| Deceased ovarian reserve refers to a pathological condition in which the number and quality of oocytes available for collection in the ovary decrease.Currently,there is no clear and effective treatment method.Our group's preliminary clinical study found that Qilin pills can improve the AMH level of DOR patients with deficiency of spleen and kidney and insufficiency of blood flow,improve their menstruation and TCM syndromes of deficiency of spleen and kidney,but there is a lack of relevant basic research,and its mechanism of action needs to be clarified urgently.Objective1.To study the effects of Qilin pills and hyperin,one of its main components,on ovarian function and fertility of DOR mice and its mechanism.2.To study the effects of hyperin on proliferation,apoptosis,mitochondrial membrane potential,autophagy and hypoxia of damaged granulosa cells.Methods1.Animal experiment:Based on hypoxia-related autophagy,the effects of Qilin pills and hyperinon ovarian function and fertility of DOR mice were studiedA total of 48 SPF female C57BL/6 mice were randomly divided into blank group,model group,hyperin group and Qilin pills group,with 12 mice in each group.Qilin pills group was intragastrically administrated with Qilin pills solution at 5.46 g·kg-1·d-1,hyperin group was intragastrically administrated with hyperin solution at 40mg·kg-1·d-1,and the other two groups were intragastrically administrated with 0.5%CMC-Na solution at the same volume for 5 consecutive weeks.After one week of drug intervention,the model group,hyperin group and Qilin pills group were intraperitoneally injected with cyclophosphamide solution75mg·kg-1,and the blank group was injected with isovolumetric sodium chloride injection,once a week,4 times in total.Body weight and estrus cycle of mice were monitored during the administration period.After the end of administration,6 mice in each group were randomly selected and killed for sampling.Record mouse ovarian wet weight,ELISA method to detect serum AMH level in mice,FSH and E2 levels,HE observe ovarian tissue structure,and the statistical number of follicles at different levels,RT-qPCR method and Western Blot method respectively to detect ovarian LC3B,P62,Bnip3,Beclin1,HIF-1?mRNA and protein level,immunohistochemical method to detect LC3B and HIF-1? positive expression in mouse ovarian.The other 6 mice in each group were fed normally for 7 weeks,and then caged with SPF male C57BL/6 mice in a ratio of 2:1 for 4 weeks.The time required for conception,the total number of conception,the number of absorbed fetuses and the number of normal offspring were recorded.2.Cell experiment:Effects of hyperin on proliferation,apoptosis,hypoxia and autophagy of damaged granulosa cells(1)Effects of different concentrations of 4-HC on proliferation,hypoxia and autophagy of KGN cellsKGN cells were treated with 2?M,4?M,8?M and 16?M 4-HC for 6h,12h,24h and 48h,respectively,and the cell proliferation level of each group was detected by CCK-8 method.KGN cells were treated with 4-HC at 2?M,4?M and 8?M concentrations for 24h.The protein levels of LC3B-?,P62 and HIF-1?were detected by Western Blot.(2)The effect of hyperinat different concentrations on the proliferation of KGN cells KGN cells were treated with 0.1?M,1?M,10?M,100?M and 1m M hyperin for 24h,respectively,and cell proliferation was detected by CCK-8.(3)Effects of hyperin at different concentrations on proliferation and apoptosis of amaged KGN cellsKGN cells were treated with 4-HC at 8?M for 24h,and then treated with hyperin at .1?M,1?M,10?M and 100?M for 24h,respectively.Cell proliferation was detected by CCK-8 method,and apoptosis level was observed by Hoechst staining.(4)The effect of hyperin on hypoxia and autophagy of damaged KGN cellsKGN cells were treated with DMSO,4-HC,hyperin,4-HC+hyperin for 24h, espectively.The protein expression levels of LC3B,P62,HIF-1?,BNIP3 and Beclin1 in each group were detected by Western Blot,and the changes of mitochondrial membrane potential were detected by JC-1 staining.After transfected with mCherry-GFP-LC3 plasmid,KGN cells were treated with DMSO,4-HC,hyperin,4-HC+hyperin for 24h,respectively,and the changes of autophagy flow were observed under confocal microscope.Results1.Animal experiments(1)Changes in general conditions of miceIn the model group,the hair was dull,the dorsal arch was curled up,the activity was educed,and there were thin stools.Compared with model group,mental state and behavioral activity of mice in hyperin roup and Qilin pills group were improved,and urine and feces were normal.(2)Changes of estrus cycle in miceFrom the first week of cyclophosphamide administration,the estrus interphase of mice in odel group was prolonged until it stopped in estrus interphase.Hyperin group and Qilin pills group showed disturbance of estrus cycle in the second eek after cyclophosphamide administration,and the proportion of estrus interphase increased,but the estrus phase was always present.(3)Changes in body weight of miceCompared with blank group,the body weight of model group was significantly decreased at D13,D17,D21 and D25(P<0.05 or P<0.01).The body weight of hyperin mice was decreased at D13,D25 and D33(P<0.05 or P<0.01).The body weight of Qilin pills group was significantly decreased at D13,D19,D21,D23,D25,D27,D31,D33 and D35(P<0.05 or P<0.01).(4)Comparison of ovarian index in miceCompared with blank group,ovarian index in model group,hyperin group and Qilin pills group was significantly decreased(P<0.01).Compared with model group,the ovarian ndex of hyperin group and Qilin pills group was increased(P<0.05).(5)Comparison of AMH,FSH and E2 levels in serum of miceCompared with blank group,AMH level in model group,hyperin group and Qilin pills group was decreased(P<0.01,P<0.01,P<0.05),and FSH level in model group and yperin group was significantly increased(P<0.01,P<0.05).Compared with model group,there were no significant differences in FSH,AMH and E2 levels in hyperin group(P>0.05).AMH was increased in Qilin pills group(P<0.05),but FSH and E2 levels were not significantly changed(P>0.05).(6)Comparison of the number and proportion of different levels of follicles in miceCompared with blank group,the number of primary follicles,primary follicles and secondary follicles in model group were decreased(P<0.01,P<0.05,P<0.01),and the number and proportion of atresia follicles were significantly increased(P<0.05 or P<0.01).Compared with model group,the number of primordia follicles in hyperin group was ncreased(P<0.01),while the number and proportion of atresia follicles were decreased(P<0.05,P<0.01).The number of primary follicles in Qilin pills group was significantly ncreased(P<0.01),the number of atretic follicles was decreased(P<0.01),and the roportion of developing follicles and atretic follicles was decreased(P<0.05,P<0.01).(7)Comparison of fertility in miceCompared with blank group,model group had longer conception time(P<0.05),and ncreased the number of absorbed fetuses(P<0.05).Compared with model group,the conception time of hyperin group and Qilin pills group as shortened(P<0.05),the number of absorbed fetuses in hyperin group was reduced(P<0.05),and the number of absorbed fetuses in Qilin pills group had no significant change(P>0.05),and there were no significant differences in the total number of pregnant mice and the number of healthy young mice among all groups(P>0.05).(8)Comparison of autophagy levels in mice ovariesCompared with blank group,LC3B protein and mRNA levels in model group were ignificantly increased(P<0.01),while P62 protein level was decreased(P<0.01).Compared with model group,LC3B mRNA and protein levels of mice in hyperin group were decreased(P<0.01,P<0.05),and P62 protein was increased(P<0.01).LC3B mRNA and protein levels in Qilin pills group were significantly decreased(P<0.01),while P62 mRNA and protein levels were increased(P<0.05).(9)Comparison of the expression levels of HIF-1?/BNIP3/Beclin1 pathway in mouse variesCompared with blank group,the mRNA and protein levels of BNIP3 and Beclin1 in ovarian of mice in model group were significantly increased(P<0.01),and the protein expression of HIF-1?was significantly increased(P<0.01).Compared with model group,BNIP3 mRNA and protein in hyperin group and Qilin pills group were decreased(P<0.01),and Beclin1mRNA expression in Qilin pills group was decreased(P<0.05).There was no significant difference in HIF-1?mRNA levels among all groups(P>0.05).2.Cell experiments(1)4-HC had significant inhibitory effect on the proliferation of KGN cells in a time and oncentration dependent manner.The inhibition rate of KGN cells could reach 50%after treated with 8?M 4-HC for 24h.8?M 4-HC significantly increased the levels of LC3B-?and HIF-1?in KGN cells and decreased the levels of P62 protein in a oncentration-dependent manner(P<0.05,P<0.05,P<0.01).(2)Hyperin at 1?M and 10?M significantly promoted the proliferation of KGN cells(P<0.01).Hyperin could significantly inhibit the proliferation of KGN cells(P<0.01).(3)Compared with 4-HC group,hyperin concentration of 10?M could significantly romote the proliferation of damaged granulosa cells(P<0.01).Hoechst staining showed that 4-HC could induce apoptosis in KGN cells.Hyperin10?M could significantly inhibit the apoptosis of damaged KGN cells.(4)Compared with DMSO group,the protein expressions of LC3B-?,HIF-1?,BNIP3 nd Beclin1 in 4-HC group were increased(P<0.05 or P<0.01),while the protein expression of P62 in 4-HC group was inhibited(P<0.01).Compared with the 4-HC group,the protein levels of LC3B-?,HIF-1?,BNIP3 and eclin1 in the 4-HC+hyperin group were decreased,and the protein levels of P62 were significantly increased(all P<0.01),suggesting that hyperin inhibited hypoxia and utophagy activation induced by 4-HC.(5)JC-1 staining showed that the mitochondrial membrane potential in 4-HC group was ignificantly lower than that in DMSO group.Compared with the 4-HC group,the level of mitochondrial membrane potential in the 4-HC+hyperin group recovered somewhat.(6)After mCherry-GFP-LC3 transfection,it was found that the cells in 4-HC group showed obvious aggregation of light spots,mainly red fluorescence,suggesting autophagy activation and patency.In the 4-HC+hyperin group,there were a few scattered autophagy spots,and the intensity of red fluorescence was similar to that of green fluorescence,suggesting that hyperin significantly inhibited the over-activation of autophagy flow induced by 4-HC.Conclusion1.Cyclophosphamide can successfully induce DOR mouse model,activate hypoxia and utophagy in mice ovaries,and lead to decreased ovarian reserve function and fertility in mice;2.Qilin pills and hyperin can restore ovarian reserve function and fertility of DOR model mice,which may be exerted by inhibiting over-activated autophagy and hypoxia.3.4-HC can inhibit the proliferation of KGN cells,reduce the mitochondrial membrane potential,induce apoptosis,hypoxia and autophagy activation.4.Hyperin can significantly promote the proliferation of KGN cells,restore mitochondrial membrane potential,inhibit cell apoptosis,reverse cell hypoxia,and inhibit over-activated autophagy. |
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