The Cardioprotective Effect Of Ginsenosides Of Total Saponins From Stems And Leaves Of Panax Ginseng And Ginsenoside Re On Cisplatin-evoked Cardiotoxicity In Mice And H9c2 Cells And Its Molecular Mechanism

Cisplatin（CDDP）,the first platinum chemotherapy drug,is still considered to be one of the most effective and commonly used chemotherapy drugs in clinic.Cisplatin has a significant antitumor effect,either alone or in combination with other drugs.However,its effectiveness is often limited by tissue toxicity,such as renal toxicity and ototoxicity,which have previously been reported to be caused by cisplatin.At present,the adverse reactions caused by cisplatin,such as acute and chronic cardiovascular complications,are also reported one after another.Its main clinical symptoms are myocarditis and cardiomyopathy.These adverse reactions greatly limited the use of cisplatin as a highly effective chemotherapy drug.Although researchers do not fully understand the cardiotoxicity of cisplatin,recent studies have shown that oxidative stress,apoptosis,and inflammation play a role in cisplatin induced injury.Saponins from the leaves of Panax quinquefolium L.（PQS）is considered as the main ingredient in the stems and leaves of Panax quinquefolium L.Recent studies have shown that panax ginseng saponins have a variety of pharmacological activities that can be used clinically,including reducing oxidative stress damage,anti-inflammatory and scavenging free radicals.In order to investigate the protective effect of PQS on cisplatin and the potential molecular mechanism,an in vivo model of cisplatin induced myocardial injury was established.Cisplatin’s cardiotoxicity is associated with increased oxidative stress,inflammation,and apoptosis,which limit its clinical use as an antitumor agent.In this study,mice in the PQS group were given different doses of PQS by gavage.Mice in cisplatin group and PQS group were given intraperitoneal injection of cisplatin(3 mg kg^-1)for four times on day 7,9,11 and 13 after PQS,respectively to induce cardiac toxicity in mice.Experimental data showed that cisplatin group presented the histopathological changes of the heart in mice and serum ALT（alamine aminotransferase）,LDH（lactic dehydrogenase）,CK（creatine kinase,CK）,CK–MB（creatine kinase MB）and c Tn T（cardiac troponin T）activity increases.Cisplatin also disrupted the antioxidant defense system,leading to increased activity of GSH（glutathione）and SOD（superoxide dismutase）and MDA（malondialdehyde）in heart tissue,and these changes were mitigated by PQS.In addition,preadministration of PQS reduced the overexpression of Tumor necrosis factor cytokines（TNF-α）and interleukin-1（IL-1β）,suggesting that PQS could reduce cisplatin induced inflammatory response.Our immunohistochemical results of TUNEL and Bcl-2 demonstrated that PQS can reduce cisplatin induced apoptosis.H&E,Hoechst 33258 and TUNEL staining results showed that PQS could effectively improve the changes of cell structure in heart tissue and significantly reduce cisplatin induced apoptosis of cardiomyocytes,indicating that PQS inhibited apoptosis and inflammatory response in mice.In conclusion,PQS may exert cardioprotective activity against cisplatin-induced cardiotoxicity by inhibiting oxidative stress,inflammation,and apoptosis.The monomer saponins in the stems and leaves of panax quefolium are similar to the monomer saponins in ginsenosides of panax quefolium,and there are about 40 species.The content in ginseng is around 4%.Ginsenoside Re（G-Re）is one of the most extensively studied saponins.Numerous studies have shown that it has higher activity to cardiac muscle cells,non-toxic side effects,and strong synergistic effect with other drugs for the treatment of heart disease.Therefore,we established a mouse model of CDDP induced cardiotoxicity to observe the protective effect of ginsenoside Re on CDDP induced cardiotoxicity.Our results showed that mice in cisplatin exposure group showed significant cardiac damage,histopathological changes,and serum LDH,CK,CK-MB and c Tn T concentrations increased significantly.In addition,we also found that Re significantly alleviated inflammation and apoptosis induced by oxidation of CDDP.The results showed that Re could improve the inflammatory response of cisplatin to cardiomyocytes by inhibiting the overexpression of inflammatory factors such as TNF-αand IL-1βcytokines.Furthermore,we used fluorescence quantitative PCR to confirm that CDDP not only increased the levels of TNF-αand IL-1βin serum,but also increased the RNA levels of these two pro-inflammatory factors in mice,while Re significantly inhibited the changes of the levels of these molecules.At the same time,Re can promote the decreased protein expression level of Bax and cleaved caspase-3 and the increased expression level of anti-apoptotic protein Bcl-2.In addition,our hypothesis from in vivo studies was validated in cisplatin-administered H9C2cells in vitro.Compared with cisplatin group,cells pretreated with 5m M/L AMPK inhibitor（Compound C）increased the expression level of cleaved caspase3 protein due to cisplatin,while pretreated with Re（10,20,30M）for 48 hours significantly reduced the increase in cleaved caspase3 protein level.In addition,we also used JNK and Erk inhibitors（SP600126 and U0126,respectively）to further test the MAPK signaling pathway.Our results show that the addition of these two inhibitors,through the inhibition of JNK and ERK phosphorylation,leads to excessive apoptosis of cardiac cells,aggravating cardiac damage.Furthermore,our Hoechst 33258,flow cytometry,immunohistochemical staining of Bcl-2,and immunofluorescence analysis of caspase-3 further confirmed that Re can effectively inhibit the apoptosis of cardiomyocytes.In conclusion,the results showed that PQS and Re had good protective activities against oxidative stress,inflammation and apoptosis induced by cisplatin in mice,and the possible molecular mechanism was to improve CDDP induced heart by inhibiting the activation of a series of signaling pathways including caspase-3/caspase-9 and MAPK and NF-k B in vivo.Traditionally,panax quinquefolium is only used in the underground part including the root and rhizome,resulting in the waste of the aboveground part.And the research proves that not only the stem leaf total saponins of American ginseng PQS has a good heart protection,and proves that its main monomer saponin play a protective role,not only to provide guidance for clinical drug use,and provide reference for developing and using American ginseng meaning,if we can better to the field of health care,will have more important clinical significance.