The Mechanism Of Ganoderic Acid Relieving Angiotensin Ⅱ-induced Cardiac Hypertrophy By Inhibiting NLRP3 Inflammasome

Background:Cardiac hypertrophy is the main adaptive response of the heart in the face of load,but long-term or excessive hypertrophy promotes myocardial interstitial fibrosis,systolic dysfunction,cardiomyocyte death,especially the sterile inflammation mediated by NLRP3 inflammasomes,which can aggravate ventricular remodeling and myocardial damage,and is an important mechanism for the progression of heart failure.Various cardiac overburdens can lead to mitochondrial damage,and mitochondrial DNA(mtDNA)released by damaged mitochondria can be found in many tissues and cells,which can further activate NLRP3 inflammasome.However,the mechanism of mtDNA homeostasis and aseptic inflammation in cardiac hypertrophy is still poorly studied.This paper will conduct a series of studies on the role of mitochondrial dynamic imbalance in myocardial hypertrophy and inflammatory response.Ganoderic acid(GA),as one of the most abundant ganoderma triterpenes in the famous traditional Chinese medicine Ganoderma lucidum,has anti-inflammatory,antioxidative stress,anti-tumor and anti-apoptosis effects.Studies have shown that GA can inhibit renal fibrosis through the MAPK pathway and exert anti-kidney damage through the NF-κB pathway anti-inflammatory.Therefore,this article discusses the effect and mechanism of GA in regulating myocardial inflammation and alleviating cardiac hypertrophy,which is intended to provide a theoretical basis for revealing the prevention and treatment role of GA in heart failure.Object:Angiotensin Ⅱ(Ang Ⅱ)was used to construct animal and cell(H9C2 rat cardiomyocytes)models of cardiac hypertrophy,observe the changes of sterile inflammation and hypertrophy phenotype,explore the role of mtDNA homeostasis balance leading to inflammatory response in the pathogenesis of pathological cardiac hypertrophy,and further explore the mechanism of GA regulating mitochondrial dynamics and inflammatory response in myocardial protective effect.Method:1.The mouse model of cardiac hypertrophy was established by injecting Ang II(1000 ng/kg/min)into the subcutaneous sustained pump for 4 weeks,the control group was given the same dose of normal saline,and the GA treatment group was given Ang II and GA(5 mg/kg/d)intraperitoneally.Echocardiography detected the cardiac function of mice,and transmission electron microscopy,HE staining,and Masson staining observed myocardial morphological changes.2.A model of H9C2 rat cardiomyocyte hypertrophy was established after 48 hours of stimulation using Ang Ⅱ(0.1 μM),and GA(1,5,25 μg/mL)was given to the GA treatment group at the same time.3.Western Blot and qPCR were used to detect changes in cardiac hypertrophy marker ANP,sterile inflammation-related molecules(NLRP3,IL-Iβ,Caspase-1),mitochondrial division fusion-related molecules(DRP1,MFN1)and other proteins and RNA levels.4.Immunofluorescence microscopy to observe α-actin(cardiomyocyte area change)and NLRP3 expression.5.Detection of oxidative stress level(DCFH-DA staining)and mitochondrial membrane potential(JC-1 staining)by flow cytometry and immunofluorescence microscopy.6.Observe dsDNA and TOM20 expression by confocal microscopy,and observe DNA and mitochondria colocalization.7.Extract cytoplasmic DNA and detect the copy number of mitochondrial DNA in the cytoplasm by qPCR.Result:Ⅰ.The role of inflammatory response induced by mitochondrial injury in the pathogenesis of Ang Ⅱ-induced cardiac hypertrophy1.Compared with the control group,after Ang Ⅱ treatment for 4 weeks,the cardiac function of mice in the model group decreased(IVSD,LVPW was significantly increased;LVIDd,EF and FS were significantly decreased),the cardiac the heart coefficient of the Ang Ⅱ group increased,the surface area and fibrotic area of cardiomyocytes increased significantly,and the expression level of mRNA and protein of the cardiac hypertrophy marker ANP increased significantly,indicating that the myocardial hypertrophy model was successfully replicated.2.After Ang Ⅱ loading,the electron microscope found that the mitochondrial structure of mouse cardiomyocytes was swollen and the cristae were reduced,and no dynamic changes of fusion/division were observed.The expression of mitochondrial fission protein DRP1 was increased,and the expression of fusion protein MFN1 was decreased,indicating that the mitochondrial function was decreased and the mitochondrial dynamics was disordered.3.After Ang Ⅱ loading,the expression of NLRP3,caspase-1,IL-1β,and IL-6related molecules increased in mouse heart tissues.4.After Ang Ⅱ treated H9C2 cells for 48 hours,the surface area of myocardial cells increased significantly,and the mRNA and protein expression levels of ANP,a marker of cardiac hypertrophy,also increased significantly.Mitochondrial membrane potential decreased,and ROS production increased.The increase of mtDNA copy number in cytoplasm and the decrease of co-location with mitochondrial marker TOM20 indicate that there is a significant mtDNA leakage.The expression of NLRP3 inflammasome and inflammatory mediators was also significantly increased.Ⅱ.GA alleviates Ang Ⅱ-induced cardiac hypertrophy by inhibiting NLRP3 inflammasome1.After Ang Ⅱ was treated with GA at the same time,the cardiac function of mice was significantly improved(IVSD,LVPW and decreased,LVIDd,FS and EF basically returned to normal),the surface area of myocardial cells and the area of fibrosis were significantly reduced,and the expression level of ANP was significantly inhibited.2.The electron microscope observation showed that GA also significantly improved the mitochondrial damage state,reduced mitochondrial swelling,and restored the crista.The dynamic process of fusion division can be observed.3.Compared with the model group,after GA treatment,the mice not only reduced the degree of cardiac hypertrophy,but also significantly decreased the expression level of NLRP3-induced sterile inflammation-related molecules such as NLRP3 and IL-Iβ.4.The inhibitory effect of GA on myocardial hypertrophy(reduced surface area,down-regulation of ANP expression)was observed in the cell model.Compared with the model,ΔΨm was restored,ROS production was inhibited,mtDNA leakage to cytoplasm was reduced,DRP1 was down-regulated,MFN1 was upregulated.And the expression level of NLRP3-induced sterile inflammation-related molecules was also significantly reduced.Conclusion:1.Ang Ⅱ can induce mitochondrial dysfunction,increased mitochondrial division and mtDNA leakage in cardiac myocytes,thus inducing NLRP3 inflammatory corpuscle-related inflammatory response and cardiac hypertrophy.2.GA may prevent the activation of NLRP3 inflammasome by restoring mitochondrial dynamic balance,inhibiting the leakage of mtDNA into the cytoplasm,thereby improving inflammation and myocardial hypertrophy,and ultimately protecting the heart.