维拉帕米对脓毒症大鼠心肌细胞自噬水平的影响

Effect of verapamil on autophagy in myocardial cells of septic rats

目的观察钙通道阻滞剂维拉帕米对脓毒症大鼠心肌细胞自噬水平的影响。方法将54只大鼠随机分为对照组、模型组、干预组,每组18只。模型组和干预组采用盲肠结扎穿孔术制作脓毒症模型,对照组取出盲肠不结扎穿孔仍放回腹腔。三组术后均给予生理盐水腹腔注射,干预组另给予盐酸维拉帕米注射液5 mg/kg腹腔注射。于术后6、12、24 h各组分别处死6只大鼠,取心肌组织,采用HE染色法观察组织形态学变化;取心脏血,离心分离血清,酶联免疫吸附法测定肌钙蛋白T(c Tn T)、肌酸激酶同工酶(CK-MB); Western blotting法、qRT-PCR法分别检测自噬相关蛋白微管相关蛋白1轻链3(LC3)、Beclin-1及凋亡相关蛋白B淋巴细胞瘤-2(Bcl-2)蛋白和mRNA表达。结果模型组、干预组心肌组织HE染色见心肌细胞排列紊乱、明显肥大;模型组、干预组CK-MB、c Tn T较对照组升高;模型组LC3、Beclin-1表达高于对照组,且随时间延长逐渐增高,Bcl-2表达低于对照组,且随时间延长逐渐降低;干预组12、24 h时LC3表达低于模型组,6、12、24 h时Beclin-1表达低于模型组,6、12、24 h时BCL-2表达高于模型组(P均<0. 05)。结论脓毒症可有效激活心肌自噬,自噬水平在24 h内呈上升趋势;维拉帕米干预可抑制自噬,保护心肌组织。

Objective To observe the effect of calcium channel blocker verapamil on autophagy in myocardial cells of septic rats.Methods Fifty-four rats were randomly divided into the control group,model group and intervention group,with 18 rats in each group.We used the cecal ligation and perforation to establish the sepsis models in the model group and the intervention group,while in the control group,we removed the cecum without ligation and placed it back into the abdominal cavity after perforation.All the three groups were given intraperitoneal injection of normal saline 30 mL/kg,and the intervention group was given intraperitoneal injection of verapamil hydrochloride 5 mg/kg.At 6,12 and 24 h after operation,6 rats were sacrificed,myocardial tissues were taken,and histomorphological changes were observed by HE staining.Blood was collected from the heart,serum was separated by centrifugation,and troponin T(cTnT)and creatine kinase isoenzyme(CK-MB)was determined by enzyme-linked immunosorbent assay.Western blotting and qRT-PCR were used to detect the protein and mRNA expression of microtubule-associated protein 1 light chain 3(LC3)and autophagy-related protein Beclin-1(Beclin-1),and B-cell lymphoma 2(Bcl-2).Results In the model group and the intervention group,HE staining showed that the myocardial cells were disordered and hypertrophied.The CK-MB and cTnT in the model group and the intervention group were higher than those in the control group.The expression of LC3 and Beclin-1 in the model group was higher than that in the control group.Over the time,the expression of Bcl-2 was lower than that of the control group,and gradually decreased with time.The expression of LC3 was lower in the intervention group than that in the model group at 12 and 24 h,the expression of Beclin-1 was lower at 6,12 and 24 h,and the expression of BCL-2 was higher than that of the model group at 6,12 and 24 h(all P<0.05).Conclusions Sepsis can effectively activate myocardial autophagy,and the level of autophagy increases within 24 h.Verapamil intervention can inhibit autophagy and protect myocardial tissues.