Mitophagy Is Involved In The Protective Effect Of Betulinic Acid Against Cardiac Injury Induced By LPS

Sepsis is a life-threatening organ dysfunction syndrome caused by the dysregulation of response to infection.Cardiac injury,a common complication in sepsis,is one of the leading cause of high mortality in patients with sepsis in intensive care units.Mitophagy plays an important role in preserving cell viability by removing damaged mitochondria,and maintaining mitochondrial homeostasis.Mitochondrial damage and insufficient mitophagy triggered by sepsis exacerbate cardiac injury in sepsis,Lipopolysaccharides(LPS)is the major factor causing the pathological and physiological outcomes of sepsis,often used to construct sepsis models.Betulinic acid(BA),a pentacyclic triterpene acid,has been shown in numerous animal and cellular models to reduce cardiac injury.Our previous studies found that BA attenuates vascular injury through anti-inflammation and anti-oxidative stress in septic rats.However,it is not clear the underlying mechanism by which BA improves cardiac injury induced by LPS.Objective: 1.To determine the protective effect of pretreatment with BA against cardiac injury induced by LPS.2.To explore mitophagy-related mechanisms by which BA attenuates cardiac injury induced by LPS using mitophagy inhibitor Mdivi-1.Methods: Male SD rats were randomly divided into four groups: Control group(Control),LPS group(LPS),BA(25 mg/kg,5d,i.g.)pretreated group(BA+LPS),mitophagy inhibitor Mdivi-1(5 mg/kg,i.p.)and BA pretreated groups(Mdivi-1+BA+LPS).After 5 days of BA pretreatment,rats were injected with LPS(10 mg/kg,i.p.)at day 6 for 8 h and the cardiac function of rats was measured by small animal ultrasound.Hematoxylin-eosin staining(HE)staining was adopted for detecting cardiac tissue morphology,enzyme-linked immunosorbent assay was used for determining myocardial injury marker including serum cardiac troponin I(c Tn I)and creatine kinase-MB(CK-MB)and inflammatory factors including interleukin-1β(IL-1β),interleukin-6(IL-6)and tumor necrosis factor α(TNF-α),colorimetric assay was used for determining the activity of myocardial mitochondrial respiratory chain complex(I-III),dihydroethidium(DHE)staining was adopted for determining cardiac reactive oxygen species(ROS),fluorescence immunolocalization was adopted for determining cardiac mitophagy,Western blot was used for detecting the expression of cardiac microtubule-associated protein light chain 3(LC3),lysosomal associated membrane protein 1(Lamp1),autophagy adaptor proteins(sequestosome 1,p62),PTEN induced putative kinase 1(PINK1),Parkin and BCL2/adenovirus E1 B 19 k Da interacting protein 3(BNIP3),and fluorescence assay kit was used for detecting the opening of mitochondrial permeability transition pore(m PTP).Results: Pretreatment with BA significantly increased left ventricular ejection fraction and left ventricular fractional shortening,improved myocardial morphology,significantly decreased serum c Tn I,CK-MB,TNF-α,IL-1β and IL-6(P<0.05),significantly increased the activity of myocardial mitochondrial respiratory chain complexes(I-III)(P<0.01),markedly inhibited the accumulation of myocardial mitochondrial ROS(P<0.01),significantly increased the expression of cardiac LC3 II and Lamp1(P<0.01),significantly increased the expression of myocardial mitochondrial PINK1,Parkin and LC3 II(P<0.05)and markedly decreased the expression of myocardial mitochondrial BNIP3,and p62(P<0.01),significantly decreased myocardial mitochondrial permeability transition pore content and opening(P<0.01)in LPS group rats.Mitophagy inhibitor Mdivi-1 significantly reversed these protective effects of BA against LPS-induced cardiac injury(P<0.05).Conclusion: 1.BA attenuates LPS-induced cardiac injury.2.BA attenuates LPS-induced cardiac injury by enhancing myocardial mitophagy and activating PINK1/Parkin pathway and inhibiting BNIP3 may be involved in this protective effect of BA.