Protective Effect And Mechanism Of Apocynum Venetum Leaves Flavonoids And Isoquercitrin On Pirarubicin-induced Cardiac Injury

Anthracyclines,including doxorubicin(DOX),epirubicin(EPI),pirarubicin(THP),are widely used in the treatment of solid malignant tumors and hematological tumors.However,the cardiotoxicity of anthracyclines limits its clinical application to a certain extent.Dexrazoxane(DZR)is the only cardiac protective drug approved by FDA.However,it has been reported that DZR may aggravate chemotherapy-induced myelosuppression and induce the occurrence of second cancer.Therefore,it is particularly important to find a drug to reduce the heart injury caused by anthracycline.Apocynum venetum leaf flavonoids(AVLE)is the main active component of Apocynum venetum leaf,which has anti-hypertension,anti-anxiety,anti-depression and heart protection pharmacological effects.With the deepening of the research on AVLE,its prevention and treatment of cardiovascular diseases has become a research hotspot.Isoquercitrin,also known as quercetin-3-o-glucoside(IQC),is a kind of natural flavonoid compound,which is one of the effective ingredients in AVLE.It widely exists in fruits,vegetables,grains and many kinds of drinks.It has many pharmacological effects,such as antioxidant stress,anti-tumor,cardiovascular protection,hypoglycemia and anti-allergy.Non coding RNA(ncRNA)plays a role in many important life activities.As the role of microRNA(miRNA)in human diseases is gradually revealed,deepening the study of miRNA has practical significance for understanding the molecular mechanism of disease occurrence and development.In this study,we first explored the protective effect and mechanism of IQC and AVLE on THP induced cardiac injury in vivo and in vitro,and then constructed mir-190a-5p/Phlpp1/AKT/Bcl-2 signaling pathway through bioinformatics analysis.Taking this signaling pathway as the breakthrough point,we used AKT blockers and miRNA overexpression/silencing transient cells to verify it at the cellular level.In order to provide scientific basis for the development and utilization of traditional Chinese medicine resources with IQC and AVLE as active ingredients.The research is divided into five parts:(1)Protective effect and mechanism of AVLE on THP induced cardiac injury in ratsThe rats were given different doses of AVLE for 1 W,then THP(3mg/kg/w,6W)was injected into tail vein to establish the chronic cardiac injury model of rats,and the rats were given AVLE by gavage.The changes of ECG and cardiac tissue morphology were observed in rats.The hemodynamics,serum BNP,CK MB,c Tn T and LDH levels,oxidative stress related indexes,myocardial mitochondrial membrane m RNA level and AKT/Bcl-2 signaling pathway related protein were detected.The protective effect of AVLE on THP induced cardiac injury in rats and its possible molecular mechanism were studied.The results were as follows:(1)In the AVLE medium and high dose groups the heart rate,QRS wave group,LVEDP,LVSP and ±dp/dtmax were increased,BNP,CK MB,c Tn T and LDH were decreased in serum,SOD activity and MDA content were increased;the pathological changes of myocardium caused by THP were improved;the mitochondrial membrane VDAC1,ANT1 and CYPD in the heart tissue were reduced.The expression of m RNA can improve the permeability of mitochondrial membrane.The above indexes were not improved in the low dose group of AVLE.(2)AVLE inhibited the release of cyt c from mitochondria to cytoplasm,increased the expression of pro-caspase-9,procaspase-3,p-Akt and Bcl-2 proteins,reduced the protein levels of Bax and cleavedcaspase-3,and reduced the apoptosis of cardiac tissue cells induced by THP.(2)Preparation and composition analysis of AVLEAVL was prepared by alcohol extraction,purified by macroporous resin and enriched AVLE.HPLC-ESI-MS/MS and HPLC were used for qualitative and quantitative analysis of AVLE.The results were as follows: there were 7 flavonoids in AVLE,and then four main monomers were quantitatively analyzed.IQC was the most abundant compound in AVLE.(3)Role of AKT in the protection of avle and IQC against THP induced H9c2 cell injuryInduce H9c2 cell damage with THP,add IQC/AVLE and AKT inhibitor,use MTT,DCFH-DA fluorescent probe,ELISA,Mito-Tracker Red CMXRos probe,JC-1probe,TUNEL,RT-qPCR,Western blot to explore the role of AKT in AVLE and IQC protecting THP-induced H9c2 cell damage.The results are as follows:(1)IQC(5?500?M)and AVLE(5?400?g/ml)have no toxic effects on H9c2 cells at different time points(6,12,24 and 36h);treatment of H9c2 cells with 5?M THP for 24 h can cause cell growth apoptosis,the survival rate is reduced;while IQC and AVLE can inhibit THP-induced H9c2 cell damage,the optimal concentration and time of administration are 70?M and 70?g/ml,24 h.Both IQC or AVLE can reduce the ROS content in H9c2 cells treated with THP,increase SOD activity,and reduce MDA content;increase mitochondrial viability in H9c2 cells,improve cell mitochondrial membrane potential,and reduce the expression of mitochondrial membrane-related genes.(2)IQC and AVLE increase the protein expression of p-AKT in H9c2 cells,inhibit the release of Cyt c from cell mitochondria to the cytoplasm,reduce the rate of apoptosis,and at the same time reduce the expression level of cleaved-caspase-3protein in the cytoplasm,and increase pro-caspase-9 and pro-caspase-3 protein level and the protein ratio of Bcl-2/Bax;application of AKT inhibitor can block the protective effect of IQC or AVLE on THP-induced cell damage,indicating that AKT is the node where IQC or AVLE exerts a cardioprotective effect molecular.(4)THP-induced rat heart injury miRNA chip bioinformatics analysis and the construction and verification of miR-190a-5p/Phlpp1/AKT/Bcl-2 signaling pathwayThe miRNA expression profile of THP induced cardiac injury in rats was analyzed,and mir-190a-5p was selected as the research object.Through the prediction of mir-190a-5p target gene and KEGG enrichment analysis,it was found that there was a potential binding site of mir-190a-5p in the 3'UTR of Phlpp1 m RNA,and Phlpp1 was located in the upstream regulatory molecules of AKT;meanwhile,AVL target gene was searched and KEGG enrichment analysis was carried out,and the results showed that AVL could participate in the regulation of AKT and its downstream Bcl-2 family proteins.Therefore,mir-190a-5p/Phlpp1/AKT/Bcl-2signaling pathway was constructed.RT-qPCR and Western blot were used to verify the expression of mir-190a-5p and its target gene Phlpp1 in THP induced cardiac injury tissues/H9c2 cells.The results showed that IQC and AVLE could increase the expression of mir-190a-5p and inhibit the expression of Phlpp1 gene and protein.Double luciferase reporter gene experiment confirmed that mir-190-5p can directly regulate Phlpp1,and there is only one binding site.(5)Role and mechanism of mir-190a-5p in THP induced H9c2 cell injuryTransient H9c2 cells by constructing miR-190a-5p overexpression/silencing transiently,using DCFH-DA probe,ELISA,Mito-Tracker Red CMXRos probe,JC-1probe,RT-qPCR,TUNEL and Western blot experiments methods to explore the role and mechanism of miR-190a-5p in the treatment of H9c2 cell injury by THP.The results are as follows:(1)miR-190a-5p mimics reduces the content of ROS in H9c2 cells,increases SOD activity,and reduces MDA content;at the same time,it increases the mitochondrial viability and membrane potential,and reduces the expression of mitochondrial membrane genes;while transfection with miR-190a-5p inhibitor did not improve the THP treatment of H9c2 cells.(2)miR-190a-5p mimics increases the expression of miR-190a-5p in H9c2 cells,inhibits Phlpp1 gene and protein,increases the expression of p-AKT protein,and inhibits the release of Cyt c from mitochondria to the cytoplasm,reducing the rate of apoptosis.At the same time,it reduces the expression of cleaved-caspase-3 protein in the cytoplasm,increases the protein levels of pro-caspase-9 and pro-caspase-3 and the ratio of Bcl-2/Bax protein;while transfection with miR-190a-5p inhibitor did not have any effects.In summary:IQC and AVLE have protective effects on THP induced Heart/H9c2 cell injury,and their protective effects are equal;the related mechanism may be that IQC and AVLE regulate mir-190a-5p/Phlpp1/AKT/Bcl-2 signaling pathway,and AKT is the nodal element of their cardioprotective effects.