南葶苈子提取液对心肌缺血/再灌注大鼠心肌自噬及MAPK/ERK1/2通路的影响

Effects of South Semen Lepidii Extraction Solution on Myocardial Autophagy and MAPK/ERK1/2 Pathway in Rats with Myocardial Ischemia/Reperfusion

目的观察南葶苈子提取液对心肌缺血/再灌注损伤(MI/RI)大鼠心肌自噬的影响并探讨其可能的作用机制。方法大鼠随机分为假手术组、模型组、阳性对照组、南葶苈子提取液低剂量组、南葶苈子提取液中剂量组、南葶苈子提取液高剂量组。采用结扎大鼠冠脉左前降支缺血30 min、再灌注120 min建立MI/RI模型。检测大鼠血清乳酸脱氢酶(LDH)、肌酸激酶同工酶(CK-MB)和肌钙蛋白Ⅰ(cTnⅠ)水平以及心肌组织丙二醛(MDA)和超氧化物歧化酶(SOD)水平,HE染色观察大鼠心肌组织病理学变化,透射电镜观察自噬小体变化,Western blotting测定心肌组织Beclin-1、LC3Ⅱ、B淋巴细胞瘤(Bcl-2)、蛋白激酶p38(p38)和p-ERK1/2蛋白水平。结果与假手术组比较,模型组大鼠心电图ST段抬高,血清LDH、CK-MB和cTnⅠ均升高,心肌组织MDA含量、Beclin-1、p38蛋白以及LC3Ⅱ/LC3Ⅰ比值升高,SOD活力、Bcl-2和p-ERK1/2蛋白表达降低(P <0.05)。与模型组比较,阳性对照组及南葶苈子提取液中、高剂量组大鼠心电图ST段、血清LDH、CK-MB和cTnⅠ、心肌组织MDA含量、Beclin-1、p38蛋白以及LC3Ⅱ/LC3Ⅰ比值降低,SOD活力、Bcl-2和p-ERK1/2蛋白表达升高(P <0.05)。与阳性对照组比较,南葶苈子提取液低、中剂量组大鼠心电图ST段、血清LDH、CK-MB和cTnⅠ、心肌组织MDA含量均升高,心肌组织SOD活力、p-ERK1/2降低,且南葶苈子提取液高剂量组大鼠血清CK-MB降低,低剂量南葶苈子提取液Beclin-1、p38及不同剂量南葶苈子提取液LC3Ⅱ/LC3Ⅰ升高,南葶苈子提取液低剂量组Bcl-2降低(P <0.05)。心电图ST段、血清LDH、CK-MB和cTnⅠ、心肌组织MDA含量、Beclin-1、p38蛋白以及LC3Ⅱ/LC3Ⅰ比值随南葶苈子提取液剂量增加呈下降趋势,心肌组织SOD活力、p-ERK1/2蛋白表达随剂量增加呈上升趋势。结论南葶苈子提取液处理可通过降低MI/RI大鼠心肌自噬保护心肌损伤,其保护机制可能与MAPK/ERK1/2通路有关。

Objective:To investigate the effect of South Semen Lepidii extraction solution on myocardial autophagy in rats with myocardial ischemia/reperfusion injury(MI/RI)and its possible mechanism. Methods:Rats wererandomly divided into sham-operated group,the model group,positive control group,low-dose group,medium-dose group and high-dose group. MI/RI model was established by ligating the left anterior descending coronary artery for 30 min and reperfusion for 120 min.The levels of LDH,CK-MB,cTnⅠ and MDA and SOD in myocardium ofrats were detected by the kit;HE staining was used to observe the histopathological changes of myocardium in rats;the changes of autophagosomes were observed by transmission electron microscopy;the protein levels of Beclin-1,LC3 Ⅱ,Bcl-2,P-38 and p-ERK1/2 in myocardium were determined by Western blot. Results:Compared withsham-operated group,ST segment elevation,serum LDH,CK-MB and cTnⅠ increased,MDA content,Beclin-1,p-38 protein and LC3 Ⅱ/LC3Ⅰ ratio increased,SOD activity,Bcl-2 and p-ERK1/2 protein expression decreased in themodel group(P < 0.05). Compared with model group,ST segment of ECG,serum LDH,CK-MB and c TnI,MDAcontent in myocardium,Beclin-1,p-38 protein and LC3 Ⅱ/LC3Ⅰ ratio in positive control group and medium andhigh dose groups decreased;SOD activity,Bcl-2 and p-ERK1/2 protein expression increased(P < 0.05). Comparedwith the positive control group,ST segment of ECG,serum LDH,CK-MB,cTnⅠ and MDA content in myocardiumwere increased and SOD activity and p-ERK1/2 in myocardium were decreased in low and medium dose groups;the serum CK-MB in high dose group was decreased. Beclin-1,p-38 in low dose group and LC3 Ⅱ/LC3Ⅰ in differ-ent dose groups increased,while Bcl-2 in low dose group decreased(P < 0.05). ST segment of ECG,LDH,CK-MBand cTnⅠ in serum,MDA content in myocardium,Beclin-1,p-38 protein and LC3 Ⅱ/LC3Ⅰ ratio showed a downwardtrend with the increase of the dosage of South Semen Lepidii extraction solution,while SOD activity and p-ERK1/2 protein expression in myocardium showed an upward trend with the increase of the dosage. Conclusion:SouthSemen Lepidii extraction solution can protect myocardial injury by reducing myocardial autophagy in MI/RI rats,and its protective mechanism may be related to MAPK/ERK1/2 pathway.