【作者】 陈力；

【导师】 唐玉涵；

【作者基本信息】 华中科技大学 ， 公共卫生， 2020， 硕士

【摘要】 目的:以细胞衰老和自噬为关键点,探讨一氧化碳(carbon monoxide,CO)在糖尿病肾病(diabetic nephropathy,DN)中的干预效应以及作用机制。方法:动物实验:SPF级成年雄性C57BL/6J小鼠通过高脂饮食(HFD)+链脲佐菌素(streptozocin,STZ)建立DN模型,随后用一氧化碳释放分子干预,具体分组如下:正常对照组(CON),糖尿病肾病组(DN),糖尿病+一氧化碳干预组(DN+CO),糖尿病+无效一氧化碳处理组(DN+i CO)。干预16周后,留取血清及肾脏样本用于检测肾脏功能、细胞衰老、自噬和自噬流等相关指标。细胞实验:通过高糖(HG)处理3种肾脏细胞(大鼠系膜细胞(HBZY-1)、人肾小管上皮细胞(HK-2)和人足细胞(HPC)),建立体外模型。联合运用HG、CO和自噬抑制剂,探讨CO抗细胞衰老的机制;联合运用HG、CO和Beclin-1-Bcl-2解离剂ABT737,探讨CO激活自噬的机制。结果:1)与CON组相比,DN组小鼠肾功能严重受损,包括空泡变性,基底膜增厚,足突融合等,肾体比和血尿素氮(BUN)显著上升(P<0.05),剪切波成像和Masson染色显示纤维化程度增强。而DN+CO组与DN组上述指标趋势相反,表明CO对DN小鼠肾损伤有良好的改善作用。2)与DN组相比,DN+CO组小鼠肾脏细胞衰老得以缓解(SA-β-Gal、p53、p21、p16和SASP表达下降)(P<0.05);同时伴随自噬水平上升(Beclin-1升高,p62和LC3II/LC3I减少)(P<0.05),且溶酶体相关蛋白LAMP2和Cathepsin B以及LC3II和LAMP2共定位增加(P<0.05)。进一步通过自噬抑制剂处理HBZY-1、HK-2和HPC,阻断了CO对细胞衰老的改善效果,即导致SA-β-Gal、p53、p21和p16上升(P<0.05),Ed U阳性细胞比例减少(P<0.05)。且在DN+CO组小鼠中观察到了IL-1β、IL-6、TGF-β和VEGF与LC3II和LAMP2共定位增多。由此表明CO可以通过改善自噬,降解SASP,从而缓解细胞衰老。3)另外,免疫共沉淀显示DN+CO组小鼠中Beclin-1和Bcl-2的结合程度比DN组低(P<0.05)。进一步通过联合使用CO和ABT737,相比于单独CO干预,进一步激活了HBZY-1的自噬(p62减少和LC3II/LC3I增多),改善了其细胞衰老(SA-β-Gal、p53、p21和p16降低),同时也降低了HK-2和HPC的SA-β-Gal水平,以上说明CO可以通过解离Beclin-1-Bcl-2复合物来激活自噬改善细胞衰老。结论:DN小鼠肾脏细胞衰老上升伴随着自噬水平降低和自噬流阻滞,而CO可以通过解离Beclin-1-Bcl-2改善自噬,降解SASP,从而缓解细胞衰老,最终改善肾损伤。更多还原

【Abstract】 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.更多还原