Exploring The Mechanism Of Moxibustion Inhibiting Apoptosis Of Granulosa Cells And Improving Ovarian Function In POI Model Rats Based On Autophagy

Objective:In this study,the rat model of premature ovarian insufficiency（POI）induced by intragastric administration of Tripterygium glycoside suspension（TGs）was used as the research object to evaluate the effect of different moxibustion intervention timings in preventing and treating POI,focusing on analyzing the effects of moxibustion on the apoptosis and autophagy of ovarian granulosa cells in POI model rats,and explore part of the biological basis of moxibustion protecting ovarian function in POI model rats from the perspective of moxibustion-mediated autophagy reducing ovarian granulosa cell apoptosis.Methods:1.Sixty-four female SD rats with normal estrous cycle were randomly divided into control group and TGs low,medium and high dose groups,with 16 rats in each group.The low-dose,middle-dose and high-dose groups were given 50,75,100 mg/kg TGs by gavage,once a day,for 14 consecutive days,and the control group was given an equal volume of normal saline by gavage.At 8:00 am every day,vaginal exfoliated cell smear was performed to observe the changes of estrous cycle of rats in each group.After gavage,10 rats in each group were randomly selected for ovarian endocrine function detection,which mainly included:weighing the wet weight of bilateral ovaries in each group,calculating the ovarian index;observing the morphological structure of the ovary by HE staining.Growing follicles and atretic follicles were counted.Serum levels of follicle-stimulating hormone（FSH）,luteinizing hormone（LH）,estradiol（E₂）,testosterone（T）and anti-Müerian hormone（AMH）were detected by ELISA in each group.A total of 24 rats,6 rats in each group,were caged with 12 male SD rats in a ratio of 2:1,and the materials were collected on the 8th day of pregnancy.The effects of different doses of TGs on the reproductive function of rats were compared by observing the pregnancy and the number of embryos in each group.2.Eighty female SD rats with normal estrous cycle were randomly divided into 5 groups:control group,model group,moxibustion group 1,moxibustion group 2 and moxibustion group 3,with 16 rats in each group.The control group was given an equal volume of normal saline by gavage,the other four groups were given TGs by gavage（the dose of TGs was based on the results of experiment 1）.Moxibustion groups 1,2,and 3 were given moxibustion intervention before modeling,at the same time,and after modeling,respectively.The moxibustion method is as follows:the two groups of acupoints "Shenshu" on both sides or "Guanyuan" and"Zhongwan" alternately performed mild moxibustion every other day,once a day,for 10 minutes per acupoint,for 14 consecutive days.By observing the effects of different times of moxibustion intervention on ovarian endocrine function（estrous cycle disorder rate,ovarian wet weight,ovarian index,tissue morphology,follicle number and serum sex hormone level）and reproductive function（pregnancy rate and embryo number）in POI model rats,to evaluate the difference in the effect of different moxibustion intervention timings in the prevention and treatment of POI.3.TUNEL staining method was used to observe the apoptosis of ovarian granulosa cells in the control group,model group and moxibustion group（the best effect group in experiment 2）,and the rats in each group were determined by immunohistochemistry,Western blot and PCR techniques.The tissue localization,protein and mRNA expression levels of apoptosis-related indicators（Bcl-2,Bax,Cyt c）in ovarian tissue of rats in each group were determined by immunohistochemistry,Western blot and PCR,respectively.To observe the effect of moxibustion on the autophagy of ovarian granulosa cells in POI model rats.4.The ultrastructure and the number of autophagosomes in the ovarian tissue of the rats in the control group,the model group and the moxibustion group were observed by transmission electron microscopy to evaluate the level of ovarian autophagy in the rats in each group.The tissue localization,protein and mRNA expression levels of autophagy-related indicators（Beclinl,p62,LC3）in ovarian tissue of rats in each group were determined by immunohistochemistry,Western blot and PCR.To observe the effect of moxibustion on the autophagy of ovarian granulosa cells in POI model rats.5.Forty female SD rats with normal estrous cycle were randomly divided into 4 groups:model group,moxibustion group,autophagy inhibitor+moxibustion group,autophagy activator+moxibustion group,10 rats in each group.The rats in the four groups were all given 75 mg/kg/d TGs by continuous intragastric administration for 14 days to prepare the POI rat model;one hour after gavage of TGs,the autophagy inhibitor+moxibustion group and autophagy activator+moxibustion group were intraperitoneally injected with 3-MA solution（10mg/kg/d）and RAPA solution（2mg/kg/d）,respectively;the model group and moxibustion group were given intraperitoneal injection of equal volume of normal saline;30 minutes later,except the model group,the other three groups were given mild moxibustion intervention,for 14 consecutive days.The ultrastructure of ovarian tissue and the number of autophagosomes were observed by transmission electron microscopy,and the apoptosis of granulosa cells was detected by TUNEL staining.Immunohistochemistry,Western blot and real-time quantitative PCR techniques were used to detect the tissue localization,protein and mRNA expression levels of apoptosis and autophagy-related indicators in ovarian tissue of rats in each group.Subsequently,by observing the estrous cycle disorder rate,ovarian wet weight and index,ovarian tissue morphology and serum sex hormone levels in each group of rats,to retrospectively verified whether moxibustion could reduce ovarian granulosa cell apoptosis by regulating autophagy and protect POI model rats ovarian function.Results:1.TGs gavage for 14 days can lead to the decline of ovarian reserve in rats,and the degree of ovarian function damage is positively correlated with the gavage dose of TGs,that is,there is a dose-dependent trend.Compared with the control group,the ovary weight and ovarian index of the rats in the low,medium and high dose groups of TGs were significantly decreased;the estrous cycle disorder rate of the rats in the low dose group of TGs was 90%,while that of the rats in the middle and high dose groups of TGs was greater than that of the control group,as all rats showed estrous cycle disorder.HE staining results showed that the ovarian cortex was wrinkled in the low,medium and high groups,and the ovarian granulosa cells were damaged to varying degrees,showing that the follicular granulosa cell layer was significantly reduced and the boundary was blurred,arrangement disorder.Follicle counts at all levels of the rats in each group showed that compared with the control group,the number of healthy follicles and total follicles in the ovaries of rats in the middle and high doses of TGs were significantly reduced,and the number of atretic follicles was significantly increased compared with the control group.ELISA results showed that compared with the control group,the serum FSH,LH and T levels of the rats in the low,medium and high dose groups of TGs were significantly increased,and the levels of E₂ and AMH were decreased.The results of reproductive function test showed that the number of embryos in the low and medium dose groups was significantly reduced,and the pregnancy rate was significantly lower than that of the control group,which were 33.3%and 16.7%,respectively.The high dose group of TGs had obvious reproductive toxicity,and the pregnancy rate was as low as 0%.In addition,the rats in the high-dose group also showed hair loss,polyuria,diarrhea,swelling of the upper limbs and irritability during the gavage period.2.Compared with the model group,the group of moxibustion intervention at the same time and the group of moxibustion intervention after the model establishment（moxibustion groups 2 and 3）can significantly increase the ovarian wet weight and ovarian index of POI model rats,while it was no significant difference in ovarian index in the group of moxibustion group 1.The results of estrous cycle showed that the disorder rate of estrous cycle of rats in moxibustion groups 1,2,and 3 was significantly decreased,which were 40%,20%,and 30%,respectively.HE staining showed that the pathological morphology of the ovaries in the moxibustion groups 1,2,and 3 was significantly improved,with abundant follicles at all levels in the ovary,more and larger corpus luteum tissue,less atretic follicles,under the high magnification microscope,the morphology of granulosa cells was normal,and the number of granule layers increased and arranged neatly.Among them,moxibustion group 2 can significantly reduce the number of atretic follicles and increase the number of growth follicles and total follicles in POI model rats.The ELISA results showed that the serum FSH level of the rats in the moxibustion group 1 was significantly lower than that of the model group,and the AMH level was significantly higher than that of the model group,while there was no significant difference in the serum LH,E₂ and T levels.Compared with the model group,the serum FSH,LH and T levels of the rats in the moxibustion group 2 were significantly lower,and the levels of E₂ and AMH were significantly higher than those in the model group.Compared with the model group,the serum FSH,LH and T levels of the rats in the moxibustion group 3 were lower,and the AMH level was higher,but there was no statistical difference in the level of E₂.The reproductive function test results showed that different times of moxibustion intervention could improve the pregnancy rate and increase the number of embryos in POI model rats.Among them,the pregnancy rate of rats in the moxibustion group 2 was 100%,and the number of embryos was（11.17±2.23）.3.Compared with the control group,the number of TUNEL-positive cells in the ovarian granulosa cells of the model group was significantly increased,the protein and mRNA levels of the anti-apoptotic protein Bcl-2 in the ovarian tissue were significantly decreased,and the pro-apoptotic proteins Bax and Cyt c were significantly decreased.Compared with the model group,the number of TUNEL-positive cells in ovarian granulosa cells in the moxibustion group was significantly decreased,the protein and mRNA levels of Bcl-2 in ovarian tissue were significantly increased,while the protein and mRNA contents of Bax and Cyt c were significantly decreased.4.The results of transmission electron microscopy showed that autophagosomes were distributed in the ovarian granulosa cells in the control group,and the mitochondrial structure was complete.While the autophagosomes were not obvious in the ovarian granulosa cells,and mitochondria were scattered and vacuolated in the model group.The number of autophagosomes in ovarian granulosa cells of rats in the moxibustion group was increased,and the mitochondrial structure was improved.Compared with the control group,the protein and mRNA levels of p62 in the ovarian tissue of model group were significantly increased,the protein and mRNA levels of Beclinl and LC3 were significantly decreased,and the ratio of LC3-Ⅱ/Ⅰ was significantly decreased.Compared with the moxibustion group,the protein and mRNA levels of Beclinl and LC3 in the ovarian tissue of the rats in the moxibustion group increased significantly,the ratio of LC3-Ⅱ/Ⅰ increased,and the protein and mRNA contents of p62 decreased significantly.5.（1）The level of autophagy in the ovaries of rats in each group:the results of transmission electron microscopy showed that autophagosomes in ovarian granulosa cells in moxibustion group increased and the number of vacuolated mitochondria decreased;autophagosomes were less common and the mitochondrial structure was incomplete in ovarian granulosa cells in the autophagy inhibitor+moxibustion group,while the number of autophagosomes in ovarian granulosa cells increased in the autophagy activator+moxibustion group,and the mitochondria were densely distributed without obvious damage to the structure.The protein and mRNA expression levels of autophagy-related indicators Beclinl,p62,and LC3 were further detected.The results showed that compared with the moxibustion group,the protein and mRNA levels of Beclin1 and LC3 in the ovarian tissue of the rats in the autophagy inhibitor+moxibustion group were significantly decreased,the ratio of LC3-Ⅱ/Ⅰ was decreased,and the protein and mRNA contents of p62 were significantly increased.While there was no significant difference in Beclin1,LC3,p62 protein and mRNA content and LC3-Ⅱ/Ⅰ ratio in the ovarian tissue of the autophagy activator+moxibustion group compared with the moxibustion group.（2）The level of apoptosis in the ovaries of rats in each group:the results of TUNEL staining showed that compared with noxibustion group,the number of TUNEL-positive cells in the granulosa cells of the autophagy inhibitor+moxibustion group increased significantly.However,there was no significant difference in the number of TUNEL-positive cells in the ovaries of the autophagy activator+moxibustion group compared with the moxibustion group.The protein and mRNA expression levels of apoptosis-related indicators Bcl-2,Bax,and Cyt c were further detected.The results showed that compared with the moxibustion group,the protein and mRNA levels of Bcl-2 in the ovarian tissue of the rats in the autophagy inhibitor+moxibustion group were significantly decreased,while the protein and mRNA levels of Bax and Cyt c were significantly increased.Compared with the moxibustion group,the mRNA content of Bcl-2 in the ovaries of the autophagy activator+moxibustion group was significantly increased,however,there was no significant difference in the protein expression level of Bcl-2;the protein expression level of Bcl-2 was not significantly different from that in the moxibustion group,and the protein and mRNA contents of Bax and Cyt c were not significantly different between the moxibustion group and the autophagy activator+moxibustion group.（3）The ovarian function of rats in each group:compared with the moxibustion group,the autophagy inhibitor+moxibustion group had a significantly higher rate of estrous cycle disorder,significantly lower ovarian wet weight and ovarian index,fewer healthy follicles at all levels in the ovarian tissue,under the high magnification microscope,the edges of granulosa cells were blurred,and the arrangement was disordered and less granulosa cell layers;besides,serum FSH and LH levels were significantly increased,and E₂ and AMH levels were significantly decreased.Compared with the moxibustion group,the autophagy activator+moxibustion group showed no significant difference in estrous cycle disorder rate,ovarian wet weight and ovarian index;the serum FSH level was significantly lower than that in the moxibustion group,while there was no significant difference in serum LH,E₂ and AMH level compared with the moxibustion group.Conclusions:1.Different doses of TGs for 14 days can cause damage to ovarian endocrine function and reproductive function in rats.The ovarian function damage caused by 75 mg/kg/d TGs is similar to the clinical manifestations and clinical symptoms of POI diseases,and can be used to establish POI rat models.2.Different times of moxibustion intervention can protect the ovarian endocrine function and reproductive function of POI model rats,and the best effect of moxibustion intervention was given during modeling.3.Moxibustion can reduce the apoptosis of ovarian granulosa cells in POI model rats.4.Moxibustion can promote the autophagy of ovarian granulosa cells in POI model rats.5.Moxibustion may protect ovarian function in TGs-induced POI model rats by promoting autophagy and reducing ovarian granulosa cell apoptosis.Hence,different times of moxibustion intervention have protective effects on ovarian endocrine function and reproductive function in TGs-induced POI model rats,and the most significant effect of moxibustion intervention was given at the same time as the model was established.Moxibustion may promote moderate autophagy,reduce ovarian granulosa cell apoptosis,protect ovarian function damage in POI model rats,and ultimately delay ovarian aging.