AIM To explore the role and mechanism of total flavone of Abelmoschus manihot(TFA) on epithelial-mesenchymal transition(EMT) progress of Crohn's disease(CD) intestinal fibrosis.METHODS First,CCK-8 assay was perfor...AIM To explore the role and mechanism of total flavone of Abelmoschus manihot(TFA) on epithelial-mesenchymal transition(EMT) progress of Crohn's disease(CD) intestinal fibrosis.METHODS First,CCK-8 assay was performed to assess TFA on the viability of intestinal epithelial(IEC-6) cells and select the optimal concentrations of TFA for our further studies.Then cell morphology,wound healing and transwell assays were performed to examine the effect of TFA on morphology,migration and invasion of IEC-6 cells treated with TGF-β1.In addition,immunofluorescence,real-time PCR analysis(q RT-PCR) and western blotting assays were carried out to detect the impact of TFA on EMT progress.Moreover,western blotting assay was performed to evaluate the function of TFA on the Smad and MAPK signaling pathways.Further,the role of co-treatment of TFA and si-Smad or MAPK inhibitors has been examined by q RTPCR,western blotting,morphology,wound healing andtranswell assays.RESULTS In this study,TFA promoted transforming growth factor-β1(TGF-β1)-induced(IEC-6) morphological change,migration and invasion,and increased the expression of epithelial markers and reduced the levels of mesenchymal markers,along with the inactivation of Smad and MAPK signaling pathways.Moreover,we revealed that si-Smad and MAPK inhibitors effectively attenuated TGF-β1-induced EMT in IEC-6 cells.Importantly,co-treatment of TFA and si-Smad or MAPK inhibitors had better inhibitory effects on TGF-β1-induced EMT in IEC-6 cells than either one of them.CONCLUSION These findings could provide new insight into the molecular mechanisms of TFA on TGF-β1-induced EMT in IEC-6 cells and TFA is expected to advance as a new therapy to treat CD intestinal fibrosis.展开更多
Background: Histone deacetylases(HDACs) inhibitors are new anti-fibrotic drugs that inhibit the activity of hepatic stellate cells. The present study focused on the anti-fibrotic function of HDAC inhibitor suberoylani...Background: Histone deacetylases(HDACs) inhibitors are new anti-fibrotic drugs that inhibit the activity of hepatic stellate cells. The present study focused on the anti-fibrotic function of HDAC inhibitor suberoylanilide hydroxamic acid(SAHA) by suppressing transforming growth factor-β1(TGF-β1) signaling. Methods: Male Sprague-Dawley rats were used to induce liver fibrosis with carbon tetrachloride(CCl 4) and LX2 cell(human hepatic stellate cell line) was stimulated by TGF-β1. Both animals and cells were treated with SAHA. The Smad7 and connective tissue growth factor(CTGF) mRNA levels were detected by real-time polymerase chain reaction(PCR). Western blotting was used to examine the protein levels of CTGF, Histone H3(H3), Smad7, Smad2/3, Acetyl-Histone H3(AH3), HDAC2, α-smooth muscle actin( α-SMA), HDAC6, p-Smad2/3 and HDAC8. In addition, the TGF-β1 and liver enzyme levels from rat serum were detected. Histopathological changes were examined by hematoxylin and eosin(HE), Sirius red and Masson trichrome staining. The α-SMA expression was detected by immumohistochemical staining. Results: Compared with control group, the TGF-β1 and liver enzyme levels from rat serum, together with the mRNA levels of CTGF and protein levels of CTGF, HDAC2, α-SMA, HDAC6, p-Smad2/3 and HDAC8 were elevated in fibrotic rats( P < 0.01). But the Smad7 mRNA and AH3 protein levels were notably suppressed in the fibrotic rats( P < 0.01). Pathological examination showed the typical changes of liver fibrosis in the fibrotic rats. After the treatment with SAHA, the levels of liver enzymes, TGF-β1, CTGF, HDAC2, α-SMA, HDAC6, p-Smad2/3 and HDAC8 were reduced( P < 0.01) and Smad7 and AH3 protein contents were elevated in liver fibrotic rats( P < 0.01). Moreover, immumohistochemistry showed that SAHA significantly suppressed the α-SMA protein content in fibrotic liver( P < 0.01). Conclusion: The HDAC inhibitor SAHA alleviated liver fibrosis by suppressing the TGF-β1 signaling.展开更多
BACKGROUND The incidence of inflammatory bowel disease,a chronic intestinal inflammatory disorder that includes Crohn’s disease(CD)and ulcerative colitis,is rising.Circular RNAs are considered valuable diagnostic bio...BACKGROUND The incidence of inflammatory bowel disease,a chronic intestinal inflammatory disorder that includes Crohn’s disease(CD)and ulcerative colitis,is rising.Circular RNAs are considered valuable diagnostic biomarkers for CD.Current evidence supports the views that epithelial-mesenchymal transition(EMT)plays an important role in CD pathogenesis,and that hsa-miR-130a-3p can inhibit transforming growth factor-β1(TGF-β1)-induced EMT.Our previous study revealed that hsa_circRNA_102610 was upregulated in CD patients.Moreover,we predicted an interaction between hsa_circRNA_102610 and hsa-miR-130a-3p.Thus,we hypothesized that hsa_circRNA_102610 may play roles in the proliferation and EMT of intestinal epithelial cells by sponging hsa-miR-130a-3p to participate in the pathogenesis of CD.AIM To explore the mechanism of hsa_circRNA_102610 in the pathogenesis of CD.METHODS The relative expression levels of hsa_circRNA_102610 and hsa-miR-130a-3p in patients were detected by quantitative reverse transcription-polymerase chain reaction.The proliferation of human intestinal epithelial cells(HIECs)and normal-derived colon mucosa cell line 460(NCM460)cells was detected by cell counting kit-8,5-ethynyl-2’-deoxyuridine staining and cell cycle assays following overexpression or downregulation of hsa_circRNA_102610.Cell proliferation assays were performed as described above in a rescue experiment with hsa-miR-130a-3p mimics.The interaction of hsa_circRNA_102610 and hsa-miR-130a-3p was verified by fluorescence in situ hybridization and dual luciferase reporter assays.The relative expression levels of CyclinD1,mothers against decapentaplegic homolog 4(SMAD4),E-cadherin,N-cadherin and Vimentin were detected by western blotting following hsa_circRNA_102610 overexpression,TGF-β1-induced EMT or hsa-miR-130a-3p mimic transfection(in rescue experiments).RESULTS Upregulation of hsa_circRNA_102610 was determined to be positively correlated with elevated fecal calprotectin levels in CD(r=0.359,P=0.007)by Pearson correlation analysis.Hsa_circRNA_102610 promoted the proliferation of HIECs and NCM460 cells,while hsa-miR-130a-3p reversed the cell proliferationpromoting effects of hsa_circRNA_102610.Fluorescence in situ hybridization and dual luciferase reporter assays showed that hsa_circRNA_102610 directly bound hsa-miR-130a-3p in NCM460 and 293T cells.An inverse correlation between downregulation of hsa-miR-130a-3p and upregulation of hsa_circRNA_102610 in CD patients was observed(r=-0.290,P=0.024)by Pearson correlation analysis.Moreover,overexpression of hsa_circRNA_102610 promoted SMAD4 and CyclinD1 protein expression validated by western-blotting.Furthermore,overexpression of hsa_circRNA_102610 promoted TGF-β1 induced EMT in HIECs and NCM460 cells via targeting of hsa-miR-130a-3p,with increased expression of Vimentin and N-cadherin and decreased expression of E-cadherin.CONCLUSION Hsa_circRNA_102610 upregulation in CD patients could promote the proliferation and EMT of intestinal epithelial cells via sponging of hsa-miR-130a-3p.展开更多
BACKGROUND The epiphyseal growth plate is an important anatomical segment localized on the ends of a long bone.Despite the abovementioned atractive reasons for alendronate’s use,few data on the effect of alendronate ...BACKGROUND The epiphyseal growth plate is an important anatomical segment localized on the ends of a long bone.Despite the abovementioned atractive reasons for alendronate’s use,few data on the effect of alendronate during epiphyseal growth exist.AIM Verify the effect of alendronate on the growth epiphyseal plate,and compare its effect with the size of the femur during the double-staining of the immunolocalization of transforming growth factor-β1(TGF-β1)and bone morphogenetic protein-2(BMP2)in endochondral ossifing in specimens that have received alendronate.METHODS Forty newborn rats were randomly divided into two groups:a control group(were given applications of 1 mg/kg physiologic saline)and a group that received Alendronate(a dose of 2.5 mg/kg).These groups were then divided into two subgroups for euthanasia in two and 12 d of life.After euthanasia,the femurs were removed,and the femoral bones were measured linearly between the apex of the greater trochanter until the lower intercondylar midlle face to verify the probable bone growth between 3 and 12 d in control and alednroanto treated rats.Posteriorly,the surgical pieces were also sent to the histopathology laboratory to produce histological slides.The obtained slides were stained with hematoxylin and eosin to measure each of the cartilage zones in endochondral development.and other slides were immunohistochemically tested for anti-TGF-β1 and BMP-2 antibodies to investigate the immunolocalization of these proteins in the epiphyseal plaque area.RESULTS On the third day,some diferences between the control group and specimens treated with alendronate were verified.Macroscopiccaly,we found similarities in size between the femoral bones when we compared the control group with the specimens that received alendronate.On the 12^th day,the bone size of the mice receiving the drug was significantly smaller than those of the control group.These results coincide with changes in the TGF-β1 and BMP-2 expression.In the specimens that received alendronate,the TGF-β1 was expressed in some sites of trabecular bone that was neoformed,peripherally to the bone marrow area.The BMP-2 was also positive in proliferative chondrocytes and hypertrofic chondrocytes.On the 12^th day,all layers of chondrocytes exhibited positivity for BMP-2 in the specimens that received alendronate.In the interface between the trabecular bone and cartilage,an area of disorganized bone deposition was evident.Neoformed bone also appeared to be different at 12 d.In the control group,BMP-2 was positive in an intense area of bone trabeculae,whereas the alendronate-treated group showed TGF-β1 positive trabeculae and a greater bone area.CONCLUSION Alendronate alters the immunolocalization of TGF-β1 and BMP-2 simultaneously,a condition that changes the usual histological aspects of the cartilage zone and impairs epiphysis growth and femur growth.展开更多
AIM:To investigate the effects of transforming growth factorβ1(TGF-β1)on the differentiation of colonic lamina propria fibroblasts(CLPF)into myofibroblasts in vitro. METHODS:Primary CLPF cultures were incubated with...AIM:To investigate the effects of transforming growth factorβ1(TGF-β1)on the differentiation of colonic lamina propria fibroblasts(CLPF)into myofibroblasts in vitro. METHODS:Primary CLPF cultures were incubated with TGF-β1 and analyzed for production ofα-smooth muscle actin(α-SMA),fibronectin(FN)and FN isoforms.Migration assays were performed in a modified 48-well Boyden chamber.Levels of total and phosphorylated focal adhesion kinase(FAK)in CLPF were analyzed after induction of migration.RESULTS:Incubation of CLPF with TGF-β1 for 2 d did not changeα-SMA levels,while TGF-β1 treatment for 6 d significantly increasedα-SMA production. Short term incubation(6 h)with TGF-β1 enhanced CLPF migration,while long term treatment(6 d)of CLPF with TGF-β1 reduced migration to 15%-37% compared to untreated cells.FN and FN isoform mRNA expression were increased after short term incubation with TGF-β1(2 d)in contrast to long term incubation with TGF-β1 for 6 d.After induction of migration, TGF-β1-preincubated CLPF showed higher amounts of FN and its isoforms and lower levels of total and phosphorylated FAK than untreated cells. CONCLUSION:Long term incubation of CLPF with TGF-β1 induced differentiation into myofibroblasts with enhancedα-SMA,reduced migratory potential and FAK phosphorylation,and increased FN production.In contrast,short term contact(6 h)of fibroblasts with TGF-β1 induced a dose-dependent increase of cell migration and FAK phosphorylation without induction ofα-SMA production.展开更多
Objective To investigate the role of transforming growth factor-β1(TGF-β1)/Smad4 pathway in development of renal fibrosis in streptozotocin(STZ)-induced diabetic nephropathy(DN) rats and explore its possible mechani...Objective To investigate the role of transforming growth factor-β1(TGF-β1)/Smad4 pathway in development of renal fibrosis in streptozotocin(STZ)-induced diabetic nephropathy(DN) rats and explore its possible mechanism.Methods Male Wistar rats weighing 180-220 g were divided into 5 groups:group A(normal control),group B [diabetes mellitus(DM) 2 weeks],group C(DM 4 weeks),group D(DM 8 weeks),and group E(DM 16 weeks).Except for the normal control group,other groups were induced DM by single injection of STZ(55 mg/kg) respectively.Blood glucose level,serum creatinine,and 24-hour urine protein were examined.Expressions of TGF-β1 and Smad4 protein and mRNA in kidney were detected using immunohistochemical technique,Western blot,and real-time PCR.mRNA expressions of stromelysin-1(MMP-3),tissue inhibitor of metalloproteinase-1(TIMP-1),and collagen Ⅲ in kidney were also detected by real-time PCR..Results The levels of blood glucose,serum creatinine,and 24-hour urine protein in rats of group B,C,D,and E were higher than those of the control group.With the progression of renal fibrosis,the expressions of TGF-β1 and Smad4 protein and mRNA in kidney of diabetic rats elevated.In addition,the renal MMP-3 mRNA expression diminished in diabetic rats,while TIMP-1 and collagen Ⅲ mRNA increased.Conclusions In STZ-induced diabetic rats,the TGF-β1/Smad4 appears to play an important role in renal fibrosis of DN.The increased expression of TGF-β1 and Smad4 might result in the transcriptional regulation of downstream target genes of TGF-β1/Smad4 pathway,which contributes to the progression of renal fibrosis in diabetic rats.展开更多
AIM: To elucidate the possible difference in two promoter polymorphisms of the transforming growth factor-β1 (TGF-β1) gene (-800G > A, -509C > T) between ulcerative colitis (UC) patients and normal subjects.ME...AIM: To elucidate the possible difference in two promoter polymorphisms of the transforming growth factor-β1 (TGF-β1) gene (-800G > A, -509C > T) between ulcerative colitis (UC) patients and normal subjects.METHODS: A total of 155 patients with established ulcerative colitis and 139 normal subjects were selected as controls. Two single nucleotide polymorphisms within the promoter region of TGF-β1 gene (-509C > T and -800G > A) were genotyped using PCR-RFLP. RESULTS: There was a statistically significant difference in genotype and allele frequency distributions between UC patients and controls for the -800G > A polymorphism of the TGF-β1 gene (P < 0.05). The frequency of the TGF-β1 gene polymorphism at position -800 showed that the AA genotype and the allele A frequencies significantly differed between the patients and healthy controls (P < 0.05). At position -509, there was no statically significant difference in genotype and allele frequency between the patients and control subjects.CONCLUSION: The results of our study indicate that there is a significant difference in both allele and genotype frequency at position -800G > A of TGF-β1 gene promoter between Iranian patients with UC and normal subjects.展开更多
In order to investigate the biological function of transforming growth factor-β1(TGF-β1) during fibrosis in denervated skeletal muscle,we recruited sciatic nerve injury model of SD rats in which denervated gastrocne...In order to investigate the biological function of transforming growth factor-β1(TGF-β1) during fibrosis in denervated skeletal muscle,we recruited sciatic nerve injury model of SD rats in which denervated gastrocnemius was isolated for analysis.At different time points after operation,denervated muscle was examined by several methods.Masson trichrome staining showed morphological changes of denervated skeletal muscle.Quantitative RT-PCR detected the rapid increase of TGF-β1 expression at mRNA level after nerve injury.It was found that a peak of TGF-β1 mRNA expression appeared one week post-operation.The expression of collagen Ⅰ(COL Ⅰ) mRNA was up-regulated in the nerve injury model as well,and reached highest level two weeks post-injury.Immunoblot revealed similar expression pattern of TGF-β1 and COL Ⅰ in denervated muscles at protein level.In addition,we found that the area of the gastrocnemius muscle fiber was decreased gradually along with increased interstitital fibrosis.Interestingly,this pathological change could be prevented,at least partly,by local injection of TGF-β1 antibodies,which could be contributed to the reduced production of COL Ⅰ by inhibiting function of TGF-β1.Taken together,in this study,we demonstrated that the expression of TGF-β1 was increased significantly in denervated skeletal muscle,which might play a crucial role during muscle fibrosis after nerve transection.展开更多
Aim To determine the effect of local simvastatin application on the mRNA expression level of transforming growth factor-β1(TGF-β1),bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor(VEGF) in...Aim To determine the effect of local simvastatin application on the mRNA expression level of transforming growth factor-β1(TGF-β1),bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor(VEGF) in the tooth sockets of rat. Methodology Forty-eight male Wistar rats were randomly divided into experimental and control groups(n=24). Polylactic acid/polyglycolic acid copolymer carriers,with or without simvastatin,were implanted into extraction sockets of right mandibular incisors.The expression of TGF-β1,BMP-2 and VEGF mRNA was determined by in situ hybridization in the tooth extraction socket at five days,one week,two weeks and four weeks after implantation. Results The fusiform stroma cells in the tooth extraction socket began to express TGF-β1,BMP-2 and VEGF mRNA in both experimental and control groups from one week after tooth extraction until the end of experiment.The expression of TGF-β1 and BMP-2 mRNA in the experimental group was significantly up-regulated after one,two and four weeks,and expression of VEGF mRNA was significantly increased after one and two weeks compared with that in the control group. Conclusion The findings indicate that local administration of simvastatin can influence alveolar bone remodeling by regulating the expression of a school of growth factors which are crucial to osteogenesis in the tooth extraction socket.展开更多
AIM: To elucidate the biological effects of transforming growth factor-β1 (TGF-β1) on intrahepatic cholan- giocarcinoma (ICC). METHODS: We investigated the effects of TGF-β1 on human ICC cell lines (HuCCT1, MEC, an...AIM: To elucidate the biological effects of transforming growth factor-β1 (TGF-β1) on intrahepatic cholan- giocarcinoma (ICC). METHODS: We investigated the effects of TGF-β1 on human ICC cell lines (HuCCT1, MEC, and HuH-28) by monitoring the influence of TGF-β1 on tumor growth and interleukin-6 (IL-6) expression in ICC cells. RESULTS: All three human ICC cell lines produced TGF-β1 and demonstrated accelerated growth in the presence of TGF-β1 with no apoptotic effect. Studies on HuCCT1 revealed a TGF-β1-induced stimulation of the expression of TGF-β1, as well as a decrease in TGF-β1 mRNA expression induced by neutralizing anti-TGF-β1 antibody. These results indicate that TGF-β1 stimulates the production and function of TGF-β1 in an autocrine fashion. Further, IL-6 secretion was observed in all three cell lines and exhibited an inhibitory response to neutralizing anti-TGF-β1 antibody. Experiments using HuCCT1 revealed a TGF-β1-induced acceleration of IL-6 protein expression and mRNA levels. These findings demonstrate a functional interaction between TGF-β1 and IL-6. All three cell lines proliferated in the presence of IL-6. In contrast, TGF-β1 induced no growth effect in HuCCT1 in the presence of small interfering RNA against a specific cell surface receptor of IL-6 and signal transducer and activator of transcription-3. CONCLUSION: ICC cells produce TGF-β1 and confer a TGF-β1-induced growth effect in an autocrine fashion.TGF-β1 activates IL-6 production, and the functional interaction between TGF-β1 and IL-6 contributes to ICC cell growth by TGF-β1.展开更多
BACKGROUND The TGF-β/SMAD3 and VEGFR-1 signaling pathways play important roles in gastric cancer metastasis.SMAD3 phosphorylation is a crucial prognostic marker in gastric cancer.AIM To determine the prognostic value...BACKGROUND The TGF-β/SMAD3 and VEGFR-1 signaling pathways play important roles in gastric cancer metastasis.SMAD3 phosphorylation is a crucial prognostic marker in gastric cancer.AIM To determine the prognostic value and relationship of SMAD3 phospho-isoforms and VEGFR-1 in gastric cancer.METHODS This was a single-center observational study which enrolled 98 gastric cancer patients and 82 adjacent normal gastric tissues from patients aged 32-84 years(median age 65)between July 2006 and April 2007.Patients were followed up until death or the study ended(median follow-up duration of 28.5 mo).The samples were used to generate tissue microarrays(TMAs)for immunohistochemical(IHC)staining.The expressions of TGF-β1,pSMAD3C(S423/425),pSMAD3L(S204),and VEGFR-1 in gastric cancer(GC)tumor tissue and normal tissue were measured by IHC staining using TMAs obtained from 98 GC patients.Prognosis and survival information of the patients was recorded by Outdo Biotech from May 2007 to July 2015.The relationship between TGF-β1,pSMAD3C(S423/425),pSMAD3L(S204),and VEGFR-1 protein expression levels was analyzed using Pearson's correlation coefficient.The relationship between protein expression levels and clinicopathological parameters was analyzed using the Chi-squared test.A survival curve was generated using the Kaplan-Meier survival analysis.RESULTS TGFβ-1 and VEGFR-1 expression was significantly upregulated in gastric cancer tissue compared to adjacent noncancerous tissue.The positive expression of phosphorylated isoforms of Smad3 varied depending on the phosphorylation site[pSMAD3C(S423/425):51.0%and pSMAD3L(S204):31.6%].High expression of pSMAD-3L(S204)was significantly correlated with larger tumors(P=0.038)and later N stages(P=0.035).Additionally,high expression of VEGFR-1 was closely correlated with tumor size(P=0.015)and pathological grading(P=0.013).High expression of both pSMAD3L(S204)and VEGFR-1 was associated with unfavorable outcomes in terms of overall survival(OS).Multivariate analysis indicated that high expression of pSMAD3L(S204)and VEGFR-1 were independent risk factors for prognosis in GC patients.VEGFR-1 protein expression was correlated with TGF-β1(r=0.220,P=0.029),pSMAD3C(S423/425)(r=0.302,P=0.002),and pSMAD3L(S204)(r=0.201,P=0.047),respectively.Simultaneous overexpression of pSMAD3L(S204)and VEGFR-1 was associated with poor OS in gastric cancer patients.CONCLUSION Co-upregulation of pSMAD3L(S204)and VEGFR-1 can serve as a predictive marker for poor gastric cancer prognosis,and pSMAD3L(204)may be involved in enhanced gastric cancer metastasis in a VEGFR-1-dependent manner.展开更多
Objective: To analyze and compare the expression pattern of the transforming growth factor-β1(TGF-β1) and its type I receptor (TGF-β RI ) in nounal human skin and various phases of post-burn hypertrophic scars (HTS...Objective: To analyze and compare the expression pattern of the transforming growth factor-β1(TGF-β1) and its type I receptor (TGF-β RI ) in nounal human skin and various phases of post-burn hypertrophic scars (HTS). Method: The immunohistochemical ABC method was employed. Results: In nounal human skin, no evident immunoreactivity of TGF-β1 and TGF-β R I was observed. In activation phase of post-burn HTS, TGF-β R I and TGF-β1 were highly expressed in most dermal fibroblasts which seemed to be the same subset. However, in remission phase, no staining was seen in der mal fibroblasts. Conclusion: The formation of all may involve the increase of TGF-β responsiveness in fibroblasts The ac cumulation at the wound site and failure of apoptosis of over-resposive fibroblasts may contribute to the formation of HTS.展开更多
基金Supported by the Natural Science Foundation of Jiangsu Province,China,No.BK2016157the National Natural Science Foundation of China,No.81673973+1 种基金Phase Ⅱ Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions,No.035062002003Developing Program for Highlevel Academic Talent in Jiangsu Hospital of TCM,No.y2018rc16
文摘AIM To explore the role and mechanism of total flavone of Abelmoschus manihot(TFA) on epithelial-mesenchymal transition(EMT) progress of Crohn's disease(CD) intestinal fibrosis.METHODS First,CCK-8 assay was performed to assess TFA on the viability of intestinal epithelial(IEC-6) cells and select the optimal concentrations of TFA for our further studies.Then cell morphology,wound healing and transwell assays were performed to examine the effect of TFA on morphology,migration and invasion of IEC-6 cells treated with TGF-β1.In addition,immunofluorescence,real-time PCR analysis(q RT-PCR) and western blotting assays were carried out to detect the impact of TFA on EMT progress.Moreover,western blotting assay was performed to evaluate the function of TFA on the Smad and MAPK signaling pathways.Further,the role of co-treatment of TFA and si-Smad or MAPK inhibitors has been examined by q RTPCR,western blotting,morphology,wound healing andtranswell assays.RESULTS In this study,TFA promoted transforming growth factor-β1(TGF-β1)-induced(IEC-6) morphological change,migration and invasion,and increased the expression of epithelial markers and reduced the levels of mesenchymal markers,along with the inactivation of Smad and MAPK signaling pathways.Moreover,we revealed that si-Smad and MAPK inhibitors effectively attenuated TGF-β1-induced EMT in IEC-6 cells.Importantly,co-treatment of TFA and si-Smad or MAPK inhibitors had better inhibitory effects on TGF-β1-induced EMT in IEC-6 cells than either one of them.CONCLUSION These findings could provide new insight into the molecular mechanisms of TFA on TGF-β1-induced EMT in IEC-6 cells and TFA is expected to advance as a new therapy to treat CD intestinal fibrosis.
基金supported by a grant from the National Natural Science Foundation of China(81870413)
文摘Background: Histone deacetylases(HDACs) inhibitors are new anti-fibrotic drugs that inhibit the activity of hepatic stellate cells. The present study focused on the anti-fibrotic function of HDAC inhibitor suberoylanilide hydroxamic acid(SAHA) by suppressing transforming growth factor-β1(TGF-β1) signaling. Methods: Male Sprague-Dawley rats were used to induce liver fibrosis with carbon tetrachloride(CCl 4) and LX2 cell(human hepatic stellate cell line) was stimulated by TGF-β1. Both animals and cells were treated with SAHA. The Smad7 and connective tissue growth factor(CTGF) mRNA levels were detected by real-time polymerase chain reaction(PCR). Western blotting was used to examine the protein levels of CTGF, Histone H3(H3), Smad7, Smad2/3, Acetyl-Histone H3(AH3), HDAC2, α-smooth muscle actin( α-SMA), HDAC6, p-Smad2/3 and HDAC8. In addition, the TGF-β1 and liver enzyme levels from rat serum were detected. Histopathological changes were examined by hematoxylin and eosin(HE), Sirius red and Masson trichrome staining. The α-SMA expression was detected by immumohistochemical staining. Results: Compared with control group, the TGF-β1 and liver enzyme levels from rat serum, together with the mRNA levels of CTGF and protein levels of CTGF, HDAC2, α-SMA, HDAC6, p-Smad2/3 and HDAC8 were elevated in fibrotic rats( P < 0.01). But the Smad7 mRNA and AH3 protein levels were notably suppressed in the fibrotic rats( P < 0.01). Pathological examination showed the typical changes of liver fibrosis in the fibrotic rats. After the treatment with SAHA, the levels of liver enzymes, TGF-β1, CTGF, HDAC2, α-SMA, HDAC6, p-Smad2/3 and HDAC8 were reduced( P < 0.01) and Smad7 and AH3 protein contents were elevated in liver fibrotic rats( P < 0.01). Moreover, immumohistochemistry showed that SAHA significantly suppressed the α-SMA protein content in fibrotic liver( P < 0.01). Conclusion: The HDAC inhibitor SAHA alleviated liver fibrosis by suppressing the TGF-β1 signaling.
基金Supported by the Suzhou Special Project of Diagnosis and Treatment for Key Clinical Disease,No.LCZX201715the Natural Science Foundation of Jiangsu Province,No.BK20161232the Science and Technology Development Fund of Nanjing Medical University,No.NMUB2018215.
文摘BACKGROUND The incidence of inflammatory bowel disease,a chronic intestinal inflammatory disorder that includes Crohn’s disease(CD)and ulcerative colitis,is rising.Circular RNAs are considered valuable diagnostic biomarkers for CD.Current evidence supports the views that epithelial-mesenchymal transition(EMT)plays an important role in CD pathogenesis,and that hsa-miR-130a-3p can inhibit transforming growth factor-β1(TGF-β1)-induced EMT.Our previous study revealed that hsa_circRNA_102610 was upregulated in CD patients.Moreover,we predicted an interaction between hsa_circRNA_102610 and hsa-miR-130a-3p.Thus,we hypothesized that hsa_circRNA_102610 may play roles in the proliferation and EMT of intestinal epithelial cells by sponging hsa-miR-130a-3p to participate in the pathogenesis of CD.AIM To explore the mechanism of hsa_circRNA_102610 in the pathogenesis of CD.METHODS The relative expression levels of hsa_circRNA_102610 and hsa-miR-130a-3p in patients were detected by quantitative reverse transcription-polymerase chain reaction.The proliferation of human intestinal epithelial cells(HIECs)and normal-derived colon mucosa cell line 460(NCM460)cells was detected by cell counting kit-8,5-ethynyl-2’-deoxyuridine staining and cell cycle assays following overexpression or downregulation of hsa_circRNA_102610.Cell proliferation assays were performed as described above in a rescue experiment with hsa-miR-130a-3p mimics.The interaction of hsa_circRNA_102610 and hsa-miR-130a-3p was verified by fluorescence in situ hybridization and dual luciferase reporter assays.The relative expression levels of CyclinD1,mothers against decapentaplegic homolog 4(SMAD4),E-cadherin,N-cadherin and Vimentin were detected by western blotting following hsa_circRNA_102610 overexpression,TGF-β1-induced EMT or hsa-miR-130a-3p mimic transfection(in rescue experiments).RESULTS Upregulation of hsa_circRNA_102610 was determined to be positively correlated with elevated fecal calprotectin levels in CD(r=0.359,P=0.007)by Pearson correlation analysis.Hsa_circRNA_102610 promoted the proliferation of HIECs and NCM460 cells,while hsa-miR-130a-3p reversed the cell proliferationpromoting effects of hsa_circRNA_102610.Fluorescence in situ hybridization and dual luciferase reporter assays showed that hsa_circRNA_102610 directly bound hsa-miR-130a-3p in NCM460 and 293T cells.An inverse correlation between downregulation of hsa-miR-130a-3p and upregulation of hsa_circRNA_102610 in CD patients was observed(r=-0.290,P=0.024)by Pearson correlation analysis.Moreover,overexpression of hsa_circRNA_102610 promoted SMAD4 and CyclinD1 protein expression validated by western-blotting.Furthermore,overexpression of hsa_circRNA_102610 promoted TGF-β1 induced EMT in HIECs and NCM460 cells via targeting of hsa-miR-130a-3p,with increased expression of Vimentin and N-cadherin and decreased expression of E-cadherin.CONCLUSION Hsa_circRNA_102610 upregulation in CD patients could promote the proliferation and EMT of intestinal epithelial cells via sponging of hsa-miR-130a-3p.
文摘BACKGROUND The epiphyseal growth plate is an important anatomical segment localized on the ends of a long bone.Despite the abovementioned atractive reasons for alendronate’s use,few data on the effect of alendronate during epiphyseal growth exist.AIM Verify the effect of alendronate on the growth epiphyseal plate,and compare its effect with the size of the femur during the double-staining of the immunolocalization of transforming growth factor-β1(TGF-β1)and bone morphogenetic protein-2(BMP2)in endochondral ossifing in specimens that have received alendronate.METHODS Forty newborn rats were randomly divided into two groups:a control group(were given applications of 1 mg/kg physiologic saline)and a group that received Alendronate(a dose of 2.5 mg/kg).These groups were then divided into two subgroups for euthanasia in two and 12 d of life.After euthanasia,the femurs were removed,and the femoral bones were measured linearly between the apex of the greater trochanter until the lower intercondylar midlle face to verify the probable bone growth between 3 and 12 d in control and alednroanto treated rats.Posteriorly,the surgical pieces were also sent to the histopathology laboratory to produce histological slides.The obtained slides were stained with hematoxylin and eosin to measure each of the cartilage zones in endochondral development.and other slides were immunohistochemically tested for anti-TGF-β1 and BMP-2 antibodies to investigate the immunolocalization of these proteins in the epiphyseal plaque area.RESULTS On the third day,some diferences between the control group and specimens treated with alendronate were verified.Macroscopiccaly,we found similarities in size between the femoral bones when we compared the control group with the specimens that received alendronate.On the 12^th day,the bone size of the mice receiving the drug was significantly smaller than those of the control group.These results coincide with changes in the TGF-β1 and BMP-2 expression.In the specimens that received alendronate,the TGF-β1 was expressed in some sites of trabecular bone that was neoformed,peripherally to the bone marrow area.The BMP-2 was also positive in proliferative chondrocytes and hypertrofic chondrocytes.On the 12^th day,all layers of chondrocytes exhibited positivity for BMP-2 in the specimens that received alendronate.In the interface between the trabecular bone and cartilage,an area of disorganized bone deposition was evident.Neoformed bone also appeared to be different at 12 d.In the control group,BMP-2 was positive in an intense area of bone trabeculae,whereas the alendronate-treated group showed TGF-β1 positive trabeculae and a greater bone area.CONCLUSION Alendronate alters the immunolocalization of TGF-β1 and BMP-2 simultaneously,a condition that changes the usual histological aspects of the cartilage zone and impairs epiphysis growth and femur growth.
文摘AIM:To investigate the effects of transforming growth factorβ1(TGF-β1)on the differentiation of colonic lamina propria fibroblasts(CLPF)into myofibroblasts in vitro. METHODS:Primary CLPF cultures were incubated with TGF-β1 and analyzed for production ofα-smooth muscle actin(α-SMA),fibronectin(FN)and FN isoforms.Migration assays were performed in a modified 48-well Boyden chamber.Levels of total and phosphorylated focal adhesion kinase(FAK)in CLPF were analyzed after induction of migration.RESULTS:Incubation of CLPF with TGF-β1 for 2 d did not changeα-SMA levels,while TGF-β1 treatment for 6 d significantly increasedα-SMA production. Short term incubation(6 h)with TGF-β1 enhanced CLPF migration,while long term treatment(6 d)of CLPF with TGF-β1 reduced migration to 15%-37% compared to untreated cells.FN and FN isoform mRNA expression were increased after short term incubation with TGF-β1(2 d)in contrast to long term incubation with TGF-β1 for 6 d.After induction of migration, TGF-β1-preincubated CLPF showed higher amounts of FN and its isoforms and lower levels of total and phosphorylated FAK than untreated cells. CONCLUSION:Long term incubation of CLPF with TGF-β1 induced differentiation into myofibroblasts with enhancedα-SMA,reduced migratory potential and FAK phosphorylation,and increased FN production.In contrast,short term contact(6 h)of fibroblasts with TGF-β1 induced a dose-dependent increase of cell migration and FAK phosphorylation without induction ofα-SMA production.
文摘Objective To investigate the role of transforming growth factor-β1(TGF-β1)/Smad4 pathway in development of renal fibrosis in streptozotocin(STZ)-induced diabetic nephropathy(DN) rats and explore its possible mechanism.Methods Male Wistar rats weighing 180-220 g were divided into 5 groups:group A(normal control),group B [diabetes mellitus(DM) 2 weeks],group C(DM 4 weeks),group D(DM 8 weeks),and group E(DM 16 weeks).Except for the normal control group,other groups were induced DM by single injection of STZ(55 mg/kg) respectively.Blood glucose level,serum creatinine,and 24-hour urine protein were examined.Expressions of TGF-β1 and Smad4 protein and mRNA in kidney were detected using immunohistochemical technique,Western blot,and real-time PCR.mRNA expressions of stromelysin-1(MMP-3),tissue inhibitor of metalloproteinase-1(TIMP-1),and collagen Ⅲ in kidney were also detected by real-time PCR..Results The levels of blood glucose,serum creatinine,and 24-hour urine protein in rats of group B,C,D,and E were higher than those of the control group.With the progression of renal fibrosis,the expressions of TGF-β1 and Smad4 protein and mRNA in kidney of diabetic rats elevated.In addition,the renal MMP-3 mRNA expression diminished in diabetic rats,while TIMP-1 and collagen Ⅲ mRNA increased.Conclusions In STZ-induced diabetic rats,the TGF-β1/Smad4 appears to play an important role in renal fibrosis of DN.The increased expression of TGF-β1 and Smad4 might result in the transcriptional regulation of downstream target genes of TGF-β1/Smad4 pathway,which contributes to the progression of renal fibrosis in diabetic rats.
基金a grant from Shiraz University of Medical Sciences, No. 83-2363a grant from Shiraz Institute for Cancer Research, ICR-8296
文摘AIM: To elucidate the possible difference in two promoter polymorphisms of the transforming growth factor-β1 (TGF-β1) gene (-800G > A, -509C > T) between ulcerative colitis (UC) patients and normal subjects.METHODS: A total of 155 patients with established ulcerative colitis and 139 normal subjects were selected as controls. Two single nucleotide polymorphisms within the promoter region of TGF-β1 gene (-509C > T and -800G > A) were genotyped using PCR-RFLP. RESULTS: There was a statistically significant difference in genotype and allele frequency distributions between UC patients and controls for the -800G > A polymorphism of the TGF-β1 gene (P < 0.05). The frequency of the TGF-β1 gene polymorphism at position -800 showed that the AA genotype and the allele A frequencies significantly differed between the patients and healthy controls (P < 0.05). At position -509, there was no statically significant difference in genotype and allele frequency between the patients and control subjects.CONCLUSION: The results of our study indicate that there is a significant difference in both allele and genotype frequency at position -800G > A of TGF-β1 gene promoter between Iranian patients with UC and normal subjects.
基金supported by a grant from the National Natural Sciences Foundation of China (No. 30872627)
文摘In order to investigate the biological function of transforming growth factor-β1(TGF-β1) during fibrosis in denervated skeletal muscle,we recruited sciatic nerve injury model of SD rats in which denervated gastrocnemius was isolated for analysis.At different time points after operation,denervated muscle was examined by several methods.Masson trichrome staining showed morphological changes of denervated skeletal muscle.Quantitative RT-PCR detected the rapid increase of TGF-β1 expression at mRNA level after nerve injury.It was found that a peak of TGF-β1 mRNA expression appeared one week post-operation.The expression of collagen Ⅰ(COL Ⅰ) mRNA was up-regulated in the nerve injury model as well,and reached highest level two weeks post-injury.Immunoblot revealed similar expression pattern of TGF-β1 and COL Ⅰ in denervated muscles at protein level.In addition,we found that the area of the gastrocnemius muscle fiber was decreased gradually along with increased interstitital fibrosis.Interestingly,this pathological change could be prevented,at least partly,by local injection of TGF-β1 antibodies,which could be contributed to the reduced production of COL Ⅰ by inhibiting function of TGF-β1.Taken together,in this study,we demonstrated that the expression of TGF-β1 was increased significantly in denervated skeletal muscle,which might play a crucial role during muscle fibrosis after nerve transection.
基金supported by grants from the National Nature Science foundation of China(Grant Nos.30872912 and 30830108)
文摘Aim To determine the effect of local simvastatin application on the mRNA expression level of transforming growth factor-β1(TGF-β1),bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor(VEGF) in the tooth sockets of rat. Methodology Forty-eight male Wistar rats were randomly divided into experimental and control groups(n=24). Polylactic acid/polyglycolic acid copolymer carriers,with or without simvastatin,were implanted into extraction sockets of right mandibular incisors.The expression of TGF-β1,BMP-2 and VEGF mRNA was determined by in situ hybridization in the tooth extraction socket at five days,one week,two weeks and four weeks after implantation. Results The fusiform stroma cells in the tooth extraction socket began to express TGF-β1,BMP-2 and VEGF mRNA in both experimental and control groups from one week after tooth extraction until the end of experiment.The expression of TGF-β1 and BMP-2 mRNA in the experimental group was significantly up-regulated after one,two and four weeks,and expression of VEGF mRNA was significantly increased after one and two weeks compared with that in the control group. Conclusion The findings indicate that local administration of simvastatin can influence alveolar bone remodeling by regulating the expression of a school of growth factors which are crucial to osteogenesis in the tooth extraction socket.
基金Supported by the Ministry of Education, Science, Sports, and Culture of Japan, through a Grant-in-Aid for Scientific Research (C)
文摘AIM: To elucidate the biological effects of transforming growth factor-β1 (TGF-β1) on intrahepatic cholan- giocarcinoma (ICC). METHODS: We investigated the effects of TGF-β1 on human ICC cell lines (HuCCT1, MEC, and HuH-28) by monitoring the influence of TGF-β1 on tumor growth and interleukin-6 (IL-6) expression in ICC cells. RESULTS: All three human ICC cell lines produced TGF-β1 and demonstrated accelerated growth in the presence of TGF-β1 with no apoptotic effect. Studies on HuCCT1 revealed a TGF-β1-induced stimulation of the expression of TGF-β1, as well as a decrease in TGF-β1 mRNA expression induced by neutralizing anti-TGF-β1 antibody. These results indicate that TGF-β1 stimulates the production and function of TGF-β1 in an autocrine fashion. Further, IL-6 secretion was observed in all three cell lines and exhibited an inhibitory response to neutralizing anti-TGF-β1 antibody. Experiments using HuCCT1 revealed a TGF-β1-induced acceleration of IL-6 protein expression and mRNA levels. These findings demonstrate a functional interaction between TGF-β1 and IL-6. All three cell lines proliferated in the presence of IL-6. In contrast, TGF-β1 induced no growth effect in HuCCT1 in the presence of small interfering RNA against a specific cell surface receptor of IL-6 and signal transducer and activator of transcription-3. CONCLUSION: ICC cells produce TGF-β1 and confer a TGF-β1-induced growth effect in an autocrine fashion.TGF-β1 activates IL-6 production, and the functional interaction between TGF-β1 and IL-6 contributes to ICC cell growth by TGF-β1.
基金Supported by National Nature Science Foundation of China,No.82060450,No.82360517,No.81460374,and No.31460304Nature Science Foundation of Jiangxi Province of China,No.20232BAB206086,No.20192BAB205072,No.20203BBGL73206,No.2017BCB23086,No.2017BAB205062,and No.20181BAB205050.
文摘BACKGROUND The TGF-β/SMAD3 and VEGFR-1 signaling pathways play important roles in gastric cancer metastasis.SMAD3 phosphorylation is a crucial prognostic marker in gastric cancer.AIM To determine the prognostic value and relationship of SMAD3 phospho-isoforms and VEGFR-1 in gastric cancer.METHODS This was a single-center observational study which enrolled 98 gastric cancer patients and 82 adjacent normal gastric tissues from patients aged 32-84 years(median age 65)between July 2006 and April 2007.Patients were followed up until death or the study ended(median follow-up duration of 28.5 mo).The samples were used to generate tissue microarrays(TMAs)for immunohistochemical(IHC)staining.The expressions of TGF-β1,pSMAD3C(S423/425),pSMAD3L(S204),and VEGFR-1 in gastric cancer(GC)tumor tissue and normal tissue were measured by IHC staining using TMAs obtained from 98 GC patients.Prognosis and survival information of the patients was recorded by Outdo Biotech from May 2007 to July 2015.The relationship between TGF-β1,pSMAD3C(S423/425),pSMAD3L(S204),and VEGFR-1 protein expression levels was analyzed using Pearson's correlation coefficient.The relationship between protein expression levels and clinicopathological parameters was analyzed using the Chi-squared test.A survival curve was generated using the Kaplan-Meier survival analysis.RESULTS TGFβ-1 and VEGFR-1 expression was significantly upregulated in gastric cancer tissue compared to adjacent noncancerous tissue.The positive expression of phosphorylated isoforms of Smad3 varied depending on the phosphorylation site[pSMAD3C(S423/425):51.0%and pSMAD3L(S204):31.6%].High expression of pSMAD-3L(S204)was significantly correlated with larger tumors(P=0.038)and later N stages(P=0.035).Additionally,high expression of VEGFR-1 was closely correlated with tumor size(P=0.015)and pathological grading(P=0.013).High expression of both pSMAD3L(S204)and VEGFR-1 was associated with unfavorable outcomes in terms of overall survival(OS).Multivariate analysis indicated that high expression of pSMAD3L(S204)and VEGFR-1 were independent risk factors for prognosis in GC patients.VEGFR-1 protein expression was correlated with TGF-β1(r=0.220,P=0.029),pSMAD3C(S423/425)(r=0.302,P=0.002),and pSMAD3L(S204)(r=0.201,P=0.047),respectively.Simultaneous overexpression of pSMAD3L(S204)and VEGFR-1 was associated with poor OS in gastric cancer patients.CONCLUSION Co-upregulation of pSMAD3L(S204)and VEGFR-1 can serve as a predictive marker for poor gastric cancer prognosis,and pSMAD3L(204)may be involved in enhanced gastric cancer metastasis in a VEGFR-1-dependent manner.
文摘Objective: To analyze and compare the expression pattern of the transforming growth factor-β1(TGF-β1) and its type I receptor (TGF-β RI ) in nounal human skin and various phases of post-burn hypertrophic scars (HTS). Method: The immunohistochemical ABC method was employed. Results: In nounal human skin, no evident immunoreactivity of TGF-β1 and TGF-β R I was observed. In activation phase of post-burn HTS, TGF-β R I and TGF-β1 were highly expressed in most dermal fibroblasts which seemed to be the same subset. However, in remission phase, no staining was seen in der mal fibroblasts. Conclusion: The formation of all may involve the increase of TGF-β responsiveness in fibroblasts The ac cumulation at the wound site and failure of apoptosis of over-resposive fibroblasts may contribute to the formation of HTS.
