Study On The Mechanism Of Xin Fu Kang Oral Liquid Regulating Mitochondrial Energy Metabolism Homeostasis In Mice With Heart Failure

IntroductionIn recent years,the role of mitochondrial dysfunction or mitochondrial damage in the pathogenesis of heart failure(HF)has gradually attracted attention.Adenosine triphosphate produced by oxidative phosphorylation of mitochondria is an important source of energy to meet the electrical activity,contraction activity and ion pump activity of cardiomyocytes,and mitochondria is also used as the main organ of reactive oxygen species production,participating in the regulation of calcium homeostasis and ion channel opening in cardiomyocytes.Imbalance of myocardial energy metabolism homeostasis is one of the important mechanisms of HF,and during HF,mitochondrial energy metabolism homeostasis is broken,resulting in increased ROS in cardiomyocytes,mitochondrial structural damage,energy depletion,etc.The "energy exhaustion theory" has become a hot issue in the field of international HF research,highlighting the key role of mitochondrial energy metabolism in the treatment of HF.Traditional Chinese medicine attaches great importance to the overall concept and dialectical treatment,and the compound of traditional Chinese medicine contains a variety of active ingredients,which has the characteristics of multiple targets and multiple pathways,and has shown great advantages in the prevention and treatment of HF and other diseases,so it has become an important field to find new ways to treat HF.ObjectBased on the theory of "invigorating qi and activating blood",XFK oral liquid is a classic compound for the clinical treatment of HF with qi deficiency and blood stasis.Previous studies have fully demonstrated that XFK oral liquid has a significant effect on improving ventricular remodeling in rats with HF.In this study,a mouse model of CHF was constructed by Transverse aortic constriction(TAC)to construct a mouse model of CHF by taking mitochondrial energy metabolism homeostasis as the starting point,combined with PE-induced hypertrophic ventricular myocytes model,and evaluating the efficacy of XFK oral liquid in regulating mitochondrial energy metabolism homeostasis from in vivo and in vitro experiments.Small molecule interference RNA technology was used to silence gene expression and elucidate the molecular mechanism and effector target of XFK oral liquid.Methods1.Evaluation of the efficacy of XFK oral liquid in improving ventricular remodeling in CHF mice:180 8-week-old male C57/BL6J mice with SPF-level were divided into blank control group(Sham)and model group,and a mouse CHF model was established by 27G needle ligation of the aorta by TAC surgery,and the Sham group was not ligated after winding.The mice were randomly divided into model group,high dose of XFK oral liquid(XFKH),middle dose of XFK oral liquid(XFHM),low dose of XFK oral liquid(XFKL)and empagliflozin group(EMPA),and the corresponding drugs were given gavage for 8 weeks.During the period,the survival and weight changes of mice in each group were recorded,the changes of serum BNP levels were detected by eye blood extraction,the ratio of heart weight to body weight was calculated,and cardiac hypertrophy was evaluated.Echocardiography measures systolic and diastolic function and measures changes in ventricular structure;HE staining to determine the pathological changes of myocardial tissue;Masson staining to determine the degree of myocardial fibrosis;WGA staining traces cardiomyocyte membranes to measure cardiomyocyte hypertrophy.2.Efficacy evaluation of XFK oral liquid in improving oxidative stress and calcium overload in CHF mice:ROS content was detected by cryosection,and serum SOD,MDA and GSH-PX activity levels were detected by Elisa.Langendorff technology was used to acute isolate mouse ventricular myocytes,and the level of mitochondrial superoxide was detected after Mito-SOX staining.Use the IonOptix single-cell contraction and ion synchronization detection system to track muscle segment length,sarcoplasmic reticulum calcium volume,and calcium leakage levels in real time.3.Evaluation of the efficacy of XFK oral liquid in improving mitochondrial energy metabolism in CHF mice:the ultrastructure of mitochondrial microscopy of myocardial tissue was observed by transmission electron microscopy;Immunofluorescence was used to detect mitochondrial membrane potential levels;The ATP content of ventricular muscle and the I-V activity level of mitochondrial respiratory chain complex were calculated spectrophotometrically,and the Seahorse XF 96 cellular energy metabolism instrument recorded the changes of mitochondrial aerobic respiratory function.Western-Blot and RT-PCR technology were used to determine the expression and gene expression of PHB1/2,the signature protein antiproliferative protein in the inner mitochondrial membrane,and the PHB1/2 was localized and displayed by immunohistochemistry.4.Study on the mechanism of XFK oral liquid on Improving Mitochondrial Energy Metabolism in Hypertrophic Ventricular Myocytes:Establishment of Hypertrophic Ventricular Myocyte Model in Vitro by PE Induction,Lyophilized Powder of XFK oral liquid,CCK-8 Method to Screen the Best Intervention Concentration of Drugs,Intracellular mitoflash and TMRM Colocalization Signal were observed after adenovirus AAV-mt-cpYFP transfection,and Flashsniper software was used to analyze mitoflash frequency.PHB1 and PHB2 siRNA silencing gene expression was treated by siRNA technology,and Western-Blot and RT-PCR technology were used to verify the transfection efficiency.MitoTracker Green staining was used to observe and measure the complete mitochondrial morphology of each group of cardiomyocytes and record the degree of mitochondrial fragmentation and mitochondrial length.The mitochondrial membrane potential level was evaluated by JC-1 staining.Immunofluorescence and laser confocal microscopy were used to observe the changes of intracellular ROS,mitochondrial superoxide levels and free Ca2+concentrations.The Seahorse XF 96 cellular energy metabolism analyzer was used to evaluate mitochondrial aerobic respiration and energy metabolism,and ATP production,basal respiration,maximal respiration,spare respiration,and mitochondrial proton leakage levels were recorded.The I-V activity of mitochondrial respiratory chain complexes and the openness of mPTP were measured by spectrophotometry.Results1.After 8 weeks of drug intervention,there was no significant difference in survival rate and body weight between the groups.The ratio of heart weight and heart weight in the Model group increased significantly,and the heart weight and heart weight ratio could be significantly reduced in each dose group of XFK oral liquid.Compared with the Sham group,BNP levels in the Model group were significantly increased,and the BNP levels in each drug group decreased to varying degrees.The echocardiography results showed that LVDs and LVDd were significantly reduced in each dose group of XFK oral liquid,and the regulatory effect on LVAW and LVPW was not obvious.XFK oral liquid significantly increased LVEF,LVFS,Ea,decreased E/Ea,and improved systolic and diastolic function.HE is staining showed that XFK oral liquid significantly improved the morphology of myocardial tissue and reduced the infiltration of inflammatory cells.Masson staining showed that XFK oral liquid reduced the degree of myocardial tissue fibrosis and reduced the number of collagen volumetric points.WGA staining results showed that cardiomyocyte hypertrophy in mice with HF could effectively inhibit cardiomyocyte hypertrophy in mice with HF.EMPA can also improve myocardial tissue morphology and reverse ventricular remodeling.2.The results of in situ ROS staining of frozen sections showed that compared with the Sham group,the fluorescence signal of myocardial ROS in the Model group was enhanced,and the level of ROS generation was significantly increased,and XFK oral liquid could significantly reduce ROS accumulation and ROS production level.The results of Mito-SOX staining by laser confocal technology showed that each dose group of XFK oral liquid could effectively reduce the level of superoxide in mitochondria.The serum ELISA test results showed that compared with the Sham group,the SOD and GSH-PX activities and MDA content in the Model group were significantly increased.The activities of SOD and GSH-PX were significantly upregulated in the XFKH group and XFKM group,and the MDA content was reduced.The above results suggest that XFK oral liquid has the effect of improving the oxidative stress state of CHF mice.The results of calcium transient and systolic function detection in a single ventricular myocyte showed that the XFKH group increased the amplitude and contraction fraction of myocardial calcium in CHF mice and shortened the recovery time of Tau and 50%.Compared with the Sham group,the calcium leakage in the Model group increased,and the calcium leakage level and sarcoplasmic reticulum calcium load in the XFKH group and XFKM group significantly reduced the calcium leakage level and sarcoplasmic reticulum calcium load in the CHF mice.EMPA can also significantly reduce ROS,mitochondrial superoxide levels,MDA content,upregulate SOD and GSH-PX activities,alleviate myocardial oxidative stress in CHF mice,reduce calcium leakage,and maintain calcium homeostasis in cardiomyocytes.3.The results of transmission electron microscopy showed that the mitochondrial structure damage was significantly improved,and the mitochondrial structure was stable in each dose group.The results of JC-1 staining showed that the mitochondrial membrane potential was lost in the Model group,and XFK oral liquid could restore the mitochondrial membrane potential and ensure the normal function of mitochondria.The spectrophotometric results showed that XFK oral liquid significantly increased the content of myocardial ATP and the activity of mitochondrial respiratory chain complex I-V in CHF mice,and maintained the normal function of ETC.Seahorse results showed that XFKH group significantly increased the maximum respiration rate and spare respiration capacity of cells,which optimized cellular energy metabolism.WesternBlot and immunohistochemical results showed that XFK oral liquid significantly upregulated the expression of PHB1 and PHB2 proteins.RT-PCR results showed that XFK oral liquid significantly upregulated PHB1 and PHB2 mRNA gene expression.EMPA also inhibits mitochondrial damage,protects mitochondrial function,improves ATP production,maintains respiratory chain activity,and ensures the normal progress of mitochondrial energy metabolism.4.CCK-8 results showed that 25mg/mL lyophilized powder of XFK oral liquid could significantly improve cardiomyocyte activity.The results of laser confocal and JC-1 staining experiments showed that the occurrence of mitoflash depended on the integrity of ETC,and with the loss of mitochondrial membrane potential and the abnormal opening of mPTP,XFK oral liquid could significantly reduce the frequency of mitoflash,restore mitochondrial membrane potential,turn off mPTP close to normal,and improve the I-V activity of mitochondrial respiratory chain complex.Western-Blot and RT-PCR results showed that PHB1 and PHB2 protein and gene levels in the PHB1 and PHB2 siRNA groups were significantly reduced.The results of MitoTracker Green staining showed that mitoflash could significantly reduce the degree of mitochondrial fragmentation,maintain mitochondrial length,and stabilize mitochondrial structure.Immunofluorescence and laser confocal microscopy showed that mitoflash could significantly reduce intracellular ROS,mitochondrial superoxide levels and free Ca2+concentrations.Seahorse results showed that mitoflash significantly increased ATP production,increased basal respiration,maximal respiration,and spare respiration,and inhibited mitochondrial proton leakage levels.EMPA can also significantly reduce mitoflash frequency,maintain mitochondrial morphological structure integrity,increase mitochondrial membrane potential,reduce mitochondrial proton leakage,reduce intracellular oxidative stress and calcium overload.Conclusion1.XFK oral liquid significantly improves myocardial tissue morphology,improves cardiac function,reduces myocardial damage,reduces the degree of fibrosis,and effectively reverses ventricular remodeling.2.XFK oral liquid can improve the state of oxidative stress,reduce the calcium overload of ventricular myocytes,restore intracellular calcium homeostasis,and improve the transient amplitude and contractile ability of myocardial calcium in CHF mice.3.XFK oral liquid can stabilize the mitochondrial structure of myocardial in CHF mice,protect mitochondrial function,upregulate the expression of mitochondrial inner membrane antiproliferative proteins PHB1 and PHB2,improve the I-V activity of mitochondrial respiratory chain complex,increase ATP synthesis,increase OXPHOS levels,and maintain mitochondrial energy metabolism homeostasis.4.XFK oral liquid may stabilize mitochondrial structural integrity and maintain mitochondrial membrane potential by regulating PHBs;Reduce intracellular oxidative stress,reduce abnormal opening of mPTP,restore calcium homeostasis,and reduce the frequency of mitoflash;Improve mitochondrial respiratory chain complex activity,maintain normal electron transport of ETC,reduce mitochondrial proton leakage level,optimize the productivity mode of cardiomyocytes,and maximize oxygen utilization.