目的 观察麝香保心丸(Shexiang baoxin pill,SBP)对缺血/再灌注损伤(Ischemia/reperfusion injury,I/R)大鼠心肌组织的保护作用及与自噬的调节关系.方法 采用左冠状动脉前降支缺血再灌注建立I/R模型.将Sprague-Dawley(SD)大鼠30只按随机数字表法分为对照组、模型组及SBP组,每组各10只.通过心脏超声多普勒检测大鼠不同时期左心室射血分数(Left ventricular ejection fraction,LVEF)、左室缩短分数(Fractional shorten-ing,FS)、左室舒张末期内径(Left ventricular end diastolic diameter,LVIDd)和左心室收缩期内径(Left ventricular end systolic dimension,LVIDs)的变化,HE染色观察心肌组织病理改变,TUNEL染色检测细胞凋亡情况,采用qRT-PCR方法检测白细胞介素-1β(Interleukin-1β,IL-1β)、白细胞介素-6(Interleukin-6,IL-6)、肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)和自噬相关基因(Autophagy-related genes,ATG)、BCL-2 关联 X 蛋白(BCL-2 associated X protein,Bax)和 B 淋巴细胞瘤-2 基因(B-cell lymphoma-2,Bcl-2)的表达水平.采用Western blot检测自噬特异性基因(Beclin-1)、泛素结合蛋白p62(p62)和微管相关蛋白质1轻链3(Microtubule-associated protein 1 light chain 3,LC3)的表达水平.结果 与对照组比较,心肌缺血/再灌注损伤后,各组大鼠心脏LVEF、FS均下降,LVIDd、LVIDs均增大.术后随访发现SBP组大鼠心脏功能指标较模型组明显改善.HE染色见SBP减轻I/R大鼠心肌细胞水肿紊乱情况.TUNEL染色显示,与对照组比较,模型组心肌组织显示凋亡细胞增多,而经SBP处理的大鼠心肌组织中凋亡细胞减少,说明SBP可以减轻I/R导致的细胞损伤.通过qRT-PCR检测发现,与对照组比较,模型组大鼠ATG12表达水平明显上调.而SBP组大鼠在1个月随访时发现ATG12表达水平较模型组降低.说明SBP抑制I/R诱导的ATG12 mRNA表达上调.与对照组比较,模型组Bax表达增加,Bcl-2表达降低.与模型组比较,SBP处理后大鼠心肌组织中 Bax表达降低,Bcl-2表达升高.表明SBP可以下调促凋亡蛋白,上调抗凋亡基因.与对照组比较,模型组Beclin-1、p62及LC3-Ⅱ/LC3-I水平均明显升高.与模型组比较,SBP下调了心肌中Beclin-1、p62和LC3-Ⅱ/LC3-Ⅰ蛋白的表达水平.结论 心肌缺血再灌注可增强自噬反应,促进细胞凋亡.麝香保心丸可能通过调节ATG12从而抑制自噬,对受损心肌发挥保护作用,改善心肌细胞凋亡.
Regulatory Effects of Shexiang Baoxin Pills on Myocardial Injury and Cellular Auto-phagy in Rats with Ischemia-Reperfusion
Objective To observe the effect of Shexiang Baoxin Pills(SBPs)on myocardial tissue in rats with is-chemia/reperfusion injury(I/R)and its regulatory relationship with autophagy.Method The I/R model was established using left anterior descending coronary artery ischemia/reperfusion.30 Sprague-Dawley(SD)rats were randomly divided into three groups:control,model,and SBP,with 10 rats in each group.Cardiac ultrasound Doppler was used to detect chan-ges in the left ventricular ejection fraction(LVEF),fractional shortening(FS),left ventricular end diastolic diameter(LVIDd),and left ventricular end systolic dimension(LVIDs)at different periods.HE staining was used to observe patho-logical changes in myocardial tissue,TUNEL staining was used to observe myocardial cell apoptosis,and qRT-PCR was used to measure the expression levels of interleukin-1β(IL-1β),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),autophagy-related genes(ATG),BCL-2 associated X protein(Bax),and B-cell lymphoma-2(Bcl-2).Western blot was used to detect the expression levels of Beclin-1,ubiquitin-binding protein p62(p62),and microtubule-associated protein 1 light chain 3(LC3).Result Compared with the control group,the LVEF and FS in each group de-creased,while the LVIDd and LVIDs increased after myocardial I/R injury.Compared with the model group,the cardiac function indexes in the SBP group significantly improved in postoperative follow-up.HE staining showed that SBP reduced edema and disorder of myocardial cells in I/R rats.TUNEL staining indicated an increase in apoptotic cells in the myocar-dial tissue of the model group compared to the control group,while SBP treatment reduced apoptosis,suggesting that SBP could mitigate I/R-induced cell damage.qRT-PCR revealed a significant upregulation of ATG12 expression in the mod-el group compared to the control group.However,a one-month follow-up showed reduced ATG12 expression in the SBP group compared to the model group,indicating that SBP inhibited the I/R-induced upregulation of ATG12 mRNA expres-sion.Bax expression increased,and Bcl-2 expression decreased in the model group compared to the control group.SBP treatment resulted in decreased Bax and increased Bcl-2 expression in myocardial tissue compared to the model group,in-dicating that SBP can downregulate pro-apoptotic protein and upregulate anti-apoptotic genes.Compared with the control group,the expression of Beclin-1,p62,and LC3Ⅱ/LC3I were significantly increased in the model group.SBP treatment decreased the expression levels of Beclin-1,p62,and LC3Ⅱ/LC3I in myocardial tissue compared to the model group.Conclusion Myocardial ischemia/reperfusion can enhance the autophagic response and promote apoptosis.SBP protects the damaged myocardium by regulating ATG12,thereby inhibiting autophagy and improving myocardial cell apoptosis.
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