延胡索乙素通过ULK1/FUNDC1通路抑制线粒体自噬减轻H9c2心肌细胞缺氧/复氧损伤

Tetrahydropalmatine inhibiting mitophagy through ULK1/FUNDC1 pathway to alleviate hypoxia/reoxygenation injury in H9c2 cells

该研究探讨了延胡索乙素(tetrahydropalmatine,THP)通过UNC-51样激酶1(UNC-51-like kinase 1,ULK1)/FUN14结构域蛋白1(FUN14 domain containing 1,FUNDC1)通路抑制线粒体自噬减轻H9c2心肌细胞缺氧/复氧(hypoxia/reoxygenation,H/R)损伤的具体机制。该研究以H9c2心肌细胞为研究对象,构建心肌细胞H/R损伤模型。首先采用细胞活力检测试剂盒检测细胞活性、微量法检测乳酸脱氢酶(lactate dehydrogenase,LDH)漏出量评估THP对H9c2心肌细胞H/R损伤的保护作用。然后采用化学荧光法检测细胞内活性氧、线粒体内活性氧、线粒体膜电位、自噬小体情况,并用萤光素法检测细胞内三磷酸腺苷(adenosine 5′-triphosphate,ATP)含量评估THP对线粒体的保护作用。进一步用免疫印记检测微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)膜型(LC3-Ⅱ)和包浆型(LC3-Ⅰ)的比值,活化的半胱氨酸天冬氨酸蛋白水解酶-3(cleaved caspase-3)表达水平、ULK1表达水平及其在Ser555位点的磷酸化程度,FUNDC1表达水平及其在Ser17位点的磷酸化程度探索其具体机制。结果显示,THP有效减轻H9c2细胞H/R后线粒体损伤,THP通过降低细胞内和线粒体内活性氧水平、升高线粒体膜电位保护线粒体,进而增加细胞ATP生成、升高细胞活性、降低细胞LDH漏出,减轻H9c2细胞H/R损伤;进一步研究发现THP能够降低自噬小体生成,降低LC3-Ⅱ/LC3-Ⅰ比值,降低凋亡相关蛋白cleaved caspase-3表达,表明THP通过抑制自噬减轻细胞凋亡;深入研究发现THP通过降低ULK1在Ser555位点和FUNDC1在Ser17位点的磷酸化程度,抑制线粒体自噬ULK1/FUNDC1通路激活,抑制线粒体自噬发生。ULK1激动剂BL-918的应用逆向验证了THP降低ULK1和FUNDC1磷酸化的作用。综上所述,THP通过ULK1/FUNDC1通路抑制线粒体自噬减轻H9c2心肌细胞H/R损伤。

This study explored the specific mechanism by which tetrahydropalmatine(THP)inhibited mitophagy through the UNC-51-like kinase 1(ULK1)/FUN14 domain containing 1(FUNDC1)pathway to reduce hypoxia/reoxygenation(H/R)injury in H9c2 cells.This study used H9c2 cells as the research object to construct a cardiomyocyte H/R injury model.First,a cell viability detection kit was used to detect cell viability,and a micro-method was used to detect lactate dehydrogenase(LDH)leakage to evaluate the protective effect of THP on H/R injury of H9c2 cells.In order to evaluate the protective effect of THP on mitochondria,the chemical fluorescence method was used to detect intracellular reactive oxygen species,intramitochondrial reactive oxygen species,mitochondrial membrane potential,and autophagosomes,and the luciferin method was used to detect intracellular adenosine 5′-triphosphate(ATP)content.Western blot was further used to detect the ratio of microtubule-associated protein 1 light chain 3(LC3)membrane type(LC3-Ⅱ)and slurry type(LC3-Ⅰ)and activated cleaved caspase-3 expression level.In addition,ULK1 expression level and its phosphorylation degree at Ser555 site,as well as the FUNDC1 expression level and its phosphorylation degree of Ser17 site were detected to explore its specific mechanism.The results showed that THP effectively reduced mitochondrial damage in H9c2 cells after H/R.THP protected mitochondria by reducing the level of reactive oxygen species in cells and mitochondria,increasing mitochondrial membrane potential,thereby increasing cellular ATP production,enhancing cellular activity,reducing cellular LDH leakage,and finally alleviating H/R damage in H9c2 cells.Further studies have found that THP could reduce the production of autophagosomes,reduce the LC3-Ⅱ/LC3-Ⅰratio,and lower the expression of the apoptosis-related protein,namely cleaved caspase-3,indicating that THP could reduce apoptosis by inhibiting autophagy.In-depth studies have found that THP could inhibit the activation of the ULK1/FUNDC1 pathway of mitophagy and the occurrence of mitophagy by reducing the phosphorylation degree of ULK1 at Ser555 and FUNDC1 at Ser17.The application of ULK1 agonist BL-918 reversely verified the effect of THP on reducing the phosphorylation of ULK1 and FUNDC1.In summary,THP inhibited mitophagy through the ULK1/FUNDC1 pathway to reduce H/R injury in H9c2 cells.