Applied Basic Study On The Effect Of 7,8-dihydroxyflavone On Rotenone-induced Myocardial Cell Injury

Objective:Brain-derived neurotrophic factor(BDNF)has been shown to regulate various biological processes by activating tyrosine kinase receptor B(TrkB)and its downstream signaling pathways such as PI3K/Akt and MAPK.Neurotrophic factor family members such as BDNF has a short half-life and is difficult to cross the blood-brain barrier,which limits their clinical application.7,8-dihydroxyflavone(7,8-DHF)is a flavonoid compound derived from plants,which can better mimic BDNF and penetrate the blood-brain barrier,and has promising effects on neurodegenerative diseases such as Parkinson’s disease.Mitochondrial dysfunction is closely related to the occurrence and development of cardiovascular diseases.Previous studies have shown that 7,8-DHF improves cardiac function and alleviates mitochondrial dysfunction caused by pathological stimulation.However,the direct regulatory effects of 7,8DHF on cardiomyocyte mitochondria remain unclear.Rotenone(Rot),a commonly used pesticide in agriculture,is widely used as an inhibitor of mitochondrial respiratory electron transport chain(ETC)complex I,blocking mitochondrial electron transport and resulting in mitochondrial function inhibition.The purpose of this study was to investigate the effect of 7,8-DHF on Rot-induced mitochondrial dysfunction of cardiomyocytes and its underlying mechanisms,and to provide new ideas for the development of cardioprotective drugs.Methods:This study is mainly divided into three parts:In the first part,the in vitro model of Rot-induced mitochondrial dysfunction of H9c2 cardiomyocytes was established,and the effects of Rot on myocardial cell injury and survival were observed by light microscope.LDH kit and LIVE/DEAD staining were used to determine the optimal concentration of Rot-induced myocardial cell injury model and the effective concentration of 7,8-DHF to inhibit Rot-induced myocardial cell injury.Mitochondrial membrane potential and ROS levels were detected by JC-1 and Mito-SOX staining.The changes of mitochondrial respiratory function were detected by a high-resolution mitochondrial respiratory measurement system,and changes of mitochondrial respiratory oxygen consumption rate(OCR),basic respiratory level(Routine),maximum mitochondrial respiratory(MMR),reserve respiratory capacity(RRC)and leak respiratory(Leak)were examined.Mitochondrial complex protein expression was detected by Western Blot.ATP and pyruvate kinase(PK)kits were used to detect energy metabolism of H9c2 cardiomyocytes.In the second part,Rot-induced mitochondrial dysfunction of HL-1 cardiomyocytes was established.The effects of 7,8-DHF on morphology,LDH release and ratio of dead cells of HL-1 cardiomyocytes treated with different Rot concentrations were detected by light microscopy,LDH kit and LIVE/DEAD staining to verify the protective effect of 7,8-DHF on Rot-induced cardiomyocyte damage.Then,mito-SOX probe was used to observe the production of mitochondrial reactive oxygen species(ROS)in cardiomyocytes.Changes of mitochondrial respiratory OCR,Routine,MMR,RRC and Leak of HL-1 cardiomyocytes were detected.The protein expression of mitochondrial respiratory complex in HL-1 cardiomyocytes was detected by Western Blot.ATP levels in HL-1 cardiomyocytes were detected.In the third part,the mechanism of 7,8-DHF inhibiting Rot induced myocardial mitochondrial damage was discussed.Antioxidant NAC and mitochondria-targeting antioxidant Mito-TEMPO were used to clarify whether 7,8-DHF plays a protective role in inhibiting Rot induced myocardial injury through an antioxidant mechanism.Further,Western blotting was used to detect adenylate activated protein kinase(AMPK),signal transduction and transcription-activating protein 3(STAT3)phosphorylation and nuclear translocation,as well as protein expressions of Akt and p38MAPK,etc.,to explore the molecular mechanism of 7,8-DHF inhibiting the protective effect of Rot induced myocardial injury.Results:7,8-DHF could effectively inhibit Rot-induced cardiomyocyte injury and cell death,and reduce LDH release.7,8-DHF significantly improved Rot-induced mitochondrial membrane potential and inhibited mitochondrial ROS levels.Acute administration of 7,8-DHF increased basal respiration and leak respiration of H9c2 cardiomyocytes.7,8-DHF incubation for 24 h reversed Rot-induced mitochondrial leak respiratory values of H9c2 cardiomyocytes,and had no significant effect on mitochondrial MMR and RRC after Rot treatment.7,8-DHF reverses protein expression of mitochondrial complex Ⅰ-Ⅳ in Rot-treated cardiomyocytes.7,8DHF increases Rot-induced ATP and PK levels in H9e2 cells.The role of 7,8-DHF in Rotinduced HL-1 cell models was consistent with that in H9c2 cells.Neither NAC nor MitoTEMPO improved Rot-induced cardiomyocyte damage.7,8-DHF inhibits AMPK phosphorylation and induces nuclear translocation of p-STAT3-Y705 cardiomyocytes.In addition,Akt and p38MAPK signaling was activated by 7,8-DHF intervention.Conclusion:7,8-DHF can effectively ameliorate Rot induced myocardial cell injury and mitochondrial dysfunction.The protective effect of 7,8-DHF May be related to the regulation of Akt,p38MAPK and the promotion of nuclear translocation of p-STAT3-Y705 by AMPK signal.This study is the first to identify 7,8-DHF as a potential compound for the prevention and treatment of mitochondrial dysfunction in heart disease.