【作者】 陈曦；

【导师】 马根山；

【作者基本信息】 东南大学 ， 心血管病学， 2022， 硕士

【摘要】 目的:心肌缺血再灌注(Ischemia/Reperfusion,I/R)损伤是指冠状动脉部分或完全急性阻塞后再通,恢复缺血心肌正常灌注后进一步加重组织损伤的病理过程,严重限制了血运重建对缺血性心脏病的治疗效果,因此,在开通血管恢复缺血心肌供血的同时避免再灌注所产生的损伤,一直是心血管领域研究的重点。川芎嗪,又称四甲基吡嗪(Tetramethylpyrazine,TMP)是从中药川芎中提取出的一种特征性生物碱,具有广泛的药理作用,对心肌I/R损伤具有一定的保护作用,然而目前很少以药物直接靶点作为切入点深入探讨川芎嗪抗心肌I/R损伤的具体机制,因此川芎嗪抗心肌I/R损伤的具体机制还有待阐明。本课题旨在基于药物亲和反应靶稳定性(DARTS)技术和液相色谱-质谱联用(LC-MS/MS)技术联用探讨川芎嗪保护心肌I/R损伤作用机制。方法:第一部分:体内实验将36只雄性Sprague-Dawley(SD)大鼠随机分为假手术(Sham)组、TMP(40mg/kg)组、I/R模型组、I/R+TMP(10mg/kg、20mg/kg和40mg/kg)组,每组6只。药物灌胃后30min后采用冠状动脉左前降支结扎的方法制备大鼠心肌I/R损伤(缺血30min,再灌注120min)模型,通过TTC染色检测心肌损伤面积,试剂盒检测血清中的心肌损伤相关标志物肌酸激酶同工酶(CK-MB)、心肌肌钙蛋白T(c Tn T)以及乳酸脱氢酶(LDH)的表达水平,HE染色观察各组的病理改变;体外实验选用H9C2细胞,分为正常(Control)组、TMP(20μM)、缺氧/复氧(Hypoxia/Reoxygenation,H/R)组、H/R+TMP(5、10、20μM)组,建立H/R(缺氧8h,复氧2h)模型后药物干预24h,CCK8检测细胞活力,流式细胞仪评估各组细胞凋亡率。第二部分:体外实验选用H9C2细胞,分为2组:H/R组和H/R+TMP(10uM)组。DARTS联合LC-MS/MS技术分析两组间的蛋白差异表达,Western Blot验证川芎嗪与目标靶蛋白两者间的直接结合力,并于第一部分体内和体外模型样本中检测筛选得到的靶蛋白。第三部分:将24只雄性SD大鼠随机分为Sham组、I/R组、I/R+TMP+siRNA-NC组、I/R+TMP+si RNA-Myl2组,每组6只,其中I/R+TMP+si RNA-NC和I/R+TMP+si RNA-Myl2组分别将si RNA-NC和si RNA-Myl2注射至心肌后行川芎嗪治疗和I/R模型构建。通过TTC染色观察心肌损伤面积,试剂盒检测CK-MB、c Tn T以及LDH的表达水平,HE染色观察病理改变;网络药理学分析川芎嗪治疗心肌缺血再灌注损伤靶点的通路富集,Western Blot评估焦亡标记物Cleaved-caspase-1、GSDMD、IL-18、IL-1β表达水平。结果:第一部分:川芎嗪能够呈剂量依赖性减少大鼠心肌I/R损伤的损伤面积和H/R后的心肌细胞凋亡率,降低I/R大鼠血清中CK-MB、c Tn T以及LDH的表达水平,减轻I/R大鼠心肌组织炎症浸润程度。第二部分:DARTS联合LC-MS/MS技术共鉴定出H/R和H/R+TMP组间存在613个差异蛋白(H/R+TMP组中上调的差异蛋白283个,下调的差异蛋白335个),其中Myl2蛋白表达变化最显著,Western Blot结果发现,川芎嗪呈浓度依赖性上调Myl2蛋白表达水平,在第一部分中体内和体外模型样本中检测Myl2蛋白水平发现,与对照组相比,I/R和H/R组Myl2蛋白表达明显下调,而川芎嗪可以拮抗其下调趋势,导致心肌Myl2表达增多。第三部分:与I/R组相比,I/R+TMP+siRNA-NC组大鼠心肌损伤面积明显减小,CK-MB、cTnT以及LDH的表达水平有效降低,炎症浸润程度减轻,而进一步抑制Myl2表达后,药物治疗效果被逆转。网络药理学分析提示川芎嗪治疗心肌I/R损伤靶点通路富集在TNF、NOD样受体等信号通路上,NOD样受体信号通路与细胞焦亡有关,Western Blot检测焦亡标记物表达发现,与I/R组相比,川芎嗪能明显下调焦亡标记物Cleaved-caspase-1、GSDMD、IL-18、IL-1β的表达,而抑制Myl2表达后逆转上述焦亡标记物的表达水平。结论:川芎嗪可减轻缺血再灌注诱导的心肌损伤,其机制与调控Myl2的表达,抑制心肌细胞焦亡有关。更多还原

【Abstract】 Objective: Myocardial ischemia/reperfusion(I/R)injury is a pathological process of further aggravating tissue damage after restoring perfusion of ischemic myocardium,which severely limits the therapeutic effect of revascularization for ischemic heart disease,therefore,it is important to avoid the damage caused by reperfusion while open the blood vessels to restore the blood supply to the ischemic myocardium.Ligustrazine,also known as Tetramethylpyrazine(TMP),is a characteristic alkaloid extracted from the traditional Chinese medicine-Ligusticum wallichii.TMP has a wide range of pharmacological effects and has a certain protective effect on myocardial I/R injury.The specific mechanism of TMP protecting against myocardial I/R injury is rarely explored in depth with the direct drug target as the entry point.Therefore,the specific mechanism of TMP protecting against myocardial I/R injury remains to be elucidated.This study aims to explore the mechanism of TMP protecting against myocardial I/R injury based on the combination of drug affinity reaction target stability(DARTS)and liquid chromatography-mass spectrometry(LC-MS/MS)technology.Methods: Section one: In the in vivo experiment,36 male Sprague-Dawley(SD)rats were randomized into Sham,TMP(40mg/kg),I/R,and I/R+TMP(10mg/kg,20mg/kg and 40mg/kg,respectively)groups,with 6 rats in each group.30 min after TMP treatment,the left anterior descending coronary artery was ligated to establish the myocardial I/R injury model(ischemia for 30 min,reperfusion for 120min).TTC staining was used to assess the area of myocardial necrosis.Kits were used to detect the contents of CK-MB,c Tn T,and LDH in serum.The level of inflammatory infiltration was evaluated using HE staining.In the in vitro experiment,H9C2 cells were divided into Control,TMP (20μM),Hypoxia/Reoxygenation(H/R),and H/R+TMP(5,10,20μM,respectively)groups.H9C2 cells induced H/R(hypoxia for 8h,reoxygenation for 2h)injury and treat by TMP for 24 h.CCK8 and flow cytometry was used to detect the cell viability and apoptosis rate in each group,respectively.Section two: In the in vitro experiment,H9C2 cells were divided into two groups: the H/R and H/R+TMP(10u M)groups.Combination of DARTS and LC-MS/MS was used to identify the differentially expression protein between of the two groups;Western blot further verified the direct binding ability between TMP with the target differential protein and assess the expression levels of target differential proteins in samples of section one.Section three: In the in vivo experiment,24 male SD rats were randomly assigned to the Sham,I/R,I/R+TMP+si RNA-NC,and I/R+TMP+si RNA-Myl2 groups,of which I/R+TMP+si RNA-Myl2 group was treated with ligustrazine after si RNA-Myl2 was injected into the myocardium and the I/R model was constructed.The area of myocardial necrosis was observed by TTC staining.The contents of CK-MB,c Tn T and LDH were detected by kits.HE staining was applied to evaluated the inflammatory infiltration level.Network pharmacology was used to analyze the pathway enrichment of the targets of TMP in the treatment of myocardial I/R injury.Western blot was used to evaluate the expression levels of pyroptosis markers Cleaved-caspase-1,GSDMD,IL-18,and IL-1β.Results: Section one: TMP dose-dependently decreased the infarct size in I/R rats and the apoptosis rate of H9C2 cells following H/R,reduced the serum levels of CK-MB,c Tn T and LDH,and alleviated the level of inflammatory infiltration in I/R rats.Section two: DARTS combined with LC-MS/MS technology identified a total of 613 differential proteins between the H/R and H/R+TMP groups,and the 283 up-regulated and 335 downregulated differential proteins in the H/R+TMP group.Among them,the expression of Myl2 protein changed most significantly.Western blot results showed that TMP up-regulated the protein expression level of Myl2 in a concentration-dependent manner,the Myl2 expression was considerably decreased following I/R and H/R,and TMP treatment was able to reverse the I/R and H/R-induced decrease in Myl2 expression.Section three: Compared with the I/R group,the myocardial infarction area of the rats in the I/R+TMP group was significantly reduced,the contents of CK-MB,c Tn T and LDH were effectively reduced,and the level of inflammatory infiltration was reduced.After further inhibiting the expression of Myl2,therapeutic effect of TMP was partial reversed.The network pharmacology analysis showed that the target of TMP protects against myocardial I/R injury were enriched in TNF,NOD-like receptor and other signaling pathways,and the NOD-like receptor signaling pathway was related to pyroptosis.Western blot results showed that compared with the I/R group,TMP could significantly down-regulate the expressions of pyroptosis markers Cleaved-caspase-1,GSDMD,IL-18,and IL-1β,while inhibiting the expression of Myl2 reversed the expression of the above pyroptosis markers.Conclusion: TMP protects against myocardial I/R injury by upregulating Myl2 and inhibiting cardiomyocyte pyroptosis following myocardial IR injury.更多还原