Luteolin Inhibits FUNDC1/BNIP3/BNIP3L Mitochondrial Autophagy Pathway And Reduces LPS-induced H9c2 Cardiomyocyte Inflammation

Objective:To study the protective effect of luteolin on LPS-induced H9c2 cardiomyocyte injury,and to explore the mechanism of mitochondrial autophagy pathway.Methods:(1)To determine the optimal concentration of LPS and treatment time of H9c2 cell injury induced by LPS,and then to detect cell mitochondrial autophagy protein LC3,expression levels of P62 and Beclin1.In order to determine the changes in mitochondrial autophagy level of LPS,mitochondrial autophagy inhibitor CsA was used to observe cardiomyocytes on mitochondrial autophagy level;cell survival rating was detected by CCK8,cell leakage rating was detected by LDH,and myocardium was detected by CK.The degree of injury was measured by qRT-PCR to detect the level of cell inflammation factors,flow cytometry was used to detect the apoptosis rate and mitochondrial membrane potential,and the expression levels of receptor-mediated mitochondrial autophagy proteins FUNDC1,BNIP3 and BNIP3 L were detected by Western blotting.(2)In order to observe the effect of luteolin on LPS-induced H9c2 cardiomyocyte injury,except these methods,the mitochondrial autophagy fluorescent marker Mtphagy Dye was used to detect the occurrence of mitochondrial autophagy,and the mitochondrial autophagy was detected by cell inflammation factors technology.Expression of phage protein FUNDC1.(3)In order to determine that luteolin plays a protective role in mitochondrial autophagy,to observe the effect of luteolin combined with mitochondrial autophagy agonist CCCP on LPS-induced H9c2 cardiomyocyte injury;in addition to the above detection methods,it is detected by cell inflammation factors technology expression of mitochondrial autophagy protein FUNDC1.Result:(1)Compared with the control group,The expression of mitochondrial autophagy proteins LC3Ⅱ and Beclin1 in H9c2 cardiomyocytes was significantly increased,p62 expression was significantly reduced;Compared with the LPS group,after treatment with the mitochondrial autophagy inhibitor CsA,the cell survival rating was significantly increased,the LDH leakage rating was significantly reduced,and the cell apoptosis rating was significantly reduced.The mitochondrial membrane potential increased,and the receptor-mediated mitochondrial autophagy protein FUNDC1 was relatively increased,and the expression of BNIP3 and BNIP3L proteins was decreased.(2)Compared with the LPS injury group,luteolin can also increase the survival rate of H9c2 cardiomyocytes after LPS induction,significantly reduce the LDH leakage rating,CK value and apoptosis rate of H9c2 cells treated with LPS,and increase mitochondrial membrane potential.At the same time,the receptor-mediated mitochondrial autophagy protein FUNDC1 increased and the expression of BNIP3 and BNIP3 L protein decreased after luteolin treated together,and mitochondrial autophagy fluorescent markers showed that autophagy was significantly increased in the LPS group,and mitochondrial autophagy fluorescence is significantly reduced after treating.(3)The autophagy agonist CCCP significantly increased the induction of LPS on H9c2 cells.However,luteolin can significantly improve the effect of mitochondrial autophagy agonist CCCP.Luteolin relatively increased the cell survival rating after co-induction of LPS and CCCP,relatively decreased the LDH leakage rating,CK value and apoptotic rate of cells after co-induction of LPS and CCCP on H9c2 cells,and increased mitochondrial membrane potential.At the same time,the receptor-mediated mitochondrial autophagy protein FUNDC1 was relatively increased,and the expression of BNIP3 and BNIP3 L proteins was relatively decreased.Conclusion:Luteolin can improve the damage of H9c2 cells induced by LPS,and its mechanism may be related to the inhibition of FUNDC1/ BNIP3/ BNIP3 L mitochondrial autophagy pathway.