【作者】 马艳；

【导师】 李海霞；

【作者基本信息】 郑州大学 ， 生药学， 2022， 硕士

【摘要】 研究背景与目的:冠心病导致的心肌缺血已成为中老年人的常见病和多发病,严重危害健康。心肌缺血预适应（Ischemic Preconditioning,IPC）是至今为止最有效的内源性心脏保护途径,可对抗随后的缺血损伤。IPC的重要进展之一,是用药物代替IPC,即药物预适应（Pharmaceutical Preconditioning,PPC）,如腺苷、缓激肽、氧自由基、儿茶酚胺、异氟醚及促炎细胞因子如TNF-α（tumor necrosis factor-α,TNF-α）等,均可诱导IPC样保护作用。但这些药物,或不良反应多、或不能在机体正常状态下使用,很难在临床广泛应用。长期以来,中医根据气血理论“气与血是一个统一整体,气为血帅,血为气母,气赖血载,血赖气行”,认为由冠心病导致的心肌缺血是气虚为本,血瘀为标,益气活血法可以标本兼治,临床上采用“益气活血”方药防治取得了较大成功,如由人参等药材组成的生脉饮、通心络胶囊、保元汤等,均有显著疗效。流行病学调查也发现气虚血瘀是冠心病的主要证型。此外,Meta分析显示,西医常规治疗基础上加用益气活血方药治疗冠心病,在临床总疗效、心绞痛疗效等方面,优于单纯的西医治疗,安全性较好,但因中药提取物质量难以控制、机制阐明不清,尤其是符合中医药整体作用观念的机制阐明不足,极大阻碍了中医药的发展。为了获得化学成分明确、质量可控的人参总皂苷提取物（Total saponin extracts of Panax ginseng,EPG）,并进一步阐明其改善心肌缺血的机制,本课题进行了以下研究:（1）利用60%乙醇连续回流提取人参中的皂苷类有效成分,通过大孔吸附树脂HPD-100进行纯化,通过喷雾干燥对纯化的浓缩液进行干燥,获得的纯化物采用高效液相色谱仪进行含量测定。（2）利用酶消化法分离乳鼠原代心肌细胞,利用厌氧产气包构建心肌细胞缺氧模型;通过CCK-8实验确定人参总皂苷提取物的最佳给药浓度;使用LDH试剂盒检测细胞上清LDH酶活力;使用ELISA试剂盒检测细胞上清BNP、c Tn I的含量,评价心肌细胞损伤的程度;通过荧光显微镜观察荧光强度和流式细胞仪测定细胞内钙离子浓度。（3）通过结扎SD大鼠冠状动脉左前降支（Left anterior descending coronary artery,LAD）构建急性心肌缺血模型,利用小动物超声仪评价心脏功能;LDH试剂盒检测血清中LDH酶活力;ELISA试剂盒检测血清中BNP、c Tn I的含量;TTC染色检测心肌梗死面积;HE染色观察组织病变;Masson染色检测心肌纤维化;Tunel法检测心肌细胞凋亡;免疫荧光法检测钙调控相关蛋白的表达;通过透射电子显微镜观察心肌的超微结构;通过Western blot和Real Time-PCR法检测钙调控相关蛋白Caveolin-1、ORAI1、TRPC1及STIM1蛋白和m RNA的表达,并通过Western blot检测磷酸化Caveolin-1的表达,初步探究人参总皂苷提取物保护心脏的作用机制。结果:（1）人参总皂苷提取物的提取率为1.78%,定量了10种人参皂苷,即人参皂苷Rg₁、Re、Rf、Rb₁、Rc、Rg₂、Rh₁、Rb₂、Rb₃、Rd,它们在人参总皂苷提取物中的总含量为57.96%,为后续实验制备了质量可控、化学成分明确的人参总皂苷提取物。（2）人参总皂苷提取物预保护给药（200μg/m L）可以在心肌细胞缺氧模型中产生保护作用,表现为细胞上清中心肌损伤标志物（BNP、c Tn I）的含量降低（P<0.01）,LDH酶活力降低（P<0.01）,钙离子阳性细胞率降低（P<0.01）,细胞存活率增加（P<0.01）;使用Caveolin-1磷酸化抑制剂PP2后,人参总皂苷提取物对心肌细胞的保护作用被减弱;表现为降低的LDH酶活力被增加（P<0.01）,降低的BNP被增加（P<0.01）,降低的c Tn I被增加（P<0.05）,降低的钙离子阳性细胞率被增加（P<0.05）,增加的细胞存活率被降低（P<0.01）,提示人参总皂苷提取物可能通过Caveolin-1磷酸化抑制钙内流而发挥心脏保护作用。（3）人参总皂苷提取物预保护给药（200 mg/kg）可以在急性心肌缺血动物模型中产生心脏保护作用,表现为反映心功能的射血分数和短轴缩短分数增加、心肌损伤标志物（BNP、c Tn I）的漏出程度减轻（P<0.01）、LDH酶活力降低（P<0.01）、心肌梗死面积减少（P<0.01）、病理形态学改善（心肌细胞炎性浸润减少、细胞肿胀减轻）、心肌纤维化程度降低（P<0.05）、心肌超微结构改善（受损的线粒体结构得到改善）、凋亡的心肌细胞数目减少（P<0.01）、Caveolin-1蛋白表达及其磷酸化水平增加（P<0.01）,ORAI1、TRPC1、STIM1蛋白表达降低（P<0.01）;Caveolin-1、ORAI1、TRPC1及STIM1的m RNA表达,具有与蛋白质表达相同的趋势,但除了ORAI1的m RNA之外,其他m RNA的变化没有统计学差异。使用Caveolin-1磷酸化抑制剂PP2后,人参总皂苷提取物对心脏的保护作用被减弱,表现为升高的射血分数和短轴缩短分数被降低,降低的LDH酶活力增加（P<0.05）、降低的BNP被增加（P<0.01）,降低的c Tn I被增加（P<0.05）,降低的心肌梗死面积被增加（P<0.01）,对病理形态学的改善被减弱,降低的心肌纤维化的程度被增加,降低的凋亡心肌细胞数目被增加（P<0.01）,升高的Caveolin-1蛋白表达及其磷酸化水平被降低（P<0.01）,降低的ORAI1、TRPC1、STIM1蛋白表达被增加（P<0.01）,降低的ORAI1的m RNA水平被升高,这些结果提示人参总皂苷提取物可能通过增加Caveolin-1及其磷酸化,降低钙调控相关蛋白（ORAI1、TRPC1、STIM1）的表达发挥心脏保护作用。结论:人参总皂苷提取物预保护给药模拟缺血预适应（IPC）,在心肌缺血损伤时,通过增加Caveolin-1蛋白表达及其磷酸化水平,降低钙调控相关蛋白（ORAI1、TRPC1、STIM1）的表达,负性调控钙库调控的钙内流（Store-operated calcium entry,SOCE）,降低细胞内钙离子浓度,缓解钙超载而发挥心脏保护作用。更多还原

【Abstract】 Background and objective:Myocardial ischemia caused by coronary heart disease has become a common and frequently-occurring disease in middle-aged and elderly people,seriously endangering health.Ischemic Preconditioning（IPC）is by far the most effective endogenous cardioprotective pathway against subsequent ischemic injury.One of the important advances in IPC is the use of medicines to replace IPC,i.e.,Pharmaceutical Preconditioning（PPC）,for example adenosine,bradykinin,oxygen free radicals,catecholamines,isoflurane and proinflammatory cytokines such as tumor necrosis factor-α（TNF-α）can induce IPC-like protective effects.However,these drugs have many adverse reactions or cann’t be used in the normal state of the body,and therefore aren’t widely used in clinical practice.For a long time,according to the theory of Qi and blood,"Qi and blood are a unified whole,the flow of blood is accompanied by the movement of Qi in the body",it is believed that Qi deficiency and blood stasis can bring on myocardial ischemia,the method of tonifying Qi combined with activating blood circulation can treat both the symptoms and the root causes,thus the use of tonifying Qi combined with activating blood prescription to prevent and treat myocardial ischemia has achieved great success in clinical,For example,Shengmai Yin,Tongxinluo Capsule,and Baoyuan Decoction composed of medicinal materials such as Panax ginseng,which have significant effects.Epidemiological surveys have also found that Qi deficiency and blood stasis is the main cause of coronary heart disease.Moreover,meta-analysis showed that compared with conventional therapy of western medicine treatment alone,the addition of tonifying Qi combined with activating blood prescription was superior to treat coronary heart disease in terms of total clinical efficacy and angina pectoris efficacy,along with better safety,However,the development of traditional chinese medicine is greatly hindered because the quality of traditional chinese medicine extracts is difficult to control and the mechanism remains unclear,especially the mechanism in line with the overall treatment concept of traditional chinese medicine is not elucidated enough.In order to obtain total saponin extracts of Panax ginseng（EPG）with clear chemical composition and controllable quality,and to further elucidate its mechanism on improving myocardial ischemia,the following studies were conducted in this thesis:（1）The ginsenosides,which are active ingredients of Panax ginseng were extracted by continuous reflux with 60%ethanol,and purified by macroporous adsorption resin HPD-100,The purified concentrate was dried by spray dryer,and the obtained purification was determined by HPLC-UV.（2）After primary neonatal rat cardiomyocytes were isolated by enzymatic digestion,the hypoxia model was induced by the method of anaerobic gas generation.The optimal administration concentration of EPG was determined by CCK-8 assay,For evaluating the degree of myocardial cell injury,the activity of LDH in cell supernatant was detected by LDH kit,the contents of BNP and c Tn I,corresponding ELISA kits respectively;the intracellular calcium concentration was measured by fluorescence microscopy and flow cytometry.（3）To preliminarily explore the mechanism of cardioprotective effect of total saponin extracts of Panax ginseng,acute myocardial infarction model was induced by ligation of left anterior descending coronary artery（LAD）,cardiac function was evaluated by Small animal ultrasound machine;LDH kit was used to detect LDH activity in serum and corresponding ELISA kits were used to detect the content of BNP and c Tn I in serum,respectively;myocardial infarct size was detected by TTC staining,myocardial tissue lesions was detected by Hematoxylin and eosin staining,myocardial fibrosis was detected by Masson staining,cardiomyocyte apoptosis was detected by Tunel assay,the expression of calmodulin-related proteins were detected by immunofluorescence,the ultrastructure of myocardium was observed by transmission electron microscopy,and the expression of calcium regulation-related proteins Caveolin-1,ORAI1,TRPC1 and STIM1 protein and m RNA were detected by Western blot and Real time-PCR,respectively;and the expression of phosphorylated Caveolin-1 was detected by Western blot.Results:（1）The extraction of total saponin extracts of Panax ginseng（EPG）was 1.78%.Ten ginsenosides i.e.Ginsenoside Rg₁,Re,Rf,Rb₁,Rc,Rg₂,Rh₁,Rb₂,Rb₃,Rd,were quantified,and their total contents in total saponin extracts of Panax ginseng were57.96%,and total ginsenoside extracts with controllable quality and clear chemical composition were prepared for subsequent experiments.（2）Preprotective administration of total saponin extracts of Panax ginseng（200μg/m L）produced protective effect on the cardiomyocyte hypoxia model,as shown by the decreased content of myocardial injury markers（BNP,c Tn I）in the supernatant（P<0.01）,the decreased LDH activity（P<0.01）,the decreased rate of calcium positive cells（P<0.01）,and the increased cell survival rate（P<0.01）;After using PP2,a Caveolin-1 phosphorylation inhibitor,the protective effect of total saponin extracts of Panax ginseng on cardiomyocytes was attenuated;as shown by the increase of LDH activity（P<0.01）,the increase of BNP（P<0.01）,the increase of c Tn I（P<0.05）,the enhanced percent of calcium positive cells（P<0.05）,and the decrease of cell survival rate（P<0.01）,suggesting that total saponin extracts of Panax ginseng may play a cardioprotective role by inhibiting calcium influx through Caveolin-1phosphorylation.（3）Preprotective administration of total saponin extracts of Panax ginseng（200mg/kg）produced cardioprotective effects on acute myocardial ischemia;it was characterized by enhancing cardiac function via increasing left ventricular ejection fraction and left ventricular fractional shortening,reducing leakage of myocardial injury markers（BNP,c Tn I）（P<0.01）,reducing LDH activity（P<0.01）,reducing myocardial infarct size（P<0.01）,pathomorphological improvement（reducing inflammatory infiltration of cardiomyocytes,reducing cell swelling）,reducing degree of myocardial fibrosis（P<0.05）,improving myocardial ultrastructure（amelioration of the damaged mitochondrial structure）,reducing the number of apoptotic cardiomyocytes（P<0.01）,increasing Caveolin-1 protein expression and its phosphorylation level（P<0.01）,and decreasing ORAI1,TRPC1,and STIM1 protein expression（P<0.01）;and m RNA expression of Caveolin-1,ORAI1,TRPC1 and STIM1 has the same tendency as corresponding protein expression,but except ORAI1 m RNA,there was no statistically significant difference in the changes of m RNAs.After using PP2,a Caveolin-1 phosphorylation inhibitor,the protective effect of total saponin extracts of Panax ginseng on the heart was attenuated,It showed that the elevated left ventricular ejection fraction and left ventricular fractional shortening were decreased,the decreased LDH activity was increased（P<0.05）,the decreased BNP was increased（P<0.01）,the decreased c Tn I was increased（P<0.05）,the decreased myocardial infarct size was increased（P<0.01）,the improvement of pathomorphology was attenuated,the degree of decreased myocardial fibrosis was increased,and the number of decreased apoptotic cardiomyocytes was increased（P<0.01）,the elevated Caveolin-1 protein expression and its phosphorylation level were decreased（P<0.01）,decreased ORAI1,TRPC1,and STIM1 protein expression were increased（P<0.01）,and the decreased expression of ORAI1 m RNA was increased,suggesting that EPG may play a cardioprotective role by increasing Caveolin-1 and its phosphorylation and decreasing the expression of calcium-regulation-related proteins（ORAI1,TRPC1,STIM1）.Conclusion:During myocardial ischemia injury,similar to IPC,total saponin extracts of Panax ginseng inhibits calcium influx through Caveolin-1 phosphorylation,and negatively regulates Store-operated calcium entry（SOCE）,which together reduces the concentration of intracellular calcium and alleviates calcium overload and thus exerts cardioprotective effects.更多还原