Investigation On HMGB1-mediated Autophagy Involved In Protective Effects Of Glycyrrhizin Against Cardiotoxicity Induced By Doxorubicin

Background&ObjectiveDoxorubicin(DOX)is a broad-spectrum anti-tumor agent among anthracycline but its clinical use is limited due to its cardiotoxicity while the mechanism has not been clarified clearly yet.Autophagy is an important way to degrade intracellular misfolded protein or damaged organelles,regulating both positively and negatively.Study suggested DOX promoted dysfunction of autophagy and HMGB1 played a vital role in autophagy.Glycyrrhizin(GL)is a natural small molecule compound,directly binding to HMGB1 and inhibit its expression.Therefore,the present study intends to explore the effects of GL on DOX-induced cardiotoxicity and explore whether GL exhibits its cardioprotective effects through inhibitng HMGB 1-mediated autophagy.Methods&ResultsH9c2 cardiomyoblasts cell viability against GL and DOX was determined using MTT assay.DOX decreased H9c2 cell viability(0-10 ?M)in a dose-dependent manner while GL below 0.8 mM showed no significant cytotoxicity.And 0.8 mM GL pretreatment for 12 h could reduce DOX-induced cell viability loss in H9c2 cardiomyoblasts.In addition,GL could significantly downregulate the increase of intracellular oxidative stress evoked by DOX in a concentration-dependent manner.JC-1 was applied to detect mitochondrial membrane potential(MMP)in H9c2.GL significantly reverted MMP depolarization caused by DOX.A single dose of 20 mg/kg DOX(i.p)was used to establish an acute cardiac toxicity model in Sprague-Dawley rats in vivo.The low dose(25 mg/kg)and high dose(50 mg/kg)of GL pretreatment groups significantly reverted serum Aspartate aminotransferase(AST),Creatine Kinase,MB Form(CK-MB)and Superoxide dismutase(SOD)activity caused by DOX.The above showed that GL could reduce myocardial cytotoxicity of DOX both in vitro and in vivo.DOX upregulated the levels of the autophagy marker LC3 ? and the selective autophagy receptor p62 in H9c2 cardiomyoblasts and primary neonatal rat cardiomyocytes(NRCMs),which further confirmed that DOX gave rise to dysfunction of autophagy in vitro,whereas GL could effectively reduce the impairment.In vivo results also showed that GL pre-treatment significantly downregulated LC3 ? and p62 expression in the presence of DOX.In addition,we also introduced the autophagy inhibitor Bafilomycin A1(50 nM,2 h)and mCherry-GFP-LC3B adenovirus dual fluorescence reporting system to monitor autophagy flux.DOX could inhibit degradation of autophagosomes and block the late stage of autophagy flux whereas GL was captable of effectively improving the obstructed autophagy flux caused by DOX.Western Blot analysis were performed to detect the effects of GL and DOX on Akt/mTOR autophagy signaling pathway and found that GL was able to downregulate the pathway expression in the presence of DOX.RNA interference(RNAi)was applied to silence HMGB1 and found that knockdown of HMGB1 could significantly increase H9c2 cell viability and LC3 ? level during exposure to DOX,which suggested that HMGB1 played a crucial role in DOX cardiac toxicity.In the other hand,compared to DOX group,the expression of p-Akt,p-mTOR and autophagy marker LC3 ?,p62 after GL pretreatment was not statistically different under DOX exposure in H9c2/HMGB 1-cells(transfeccted with HMGB1 siRNA),whereas in the non-silencing H9c2 cells(transfected with Negative Control siRNA),GL pretreatment could significantly downregulate the expression of p-Akt,p-mTOR,LC3 ? and p62.This indicated that HMGB 1-dependent Akt/mTOR autophagy signaling pathway was involved in protective effects of GL against cardiotoxicity induced by DOX.ConclusionThis study illuminates that HMGB 1-mediated Akt/mTOR signaling pathway is a novel mechanism of GL in regulating DOX-induced cardiotoxicity by improving the obstructed autophagy flux.GL has the potential to be applied in the prevention and treatment in DOX-induced cardiac toxicity.