| ObjectiveThe disease model of premature ovarian insufficiency(POI)in rats was constructed with 4-vinylcyclonhexene diepoxide(VCD),to explore the mechanism of Huyang Yangkun Formula in protecting and improving the ovarian function of POI by regulating the expression of aquaporins(AQPs),Fas,TNF and Bcl-2 signaling pathways.MethodsExperiment 1:The protective effect of Huyang yangkun Formula on the ovarian function of VCD-induced premature ovarian insufficiency model of ratsSPF female SD rats were randomly divided into 5 groups using a random number table method,10 rats per group:normal control group(NCN),adjuvant control group(SCN),model group(MOD),and Huyang Yangkun Formula intervention group(HYF),and estradiol valerate tablets intervention group(EVT).Normal control group(NCN):the animal experiments were performed from day 1 to 15 without intervention;from day 16 to the end of the experiment,normal saline was administered by gavage.The standard volume of gavage was1 m L per 100g of body weight.Adjuvant control group(SCN):from the first day to the 15th day of the animal experiment,the same volume of sesame oil as the VCD solution in the model group was injected intraperitoneally;from the 16th day to the end of the experiment,saline was administered to the stomach,the volume standard is 1m L per 100g body weight.Model group(MOD):from the first day to the 15th day of the animal experiment,the VCD solution was given intraperitoneally,and the VCD concentration was 160mg/kg according to the reference,which was converted into a solution volume of0.59m L per 100g of body weight;from the 16th day to the end,gavage was performed with saline,and the standard volume of gavage was 1m L per 100g of body weight.Huyang Yangkun Formula intervention group(HYF):from the first day to the 15th day of the animal experiment,the VCD solution was given intraperitoneally,and the VCD concentration was 160mg/kg according to the reference,which was converted into a solution volume of 0.3m L per100g of body weight;From the 16th day to the end of the test,Huyang Yangkun Formula water extract was administered to the stomach,and the standard volume of the stomach was 1m L per 100g of body weight.Estradiol valerate intervention group(EVT):VCD solution was given intraperitoneally from day1 to day 15 of the animal experiment.According to the reference,VCD concentration was 160mg/kg,which was converted into a solution volume of0.59m L per 100g body weight.From the 16th day to the end of the test,the estradiol valerate aqueous solution was administered by gavage,and the standard volume of gavage was 1m L per 100g of body weight.At the end of the test,the body weight,ovarian and uterine weights of rats were measured to calculate the organ coefficients,the serum levels of liver and kidney function were measured,the ovarian tissue was made into pathological sections and the ovarian morphology was observed after HE staining,and the follicles were counted,serum levels of FSH,AMH and E2were measured by Elisa.Experiment 2:Study on the mechanism of protective effect of Huyang Yangkun Formula on the ovarian function of rats POI model mediated by aquaporinsThe ovarian tissues of rats in all groups were obtained from experiment1.Part of ovarian tissue was taken to extract total protein,using Western-Blot method to detect protein expression in the ovaries,including AQP1,AQP5 and AQP8,and Bcl-2,Bcl-XL,Mcl-1,Bax,Bak,Bim,caspase3,caspase9,FAS,FASL,TNFR1,NF?B(P65),P-P65 in classical apoptosis signaling pathways.Pathological sections and immunohistochemical staining were performed on some ovarian tissues,and the protein expressions of AQP1,AQP5,AQP8,and AQP9 among the groups were observed under a light microscope,and quantitative analysis was performed by filming.Pathological sections and TUNEL staining of some ovarian tissues were used to observe the apoptosis of rat ovarian sections.ResultsExperiment 1:The protective effect of Huyang yangkun Formula on the ovarian function of VCD-induced premature ovarian insufficiency model of rats1.Changes of serum liver and kidney function in rats:the analysis results of serum ALT,AST,ALT/AST,bun and Cr showed that there was no statistical difference between each group(P values were 0.064,0.278,0.643,0.626 and 0.134,respectively,which were greater than 0.05).2.Ovarian organ index of the rats:the analysis results of ovarian organ index showed that there was significant difference in the mean number of each group(F=2.951,P=0.035,P<0.05).The difference was mainly reflected between the adjuvant control group and the model group(P=0.021,P<0.05).3.The change of ovarian volume and mean ovarian product:the analysis of ovarian volume showed that there was significant difference in the mean of each group(F=3.726,P=0.009,P<0.01).Compared with the normal control group,the ovarian volume of the model group was smaller,but the difference was not statistically significant;compared with the model group,the ovarian volume of the Huayang yangkun Formula group was significantly increased,the difference was statistically significant(P=0.028,P<0.05).There was significant difference in the mean ovarian product of rats(F=3.914,P=0.007,P<0.01).Compared with the normal control group,the mean ovarian product of the model group was smaller;compared with the model group,the mean ovarian product of the Huyang yangkun Formula group increased,but there was no statistical significance.4.Morphological changes of the rat ovaries:under light microscope,follicles of different development stages could be seen in the cortex of normal control group,there were abundant blood vessels in medulla;follicles and blood vessels at different development stages could be observed in ovarian tissue of adjuvant control group;ovarian tissue of model group was atrophic at different degrees,and the number of follicles at development stage was significantly reduced;ovarian tissue of Huyang yangkun Formula intervention group could be observed at different development stages and abundant blood vessels;in estradiol valerate intervention group,the number of follicles in the development stage is less.5.The number of follicles at all levels in the ovaries of rats:the analysis results of the number of primordial follicles showed that there was a significant difference between each group(H=17.044,P=0.002,P<0.01).Compared with the normal control group,the number of primordial follicles in the model group decreased;compared with the model group,the number of primordial follicles in the Huyang yangkun Formula group increased.Among them,the number of primordial follicles in estradiol valerate group decreased significantly compared with that in normal control group and adjuvant control group(P<0.05).The results of primary follicle analysis showed that there was no significant difference between each group(F=0.778,P=0.564,P>0.05).There was no significant difference between the secondary follicle values(F=3.189,P=0.070,P>0.05).The results of antral follicles showed that there was a very significant difference between each group(F=25.308,P=0.000,P<0.01).Compared with normal control group and adjuvant control group,the number of antral follicles in model group decreased significantly(P<0.01).The analysis results of total follicle number showed that there was a significant difference between each group(F=11.688,P=0.000,P<0.01).Compared with the normal control group and adjuvant control group,the total number of follicles in the model group decreased significantly(P<0.01).Compared with the model group,the total number of follicles in Huyang yangkun Formula group increased significantly(P=0.045,P<0.05).6.Uterine organ index of the rats:the analysis results of uterine organ index showed that there was no statistical difference between the values of each group(H=4.619,P=0.329,P>0.05).7.Pathological changes of rat uterus:the results show that there is no visible difference between the groups from the endometrial thickness,the number of glands in the lamina propria and the size of the gland cavity.However,compared with other groups,the myometrium and adventitia of estradiol valerate group were denser.8.Detection of serum hormone level of rats:the analysis results of serum AMH value of rats showed that there was statistical difference between the values of each group(H=13.307,P=0.010,P<0.05).Compared with the normal control group,the AMH level of the model group decreased;compared with the model group,the AMH of the Huyang yangkun formula group increased significantly,with statistical difference(P=0.011,P<0.05).The results of the analysis of serum E2showed that there were statistical differences between the groups(H=16.254,P=0.003,P<0.01).There were significant differences between the model group and the normal control group,and between the estradiol valerate group and the model group(P values were 0.042 and 0.010,respectively,less than 0.05).Experiment 2:Study on the mechanism of protective effect of Huyang Yangkun Formula on the ovarian function of rats POI model mediated by aquaporins1.The result of TUNEL staining in rat ovarian sections:there were follicles of different developmental stages in the normal control group,and a few apoptotic signals were scattered in the ovaries;there were fewer tfollicles in the model group and more apoptotic signals were detected;the follicles at different developmental stages were also observed in the Huyang Yangkun Formula group,and the signal of apoptosis was occasionally seen in the ovary;fewer developing follicles and fewer apoptotic signals were observed in the estradiol valerate group.2.In the death receptor pathway of apoptosis,there was no significant difference in the protein expression of FAS,Fasl,TNFR1,NF?B P65 and p-P65 in rat ovaries.3.The expression of Bcl-2 family proteins in the mitochondrial pathway of apoptosis:BCL-2and BCL-XL was decreased in the MOD group;while compared to the MOD group,BCL-XL was significantly upregulated in the HYF group and EVT group.MCL-1was increased in the MOD group and downregulated in the HYF group.BAX and BIM were significantly upregulated in the MOD group and down regulated in the HYF/EVT group.There was no significant result among four groups about BAK.The ratio of BCL-2/BAK and BCL-XL/BAX could reflect the anti-apoptosis:both BCL-2/BAX and BCL-XL/BAX were decreased in the MOD group,and only BCL-XL/BAX was increased in the HYF group and EVT group.All these results indicated that BCL-2 family members from the mitochondrial apoptosis pathway were involved in the inhibition of apoptosis by HYF treatment.There was no significant difference among the four groups in terms of Pro-caspase3,but the expression of Cleaved-caspase3 was up-regulated in the MOD group and down-regulated in the HYF group.Although there was no difference in the expression of Caspase9 between the CON group and the MOD group,the expression of Caspase9 in the HYF group was obviously down-regulated,and there was also a down-regulation in the EVT group when compared to the MOD group.4.Detection and analysis of AQPs protein in rat ovaries by WB:The expression of AQP8 in the model group was higher than that in the normal group.Compared with the model group,the expression of AQP8 in the Huyang Yangkun Formula Group and the estradiol valerate group were decreased,but there was no significant difference among the groups(F=2.808,P=0.108,P>0.05).5.IHC analysis of AQPs protein in rat ovaries:Welch's variance analysis of AQP1 values in each group showed that there was a statistical difference among the groups(F=8.027,P=0.000,P<0.01).Compared with normal group and model group,the expression of AQP1 protein in Huyang Yangkun Formula group was significantly decreased(p=0.003,P<0.01),which indicated that Huyang Yangkun Formula had a significant effect on AQP1expression.The results of Kruskal-Wallis test of AQP5 in each group showed that there was significant difference among the groups(H=9.267,P=0.026,P<0.05).The results showed that AQP5 protein expression was increased in model group and decreased in Huyang Yangkun Formula group.The results of Kruskal-Wallis test of AQP8 showed that there was significant difference among the groups(F=17.076,P=0.001,P<0.01).The results showed that the expression of AQP8 in model group was significantly higher than that in normal control group(P=0.001,P<0.01).Compared with the model group,the expression of AQP8 in Huyang Yangkun Formula group was significantly decreased(P=0.01,P<0.05).Conclusion1.In this study,VCD modeling method was feasible,and the establishment of premature ovarian insufficiency SD rat model was completed;2.Huyang Yangkun Formula could improve the serum AMH level of POI rats,promote the development of follicles and protect the ovaries.3.Huyang Yangkun Formula regulated the expression of apoptosis signaling pathway especially Bcl-2family in ovaries.4.Huyang Yangkun Formula reduced the over-expression of AQPs caused by VCD.5.The expression of AQPS may be related to the apoptosis signaling pathway.Huyang Yangkun Formula may slow down the atresia of follicles and enable more follicles to mature by decreasing the expression of AQPs to regulate the apoptosis signaling pathway. |
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