Aconitine Activates Sirt3 To Improve Mitochondrial Function In Cardiomyocytes

Aims:Fuzi is a processed product of the roots of Aconitum and belongs to the Ranunculus,which is a commonly used drug in traditional Chinese medicine.Shenfu injection with Fuzi as its main component is clinically used for the treatment of cardiovascular diseases.Aconitine as a major active ingredient in aconite and Shenfu injection has cardiovascular protection.Clinical studies have shown that myocardial energy metabolism is disordered in cardiac dysfunction,and ATP production is insufficient,leading to ventricular remodeling and aggravating myocardial damage.Therefore,improving cardiac energy metabolism is a new strategy for the treatment of cardiovascular disease.Studies have shown that aconitine can protect the ischemic myocardium by regulating energy metabolism and signal transduction of ischemic myocardium.However,the mechanism of the aconitine produces myocardial protection by improving energy metabolism has not been elucidated.This study explored the pharmacological effects of aconitine from myocardial protection and its possible mechanism of action from the perspective of energy metabolism.The aim of this study was to investigate the mechanism of the aconitine produces cardioprotection from the perspective of energy metabolism.Methods:?1?H9c2 cells were treated with different doses of Ang II and aconitine.The cell viability and concentrations of the Ang II and aconitine were determined using the CCK-8 assay.The mRNA and protein expressions of related molecules were measured using quantitative PCR and western-blot.?2?The effect of aconitine on the expressions of F-actin and Sirt3 wered detected by immunofluorescence assay.?3?The effect of aconitine on mitochondrial ultrastructure in H9c2 cells was detected by transmission electron microscope.?4?The effect of aconitine on mPTP and NAD⁺/NADH were detected by luciferase method.?5?The influence of aconitine on mitochondrial membrane potential was detected by flow cytometry.?6?The effect of aconitine on mitochondrial oxidative phosphorylation was determined using the Seahorse technology.?7?The effect of aconitine on the acetylation of Cyp-D and the interaction between Cyp-D and ANT-1 were detected using immunoprecipitation technique.?8?The Sirt3 siRNA was transfected into H9c2 cells to knockdown the expression of Sirt3.The effect of aconitine on mitochondrial oxidative phosphoric acid level was detected by Seahorse technology after siRNA transfection.The effect of aconitine on the acetylation of Cyp-D and the interaction between cyp-d and ant-1 were detected by immunoprecipitation technology after Sirt3 knocking down.?9?The TAC method was applied to simulate the heart failure rat model.The HF rats were oral administration of aconitine for two weeks.The serum and heart were separated after the last administration.The biochemical indexesa,pathological analysis of organs and the contents of ATP in rats were observed and detected.Results:?1?The cell viability showed that Ang II(1-100?mol?L^-1)and aconitine(0.1-100nmol?L^-1)had no significant damage to H9c2cells.Aconitine has an anti-cardiac hypertrophy effect by reducing the synthesis of total proteins and myofibrils in cardiac mast cells.?2?The results of transmission electron microscopy signified that aconitine could protect the ultrastructure of mitochondria in cardiac myocytes;The results of flow cytometry showed that aconitine promoted mitochondrial membrane potential in myocardial mast cells.?3?The expression of Sirt3increased after aconitine treatment.Aconitine promoted deacetylation of Sirt3 to reduce the degree of acetylation of cyp-d and the binding of cyp-d to ANT-1,and inhibit the opening of mPTP.?4?The mitochondrial stress results showed that aconitine increased the oxidative phosphorylation level of H9c2 cells in a dose-dependent manner.The aconitine promoted the expression of mitochondrial respiratory chain complex I,III and V.?5?The inhibitory effect of aconitine on Cyp-D acetylation disappeared when Sirt3expression was knocked down.?6?The serum biochemical test results showed that the levels of cTn,CK,AST and LDH in the aconitine group were significantly lower than those in the TAC group,especially in the high-dose group.The results of cardiac pathological examination showed that aconitine improved the swelling,degeneration and inflammatory infiltration of cardiac tissue in HF rats group.The results of transmission electron microscopy showed that aconitine increased the contents of mitochondria in heart failure rats and improved the phenomenon of mitochondrial rupture and vacuolation.Conclusion:This study finds that aconitine improves energy metabolism disorder by regulating mitochondrial function for the first time.Furthermore,the protection mentioned above is closely related to the activation of Sirt3.The molecular biology technology is used to investigate indicators related to mitochondrial function to verify whether the protective effect of aconitine on cardiac myocytes is related to the improvement of mitochondrial structure and function.The results demonstrates that the increase of mitochondrial membrane potential,the protection of mitochondrial ultrastructure,inhibition of mPTP opening,activation of Sirt3 are related to aconitine.Aconitine enhancs mitochondrial oxidative phosphorylation in a dose dependent manner.Sirt3siRNA transfection results display that the inhibition of mPTP opening and promotion of oxidative phosphorylation disappeared after aconitine treatment.The results indentified the relationship between Sirt3 activation and the inhibition of mPTP opening by aconitine.The activation of Sirt3by aconitine is an important reason for improving mitochondrial energy metabolism.The mechanism may be related the role of Sirt3 in deacetylation.Sirt3 reduces the degree of acetylation of Cyp-D and inhibits the opening of mPTP.The results showed that Sirt3 plays an importany role in the mitochondrial energy metabolism improvemnt of aconitine.In vivo tests verifies that aconitine has good protective effect on myocardium and mitochondria.