Caspase-3抑制剂联合小剂量GC对脓毒症小鼠AKI TNF-α、NGAL及Bax、Bcl-2表达的影响

发布时间：2018-04-15 08:29

  本文选题：脓毒症急性肾损伤 + 糖皮质激素　； 参考：《昆明医科大学》2017年硕士论文

【摘要】：[目的]脓毒症急性肾损伤(septic acute kidney injury,SAKI)是重症监护室中常见的一种危重疾病,其发病率及死亡率居高不下,但发病机制尚未明确。其中,细胞凋亡学说在脓毒症急性肾损伤中的作用越来越受到重视,多项临床及动物实验显示,脓毒症发生时,机体处于相对肾上腺皮质功能不全(relative adrenal insufficiency,RAI)的状态,小剂量糖皮质激素可以保护SAKI中的肾功能。本实验拟构建SAKI小鼠模型,采用Ac-DEVD-CHO(Caspase-3特异性抑制剂)联合小剂量甲强龙早期干预,通过ELISA检测小鼠血清中炎症因子TNF-α、急性肾损伤标志物NGAL(中性粒细胞明胶酶相关载脂蛋白,载脂蛋白-2)以及Western Blot检测肾脏凋亡相关蛋白Bax、Bcl-2表达,比较二者单独干预与联合干预措施对SAKI小鼠模型的肾保护作用及机制,为临床治疗SAKI提供新的治疗靶点和理论依据。[方法]清洁级雄性C57BL/6小鼠72只,采用盲肠结扎穿孔(Cecal Ligation and Puncture,CLP)法,构建SAKI模型,随机分为6组(n=12),①假手术对照组(sham group);②盲肠结扎穿孔组(CLP group);③DMSO对照组(DMSO group);④小剂量甲泼尼龙早期干预组(GC group);⑤Caspase-3抑制剂Ac-DEVD-CHO早期干预组(CHO group);⑥Caspase-3抑制剂Ac-DEVD-CHO联合小剂量甲强龙组(CHO+GC group)。各组术后都给予37℃生理盐水0.3ml/10g股部皮下注射行液体复苏。六组小鼠分别于术后6h、12h、24h通过摘眼球法取血,使用ELISA方法测定血清中的TNF-α、NGAL浓度;同时各组使用磷酸盐缓冲液灌注后取出小鼠肾脏,使用Western-Blot方法测定肾脏中凋亡蛋白Bax、Bcl-2的表达。ELISA结果分析使用酶标仪在450nm波长下测量各孔的光密度(optical density,OD)值,Western-Blot条带图像灰度值测量分析采用Gel-pro软件包分析。数据统计分析采用SPSS20.0版软件包(SPSS Company,Chicago,IllinoisUSA)进行;数据均以均数土标准差(x±s)表示,统计方法采用重复测量资料的方差分析,P≤0.05为差异有统计学意义。[结果]1.各组小鼠血清TNF-α及血清NGAL结果:6h、12h、24h小鼠CLP组和DMSO对照组血清TNF-α及血清NGAL表达无明显差异(P0.05)。6hCLP组、CHO组TNF-α较Sham组有明显升高(P0.01);GC组较CLP组TNF-α有明显降低(P0.01),GC+CHO组较GC组有降低趋势,较Sham组有升高趋势,但无统计学差异(PO.05);12h、24hSham组血清TNF-α较6h组有升高趋势,但无统计学差异(P0.05);12hCLP组较6hCLP组血清TNF-α有明显升高,24hCLP组较12h CLP组血清TNF-α有明显升高(P0.01);12hGC组较12hCLP组有明显降低(P0.01);12hGC+CHO组较12hGC组有明显降低(P0.01);12hGC组和GC+CHO组较6h组有升高趋势,但无统计学差异(P0.05);24hGC组较24hCLP组有明显降低(P0.01);24hGC+CHO组较24h GC组有明显降低(P0.01);24hGC组和GC+CHO组较12h组有升高趋势,但无统计学差异(P0.05)。12hCLP组较6hCLP组血清NGAL有明显升高(P0.01),24hCLP组较12hCLP组血清NGAL有明显降低(P0.01),与6hCLP手术组无统计学差异(P0.05),各个时间组CLP组较Sham组NGAL都有明显升高(P0.01),GC组较CLP组血清NGAL有降低趋势,但无统计学差异(P0.05);CHO组较CLP组血清NGAL有明显降低(P0.01),GC+CHO组较CHO组有降低趋势,但无统计学差异(P0.05)。2.各组小鼠肾组织凋亡相关蛋白Bax及Bcl-2表达结果:6h和12h的Bax/actin比值比较中,CLP组最高,GC+CHO组最低,其中CLP组与Sham组比较Bax表达升高有统计学意义(P0.05)。CHO组及GC+CHO组与CLP组比较Bax表达降低差异有统计学意义(P0.05)。GC+CHO组与DMSO组比较Bax表达降低有统计学意义(P0.05)。GC+CHO组较CHO组有降低趋势,但差异无统计学意义(P0.05)。24h的Bax/β-actin比值比较中,Sham组最高,CLP组其次,GC+CHO组较CLP组升高有统计学意义(P0.05),CHO组较CLP组降低趋势,其余各组间均无统计学差异(P0.05)。6h和12h的Bcl-2/β-actin比值比较中,GC+CHO组最高,CHO组其次,DMSO组最低。CHO组及GC+CHO组与DMSO组比Bcl-2表达增高均有统计学意义(P0.05);GC+CHO组较CHO组有升高趋势,但差异无统计学意义(P0.05)。24h的Bcl-2/β-actin比值比较中,GC+CHO组最高,CHO组其次,CLP组最低。各组间均无统计学差异(P0.05),GC+CHO组较CHO组有升高趋势,但差异无统计学意义(P0.05)。[结论]1.单独使用小剂量甲强龙早期干预脓毒症相关肾损伤小鼠模型,可以减少炎性因子TNF-α的释放,减轻小鼠SAKI模型的肾损伤;2.单独使用Caspase-3抑制剂Ac-DEVD-CHO早期干预小鼠脓毒症急性肾损伤模型,可以减轻小鼠肾脏细胞凋亡,进而减轻小鼠SAKI模型的肾损伤;3.Caspase-3抑制剂Ac-DEVD-CHO和小剂量甲强龙联合应用时,比单独使用其中一种药物能更有效的减少小鼠脓毒症急性肾损伤模型肾脏细胞凋亡、炎症因子TNF-α释放,降低血清NGAL,更好的减轻小鼠SAKI模型的肾损伤。
[Abstract]:[Objective] sepsis acute kidney injury (septic acute kidney injury, SAKI) is a common disease in critically ill ICU, high morbidity and mortality, but the pathogenesis is still unclear. Among them, the theory of apoptosis in sepsis and acute kidney injury in the clinical display more attention. And a number of animal experiments, sepsis occurs when the body is in relative adrenal insufficiency (relative adrenal, insufficiency, RAI) of the state, a small dose of glucocorticoid can protect renal function in SAKI. This study intends to construct a mouse model of SAKI, with Ac-DEVD-CHO (Caspase-3 inhibitor) combined with low-dose methylprednisolone early intervention by inflammatory cytokines TNF- ELISA detection in serum alpha, NGAL markers of acute kidney injury (neutrophil gelatinase associated lipocalin, apolipoprotein -2) and Western Blot detection of renal The dirty of apoptosis related protein Bax, Bcl-2 expression, compared with the two individual intervention and combined interventions on a mouse model of SAKI renal protective effect and mechanism for the treatment of SAKI to provide therapeutic targets and theoretical basis. Methods: male C57BL/6 mice were new 72, cecal ligation perforation (Cecal Ligation and Puncture. CLP) method to construct the model of SAKI, were randomly divided into 6 groups (n=12), sham operation control group (sham group); the cecal ligation and puncture group (CLP group); the DMSO control group (DMSO group); the early small dose methylprednisolone intervention group (GC group) and Caspase-3 inhibitor Ac-DEVD-CHO; early intervention group (CHO group); the Caspase-3 inhibitor Ac-DEVD-CHO combined with low dose methylprednisolone group (CHO+GC group). Each group were given normal saline 0.3ml/10g 37 C femoral subcutaneous injection for fluid resuscitation. The small group of six rats respectively after 6h, 12h, 24h by eyeball method Blood serum levels of TNF- were determined using the method of ELISA alpha, NGAL concentration; at the same time the groups using phosphate buffer after reperfusion kidney was removed, the determination of apoptosis protein Bax in kidney using the Western-Blot method, the optical density using eliasa measurement of each hole in the 450nm wavelength analysis results the expression of.ELISA Bcl-2 (optical density OD) the value of Western-Blot band image gray value measurement and analysis using Gel-pro software package. The analysis using SPSS20.0 software package for statistical analysis of data (SPSS Company, Chicago, IllinoisUSA); data are presented as mean + standard deviation (x + s), variance analysis using statistical methods for repeated measurement data, P is less than or equal to 0.05 for the difference there was statistical significance. Results the serum]1. TNF- mice and serum NGAL results: 6h, 12h, 24h in CLP group and DMSO control group of serum TNF- and serum NGAL expression had no significant difference (P0.05) in.6hCLP group, CHO group, TNF- alpha 杈僑ham缁勬湁鏄庢樉鍗囬珮(P0.01);GC缁勮緝CLP缁凾NF-伪鏈夋槑鏄鹃檷浣，

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