香芹酚通过增强PTEN诱导激酶1/Parkin介导的自噬而减轻心肌缺血再灌注损伤的动物及细胞实验

Carvacrol Reduces Myocardial Ischemia-reperfusion Injury by Activating PTEN Induced Putative Kinase 1/Parkin-mediated Autophagy: Animal and Cell Experiments

背景在心血管疾病中,PTEN诱导激酶1(PINK1)/Parkin介导的自噬通过有效地清除受损的线粒体和过量的活性氧(ROS)来维持细胞内线粒体的动态平衡。香芹酚具有减少心肌细胞中ROS生成和降低线粒体损伤的作用。然而,目前尚不清楚香芹酚对心肌缺血再灌注损伤(MIRI)中自噬的影响。目的通过动物及细胞实验探讨香芹酚对MIRI的治疗作用及其机制。方法本研究时间为2020年5月至2021年5月。动物实验:将成年雄性Sprague-Dawley大鼠随机分为Sham组(A组)、MIRI组(B组)、MIRI+20 mg/kg香芹酚组(C组)和MIRI+60 mg/kg香芹酚组(D组),每组12只。B、C、D组大鼠建立MIRI模型,A组大鼠接受相同的手术,但不结扎冠状动脉左前降支;术前15 min,C组和D组大鼠腹腔注射相应剂量的香芹酚,A组和B组大鼠给予等体积的0.9%氯化钠溶液。检测A、B、C、D组大鼠血清心肌肌钙蛋白I(cTnI)、肌酸激酶同工酶(CK-MB)、天冬氨酸氨基转移酶(AST)、乳酸脱氢酶(LDH)水平,心肌梗死面积,心肌病变情况,心肌组织中PINK1、Parkin、Beclin1蛋白表达水平及微管相关蛋白轻链3(LC3)-Ⅱ/LC3-Ⅰ。细胞实验:将H9C2细胞分为对照组(E组)、缺氧/复氧(H/R)组(F组,进行H/R处理)、H/R+香芹酚组(G组,用100μmol/L香芹酚预处理6 h后进行H/R处理)、H/R+香芹酚+阴性对照小干扰RNA(NC-siRNA)组(H组,转染NC-siRNA后用100μmol/L香芹酚预处理6 h,然后进行H/R处理)和H/R+香芹酚+靶向PINK1的小干扰RNA(PINK1-siRNA)组(I组,转染PINK1-siRNA后用100μmol/L香芹酚预处理6 h,然后进行H/R处理)。检测E、F、G、H、I组细胞活力、细胞凋亡率,H9C2细胞中PINK1、Parkin、Beclin1蛋白表达水平及LC3-Ⅱ/LC3-Ⅰ。结果动物实验:B、C、D组大鼠血清cTnI、CK-MB、AST、LDH水平高于A组(P<0.05);C、D组大鼠血清cTnI、CK-MB、AST、LDH水平低于B组(P<0.05);D组大鼠血清cTnI、CK-MB、AST、LDH水平低于C组(P<0.05)。A组大鼠心肌梗死面积为0。C、D组大鼠心肌梗死面积小于B组(P<0.05);D组大鼠心肌梗死面积小于C组(P<0.05)。HE染色结果显示,A组大鼠心肌细胞排列整齐,细胞核明显,无炎性细胞浸润;B组大鼠心肌组织广泛坏死,心肌纤维排列紊乱,大量炎性细胞浸润;C、D组大鼠心肌细胞排列较整齐,细胞坏死程度和范围明显减轻。Masson三色染色结果显示,A组大鼠心肌组织以心肌细胞为主,没有明显的胶原成分;B组大鼠心肌组织心肌纤维化明显,仅有少量心肌细胞存在;C、D组大鼠心肌组织的胶原纤维含量明显减少。定量分析结果显示,B、C、D组大鼠心肌纤维化百分比高于A组(P<0.05);C、D组大鼠心肌纤维化百分比低于B组(P<0.05);D组大鼠心肌纤维化百分比低于C组(P<0.05)。TUNEL染色结果显示,B、C、D组大鼠心肌组织TUNEL阳性率高于A组(P<0.05);C、D组大鼠心肌组织TUNEL阳性率低于B组(P<0.05);D组大鼠心肌组织TUNEL阳性率低于C组(P<0.05)。B、C、D组大鼠心肌组织中PINK1、Beclin1蛋白表达水平低于A组,B、C组大鼠心肌组织中Parkin蛋白表达水平低于A组,B组大鼠心肌组织中LC3-Ⅱ/LC3-Ⅰ低于A组,C、D组大鼠心肌组织中LC3-Ⅱ/LC3-Ⅰ高于A组(P<0.05);C、D组大鼠心肌组织中PINK1、Parkin、Beclin1蛋白表达水平及LC3-Ⅱ/LC3-Ⅰ高于B组(P<0.05);D组大鼠心肌组织中Parkin、Beclin1蛋白表达水平高于C组,(P<0.05)。细胞实验:F、G、H、I组细胞活力小于E组(P<0.05);G、H组细胞活力大于F组(P<0.05);I组细胞活力小于G、H组(P<0.05)。F、G、H、I组细胞凋亡率高于E组(P<0.05);G、H、I组细胞凋亡率低于F组(P<0.05);I组细胞凋亡率高于G、H组(P<0.05)。F组H9C2细胞中PINK1、Parkin、Beclin1蛋白表达水平及LC3-Ⅱ/LC3-Ⅰ低于E组(P<0.05);G、H组H9C2细胞中PINK1、Beclin1蛋白表达水平低于E组、高于F组,Parkin蛋白表达水平高于F组,LC3-Ⅱ/LC3-Ⅰ高于E、F组(P<0.05);I组H9C2细胞中PINK1、Parkin、Beclin1蛋白表达水平及LC3-Ⅱ/LC3-Ⅰ低于E、G、H组,Beclin1蛋白表达水平高于F组(P<0.05)。结论动物及细胞实验均表明,香芹酚通过激活PINK1/Parkin通路增强自噬,从而减轻MIRI。

Background In cardiovascular diseases, PTEN-induced putative kinase 1(PINK1)/Parkin-mediated autophagy maintains the dynamic balance of intracellular mitochondria by effectively scavenging damaged mitochondria and excess reactive oxygen species(ROS). Carvanol can reduce the production of ROS and mitochondrial damage in cardiomyocytes.However, the effect of carvanol on autophagy in myocardial ischemia-reperfusion injury(MIRI) is not clear. Objective To investigate the therapeutic effect of carvanol on MIRI and its mechanism through animal and cell experiments. Methods The time of this study is from May 2020 to May 2021. Animal experiment: adult male Sprague-Dawley rats were randomly divided into Sham group(group A), MIRI group(group B), MIRI+20 mg/kg carvacrol group(group C) and MIRI+60 mg/kg carvacrol group(group D), with 12 rats in each group. The MIRI model was established in groups B, C and D, and the rats in group A received the same operation without ligating the left anterior descending branch of coronary artery. Fifteen min before operation, the rats in groups C and D were injected intraperitoneally with corresponding dose of carvanol, and rats in groups A and B were given the same volume of 0.9% sodium chloride solution. The levels of serum cardiac troponin I(cTnI), creatine kinase isoenzyme(CK-MB), aspartate aminotransferase(AST), lactate dehydrogenase(LDH), myocardial infarction area, myocardial pathological changes, expression level of PINK1, Parkin and Beclin1 protein and microtubule-associated protein 3(LC3)-Ⅱ/LC3-Ⅰ in groups A, B, C and D were measured. Cell experiment: H9 C2 cells were divided into control group(group E), hypoxia/reoxygenation(H/R) group(group F, treated with H/R), H/R + carvacrol group(group G, pretreated with 100 μmol/L carvanol for 6 h and then treated with H/R),H/R + carvacrol + negative control siRNA(NC-siRNA) group(group H, after transfection of NC-siRNA, 100 μmol/L carvacrol was pretreated for 6 h, and then treated with H/R) and H/R + carvacrol + small interfering RNA targeting PINK1(PINK1-siRNA)group(group I, after transfection of PINK1-siRNA, 100 μmol/L carvacrol was pretreated for 6 h, and then treated with H/R). The cell viability, apoptosis rate, the expression levels of PINK1, Parkin and Beclin1 protein and LC3-Ⅱ/LC3-Ⅰ in H9 C2 cells in groups E, F, G, H and I were detected. Results Animal experiments: the levels of serum cTnI, CK-MB, AST and LDH in groups B, C and D were higher than those in group A(P < 0.05);the levels of serum cTnI, CK-MB, AST and LDH in groups C and D were lower than those in group B(P < 0.05);the levels of serum cTnI, CK-MB, AST and LDH in group D were lower than those in group C(P < 0.05). The myocardial infarction area in group A was 0. The myocardial infarction area in groups C and D was smaller than that in group B(P < 0.05);the myocardial infarction area in group D was smaller than that in group C(P < 0.05).HE staining results showed that myocardial cells in group A were arranged neatly, with obvious nuclei and no inflammatory cell infiltration;rats in group B had extensive necrosis of myocardial tissue, disordered myocardial fiber arrangement, and a large number of inflammatory cell infiltration;the cardiomyocytes of rats in groups C and D were arranged neatly, and the degree and scope of cell necrosis were significantly reduced. The results of Masson’s tricolor staining showed that the myocardial tissues of rats in group A were mainly composed of cardiomyocytes without obvious collagen components;the myocardial tissues of rats in group B had obvious myocardial fibrosis, with only a few cardiomyocytes present;the content of collagen fibers in myocardial tissue of rats in groups C and D were significantly reduced. The results of quantitative analysis showed that the myocardial fibrosis rate of rats in groups B, C and D was higher than that in group A(P < 0.05);the rate of myocardial fibrosis of rats in groups C and D was lower than that in group B(P < 0.05);the rate of myocardial fibrosis of rats in group D was lower than that in group C(P < 0.05).TUNEL staining results showed that the positive rate of TUNEL in myocardial tissue of rats in groups B, C and D was higher than that in group A(P < 0.05);the positive rate of TUNEL in myocardial tissue of rats in groups C and D was lower than that in group B(P < 0.05);the positive rate of TUNEL in myocardial tissue of rats in group D was lower than that in group C(P < 0.05). The expression levels of PINK1 and Beclin1 protein in myocardial tissues of rats in groups B, C and D were lower than those in group A,the expression levels of Parkin protein in myocardial tissues of rats in groups B and C were lower than those in group A, LC3-Ⅱ/LC3-Ⅰ in myocardial tissues of rats in group B was lower than that in group A, LC3-Ⅱ/LC3-Ⅰ in myocardial tissues of rats in groups C and D was higher than that in group A(P < 0.05);the expression levels of PINK1, Parkin and Beclin1 protein and LC3-Ⅱ/LC3-Ⅰ in myocardial tissues of rats in groups C and D were higher than those in group B(P < 0.05);the expression levels of Parkin and Beclin1 protein in myocardial tissues of rats in group D were higher than those in group C(P < 0.05). Cell experiment:the cell viability of groups F, G, H and I was lower than that of group E(P < 0.05);the cell viability of groups G and H was higher than that of group F(P < 0.05);the cell viability of group I was lower than that of groups G and H(P < 0.05). The apoptotic rate of groups F, G, H and I was higher than that of group E(P < 0.05);the apoptotic rate of groups G, H and I was lower than that of group F(P < 0.05);the apoptotic rate of group I was higher than that of groups G and H(P < 0.05). The expression levels of PINK1, Parkin, Beclin1 protein and LC3-Ⅱ/LC3-Ⅰ in H9 C2 cells in group F were lower than those in group E(P < 0.05);the expression levels of PINK1 and Beclin1 protein in H9 C2 cells in groups G and H were lower than those in group E, and higher than those in group F, the expression level of Parkin protein was higher than that in group F, LC3-Ⅱ/LC3-Ⅰ was higher than that in groups E and F(P < 0.05);the expression levels of PINK1, Parkin and Beclin1 protein and LC3-Ⅱ/LC3-Ⅰ in H9 C2 cells in group I were lower than those in groups E, G and H, the expression levels of Beclin1 protein were higher than those in group F(P < 0.05). Conclusion Animal and cell experiments have shown that carvacrol enhances autophagy by activating the PINK1/Parkin pathway, thereby reducing MIRI.