| ObjectiveAfter established VCD-induced POI rat model(in vivo)and the VCD-induced COV434 apoptosis model(in vitro),the efficacy of Huyang Yangkun Formula(HYF)was evaluated through certain methods for the two models above.From the perspective of m6A RNA methylation modification,the m6A level and the protein expression of four methylases(or demethylases)were detected in rat ovarian tissues,and search for(de)methylases that may play a role in HYF’s effects;and the major protein factors in the JNK/HO1/P53-related BCL2 mitochondrial apoptosis pathway.The COV434 cell line was stably transfected with certein(de)methylase’ shRNA to further explore the anti-apoptotic molecular mechanism of HYF based on m6A RNA modification..MethodsBased on the previous experiments of our lab,the VCD-induced POI rat model was continuously used in this study.And a VCD-induced COV434 apoptosis model was established in vitro.Both in vivo and in vitro experiments were carried out for three gradations:pharmacodynamic phenotype,m6A RNA modification,and(anti)apoptotic molecular mechanisms.According to the resutlts of in vivo/vitro experiments,stable transfected cell lines were constructed and the mechanism verification experiments were carried out..1.Animal experiment:(1)Grouping and administration:28-day-old SD female rats were randomly divided into three groups:normal control group(CON),POI model group(VCD),and Huyang Yangkun Formula gavage group(HYF).At the starting,rats in VCD group and HYF group were given intraperitoneal injection of VCD(160mg/kg)for 15 days,and then rats in HYF group were given with HYF decoration for 100 days by gavage,while rats in CON group and VCD group were given an equal volume of 0.9%saline.(2)Specimen collection:During the experiment,blood serum was collected and vaginal exfoliation cytology were performed on 30 days,60 days and 100 days after administration of HYF;near the end of the experiment,blood serum was collected for five consecutive days by tail vein.Abdominal aorta serum and ovaries(including diameters and weight)of all rats were collected on the sacrifice days.(3)Tests and assays:① Pharmacodynamic phenotype:follicular counts,serum AMH/FSH/E2,etc.;② Targets detection:m6A modification levels(by m6A-ELISA,Dot blot and LC-MS/MS)and m6A-related catalytic enzymes(METTL3,METTL14,ALKBH5,FTO),upstream protein candidates(pJNK,P38,pP38,NRF2,HO1,NQO1,P53)and downstream members which related to mitochondrial apoptosis signaling pathway(BCL2,BCLXL,MCL1,BAX,BAK,pBIM,PUMA,Pro-caspase9,Cleaved-caspase9,Caspase3,Cleaved-caspase3);2.Cell experiment:COV434(human ovarian granulosa cells)were cultured according to the proper conditions,and cells which were in the logarithmic phase of growth were used for seeding.They were divided into five groups:blank control group(CON),model group(VCD),low/medium and high concentration groups of HYF(1HYF,2HYF,3HYF).In the in vitro experiment,HYF was given for pre-intervention and then VCD was given to induce apoptosis.m6A modification level and expressions of proteins mentioned in vivo experiment part.3.Molecular mechanism exploration:According to the results of m6A modification catalytic enzymes in vivo/in vitro experiments,certain shRNA-COV434 cell line was constructed for the exploration of molecular mechanism;m6A modification was verified and then the candidate targets regulated by m6A modification were selected,the potential targets were finally verified by MeRIP-qPCR.Results1.Animal experiment:Under the intervention of HYF,the ovarian volume was increased,the total follicle number didn’t show significant changes,but the number of mature follicles increased;serum AMH also increased,but there was no significant difference in serum FSH and E2 of both basal level and five consecutive days.But serum FSH and E2 showed a cyclical trend of recovery.The level of m6A RNA modification level was decreased in VCD group and increased in the HYF group by ELISA assay;the results of Dot blot and LC-MS/MS also showed same tendency,but there was no statistical difference.As for the expression of(de)methylases,METTL3 was down-regulated in VCD group but unchanged in HYF group,METTL14 had no significant change among the three groups,ALKBH5 was down-regulated in VCD group and up-regulated in HYF group,FTO was up-regulated in VCD group and down-regulated in HYF group.Under the intervention of HYF,there were several protein targets showed differences in groups:JNK,pJNK,NQO1,P53,BCLXL,BAK,Cleaved-caspase9,Caspase3 and Cleaved-caspase3.2.Cell experiment:Under the intervention of HYF,the m6A RNA modification level of cells was obvious down-regulated in VCD group and up-regulated in HYF groups,detected by Dot blot method.As for(de)methylases,only the expression of FTO was up-regulated in the model group and down-regulated in HYF groups,and the expression of METTL3 showed a downward trend but no statistical difference;there was no difference about METTL14 among the three groups,and nor was ALKBH5.Under the intervention of HYF in vitro,there also were several protein targets showed differences in groups:JNK,pJNK,NRF2,pP38,P53,BCLXL,BAX,BAK,pBIM,PUMA,Cleaved-caspase9 and Cleaved-caspase3.3.Molecular mechanism exploration:(1)According to the results of m6A modification catalytic enzymes in vivo/in vitro experiments,the gene of FTO was stably knockdown in COV434 cell line(kdFTO cell)for the further exploration of molecular mechanism.m6A modification level was verified in the kdFTO cell,and all the protein candidates were detected in kd FTO cell.m6A level was up-regulated after FTO knockdown;JNK,pJNK and P53 proteins were down-regulated and HO1 protein was up-regulated after FTO knock-down,BAX and PUMA were down-regulated,and Pro-Caspase9 was up-regulated and Cleaved-caspase9 was down-regulated.(2)Summarizing the above experimental results,the potential targets pool(Tp53,Jnk,Puma,Bax,Bak,etc.)that might be regulated by m6A RNA modification,the results of the qPCR/MeRIP-qPCR expreiments showedt that there were two tagets were verified to be associated with HYF or POI:there was no statistical difference in the total transcript of Tp53 among the three groups,but the m6A-Tp53 transcript was up-regulated in the VCD group and down-regulated in the HYF group,which was consistent with the results of the expression of P53 protein;the total transcript of Jnk was up-regulated in the VCD group and down-regulated in the HYF group,Consistent with the pJNK protein results,m6A-Jnk transcripts were down-regulated in the VCD group,but not in the HYF group.Besides,there was no specific result in other candidate targets.ConclusionThe FTO-mediated increasement of m6A modification on Tp53 transcript(m6A-Tp53)may promotes the translation of P53 protein,which might be the switch on P53 triggered-mitochondrial apoptosis pathway(BCL2 family signaling pathway)in ovarian granulosa cells.It might be a potential mechanism by which HYF exerts its effect of anti-apoptosis. |
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