TDCPP Attenuates H₂O₂ And Hypoxia/Reoxygenation-induced Injuries In Myocardiocytes Via Activating PI3K/Akt/GSK3β Signaling Pathway

Myocardial ischemia reperfusion（IR）injury refers to the more obvious and more serious injury and dysfunction than before after recovering myocardial blood perfusion.It is one of the main complications in the clinical treatment process such as coronary artery bypass surgery,cardiac extracorporeal circulation and macro vascular surgery,etc.Reactive oxygen species（ROS）and Ca²⁺overload produced by IR are important factors that cause oxidative stress injury in myocardial cells.In addition to endoplasmic reticulum/sarcoplasmic reticulum calcium pumps（SERCA）,reverse sodium calcium exchangers（NCX）,L-type Ca²⁺channels,and Ca²⁺uniporters（MCU）on mitochondrial inner membrane,myocardial cells also express store-operated calcium entry（SOCE）channels,but the molecular basis and regulatory pathway of myocardial cells Ca²⁺overload and oxidative stress injury caused by IR are still lack of in-depth research.Tris（2,3-dichloropropyl）phosphate（TDCPP）is a widely used organophosphorus flame retardant.Toxicological studies have found that TDCPP has endocrine disrupting toxicity,reproductive and developmental toxicity,neurotoxicity and potential carcinogenicity.David.W.Killilea and his colleagues have proven that its toxic effects are concentration-dependent.Killilea and his colleagues reported that under normal physiological conditions,low concentration of TDCPP only causes cell cycle arrest and inhibits cell growth（IC50 27μM）,while high concentration of TDCPP causes cell viability decrease（IC50 171μM）and cell toxic effects（IC50 168μM）;However,in pathological conditions,low concentration of TDCPP can also play a protective role.Ca²⁺overload and ROS can influence and promote each other.Armstrong et al.（Personal Communications）confirmed that TDCPP can inhibit thapsigargin-induced Ca²⁺release and Ca²⁺entry,And TDCPP has the function of directly blocking SOCE channel not its signal pathway.This present study shows that 50μM TDCPP can activate PI3K-AKT-GSK3βsignaling pathway,PI3K-AKT-GSK3βsignaling pathway promotes myocardial cell survival and proliferation,and participates in the protection of myocardial cell apoptosis induced by IR.Autophagy is a conservative process of self-degradation of intracellular components,which can degrade intracellular longevity protein and damaged organelles through lysosomal-dependent pathway,thus maintaining cytoplasmic homeostasis.Autophagy plays an important role in myocardial cell damages caused by IR.H₂O₂ can activate myocardial autophagy,which can degrade damaged organelles,recycle energy generation and play a protective role in the ischemic stage.When reperfusion occurs,autophagy is excessively activated,which leads to autop hagic cell death and exacerbates myocardial cell damage.To our knowledge,the effect of TDCPP on H₂O₂-induced autophagy of myocardial cel s has not been reported so far.This experiment studied the effect of TDCPP pretreatment on oxidative stress injury of myocardial cells induced by H₂O₂ and hypoxia/reoxygenation（H/R）.Our study found that TDCPP reduced the intracellular Ca²⁺flow mediated by toxic carotene（Thapsigargin,Tg）,and reduced the Ca²⁺overload induced by H/R and H₂O₂ by reducing the expression of Stim1 in H/R-treated cardiomyocytes and affecting the expression of TRPC6 in H₂O₂-treated cells.TDCPP pretreatment can relieve the morphological change of apoptosis such as shrinkage,rounding,disorder of arrangement and loose connections of cardiac myocytes treated with H/R and H₂O₂.TDCPP can significantly reduce LDH release level of H₂O₂-treated cells,reduce intracellular ROS and MDA levels,and increase the viability of H/R and H₂O₂-treated myocardial cells.Mitochondrial membrane potential of cardiomyocytes treated with H/R decreased rapidly and mitochondrial function was lost.TDCPP partially recovers the reduction of mitochondrial membrane potential induced by H/R,protects mitochondrial function and reduces myocardial cell apoptosis;TDCPP alleviates the increase of BAX and CC3 expression and the decrease of Bcl-2 expression,and the increase of BAX/Bcl-2 expression ratio induced by H/R and H₂O₂.TDCPP reverses H₂O₂-induced increase in LC3-Ⅱexpression in H₂O₂-treated cardiomyocytes.TDCPP can increase the expression of p-AKT（ser473）and p-GSK-3β（ser-9）in H/R and H₂O₂ treated cells.Further western blotting detection showed that pretreatment of H/R and H₂O₂ cardiomyocytes with PI3K inhibitors LY294002 and TDCPP at the same time eliminated the protective effect of TDCPP.In a word,TDCPP inhibits SOCE,alleviates calcium overload of cardiomyocytes induced by H/R and H₂O₂,alleviates apoptosis and autophagy of cardiomyocytes induced by H/R and H₂O₂,and protection of cardiomyocytes of TDCPP depend on PI3K-AKT-GSK3β signaling pathway.