC mice ameliorated hepatic inflammation by reducing NF-κB,interleukin 1 beta and TNF-αexpression(P<0.05,respectively)and abrogated oxidative stress by attenuating 4-hydroxynonenal expression(P<0.01).Molecular docking studies further confirmed that nimbolide interacts with ZO-1,NF-κB,and TNF-α.CONCLUSION Our current study showed for the first time that nimbolide exhibits anticancer effect by reducing tumor size,tumor burden and by suppressing cell cycle progression in HCC mice.Furthermore,nimbolide treatment to HCC mice ameliorated inflammation and oxidative stress,and improved TJ proteins expression.Consequently,nimbolide could be potentially used as a natural therapeutic agent for HCC treatment,however further human studies are warranted.展开更多
Tight junctions are mainly formed by two types of proteins;claudins and occludin, both of which are fundamental to maintain the integrity and barrier function of the intestinal epithelium. This barrier function allows...Tight junctions are mainly formed by two types of proteins;claudins and occludin, both of which are fundamental to maintain the integrity and barrier function of the intestinal epithelium. This barrier function allows for the absorption of nutrients, mainly by transcytosis;however, in birds, 90% of the substances are absorbed by paracellular mechanisms. Despite this, claudins present in th<span style="font-family:Verdana;"></span><span style="font-family:;" "="">e different parts of the intestinal tract of adult chickens are not known, much less their functional role. This study aimed to determine the presence of mRNA of claudins 1, 2, 3, 5, 10, 12, 16 and occludin, in the different regions of the intestine (duodenum, jejunum, ileum, cecum, and rectum) in chickens (<i>Gallus gallus domesticus</i>) through RT-PCR. To meet this goal, 7 weeks old roosters destined for slaughter and chicken embryos of 16 days of incubation (positive control) were used. For all the processed samples, amplicons of the expected size were obtained;claudin 1 (662 pb), claudin 2 (162 pb), claudin 3 (185 pb), claudin 5 (224 pb), claudin 10 (687 pb), claudin 12 (738 pb), claudin 16 (191 pb) and occludin (430 pb). To corroborate these findings, obtained amplicons were sequenced and, subsequently, a basic alignment was performed on the NCBI, obtaining a correlation of 100% with the original sequences in all analyzed samples. To our knowledge, the present work represents the first written report regarding the presence of mRNA of the main proteins involved in tight junction formation throughout the intestinal tract of domestic chickens of 7 weeks of age. These findings will allow elucidating the specific function of each of the reported proteins in the process of paracellular absorption in chickens.</span>展开更多
We aimed to examine paracellular barrier function in cultured mouse lung microvascular endothelial cells (LMECs). The transcellular resistance of LMEC monolayers yielded an electrical resistance of approximately 19 Ω...We aimed to examine paracellular barrier function in cultured mouse lung microvascular endothelial cells (LMECs). The transcellular resistance of LMEC monolayers yielded an electrical resistance of approximately 19 Ω × cm<sup>2</sup> at days 6 - 7 in culture when the cells reached confluence, and paracellular permeable clearance of sodium fluorescein was the lowest on day 6 in culture, suggesting the formation of tight junctions (TJs) in cultured LMECs. Moreover, the expression of TJ-associated proteins, occludin, claudin-1 and -4 and zonula occludents 1 (ZO-1) was detected in LMECs at day 6 in culture. However, mRNAs of occludin, claudin-1 and -4 and ZO-1 were already expressed on day 1 after culture, and large variations were absent in the mRNA levels of occludin, claudin-4 and ZO-1 between days 1 and 7 in culture, when the level of each mRNA on day 1 in culture was used as a basal level. However, the claudin-1 mRNA level gradually increased up to approximately 7-fold on day 7 in culture over the basal level. These results indicate that the drastic increase in the mRNA expression level of claudin-1 leads to the strong formation of TJs.展开更多
Background:Cells are influenced by their environment.In vivo,the corneal endothelium is subjected to intraocular pressure(IOP).The purpose of this project was to evaluate in vitro,the effect of the IOP on the formatio...Background:Cells are influenced by their environment.In vivo,the corneal endothelium is subjected to intraocular pressure(IOP).The purpose of this project was to evaluate in vitro,the effect of the IOP on the formation of tight junctions in the corneal endothelium.Methods:Cultivated corneal endothelial cells(P2-P3;n=6 populations)were seeded on devitalized on corneas(n=10 pairs).Native corneas and devitalized corneas were respectively used as positive(n=2 pairs)and negative controls(n=3 pairs).Corneas were placed in artificial anterior chambers and subjected to a hydrostatic pressure between 0.3 and 0.4 psi during 4-5 days.Unpressured control corneas were maintained in cell culture dishes.Pictures of the corneas were taken following the experiment to assess stromal transparency.Morphology,corneal thickness and distribution of ZO-1,n-cadherin,b-catenin,NaK ATPase pump and HCO3-cotransporter were evaluated by electron microscopy,histological staining and immunofluorescences.Results:Pressure treated corneas were more transparent than the controls.Thickness was accordingly reduced by 38.4%±4.9%for cultivated endothelium and 32.2%±2.7%for native endothelium.Negative controls change in transparency and thickness were marginal.Pressure treated cells showed none or at most marginal difference in morphology and expression of ZO-1,n-cadherin,b-catenin,NaK ATPase pump and HCO3-cotransporters and failed to recreate a phenotype similar to native corneas.Pressure however increased cortical localisation of the protein ZO-1 in both cultivated and native endothelium.Conclusions:These results suggest that anterior chamber hydrostatic pressure may enhance endothelial functionality by modulating the distribution of tight junction’s proteins.展开更多
Inflammatory bowel diseases are characterised by inflammation that compromises the integrity of the epithelial barrier. The intestinal epithelium is not only a static barrier but has evolved complex mechanisms to cont...Inflammatory bowel diseases are characterised by inflammation that compromises the integrity of the epithelial barrier. The intestinal epithelium is not only a static barrier but has evolved complex mechanisms to control and regulate bacterial interactions with the mucosal surface. Apical tight junction proteins are critical in the maintenance of epithelial barrier function and control of paracellular permeability. The characterisation of alterations in tight junction proteins as key players in epithelial barrier function in inflammatory bowel diseases is rapidly enhancing our understanding of critical mechanisms in disease pathogenesis as well as novel therapeutic opportunities. Here we give an overview of recent literature focusing on the role of tight junction proteins, in particular claudins, in inflammatory bowel diseases and inflammatory bowel disease associated colorectal cancer.展开更多
AIM:To investigate the effect of herb-partitioned moxibustion combined with acupuncture on the expression of intestinal epithelial tight junction(TJ) proteins.METHODS:Sixty patients diagnosed with mild to moderate Cr...AIM:To investigate the effect of herb-partitioned moxibustion combined with acupuncture on the expression of intestinal epithelial tight junction(TJ) proteins.METHODS:Sixty patients diagnosed with mild to moderate Crohn’s disease(CD)were allocated into the herb-partitioned moxibustion combined with acupuncture(HMA)group(n=30)or the mesalazine(MESA)group(n=30)using a parallel control method.There were 2 sets of acupoints used alternately for HMA treatment.The following points were included in Set A:ST25(Tianshu),RN6(Qihai),and RN9(Shuifen)for herb-partitioned moxibustion and ST36(Zusanli),ST37(Shangjuxu),LI11(Quchi),and LI4(Hegu)for acupuncture.The points for Set B included BL23(Shenshu)and BL25(Dachangshu)for herb-partitioned moxibustion and EX-B2 of T6-T1(Jiajixue)fo r acupuncture.The patients received the same treatment6 times a week for 12 consecutive weeks.The MESA group received 1 g of mesalazine enteric coated tablets4 times daily for 12 consecutive weeks.Intestinaltissues were stained and examined to compare the morphological and ultrastructural changes before and after the treatment session.Immunohistochemistry and in situ hybridization assays were used to detect the expression of intestinal epithelial TJ proteins zonula occludens-1(ZO-1),occludin,and claudin-1.The m RNA levels were also evaluated.RESULTS:After the treatment,both herb-partitioned moxibustion combined with acupuncture and mesalazine improved intestinal morphology and ultrastructure of CD patients;the patients treated with HMA showed better improvement.HMA significantly increased the expression of ZO-1(P=0.000),occludin(P=0.021),and claudin-1(P=0.016).MESA significantly increased the expression of ZO-1(P=0.016)and occludin(P=0.026).However,there was no significant increase in the expression of claudin-1(P=0.935).There was no statistically significant difference between the two groups for the expression of occludin and claudin-1(P>0.05).The HMA group showed a significant improvement in ZO-1 expression compared to the MESA group(2333.34±352.51 vs 2160.38±307.08,P=0.047).HMA significantly increased the expression of ZO-1 m RNA(P=0.000),occludin m RNA(P=0.017),and claudin-1 m RNA(P=0.017).MESA significantly increased the expression of ZO-1 m RNA(P=0.000),occludin m RNA(P=0.042),and claudin-1 m RNA(P=0.041).There was no statistically significant difference between the two groups in the expression of occludin and claudin-1 m RNA(P>0.05).However,the HMA group showed a significant improvement in ZO-1 m RNA expression compared with the MESA group(2378.17±308.77 vs 2200.56±281.88,P=0.023).CONCLUSION:HMA can repair intestinal epithelial barrier lesions and relieve inflammation by upregulating the expression of TJ proteins and their m RNAs.展开更多
AIM: To investigate the effects of moxibustion on down-regulation of the colonic epithelial cell apoptosis and repair of the tight junctions in rats with Crohn's disease (CD). METHODS: Sixty male Sprague-Dawley ra...AIM: To investigate the effects of moxibustion on down-regulation of the colonic epithelial cell apoptosis and repair of the tight junctions in rats with Crohn's disease (CD). METHODS: Sixty male Sprague-Dawley rats were randomly divided into a normal control (NC) group, a model control (MC) group, an herbs-partitioned moxibustion (HPM) group, a mild-warm moxibustion (MWM) group and a salicylazosulphapyridine (SASP) group, with 12 rats in each group. The CD model rats were treated with trinitrobenzene sulphonic acid to induce intestinal inflammation. The rats in the HPM and MWM groups were treated at the Tianshu (ST25) and Qihai (CV6) acupoints once daily for 14 d, and the SASP group was fed SASP twice daily for 14 d. No additional treatment was given to the MC and NC groups. Themicrostructure of the colonic epithelium was observed under a transmission electron microscope, the transepithelial resistance was measured using a shortcircuit current, colonic epithelial cell apoptosis was determined by terminal deoxynucleotidyl transferasemediated dUTP-biotin nick end labelling assay, and the expression of occludin, claudin-1 and zonula occludens-l (ZO-1) in the colonic epithelial junction was determined by Western blotting and immunofluorescence staining. RESULTS: Compared with the MC group, the microstructure of the colonic epithelial barrier was signifi-cantly improved in rats treated with HPM, MWM or SASP, meanwhile, the current flow was reduced signifi-cantly, with values of 168.20 ± 6.14 vs 99.70 ± 3.13, 99.10 ± 4.28 and 120.30 ± 3.65 mA, respectively (P = 0.001). However, the HPM and MWM groups had higher current flow rates than the SASP group (99.70 ± 3.13, 99.10 ± 4.28 vs 120.30 ± 3.65 mA, P = 0.001). The number of the apoptotic colonic epithelial cells in HPM, MWM and SASP groups was largely reduced (61.5 ± 16.91 vs 15.5 ± 8.89, 14.8 ± 6.27 and 24.7 ± 9.68, respectively (P = 0.001); and the expression of occlu- din, claudin-1 and ZO-1 in the MWM and HPM groups was signifi cantly enhanced (0.48 ± 0.10, 0.64 ± 0.09 vs 0.18 ± 0.05 for occludin, 0.12 ± 0.02, 0.17 ± 0.03 vs 0.05 ± 0.01 for claudin-1, and 0.08 ± 0.01, 0.11 ± 0.01 vs 0.02 ± 0.01 for ZO-1). And in SASP group, the expression of occludin and ZO-1 was also signifi cantly increased (0.27 ± 0.04 vs 0.18 ± 0.05 for occludin and 0.05 ± 0.01 vs 0.02 ± 0.01 for ZO-1), but there was no significant difference for claudin-1. The HPM and MWM groups had higher expression of occludin, claudin-1 and ZO-1 than the SASP group. CONCLUSION: HPM and MWM treatment can down-regulate apoptosis of colonic epithelial cells, repair tight junctions and enhance colonic epithelial barrier function in rats with CD.展开更多
AIM: To investigate the effects of rhein on intestinal epithelial tight junction proteins in rats with IgA nephropathy (IgAN). METHODS: Twenty-eight female Sprague-Dawley rats were randomly divided into four groups (7...AIM: To investigate the effects of rhein on intestinal epithelial tight junction proteins in rats with IgA nephropathy (IgAN). METHODS: Twenty-eight female Sprague-Dawley rats were randomly divided into four groups (7 per group): Control, IgAN, Rhein-treated, and Rheinprevented. Bovine serum albumin, lipopolysaccharide and CCl4 were used to establish the rat model of IgA nephropathy. The Rhein-treated group was given rhein from week 7 until the rats were sacrificed. The Rheinprevented group was given rhein from week 1. Animals were sacrificed at the end of week 10. We observed the changes in the intestinal epithelial tight junctions using transmission electron microscopy, and expression of intestinal epithelial tight junction proteins zona occludens protein (ZO)-1 and occludin by immunofluorescence using laser confocal microscopy. Changes in mRNA and protein expression of ZO-1 and occludin were measured by reverse transcriptase polymerase chain reaction and Western blotting. The ratio of urinary lactulose/mannitol was measured by high performance liquid chromatography (HPLC) for assessing the intestinal permeability. RESULTS: In the control group, the tight junctions lied between epithelial cells on the top of the outer side of the cell membrane, and appeared in dense dotted crystal structures, the neighboring cells were binded tightly with no significant gap, and the tight junction protein ZO-1 and occludin were evenly distributed in the intestinal epithelial cells at the top of the junction. Compared with the control group, in the IgAN group, the structure of the tight junction became obscured and the dotted crystal structures had disappeared; the fluorescence of ZO-1 and occludin was uneven and weaker (5.37 ± 1.27 vs 10.03 ± 1.96, P < 0.01; 4.23 ± 0.85 vs 12.35 ± 4.17, P < 0.01); the mRNA expression of ZO-1 and occludin decreased (0.42 ± 0.19 vs 0.92 ± 0.24, P < 0.01; 0.40 ± 0.15 vs 0.97 ± 0.25, P < 0.01); protein expression of ZO-1 and occludin was decreased (0.85 ± 0.12 vs 1.98 ± 0.43, P < 0.01; 0.72 ± 0.15 vs 1.38 ± 0.31, P < 0.01); and the ratio of urinary lactulose/mannitol increased (3.55 ± 0.68 vs 2.72 ± 0.21, P < 0.01). In the Rheinprevented and Rhein-treated groups, compared with the IgAN group, the intestinal epithelial tight junctions were repaired; fluorescence of ZO-1 and occludin was stronger (11.16 ± 3.52 and 8.81 ± 2.30 vs 5.37 ± 1.27, P < 0.01; 10.97 ± 3.40 and 9.46 ± 2.40 vs 4.23 ± 0.85, P < 0.01); mRNA of ZO-1 and occludin increased (0.81 ± 0.17 and 0.64 ± 0.16 vs 0.42 ± 0.19, P < 0.01; 0.82± 0.22 and 0.76 ± 0.31 vs 0.40 ± 0.15, P < 0.01); protein expression of ZO-1 and occludin was increased (2.07 ± 0.41 and 1.57 ± 0.23 vs 0.85 ± 0.12, P < 0.01; 1.34 ± 0.21 and 1.15 ± 0.17 vs 0.72 ± 0.15, P < 0.01); and the ratio of urinary lactulose/mannitol decreased (2.83 ± 0.43 and 2.87 ± 0.18 vs 3.55 ± 0.68, P < 0.01). CONCLUSION: Rhein can enhance the expression of ZO-1 and occludin, repair damaged tight junctions, and protect the intestinal barrier.展开更多
基金supported by the National Natural Science Foundation of China(31501977)the Sichuan Provincial Key R&D Project China(22ZDYF0194)the Double World-Class Project of Southwest Minzu University China(XM2023010)。
文摘This study aimed to investigate the dose-effect of iron on growth performance,antioxidant function.intestinal morphology,and mRNA expression of jejunal tight junction protein in 1-to21-d-old yellow-feathered broilers.A total of 7201-d-old yellow-feathered maleb roilers were allocated to 9 treatments with 8 replicate cages of 10 birds per cage.The dietary treatments were consisted of a basal diet(contained 79.6 mg Fe kg^(-1))supplemented with 0,20,40,60,80,160,320,640,and 1,280 mg Fe kg^(-1)in the form of FeSO_(4)·7H_(2)O.Compared with the birds in the control group,birds supplemented with 20mg Fe kg^(-1)had higher average daily gain(ADG)(P<0.0001).Adding 640 and 1,280 mg Fe kg^(-1)significantly decreased ADG(P<0.0001)and average daily feed intake(ADFI)(P<0.0001)compared with supplementation of 20mg Fe kg^(-1).Malondialdehyde(MDA)concentration in plasma and duodenum increased linearly(P<0.0001),but MDA concentration in liver and jejunum increased linearly(P<0.05)or quadratically(P<0.05)with increased dietary Fe concentration.The villus height(VH)in duodenum and jejunum,and the ratio of villus height to crypt depth(V/C)in duodenum decreased linearly(P?0.05)as dietary Feincreased.As dietary Fe increased,the jejunal relative mRNA abundance of claudin-1 decreased linearly(P=0.001),but the jejunal relative mRNA abundance of zona occludens-1(ZO-1)and occludin decreased linearly(P?0.05)or quadratically(P?0.05).Compared with the supplementation of 20 mg Fe kg^(-1),the supplementation of640 mg Fe kg^(-1)or higher increased(P?0.05)MDA concentrations in plasma,duodenum,and jejunum,decreased VH in the duodenum and jejunum,and the addition of 1,280 mg Fe kg^(-1)reduced(P?0.05)the jejunal tight junction protein(claudin-1,ZO-1,occludin)mRNA abundance.In summary,640 mg of supplemental Fe kg^(-1)or greater was associated with decreased growth performance,increased oxidative stress,disrupted intestinal morphology,and reduced mRNA expression of jejunal tight junction protein.
基金the following funds:the National Key R&D Program of China(Project No.2017YFD0502200)the National Natural Science Foundation of China(Project No.31960721)the National Natural Science Foundation of China(Project No.31873034)。
文摘Background:Bacillus cereus is an important pathogen that causes human food poisoning,specifically diarrhea and vomiting.B.cereus can also induce mastitis in dairy cows and has a strong survival ability in milk,as it cannot be inactivated by high-temperature short-time pasteurization.Therefore,B.cereus can enter the market through pasteurized milk and other dairy products,imposing enormous hidden dangers on food safety and human health.Results:In this study,B.cereus 2101(BC)was isolated from milk samples of cows with mastitis.BC grew rapidly with strong hemolysis,making it difficult to prevent mastitis and ensure food security.MAC-T cells were treated with BC and/or Lactobacillus rhamnosus GR-1(LGR-1).Pretreatment with LGR-1 protected the integrity of tight junctions and the expression of zonula occludens-1(ZO-1)and occludin destroyed by BC.Furthermore,LGR-1 pretreatment reduced the expression of NOD-like receptor family member pyrin domain-containing protein 3(NLRP3),caspase recruitment and activation domain(ASC),Caspase-1 p20,gasdermin D(GSDMD)p30,inflammatory factors(interleukin(IL)-1βand IL-18),and cell death induced by BC.Moreover,LGR-1 pretreatment reduced NLRP3 inflammasome activity and increased expressions of ZO-1 and occludin induced by lipopolysaccharides(LPS)+ATP stimulation.MAC-T cells were transfected with NLRP3 si RNA or MCC950 and/or treated with BC and/or LGR-1.NLRP3-si RNA transfection and MCC950 attenuated BC-induced NLRP3 inflammasome activity.Expression of inflammatory cytokines and cell death suggested that the inflammatory pathway might play an important role in the induction of the NLRP3 inflammasome by BC and the protection of LGR-1.Conclusions:These results suggest that LGR-1 might be a probiotic alternative to antibiotics and could be administered to prevent mastitis in dairy cows,thus ensuring food security.
文摘The chronic complications of diabetes mellitus constitute a major public health problem.For example,diabetic eye diseases are the most important cause of blindness,and diabetic nephropathy is the most frequent cause of chronic kidney disease worldwide.The cellular and molecular mechanisms of these chronic complications are still poorly understood,preventing the development of effective treatment strategies.Tight junctions(TJs)are epithelial intercellular junctions located at the most apical region of cell-cell contacts,and their main function is to restrict the passage of molecules through the paracellular space.The TJs consist of over 40 proteins,and the most important are occludin,claudins and the zonula occludens.Accumulating evidence suggests that TJ disruption in different organs,such as the brain,nerves,retina and kidneys,plays a fundamental pathophysiological role in the development of chronic complications.Increased permeability of the blood-brain barrier and the blood-retinal barrier has been demonstrated in diabetic neuropathy,brain injury and diabetic retinopathy.The consequences of TJ disruption on kidney function or progression of kidney disease are currently unknown.In the present review,we highlighted the molecular events that lead to barrier dysfunction in diabetes.Further investigation of the mechanisms underlying TJ disruption is expected to provide new insights into therapeutic approaches to ameliorate the chronic complications of diabetes mellitus.
基金This work was financially supported by The Jiangsu Provincial Maternal and Child Health Key Talents Project(F202042).
文摘The placenta plays an important role in nutrient transport to maintain the growth and development of the embryo.Gestational diabetes mellitus(GDM),the most common complication during pregnancy,highly affects placental function in late gestation.Advanced glycation end-products(AGEs),a complex and heterogeneous group of compounds engaged by the receptor for AGEs(RAGE),are closely associated with diabetes-related complications.In this study,AGEs induced a decrease in the expression of tight junction(TJ)proteins in BeWo cells and increased the paracellular permeability of trophoblast cells by regulating RAGE/NF-κB.Sprague-Dawley(SD)rats injected with 100 mg/kg AGEs-rat serum albumin(RSA)via the tail vein from embryo day 2 were set as the placental barrier dysfunction model group(n=10).The effect of AGEs on placental permeability was determined using the Evans-Blue dye extravasation method.The ultrastructure of the placenta samples was observed by transmission electron microscopy.The effects of AGEs on the placenta were confirmed by treating rats with RAGE antagonist FPS-ZM1 and soluble forms of RAGE(sRAGE).AGEs treatment increased placental permeability and disrupted the tight junctions in pregnant rat placenta,but has no effect on blood glucose.The expression of TJ-related proteins,including ZO-1,Occludin,and Claudin 5,were downregulated after AGEs treatment.Further,AGEs treatment increased the expression of RAGE and nuclear factor-κB in the placenta of rats and upregulated the levels of vascular endothelial growth factor.The effects of AGEs on the placenta were blocked by RAGE antagonist FPS-ZM1 and sRAGE.This study demonstrates the mechanism underlying AGEs-induced disturbance in placental function in pregnant rats and highlights the potential of AGEs in the treatment of GDM.
文摘AIM To examine the effects of Acanthopanax senticosus polysaccharides(ASPS) on intestinal tight junction(TJ) disruption and nuclear factor-kappa B(NF-κB)/myosin light chain kinase(MLCK) activation in endotoxemia.METHODS BALB/C mice(6-8-weeks-old) received continuous intragastric gavage of ASPS for 7 d before injection of lipopolysaccharide(LPS), or received ASPS once after LPS injection. Blood and intestinal mucosal samples were collected 6 h after LPS challenge. Clinical symptoms, histological injury, intestinal permeability,TJ ultrastructure, and TJ protein expression were determined.RESULTS Compared with mice in the LPS group, pretreatment with ASPS improved clinical and histological scores by 390.9%(P < 0.05) and 57.89%(P < 0.05), respectively, and gut permeability change in endotoxemic mice was shown by a 61.93% reduction in reduced leakage of fluorescein isothiocyanate-dextran 6 h after LPS injection(P < 0.05). ASPS pretreatment also prevented LPS-induced TJ ultrastructure breakdown supported by increased electron dense materials between adjoining cells, sustained redistribution and expression of occludin(0.597 ± 0.027 vs 0.103 ± 0.009, P < 0.05) and zonula occludens-1(0.507 ± 0.032 vs 0.125 ± 0.019, P < 0.05), and suppressed activation of the NF-κB/MLCK pathway indicated by reduced expression of NF-κB, phospho-inhibitor kappa B-alpha, MLCK and phospho-myosin light-chain-2 by 16.06%(P < 0.05), 54.31%(P < 0.05), 66.10%(P < 0.05) and 64.82%(P < 0.05), respectively. CONCLUSION ASPS pretreatment may be associated with inhibition of the NF-κB/MLCK pathway and concomitant amelioration of LPS-induced TJ dysfunction of intestinal epithelium in endotoxemia.
基金partially supported by the funds from the National Natural Science Foundation of China (31772819, 31741115)Hunan Provincial Natural Science Foundation for Distinguished Young Scholars (2019JJ30012)Double-First-Class Construction Project of Hunan Province (kxk201801004)。
文摘Background: Weaning is one of the major factors that cause stress and intestinal disease in piglets. Protocatechuic acid(PCA) is an active plant phenolic acid which exists in Chinese herb, Duzhong(Eucommia ulmoides Oliver), and is also considered as the main bioactive metabolite of polyphenol against oxidative stress and inflammation. This study aimed to investigate the effect of PCA on growth performance, intestinal barrier function, and gut microbiota in a weaned piglet model challenged with lipopolysaccharide(LPS).Methods: Thirty-six piglets(Pig Improvement Company line 337 × C48, 28 d of age, 8.87 kg ± 0.11 kg BW) were randomly allocated into 3 treatments and fed with a basal diet(CTL), a diet added 50 mg/kg of aureomycin(AUR), or a diet supplemented with 4000 mg/kg of PCA, respectively. The piglets were challenged with LPS(10 μg/kg BW) on d 14 and d 21 by intraperitoneal injection during the 21-d experiment. Animals(n = 6 from each group) were sacrificed after being anesthetized by sodium pentobarbital at 2 h after the last injection of LPS. The serum was collected for antioxidant indices and inflammatory cytokines analysis, the ileum was harvested for detecting mRNA and protein levels of tight junction proteins by PCR and immunohistochemical staining, and the cecum chyme was collected for intestinal flora analysis using 16 S rRNA gene sequencing.Results: Dietary supplementation of PCA or AUR significantly increased the expression of tight junction proteins including ZO-1 and claudin-1 in intestinal mucosa, and decreased the serum levels of thiobarbituric acid reactive substances(TBARS) and IL-6, as compared with CTL group. In addition, PCA also decreased the serum levels of IL-2 and TNF-α(P < 0.05). Analysis of gut microbiota indicated that PCA increased the Firmicutes/Bacteroidetes ratio(P < 0.05). Spearman's correlation analysis at the genus level revealed that PCA reduced the relative abundance of Prevotella 9, Prevotella 2, Holdemanella, and Ruminococcus torques group(P < 0.05), and increased the relative abundance of Roseburia and Desulfovibrio(P < 0.05), whereas AUR had no significant effect on these bacteria.Conclusions: These results demonstrated that both PCA and AUR had protective effect on oxidative stress, inflammation and intestinal barrier function in piglets challenged with LPS, and PCA potentially exerted the protective function by modulating intestinal flora in a way different from AUR.
基金Supported by A GRF Grant from the Research Grants Council of Hong Kong to Luk JM,No.771607M
文摘The tight junction (TJ) is a critical cellular component for maintenance of tissue integrity, cellular interactions and cell-cell communications, and physiologically functions as the "great wall" against external agents and the surrounding hostile environment. During the host-pathogen evolution, viruses somehow found the key to unlock the gate for their entry into cells and to exploit and exhaust the host cells. In the liver, an array of TJ molecules is localized along the bile canaliculi forming the blood-biliary barrier, where they play pivotal roles in paracellular permeability, bile secretion, and cell polarity. In pathology, certain hepatic TJ molecules mediate virus entry causing hepatitis infection; deregulation and functional abnormality of the TJ have also been implicated in triggering liver cancer development and metastasis. All these findings shed new insights on the understanding of hepatic TJs in the development of liver disease and provide new clues for potential intervention.
基金Supported by Sanming Project of Medicine in Shenzhen of China,No.SZSM201612074
文摘BACKGROUND Progressive familial intrahepatic cholestasis(PFIC)encompasses a group of autosomal recessive disorders with high morbidity and mortality.Variants in the gene encoding tight junction protein-2(TJP2)have been linked to PFIC type 4(PFIC4),which predominantly presents in childhood.However,there are only limited data from adults with TJP2-related PFIC4.We report a family with an autosomal recessive disorder with a novel variant in the TJP2 gene in adults with very variable expression of PFIC4.CASE SUMMARY The index patient presented at 19 years old with liver cirrhosis and variceal bleeding and was treated with endoscopic banding and beta-blockers.In 2018,he developed primary liver cancer that was treated with radiofrequency ablation followed by liver transplantation in 2019.Genetic testing revealed a novel homozygous TJP2 variant causing PFIC4(TJP2([NM_004817.3]:c.[3334C>T];[3334C>T])).The consanguineous family consists of the father and mother(both heterozygous)and their 12 children,of which five carry the variant in a homozygous state;however,these five siblings have highly variable expression of PFIC4.Two homozygous brothers had cirrhosis and portal hypertension at diagnosis at the ages of 19 and 36.Two other homozygous brothers,age 23 and 19,and the homozygous sister,age 21,have elevated liver enzymes but presently no cirrhosis,which may suggest an age-dependent penetrance.In addition,five sisters had severe and mild intrahepatic cholestasis of pregnancy and carry the TJP2 variant in a homozygous and heterozygous state,respectively.CONCLUSION This novel TJP2 variant is associated with PFIC4 causing severe liver disease with cirrhosis and primary liver cancer in adolescents/adults.
文摘Tight junctions(TJs) are the most apical intercellular junctions of epithelial cells formed by occludin, claudins, junctional adhesion molecules(JAMs), and zonula occludens(ZO). Tight junction proteins can sense the presence of bacteria and regulate the transcription of target genes that encode effectors and regulators of the immune response. The aim of this study was to determine the impact of TJ proteins in response to Porphyromonas gingivalis(P. gingivalis), P. gingivalis lipopolysaccharide(P. gingivalis LPS), and extracellular adenosine triphosphate(ATP) in the oral epithelial cell culture model. Quantified real timepolymerase chain reaction(RT-PCR), immunoblots, and immunostaining were performed to assess the gene and protein expression in TJs. It was found that P. gingivalis infection led to transient upregulation of the genes encoding occludin, claudin-1, and claudin-4 but not JAM-A, claudin-15, or ZO-1, while P. gingivalis LPS increased claudin-1, claudin-15, and ZO-1 and decreased occludin, JAM-A, and claudin-4. Tight junction proteins showed significant upregulation in the above two groups when cells were pretreated with ATP for 3 h. The findings indicated that P. gingivalis induced the host defence responses at an early stage. P. gingivalis LPS exerted a more powerful stimulatory effect on the disruption of the epithelial barrier than P. gingivalis.ATP stimulation enhanced the reaction of TJ proteins to P. gingivalis invasion and LPS destruction of the epithelium.
文摘Objective:In this study,the influence of puerarin,paeoniflorin,and menthol on the structure and barrier function of tight junctions(TJs)in MadineDarby canine kidney epithelial(MDCK)and MDCK-multi-drug resistance 1(MDR1)cells was evaluated to determine the mechanisms by which the drugs cross the bloodebrain barrier(BBB).Method:Cells were treated with puerarin,paeoniflorin,and menthol followed by immunohistochemical staining with occludin,claudin-1,and F-actin.The cells were then observed using laser-scanning confocal microscopy.Average optical density(AOD)of the immunofluorescence images of the proteins were analyzed using ImageJ software while Transepithelial electrical resistance(TEER)was measured using an epithelial voltohmmeter.Results:Confocal microscopy revealed that puerarin-and paeoniflorin-treated tight junction proteins were conspicuous while menthol suppressed their expression.Correspondingly,AOD values of cells treated with puerarin or paeoniflorin,or both showed no difference compared to the control group(P>.05)while the menthol group value was downregulated.In 3 h,TEER of cells not treated with menthol were similar to the control group,while treatment with menthol significantly decreased TEER value(P<.05).In addition,application of menthol decreased TEER in MDCK cells earlier than in MDCK-MDR1 cells.Conclusion:Menthol but not puerarin and paeoniflorin may enhance paracellular transport and improve drug penetration of the BBB by disrupting the structure and,thereby,weakening the barrier function of TJs.
文摘AIM:To investigate the mechanism of the tight junction(TJ) disruption and the association between tumor necrosis factor(TNF)-α and matrix metalloproteinase(MMPs) under hyperosmotic condition in primary human corneal epithelial cells(HCECs).METHODS:The cultured HCECs were exposed to media which adding sodium chloride(Na Cl) for hyperosmolar stress or adding rh-TNF-α(10 ng/m L). NF-κB inhibitor(5 μmol/L) or GM-6001(potent and broad spectrum MMP inhibitor, 20 μmol/L)was added 1 h before that treatment. The integrity of TJ proteins was determined by immunofluorescent(IF) staining. The m RNA levels of TNF-α and MMPs were evaluated by quantitative reverse transcription polymerase chain reaction(RT-q PCR) and the protein expression by enzyme-linked immunosorbent assay(ELISA).RESULTS:TJ proteins ZO-1 and Occludin were disrupted in primary HCECs exposed to hyperosmotic medium. The m RNA expression and protein production of TNF-α increased significantly in hyperosmotic media at 500 m Os M. TNF-α mediated the expression and production of MMP-1, MMP-13, MMP-9, and MMP-3 stimulated by hyperosmotic stress. The production of MMPs in hyperosmolar media were increased through the increase of TNF-α. GM-6001 prevent the destruction of ZO-1 and Occludin in hyperosmolar stress and rh-TNF-α treated medium. TNF-α induced activation of MMPs was involved in the TJ disruption by hyperosmolarity.CONCLUSION:TJ proteins ZO-1 and Occludin are disrupted by hyperosmolar stress and TNF-α, but protected by MMP inhibitor(GM-6001). It suggests that TNF-α/MMP pathway mediates the TJ disruption in primary HCECs exposed to hyperosmotic stress.
文摘The differentiation of cells composing mature human hairs produces layers with different corneous characteristics that would tend to flake away one from another,as in the corneous layer of the epidermis,without anchoring junctions.It is likely that cell junctions established in the forming cells of the hair bulb are not completely degraded like in the corneous layer of the epidermis but instead remain in the hair shaft to bind mature cuticle,cortex,and medulla cells into a compact hair shaft.During cell differentiation in hairs,cell junctions seem to disappear,and little is known about the fate of junctional proteins present in the mature human hair shaft.The present ultrastructural immunogold study has detected some marker proteins of adhesion junction(cadherin and beta-catenin)and tight junctions(occludin and cingulin)that are still present in cornified hairs where numerous isopeptide bonds are detected,especially in the medulla.This qualitative ultrastructural study indicates that aside from the cell membrane complex,a long corneo-desmosome bonding cortex and cuticle cells,also sparse adherens and tight junction remnants are present.It is suggested that the cornification of these junctions with the incorporation of their proteins within the mature corneous material of the hair shaft likely contributes to maintaining the integrity of the mature hair.This information will also allow us to evaluate the effects of different chemical components present in hair formulations and stains on these junctional proteins and the consequent integrity of the hair shaft.
基金Supported by JIPMER intramural research grantIndian Council of Medical Research(ICMR),New Delhi,India,No.3/1/3 J.R.F.-2016/LS/HRDDepartment of Biotechnology,Government of India,No.102/IFD/SAN/22/2013-14.
文摘BACKGROUND Altered tight junction(TJ)proteins are correlated with carcinogenesis and tumor development.Nimbolide is a tetranotriterpenoid that has been shown to have antioxidant and anti-proliferative properties;however,its anticancer effects and molecular mechanism in hepatocellular carcinoma(HCC)remains obscure.AIM To investigate the effect of nimbolide on TJ proteins,cell cycle progression,and hepatic inflammation in a mouse model of HCC.METHODS HCC was induced in male Swiss albino mice(CD-1 strain)by a single intraperitoneal injection of 100 mg/kg diethylnitrosamine(DEN)followed by 80 ppm N-nitrosomorpholine(NMOR)in drinking water for 28 wk.After 28 wk,nimbolide(6 mg/kg)was given orally for four consecutive weeks in DEN/NMOR induced HCC mice.At the end of the 32nd week,all the mice were sacrificed and blood and liver samples were collected for various analyses.Macroscopic examinations of hepatic nodules were assessed.Liver histology and HCC tumor markers such as alpha-fetoprotein(AFP)and glypican-3 were measured.Expression of TJ proteins,cell proliferation,and cell cycle markers,inflammatory markers,and oxidative stress markers were analyzed.In silico analysis was performed to confirm the binding and modulatory effect of nimbolide on zonula occludens 1(ZO-1),nuclear factor of kappa light polypeptide gene enhancer in B-cells(NF-κB),and tumor necrosis factor alpha(TNF-α).RESULTS We found nimbolide treatment at a concentration of 6 mg/kg to HCC mice reduced hepatic tumor size by 52.08%and tumor volume(P<0.01),and delayed tumor growth in HCC mice with a concomitant reduction in tumor markers such as AFP levels(P<0.01)and glypican-3 expression(P<0.05).Furthermore,nimbolide treatment increased tight junction proteins such as ZO-1 and occludin expression(P<0.05,respectively)and reduced ZO-1 associated nucleic acid binding protein expression(P<0.001)in HCC mice liver.Nimbolide treatment to HCC mice also inhibited cell proliferation and suppressed cell cycle progression by attenuating proliferating cell nuclear antigen(P<0.01),cyclin dependent kinase(P<0.05),and CyclinD1(P<0.05)expression.In addition,nimbolide treatment to HCC mice ameliorated hepatic inflammation by reducing NF-κB,interleukin 1 beta and TNF-αexpression(P<0.05,respectively)and abrogated oxidative stress by attenuating 4-hydroxynonenal expression(P<0.01).Molecular docking studies further confirmed that nimbolide interacts with ZO-1,NF-κB,and TNF-α.CONCLUSION Our current study showed for the first time that nimbolide exhibits anticancer effect by reducing tumor size,tumor burden and by suppressing cell cycle progression in HCC mice.Furthermore,nimbolide treatment to HCC mice ameliorated inflammation and oxidative stress,and improved TJ proteins expression.Consequently,nimbolide could be potentially used as a natural therapeutic agent for HCC treatment,however further human studies are warranted.
文摘Tight junctions are mainly formed by two types of proteins;claudins and occludin, both of which are fundamental to maintain the integrity and barrier function of the intestinal epithelium. This barrier function allows for the absorption of nutrients, mainly by transcytosis;however, in birds, 90% of the substances are absorbed by paracellular mechanisms. Despite this, claudins present in th<span style="font-family:Verdana;"></span><span style="font-family:;" "="">e different parts of the intestinal tract of adult chickens are not known, much less their functional role. This study aimed to determine the presence of mRNA of claudins 1, 2, 3, 5, 10, 12, 16 and occludin, in the different regions of the intestine (duodenum, jejunum, ileum, cecum, and rectum) in chickens (<i>Gallus gallus domesticus</i>) through RT-PCR. To meet this goal, 7 weeks old roosters destined for slaughter and chicken embryos of 16 days of incubation (positive control) were used. For all the processed samples, amplicons of the expected size were obtained;claudin 1 (662 pb), claudin 2 (162 pb), claudin 3 (185 pb), claudin 5 (224 pb), claudin 10 (687 pb), claudin 12 (738 pb), claudin 16 (191 pb) and occludin (430 pb). To corroborate these findings, obtained amplicons were sequenced and, subsequently, a basic alignment was performed on the NCBI, obtaining a correlation of 100% with the original sequences in all analyzed samples. To our knowledge, the present work represents the first written report regarding the presence of mRNA of the main proteins involved in tight junction formation throughout the intestinal tract of domestic chickens of 7 weeks of age. These findings will allow elucidating the specific function of each of the reported proteins in the process of paracellular absorption in chickens.</span>
文摘We aimed to examine paracellular barrier function in cultured mouse lung microvascular endothelial cells (LMECs). The transcellular resistance of LMEC monolayers yielded an electrical resistance of approximately 19 Ω × cm<sup>2</sup> at days 6 - 7 in culture when the cells reached confluence, and paracellular permeable clearance of sodium fluorescein was the lowest on day 6 in culture, suggesting the formation of tight junctions (TJs) in cultured LMECs. Moreover, the expression of TJ-associated proteins, occludin, claudin-1 and -4 and zonula occludents 1 (ZO-1) was detected in LMECs at day 6 in culture. However, mRNAs of occludin, claudin-1 and -4 and ZO-1 were already expressed on day 1 after culture, and large variations were absent in the mRNA levels of occludin, claudin-4 and ZO-1 between days 1 and 7 in culture, when the level of each mRNA on day 1 in culture was used as a basal level. However, the claudin-1 mRNA level gradually increased up to approximately 7-fold on day 7 in culture over the basal level. These results indicate that the drastic increase in the mRNA expression level of claudin-1 leads to the strong formation of TJs.
文摘Background:Cells are influenced by their environment.In vivo,the corneal endothelium is subjected to intraocular pressure(IOP).The purpose of this project was to evaluate in vitro,the effect of the IOP on the formation of tight junctions in the corneal endothelium.Methods:Cultivated corneal endothelial cells(P2-P3;n=6 populations)were seeded on devitalized on corneas(n=10 pairs).Native corneas and devitalized corneas were respectively used as positive(n=2 pairs)and negative controls(n=3 pairs).Corneas were placed in artificial anterior chambers and subjected to a hydrostatic pressure between 0.3 and 0.4 psi during 4-5 days.Unpressured control corneas were maintained in cell culture dishes.Pictures of the corneas were taken following the experiment to assess stromal transparency.Morphology,corneal thickness and distribution of ZO-1,n-cadherin,b-catenin,NaK ATPase pump and HCO3-cotransporter were evaluated by electron microscopy,histological staining and immunofluorescences.Results:Pressure treated corneas were more transparent than the controls.Thickness was accordingly reduced by 38.4%±4.9%for cultivated endothelium and 32.2%±2.7%for native endothelium.Negative controls change in transparency and thickness were marginal.Pressure treated cells showed none or at most marginal difference in morphology and expression of ZO-1,n-cadherin,b-catenin,NaK ATPase pump and HCO3-cotransporters and failed to recreate a phenotype similar to native corneas.Pressure however increased cortical localisation of the protein ZO-1 in both cultivated and native endothelium.Conclusions:These results suggest that anterior chamber hydrostatic pressure may enhance endothelial functionality by modulating the distribution of tight junction’s proteins.
基金Supported by The Association for International Cancer Research(AICRto Dr.Al-Hassi HO)+6 种基金ScotlandFunded by the AICRgrant No.120234a BBSRC Strategic Research Grant(to English N and Knight SCWMNIP33458)the St Mark’s Hospital FoundationUnited Kingdom
文摘Inflammatory bowel diseases are characterised by inflammation that compromises the integrity of the epithelial barrier. The intestinal epithelium is not only a static barrier but has evolved complex mechanisms to control and regulate bacterial interactions with the mucosal surface. Apical tight junction proteins are critical in the maintenance of epithelial barrier function and control of paracellular permeability. The characterisation of alterations in tight junction proteins as key players in epithelial barrier function in inflammatory bowel diseases is rapidly enhancing our understanding of critical mechanisms in disease pathogenesis as well as novel therapeutic opportunities. Here we give an overview of recent literature focusing on the role of tight junction proteins, in particular claudins, in inflammatory bowel diseases and inflammatory bowel disease associated colorectal cancer.
基金Supported by National Natural Science Foundation of China,No.30772831,No.81473757the National Basic Research Program of China,973 Program,No.2009CB522900
文摘AIM:To investigate the effect of herb-partitioned moxibustion combined with acupuncture on the expression of intestinal epithelial tight junction(TJ) proteins.METHODS:Sixty patients diagnosed with mild to moderate Crohn’s disease(CD)were allocated into the herb-partitioned moxibustion combined with acupuncture(HMA)group(n=30)or the mesalazine(MESA)group(n=30)using a parallel control method.There were 2 sets of acupoints used alternately for HMA treatment.The following points were included in Set A:ST25(Tianshu),RN6(Qihai),and RN9(Shuifen)for herb-partitioned moxibustion and ST36(Zusanli),ST37(Shangjuxu),LI11(Quchi),and LI4(Hegu)for acupuncture.The points for Set B included BL23(Shenshu)and BL25(Dachangshu)for herb-partitioned moxibustion and EX-B2 of T6-T1(Jiajixue)fo r acupuncture.The patients received the same treatment6 times a week for 12 consecutive weeks.The MESA group received 1 g of mesalazine enteric coated tablets4 times daily for 12 consecutive weeks.Intestinaltissues were stained and examined to compare the morphological and ultrastructural changes before and after the treatment session.Immunohistochemistry and in situ hybridization assays were used to detect the expression of intestinal epithelial TJ proteins zonula occludens-1(ZO-1),occludin,and claudin-1.The m RNA levels were also evaluated.RESULTS:After the treatment,both herb-partitioned moxibustion combined with acupuncture and mesalazine improved intestinal morphology and ultrastructure of CD patients;the patients treated with HMA showed better improvement.HMA significantly increased the expression of ZO-1(P=0.000),occludin(P=0.021),and claudin-1(P=0.016).MESA significantly increased the expression of ZO-1(P=0.016)and occludin(P=0.026).However,there was no significant increase in the expression of claudin-1(P=0.935).There was no statistically significant difference between the two groups for the expression of occludin and claudin-1(P>0.05).The HMA group showed a significant improvement in ZO-1 expression compared to the MESA group(2333.34±352.51 vs 2160.38±307.08,P=0.047).HMA significantly increased the expression of ZO-1 m RNA(P=0.000),occludin m RNA(P=0.017),and claudin-1 m RNA(P=0.017).MESA significantly increased the expression of ZO-1 m RNA(P=0.000),occludin m RNA(P=0.042),and claudin-1 m RNA(P=0.041).There was no statistically significant difference between the two groups in the expression of occludin and claudin-1 m RNA(P>0.05).However,the HMA group showed a significant improvement in ZO-1 m RNA expression compared with the MESA group(2378.17±308.77 vs 2200.56±281.88,P=0.023).CONCLUSION:HMA can repair intestinal epithelial barrier lesions and relieve inflammation by upregulating the expression of TJ proteins and their m RNAs.
基金Supported by National Natural Science Foundation of China,No. 30772831National Basic Research Program of China, 973program, No. 2009CB522900Shanghai Leading Discipline Project, No. S30304
文摘AIM: To investigate the effects of moxibustion on down-regulation of the colonic epithelial cell apoptosis and repair of the tight junctions in rats with Crohn's disease (CD). METHODS: Sixty male Sprague-Dawley rats were randomly divided into a normal control (NC) group, a model control (MC) group, an herbs-partitioned moxibustion (HPM) group, a mild-warm moxibustion (MWM) group and a salicylazosulphapyridine (SASP) group, with 12 rats in each group. The CD model rats were treated with trinitrobenzene sulphonic acid to induce intestinal inflammation. The rats in the HPM and MWM groups were treated at the Tianshu (ST25) and Qihai (CV6) acupoints once daily for 14 d, and the SASP group was fed SASP twice daily for 14 d. No additional treatment was given to the MC and NC groups. Themicrostructure of the colonic epithelium was observed under a transmission electron microscope, the transepithelial resistance was measured using a shortcircuit current, colonic epithelial cell apoptosis was determined by terminal deoxynucleotidyl transferasemediated dUTP-biotin nick end labelling assay, and the expression of occludin, claudin-1 and zonula occludens-l (ZO-1) in the colonic epithelial junction was determined by Western blotting and immunofluorescence staining. RESULTS: Compared with the MC group, the microstructure of the colonic epithelial barrier was signifi-cantly improved in rats treated with HPM, MWM or SASP, meanwhile, the current flow was reduced signifi-cantly, with values of 168.20 ± 6.14 vs 99.70 ± 3.13, 99.10 ± 4.28 and 120.30 ± 3.65 mA, respectively (P = 0.001). However, the HPM and MWM groups had higher current flow rates than the SASP group (99.70 ± 3.13, 99.10 ± 4.28 vs 120.30 ± 3.65 mA, P = 0.001). The number of the apoptotic colonic epithelial cells in HPM, MWM and SASP groups was largely reduced (61.5 ± 16.91 vs 15.5 ± 8.89, 14.8 ± 6.27 and 24.7 ± 9.68, respectively (P = 0.001); and the expression of occlu- din, claudin-1 and ZO-1 in the MWM and HPM groups was signifi cantly enhanced (0.48 ± 0.10, 0.64 ± 0.09 vs 0.18 ± 0.05 for occludin, 0.12 ± 0.02, 0.17 ± 0.03 vs 0.05 ± 0.01 for claudin-1, and 0.08 ± 0.01, 0.11 ± 0.01 vs 0.02 ± 0.01 for ZO-1). And in SASP group, the expression of occludin and ZO-1 was also signifi cantly increased (0.27 ± 0.04 vs 0.18 ± 0.05 for occludin and 0.05 ± 0.01 vs 0.02 ± 0.01 for ZO-1), but there was no significant difference for claudin-1. The HPM and MWM groups had higher expression of occludin, claudin-1 and ZO-1 than the SASP group. CONCLUSION: HPM and MWM treatment can down-regulate apoptosis of colonic epithelial cells, repair tight junctions and enhance colonic epithelial barrier function in rats with CD.
基金Supported by National Natural Science Foundation of China,No. 81160050Science and Technology Support Program of China, No. 2008BAI68B01+1 种基金Science and Technology Support Program of Jiangxi Province, No. 20111BBG70015-3Natural Science Foundation of Jiangxi Province, No. 2007GQY0997
文摘AIM: To investigate the effects of rhein on intestinal epithelial tight junction proteins in rats with IgA nephropathy (IgAN). METHODS: Twenty-eight female Sprague-Dawley rats were randomly divided into four groups (7 per group): Control, IgAN, Rhein-treated, and Rheinprevented. Bovine serum albumin, lipopolysaccharide and CCl4 were used to establish the rat model of IgA nephropathy. The Rhein-treated group was given rhein from week 7 until the rats were sacrificed. The Rheinprevented group was given rhein from week 1. Animals were sacrificed at the end of week 10. We observed the changes in the intestinal epithelial tight junctions using transmission electron microscopy, and expression of intestinal epithelial tight junction proteins zona occludens protein (ZO)-1 and occludin by immunofluorescence using laser confocal microscopy. Changes in mRNA and protein expression of ZO-1 and occludin were measured by reverse transcriptase polymerase chain reaction and Western blotting. The ratio of urinary lactulose/mannitol was measured by high performance liquid chromatography (HPLC) for assessing the intestinal permeability. RESULTS: In the control group, the tight junctions lied between epithelial cells on the top of the outer side of the cell membrane, and appeared in dense dotted crystal structures, the neighboring cells were binded tightly with no significant gap, and the tight junction protein ZO-1 and occludin were evenly distributed in the intestinal epithelial cells at the top of the junction. Compared with the control group, in the IgAN group, the structure of the tight junction became obscured and the dotted crystal structures had disappeared; the fluorescence of ZO-1 and occludin was uneven and weaker (5.37 ± 1.27 vs 10.03 ± 1.96, P < 0.01; 4.23 ± 0.85 vs 12.35 ± 4.17, P < 0.01); the mRNA expression of ZO-1 and occludin decreased (0.42 ± 0.19 vs 0.92 ± 0.24, P < 0.01; 0.40 ± 0.15 vs 0.97 ± 0.25, P < 0.01); protein expression of ZO-1 and occludin was decreased (0.85 ± 0.12 vs 1.98 ± 0.43, P < 0.01; 0.72 ± 0.15 vs 1.38 ± 0.31, P < 0.01); and the ratio of urinary lactulose/mannitol increased (3.55 ± 0.68 vs 2.72 ± 0.21, P < 0.01). In the Rheinprevented and Rhein-treated groups, compared with the IgAN group, the intestinal epithelial tight junctions were repaired; fluorescence of ZO-1 and occludin was stronger (11.16 ± 3.52 and 8.81 ± 2.30 vs 5.37 ± 1.27, P < 0.01; 10.97 ± 3.40 and 9.46 ± 2.40 vs 4.23 ± 0.85, P < 0.01); mRNA of ZO-1 and occludin increased (0.81 ± 0.17 and 0.64 ± 0.16 vs 0.42 ± 0.19, P < 0.01; 0.82± 0.22 and 0.76 ± 0.31 vs 0.40 ± 0.15, P < 0.01); protein expression of ZO-1 and occludin was increased (2.07 ± 0.41 and 1.57 ± 0.23 vs 0.85 ± 0.12, P < 0.01; 1.34 ± 0.21 and 1.15 ± 0.17 vs 0.72 ± 0.15, P < 0.01); and the ratio of urinary lactulose/mannitol decreased (2.83 ± 0.43 and 2.87 ± 0.18 vs 3.55 ± 0.68, P < 0.01). CONCLUSION: Rhein can enhance the expression of ZO-1 and occludin, repair damaged tight junctions, and protect the intestinal barrier.
