Protective Effect Of Rutin On Myocardial Injury Induced By Pirarubicin Based On MiR-22-5p/RAP1/ERK Signaling Pathway

Anthracyclines,including doxorubicin(DOX),epirubicin(EPI),pirarubicin(THP),are widely used in the treatment of solid malignant tumors and hematological tumors,but anthracyclines drugs have many adverse reactions,of which cardiotoxicity is the most serious adverse reaction of anthracyclines,which limits its application to a certain extent.Dexrazoxane is the only cardioprotective drug approved by the FDA,but there are reports that dexrazoxane may aggravate bone marrow suppression caused by chemotherapy drugs.Therefore,there is an urgent need to find a safer and more effective cardioprotective drug in clinic.Rutin(RUT),also known as rutin,vitamin P,lutein,and paclitaxel,is a flavonoid compound with a wide range of sources.It is mainly found in various plants such as rutin leaves and tobacco leaves.It has many important biological activities such as anti-inflammatory,anti-oxidation and anti-tumor.The previous research of this research group has confirmed that RUT can improve the myocardial injury caused by THP,and made a miRNA chip that RUT interferes with THP-induced myocardial injury in rats.Starting with miRNA chips,this study constructed the miR-22-5p/RAP1/ERK signaling pathway through bioinformatics analysis,and using this signaling pathway as an entry point to study its role and mechanism in RUT on myocardial damage induced by THP.To elucidate the molecular mechanism of THPinduced myocardial damage,and provide a new target for RUT to improve THPinduced myocardial damage.This study is divided into 3 parts:(1)miRNA chip bioinformatics analysis of RUT protecting rat myocardial injury caused by THP and construction of miR-22-5p/RAP1/ERK signaling pathway.First analyze the chip results.Set the condition of the differential gene to p<0.05 and |log2 fold change|>0.7,then compare the RUT+THP group/THP group and THP group/CON group,select the miRNAs that RUT can recall the abnormal changes of THP and have better homology in rats and humans.Consult relevant literature to determine the miRNA.Then use Target Scan database and miRDB database to predict their target genes,use omicshare database and DAVID database to perform GO analysis and KEGG analysis on the predicted target genes,and construct signaling pathways.The results showed:(1)A total of 18 miRNAs met the above conditions,including 8 up-regulations and 10 down-regulations(RUT+THP group/THP group);(2)miR-22-5p,which is related to heart protection studies and has not been reported to be associated with myocardial injury caused by anthracyclines,was selected as the study object.(3)Through target gene prediction and KEGG enrichment analysis,it was found that there is a potential binding site of miR-22-5p at the 3'UTR of RAP1 A m RNA,which has good targeting and RAP1 A is located in the MAPK signaling pathway,thereby constructing miR-22-5p/RAP1/ERK signaling pathway,and based on this pathway,the protective effect of RUT on myocardial injury induced by THP is discussed.(2)The mediating role of miR-22-5p/RAP1/ERK signaling pathway in RUT improving THP-induced myocardial injury in rats.The rat myocardial injury model was replicated by tail vein injection of THP(18mg/kg,divided into 6 times),and pre-protected with RUT(50mg/kg).Observe the electrocardiogram,cardiac function parameters and myocardial pathological changes of rats,and use qPCR method to detect the expression of miR-22-5p and RAP1 A m RNA in rat myocardial tissue,and Western blot method was used to detect the expression of RAP1/ERK pathway related proteins in rat myocardial injury tissue.The results showed that:(1)RUT can reduce the changes of ECG,cardiac function and myocardial pathology caused by THP;(2)RUT can reverse the decrease of miR-22-5p expression caused by THP,which is consistent with the results of the chip;RAP1A protein expression and increased phosphorylation levels of BRAF,MEK1/2,and ERK1/2 proteins suggest that RUT may regulate RAP1 A and downstream ERK signaling pathway through miR-22-5p to mediate its protective effect on THP-induced myocardial injury.(3)The role and mechanism of miR-22-5p in THP treatment of H9c2 cell injury.A model of THP-induced H9c2 cell damage was established,pre-protected with RUT,and the effect of RUT on miR-22-5p and RAP1 A m RNA expression was detected by qPCR.Construct miR-22-5p overexpression/silent transient H9c2 cell line,use qPCR method to detect miR-22-5p and RAP1 A m RNA expression,and use ROS,TUNEL,Western blot experiments to detect intracellular ROS level,apoptosis status and changes in related proteins on the RAP1/ERK pathway.The results showed that:(1)RUT can reverse the down-regulation of miR-22-5p caused by THP,which is consistent with the results of the chip;(2)RAP1A m RNA expression is downregulated when miR-22-5p is overexpressed;When miR-22-5p is silenced,RAP1 A m RNA expression is up-regulated,miR-22-5p can target and regulate RAP1A;(3)miR-22-5p can reduce the production of ROS in H9c2 cells caused by THP,reduce apoptosis,and inhibit the up-regulation of RAP1 A protein expression level and BRAF,MEK1/2,ERK1/2 protein phosphorylation levels caused by THP.The up-regulation of protein phosphorylation level is inhibited and plays the same role as RUT.The above results indicate that RUT may play a cardioprotective role by up-regulating miR-22-5p and inhibiting the activation of RAP1/ERK signaling pathway.In summary,in this study,the rat myocardial tissue/H9c2 cell injury model was replicated by THP,RUT was used for pre-protection,and the miR-22-5p overexpression/silent transient cell line was constructed by transfection technology,using ROS,TUNEL,qPCR,Western blot and morphological observation methods,in vivo and in vitro experiments were used to systematically explore the role and mechanism of RUT against myocardial injury caused by THP from the perspective of miR-22-5p/RAP1/ERK pathway,and the following conclusions were drawn:(1)RUT can improve THP-induced changes in electrocardiogram,cardiac function and myocardial morphology of rats,indicating that RUT has a protective effect on THP-induced myocardial toxicity in rats.(2)RUT could reverse the down-regulation of miR-22-5p expression in THPinduced rat cardiac tissue/H9c2 cells,reverse the increase of THP-induced RAP1 A gene and protein expression,and reverse the increase of THP-induced phosphorylation of BRAF,MEK1/2 and ERK1/2 proteins.And it can reduce the production of ROS and apoptosis in H9c2 cells caused by THP.Overexpression of miR-22-5p in H9c2 cells has the same effect as RUT.It is suggested that the protective effect of RUT on myocardial tissue/H9c2 cell damage caused by THP may be achieved by up-regulating miR-22-5p,thereby inhibiting the RAP1/ERK signaling pathway.(3)After transfection of miR-22-5p in H9c2 cells,miR-22-5p overexpression can down-regulate RAP1 A m RNA expression,and miR-22-5p silencing can up-regulate RAP1 A m RNA expression,it was demonstrated that miR-22-5p can target and regulate RAP1A.