Study On The Effect And Mechanism Of Reverse Moxibustion In Preventing And Treating Premature Ovarian Failure In Mice Based On The Theory Of Preventing Disease Before It Occur

Objective:This study is based on the prevention treatment of disease theory,this study used reverse moxibustion to early intervention on premature ovarian failure（POF）mice,namely,preventive moxibustion was selected at Guanyuan（CV 4）,Xinshu（BL 15）and Shenshu（BL23）.To observe the changes in estrus cycle and ovarian morphology,determine the changes in hormone levels and apoptosis factor protein content in mice,and explore the effect and mechanism of reverse moxibustion on the prevention and treatment of POF mice,to provide new experimental support for clinical treatment.Materials and methods:Thirty-five Balb/c mice were observed for estrous cycle,and 30 mice were included according to the results of vaginal smear microscopy.According to the random number table,they were randomly divided into 5 groups,namely the blank group（n=6）,the model group（n= 6）,the Reverse moxibustion group（n=6）,the moxibustion group（n=6）and the medication group（n=6）.Fourteen days before modeling in the reverse moxibustion group,mild moxibustion was applied at Guanyuan（CV 4）,Xinshu（BL 15）and Shenshu（BL 23）,once every other day,10 min each time,totally 7 times.Mice in the other groups were fixed with a fixator,once every other day,10 min each time,for a total of 7 times.After the inverse moxibustion,except for the blank group,the POF model was established in the other four groups by a single intraperitoneal injection of Cyclophosphamide（CTX）.After the model was successfully established,Mice in the inverse moxibustion group and the moxibustion group were treated with mild moxibustion at Guanyuan（CV 4）,Xinshu（BL 15）and Shenshu（BL 23）,once a day,10 min each time for two weeks.Mice in the medication group were treated with estradiol valerate by gavage once a day for two weeks.Vaginal smears were collected every day after modeling,and the estrous cycle was observed under a microscope.At the end of the last treatment,body weight and ovarian coefficient were calculated by weighing.ELISA was used to detect the levels of FSH,LH,E₂ and AMH in serum.Western blotting was used to detect the protein expression of Bcl-2,Bax and Caspase-3 in ovarian tissue.Results:1.Estrous cycle: Before modeling,the estrous cycle of mice in each group was normal,and the cell smear showed periodic regular changes.After modeling,compared with blank group,the estrous cycle in model group was disordered,interestrous period was prolonged or stagnated,and cell characteristics were not obvious.Compared with the model group,one week after moxibustion intervention,The estrus cycle of mice in the reverse moxibustion group was effectively improved,and 3 days after western medicine intervention,showed persistent estrus period.2.Ovarian morphology: compared with the blank group,the model group showed many vacuole-like structures,the number of primordial follicles and primary follicles decreased,the number of atretic follicles increased,the arrangement of granulosa cells was irregular,and the zona pellucida and corona radiata were not clear.Compared with the model group,the number of follicles at all levels increased in the retrograde moxibustion group,moxibustion group and medication group.Compared with the moxibustion group,the number of primary follicles in the reverse moxibustion cell vessel group increased,and the corpus luteum structure was obvious.Compared with the medication group,the number of primary follicles was increased and the level of granulosa cells was decreased in the retrograde moxibustion group.3.Ovarian coefficient: compared with the blank group,the model group was significantly decreased（P<0.01）;Compared with the model group,the levels in the reverse moxibustion groups and medication groups were significantly increased（P<0.01）.4.ELISA serum hormone levels:Compared with the blank group,the levels of FSH and LH were increased（P<0.01）and the levels of E₂ and AMH were decreased（P<0.01）in the model group;Compared with the model group,the levels of FSH and LH in the inverse moxibustion group were decreased（P<0.01）and the levels of E₂ and AMH were increased（P<0.01）,the levels of FSH and LH in the moxibustion group were decreased（P<0.05,P<0.01）and the levels of E₂ were increased（P<0.05）.FSH level was decreased and E₂ level was increased in western medicine group（P<0.01）.Compared with moxibustion group,FSH level in reverse moxibustion group was decreased（P<0.05）,and E₂ level in western medicine group was increased（P<0.05）.5.WB :Compared with the blank group,the expression level of Bcl-2 protein was decreased（P<0.01）,the expression level of Bax protein was increased（P<0.01）,the expression level of Bcl-2/Bax protein was decreased（P<0.01）,and the expression level of Caspase-3 protein was increased（P<0.01）in the model group.Compared with the model group,the expression of Bcl-2 protein was increased（P<0.01）,the expression of Bax protein was decreased（P < 0.01）,the expression of Bcl-2/Bax was increased（P<0.01）,and the expression of Caspase-3 was decreased（P<0.05）in the intervention groups.Compared with the moxibustion group,the expression of Bax was decreased（P<0.05）,and the expression of Bcl-2/Bax was increased in the reverse-moxibustion group and the medication group（P<0.05）.Conclusion:1.The method of reverse moxibustion can effectively reduce the levels of FSH and LH,increase the levels of E₂ and AMH in mice with POF,and regulate the physiological function and morphological structure of mice ovary,and the effect is better than that of single moxibustion.2.Reverse moxibustion on may play a role in the prevention and treatment of POF by reducing the expression of pro-apoptotic protein Bax,increasing the expression of anti-apoptotic protein Bcl-2,increasing the ratio of Bcl-2/Bax,inhibiting the activity of Caspase-3 protease,and inhibiting the abnormal apoptosis process of ovarian cells.