Effects And Mechanisms Of Sirt1 On High Glucose Induced Cell Proliferation And TGF-?1 Expression In Human Glomerular Mesangial Cells

Objective To investigate the effects of silent information regulator 1(Sirt1)over-expression on high glucose induced proliferation and transforming growth factor-?1(TGF-?1)expression in human glomerular mesangial cells(HMC)and further explore the potential mechanisms.Methods 1.HMCs were transfected with recombinant lentiviral vectors containing the Sirt1 cDNA(LV-Sirt1),Sirt1-RNAi sequence or related control recombinant lentiviral vectors(LV-CTL or LV-CTL-RNAi),respectively.Those cells were selected by adding puromycin in culture medium for three days.2.Those HMCs transfected with recombinant lentiviral vectors were divided into eight groups,including normal glucose plus LV-CTL(NG+LV-CTL),high glucose plus LV-CTL(HG+ LV-CTL),normal glucose plus LV-Sirt1(NG+LV-Sirt1)and high glucose plus LV-Sirt1(HG+LV-Sirt1),normal glucose plus LV-CTL-RNAi(NG+LV-CTL-RNAi),high glucose plus LV-CTL-RNAi(HG+LV-CTL-RNAi),normal glucose plus LV-Sirt1-RNAi(NG+LV-Sirt1-RNAi)and high glucose plus LV-Sirt1-RNAi(HG+LV-Sirt1-RNAi)groups.Those untransfected HMC cultured at normal glucose(5.5mmol/L)(NG),normal glucose plus mannitol(5.5 mmol/L glucose+24.5mmol/L mannitol)(NM)or high glucose(30mmol/L)(HG)were set as control,respectively.After treatment for different periods,the cell proliferation of each group was examined using CCK-8 detection kit,the levels of mRNA and protein of TGF-?1from each group were measured through real-time polymerase chain reaction(RT-PCR)and Western blot,respectively.3.The HMCs transfected with recombinantlentiviral control vectors were treated with normal glucose(NG)or high glucose in different time,and thus,the levels of phosphorylation and acetylation of STAT1 and STAT3 from each group were assessed with Western blot or using immune coprecipitation plus Western blot,respectively.4.The levels of phosphorylation and acetylation of STAT1 from LV-CTL,LV-Sirt1 LV-Sirt1-RNAi groups treated with normal of high glucose for 24 h were explored.5.The interaction between Sirt1 protein and STAT1 protein was assayed by using Co-immunoprecipitation kit.Results 1.The HMCs which was stably over-expressed or silenced Sirt1 protein was obtained after recombinant lentivirus transfection and puromycin selection.2.Compared with NG group,the proliferation levels of HMC from HG,HG+LV-CTL,HG+LV-CTL-RNAi and HG+LV-Sirt1 groups on 12,24 and 48 hours were increased significantly(each P<0.05).However,the proliferation levels of HG+LV-Sirt1 group were significantly lower than those in HG and HG+LV-CTLgroups on 24 and 48 hours,on the other hand,the proliferation levels of HG+LV-Sirt1-RNAi were higher that those in HG and HG+LV-CTL-RNAi groups(P<0.05).3.The levels of m RNA and protein of TGF-?1 from HG treated groups were higher than those of NG group(each P<0.05).As compared to counterparts of HG and HG+LV-CTL groups,the levels of mRNA and protein of TGF-?1 from HG+LV Sirt1 group were decreased significantly(each P<0.05).However,the levels of mRNA and protein of TGF-?1from HG+LV Sirt1-RNAi were higher than those in those in HG and HG+LV-CTL-RNAi groups.In addition,there were no difference on the levels of mRNA and protein of TGF-?1 among NG,NM,NG+LV-CTL and NG+LV Sirt1 groups(P>0.05).3.High glucose increased those levels of acetylation and phosphorylation of STAT1 and STAT3 in time-dependent manner.4.Compared with counterparts in NG+LV-CTL group,the levels of acetylation and phosphorylation of STAT1 from HG+LV-CTL and HG+LV-Sirt1-RNAi group were significantly increased.Comparedwith those from HG+LV-CTL group,the levels of acetylation of STAT1 from HG+LV-Sirt1 group was decreased,however,it was increased in HG+LV-Sirt1-RNAi group.In addition,there were no differences on the levels of phosphorylation of STAT1 among HG+LV-CTL,HG+LV-Sirt1 as well as HG+LV-Sirt1-RNAi groups.5.The interaction between Sirt1 protein and STAT1 protein was detected by Co-immunoprecipitation assay.Conclusions Overexpression of Sirt1 ameliorates cell proliferation and TGF-?1expression of HMC induced by high glucose treatment,as well as acetylation of STAT1 and STAT3.Instead,silencing Sirt1 expression enhanced the effect of high glucose treatment.Sirt1 plays the role in conteracting high glucose induced effects by deacetylateing STAT1 in directly or indirectly manner.The Sirt1 gene may become a new therapeutic target of diabetic nephropathy.