CO Alleviates Cellular Senescence In Diabetic Nephropathy By Improving Autophagy

Objective: This research was designed to explore the effect and mechanism of carbon monoxide(CO)on senescence and autophagy in diabetic nephropathy(DN).Methods: Animal experiment: the SPF adult male C57BL/6J mice fed with HFD and injected with STZ to induce type 2 diabetes,and then treated with CORM-2.The grouping was as follows: normal control group(CON),diabetic nephropathy group(DN),diabetic nephropathy+carbon monoxide group(DN+ CO),diabetic nephropathy+invalid carbon monoxide group(DN + i CO).After 16 weeks,serum and kidney samples were collected to measure renal function,senescence,autophagy,autophagy flow and other related markers.Cell experiment: Three types of kidney cells(rat mesangial cells(HBZY-1),human tubular epithelial cells(HK-2)and human podocytes(HPC))were treated with high glucose(HG)to establish a vitro model.HG,CO and autophagy inhibitor were used in combination to explore the mechanism of CO against senescence.HG,CO and Beclin-1-Bcl-2 dissociation agent ABT737 were combined to explore the mechanisms of CO in activating autophagy.Results:1)Compared with CON group,mice in DN group had severe kidney injury,including vacuolar degeneration,thickening of basement membrane and fusion of foot processes.Kidney-body ratio and BUN significantly increased(P < 0.05),and shear wave imaging and Masson staining showed more severe fibrosis.The DN+CO group was opposite to the DN group in the above markers,indicating that CO was good for DN.2)Compared with DN group,kidney senescence was alleviated in DN+CO group(SA-β-Gal,p53,p21,p16 and SASP were dramatically down-regulated)(P < 0.05).Simultaneously,autophagy significantly improved(Beclin-1 increased,p62 and LC3II/LC3 I decreased)(P <0.05),and lysosomal related proteins LAMP2 and Cathepsin B and the colocalization of LC3 II and LAMP2 increased(P <0.05).Further,the autophagy inhibitors blocked the improvement of CO on the senescence of HBZY-1,HK-2 and HPC,which led to SA-β-Gal,p53,p21 and p16 increased and the proportion of Ed U positive cells decreased(P <0.05).Moreover,co-localization of IL-1β,IL-6,TGF-β and VEGF with LC3 II and LAMP2 increased in the DN+CO group mice.The above indicated that CO can alleviate senescence by improving autophagy and degrading SASP.3)In addition,immunoprecipitation showed that Beclin-1 and Bcl-2 binding in DN+CO group was less than that in DN group.Furthermore,the combined use of CO and ABT737 further activated autophagy(p62 decreased and LC3II/LC3 I increased)(P<0.05)and alleviated senescence(SA-β-Gal,p53,p21 and p16 decreased)(P < 0.05)of HBZY-1,and also reduced the SA-β-Gal levels of HK-2 and HPC,compared with the treatment of CO alone.This indicated that CO can alleviate senescence by dissociating Beclin-1-Bcl-2 complex to activate autophagy.Conclusions: In DN,severe senescence of kidney was accompanied by the decrease of autophagy and the blockage of autophagy flow.CO effectively improved autophagy by dissociating Beclin-1-Bcl-2 complex and degrade SASP,thereby alleviating senescence and ultimately improving renal injury.