Protective Effect Of Geniposide On Cisplatin-Induced Cardiotoxicity And Discussion Of Its Related Mechanism

Background:Cisplatin is one of the most effective chemotherapeutic drugs in the treatment of many kinds of solid t umors,including gynecological malignant t umors.In recent years,more and more attention has been paid to the clinical reports of cisplatin-induced cardiotoxicity.Existing studies suggest that the cardiotoxicity of cisplatin is the result of many factors,such as the role of membrane transporters,metabolic toxicity,DNA damage,imbalance of intracellular ion homeostasis,excessive oxidative stress,inflammatory response,activation of apoptosis pathway and so on.Oxidative stress may be the main cause of cardiovascular injury.Cardiotoxicity seriously affects the quality of life of t umor patients,so to explore effective myocardial protective agents,optimize cardiac protection strategies,and improve cardiovascular events of t umor patients,at the same time,ensuring the anti-tumor efficacy of cisplatin is the focus of cisplatin research,but also faces many challenges.Geniposide,as a plant-derived antioxidant,has been used in neuroprotection,regulation of blood glucose,anti-hepatitis injury and other related treatments for many years because of its high pharmacological activity and low toxicity.However,the study of geniposide as a prevention of chemotherapy-related cardiotoxicity of antineoplastic drugs is just in its infancy.Some studies have suggested that geniposide can all eviate doxorubicin-induced myocardial injury by activating the classic antioxidant stress pathway Kelch-like ECH-associated protein 1(Keap1)/ Nuclear factor erythroid-2 related factor 2(Nrf2)/ Antioxidant responsive element(ARE).In addition,it has been reported that cisplatin-induced myocardial injury is also related to oxidative stress regulated by this signal pathway.Therefore,we speculate that geniposide may have a protective effect on cisplatin-induced cardiotoxicity,which may be related to the regulation of oxidative stress by up-regulating the expression of Nrf2 gene and promoting the expression of downstream antioxidant enzymes.At present,there are no reports on geniposide as a potential drug to protect cisplatin chemotherapy-related cardiotoxicity,and the upstream regulatory pathway of Nrf2 activation initiated by geniposide is unknown.Some studies have shown that phosphorylated endothelial nitric oxide synthase(e NOS)is a prerequisite for Nrf2 to play an oxidative stress role,and the phosphorylation of e NOS cannot be separated from the recruitment of protein kinase B(AKT)by heat shock protein 90 subtype α(HSP90AA1).The effective combination of HSP90 AA1,e NOS and AKT promotes the activation of downstream Nfr2,which regulates t he oxidative stress response.In this study,whether geniposide has protective effect on cisplatin-induced cardiotoxicity,and whether geniposide can protect cisplatin-induced cardiotoxicity by regulating HSP90AA1/e NOS/Nrf2 signal pathway is the focus of o ur attention and research.In recent years,the emerging methods of network pharmacology may provide a valuabl e theoretical basis for our scientific research.The purpose of this study is to find effective protective drugs for cisplatin-induced cardiotoxicity.Objective(1)To investigate the effect of geniposide on cisplatin-induced cardiomyocyte toxicity and anti-tumor effect,and select the appropriat e drug concentration for follow-up experiments.(2)To explore the mechanism of HSP90AA1/e NOS/Nrf2 signal pathway in the protective effect of geniposide on cisplatin-induced cardiotoxicity in vivo and in vitro.(3)To look for potential protective drugs for cisplatin-induced cardiotoxicity.Materials and Methods:(1)Based on the rat cardiomyocyte line H9C2 and ovarian epithelial cancer cell line SKOV3,the control group,different concentrations of cisplatin,geniposide group and cisplatin + geniposide group were set up.The cell viability of different groups was detected by CCK-8 method,the cell morphology was observed by crystal violet staining,and the apoptosis rate was detected by flow cytometry.(2)Based on Stitch database,Pharm Mapper database,Swiss target prediction database,Pubmed database,TCMID database,Uniprot database,OMIIM database and Genecards database,the material basis and mechanism of GE in the treatment of cardiotoxicity were discussed by means of network pharmacology,and the theoretical basis of HSP90AA1/e NOS/Nrf2 signal pathway in the protec tive effect of geniposide on cisplatin-induced cardiotoxicity was discussed.(3)HSP90AA1 plasmid and si RNA-HSP90AA1 were transfected into H9C2 cells by liposome transfection,respectively,to construct H9 C2 cells with overexpression of HSP90AA1 and H9C2 cells with knockdown of hsp90AA1 gene expression.(4)Normal H9C2 cells,H9C2 cells with overexpression of HSP90AA1 and H9C2 cells with low HSP90AA1 gene expression were divided into four groups(control group,cisplatin group,geniposide group,cisplatin + geniposide group),and healthy male Wistar rats were divided into control group,cisplatin group,geniposide group,cisplatin +geniposide group,cisplatin + geniposide + HSP90 specific inhibitor group.Fluorescence quantitative PCR was used to detect the expression of related genes involved in HSP90AA1/e NOS/Nrf2 signal pathway.The expression of proteins related to the signal pathway was detected by Western Blot.The activities and contents of oxidative stress molecules and myocardial enzymes were detected by spectrophotometer and flow cytometry.The weight and heart weight of experimental animals were measured by weighing method.The injury of cardiomyocytes was observed by paraffin embedding and section by hematoxylin-eosin(HE)staining.(5)Graph Pad statistical software package was used for data analysis.Statistical analysis methods included t-test and analysis of variance,P <0.05 was regarded as statistically significant.Results:The main results of the first chapter:(1)Compared with the H9C2 cell activity of the control group,the cell activity of different concentrations of cisplatin(2.5,5,10,20μM)were87.86±1.57%,75.11±1.63%,54.95±2.20% and 36.02±1.69% respectively under the condition of 24 hours culture.and the c ell activity of the corresponding concentration decreased gradually with the extension of culture time.Different concentrations of geniposide(25,50,100,200,250μM)had no inhibitory effect on the activity of H9C2 cells,and when the concentration was 100 μM and the culture time was 24,48,72 hours,the cell activity was 114.69±0.77%,109.52±0.86% and 105.01±0.25%,respectively,which was significantly higher than that of other concentrations at the same time.There were significant differences in the above data(P < 0.05).(2)Compared with the H9C2 cell activity of cisplatin(5μM)group,the cell activity of cisplatin + geniposide(50,100μM)group was significantly higher than that of cisplatin group.In particular,the cell activity increased most signifi cantly in the cisplatin + geniposide(100μM)group,which increased the cell activity of 75.53±0.97% in the cisplatin group to 96.60±1.40% in the cisplatin group in 24 hours and 88.67±1.07%in the cisplatin group in 48 hours,which was significantly better than the protective effect of geniposide at the concentration of 50μM on the cardiotoxicity of cisplatin.There were significant differences in the above data(P < 0.05).(3)Compared with the SKOV3 cell activity of different concentrations of cisplatin(5,10,20μM),the cell activity of cisplatin(5,10,20μM)+ geniposide(100μM)group(65.91±1.45%,28.84±0.35%,17.62±1.62%)was significantly higher than that of cisplatin(5,10,20μM)group(77.57±2.41%,44.82±1.89%,25.78±1.42%).We selected cisplatin(5μM)and geniposide(100μM)as the experimental concentrations for subsequent cell experiments.When SKOV3 cells were cultured for 24 hours,the apoptosis rate of cisplatin + geniposide group(18.16±0.75%)was significantly higher than that of cisplatin group(16.17±1.07).There were significant differences in the above data(P < 0.05).The main results of the second chapter:(1)The results of network pharmacological studies show that the overlapping targets of geniposide on cardiotoxicity include HSP90AA1 and AKT genes.The biological process of geniposide in the treatment of cardiotoxicity is mainly related to the response to ROS.The results of the first 20 signal pathways with the strongest enrichment correlation showed that HSP90,e NOS,AKT,Nrf2,NQO1 and HO-1 all formed the material basis of fluid shear stress and atherosclerosis signal pathway arranged in the second place of enrichment correlatio n,and participated in the transcriptional regulation of this signal pathway.(2)In normal and overexpressing HSP90AA1 cells,compared with cisplatin group,the m RNA amplification multiples of HSP90AA1(2.10±0.19 vs.1.13±0.10;3.28±0.12 vs.1.98±0.05),e NOS(3.94±0.18 vs.2.02±0.13;5.18±0.22 vs.3.53±0.10),AKT(2.22±0.22 vs.1.24±0.11;2.86±0.07 vs.1.33±0.46),Nrf2(2.08±0.15 vs.1.21±0.16;3.88 ±0.14 vs.1.84 ±0.07),NQO1(2.09±0.18 vs.1.20±0.08;2.95±0.10 vs.1.63±0.11)and HO-1(2.84±0.34 vs.1.19±0.11;3.96±0.11 vs.2.05±0.11)were significantly increased in cisplatin + geniposide group;and the protein expressions of HSP90AA1,pe NOS,p AKT,Nrf2,NQO1 and HO-1 were also significantly increased,while the expression of e NOS and AKT proteins did not change significantly.After knocking down the expression of HSP90AA1,the expression of these genes and proteins decreased significantly in cisplatin + geniposide group,and the expression of pe NOS could hardly be detected.The above differences were statistically significant(P < 0.05).(3)In normal and overexpressing HSP90AA1 cells,compared wit h cisplatin group,the contents of SOD(252.23±4.86U/mg vs.152.07±3.86U/mg;304.19±13.80U/mg vs.172.82±3.51U/mg)and CAT(3.01±0.16U/g vs.1.54±0.11U/g;3.67±0.09U/g vs.1.78±0.10U/g)in cisplatin + geniposide group increased significantly,while the contents of ROS(29.93±4.83% vs.51.70±5.91%;25.17±1.50% vs.36.17±1.45%),MDA(10.46±0.25nmol/mg vs.17.46±0.48nmol/mg;8.63±1.13nmol/mg vs.12.56±0.49nmol/mg),and LDH(4117.56±114.27U/g vs.5821.62±176.64U/g;3229.40±94.06U/g vs.4770.65±127.94U/g)decreased significantly.After knocking down the expression of HSP90AA1,SOD and CAT in cisplatin + geniposide group were significantly lower than those in cisplatin + geniposi de group,while ROS,MDA and LDH in cisplatin + geniposide group were significantly higher than those in normal cells.The above differences were statistically significant(P < 0.05).The main results of the third chapter:(1)On the last day of the animal experiment,the average body weight(327.67±22.21 g vs.225.60±22.21g)and average heart weight(0.88±0.02 g vs.0.57±0.04g)in the cisplatin + geniposide group were significantly higher than those in the cisplatin group,while the average body weight(264.50±25.57g)and heart weight(0.77±0.01g)in the cisplatin +geniposide + geldanamycin group were significantly lower than those in the cisplatin + geniposide group.The above differences were statistically significant(P < 0.05).(2)Cisplatin + geniposide group compared with cisplatin group,the contents of CK(569.79±23.58U/L vs.1325.75±52.51U/L),CK-MB(68.76±3.94U/L vs.122.52±3.93U/L),LDH(454.35±7.75U/L vs.784.38±13.03U/L),MDA(17.56 ±0.74U/L vs.29.92±1.19U/L)decreased significantly,while the contents of SOD(608.10±9.79U/L vs.478.16±12.55U/L),CAT(8.55 ±0.32U/L vs.4.76±0.21U/L),NO(1.47±0.11U/L vs.0.43±0.03U/L),T-AOC(180.82±4.00U/L vs.76.47±1.73U/L)increased significantly.The content s of CK,CK-MB,LDH and MDA in cisplatin + geniposide + geldanamycin group were significantly higher than those in cisplatin + geniposide group,while the contents of SOD,CAT,NO and T-AOC were significantly decreased.The above differences were statistic ally significant(P < 0.05).(3)Compared with cisplatin group,HSP90AA1(2.44±0.08 vs.1.10±0.08),e NOS(4.58±0.25 vs.2.63±0.10),AKT(2.32±0.14 vs.1.24±0.06),Nrf2(2.84±0.06 vs.1.23±0.09),NQO1(2.86±0.09 vs.1.36±0.07),HO-1(3.07±0.11 vs.1.13±0.07)m RNA amplification multiples increased significantly in cisplatin + geniposide group.The expression of these genes in cisplatin + geniposide + geldanamycin group was significantly lower than that in cisplatin + geniposide group.The protein expression of HSP90AA1,e NOS,pe NOS,p AKT,Nrf2,NQO-1 and HO-1 in cisplatin + geniposide group was significantly higher than that in cisplatin group,but there was no significant change in AKT protein expression.The protein expression in cisplatin + geniposide +geldanamycin group was significantly lower than that in cisplatin +geniposide group,and the protein expression of pe NOS was the most significant.The above differences were statistically significant(P < 0.05).(4)There was no obvious injury reaction in myocardial HE staining in control group and geniposide group,while HE staining in cisplatin group and cisplatin + geniposide + geldanamycin group showed severe injury reaction,while cisplatin + geniposide group showed only slight injury reaction.Conclusion:1.Geniposide can protect the cardiotoxicity induced by cisplatin without affecting the anti-tumor effect of cisplatin.2.Oxidative stress may be the main mechanism of cisplatin-induced cardiotoxicity.Geniposide has a protective effect on cisplatin-induced cardiotoxicity in vivo and in vitro by up-regulating the expression of HSP90AA1,recruiting phosphorylated AKT,to furth er activate e NOS,and then activating downstream Nrf2/NQO-1/HO-1 signal pathway to regulat e oxidative stress.3.Geniposide can be used as one of the potentially effective drugs to protect cisplatin-induced cardiotoxicity.