The Role And Mechanism Of SIRT2 Through Smad2/3 Pathway In Idiopathic Pulmonary Fibrosis

Research backgroundIdiopathic pulmonary fibrosis(IPF)is a chronic and progressive pulmonary interstitial disease with poor prognosis.Its pathogenesis is very complex and unclear at present.Accumulating evidences have been shown that epigenetic modification plays an important role in the occurrence and development of IPF.SIRT2 is a nicotinamide adenine dinucleotide(NAD)-dependent deacetylase.Studies have confirmed that SIRT2 is related to fibrosis in liver,kidney and myocardium,but its role in IPF has not been studied.ObjectiveTo investigate the role of SIRT2 in transforming growth factorβ1(TGF-β1)induced differentiation of pulmonary fibroblast and bleomycin-induced pulmonary fibrosis,hoping to provide a new target and theoretical basis for the treatment of IPF.Method1.MRC-5 cells were treated with TGF-β1 of various concentration and incubated for different time period.Fibronectin,α-SMA and SIRT2 protein expression were detected by western blot.2.MRC-5 cells were pretreated with 2 ng/ml TGF-β1 for 24 hours,and then SIRT2 inhibitor AKG2 plus TGF-β1 for another 24 hours,cells were collected and protein and RNA were extracted.The protein and RNA expressions of SIRT2,Fibronectin,α-SMA,p-Smad2/3 and Smad2/3 were detected by western blot,immunofluorescence and RTPCR.3.MRC-5 cells and IPF lung fibroblasts were transfected with SIRT2 si RNA or control si RNA,and then treated with 2 ng/ml TGF-β1for 24 h.Protein and m RNA expression of Fibronectin,α-SMA,pSmad2/3 and Smad2/3 were detected by western blot and RT-PCR.4.Healthy C57BL/6 mice were randomly divided into three groups:normal saline treated control group(n=6,saline group),bleomycin treated group(n=6,BLM group)and bleomycin plus AGK2 treated group(n=6,BLM+AGK2 group).Mice were sacrificed on day 21 post bleomycin injury,and the lung tissues were prepared for western blot and histology.5.MRC-5 cells were treated with 0,2 and 5 ng/ml TGF-β1.The total protein was co-immunoprecipitated with anti SIRT2 antibody or control Ig G to detect the interaction between SIRT2 and Smad3.Result1.After treating with different concentrations of TGF-β1,or 2 ng/ml TGF-β1 for different time period,the expression of Fibronectin,α-SMA and SIRT2 protein or m RNA was increased significantly than control.2.After treating with 2 ng/ml TGF-β1 for 24 hours and then AGK2 for another 24 hours,the protein and RNA expression of SIRT2 were significantly decreased,the increased Fibronectin,α-SMA,p-Smad2/3and Smad2/3 protein and m RNA induced by TGF-β1 were significantly downregulated.Immunofluorescence staining further confirmed that AGK2 reduced the increased Fibronectin and α-SMA fluorescence intensity induced by TGF-β1 treatment.3.Silencing SIRT2 significantly reduced the increased protein and m RNA expression of Fibronectin,α-SMA,p-Smad2/3 and Smad2/3induced by TGF-β1.4.IPF lung fibroblasts were transfected with SIRT2 si RNA and control si RNA,then incubated with 2 ng/ml TGF-β1 for 24 h.Silencing SIRT2 significantly reduced the increased Fibronectin and α-SMA protein and m RNA induced by TGF-β1.5.In bleomycin induced pulmonary fibrosis mouse model,H&E and Masson staining showed that AGK2 reduce bleomycin induced alveolitis,alveolar septal thickening and pulmonary fibrosis.6.Immunohistochemical staining of SIRT2,Fibronectin and α-SMA were seen in large areas in the bleomycin group,while little staining was observed in the saline group and BLM+AGK2 group.AGK2 treatment decreased the expression of SIRT2,Fibronectin and α-SMA.7.Compared with saline group,the protein expression of SIRT2,Fibronectin,α-SMA,p-Smad2/3 and Smad2/3 in lung tissue is increased in BLM group.Compared with BLM group,the expression of SIRT2,Fibronectin,α-SMA,p-Smad2/3 and Smad2/3 is decreased in BLM+AGK2 group.8.Co-immunoprecipitation showed there was interaction between SIRT2 and Smad3.ConclusionExperimental results confirm that SIRT2 is involved in the differentiation of lung fibroblasts induced by TGF-β1 and the formation of pulmonary fibrosis induced by bleomycin.SIRT2 inhibitors or silencing SIRT2 can reduce the differentiation of fibroblasts and the formation of lung fibrosis.Both at the cellular and overall levels,it is confirmed that,SIRT2 regulates the TGF-β1 induced differentiation of pulmonary fibroblasts and the formation of pulmonary fibrosis through Smad2/3 pathway,which provides a basic theoretical basis for the treatment of idiopathic pulmonary fibrosis,and is expected to become a new therapeutic target.