Study On Danqi Tablet And Tanshinone ?A Protecting Doxorubicin-induced Myocardial Injury Through MTOR-ULK1/TFEB Autophagy Pathway

Objective:Doxorubicin(DOX),an anthracycline drug,is a first-line chemotherapeutic agent against solid tumors and leukemias.However,its serious side effects on heart and even heart failure(HF)have attracted great attention of clinicians and researchers in recent decades.Previous studies have found that autophagy plays an important role in DOX-induced myocardial injury(DIMI).At the same time,a large number of evidences show that mammalian rapamycin target(mTOR)and transcription factor EB(TFEB)are key regulators of autophagy.Danqi tablet(composed of Danshen and Sanqi)is widely used to treat heart failure in clinic.Tanshinone ?A comes from the root of Danshen.Its clinical application shows that it has a wide range of cardiovascular protective effects.At the same time,it has also been reported that tanshinone IIA has a protective effect on DIMI.The aim of this study was to investigate the protective effects of Danqi tablet and tanshinone IIA on DIMI and its internal regulatory mechanism on autophagyMethods:1.The in vivo model was established stably and the overall efficacy was evaluated:DOX was injected into the tail vein of mice after fixation,the injection concentration was 10 mg/mL,the injection amount was 5 mg/kg,once every 7 days,for 4 weeks.The normal saline group was injected with the same dose of normal saline.The successful evaluation indexes include echocardiography,hematoxylin eosin(HE)and Masson staining,NT proBNP,creatine kinase isozymes(CKMB)and lactate dehydrogenase(LDH).In conclusion,a stable model of DIMI in mice was established.Sixty C57 male mice were randomly divided into six groups:blank group,normal saline group,DOX group,DOX+tanshinone IIA group,DOX+ pravastatin group,DOX+Danqi tablet group(Danshen:Sanqi=1:1).The dosage of Danqi tablet was 1.5 g/kg/d,tanshinone IIA was 10 mg/kg,pravastatin was 750 ?g/kg,all of them were given by Sodium carboxymethyl cellulose water-soluble way every day for 28 days,blank group,normal saline group and DOX group were given the same volume of CMC-Na water soluble.Combined with dynamic image observation and instantaneous stop frame,the indexes of ejection fraction(EF),fractional shortening(FS),left ventricular end systolic diameter(LVEDs)and left ventricular end diastolic diameter(LVED;d)were evaluated by echocardiography.The heart was detected by HE and Masson staining.The levels of CKMB and LDH in serum were measured by the related kit.TUNEL method was used to detect cardiomyocyte apoptosis,and Western blot(WB)method was used to detect the expression of apoptosis related proteins B-cell lymphama-2(Bcl-2)and Bcl-2-associated x(Bax).To sum up,the effects of Danqi tablet and tanshinone IIA on reducing DIMI and protecting cardiac function were evaluated.2.To detect the regulatory effects of Danqi tablet and tanshinone IIA on autophagy:the number of autophagosome and autolysosome in the left ventricular cardiomyocytes was observed and counted by electron microscope;the activity of lysosome related cathepsin was detected by using the related kit.WB method was used to detect the expression levels of autophagy indicator proteins,including LC3 and p62;autophagy related protein Beclinl;lysosome related protein lampl;autophagy pathway regulatory proteins mTOR,4E-binding protein 1(4E-BP-1),UNC-51 like kinase 1(ULK1)and TFEB.3.Stable construction of in vitro cell model and detection of the effect of DOX-stimulation on autophagy flux:H9C2 cardiomyocytes were stimulated with different concentrations of DOX below 2 ?mol/L(DOX>2 ?mol/L has no clinical significance)for 24 hours,cell counting kit-8(CCK-8)was used to detect the cell proliferation rate,and WB method was used to detect the expression level of apoptotic protein.At the same time,WB method was used to detect the level of autophagy indicator protein LC3,p62 and Beclin 1.Baflomycin A1(BafAl),an autophagy lysosomal fusion inhibitor,was used to treat H9c2 cardiomyocytes to detect the level of LC3 expression.Furthermore,GFP-mRFP-LC3 adenovirus was used as the vector to transiently transfect H9C2 cells to monitor and quantify the number of autophagosomes and autolysosomes.To sum up,the injury model of H9C2 cardiomyocytes stimulated by DOX was constructed and the effect of DOX on autophagy flux of cardiomyocytes was further detected.4.To study the pharmacological mechanism of the cell model in vitro:divided into normal group,DOX group,DOX+tanshinone IIA group,DOX+Danqi tablet group,CCK-8 method was used to detect the cell proliferation rate,BafAl was used to detect the level of LC3 and p62,lysosome related tissue proteinase activity,GFP-mRFP-LC3 double fluorescent labeling cardiomyocytes were used to monitor the changes of autophagy flux.The expression of Beclin I,lamp1 and other regulatory proteins upstream of autophagy such as mTOR,ULK1 and TFEB were detected.The cardiomyocytes were stably transfected with slow virus labeled with GFP-TFEB.5.Construction of tumor cell model to verify the effect of drugs on the anti-tumor activity of DOX:1 ?M DOX combined with different concentrations of Danqi tablets and tanshinone?A stimulated different tumor cells,including hepatocarcinoma cells(HepG2),breast cancer cells(MCF-7),human glioblastoma(U87),and the cell proliferation rate was detected 24 hours,48 hours and 72 hours later,respectively.Results:1.Pharmacodynamic evaluation of Danqi tablet and tanshinone ?A in DIMI mice modelThe results of ultrasound showed that EF%and FS%of DOX group decreased,LVEDs and LUED;d increased,indicating that the in vivo model of DIMI was established successfully;there were almost no difference in the indexes of normal saline group and blank group.Compared with DOX group,Danqi tablet and tanshinone ?A treatment significantly enhanced cardiac function and improved left ventricular dilation.The results of HE and Masson staining showed that compared with DOX group,Danqi tablet and tanshinone ?A.could improve the disorder of myocardial tissue structure,the breaking and dissolving of muscle fibers and myocardial fibrosis.The results showed that compared with DOX group,Danqi tablet and tanshinone ?A could reduce the weight loss and growth retardation caused by DOX.The results of serum biochemistry indicated that Danqi tablet and tanshinone ?A could reduce the release of LDH and CKMB in serum compared with DOX group.The results of TUNEL and WB indicated that Danqi tablet and tanshinone ?A could regulate apoptosis-related proteins and improve the cardiomyocytes apoptosis induced by DOX2.Study on the regulation of Danqi tablet and tanshinone ?A on autophagyThe results of electron microscopy showed that Danqi tablet and tanshinone ?A could significantly improve the accumulation of autolysosomes induced by DOX.The WB results of autophagy indicator protein LC3 and p62 further confirmed that Danqi tablet and tanshinone?A can improve the dysregualtion of autophagy caused by DOX.Danqi tablet and tanshinone?A could also significantly enhance the activity of lysosomal cathepsin B.WB results showed that Danqi tablet and tanshinone ?A could upregulate Beclinl,the key protein of autophagosome formation process,and LAMP1,the key protein of autolysosome degradation process.In the upstream pathway of regulating autophagy,Danqi tablet and tanshinone ?A could inhibit the phosphorylation level of mTOR,4E-BP-1 and ULK1,and increase the expression of TFEB in the nucleus.3.Construction of in vitro DIMI H9C2 cell model and evaluation of autophagy fluxCCK-8 results showed that DOX significantly reduced the cell proliferation rate in a dose-dependent manner.WB results showed that 0.1-1 ?M DOX stimulated the apoptosis indicator protein Bax and Bcl-2 in a dose-dependent manner,which indicated that DOX could promote apoptosis in H9C2 cardiomyocytes,especially in the concentration of 1 ?M.To sum up,it was confirmed that the injury model of H9C2 cardiomyocytes stimulated by 1 DOX was constructed successfully.Based on this model,WB results showed that compared with the normal group,DOX-stimulation significantly increased the expression levels of LC3? and p62,while Beclinl decreased.Bafal alone induced significant accumulation of LC3II in H9C2 cardiomyocytes,but when BafAl combined with DOX,the accumulation of LC3? decreased.The fluorescent results of H9C2 cells transfected with GFP-mRFP-LC3 adenovirus showed that compared with the normal group,the yellow spots of cells treated with DOX decreased and the red spots increased significantly.The results showed that DOX stimulated autophagy lysosome accumulation,decreased the number of autophagosomes,and finally blocked autophagy flux 4.Study on the mechanism of Danqi tablet and tanshinone ?A regulating mTOR-ULK1/TFEB autophagy pathway to reduce DIMICCK-8 results showed that compared with DOX group,Danqi tablet(25-100?g/ml)and tanshinone ?A(1-20 ?M)could significantly improve cell viability.Finally,the concentrations of Danqi tablet and tanshinone IIA were 25 ?g/mL and 1?M respectively.WB results showed that compared with DOX group,Danqi tablet and tanshinone ?A decreased the expression level of LC3? and p62,while Danqi tablet and tanshinone ?A increased the expression level of LC3?when BafAl was added.In addition,Danqi tablet and tanshinone IIA can increase the activity of lysosomal cathepsin B.Compared with DOX group,Danqi tablet and tanshinone ?A decreased the accumulation of autolysosomes,increased the number of autophagosomes and finally recovered autophagic flux.WB results showed that Danqi tablet and tanshinone IIA could upregulate Beclinl and LAMP1,downregulate mTOR,S6K and ULK1 phosphorylation,and increase the expression of TFEB in the nucleus.After mTOR agonist MHY1485 was added,the regulatory effects of Danqi tablet and tanshinone ?A on mTOR,S6K,TFEB,Beclinl,LAMP1 and other proteins were weakened or disappeared,which indicated that Danqi tablet and tanshinone ?A regulated key proteins in different stages of autophagy by inhibiting mTOR.In addition,the addition of MHY1485 weakened the regulative effect of Danqi tablet and tanshinone ?A on autophagy flux,further indicating that Danqi tablet and tanshinone ?A regulated autophagy key protein by inhibiting mTOR,and finally restored autophagy flux.5.Effect of Danqi tablet and tanshinone ?A on anti-tumor activity of DOXCCK-8 results showed that compared with single DOX,combining with 25 ?g/mL,50 ?g/mL,100 ?g/mL Danqi tablets to stimulate HepG2 cells,MCF-7 cells,U87 cells for 24 hours,48 hours and 72 hours respectively didn't compromise anti-tumor activity of DOX;but when compared with single DOX,combining with 1 ?M,5 ?M and 20 ?M tanshinone ?A to stimulate HepG2 cells,MCF-7 cells and U87 cells for 24 hours,48 hours and 72 hours respectively helps to improve the anti-tumor activity of DOX.Conclusions:1.The pharmacodynamic study indicated that Danqi tablet and tanshinone ?A can effectively improve the left ventricular dilation caused by DOX,improve the cardiac function,improve the pathological structural damage,and reduce the apoptosis of myocardial cells.2.Mechanism research showed that Danqi tablet and tanshinone ?A can restore autophagy flux by regulating different stages of autophagy through mTOR related signal pathway.Specifically,they can promote the degradation of autolysosomes through mTOR-TFEB pathway,and promote the formation of autophagy through mTOR-ULK1 pathway.3.Danqi tablet and tanshinone ?A don't compromise the anti-tumor activity of DOX.