The Role Of MAPK Pathway In MC-LR-induced Apoptosis Of Mice Ovarian Granulosa Cells

ObjectiveThe purpose of this study is to explore the role of apoptosis signal-regulating kinase 1(ASK1)-mediated MAPK signaling pathway in microcystin-leucine arginine(MC-LR)-induced apoptosis of mice ovarian cells and the activation mechanism of ASK1 after exposed to MC-LR.It will provide a new research idea for further exploring the reproductive toxicity of MC-LR,and also provides a reference for comprehensive evaluation of MC-LR-induced female reproductive system damage.MethodsThe 6-week-old female C57BL/6 mice were used as animal models and the mices were randomly divided into 4 groups with 5 mice in each group.Mices were injected with different concentrations of MC-LR(0,40 μg/kg·bw),ASK1 inhibitor NQDI-1(20 mg/kg·bw)and MC-LR combined with NQDI-1(40 μg/kg·bw MC-LR+20 mg/kgμbw NQDI-1)for 14 days.The content of MC-LR in serum was detected and the gonadal index was calculated in each group.The pathological changes of ovarian tissues were observed by H&E staining.TUNEL staining was used to observe the apoptosis rate of granulosa cells in ovarian tissues of mice.The mice ovarian granulosa cells KK-1 was used as the research object and randomly into control group,exposure group(MC-LR,34 μg/mL),NC group,NC+MC-LR(34 μg/mL),siASK1 group and siASK1+MC-LR(34 μg/mL)group.After cells were cultured for 24 h,the mitochondrial membrane potential level and apoptosis rate were detected.RT-qPCR and western blotting were used to detect the relative expression levels of apoptosis-related genes and proteins in ASK 1-mediated MAPK pathway.In order to analyze the regulation of MC-LR on the transcription level of ASK1,according to the promoter sequence of ASK1,the apoptosis-related transcription factors regulating ASK1 were predicted and screened on the ALGGEN-PROMO website.KK-1 cells were exposed to MC-LR at 0,8.5,17,34μg/mL.RT-qPCR was used to detect the mRNA levels of these transcription factors.Finally,according to the comprehensive analysis of the results,the most likely transcription factors regulating ASK1 was found.In order to reveal the regulation of oxidative stress induced by MC-LR on the expression of phosphorylated ASK1,the KK-1 cells were used as the research object.The cells were exposured to different concentrations of MC-LR(0,34 μg/mL),NAC(10 mmol/L),and MC-LR combined with NAC(34 μg/mL MC-LR+10 mmol/L NAC)for 24 h.After 24 h of exposure,the relative expression of pASK1 protein was detected by western blotting.ResultsMC-LR can induce the decrease of gonadal index and pathological damage of ovarian tissues in mice after the adolescent mice exposure to MC-LR,while the ASK1 inhibitor NQDI-1 can effectively alleviate the phenomenon caused by MC-LR,and the difference was statistically significant compared with the group exposed alone(P<0.05).In addition,MC-LR can also increase the number of apoptosis positive cells in ovarian tissues of adolescent mice.However,with the exposure group alone,the number of apoptosis positive cells were relatively reduced in the NQDI-1 pretreated group,and the difference was statistically significant(P<0.05).MC-LR can cause the decrease of mitochondrial membrane potential and the increase of apoptosis rate in KK-1 cells,when compared with the control group,the difference was statistically significant(P<0.05).However,siASK1 pretreatment can ameliorate the changes of mitochondrial membrane potential level and apoptosis rate in KK-1 cells caused by MC-LR.Afrer exposure to MC-LR,the mRNA levels of apoptosis-related genes JNK,P38,MEF2C,Fasl and DDIT3 were increased,and the expressions of apoptosis-related proteins p-JNK,p-P38,Fas and P53 were also increased.Compared with the exposure group,MC-LR-induced the increase in these genes and proteins were suppressed by ASK1 inhibitor and the difference was statistically significant(P<0.05).Based on the bioinformatics analysis of the ASK1 promoter,some potential transcription factors were found.Of them,JunD,c-Jun,JunB and Nrf2 were related to apoptosis.PCR results showed that MC-LR can increase the mRNA expression levels of transcription factors JunD,c-jun,Nrf2 and JunB.Moreover,the mRNA levels of Nrf2 and JunB were gradually increased with the increase of the MC-LR concentration,and the differences were statistically significant(P<0.05).Based on the above results and previous research results,it is preliminarily inferred that Nrf2 participates in the process of ASK1 upregulation by MC-LR.The results of the study on the regulation of MC-LR-induced oxidative stress on ASK1 phosphorylation show that MC-LR can obviously increase the relative expression level of phosphorylated ASK1,and the difference was statistically significant when compared to control group(P<0.05).After the pretreatment with antioxidant NAC,the expression level of phosphorylated ASK1 protein was significantly reduced compared with the exposure group,and the difference was statistically significant(P<0.05).Conclusions1.MAPK signaling pathway is involved in MC-LR-induced apoptosis of mice ovarian granulosa cells,and ASK1 has the key regulatory role in MAPK pathway.2.MC-LR can regulate the function of ASK1 in the level of protein phosphorylation and transcription,and then activate the ASK1-mediated MAPK pathway.