目的 旨在阐明芪参颗粒(Qishen Granules，QSG)调控线粒体自噬，干预炎症小体活化抗心肌细胞损伤的作用机制。方法 培养HL-1心肌细胞，通过血管紧张素Ⅱ(Angiotensin Ⅱ，AngⅡ)诱导心肌细胞损伤模型，将细胞分为对照组、模型组，QSG 2。5％、5％、10％组，QSG组分别给予2。5％、5％、10％QSG含药血清MEM培养基培养细胞，对照组及模型组给予10％普通大鼠血清MEM培养基培养，培养6 h后，每组细胞加入150 nmol/L Ang Ⅱ孵育24 h。采用CCK8法检测QSG含药血清对HL-1细胞活力的影响，采用比色法测定各组乳酸脱氢酶(Lactate Dehydrogenase，LDH)、丙二醛(Malondialdehyde，MDA)和超氧化物歧化酶(Super Oxide Dismutase，SOD)水平，Western Blot 法检测 QSG 含药血清对HL-1细胞NOD样受体蛋白3(NOD-like receptor protein 3，NLRP3)炎症小体活化以及线粒体自噬水平的影响，检测NLRP3 及半胱氨酸天冬氨酸特异性蛋白酶-1(Cysteinyl Aspartate Specific Protease-1，Caspase-1)、白细胞介素-1β(Interleukin-1 beta，IL-1β)蛋白表达水平及 PTEN 诱导激酶 1(PTEN-induced putative kinase 1，PINK1)、帕金森蛋白2，E3 泛素蛋白连接酶(Parkinson Protein 2，E3 Ubiquitin Protein Ligase，Parkin)、隔离蛋白 1(Sequestosome-1，P62)蛋白表达水平，免疫荧光法观察各组心肌细胞NLRP3及微管相关蛋白1轻链3(Microtubule-Associated Protein 1 Light Chain 3，LC3)表达情况。结果 与模型组比较，QSG含药血清各剂量组细胞活力显著增强(P＜0。01，P＜0。05);与假手术组相比，模型组LDH、MDA水平显著升高，SOD水平显著降低(P＜0。01)，与模型组比较，QSG含药血清各剂量组LDH及MDA表达显著降低，SOD水平显著升高(P＜0。01或P＜0。05);与对照组比较，模型组NLRP3、Caspase-1、IL-1β、P62蛋白表达水平显著升高(P＜0。01)，PINK1、Parkin蛋白表达水平显著降低(P＜0。01)，与模型组比较，QSG含药血清各剂量组NLRP3、Caspase-1、IL-1β、P62蛋白表达水平显著降低(P＜0。01或P＜0。05)，PINK1、Parkin蛋白表达水平显著升高(P＜0。01或P＜0。05)。荧光染色结果显示，与对照组比较，模型组心肌细胞内NLRP3及LC3表达升高，与模型组比较，QSG组心肌细胞内NLRP3、LC3表达水平降低。结论 QSG能有效保护心肌细胞损伤，其作用机制可能是通过调控线粒体自噬干预炎症小体活化实现的。
Mechanism of Qishen Granules(芪参颗粒)Regulating Mitophagy and Intervening in Inflammasome Activation to Resist Cardiomyocyte Injury
Objective To elucidate the mechanism by which Qishen Granules(芪参颗粒,QSG)regulate mitochondrial autoph-agy,intervene in inflammasome activation and counteract cardiomyocyte injury.Methods The experiment cultured HL-1 cardio-myocytes and induced cardiomyocyte injury model through angiotensin Ⅱ(AngⅡ).The cells were divided into control group,mod-el group,QSG 2.5％,5％and 10％groups.The QSG group was cultured in serum MEM medium with 2.5％,5％and 10％QSG respectively,while the control group and model group were cultured in 10％ordinary rat serum MEM medium.After 6 hours of culture,the cells in each group were added with 150 nmol/L AngⅡ and incubated for 24 hours.The CCK8 method was used to detect the effect of QSG-containing serum on the viability of HL-1 cells.The colorimetric method was used to determine lac-tate dehydrogenase(LDH),malondialdehyde(MDA)and super oxide dismutase(SOD)in each group,and the Western Blot method was used to detect the effect of QSG-containing serum on HL-1 cells NLRP3 inflammation.The effects of body activa-tion and mitophagy levels were detected,and the protein expression levels of NOD-like receptor protein 3(NLRP3),Cysteinyl Aspartate Specific Protease-1(Caspase-1),Interleukin-1 beta(IL-1β),and PTEN-induced putative kinase 1(PINK1),Parkinson Protein 2,E3 Ubiquitin Protein Ligase(Parkin)and Sequestosome-1(P62)proteins were detected.The expressions of NLRP3 and LC3 in cardiomyocytes in each group was observed by immunofluorescence method.Results The results studies show that compared with that of the model group,the cell viability of each dose group of QSG-containing serum was significantly enhanced(P＜0.01,P＜0.05).Compared with those of the sham operation group,the levels of LDH and MDA in the model group were significantly increased,and the level of SOD was significantly decreased(P＜0.01).Compared with those of the mod-el group,the expressions of LDH and MDA in each dose group of QSG-containing serum were significantly reduced,and the SOD level was significantly increased(P＜0.01,P＜0.05).Compared with those of the control group,the protein expression levels of NLRP3,Caspase-1,IL-1β and P62 in the model group were significantly increased(P＜0.01),and the protein expression lev-els of PINK1 and Parkin were significantly decreased(P＜0.01).Compared with those of the model group,the protein expression levels of NLRP3,Caspase-1,IL-1β and P62 in each dose of QSG-containing serum group were significantly reduced(P＜0.01,P＜0.05),and the protein expression levels of PINK1 and Parkin were significantly increased(P＜0.01,P＜0.05).Fluo-rescent staining results showed that compared with those of the control group,the expression levels of NLRP3 and LC3 in the car-diomyocytes of the model group increased.Compared with those of the model group,the expression levels of NLRP3 and LC3 in the cardiomyocytes of the QSG-containing serum groups decreased.Conclusion Qishen Granules can effectively protect cardio-myocyte injury,and its mechanism of action may be to interfere with inflammasome activation by regulating mitophagy.
万方数据
维普
