| PartⅠ The Inhibitory effect of Insl6 on UUO model-induced renal fibrosis and its related mechanismObjective: To investigate the role of Insl6 in Unilateral ureteral obstruction-induced renal fibrosis and its underlying mechanisms.Methods: C57BL/6 wild type mice were used to establish the left ureteral obstruction model,and were sacrificed 3,7 and 10 days respentively after operation.The severity of renal injury and fibrosis were determined by hematoxylin-eosin(HE)and Masson staining on the surgical side of the kidney.Twenty-four male C57BL/6J mice were randomly distributed into the sham,UUO + saline and UUO+Insl6 groups.Soluble Insl6 protein and saline were infused via subcutaneous micro-osmotic pump.Mice were sacrificed 10 days after operation and surgical side kidneys were sampled.Renal injury and the degree of renal interstitial fibrosis were determined by HE and Masson staining.The expression levels of α-SMA,E-cadherin,FN and TGF-β1 in kidney tissues was determining by immunohistochemical staining.Real-time PCR were used for determine α-SMA,E-cadherin,FN and TGF-β1 m RNA expression.TGF-β1 protein,Smad2 and Smad3 phosphorylation and non-phosphorylation protein were determined by Western blotting.Results:(1)The severity of renal injury and fibrosis gradually increased along with the extension of UUO,day 10 was the most severe time point(P <0.05).(2)Compared with the sham group,the severity of renal injury and fibrosis of mice in UUO+NS groups increased significantly 10 days after surgery(P<0.05).Mice in UUO+Insl6 treatment group exhibited alleviated renal injury and interstitial fibrosis compared with the UUO+NS group(P<0.05).(3)Compared with the sham group,the protein expression of α-SMA,TGF-β1 and FN in UUO+NS group significantly increased(P<0.05),while the expression of E-cadhein dramatically decreased(P <0.05).As compared with the UUO+NS group,the expression levels of α-SMA,TGF-β1 and FN significantly decreased while the E-cadherin expression significantly increased in UUO+Insl6 group(P<0.05).(4)E-cadherin,α-SMA,TGF-β1 and FN expression in renal tissues were also consistent with the expression of m RNA by immunohistochemical staining(P<0.05).(5)In contrast to the Sham group,the renal protein expression of TGF-β1,phosphorylated Smad2 and Smad3 in UUO+NS group significantly increased(P<0.05).The renal tissues phosphorylation of Smad2 and Smad3 protein in UUO + Insl6 group was significantly dereased than that in UUO + NS group(P<0.05).Conclusions: Insl6 has anti-fibrotic effect in kidney,which might be associated with its inhibitory effect on TGF-B1/Smad2/3 signalling pathway.PartⅡ Effect of Insl6 on TGFβ1-induced EMT in renal proximal tubular epithelial cells and its underlying mechanismsObjective: To investigate the effect of Insl6 on TGF-β1-induced epithelial-mesenchymal-transition in renal proximal tubular epithelial cells and its underlying mechanisms.Methods: Human proximal tubular epithelial cells(HK-2)were stimulated with with different concentrations of TGF-β1(0,1,2,5,10ng/ml).And after 72 hours,the expression of E-cadherin and α-SMA were determined by western blotting.HK-2 cells divided into four groups:control group,TGF-β1 group,Insl6 group and TGF-β1+Insl6 group.The m RNA and protein expression of E-cadherin,α-SMA and FN were determined respectively by quantitative real-time polymerase chain reaction and western blotting.The phosphorylation and non-phosphorylation level of Smad2 and Smad3 were determined by western blotting.Results:(1)With the increase of TGF-β1 concentration,the expression of E-cadherin protein in HK-2 cells decreased gradually while the α-SMA protein increased gradually,the most significant TGF-β1 concentration was 10ng/ml(P<0.05).(2)Compared with the control group,the E-cadherin m RNA and protein expression in HK-2 cells were significantly decreased in TGF-β1 group(P<0.05),α-SMA and FN m RNA and protein expression in TGF-β1 group were significantly increased(P<0.05).Compared with TGF-β1 group,the expression of E-cadherin m RNA and protein in HK-2 cells were significantly increased in TGF-β1 +Insl6 group(P<0.05),the m RNA and protein expression of α-SMA and FN were significantly increased in TGF-β1+Insl6 group.(3)Compared with the control group,p-Smad2 and p-Smad3 protein expression in HK-2 cells significantly increased in TGF-β1 group(P<0.05).Compared with TGF-β1 group,the expression of p-Smad2 and p-Smad3 protein in HK-2 cells significantly decreased in TGF-β1+Insl6 group(P<0.05).Conclusions: TGF-β1 can induces the EMT in HK-2 cells in a dose-dependent manner.Insl6 can inhibit TGF-β1-induced EMT and might be associated with its effect on inactivation of Smad2/3 signalling pathway. |
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