【作者】 李林；

【导师】 夏中元；

【作者基本信息】 武汉大学 ， 麻醉学， 2022， 博士

【摘要】 第一部分MA预处理对H9c2心肌细胞H/R损伤的影响目的探讨不同浓度山楂酸(maslinic acid,MA)对H9c2心肌细胞的毒性作用及对缺氧/复氧(hypoxia/reoxygenation,H/R)损伤的影响。方法体外培养H9c2心肌细胞,其中不同浓度MA对H9c2心肌细胞毒性作用分组:分别用浓度为0、1、5、10、20和40 μM的MA处理H9c2细胞24 h;MA预处理对H9c2心肌细胞H/R损伤的影响分组:对照组(Control组)、缺氧/复氧组(H/R组)和缺氧/复氧+MA预处理组(H/R+MA组),H/R模型采用缺氧4h复氧4h的方法制备,MA的浓度分为1、5、10、20和40 μM。采用CCK-8试剂盒检测细胞活力;乳酸脱氢酶(lactic dehydrogenase,LDH)试剂盒检测细胞培养基上清中LDH释放水平;TUNEL法检测H9c2心肌细胞的凋亡水平;DAPI/PI双染检测H9c2心肌细胞发生程序性坏死的程度;Western blot检测细胞凋亡相关蛋白(Caspase-3、Caspase-9、Bcl-2及Bax)和程序性坏死相关蛋白(RIPK1、RIPK3 及 MLKL)表达量。结果1.不同浓度的MA处理H9c2心肌细胞24 h后,对各组细胞活力没有影响,提示MA预处理对H9c2心肌细胞没有药物毒性。2.与Control组相比,H/R组的细胞活力显著降低,LDH水平显著上升;而与H/R组相比,H/R+MA组中在MA剂量为5、10、20 μM时,细胞活力显著上升,LDH水平显著下降,且呈剂量依赖性;当MA剂量上升至40 μM时,与20 μM组对比,细胞活力和LDH水平没有显著变化。因此,我们在后续实验中选取浓度为5、10、20 μM的MA进行试验。3.与Control组相比,H/R组的细胞凋亡数量显著升高;而与H/R组相比,H/R+MA组的细胞凋亡数量显著下降,且呈剂量依赖性。与Control组相比,H/R组H9c2心肌细胞中的凋亡相关蛋白Bax、cleaved Caspase-3及cleaved Caspase-9表达水平显著增加,而Bcl-2表达显著降低;与H/R组相比,H/R+MA组H9c2心肌细胞中的凋亡相关蛋白Bax、cleaved Caspase-3及cleaved Caspase-9表达水平显著降低,而Bcl-2表达显著增加,且与MA的剂量呈依赖性。4.与Control组相比,H/R组的细胞坏死率显著增加;而与H/R组相比,H/R+MA组的细胞坏死率显著降低,且呈剂量依赖性。与Control组相比,H/R损伤显著上调了 H9c2心肌细胞中的p-RIPK1、p-RIPK3及p-MLKL蛋白表达;与H/R组相比,H/R+MA组中MA预处理能够显著下调p-RIPK1、p-RIPK3及p-MLKL蛋白表达,且与MA的剂量呈依赖性。结论MA预处理对H9c2心肌细胞H/R损伤的具有保护作用,且与MA的剂量呈依赖性,其机制可能与抑制细胞凋亡和程序性坏死有关。第二部分MA预处理对大鼠心肌I/R损伤的影响目的探讨MA预处理在大鼠心肌缺血/再灌注(ischemia/reperfusion,I/R)损伤的保护作用及其与细胞凋亡和程序性坏死的相关机制。方法使用随机数字表法将48只SPF级健康成年雄性SD大鼠均分成4组(n=12):对照组(Sham+DMSO组)、对照+MA预处理组(Sham+MA组)、大鼠缺血/再灌注组(I/R+DMSO组)及大鼠缺血/再灌注+MA预处理组(I/R+MA组)。结扎冠状动脉左前降支使其缺血30 min后剪断结扎线后行再灌注180 min的方法构建I/R模型。MA预处理组大鼠在手术前48 h腹腔注射20 mg/kg MA,并在手术前24 h重复注射。采用HE染色观察心肌组织形态结构变化;TTC染色法检测心肌组织的梗死面积;LDH和CK-MB试剂盒检测大鼠血清LDH和CK-MB含量;通过α-肌动蛋白+TUNEL荧光染色检测大鼠I/R诱导的心肌细胞凋亡水平;利用免疫组化检测程序性坏死相关蛋白MLKL表达水平;Western blot检测大鼠心肌凋亡相关蛋白(Caspase-3和Caspase-9)和程序性坏死相关蛋白(RIPK1、RIPK3及MLKL)活化水平。结果1.与Sham+DMSO组相比,I/R+DMSO组中心肌组织结构紊乱且炎症浸润改变明显,血清中相关心肌损伤指标LDH和CK-MB水平显著升高,心肌梗死面积明显增大;与I/R+DMSO组相比,I/R+MA组中心肌形态结构改变减轻,血清中相关心肌损伤指标LDH和CK-MB的含量均降低,心肌梗死面积明显缩小。2.与Sham+DMSO组相比,I/R+DMSO组中TUNEL阳性染色明显增加,还可以发现TUNEL阳性染色不仅出现在心肌细胞中,在心肌细胞之间也发现其存在,提示大鼠I/R诱导的细胞凋亡发生在心肌细胞、内皮细胞和成纤维细胞等细胞中。与I/R+DMSO组对比,I/R+MA组中TUNEL阳性染色显著减少。Western blot检测发现,与Sham+DMSO组相比,大鼠I/R+DMSO组中凋亡相关蛋白cleaved Caspase-3及cleaved Caspase-9裂解水平显著增加;而与I/R+DMSO组对比,I/R+MA组中凋亡相关蛋白cleaved Caspase-3及cleaved Caspase-9裂解水平水平显著降低。3.免疫组化结果提示,与Sham+DMSO组相比,I/R+DMSO组中MLKL蛋白丰度增加;而与I/R+DMSO组对比,I/R+MA组中MLKL蛋白丰度降低。Western blot检测发现,与 Sham+DMSO 组相比,I/R+DMSO 组中 p-RIPK1、p-RIPK3 以及p-MLKL蛋白表达明显增加;而与I/R+DMSO组对比,I/R+MA组中p-RIPK1、p-RIPK3以及p-MLKL蛋白表达明显降低。结论MA预处理可能通过抑制细胞凋亡和程序性坏死相关通路,发挥减轻心肌缺血再灌注损伤(myocardial ischemia reperfusion injury,MIRI)的保护作用。第三部分MA预处理对大鼠心肌I/R损伤的保护作用与溶酶体自噬的关系目的探索溶酶体-自噬途径在大鼠心肌I/R诱导的细胞凋亡与程序性坏死中的作用及相关机制,并观察MA预处理对其的影响。方法使用随机数字表法将48只SPF级健康成年雄性SD大鼠均分成4组(n=12):对照组(Sham+DMSO组)、大鼠缺血/再灌注组(I/R+DMSO组)、大鼠缺血/再灌注+MA预处理组(I/R+MA组)及大鼠缺血/再灌注+MA预处理+溶酶体抑制剂组(I/R+MA+B afA1组)。大鼠I/R模型构建方法和MA预处理同第二部分,I/R+MA+BafA1组大鼠在手术前48 h腹腔注射20 mg/kg MA和1 mg/kg BafA1,并在手术前24 h重复注射。利用分子对接技术研究MA与溶酶体膜相关蛋白2(lysosomal associated membrane protein 2,LAMP2)之间关系。采用 Western blot检测大鼠心肌自噬相关蛋白(LAMP2、Beclin-1、P62、LC3-I及LC3-Ⅱ)、凋亡相关蛋白(Caspase-3和Caspase-9)及程序性坏死相关蛋白(RIPK1、RIPK3及MLKL)表达量或活化水平。结果1.与Sham+DMSO组相比,大鼠I/R+DMSO组中自噬相关蛋白Beclin-1、P62及LC3-Ⅱ表达水平显著增加,而LAMP2表达显著降低;与I/R+DMSO相比,I/R+MA组中的LC3-Ⅱ和LAMP2表达水平显著增加,而Beclin-1和P62表达显著降低;在联合应用了溶酶体抑制剂BafA1后,与I/R+MA组相比,I/R+MA+BafA1组中的LC3-Ⅱ和LAMP2表达水平显著降低,而Beclin-1和P62表达显著增加。提示MA的应用可以增加LAMP2膜蛋白的数量,保护溶酶体免受I/R损伤的影响,并进一步促进自噬通量。2.分子对接结果显示,LAMP2蛋白和MA分子的结合能值为-5.98 kcal/mol,表明该结合显著且稳定,提示LAMP2蛋白可能是MA潜在的治疗靶点。3.与Sham+DMSO组相比,大鼠I/R+DMSO组中凋亡相关蛋白(cleaved Caspase-3 和 cleaved Caspase-9)及程序性坏死相关蛋白(p-RIPK1、p-RIPK3 及p-MLKL)表达水平显著增加;与I/R+DMSO组对比,I/R+MA组中凋亡相关蛋白(cleaved Caspase-3 和 cleaved Caspase-9)及程序性坏死相关蛋白(p-RIPK1、p-RIPK3及p-MLKL)含量显著减少;在联合应用了溶酶体抑制剂BafA1后,与I/R+MA 组相比,I/R+MA+B afA1 组中凋亡相关蛋白(cleaved Caspase-3 和 cleaved Caspase-9)及程序性坏死相关蛋白(p-RIPK1、p-RIPK3及p-MLKL)表达水平显著上升。结论MA预处理可通过靶向结合LAMP2以保护溶酶体功能进而促进自噬活性,并抑制凋亡和程序性坏死的发生,发挥心肌保护作用。更多还原

【Abstract】 Part 1:Effects of MA pretreatment on hypoxia/reoxygenation injury of H9c2 cardiomyocytesObjectiveTo investigate the toxic effects of different concentrations of maslinic acid(MA)on H9c2 cardiomyocytes and its effect on hypoxia/reoxygenation(H/R)injury.MethodsH9c2 cardiomyocytes were cultured in vitro.To investigate the toxic effects of MA on H9c2 cardiomyocytes,H9c2 cells were treated with different concentrations of MA(0,1,5,10,20 and 40μM)for 24h;The effects of MA pretreatment on H/R injury of H9c2 cardiomyocytes were divided into groups:control group,hypoxia/reoxygenation group(H/R group)and hypoxia/reoxygenation+MA pretreatment group(H/R+MA group).The H/R model was established by hypoxia 4h and reoxygenation 4h.The concentrations of MA were divided into 1,5,10,20 and 40μM.The cell viability was detected by CCK-8 kit;LDH kit was used to detect the level of lactic dehydrogenase(LDH)in supernatant;The apoptosis level of H9c2 cardiomyocyte was detected by TUNEL method;The DAPI/PI double staining was used to detect the changes of necrosis rate to H9c2 cardiomyocyte;Western blot was used to detect the expression levels of apoptosis related proteins(Caspase-3,Caspase-9,Bcl-2 and Bax)and necroptosis related proteins(RIPK1,RIPK3 and MLKL).Results1.Different concentrations of MA pretreatment had no significant toxic effect on the viability of H9c2 cardiomyocytes.2.Compared with control group,the cell viability of H/R group was decreased and the level of LDH was increased;Compared with H/R group,cell viability was increased and LDH level were decreased significantly with dose-dependent manner in the H/R+MA(5,10 and 20μM)groups;When the MA dose was up to 40μM,there were no significant compared with 20μM group.Therefore,we selected MA with concentrations of 5,10 and 20μM in the follow-up experiment.3.Compared with control group,the H9c2 cell apoptosis in H/R group was significantly higher;Compared with H/R group,the H9c2 cell apoptosis in H/R+MA group was decreased significantly with dose-dependent manner.Compared with control group,the expression levels of apoptosis related proteins Bax,cleaved Caspase-3 and cleaved Caspase-9 in H9c2 cardiomyocytes in H/R group were increased,while the expression of Bcl-2 was decreased significantly;Compared with H/R group,the expression levels of apoptosis related proteins Bax,cleaved Caspase-3 and cleaved Caspase-9 in H/R+MA group were decreased,while the expression of Bcl-2 was increased significantly,which was dose-dependent with MA.4.Compared with control group,the cell necrosis rate in H/R group was significantly increased;Compared with H/R group,the cell necrosis rate in H/R+MA group was decreased significantly with dose-dependent manner.Compared with control group,H/R injury significantly increased the expressions of p-RIPK1,p-RIPK3 and p-MLKL proteins in H9c2 cardiomyocytes;Compared with H/R group,MA pretreatment in H/R+MA group could significantly down-regulate the expressions of p-RIPK1,p-RIPK3 and p-MLKL proteins with dose-dependent manner.ConclusionThe protective effect of MA pretreatment on H/R injury of H9c2 cardiomyocytes is dose-dependent and the mechanism may be related to the inhibition of apoptosis and necroptosis.Part 2:Effects of MA pretreatment on myocardial I/R injury in rats ObjectiveTo investigate the protective effects of MA pretreatment on myocardial ischemia/reperfusion(I/R)injury in rats and confirm its mechanism.MethodsIn total,48 SPF healthy adult male SD rats were randomized to four groups(n=12)with random number table method:control group(Sham+DMSO group),control+MA pretreatment group(Sham+MA group),rat ischemia/reperfusion group(I/R+DMSO group)and rat ischemia/reperfusion+MA pretreatment group(I/R+MA group).The left anterior descending coronary artery ligation was performed to induce myocardial ischemia for 30min,and the ligature was loosened for reperfusion 180min to construct the I/R model.Rats in MA pretreatment group were injected with 20mg/kg MA intraperitoneally 48h before operation,and repeated 24h before operation.HE staining was used to observe the morphological changes of myocardial tissue;TTC staining was used to detect the infarct size of myocardial tissue;LDH and CK-MB kits were used to detect the contents of LDH and CK-MB in rat serum;α-actinin+TUNEL fluorescent staining were used to detect the level of cardiomyocyte apoptosis induced by I/R;The expression level of necroptosis associated protein MLKL was detected by immunohistochemistry;The expressions of apoptosis related proteins(Caspase-3 and Caspase-9)and necroptosis related proteins(RIPK1,RIPK3 and MLKL)were detected by Western blot.Results1.Compared with Sham+DMSO group,the morphological structure of myocardium in I/R+DMSO group changed significantly,while levels of LDH and CK-MB in serum were significantly increased,and the area of myocardial infarction was significantly increased;Compared with I/R+DMSO group,the changes of myocardium morphological structure in I/R+MA group was reduced,the levels of LDH and CK-MB in serum were decreased,and the area of myocardial infarction was decreased.2.Compared with Sham+DMSO group,TUNEL positive cells in I/R+DMSO group were significantly increased.It would also be found that TUNEL positive cells not only appeared in cardiomyocytes,but also between cardiomyocytes,suggesting that I/R-induced apoptosis occurred in cardiomyocytes,endothelial cells,fibroblasts and other cardiac tissue cells.Compared with I/R+DMSO group,TUNEL positive cells in I/R+MA group was decreased.Western blot showed that the expression levels of apoptosis related proteins cleaved Caspase-3 and cleaved Caspase-9 in I/R+DMSO group were higher than Sham+DMSO group;Compared with I/R+DMSO group,the expression levels of apoptosis related proteins cleaved Caspase-3 and cleaved Caspase-9 were decreased in I/R+MA group.3.Immunohistochemical results showed that the expression of MLKL protein in I/R+DMSO group was increased compared with Sham+DMSO group;Compared with I/R+DMSO group,the expression of MLKL protein was decreased in I/R+MA group.Western blot showed that compared with Sham+DMSO group,the expressions of p-RIPK1,p-RIPK3 and p-MLKL proteins in I/R+DMSO group were increased;Compared with I/R+DMSO group,the expressions of p-RIPK1,p-RIPK3 and p-MLKL proteins in I/R+MA group were decreased.ConclusionMA pretreatment can play a protective role on myocardial ischemia reperfusion injury(MIRI)by inhibiting apoptosis and necroptosis signalling pathways simultaneously.Part 3:The role of lysosomal autophagy in the protective effect of MA pretreatment on MIRIObjectiveTo explore the role and mechanism of lysosomal autophagy in myocardial I/R-induced apoptosis and necroptosis,and to observe the effect of MA pretreatment on it.MethodsA total of 48 SPF healthy adult male SD rats were randomized to four groups(n=12)with random number table method:control group(Sham+DMSO group),rat ischemia/reperfusion group(I/R+DMSO group),rat ischemia/reperfusion+MA pretreatment group(I/R+MA group)and rat ischemia/reperfusion+MA pretreatment+lysosomal inhibitor group(I/R+MA+BafAl group).The I/R model and MA pretreatment group were the same as the second part.Rats in lysosomal inhibitor group were injected with 20mg/kg MA and lmg/kg BafAl intraperitoneally 48h before operation,and repeated 24h before operation.The relationship between MA and lysosomal associated membrane protein 2(LAMP2)protein was studied by AutoDock software.Western blot was used to detect the expressions of autophagy related proteins(LAMP2,Beclin-1,P62,LC3-Ⅰ and LC3-Ⅱ),apoptosis related proteins(Caspase-3 and Caspase-9)and necroptosis related proteins(RIPK1,RIPK3 and MLKL).Results1.Compared with Sham+DMSO group,the expressions of autophagy related proteins Beclin-1,P62 and LC3-Ⅱ in I/R+DMSO group were increased,while the expression of LAMP2 was decreased;Compared with I/R+DMSO,the expressions of LC3-II and LAMP2 in I/R+MA group were increased,while the expression levels of Beclin-1 and P62 were decreased;After combined application of lysosomal inhibitor BafA1,compared with I/R+MA group,the expression levels of LC3-Ⅱ and LAMP2 in I/R+MA+BafAl group were decreased,while the expression levels of Beclin-1 and P62 were increased.It was suggested that the application of MA would increase the number of LAMP2,protect lysosomes from I/R,and further promote autophagy flux.2.The results of molecular docking showed that the binding energy value of LAMP2 and MA was-5.98kcal/mol,indicating that the binding was significant and stable.It suggested that LAMP2 might be a potential therapeutic target of MA.3.Compared with Sham+DMSO group,the expressions of apoptosis related proteins(cleaved Caspase-3 and cleaved Caspase-9)and necroptosis related proteins(p-RIPK1,p-RIPK3 and p-MLKL)in I/R+DMSO group were increased;Compared with I/R+DMSO group,the expressions of apoptosis related proteins(cleaved Caspase-3 and cleaved Caspase-9)and necroptosis related proteins(p-RIPK1,p-RIPK3 and p-MLKL)in I/R+MA group were reduced;After combined application of lysosomal inhibitor BafAl,compared with I/R+MA group,apoptosis related proteins(cleaved Caspase-3 and cleaved Caspase-9)and necroptosis related proteins(p-RIPK1,p-RIPK3 and p-MLKL)were increased in I/R+MA+BafA1 group.ConclusionThe protective effect of MA pretreatment on MIRI may be related to the inhibition of apoptosis and necroptosis via protecting lysosomal function and promoting autophagic flux.更多还原