Study On The Mechanism Of Sophoricoside Attenuating Doxorubicin-Induced Cardiotoxicity

ObjectiveTo determine the protective effect of sophoroside(SOPH)on cardiotoxicity induced by doxorubicin.MethodsH9c2 cells were pretreated with sophoroside(20μmol/L,80μmol/L)for 6hours,then treated with doxorubicin at the final concentration of 1μmol/L for 24 hours,then the cell survival was detected by MTT method.The level of ROS was detected by DCFH-DA probe.Apoptosis was detected by Hoechst 33342 staining and flow cytometry.The expression of apoptosis-related proteins Bax,Bcl-2,Cleavedcaspase3 and signal pathway-related proteins p38,p-p38,ERK,p-ERK,JNK,p-JNK and NF-κB were detected by Western Blot.P79350,a p38 agonist,was used to verify that sophoroside attenuated cardiomyocyte injury induced by adriamycin by inhibiting the expression of NF-κB signal pathway.Results1.H9c2 cells were treated with sophoroside at different concentrations(20μmol/L,40μmol/L,60μmol/L,80μmol/L,100μmol/L)for 24 hours,the cardio-myocyte viability of each group was(100.62±3.18)%,(100.19±1.63)%,(99.94±2.27)%,(99.94±2.27)%,(100.99±6.55)% and(90.76±1.63)%,indicating that sophoroside at the concentration of 20μmol/L to 80μmol/L had no obvious cardiomyocytotoxicity,and at 100μmol/L had cardiomyocytotoxicity.The difference was not statistically significant.2.The results of MTT showed that the cardiomyocyte activity of H9c2 cardiomyocytes treated with 1μmol/L doxorubicin for 24 hours was(53.89±2.27)%.After different concentrations of sophoroside and treatment,the cardiomyocyte activity of H9c2 was(61.36±3.27)% and(75.54±2.71)%,respectively.3.The results of Hoechst 33342 staining showed that after the H9c2 cardiomyocytes were treated according to the pre-designed treatment method,the nucleus of the blank control group was almost invisible;the nucleus of the positive control group was bright,and the chromatin of the positive control group was condensed and divided;compared with the positive control group,the bright blue spots decreased in the sophoroside pretreatment group.4.The results of flow cytometry showed that after treatment,compared with the model control group,the apoptosis of cardiomyocytes decreased significantly after sophoroside pretreatment,and the apoptosis rate of sophoroside high concentration group was lower than that of sophoroside low concentration group,and the difference was statistically significant.5.The results of DCFH-DA probe staining showed that ROS production was significantly increased after H9c2 cells were treated with doxorubicin for 24 hours,while ROS production after sophoroside pretreatment was significantly lower than that after doxorubicin treatment.6.Western Blot results showed that doxorubicin could increase the expression of apoptosis-related proteins Bax and Cleaved-caspase3,decrease the expression of Bcl-2,increase the phosphorylation of p38 MAPK and NF-κB signal pathway related proteins p38,ERK,JNK,and increase the expression of NF-κB.Sophoroside could alleviate the changes induced by doxorubicin.7.After the addition of p38 agonist P79350,the effect of sophoroside disappeared,which proved that sophoroside attenuated the injury of H9c2 cardiomyocytes induced by doxorubicin by inhibiting p38/MAPK/NF-κB signal pathway.ConclusionSophoroside down-regulates the expression of proteins related to the p38/MAPK/NF-κB signal pathway,thus reducing the production of ROS and alleviating the cardiomyocyte injury induced by doxorubicin.