Study On Rutin Protecting Myocardium And Sensitizing Pirarubicin Effect And Mechanism Against Breast Cancer

Anthracyclines including Doxorubicin,Epirubicin,Pirarubicin and Aclarubicin free Base etc.is widely used in the treatment of hematological malignancies and solid tumors,such as breast cancer,lymphoma,acute leukemia and so on.The combined treatment based on anthracyclines is usually the standard of first-line treatment,which is effective and indispensable,while cardiac toxicity is the most serious side effect of anthracyclines and restricts its clinical application.Clinical research and practical observation showed that the cardiac toxicity caused by anthracyclines is progressive and irreversible,and often the first use of anthracyclines can cause heart damage,so it is particularly important to actively prevent the cardiotoxicity caused by anthracyclines.Eukaryotic cells respond to external stimuli(such as growth factors,cytokines,physical or chemical stresses,etc.)by activating intracellular signaling mechanisms,and a major system of mediating stimulus signals transmitted from the cell membrane to the nucleus or other intracellular targets is mitogen-activated protein kinase MAPK super family.p38 MAPK is one of the three main MAPK cascade signaling pathways in myocardium,p38 MAPK cascade signals play an important role in the processes of cell growth,differentiation and apoptosis,which affect many enzymes,regulatory proteins and transcription factors,is a popular target in the pharmacological study of cardiovascular system.The research group has been engaged in the study of the protective effect of Rutin on cardiovascular system in recent years,it has been proved that it can inhibit the myocardial fibrosis induced by ISO,and the mechanism may be concerned with MAPK signaling pathways inhibiting active oxygen ROS mediated.Rutin has been proved to have many biological activities,such as anti-oxidation and protecting cardio-cerebral vessels,but it is less studied in inhibiting the cardiac toxicity and related mechanisms of anthracyclines(e.g.Pirarubicin).Based on the purified rutin,in vivo and in vitro experiments,the systematic study of its anti-anthracyclines in the cadiotoxicity and related mechanisms,is the necessary supplement and reasonable extension of rutin on the study of anti-cardiovascular disease,which will further provide a theoretical basis to research and development of Rutin-based anticardiotoxicity drugs.First,we need to determine the effect of RUT on H9c2 cell injury induced by THP.By MTT,Hoechst 33258,flow cytometry apoptosis/cycle and ROS analysis,THP inhibited the proliferation of H9c2 cells,decreased cell viability,increased the number of cardiomyocyte apoptotic cells and early/late apoptosis proportion,slowed down progress of the H9c2 cell cycle and increased the percentage of cells in S-phase and G2/M phase,reducing cell percentage and ROS formation in G0/G1period.After the intervention of RUT,DZR,SB203580 and SB203580+RUT group,the above indexes can be significantly improved,which shows that RUT has myocardial protective effect,and all the indexes of RUT are better than DZR.In order to determine whether RUT has the same myocardial protection in vivo,we establish acute and chronic myocardial injury model in rats by using tail intravenous injection THP simulate the process of chemotherapy delivery.In the model of acute myocardial injury,high dose THP can induce the increase of cardiac coefficient,decrease of heart rate,prolongation of Q-T period and decrease of QRS wave group voltage,LVEDP increased markedly,LVSP and±dp/dtmax,serum LDH,CK-MB,cTn-T and BNP levels increased markedly,SOD activity decreased,MDA content increased,the number of cells in the microscope was different or only involved several muscle pulp red staining,some cell nucleus was concentrated,irregular,part of nucleus disappeared.Prevention of rat gastric lavage RUT 7 d,can significantly improve the above indicators of rats in model group,and each test index of RUT is superior to DZR.Similarly,we obtained a similar result in the experimental study of the protective effect of RUT on chronic myocardial injury induced by THP,that is,prophylactic giving rats gastric lavage RUT 7 w,can significantly improve the heart coefficient,ECG,cardiac function,myocardial enzymes and myocardial tissue morphological changes,and RUT for the improvement of the detection indexes are better than DZR,It was suggested that RUT had better myocardial protective effect than DZR.RUT has a protective effect on THP induced myocardial injury,so does RUT have any effect on tumor cells?If combined RUT with THP,the THP will have an impact,how will it affect?Therefore,we selected human triple negative breast cancer MDA-MB-231 cells to study the effects of RUT and THP alone or in combination on the biological behavior of triple negative breast cancer MDA-MB-231 cells.The results showed that RUT inhibited the proliferation of MDA-MB-231 cells,blocked cell cycle,increased the number of apoptotic cells and the proportion of early/late apoptosis,reduced the rate of migration,repair ability and invasion.The above effect of RUT combined THP was better than that of THP alone and RUT alone,and RUT all indexes were better than DZR.It is suggested that RUT not only has the effect of resisting breast cancer,but also can increase the sensitivity of THP to treat breast cancer.To confirm whether p38 MAPK signaling pathway mediated RUT inhibit THP induced myocardial cell injury,we studied the effect of p38 MAPK inhibitor SB203580 on THP induced myocardial injury.In this study,the upstream mediated TGF-?₁ of p-p38 MAPK in THP treated cells significantly increased,indicating that activation of the TGF-?₁/p38 MAPK signaling pathway may have facilitated the apoptosis of cardiac myocytes.SB203580,RUT pretreatment with specific inhibitor SB203580 of p38 MAPK can effectively inhibit the expression of TGF-?₁ and p-p38MAPK.It was found that RUT has same effect with the SB203580 of p38 MAPK inhibitor,and blocked p38 MAPK pathway,and the synergistic effect of RUT and p38 MAPK inhibitor SB203580 was also demonstrated.The above results show that p38 MAPK signaling pathway was involved in RUT inhibiting THP induced myocardial injury,however,p38 MAPK signaling pathway had unpredictable interactions with the genes associated with the cardiac injury of anthracyclines.Therefore,through mi RNA chip technology,we first completed the analysis of RUT inhibiting miRNA expression spectrum of chronic myocardial injury induced by THP in rats,and screened the mi RNAs of THP induced myocardial injury with normal control group and Rutin group,and defined P<0.05 and|log₂ Fold change|>0.585 for differentially expressed mi RNA with a total of 104 miRNA appeared differential expression,Compared with Con group,miRNA expression in THP group have 50 increase and 28 decrease.Compared with Con group,miRNA expression in RUT grouphave14 increase and 2 decrease;Compared with THP group,miRNA expression in RUT group have 32 increase and 48 decrease.According to miRNA chip data,we define p<0.05 and|log₂ Fold change|>1.53 as 12mi RNAs with significant difference expression,and perform RT-PCR verification,mi RNA chip is in accordance with RT-PCR result,compared with normal control group,The expressions of rno-miR-451-5p,rno-miR-125b-1-3p,rno-mi R-667-3p,rno-miR-760-5p,rno-miR-185-3p,rno-miR-204-3p,rno-miR-207,rno-miR-466b-1-3p,rno-miR-494-3p,rno-miR-6216 in the model control group was increased,the expression of rno-miR-451-5p,rno-miR-207 and rno-miR-144-3p were raised in the Rutin group.Compared with the model control group,the expression of rno-miR-125b-1-3p,rno-miR-667-3p,rno-mi R-204-3p,rno-miR-3588,rno-miR-466b-1-3p,rno-miR-494-3p,rno-miR-6216 in Rutin group decreased.This concludes:1.Rutin can significantly promote the proliferation of H9c2 cells induced by THP in vitro,improve cell survival rate,accelerate cell cycle and inhibit ROS formation;and in vivo,rutin can significantly improve cardiac function in rats with myocardial injury caused by THP,decrease myocardial enzyme content and reverse myocardial tissue morphology changes,It was proved that Rutin had protective effect on the myocardial injury induced by the Pirarubicin.2.Rutin can inhibit the proliferation of breast cancer cells,block cell cycle,reduce migration speed,repair and invasion ability,combing use of Rutin and THP,the above effect is superior to the single use of Rutin,THP,which shows Rutin can sensitizing Pirarubicin against breast cancer.3.Rutin can lower the expression level of TGF-?₁,p-p38 MAPK,which shows that Rutin can inhibit THP-induced myocardial cell damage,inhibit MDA-MB-231cell proliferation achieved by blocking the TGF-?₁/p38 MAPK signaling pathway.4.Comparing myocardium tissue miRNA expression spectrum of rat in Con/THP/RUT group,screened out 12 miRNAs;target gene prediction was made by Target Scan software,common target genes of 6 rno-mir-667-3p miRNAs were identified as some members of the MAPK family by joint analysis;in the KEGG analysis that follows,a signal transduction pathway associated with rut inhibiting THP-induced myocardial damage include cancer,MAPK signaling pathways and etc.It is speculated that Cancer,MAPK and other signaling pathways are involved in the pathogenesis of RUT inhibiting THP induced myocardial injury,which may be achieved through the change of specific miRNA expression level.