| Background: Our previous studies showed that activated islet stellate cell(ISC)have a strong ability to cause islet function impairment and islet fibrosis in T2 DM.Inhibition of ISCs activation is beneficial for preserving islet function,and is potentially a pivotal strategy in the prevention of diabetes.However,the molecular mechanism of ISC activation is not clear,and there is a lack of clear intervention targets.Irisin can suppress the development of liver and kidney fibrosis by inhibit the activation of hepatic stellate cells and renal tubular cells.Therefore,irisin may suppress the development of islet fibrosis and improve islet function by decreasing the activation level of ISC.but its mechanism is still unclear.Objective: This study aimed to explore the effect of irisin on ISC activation and clarify its related pathways,so as to provide theory basis for improving islet function via irisin.Methods: 1.The ISCs from GK and Wistar rat islets were extracted by density gradient centrifugation to get GK-ISC and Wistar-ISC;The high glucose and inflammation environment were simulated by AGEs and TGF-β;Then,the TGF-β receptor 2(TGFBR2)overexpression plasmid was transfected in Wistar-ISCs.These cell models were divided into control group and irisin intervention group.2.Cell viability of the GK-ISCs were detected by the CCK8 experiment;Migration rates were compared in the Wound Healing and Transwell experiment;The disappearance rate of lipid droplets was compared by Oil red O staining;RT-PCR and immunofluorescence experiments were applied to observe the changes of ISC activation marker α-SMA and ECM components;Western Blotting was applied to observe the changes of α-SMA,ECM components,TGFBR2 and the phosphorylation levels of TGF-β/Smad pathway-related proteins.3.All data were presented as mean ± standard error,Student’s T test was used for comparisons between two groups,and univariate ANOVA analysis was used for comparisons with three or more groups.P<0.05 was a significant difference.Results: 1.Irisin intervention can reduce the activity and migration rate of GK-ISC.2.Irisin intervention can reduce the lipid droplet disappearance rate of Wistar-ISC.3.AGEs intervention can increase the migration rate and the expression levels of Wistar-ISC activation markers α-SMA and Col-I at the m RNA level and protein level,and irisin can inhibit the increase of Wistar-ISC migration rate induced by AGEs intervention,and reduce the expression levels of AGEs-induced Wistar-ISC activation markers α-SMA and Col-I at the m RNA level and protein level.4.TGF-β intervention can increase the expression levels of Wistar-ISC activation markers α-SMA and ECM(Col-I,FN)at the m RNA level and protein level and irisin can inhibit the increase of Wistar-ISC,and reduce the expression levels of TGF-β-induced Wistar-ISC activation markers α-SMA and ECM(Col-I,FN)at the m RNA level and protein level.5.Moreover,irisin can inhibit the phosphorylation level of Smad2/3 protein in a concentrationdependent manner.6.When TGFBR2 is overexpressed in Wistar-ISC,the inhibitory effect of irisin on the phosphorylation level of Smad2/3 is significantly weakened.Conclusion: Irisin can inhibit the cell viability,migration and lipid droplet disappearance rate of activated ISCs.It can also prevent the AGEs-induced and TGF-β-induced activation of ISCs.Irisin can suppress the activation of ISC by inhibiting the phosphorylation of Smad2/3 proteins by competing with TGFBR2. |
