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Objective To detect the expression of heat shock protein 47(HSP47) in renal proximal epithelial cell lines (HK-2) and to investigate the role of HSP47 in the progress of transforming growth factor β1 (TGF-β1) induced epithelial-mesenchymal transdifferentiation (EMT) in HK-2 cells.Methods HK-2 cells were exposed to TGF-β1 (0,2.5,5,10 μg/L) for different time (0,12,24,48 h).The expression of HSP47 was examined by Western blotting.Then HK-2 cells were exposed to 10 μg/L TGF-β1,the expressions of vimentin,zona occludens-1 (ZO-1) were examined by Western blotting and real-time PCR.Furthermore,the expressions of p-Smad3 and Smad3 were examined by Western blotting.HK-2 cells were transfected with HSP47 siRNA and siRNA negative control before exposing to TGF-β1.Then the expressions of vimentin,ZO-1 were detected by Western blotting and real-time PCR,meanwhile Western blotting for HSP47,p-Smad3 and Smad3.Results Stimulating HK-2 with TGF-β1 resulted in a significant increased expression of HSP47 in time-and concentration-dependent manner (P ＜ 0.05).Meanwhile,TGF-β1 up-regulated the protein and mRNA expression of vimentin (P ＜ 0.05),and down-regulated the protein and mRNA expression of ZO-1 (P ＜ 0.05),all in time-dependent manner.Stimulating HK-2 with TGF-β1 resulted in phosphorylation of Smad3,which was peaked at 30 min,slightly decreased at 1 h,and then increased again between 24 and 48 h (P ＜ 0.05).Compared to the TGF-β1 group,inhibition of HSP4.7 expression in HK-2up-regulated the protein and mRNA expression of ZO-1,down-regulated the protein and mRNA expression of vimentin (P ＜ 0.05) and down-regulated the ratio of p-Smad3/Smad3.HSP47 siRNA negative control had no significant effect on the expressions of ZO-1,vimentin and p-Smad3/Smad3 (P ＞ 0.05).Conclusion HSP47 can promote the EMT of renal tubular epithelial cell which is possibly via the TGF-β1-Smad3 pathway.
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OBJECTIVE@#To investigate the role of phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) inducing epithelial mesenchymal transition in human peritoneal mesothelial cells (HPMC).@*METHODS@#Primary HPMC was harvested from human omental tissue and maintained under defined in vitro conditions. The expression of p-GSK-3beta and total GSK-3beta in HMPC was detected by Western blot after incubation with different concentrations (0, 5, 10, 20, and 40 mmol/L)of LiCl at different time points (0, 1, 3, 6, and 12 h). The protein expression of E-cadherin and alpha-SMA was also examined after treatment with 20 mmol/L LiCl according to different time courses. The intracellular distribution and expression of alpha-SMA were determined by indirect immunofluorescence.@*RESULTS@#LiCl stimulated phosphorylation of GSK-3beta and the effect was time-dependent and concentration-dependent to limited extent (P<0.05). The expression of alpha-SMA increased (P<0.05) and the expression of E-cadherin decreased significantly (P<0.05) after 24 h stimulation by 20 mmol/L LiCl. The indirect immunoflurescence showed that the expression of alpha-SMA in HPMC increased significantly after 24 h incubation with 20 mmol/L LiCl.@*CONCLUSION@#The phosphorylation of GSK-3beta leads HMPC to epithelial mesenchymal transition and provides new clue for the treatment of peritoneal fibrosis.
Subject(s)
Humans , Actins , Metabolism , Cadherins , Metabolism , Epithelial Cells , Cell Biology , Epithelial-Mesenchymal Transition , Glycogen Synthase Kinase 3 , Metabolism , Glycogen Synthase Kinase 3 beta , Lithium Chloride , Pharmacology , Mesoderm , Cell Biology , Peritoneum , Cell Biology , PhosphorylationABSTRACT
Objective To study the relationship between blood uric acid(BUA)level and oxidative stress in the elderly. Methods One hundred and eighteen healthy subjects who aged 60years and over were recruited.They were divided into 3 groups according to the serum uric acid level:(1)Control group:BUA 142-416 μmol/L;(2)Hyperuricemia group Ⅰ:BUA 417-470 μmol/L;(3)Hyperuricemia group Ⅱ:BUA＞470 μmol/L.Serum xanthine oxidase(XO)activity,superoxide dismutase(SOD)activity,total antioxidation capacity(TAOC)and peroxide concentration were examined. Results (1)No significant differences in TAOC[control group:(8.40±2.09),Hyperuricemia group Ⅰ:(8.13±1.92)and Hyperuricemia group Ⅱ:(7.95±1.54)U/ml,all P＞O.05)and in SOD activity[control group:(80.57±15.44),Hyperuricemia group Ⅰ:(81.37±11.92)and Hyperuricemia group Ⅱ:(87.45±11.38)U/ml,all P＞0.05)among the 3 groups were found.The serum peroxide level and XO activity were significantly higher in hyperuricemia group Ⅰ and Ⅱ[XO activity:(9.17±1.68) vs.(9.32±1.87)U/ml,all P＜0.05;serum peroxide:(149.5±25.7)vs.(150.7±23.1)μmol/L,all P＜0.05]than in group control[XO activity:(7.81±1.84),serum peroxide:(122.5±33.8)].(2)Multifactorial regression analysis showed TAOC was related to age,triglyceride and uric acid(all P＜0.05),XO activity was related to uric acid(P＜0.05)and peroxide concentration was related to age and uric acid(all P＜0.05). Conclusions This study supports the presence of the xanthine oxidase-related oxidative stress in the elderly with hyperuricemia.
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Objective To observe the influence of recombinant human brain natriuretic peptide(rhBNP)treatment on serum C-reactive protein(CRP)level in patients with congestive heart failure.Methods Forty patients with congestive heart failure(NYHA class Ⅳ)were divided into experimental group(n=20)receiving rhBNP and control group(n=20)receiving isosorbide dinitrate.Blood pressure and central venous pressure were observed and hypersensitive CRP was checked before and one week after treatment in both groups.Results No obvious change of blood pressure at each time point was found after BNP treatment(systolic blood pressure:P=0.804;diastolic blood pressure:P=0.492)while significant decrease of CVP was noted(P=0.016).Serum CRP level was significantly decreased one week after BNP treatment(10.1?7.4 mg/L vs 7.7?5.0 mg/L;P=0.013)in experimental group,obviously different from that in the control group(P=0.044).hsCRP change was positively related to CVP change(Pearson correlation:0.469,P=0.043).Conclusion Brain natriuretic peptide effectively decreases serum C-reactive protein level in the patients with congestive heart failure,indicating its possible role in relieving inflammation in heart failure.