| Part OneClinical effect and mechanism of melatonin in the treatment of ovarian insufficiencyObjective:To investigate the clinical efficacy and mechanism of melatonin(MT)in patients with early-onset ovarian insufficiency.Method:(1)All patients selected in this study were diagnosed with ovarian insufficiency in the obstetrics and gynecology department of 4 tertiary medical institutions from June 2014 to August 2016,a total of 322 cases.According to the principle of randomized control,the selected patients were divided into 2 groups,the observation group and the control group.Among them,patients in the observation group were treated with melatonin tablets(1 mg/day),and patients in the control group were treated with placebo.(2)We detected the levels of FSH and E2 in 1 day before treatment,1 month,3 months and 6 months after treatment.The blood collection time of FSH and E2 before treatment was 2-4 days of menstruation.When reviewing FSH and E2 in 1 month,3 months and 6 months after the treatment,B-ultrasound examination was required first,the intima was less than 5 mm,and there were no follicles? 10 mm in bilateral ovaries.If the above conditions are not met,all FSH and E2 data of the patient will be excluded.The levels of T lymphocyte,ROS,VEGF and serum IL-1,IL-8,INH-B and AMH in the two groups were detected 1 day before treatment,1 month,3 months and 6 months after treatment;the morning urine was collected 1 day before treatment and 6 months after treatment to measure the urinary 8-OHdG and 8-isoPGF2a levels;during the 6-month treatment period,the ovulation of the two groups was monitored.Except FSH and E2,other items were not affected by menstrual cycle.The corresponding results were statistically analyzed.(3)Statistical methods:spss19.0 software was used to analyze the data.Mean and standard deviation are used to describe continuous variables.If continuous variables are skewed,median and quartile range are used.Categorical variables use absolute percentages.Continuous variables were tested by student's t test.Chi square test was used to compare categorical variables.When p<0.05,the difference was statistically significant.Result:There was no significant difference in age,BMI,menstrual cycle between the observation group and the control group(P>0.05),indicating that the two groups of patients are comparable.After treatment:(1)The FSH level of the observation group was significantly lower than that of the control group(P<0.05),and the estrogen level gradually increased(P<0.05),which was time-dependent;(2)The menstrual cycle of the patients before and after treatment There was no significant difference(P>0.05);(3)The levels of ROS,VEGF,IL-1 and IL-8 in the observation group were significantly lower than those in the control group(P<0.05),and were time-dependent;Serum INH-B and AMH levels were significantly higher than those of the control group(P<0.05),which was time-dependent;(4)The percentage of G1/M lymphocytes in the observation group was significantly lower than that of the control group,which was time-dependent(P<0.05).0.05);(5)After 6 months of treatment,urine 8-OHdG and 8-isoPGF2a levels in the observation group were significantly lower than those in the control group(P<0.05);(6)During the treatment period,the number of patients in the observation group who had ovulation was significantly higher In the control group(P<0.05);(7)The serum melatonin level of the observation group was negatively correlated with the ROS level(rs=-0.427,P<0.05).Conclusion:Melatonin has a good therapeutic effect on early-onset ovarian insufficiency.This effect may be achieved by inhibiting the production of ROS and VEGF and protecting the normal development of follicles.Part TwoStudy on the mechanism of melatonin in the treatment of premature ovarian insufficiency based on PI3K/AKT/mTOR autophagy pathwayObjective:Taking PI3K/Akt/mTOR pathway as the breakthrough point,the molecular mechanism of melatonin intervention on POI was revealed from multi-level and multi angle through cell experiment in vitro,providing new basis and new direction for clinical prevention and treatment of POI.Method:Ovarian granulosa cell cov434 was selected as the research object.Serum starvation method was used to induce autophagy in vitro,and different concentrations of MT were used to intervene.Then flow cytometry was used to detect cell apoptosis.Transmission electron microscopy was used to observe the formation of autophagy.Western blot was used to detect the expression of LC3II?LC3Iand agt5,and the activation of PI3K/Akt/mTOR signaling pathway was detected.Result:(1)The results of apoptosis detection showed that compared with the control group,the apoptosis rate of model group and each MT concentration group was significantly increased(P<0.05);compared with the model group,the apoptosis rate of each MT concentration group was significantly decreased,the difference was statistically significant(P<0.05),and with the increase of MT concentration,the apoptosis rate of cov434 cells gradually decreased;(2)transmission electron microscope observation results showed that the apoptosis rate of cov434 cells was significantly increased in the model group and each MT concentration group(P<0.05)The results showed that the typical autophagy structure appeared in the cytoplasm of cov434 cells after serum starvation treatment.Some of the cells were seriously damaged,leading to cell death.Autophagy structure could be observed in these cells.The results showed that compared with the control group,LC3?/? ratio and agt5 protein expression level of cov434 cells in the model group and each MT concentration group were significantly increased(P<0.05);compared with the model group,LC3I in each MT concentration group was significantly increased Compared with the control group,the expression levels of p-pI3k,p-Akt and p-mTOR protein in cov434 cells of model group and MT concentration groups were significantly decreased(P<0.05);compared with the model group,the protein levels of p-pI3k,p-Akt and p-mTOR in each MT concentration group were significantly decreased(P<0.05)The expression levels of P<0.05 were significantly increased in a dose-dependent manner.ConclusionMT protects POI by activating PI3K/Akt/mTOR signaling pathway,inhibiting the expression of autophagy related proteins,and then inhibiting the autophagy of ovarian granulosa cells.Part Three Study on the protective effect of melatonin on ovary function in POI ratsObjective:In this study,we intend to establish a rat model of POI to explore whether MT can protect the ovary by activating PI3K/Akt/mTOR signaling pathway in vivo.The purpose of this study is to further verify the protective mechanism of MT on POI through animal model experiments,and lay a theoretical and experimental foundation for finding a new way to treat POI.Method:Female SD rats were continuously given Tripterygium wilfordii polyglycoside tablets to establish rat POI model.Then the rats were randomly divided into model group,3-MA group,RAPA group and MT group.Normal female SD rats were taken as control.The levels of AMH,INH-B,FSH,E2,LH.P and T were detected by ELISA.The number of primordial follicles,primary follicles,sinus follicles and atresia follicles were counted.Western blot was used to detect the expression of p-pI3k,p-Akt and p-mTOR protein in ovarian tissue.Result:(1)The results of ovarian wet weight and ovarian index showed that the wet weight of left and right ovaries in the control group was the highest(P<0.05),while that of the model group was the lowest(P<0.05).Compared with the model group,the wet weight of left and right ovaries in RAPA group was significantly higher than that in RAPA group(P<0.05).The wet weight of ovary in 3-MA group and MT group was significantly higher than that in RAPA group(P<0.05).There was no significant difference between 3-MA group and MT group(P>0.05).Compared with the model group,the ovarian index of RAPA group was significantly higher(P<0.05),and that of 3-MA group and MT group was significantly higher than that of RAPA group(P<0.05),but there was no significant difference between 3-MA group and MT group(P>0.05).(2)The results of serum AMH and INH-B showed that the levels of AMH and INH-B in the control group were the highest,while those in the model group were the lowest;compared with the model group,the serum levels of AMH and INH-B in RAPA group were significantly higher(P<0.05);the levels of AMH and INH-B in 3-MA group and MT group were higher than those in RAPA group(P<0.05),but there was no significant difference between 3-MA group and MT group(P>0.05).(3)The results of serum FSH,LH,E2.P and T levels showed that the levels of FSH and LH in the control group were the lowest(P<0.05),while the levels of FSH and LH in the model group were the highest(P<0.05);compared with the model group,the serum FSH and LH levels of RAPA group were significantly lower(P<0.05);the levels of FSH and LH in 3-MA group and MT group were significantly lower than those in RAPA group(P<0.05)There was no significant difference in FSH and LH levels(P>0.05).Compared with the model group,the serum E2 level of RAPA group was significantly higher(P<0.05);the E2 level of MT group and 3-MA group was higher than that of RAPA group(P<0.05);while the E2 level of 3-MA group and MT group had no significant difference(P>0.05).There was no significant difference in T and P levels among the groups(P>0.05).(4)The number of primordial follicles,primary follicles and sinus follicles in the control group was the largest(P<0.05),and the number of primordial follicles,primary follicles and sinus follicles in the model group was the least(P<0.05);compared with the model group,the number of primordial follicles,primary follicles and sinus follicles in RAPA group was increased(P<0.05),and the number of primordial follicles,primary follicles and sinus follicles in 3-MA group and MT group were higher than those in RAPA group The number of atresia follicles in the model group was the largest(P<0.05),while that in the control group was the least(P<0.05).Compared with the model group,the number of atresia follicles in Rapa group was decreased(P<0.05),and the number of atresia follicles in 3-MA group and MT group was less than that in Rapa group(P<0.05)There was no significant difference between MT and MT groups(P>0.05).(5)The expression of p-pI3k,p-Akt and p-mTOR proteins in the control group was the highest(P<0.05),while that of the model group was the lowest(P<0.05).Compared with the model group,the expression levels of p-pI3k,p-Akt and p-mTOR in the RAPA group were significantly higher than those in the model group(P<0.05);the expression levels of p-pI3k,p-Akt and p-mTOR protein in the ovary tissue of RAPA group were significantly higher than those in the model group(P<0.05);the expression levels of p-pI3k,p-Akt and p-mTOR protein in the RAPA group were significantly higher than those in the 3-mA group and MT group The expression levels of p-pI3k,p-Akt and p-mTOR protein in RAPA group were significantly higher than those in RAPA group(P<0.05).However,there was no significant difference in the expression levels of p-pI3k,p-Akt and p-mTOR between 3-MA group and MT group(P>0.05).ConclusionMT has protective effect on ovaries of rats with POI,and the effect is similar to that of 3-MA,which is achieved by activating PI3K/Akt/mTOR signaling pathway,regulating sex hormones,increasing the number of primordial follicles,primary follicles and antral follicles,and reducing the number of atresia follicles. |
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