The development of receptive endometrium(RE) from pre-receptive endometrium(PE) for successful embryo implantation is a complex dynamic process in which the morphology and physiological states of the endometrial epith...The development of receptive endometrium(RE) from pre-receptive endometrium(PE) for successful embryo implantation is a complex dynamic process in which the morphology and physiological states of the endometrial epithelium undergo a series of significant changes, including cell proliferation and apoptosis. However, the molecular mechanisms are not yet fully understood. In this study, a higher circRNA3669 level was observed in PE than in RE of goats. Functional assays revealed that this overexpression promoted the proliferation of goat endometrial epithelial cells(GEECs) by activating the expression of genes related to the PI3K/AKT-mTOR and MAPK pathways,thereby inhibiting apoptosis in vitro. Furthermore, circRNA3669 functioned as a competing endogenous RNA(ceRNA) to upregulate Reticulocalbin-2(RCN2) expression at the post-transcriptional level by interacting with and downregulating miR-26a in GEECs. In addition, RCN2, which is highly expressed in the PE of goats, was found to be regulated by β-estradiol(E2) and progesterone(P4). Our results demonstrated that RCN2 also affected the key proteins PI3K, AKT, mTOR, JNK, and P38 in the PI3K/AKT-mTOR and MAPK pathways, thereby facilitating GEECs proliferation and suppressing their apoptosis in vitro. Collectively, we constructed a new circRNA3669-miR-26aRCN2 regulatory network in GEECs, which further provides strong evidence that circRNA could potentially play a crucial regulatory role in the development of RE in goats.展开更多
●AIM:To explore whether autophagy functions as a cellular adaptation mechanism in lens epithelial cells(LECs)under hyperosmotic stress.●METHODS:LECs were treated with hyperosmotic stress at the concentration of 270,...●AIM:To explore whether autophagy functions as a cellular adaptation mechanism in lens epithelial cells(LECs)under hyperosmotic stress.●METHODS:LECs were treated with hyperosmotic stress at the concentration of 270,300,400,500,or 600 mOsm for 6,12,18,24h in vitro.Polymerase chain reaction(PCR)was employed for the mRNA expression of autophagyrelated genes,while Western blotting detected the targeted protein expression.The transfection of stub-RFP-sens-GFPLC3 autophagy-related double fluorescence lentivirus was conducted to detect the level of autophagy flux.Scanning electron microscopy was used to detect the existence of autolysosome.Short interfering RNA of autophagy-related gene(ATG)7,transient receptor potential vanilloid(TRPV)1 overexpression plasmid,related agonists and inhibitors were employed to their influence on autophagy related pathway.Flow cytometry was employed to test the apoptosis and intracellular Ca^(2+)level.Mitochondrial membrane potential was measured by JC-1 staining.The cell counting kit-8 assay was used to calculate the cellular viability.The wound healing assay was used to evaluate the wound closure rate.GraphPad 6.0 software was utilized to evaluate the data.●RESULTS:The hyperosmotic stress activated autophagy in a pressure-and time-dependent manner in LECs.Beclin 1 protein expression and conversion of LC3B II to LC3B I increased,whereas sequestosome-1(SQSTM1)protein expression decreased.Transient Ca^(2+)influx was stimulated caused by hyperosmotic stress,levels of mammalian target of rapamycin(mTOR)phosphorylation decreased,and the level of AMP-activated protein kinase(AMPK)phosphorylation increased in the early stage.Based on this evidence,autophagy activation through the Ca^(2+)-dependent AMPK/mTOR pathway might represent an adaptation process in LECs under hyperosmotic stress.Hyperosmotic stress decreased cellular viability and accelerated apoptosis in LECs and cellular migration decreased.Inhibition of autophagy by ATG7 knockdown had similar results.TRPV1 overexpression increased autophagy and might be crucial in the occurrence of autophagy promoted by hyperosmotic stress.●CONCLUSION:A combination of hyperosmotic stress and autophagy inhibition may be a promising approach to decrease the number of LECs in the capsular bag and pave the way for improving prevention of posterior capsular opacification and capsular fibrosis.展开更多
Background Mastitis not only deteriorates the composition or quality of milk,but also damages the health and pro-ductivity of dairy goats.Sulforaphane(SFN)is a phytochemical isothiocyanate compound with various pharma...Background Mastitis not only deteriorates the composition or quality of milk,but also damages the health and pro-ductivity of dairy goats.Sulforaphane(SFN)is a phytochemical isothiocyanate compound with various pharmacologi-cal effects such as anti-oxidant and anti-inflammatory.However,the effect of SFN on mastitis has yet to be elucidated.This study aimed to explore the anti-oxidant and anti-inflammatory effects and potential molecular mechanisms of SFN in lipopolysaccharide(LPS)-induced primary goat mammary epithelial cells(GMECs)and a mouse model of mastitis.Results In vitro,SFN downregulated the mRNA expression of inflammatory factors(tumor necrosis factor-α(TNF-α),interleukin(IL)-1βand IL-6),inhibited the protein expression of inflammatory mediators(cyclooxygenase-2(COX2),and inducible nitric oxide synthase(iNOS))while suppressing nuclear factor kappa-B(NF-κB)activation in LPS-induced GMECs.Additionally,SFN exhibited an antioxidant effect by increasing Nrf2 expression and nuclear translocation,up-regulating antioxidant enzymes expression,and decreasing LPS-induced reactive oxygen species(ROS)produc-tion in GMECs.Furthermore,SFN pretreatment promoted the autophagy pathway,which was dependent on the increased Nrf2 level,and contributed significantly to the improved LPS-induced oxidative stress and inflammatory response.In vivo,SFN effectively alleviated histopathological lesions,suppressed the expression of inflammatory factors,enhanced immunohistochemistry staining of Nrf2,and amplified of LC3 puncta LPS-induced mastitis in mice.Mechanically,the in vitro and in vivo study showed that the anti-inflammatory and anti-oxidative stress effects of SFN were mediated by the Nrf2-mediated autophagy pathway in GMECs and a mouse model of mastitis.Conclusions These results indicate that the natural compound SFN has a preventive effect on LPS-induced inflam-mation through by regulating the Nrf2-mediated autophagy pathway in primary goat mammary epithelial cells and a mouse model of mastitis,which may improve prevention strategies for mastitis in dairy goats.展开更多
AIM:To investigate the effect of morroniside(Mor)on lipopolysaccharide(LPS)-treated iris pigment epithelial cells(IPE).METHODS:IPE cells were induced by LPS and treated with Mor.Cell proliferation was detected by cell...AIM:To investigate the effect of morroniside(Mor)on lipopolysaccharide(LPS)-treated iris pigment epithelial cells(IPE).METHODS:IPE cells were induced by LPS and treated with Mor.Cell proliferation was detected by cell counting kit(CCK)-8,apoptosis was detected by flow cytometry,the levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-6,and IL-8 were measured by enzyme-linked immunosorbent assay(ELISA)kits,and the protein expression of TLR4,JAK2,p-JAK2,STAT3,and p-STAT3 was analyzed by Western blotting.In addition,overexpression of TLR4 and Mor treatment of LPS-stimulated IPE cells were also tested for the above indices.RESULTS:Mor effectively promoted the proliferation and inhibited the apoptosis of LPS-treated IPE cells.In addition,Mor significantly reduced the levels of TNF-α,IL-6,and IL-8 and significantly inhibited the expression of TLR4,p-JAK2,and p-STAT3 in LPS-treated IPE cells.The effect of Mor on LPS-treated IPE cells was markedly attenuated after overexpression of TLR4.CONCLUSION:These findings suggest that Mor may ameliorate LPS-induced inflammatory damage and apoptosis in IPE through inhibition of TLR4/JAK2/STAT3 pathway.展开更多
AIM:To explore the effects of hepatocyte growth factor(HGF)on retinal pigment epithelium(RPE)cell behaviors.METHODS:The human adult retinal pigment epithelial cell line-19(ARPE-19)were treated by HGF or mesenchymalepi...AIM:To explore the effects of hepatocyte growth factor(HGF)on retinal pigment epithelium(RPE)cell behaviors.METHODS:The human adult retinal pigment epithelial cell line-19(ARPE-19)were treated by HGF or mesenchymalepithelial transition factor(MET)inhibitor SU11274 in vitro.Cell viability was detected by a Cell Counting Kit-8 assay.Cell proliferation and motility was detected by a bromodeoxyuridine incorporation assay and a wound healing assay,respectively.The expression levels of MET,phosphorylated MET,protein kinase B(AKT),and phosphorylated AKT proteins were determined by Western blot assay.The MET and phosphorylated MET proteins were also determined by immunofluorescence assay.RESULTS:HGF increased ARPE-19 cells’viability,proliferation and migration,and induced an increase of phosphorylated MET and phosphorylated AKT proteins.SU11274 significantly reduced cell viability,proliferation,and migration and decreased the expression of MET and AKT proteins.SU11274 suppressed HGF-induced increase of viability,proliferation,and migration in ARPE-19 cells.Additionally,SU11274 also blocked HGF-induced phosphorylation of MET and AKT proteins.CONCLUSION:HGF enhances cellular viability,proliferation,and migration in RPE cells through the MET/AKT signaling pathway,whereas this enhancement is suppressed by the MET inhibitor SU11274.HGF-induced MET/AKT signaling might be a vital contributor of RPE cells survival.展开更多
In order to improve the bioavailability of lutein(LUT),a novel lutein-stevio side nanoparticle(LUT-STE)were prepared previously,but the information about LUT-STE on protecting of eye health was limited.This study inve...In order to improve the bioavailability of lutein(LUT),a novel lutein-stevio side nanoparticle(LUT-STE)were prepared previously,but the information about LUT-STE on protecting of eye health was limited.This study investigated the effect of LUT-STE on antioxidant activity of H_(2)O_(2)-induced human retinal pigment epithelial(ARPE)cells.LUT and LUT-STE(final concentration of 5μg/mL)significantly enhanced cell viability from(74.84±5.10)%to(81.92±10.01)%(LUT)and(89.33±4.34)%(LUT-STE),and inhibited the cell apoptosis(P<0.05).After pretreatment with LUT-STE in ARPE cells,the levels of superoxide dismutase(SOD),catalase(CAT)and glutathion peroxidase(GSH-Px)in ARPE cells were significantly increased(P<0.05),the contents of reactive oxygen species(ROS)and malondialdehyde(MDA)were decreased.In addition,the vascular endothelial growth factor(VEGF)levels were inhibited by 13.61%and 17.39%,respectively,pretreatment with LUT and LUT-STE.Western blotting results showed that the pretreatment with LUT-STE inhibited the expression of caspase-9 and caspase-3 and up-regulated Bcl-2/Bax pathway to inhibit H_(2)O_(2)-induced apoptosis.In summary,the novel delivery LUT-STE had more pronounced inhibitory effect on H_(2)O_(2)-induced damage in human ARPE cells.展开更多
Background:Inflammatory bowel disease(IBD)is a chronic inflammatory disease of the gastrointestinal tract.The destruction of the intestinal epithelial barrier is one of the major pathological processes in IBD patholog...Background:Inflammatory bowel disease(IBD)is a chronic inflammatory disease of the gastrointestinal tract.The destruction of the intestinal epithelial barrier is one of the major pathological processes in IBD pathology.Growing evidence indicated that epithelial cell ferroptosis is linked to IBD and is considered a target process.Methods:RAS-selective lethal 3(RSL3)was used to induce ferroptosis in intestinal epithelial cell line No.6(IEC-6)cells,and cell ferroptosis and the effects of tanshinone IIA(Tan IIA)were determined by cell counting kit-8(CCK-8),reactive oxygen species(ROS)staining,Giemsa staining and transmission electron microscope(TEM).The cell viability of natural product library compounds was determined by CCK-8.The expression of ferroptosis-related genes were detected by real-time quantitative polymerase chain reaction(RT-qPCR)and western blot.Results:Treatment of IEC-6 cells results in the accumulation of ROS and typical morphological characteristics of ferroptosis.RSL3 treatment caused rapid cellular cytotoxicity which could be reversed by ferrostatin-1(Fer-1)in IEC-6 cells.Natural product library screening revealed that Tan IIA is a potent inhibitor of IEC-6 cell ferroptosis.Tan IIA could significantly protect the RSL3-induced ferroptosis of IEC-6 cells.Furthermore,the ferroptosis suppressors,glutathione peroxidase 4(GPX4),solute carrier family 7 member 11(SLC7A11),and miR-17-92 were found to be early response genes in RSL3-treated cells.Treatment of IEC-6 cells with Tan IIA resulted in upregulation of GPX4,SLC7A11,and miR-17-92.Conclusion:Our study demonstrated that Tan IIA protects IEC-6 cells from ferroptosis through the upregulation of GPX4,SLC7A11,and miR-17-92.The findings might provide a theoretical grounding for the future application of Tan IIA to treat or prevent IBD.展开更多
Objective:To explore the regulatory mechanism of transient receptor potential melastatin-7(TRPM7)in high glucose-induced renal tubular epithelial cell injury.Methods:The expression of TRPM7 in the serum of diabetic ne...Objective:To explore the regulatory mechanism of transient receptor potential melastatin-7(TRPM7)in high glucose-induced renal tubular epithelial cell injury.Methods:The expression of TRPM7 in the serum of diabetic nephropathy patients and high glucose-induced HK-2 cells was detected by RT-qPCR.Then,the TRPM7 interference vector was constructed,and the downstream high mobility group box 1(HMGB1)/Toll-like receptor 4(TLR4)signaling pathway proteins were detected.Next,in addition to interference with TRPM7 expression,overexpression of HMGB1 in high glucose-induced HK-2 cells was performed.Cell activity,apoptosis,oxidative stress levels,and inflammation levels were determined by CCK8,TUNEL,Western blotting,immunofluorescence and related kits.Results:TRPM7 expression was upregulated in the serum of diabetic nephropathy patients and high glucose-induced HK-2 cells.Interference with TRPM7 reduced cell damage,epithelial-mesenchymal transition,oxidative stress,and inflammatory response in high glucose-induced HK-2 cells via inhibiting the HMGB1/TLR4 signaling pathway.However,the effects induced by TRPM7 silencing were abrogated by HMGB1 overexpression.Conclusions:Decreased TRPM7 alleviates high glucose-induced renal tubular epithelial cell injury by inhibiting the HMGB1/TLR4 signaling pathway.Further animal experiments and clinical trials are warranted to verify its effect.展开更多
AIM:To investigate the role of reactive oxygen species(ROS)in epithelial–mesenchymal transition(EMT)and apoptosis of human lens epithelial cells(HLECs).METHODS:Flow cytometry was used to assess ROS production after t...AIM:To investigate the role of reactive oxygen species(ROS)in epithelial–mesenchymal transition(EMT)and apoptosis of human lens epithelial cells(HLECs).METHODS:Flow cytometry was used to assess ROS production after transforming growth factorβ2(TGF-β2)induction.Apoptosis of HLECs after H_(2)O_(2) and TGF-β2 interference with or without ROS scavenger N-acetylcysteine(NAC)were assessed by flow cytometry.The corresponding protein expression levels of the EMT markerα-smooth muscle actin(α-SMA),the extracellular matrix(ECM),marker fibronectin(Fn),and apoptosis-associated proteins were detected by using Western blotting in the presence of an ROS scavenger(NAC).Wound-healing and Transwell assays were used to assess the migration capability of HLECs.RESULTS:TGF-β2 stimulates ROS production within 8h in HLECs.Additionally,TGF-β2 induced HLECs cell apoptosis,EMT/ECM synthesis protein markers expression,and pro-apoptotic proteins production;nonetheless,NAC treatment prevented these responses.Similarly,TGF-β2 promoted HLECs cell migration,whereas NAC inhibited cell migration.We further determined that although ROS initiated apoptosis,it only induced the accumulation of the EMT markerα-SMA protein,but not COL-1 or Fn.CONCLUSION:ROS contribute to TGF-β2-induced EMT/ECM synthesis and cell apoptosis of HLECs;however,ROS alone are not sufficient for EMT/ECM synthesis.展开更多
Objective:To study the effects of Lycium barbarum polysaccharide(LBP)on the proliferation,apoptosis,and autophagy of retinal pigment epithelial(RPE)cells cultured under high-glucose conditions.Methods:The ARPE-19 cell...Objective:To study the effects of Lycium barbarum polysaccharide(LBP)on the proliferation,apoptosis,and autophagy of retinal pigment epithelial(RPE)cells cultured under high-glucose conditions.Methods:The ARPE-19 cell line was randomly divided into a control group(normally cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12[DMEM/F-12]medium),a high-glucose group(HG;50 mmol/L glucose added to DMEM/F-12 medium),and a HG+LBP group(incubated in DMEM/F-12 medium containing 1 mg/mL LBP for 24 h,and then treated with 50 mmol/L glucose for 24 h).Following Ad-mCherry-GFP-LC3B infection,cell proliferation,apoptosis,mammalian target of rapamy-cin(mTOR)expression,and autophagic flux were determined by Cell Counting Kit-8(CCK-8),AnnexinV-APC/7-AAD Apoptosis Detection Kit,Western blot,and laser confocal microscopy,respectively.Results:The proliferation rate of ARPE-19 cells in the HG group was significantly lower than that in the control group(P<0.05),while the proliferation rate of ARPE-19 cells in the HG+LBP group was significantly higher than that in the HG group(P<0.05).The apoptosis rate of ARPE-19 cells in the HG group was significantly higher than that in the control group(P<0.05),while the apoptosis rate of ARPE-19 cells in the HG+LBP group was significantly lower than that in the HG group(P<0.05).The relative expression of phosphorylated mTOR(p-mTOR)of ARPE-19 cells in the HG group was significantly lower than that in the control group(P<0.05),with enhanced autophagic flux;when compared with the HG group,the HG+LBP group had significantly higher expression of p-mTOR(P<0.05),with diminished autophagic flux.Conclusion:LBP has a protective effect on RPE cells with high glucose-induced injury,and its mechanism may be related to LBP inhibition of high glucose-induced abnormal autophagy.展开更多
AIM: To determine whether the PI3K/AKT/mTOR pathway is activated in proliferative vitreoretinopathy (PVR) in homo- sapiens. METHODS: The retina of controls and patients with PVR were collected and their levels of PI3K...AIM: To determine whether the PI3K/AKT/mTOR pathway is activated in proliferative vitreoretinopathy (PVR) in homo- sapiens. METHODS: The retina of controls and patients with PVR were collected and their levels of PI3K, phospho-AKT, phospho-mTOR, phospho-p70S6k and phospho-4EBP-1 were determined by Western blot. The cultured human retinal pigment epithelial cell line D407 was treated with a specific mTOR inhibitor, rapamycin (RAPA) or a PI3K inhibitor, LY294002, of various concentrations and durations. Cell morphology was observed by phase contrast microscopy and the proliferation and apoptosis of treated cells were determined by MTT assay and flow cytometry. RESULTS: Levels of PI3K, phospho-AKT, phospho-mTOR, phospho-P70S6K and phospho-4EBP1 was increased in the retina in PVR (P <0.05). In D407 cells, both RAPA and LY294002 significantly inhibited cell proliferation and cell cycle progression, and promoted apoptosis (P <0.05); mor- phologically, the cells became smaller. Both RAPA and LY294002 reduced levels of phospho-AKT, phospho-mTOR, phospho-p70S6k and phospho-4EBP1 expression (P <0.05). RAPA, but not LY294002, had no significant effect on PI3K expression. CONCLUSION: PI3K/AKT/mTOR signaling pathway is highly activated in the retinal pigment epithelial cells of PVR. The inhibitors of PI3K/AKT/mTOR signaling pathway, RAPA and LY294002, could inhibited the PI3K/AKT/mTOR signaling pathway by reducing the levels of phosphorylation of mTOR pathway components.展开更多
Treatment and functional reconstruction after central nervous system injury is a major medical and social challenge. An increasing number of researchers are attempting to use neural stem cells combined with artificial...Treatment and functional reconstruction after central nervous system injury is a major medical and social challenge. An increasing number of researchers are attempting to use neural stem cells combined with artificial scaffold materials, such as fibroin, for nerve repair. However, such approaches are challenged by ethical and practical issues. Amniotic tissue, a clinical waste product, is abundant, and amniotic epithelial cells are pluripotent, have low immunogenicity, and are not the subject of ethical debate. We hypothesized that amniotic epithelial cells combined with silk fibroin scaffolds would be conducive to the repair of spinal cord injury. To test this, we isolated and cultured amniotic epithelial cells, and constructed complexes of these cells and silk fibroin scaffolds. Implantation of the cell-scaffold complex into a rat model of spinal cord injury resulted in a smaller glial scar in the damaged cord tissue than in model rats that received a blank scaffold, or amniotic epithelial cells alone. In addition to a milder local immunological reaction, the rats showed less inflammatory cell infiltration at the transplant site, milder host-versus-graft reaction, and a marked improvement in motor function. These findings confirm that the transplantation of amniotic epithelial cells combined with silk fibroin scaffold can promote the repair of spinal cord injury. Silk fibroin scaffold can provide a good nerve regeneration microenvironment for amniotic epithelial cells.展开更多
AIM: To investigate the effect of high concentration of glucose(HCG) on double stranded RNA-activated protein kinase-like ER kinase(PERK)-eukaryotic initiation factor-2α(eIF2α)-transcription factor C/EBP homologous ...AIM: To investigate the effect of high concentration of glucose(HCG) on double stranded RNA-activated protein kinase-like ER kinase(PERK)-eukaryotic initiation factor-2α(eIF2α)-transcription factor C/EBP homologous protein(CHOP)-cysteine aspartate specific proteinase(caspase-12) signaling pathway activation and apoptosis in rabbit corneal epithelial cells(RCECs). METHODS: RCECs were treated by different concentrations of glucose for 0-48 h. The expressions of PERK, p-PERK, eIF2α, p-eIF2α, 78 k Da glucose-regulated protein 78(GRP78), CHOP, B-cell lymphoma 2(Bcl-2), B-cell lymphoma-2-associated X protein(Bax) and caspase-12 were determined by Western blot. Apoptosis was detected by TUNEL assay. Meanwhile, the function of PERK-eI F2α-CHOP-caspase-12 signaling pathway activation in high glucose-induced apoptosis was evaluated using PERK inhibitor, GSK2606414. RESULTS: HCG significantly promoted the expression of p-PERK, p-eIF2α, GRP78, CHOP, Bax and cleaved caspase-12 in RCECs(P<0.05), while remarkably decreased the expression of Bcl-2 and caspase-12(P<0.05), and the alterations caused by glucose were in concentration-and time-dependent manners. Meanwhile, PERK and eIF2α expressions were not affected in all groups(P>0.05). TUNEL assay showed that the apoptosis rate of RCECs in the HCG group increased significantly in contrast with that in the normal concentration of glucose or osmotic pressure control group(P<0.05), and the apoptosis rate increased with the increase of glucose concentration within limits(P<0.05). GSK2606414 down-regulated the expression of p-PERK and p-eI F2α in the HCG group(P<0.05), while still did not affect the expression of PERK and eIF2α among groups(P>0.05). Correspondingly, GSK2606414 also significantly reduced the apoptosis rate induced by high glucose(P<0.05). CONCLUSION: HCG activates PERK-eIF2α-CHOPcaspase-12 signaling pathway and promotes apoptosis of RCECs.展开更多
Background: Endoplasmic reticulum(ER) stress is associated with multiple pathological processes of intestinal diseases. Despite a critical role of mechanistic target of rapamycin complex 1(m TORC1) in regulating cellu...Background: Endoplasmic reticulum(ER) stress is associated with multiple pathological processes of intestinal diseases. Despite a critical role of mechanistic target of rapamycin complex 1(m TORC1) in regulating cellular stress response, the crosstalk between m TORC1 and ER stress signaling and its contribution to the intestinal barrier function is unknown.Results: In the present study, we showed that intestinal epithelial cells(IEC-6) incubated with tunicamycin led to caspase-3-dependent apoptotic cell death. The induction of cell death was accompanied by activation of unfolded protein response as evidenced by increased protein levels for Bi P, p-IRE1α, p-e IF2α, p-JNK, and CHOP. Further study demonstrated that tunicamycin-induced cell death was enhanced by rapamycin, a specific inhibitor of m TORC1.Consistently, tunicamycin decreased transepithelial electrical resistance(TEER) and increased permeability of the cells. These effects of tunicamycin were exacerbated by m TORC1 inhibitor.Conclusions: Taken together, the data presented here identified a previously unknown crosstalk between an unfold protein response and m TORC1 signaling in the intestinal epithelium. This feed-back loop regulation on ER stress signaling by m TORC1 is critical for cell survival and intestinal permeability in epithelial cells.展开更多
Objective To investigate whether exposure to particulate matter of diameter equal to or less than 2.5μm(PM2.5)alters the response of lung epithelial cells to extrinsic regulation by globally profiling cell surface li...Objective To investigate whether exposure to particulate matter of diameter equal to or less than 2.5μm(PM2.5)alters the response of lung epithelial cells to extrinsic regulation by globally profiling cell surface ligands and quantifying their binding activity.Methods Human A549 lung epithelial cells(LECs)were treated with or without PM2.5.Ligandomic profiling was applied to these cells for the global identification of LEC-binding ligands with simultaneous quantification of binding activity.Quantitative comparisons of the entire ligandome profiles systematically identified ligands with increased or decreased binding to PM2.5-treated LECs.Results We found 143 ligands with increased binding to PM2.5-treated LECs and 404 ligands with decreased binding.Many other ligands showed no change in binding activity.For example,apolipoprotein E(ApoE),Notch2,and growth arrest-specific 6(Gas6)represent ligands with increased,decreased,or unchanged binding activity,respectively.Both ApoE and Gas6 are phagocytosis ligands,suggesting that phagocytic receptors on LECs after stimulation with PM2.5 were differentially upregulated by PM2.5.Conclusion These results suggest that the newly-developed ligandomics is a valuable approach to globally profile the response of LECs to PM2.5 in terms of regulating the expression of cell surface receptors,as quantified by ligand binding activity.This quantitative ligandome profiling will provide indepth understanding of the LEC molecular response on the cell surface to particulate matter air pollution.展开更多
AIM:To explore that if peroxynitrite induced the expression of inducible nitric oxide synthase(iNOS)via nuclear factor-kappa B(NF-κB)pathway in retinal pigment epithelial(RPE) cells and the antagonism of cholecystoki...AIM:To explore that if peroxynitrite induced the expression of inducible nitric oxide synthase(iNOS)via nuclear factor-kappa B(NF-κB)pathway in retinal pigment epithelial(RPE) cells and the antagonism of cholecystokinin octapeptide-8(Melatonin,CCK-8) in vitro.METHODS:RPE cells were obtained from eyes of C57BL/6 mouse and divided into control,peroxynitrite and CCK-8 groups.Control group was treated with saline,peroxynitrite group was treated with peroxynitrite,and CCK-8 group was treated with CCK-8 after added with peroxynitrite.All changes were observered at 6,12 and 24 hours after treatment.Gene array analysis,Reverse Transcription Polymerase Chain Reaction(RT-PCR) were used to determine the expression of inducible nitric oxide synthase(iNOS)mRNA in RPE cells.Western blotting was used to test the apoptosis of RPE cells.Immunofluorescent staining was used to determine the NF-κB pathway signal transduction.RESULTS:Compared to the control group,the expression of iNOS mRNA was up-regulated in peroxynitrite group and down-regulated in CCK-8 group with gene array analysis.Apoptosis was increased in peroxynitrite group and decreased in CCK-8 group with western blotting.The NF-κB pathway signal transduction was more and more stronger in the peroxynitrite group.But in CCK-8 group,little stronger could be observed at 12 hours,then weak at 24 hours with immunofluorescent staining(P <0.001).CONCLUSION:This study suggested that apoptosis of RPE cells was partly induced by peroxynitrite,which may be the new way of oxidative damage to the RPE cells.The NF-κB signal transduction may affect and reinforce apoptosis mediated by peroxynitrite.CCK-8 decreased apoptosis of RPE cells induced by peroxynitrite and is a potential agent for therapy of retinopathy.The mechanism of CCK-8 dealing with RPE cells may be related to its direct inhibition of the formation of iNOS to produce peroxynitrite and antagnism of damage of peroxynitrite to the RPE cells.展开更多
Colorectal cancer is the most common malignant complication in patients with chronic inflammatory bowel disease (IBD). In addition, these patients are at risk for developing painful complications during chemotherapy d...Colorectal cancer is the most common malignant complication in patients with chronic inflammatory bowel disease (IBD). In addition, these patients are at risk for developing painful complications during chemotherapy due to cytotoxic effects of drugs currently in use. Past studies have suggested a protective effect of tea consumption on gastrointestinal (GI) malignancies. Green tea polyphenols (GrTP) inhibited carcinogen-induced GI tumors in rodents and induced apoptosis in various carcinoma cell lines. We hypothesized that GrTP and its polyphenolic compounds regulate apoptosis in the intestinal epithelia. In this study, the effects of GrTP and its polyphenolics on apoptosis was evaluated in intestinal epithelial, IEC-6, cells grown to 85% confluency. GrTP (400-800 mg/ml) induced DNA fragmentation in a dose dependent fashion. Higher concentrations (> 800 mg/ml) induced a mixed apoptosis and cytolysis. Epithelial cells exposed to GrTP and a major polyphenol, EGCG, but not EGC or EC, increased caspase activities in a time and dose dependent manner. The caspase inhibitors rescued cells from GrTP and EGCG-induced cell death. Concomitantly, GrTP resulted in activation of fatty acid synthase (Fas)-associated protein with death domain (FADD) and recruitment to Fas/CD95 domain 30 minutes following treatment. While GrTP also blocked NF-?B activation, an NF-?B inhibitor (MG132) only promoted cytolysis. In conclusion, these data demonstrated GrTP and EGCG induced apoptosis in intestinal epithelia mediated by caspase-8 through a FADD dependent pathway. Future investigation may warrant preventive as well as therapeutic strategies for GrTP in GI malignancy.展开更多
AIM:To study the differences of fibroblast growth factor receptor 1(FGFR1) gene on human lens epithelial cells(HLECs) of adults and fetuses.METHODS:Indirect in situ RT-PCR was adopted for detection of FGFR1 gene.The c...AIM:To study the differences of fibroblast growth factor receptor 1(FGFR1) gene on human lens epithelial cells(HLECs) of adults and fetuses.METHODS:Indirect in situ RT-PCR was adopted for detection of FGFR1 gene.The cDNA of the mRNA in the paraffin sections of fetus and adult HLEC was synthesized by reverse transcription reaction.After PCR amplification,in situ hybridization test was performed with synthesized oligonucleotide probe and relative quantification was carried out using image analysis.RESULTS:HLECs of adults and fetuses expressed FGFR1 gene,the expression level was higher in fetuses than in adults.The difference between them had significance(P <0.05).CONCLUSION:FGFR1 exist in HLEC and the expression is age-related,which could be one of causes of the high occurrence of post operational after-cataract in children.展开更多
Objective:To investigate the role of oxidative stress in human renal tubular epithelial cells(HK-2)induced by high glucose and the underlying signal pathway in vitro.Methods:MYPT1,pro-caspase-3,PGC-1α,and Drpl protei...Objective:To investigate the role of oxidative stress in human renal tubular epithelial cells(HK-2)induced by high glucose and the underlying signal pathway in vitro.Methods:MYPT1,pro-caspase-3,PGC-1α,and Drpl protein expressions were measured by Western blot.MnSOD2,Drp1 and PGC-1αmRNA expressions were detected by real time PCR.Results:Results showed that high glucose significantly up-regulated the protein expressions of MYPT1,pro-caspase-3 and the mRNA expression of MnSOD2 in HK-2 cells;while Rho kinase inhibitor fasudil and ROCK1 siRNA inhibited protein expressions of pro-caspase-3 and the mRNA expression of MnSOD2 in HK-2 cells induced by high glucose.Importantly,fasudil and ROCK1 siRNA markedly inhibited the expressions of mitochondrial motor proteins Drp1 and mitochondrial gene PGC-la in HK-2 cell=s induced by high glucose.Conclusions:Our findings suggest that Rho kinase signal pathway is involved in mitochondrial oxidative damage and apoptosis in high glucose-induced renal tubular epithelial cells by regulating mitochondrial motor proteins Drp1 and mitochondrial gene PGC-1α.Targeting Rho kinase signal pathway might be a potential strategy for the treatment of diabetic nephropathy.展开更多
AIM: To investigate the cytotoxic effect on human corneal epithelial cells(HCECs) and the ability to faciliate corneal epithelial wound healing of carboxymethylcellulose(CMC) and hyaluronic acid(HA).METHODS: HCECs wer...AIM: To investigate the cytotoxic effect on human corneal epithelial cells(HCECs) and the ability to faciliate corneal epithelial wound healing of carboxymethylcellulose(CMC) and hyaluronic acid(HA).METHODS: HCECs were exposed to 0.5% CMC(Refresh plus, Allergan, Irvine, California, USA) and 0.1% and 0.3%HA(Kynex , Alcon, Seoul, Korea, and Hyalein mini,Santen, Osaka, Japan) for the period of 30 min, and 4, 12,and 24 h. Methyl thiazolyl tetrazoiun(MTT)-based calorimetric assay was performed to assess the metabolic activity of cellular proliferation and lactate dehydrogenase(LDH) leakage assay to assess the cytotoxicity. apoptotic response was evaluated with flow cytometric analysis and fluorescence staining with Annexin V and propiodium iodide. Cellular morphology was evaluated by inverted phase-contrast light microscopy and electron microscopy. The wound widths were measured 24 h after confluent HCECs were scratch wounded.RESULTS: The inhibitory effect of human corneal epithelial proliferation and cytotoxicity showed the time-dependent response but no significant effect. Apoptosis developed in flow cytometry and apoptotic cells weredemonstrated in fluorescent micrograph. The damaged HCECs were detached from the bottom of the dish and showed the well-developed vacuole formations. Both CMC and HA stimulated reepithehlialization of HCECs scratched, which were more observed in CMC.CONCLUSION: CMC and HA, used in artificial tear formulation, could be utilized without any significant toxic effect on HCECs. Both significantly stimulated HCEC reepithelialization of corneal wounds.展开更多
基金supported by the China Postdoctoral Science Foundation(2019M653776 and 2020M673516)the Natural Science Basis Research Plan in Shaanxi Province of China(2023-JC-QN-0181)+1 种基金the Shaanxi Livestock and Poultry Breeding Double-chain Fusion Key Project,China(2022GD-TSLD-46-0202)the Natural Science Fundation of Tibet Autonomous Region,China(XZ202101ZR0063G)。
文摘The development of receptive endometrium(RE) from pre-receptive endometrium(PE) for successful embryo implantation is a complex dynamic process in which the morphology and physiological states of the endometrial epithelium undergo a series of significant changes, including cell proliferation and apoptosis. However, the molecular mechanisms are not yet fully understood. In this study, a higher circRNA3669 level was observed in PE than in RE of goats. Functional assays revealed that this overexpression promoted the proliferation of goat endometrial epithelial cells(GEECs) by activating the expression of genes related to the PI3K/AKT-mTOR and MAPK pathways,thereby inhibiting apoptosis in vitro. Furthermore, circRNA3669 functioned as a competing endogenous RNA(ceRNA) to upregulate Reticulocalbin-2(RCN2) expression at the post-transcriptional level by interacting with and downregulating miR-26a in GEECs. In addition, RCN2, which is highly expressed in the PE of goats, was found to be regulated by β-estradiol(E2) and progesterone(P4). Our results demonstrated that RCN2 also affected the key proteins PI3K, AKT, mTOR, JNK, and P38 in the PI3K/AKT-mTOR and MAPK pathways, thereby facilitating GEECs proliferation and suppressing their apoptosis in vitro. Collectively, we constructed a new circRNA3669-miR-26aRCN2 regulatory network in GEECs, which further provides strong evidence that circRNA could potentially play a crucial regulatory role in the development of RE in goats.
文摘●AIM:To explore whether autophagy functions as a cellular adaptation mechanism in lens epithelial cells(LECs)under hyperosmotic stress.●METHODS:LECs were treated with hyperosmotic stress at the concentration of 270,300,400,500,or 600 mOsm for 6,12,18,24h in vitro.Polymerase chain reaction(PCR)was employed for the mRNA expression of autophagyrelated genes,while Western blotting detected the targeted protein expression.The transfection of stub-RFP-sens-GFPLC3 autophagy-related double fluorescence lentivirus was conducted to detect the level of autophagy flux.Scanning electron microscopy was used to detect the existence of autolysosome.Short interfering RNA of autophagy-related gene(ATG)7,transient receptor potential vanilloid(TRPV)1 overexpression plasmid,related agonists and inhibitors were employed to their influence on autophagy related pathway.Flow cytometry was employed to test the apoptosis and intracellular Ca^(2+)level.Mitochondrial membrane potential was measured by JC-1 staining.The cell counting kit-8 assay was used to calculate the cellular viability.The wound healing assay was used to evaluate the wound closure rate.GraphPad 6.0 software was utilized to evaluate the data.●RESULTS:The hyperosmotic stress activated autophagy in a pressure-and time-dependent manner in LECs.Beclin 1 protein expression and conversion of LC3B II to LC3B I increased,whereas sequestosome-1(SQSTM1)protein expression decreased.Transient Ca^(2+)influx was stimulated caused by hyperosmotic stress,levels of mammalian target of rapamycin(mTOR)phosphorylation decreased,and the level of AMP-activated protein kinase(AMPK)phosphorylation increased in the early stage.Based on this evidence,autophagy activation through the Ca^(2+)-dependent AMPK/mTOR pathway might represent an adaptation process in LECs under hyperosmotic stress.Hyperosmotic stress decreased cellular viability and accelerated apoptosis in LECs and cellular migration decreased.Inhibition of autophagy by ATG7 knockdown had similar results.TRPV1 overexpression increased autophagy and might be crucial in the occurrence of autophagy promoted by hyperosmotic stress.●CONCLUSION:A combination of hyperosmotic stress and autophagy inhibition may be a promising approach to decrease the number of LECs in the capsular bag and pave the way for improving prevention of posterior capsular opacification and capsular fibrosis.
基金supported by Fuping County Dairy Goat High-efficiency Breeding Technology R&D and Extension Application Project(No.K3380216101)the Dairy Goat High-efficiency Breeding Technology Research and Application Project(No.K4040121023).
文摘Background Mastitis not only deteriorates the composition or quality of milk,but also damages the health and pro-ductivity of dairy goats.Sulforaphane(SFN)is a phytochemical isothiocyanate compound with various pharmacologi-cal effects such as anti-oxidant and anti-inflammatory.However,the effect of SFN on mastitis has yet to be elucidated.This study aimed to explore the anti-oxidant and anti-inflammatory effects and potential molecular mechanisms of SFN in lipopolysaccharide(LPS)-induced primary goat mammary epithelial cells(GMECs)and a mouse model of mastitis.Results In vitro,SFN downregulated the mRNA expression of inflammatory factors(tumor necrosis factor-α(TNF-α),interleukin(IL)-1βand IL-6),inhibited the protein expression of inflammatory mediators(cyclooxygenase-2(COX2),and inducible nitric oxide synthase(iNOS))while suppressing nuclear factor kappa-B(NF-κB)activation in LPS-induced GMECs.Additionally,SFN exhibited an antioxidant effect by increasing Nrf2 expression and nuclear translocation,up-regulating antioxidant enzymes expression,and decreasing LPS-induced reactive oxygen species(ROS)produc-tion in GMECs.Furthermore,SFN pretreatment promoted the autophagy pathway,which was dependent on the increased Nrf2 level,and contributed significantly to the improved LPS-induced oxidative stress and inflammatory response.In vivo,SFN effectively alleviated histopathological lesions,suppressed the expression of inflammatory factors,enhanced immunohistochemistry staining of Nrf2,and amplified of LC3 puncta LPS-induced mastitis in mice.Mechanically,the in vitro and in vivo study showed that the anti-inflammatory and anti-oxidative stress effects of SFN were mediated by the Nrf2-mediated autophagy pathway in GMECs and a mouse model of mastitis.Conclusions These results indicate that the natural compound SFN has a preventive effect on LPS-induced inflam-mation through by regulating the Nrf2-mediated autophagy pathway in primary goat mammary epithelial cells and a mouse model of mastitis,which may improve prevention strategies for mastitis in dairy goats.
基金Supported by the Natural Science Foundation of Gansu Province(No.23JRRA0942)Innovation Fund for Colleges and Universities in Gansu Province(No.2021B-23).
文摘AIM:To investigate the effect of morroniside(Mor)on lipopolysaccharide(LPS)-treated iris pigment epithelial cells(IPE).METHODS:IPE cells were induced by LPS and treated with Mor.Cell proliferation was detected by cell counting kit(CCK)-8,apoptosis was detected by flow cytometry,the levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-6,and IL-8 were measured by enzyme-linked immunosorbent assay(ELISA)kits,and the protein expression of TLR4,JAK2,p-JAK2,STAT3,and p-STAT3 was analyzed by Western blotting.In addition,overexpression of TLR4 and Mor treatment of LPS-stimulated IPE cells were also tested for the above indices.RESULTS:Mor effectively promoted the proliferation and inhibited the apoptosis of LPS-treated IPE cells.In addition,Mor significantly reduced the levels of TNF-α,IL-6,and IL-8 and significantly inhibited the expression of TLR4,p-JAK2,and p-STAT3 in LPS-treated IPE cells.The effect of Mor on LPS-treated IPE cells was markedly attenuated after overexpression of TLR4.CONCLUSION:These findings suggest that Mor may ameliorate LPS-induced inflammatory damage and apoptosis in IPE through inhibition of TLR4/JAK2/STAT3 pathway.
基金the Natural Science Foundation of Shaanxi Province(No.2022JM-521)the Science and Technology Plan Project of Xi’an(No.21YXYJ0031).
文摘AIM:To explore the effects of hepatocyte growth factor(HGF)on retinal pigment epithelium(RPE)cell behaviors.METHODS:The human adult retinal pigment epithelial cell line-19(ARPE-19)were treated by HGF or mesenchymalepithelial transition factor(MET)inhibitor SU11274 in vitro.Cell viability was detected by a Cell Counting Kit-8 assay.Cell proliferation and motility was detected by a bromodeoxyuridine incorporation assay and a wound healing assay,respectively.The expression levels of MET,phosphorylated MET,protein kinase B(AKT),and phosphorylated AKT proteins were determined by Western blot assay.The MET and phosphorylated MET proteins were also determined by immunofluorescence assay.RESULTS:HGF increased ARPE-19 cells’viability,proliferation and migration,and induced an increase of phosphorylated MET and phosphorylated AKT proteins.SU11274 significantly reduced cell viability,proliferation,and migration and decreased the expression of MET and AKT proteins.SU11274 suppressed HGF-induced increase of viability,proliferation,and migration in ARPE-19 cells.Additionally,SU11274 also blocked HGF-induced phosphorylation of MET and AKT proteins.CONCLUSION:HGF enhances cellular viability,proliferation,and migration in RPE cells through the MET/AKT signaling pathway,whereas this enhancement is suppressed by the MET inhibitor SU11274.HGF-induced MET/AKT signaling might be a vital contributor of RPE cells survival.
基金the National Natural Science Foundation of China (31801541)the Independent Innovation Fund Project of Agricultural Science and Technology in Jiangsu Province (CX (22)3065)+1 种基金Major Scientific and Technological Achievements Transformation Project of Taizhou (SCG 202105)the Taizhou Science and Technology Support Plan (TN202106)。
文摘In order to improve the bioavailability of lutein(LUT),a novel lutein-stevio side nanoparticle(LUT-STE)were prepared previously,but the information about LUT-STE on protecting of eye health was limited.This study investigated the effect of LUT-STE on antioxidant activity of H_(2)O_(2)-induced human retinal pigment epithelial(ARPE)cells.LUT and LUT-STE(final concentration of 5μg/mL)significantly enhanced cell viability from(74.84±5.10)%to(81.92±10.01)%(LUT)and(89.33±4.34)%(LUT-STE),and inhibited the cell apoptosis(P<0.05).After pretreatment with LUT-STE in ARPE cells,the levels of superoxide dismutase(SOD),catalase(CAT)and glutathion peroxidase(GSH-Px)in ARPE cells were significantly increased(P<0.05),the contents of reactive oxygen species(ROS)and malondialdehyde(MDA)were decreased.In addition,the vascular endothelial growth factor(VEGF)levels were inhibited by 13.61%and 17.39%,respectively,pretreatment with LUT and LUT-STE.Western blotting results showed that the pretreatment with LUT-STE inhibited the expression of caspase-9 and caspase-3 and up-regulated Bcl-2/Bax pathway to inhibit H_(2)O_(2)-induced apoptosis.In summary,the novel delivery LUT-STE had more pronounced inhibitory effect on H_(2)O_(2)-induced damage in human ARPE cells.
基金supported by the National Key Research and Development Program(Grant Number:2017YFA0105303)the Natural Science Foundation of Shandong Province(Grant Number:ZR2020MH327).
文摘Background:Inflammatory bowel disease(IBD)is a chronic inflammatory disease of the gastrointestinal tract.The destruction of the intestinal epithelial barrier is one of the major pathological processes in IBD pathology.Growing evidence indicated that epithelial cell ferroptosis is linked to IBD and is considered a target process.Methods:RAS-selective lethal 3(RSL3)was used to induce ferroptosis in intestinal epithelial cell line No.6(IEC-6)cells,and cell ferroptosis and the effects of tanshinone IIA(Tan IIA)were determined by cell counting kit-8(CCK-8),reactive oxygen species(ROS)staining,Giemsa staining and transmission electron microscope(TEM).The cell viability of natural product library compounds was determined by CCK-8.The expression of ferroptosis-related genes were detected by real-time quantitative polymerase chain reaction(RT-qPCR)and western blot.Results:Treatment of IEC-6 cells results in the accumulation of ROS and typical morphological characteristics of ferroptosis.RSL3 treatment caused rapid cellular cytotoxicity which could be reversed by ferrostatin-1(Fer-1)in IEC-6 cells.Natural product library screening revealed that Tan IIA is a potent inhibitor of IEC-6 cell ferroptosis.Tan IIA could significantly protect the RSL3-induced ferroptosis of IEC-6 cells.Furthermore,the ferroptosis suppressors,glutathione peroxidase 4(GPX4),solute carrier family 7 member 11(SLC7A11),and miR-17-92 were found to be early response genes in RSL3-treated cells.Treatment of IEC-6 cells with Tan IIA resulted in upregulation of GPX4,SLC7A11,and miR-17-92.Conclusion:Our study demonstrated that Tan IIA protects IEC-6 cells from ferroptosis through the upregulation of GPX4,SLC7A11,and miR-17-92.The findings might provide a theoretical grounding for the future application of Tan IIA to treat or prevent IBD.
文摘Objective:To explore the regulatory mechanism of transient receptor potential melastatin-7(TRPM7)in high glucose-induced renal tubular epithelial cell injury.Methods:The expression of TRPM7 in the serum of diabetic nephropathy patients and high glucose-induced HK-2 cells was detected by RT-qPCR.Then,the TRPM7 interference vector was constructed,and the downstream high mobility group box 1(HMGB1)/Toll-like receptor 4(TLR4)signaling pathway proteins were detected.Next,in addition to interference with TRPM7 expression,overexpression of HMGB1 in high glucose-induced HK-2 cells was performed.Cell activity,apoptosis,oxidative stress levels,and inflammation levels were determined by CCK8,TUNEL,Western blotting,immunofluorescence and related kits.Results:TRPM7 expression was upregulated in the serum of diabetic nephropathy patients and high glucose-induced HK-2 cells.Interference with TRPM7 reduced cell damage,epithelial-mesenchymal transition,oxidative stress,and inflammatory response in high glucose-induced HK-2 cells via inhibiting the HMGB1/TLR4 signaling pathway.However,the effects induced by TRPM7 silencing were abrogated by HMGB1 overexpression.Conclusions:Decreased TRPM7 alleviates high glucose-induced renal tubular epithelial cell injury by inhibiting the HMGB1/TLR4 signaling pathway.Further animal experiments and clinical trials are warranted to verify its effect.
基金Supported by the National Natural Science Foundation of China(No.82201163,No.81800812)Natural Science Foundation Youth Foundation of Shaanxi Province(No.2023-JC-QN-0861)Shaanxi Province Key Research and Development Program(No.2023-YBSF-483).
文摘AIM:To investigate the role of reactive oxygen species(ROS)in epithelial–mesenchymal transition(EMT)and apoptosis of human lens epithelial cells(HLECs).METHODS:Flow cytometry was used to assess ROS production after transforming growth factorβ2(TGF-β2)induction.Apoptosis of HLECs after H_(2)O_(2) and TGF-β2 interference with or without ROS scavenger N-acetylcysteine(NAC)were assessed by flow cytometry.The corresponding protein expression levels of the EMT markerα-smooth muscle actin(α-SMA),the extracellular matrix(ECM),marker fibronectin(Fn),and apoptosis-associated proteins were detected by using Western blotting in the presence of an ROS scavenger(NAC).Wound-healing and Transwell assays were used to assess the migration capability of HLECs.RESULTS:TGF-β2 stimulates ROS production within 8h in HLECs.Additionally,TGF-β2 induced HLECs cell apoptosis,EMT/ECM synthesis protein markers expression,and pro-apoptotic proteins production;nonetheless,NAC treatment prevented these responses.Similarly,TGF-β2 promoted HLECs cell migration,whereas NAC inhibited cell migration.We further determined that although ROS initiated apoptosis,it only induced the accumulation of the EMT markerα-SMA protein,but not COL-1 or Fn.CONCLUSION:ROS contribute to TGF-β2-induced EMT/ECM synthesis and cell apoptosis of HLECs;however,ROS alone are not sufficient for EMT/ECM synthesis.
基金supported by the Supporting Fund of the First Affiliated Hospital of Xi'an Medical University(XYFYPT-2023-01).
文摘Objective:To study the effects of Lycium barbarum polysaccharide(LBP)on the proliferation,apoptosis,and autophagy of retinal pigment epithelial(RPE)cells cultured under high-glucose conditions.Methods:The ARPE-19 cell line was randomly divided into a control group(normally cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12[DMEM/F-12]medium),a high-glucose group(HG;50 mmol/L glucose added to DMEM/F-12 medium),and a HG+LBP group(incubated in DMEM/F-12 medium containing 1 mg/mL LBP for 24 h,and then treated with 50 mmol/L glucose for 24 h).Following Ad-mCherry-GFP-LC3B infection,cell proliferation,apoptosis,mammalian target of rapamy-cin(mTOR)expression,and autophagic flux were determined by Cell Counting Kit-8(CCK-8),AnnexinV-APC/7-AAD Apoptosis Detection Kit,Western blot,and laser confocal microscopy,respectively.Results:The proliferation rate of ARPE-19 cells in the HG group was significantly lower than that in the control group(P<0.05),while the proliferation rate of ARPE-19 cells in the HG+LBP group was significantly higher than that in the HG group(P<0.05).The apoptosis rate of ARPE-19 cells in the HG group was significantly higher than that in the control group(P<0.05),while the apoptosis rate of ARPE-19 cells in the HG+LBP group was significantly lower than that in the HG group(P<0.05).The relative expression of phosphorylated mTOR(p-mTOR)of ARPE-19 cells in the HG group was significantly lower than that in the control group(P<0.05),with enhanced autophagic flux;when compared with the HG group,the HG+LBP group had significantly higher expression of p-mTOR(P<0.05),with diminished autophagic flux.Conclusion:LBP has a protective effect on RPE cells with high glucose-induced injury,and its mechanism may be related to LBP inhibition of high glucose-induced abnormal autophagy.
基金Scientific Research Project of Education Department of Liaoning Province, China (No.L2010676)Project of Science and Technology Commission of Shenyang City,China(No.F10-149-9-58)Doctoral Foundation of Ministry of Education of China (No.20102104120027)
文摘AIM: To determine whether the PI3K/AKT/mTOR pathway is activated in proliferative vitreoretinopathy (PVR) in homo- sapiens. METHODS: The retina of controls and patients with PVR were collected and their levels of PI3K, phospho-AKT, phospho-mTOR, phospho-p70S6k and phospho-4EBP-1 were determined by Western blot. The cultured human retinal pigment epithelial cell line D407 was treated with a specific mTOR inhibitor, rapamycin (RAPA) or a PI3K inhibitor, LY294002, of various concentrations and durations. Cell morphology was observed by phase contrast microscopy and the proliferation and apoptosis of treated cells were determined by MTT assay and flow cytometry. RESULTS: Levels of PI3K, phospho-AKT, phospho-mTOR, phospho-P70S6K and phospho-4EBP1 was increased in the retina in PVR (P <0.05). In D407 cells, both RAPA and LY294002 significantly inhibited cell proliferation and cell cycle progression, and promoted apoptosis (P <0.05); mor- phologically, the cells became smaller. Both RAPA and LY294002 reduced levels of phospho-AKT, phospho-mTOR, phospho-p70S6k and phospho-4EBP1 expression (P <0.05). RAPA, but not LY294002, had no significant effect on PI3K expression. CONCLUSION: PI3K/AKT/mTOR signaling pathway is highly activated in the retinal pigment epithelial cells of PVR. The inhibitors of PI3K/AKT/mTOR signaling pathway, RAPA and LY294002, could inhibited the PI3K/AKT/mTOR signaling pathway by reducing the levels of phosphorylation of mTOR pathway components.
基金supported by the Scientific Research Project Fund of Wuxi Municipal Health and Family Planning Commission,No.MS201402
文摘Treatment and functional reconstruction after central nervous system injury is a major medical and social challenge. An increasing number of researchers are attempting to use neural stem cells combined with artificial scaffold materials, such as fibroin, for nerve repair. However, such approaches are challenged by ethical and practical issues. Amniotic tissue, a clinical waste product, is abundant, and amniotic epithelial cells are pluripotent, have low immunogenicity, and are not the subject of ethical debate. We hypothesized that amniotic epithelial cells combined with silk fibroin scaffolds would be conducive to the repair of spinal cord injury. To test this, we isolated and cultured amniotic epithelial cells, and constructed complexes of these cells and silk fibroin scaffolds. Implantation of the cell-scaffold complex into a rat model of spinal cord injury resulted in a smaller glial scar in the damaged cord tissue than in model rats that received a blank scaffold, or amniotic epithelial cells alone. In addition to a milder local immunological reaction, the rats showed less inflammatory cell infiltration at the transplant site, milder host-versus-graft reaction, and a marked improvement in motor function. These findings confirm that the transplantation of amniotic epithelial cells combined with silk fibroin scaffold can promote the repair of spinal cord injury. Silk fibroin scaffold can provide a good nerve regeneration microenvironment for amniotic epithelial cells.
基金Supported by Shanghai Natural Science Foundation (No.19ZR1450500)National Foundation Cultivation Project of Tongji University (No.22120180285)the Good Physician Training Project of Yangpu District, Shanghai
文摘AIM: To investigate the effect of high concentration of glucose(HCG) on double stranded RNA-activated protein kinase-like ER kinase(PERK)-eukaryotic initiation factor-2α(eIF2α)-transcription factor C/EBP homologous protein(CHOP)-cysteine aspartate specific proteinase(caspase-12) signaling pathway activation and apoptosis in rabbit corneal epithelial cells(RCECs). METHODS: RCECs were treated by different concentrations of glucose for 0-48 h. The expressions of PERK, p-PERK, eIF2α, p-eIF2α, 78 k Da glucose-regulated protein 78(GRP78), CHOP, B-cell lymphoma 2(Bcl-2), B-cell lymphoma-2-associated X protein(Bax) and caspase-12 were determined by Western blot. Apoptosis was detected by TUNEL assay. Meanwhile, the function of PERK-eI F2α-CHOP-caspase-12 signaling pathway activation in high glucose-induced apoptosis was evaluated using PERK inhibitor, GSK2606414. RESULTS: HCG significantly promoted the expression of p-PERK, p-eIF2α, GRP78, CHOP, Bax and cleaved caspase-12 in RCECs(P<0.05), while remarkably decreased the expression of Bcl-2 and caspase-12(P<0.05), and the alterations caused by glucose were in concentration-and time-dependent manners. Meanwhile, PERK and eIF2α expressions were not affected in all groups(P>0.05). TUNEL assay showed that the apoptosis rate of RCECs in the HCG group increased significantly in contrast with that in the normal concentration of glucose or osmotic pressure control group(P<0.05), and the apoptosis rate increased with the increase of glucose concentration within limits(P<0.05). GSK2606414 down-regulated the expression of p-PERK and p-eI F2α in the HCG group(P<0.05), while still did not affect the expression of PERK and eIF2α among groups(P>0.05). Correspondingly, GSK2606414 also significantly reduced the apoptosis rate induced by high glucose(P<0.05). CONCLUSION: HCG activates PERK-eIF2α-CHOPcaspase-12 signaling pathway and promotes apoptosis of RCECs.
基金supported the National Natural Science Foundation of China(No.31272451,31272450,31572410)Chinese University Scientific Fund(2015DK001)+3 种基金the 111 Project(B16044)the Program for New Century Excellent Talents in University(NCET-12-0522)the Agriculture and Food Research Initiative Competitive Grant from the USDA National Institute of Food and Agriculture(No.2014–67015-21770)Texas A&M Agri Life Research(H-8200)
文摘Background: Endoplasmic reticulum(ER) stress is associated with multiple pathological processes of intestinal diseases. Despite a critical role of mechanistic target of rapamycin complex 1(m TORC1) in regulating cellular stress response, the crosstalk between m TORC1 and ER stress signaling and its contribution to the intestinal barrier function is unknown.Results: In the present study, we showed that intestinal epithelial cells(IEC-6) incubated with tunicamycin led to caspase-3-dependent apoptotic cell death. The induction of cell death was accompanied by activation of unfolded protein response as evidenced by increased protein levels for Bi P, p-IRE1α, p-e IF2α, p-JNK, and CHOP. Further study demonstrated that tunicamycin-induced cell death was enhanced by rapamycin, a specific inhibitor of m TORC1.Consistently, tunicamycin decreased transepithelial electrical resistance(TEER) and increased permeability of the cells. These effects of tunicamycin were exacerbated by m TORC1 inhibitor.Conclusions: Taken together, the data presented here identified a previously unknown crosstalk between an unfold protein response and m TORC1 signaling in the intestinal epithelium. This feed-back loop regulation on ER stress signaling by m TORC1 is critical for cell survival and intestinal permeability in epithelial cells.
基金supported by the National Institute of Health under Grant#R01EY027749-01A1,#R24EY028764-01A1,#R21EY027065-02,#R41EY027665-01A1 to LW,and#P30-EY014801The American Diabetes Association under Grant#1-18-IBS-172 to LW+1 种基金An institutional grant from Research to Prevent BlindnessThe National Natural Science Foundation of China under Grant#81573112 and#81373030 to WWD.
文摘Objective To investigate whether exposure to particulate matter of diameter equal to or less than 2.5μm(PM2.5)alters the response of lung epithelial cells to extrinsic regulation by globally profiling cell surface ligands and quantifying their binding activity.Methods Human A549 lung epithelial cells(LECs)were treated with or without PM2.5.Ligandomic profiling was applied to these cells for the global identification of LEC-binding ligands with simultaneous quantification of binding activity.Quantitative comparisons of the entire ligandome profiles systematically identified ligands with increased or decreased binding to PM2.5-treated LECs.Results We found 143 ligands with increased binding to PM2.5-treated LECs and 404 ligands with decreased binding.Many other ligands showed no change in binding activity.For example,apolipoprotein E(ApoE),Notch2,and growth arrest-specific 6(Gas6)represent ligands with increased,decreased,or unchanged binding activity,respectively.Both ApoE and Gas6 are phagocytosis ligands,suggesting that phagocytic receptors on LECs after stimulation with PM2.5 were differentially upregulated by PM2.5.Conclusion These results suggest that the newly-developed ligandomics is a valuable approach to globally profile the response of LECs to PM2.5 in terms of regulating the expression of cell surface receptors,as quantified by ligand binding activity.This quantitative ligandome profiling will provide indepth understanding of the LEC molecular response on the cell surface to particulate matter air pollution.
基金Supported by Hebei Province Science foundation,China(No.07276101D-3)Clinical Science Project Fund of the Ministry of Health in Hebei Province,China(No. 03078)Foreign Studying Project Fund in Hebei Province,China
文摘AIM:To explore that if peroxynitrite induced the expression of inducible nitric oxide synthase(iNOS)via nuclear factor-kappa B(NF-κB)pathway in retinal pigment epithelial(RPE) cells and the antagonism of cholecystokinin octapeptide-8(Melatonin,CCK-8) in vitro.METHODS:RPE cells were obtained from eyes of C57BL/6 mouse and divided into control,peroxynitrite and CCK-8 groups.Control group was treated with saline,peroxynitrite group was treated with peroxynitrite,and CCK-8 group was treated with CCK-8 after added with peroxynitrite.All changes were observered at 6,12 and 24 hours after treatment.Gene array analysis,Reverse Transcription Polymerase Chain Reaction(RT-PCR) were used to determine the expression of inducible nitric oxide synthase(iNOS)mRNA in RPE cells.Western blotting was used to test the apoptosis of RPE cells.Immunofluorescent staining was used to determine the NF-κB pathway signal transduction.RESULTS:Compared to the control group,the expression of iNOS mRNA was up-regulated in peroxynitrite group and down-regulated in CCK-8 group with gene array analysis.Apoptosis was increased in peroxynitrite group and decreased in CCK-8 group with western blotting.The NF-κB pathway signal transduction was more and more stronger in the peroxynitrite group.But in CCK-8 group,little stronger could be observed at 12 hours,then weak at 24 hours with immunofluorescent staining(P <0.001).CONCLUSION:This study suggested that apoptosis of RPE cells was partly induced by peroxynitrite,which may be the new way of oxidative damage to the RPE cells.The NF-κB signal transduction may affect and reinforce apoptosis mediated by peroxynitrite.CCK-8 decreased apoptosis of RPE cells induced by peroxynitrite and is a potential agent for therapy of retinopathy.The mechanism of CCK-8 dealing with RPE cells may be related to its direct inhibition of the formation of iNOS to produce peroxynitrite and antagnism of damage of peroxynitrite to the RPE cells.
文摘Colorectal cancer is the most common malignant complication in patients with chronic inflammatory bowel disease (IBD). In addition, these patients are at risk for developing painful complications during chemotherapy due to cytotoxic effects of drugs currently in use. Past studies have suggested a protective effect of tea consumption on gastrointestinal (GI) malignancies. Green tea polyphenols (GrTP) inhibited carcinogen-induced GI tumors in rodents and induced apoptosis in various carcinoma cell lines. We hypothesized that GrTP and its polyphenolic compounds regulate apoptosis in the intestinal epithelia. In this study, the effects of GrTP and its polyphenolics on apoptosis was evaluated in intestinal epithelial, IEC-6, cells grown to 85% confluency. GrTP (400-800 mg/ml) induced DNA fragmentation in a dose dependent fashion. Higher concentrations (> 800 mg/ml) induced a mixed apoptosis and cytolysis. Epithelial cells exposed to GrTP and a major polyphenol, EGCG, but not EGC or EC, increased caspase activities in a time and dose dependent manner. The caspase inhibitors rescued cells from GrTP and EGCG-induced cell death. Concomitantly, GrTP resulted in activation of fatty acid synthase (Fas)-associated protein with death domain (FADD) and recruitment to Fas/CD95 domain 30 minutes following treatment. While GrTP also blocked NF-?B activation, an NF-?B inhibitor (MG132) only promoted cytolysis. In conclusion, these data demonstrated GrTP and EGCG induced apoptosis in intestinal epithelia mediated by caspase-8 through a FADD dependent pathway. Future investigation may warrant preventive as well as therapeutic strategies for GrTP in GI malignancy.
文摘AIM:To study the differences of fibroblast growth factor receptor 1(FGFR1) gene on human lens epithelial cells(HLECs) of adults and fetuses.METHODS:Indirect in situ RT-PCR was adopted for detection of FGFR1 gene.The cDNA of the mRNA in the paraffin sections of fetus and adult HLEC was synthesized by reverse transcription reaction.After PCR amplification,in situ hybridization test was performed with synthesized oligonucleotide probe and relative quantification was carried out using image analysis.RESULTS:HLECs of adults and fetuses expressed FGFR1 gene,the expression level was higher in fetuses than in adults.The difference between them had significance(P <0.05).CONCLUSION:FGFR1 exist in HLEC and the expression is age-related,which could be one of causes of the high occurrence of post operational after-cataract in children.
基金supported by National Natural Science Foundation of China(No.81560124)Hainan Key Research and Development Projects(ZDYF2018131,ZDYF2017113,ZDYF2017114)+1 种基金Hainan Science and Technology Planned Project of Youth Outstanding Ability of Innovation(201704)Hainan Health Family Planning Industry Project(13A210277)
文摘Objective:To investigate the role of oxidative stress in human renal tubular epithelial cells(HK-2)induced by high glucose and the underlying signal pathway in vitro.Methods:MYPT1,pro-caspase-3,PGC-1α,and Drpl protein expressions were measured by Western blot.MnSOD2,Drp1 and PGC-1αmRNA expressions were detected by real time PCR.Results:Results showed that high glucose significantly up-regulated the protein expressions of MYPT1,pro-caspase-3 and the mRNA expression of MnSOD2 in HK-2 cells;while Rho kinase inhibitor fasudil and ROCK1 siRNA inhibited protein expressions of pro-caspase-3 and the mRNA expression of MnSOD2 in HK-2 cells induced by high glucose.Importantly,fasudil and ROCK1 siRNA markedly inhibited the expressions of mitochondrial motor proteins Drp1 and mitochondrial gene PGC-la in HK-2 cell=s induced by high glucose.Conclusions:Our findings suggest that Rho kinase signal pathway is involved in mitochondrial oxidative damage and apoptosis in high glucose-induced renal tubular epithelial cells by regulating mitochondrial motor proteins Drp1 and mitochondrial gene PGC-1α.Targeting Rho kinase signal pathway might be a potential strategy for the treatment of diabetic nephropathy.
基金Supported by Biomedical Research Institute Grant(No.2009-39)Pusan National University Hospital
文摘AIM: To investigate the cytotoxic effect on human corneal epithelial cells(HCECs) and the ability to faciliate corneal epithelial wound healing of carboxymethylcellulose(CMC) and hyaluronic acid(HA).METHODS: HCECs were exposed to 0.5% CMC(Refresh plus, Allergan, Irvine, California, USA) and 0.1% and 0.3%HA(Kynex , Alcon, Seoul, Korea, and Hyalein mini,Santen, Osaka, Japan) for the period of 30 min, and 4, 12,and 24 h. Methyl thiazolyl tetrazoiun(MTT)-based calorimetric assay was performed to assess the metabolic activity of cellular proliferation and lactate dehydrogenase(LDH) leakage assay to assess the cytotoxicity. apoptotic response was evaluated with flow cytometric analysis and fluorescence staining with Annexin V and propiodium iodide. Cellular morphology was evaluated by inverted phase-contrast light microscopy and electron microscopy. The wound widths were measured 24 h after confluent HCECs were scratch wounded.RESULTS: The inhibitory effect of human corneal epithelial proliferation and cytotoxicity showed the time-dependent response but no significant effect. Apoptosis developed in flow cytometry and apoptotic cells weredemonstrated in fluorescent micrograph. The damaged HCECs were detached from the bottom of the dish and showed the well-developed vacuole formations. Both CMC and HA stimulated reepithehlialization of HCECs scratched, which were more observed in CMC.CONCLUSION: CMC and HA, used in artificial tear formulation, could be utilized without any significant toxic effect on HCECs. Both significantly stimulated HCEC reepithelialization of corneal wounds.
