冬凌草甲素对大鼠心肌梗死后心肌细胞凋亡和纤维化及NLRP3炎性通路的影响

Effects of oridonin on myocardial cell apoptosis,fibrosis and NLRP3 inflammatory pathway after myocardial infarction in rats

目的探讨冬凌草甲素对大鼠心肌梗死后心肌细胞凋亡和纤维化及核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)炎性通路的影响。方法选取15只SD雄性大鼠作为假手术组,选取60只SD雄性大鼠采用结扎冠状动脉左前降支的方法建立心肌梗死模型,将造模成功大鼠随机分为模型组、冬凌草甲素低剂量组、冬凌草甲素高剂量组、卡托普利组,每组15只。冬凌草甲素低、高剂量组大鼠分别腹腔注射10 mg/kg和20 mg/kg冬凌草甲素,卡托普利组大鼠腹腔注射9.322 mg/kg卡托普利,假手术组和模型组大鼠均腹腔注射等量生理盐水,均1次/d,连续干预4周。采用超声心动图测定大鼠心功能参数[左心室射血分数(LVEF)、左心室收缩末期内径(LVESd)、左心室舒张末期内径(LVEDd)、左心室短轴缩短率(LVFS)],采用TTC染色、HE染色、Masson染色和TUNEL法分别观察大鼠心肌梗死面积比、心肌组织病理形态学、心肌纤维化和心肌细胞凋亡情况,Wertern blot法检测心肌纤维化相关蛋白[转化生长因子-β_(1)(TGF-β_(1))、结缔组织生长因子(CTGF)、Ⅰ型胶原蛋白(collagenⅠ)、Ⅲ型胶原蛋白(collagenⅢ)]、细胞凋亡相关蛋白[B淋巴细胞瘤-2(Bcl-2)蛋白、Bcl-2相关X蛋白(Bax)、天冬氨酸特异性半胱氨酸蛋白酶剪切体(Cleaved Caspase-3)蛋白]及NLRP3炎性通路相关蛋白、凋亡相关斑点样蛋白(ASC)、半胱氨酸天冬氨酸蛋白酶-1(Caspase-1)、白细胞介素-1β(IL-1β)、白细胞介素-18(IL-18)]表达情况。结果与模型组比较,冬凌草甲素低、高剂量组和卡托普利组大鼠LVEF、LVFS及心肌组织中Bcl-2蛋白相对表达量均显著升高(P均<0.05),LVESd、LVEDd、心肌梗死面积比、心肌细胞凋亡率及心肌组织中TGF-β_(1)、CTGF、collagenⅠ、collagenⅢ、Bax、Cleaved Caspase-3、NLRP3、ASC、Caspase-1、IL-1β、IL-18蛋白相对表达量均显著降低(P均<0.05),心肌损伤明显减轻,心肌组织胶原纤维明显减少,且冬凌草甲素高剂量组和卡托普利组各项指标改善情况均显著优于冬凌草甲素低剂量组(P均<0.05),冬凌草甲素高剂量组和卡托普利组各指标比较差异均无统计学意义(P均>0.05)。结论冬凌草甲素可能通过抑制NLRP3炎性通路减轻大鼠心肌梗死后心肌细胞凋亡和心肌纤维化。

Objective It is to explore the effect of oridonin on myocardial cell apoptosis,fibrosis and NLRP3 inflammatory pathway after myocardial infarction in rats.Methods Fifteen SD male rats were selected as the sham operation group,and another 60 SD male rats were selected to establish myocardial infarction models by ligating the left anterior descending coronary artery.The successfully modeled rats were randomly divided into model group,oridonin low-dose group,oridonin high-dose group,and captopril group,with 15 rats in each group.The rats in the low-dose and high-dose groups of oridonin were intraperitoneally injected with 10 mg/kg and 20 mg/kg oridonin,the rats in the captopril group were intraperitoneally injected with 9.322 mg/kg captopril,and the rats in the sham operation group and model group were intraperitoneally injected with the same amount of normal saline,all once per day,continuously treated for 4 weeks.The cardiac function parameters[left ventricular ejection fraction(LVEF),left ventricular end-systolic diameter(LVESd),left ventricular end-diastolic diameter(LVEDd),left ventricular short-axis shortening rate(LVFS)]were detected by echocardiography.The myocardial infarction area ratio,myocardial histopathology,myocardial fibrosis and myocardial cell apoptosis in the rats were detected by TTC staining,HE staining,Masson staining and TUNEL method,and the expressions of myocardial fibrosis-related proteins[transforming growth factor-β_(1)(TGF-β_(1)),connective tissue growth factor(CTGF),collagenⅠ,collagenⅢ],cell apoptosis-related proteins[B-lymphoma-2(Bcl-2)protein,Bcl-2-related X protein(Bax),cleaved caspase-3 protein]and NLRP3 inflammatory pathway-related protein[nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3),apoptosis associated speck like protein containing a CARD(ASC),Caspase-1,interleukin-1β(IL-1β),interferon-18(IL-18)]were detected by Wertern blot method.Results Compared with the model group,the LVEF,LVFS and the relative expressions of Bcl-2 protein in myocardial tissue of the rats in the oridonin low-dose group,oridonin high-dose group and captopril group were significantly increased(all P<0.05),LVESd,LVEDd,myocardial infarction area ratio,myocardial cell apoptosis rate and the protein relative expressions of TGF-β_(1),CTGF,collagenⅠ,collagenⅢ,Bax,Cleaved Caspase-3,NLRP3,ASC,Caspase-1,IL-1β,IL-18 in myocardial tissue were significantly decreased(all P<0.05),myocardial injury was significantly relieved,and collagen fibers in myocardial tissue were significantly reduced,and the improvement of these indicators in the oridonin low-dose group and captopril group was significantly better than that in the captopril group(P<0.05),there was no significant difference in each index between the oridonin high-dose group and captopril group(P>0.05).Conclusion Oridonin may alleviate myocardial cell apoptosis and myocardial fibrosis after myocardial infarction in rats by inhibiting the NLRP3 inflammatory pathway.