Effects Of Electrothermal Acupuncture At Sanyinjiao On Serum Sex Hormone Levels And Expression Of GDF9 In Ovarian Tissue In Premature Ovarian Insufficiency Model Rats

ObjectiveTo observe the effect of electrothermal acupuncture Sanyinjiao on serum hormone and expression of GDF9 in ovarian tissue of premature ovarian insufficiency（POI）model rats,to discuss the specific function of Sanyinjiao acupoint and the effect of electrothermal acupuncture Sanyinjiao on ovarian function in POI model rats,to provide more scientific basis for the clinical application of Sanyinjiao acupoint in POI.MethodsForty SPF female SD rats with regular estrous cycle were selected and randomly divided into blank group（n=10）and model group（n=30）.The blank group was given 0.9%physiological saline by gastric irrigation,and the model group was given 50 mg/kg Tripterygium glycosides tablet suspension by gastric irrigation.Both groups were given once a day for 14 days.24 rats with successful modeling were randomly divided into model group,non-acupoint electrothermal acupuncture group and Sanyinjiao electrothermal acupuncture group,with 8 rats in each group（n=8）and 8 rats in blank group.On the next day after the end of the model,electrothermal acupuncture intervention was started,the Sanyinjiao electrothermal acupuncture group took Sanyinjiao points,connected with the electrothermal acupuncture instrument,frequency was 2Hz,continuous wave,20 minutes each time,once a day,continuous intervention foe 14 days,7 days before the end of the intervention,vaginal exfoliated cell smears were performed for 7 continuous days.The non-meridian and non-acupoints were selected in the non-acupoint electrothermal acupuncture group,and the manipulation was the same as that in the Sanyinjiao group electrothermal acupuncture.The blank group and the model group were given the same time of capture,fixation and vaginal exfoliative cell smears without any treatment.After the intervention,the samples were collected according to the estrous interval,and the body weight and ovarian wet weight of rats in each group were measured and recorded.The contents of follicle-stimulating hormone（FSH）,estradiol（E₂）and inhibin B（INHB）in serum of rats in each group were detected by enzyme-linked immunosorbent assay（ELISA）.Immunohistochemistry and real-time fluorescence quantitative PCR were used to detect the expression of GDF9 in ovarian tissues of rats in each group.HE staining was used to observe the morphological changes of ovaries of rats in each group.Results1.Ovarian organ index:Compared with the blank group,the ovarian organ indexes of the model group,the non-acupoint electrothermal acupuncture group and the Sanyinjiao electrothermal acupuncture group were significantly decreased,and the differences were statistically significant（P<0.001,P<0.001,P<0.01）.Compared with the model group,the ovarian organ index in the non-acupoint electrothermal acupuncture group showed an increasing trend,and the difference was not statistically significant（P>0.05）.The ovarian organ index in the Sanyinjiao electrothermal acupuncture group was significantly increased,and the difference was statistically significant（P<0.05）.Compared with the non-acupoint electrothermal acupuncture group,the ovarian organ index in the Sanyinjiao electrothermal acupuncture group was significantly increased,and the difference was statistically significant（P<0.05）.2.Serum sex hormone levels:（1）FSH:Compared with the blank group,the serum FSH levels in the model group,the non-acupoint electrothermal acupuncture group and the Sanyinjiao electrothermal acupuncture group were significantly increased,and the difference were statistically significant（P<0.001）.Compared with the model group,the serum FSH in the non-acupoint electrothermal acupuncture group showed a decreasing trend,and the difference was not statistically significant（P>0.05）.The serum FSH content in the Sanyinjiao electrothermal acupuncture group was significantly decreased,and the difference was statistically significant（P<0.001）.Compared with the non-acupoint electrothermal acupuncture group,the serum FSH content in the Sanyinjiao electrothermal acupuncture group was significantly lower,and the difference was statistically significant（P<0.001）.（2）E₂:Compared with the blank group,the serum E₂content in the model group,the non-acupoint electrothermal acupuncture group and the Sanyinjiao electrothermal acupuncture group were significantly decreased,and the difference were statistically significant（P<0.001,P<0.001,P<0.05）.Compared with the model group,the serum E₂ content in the non-acupoint electrothermal acupuncture group showed an increasing trend,and the difference was not statistically significant（P>0.05）.The serum E₂ content in the Sanyinjiao electrothermal acupuncture group was significantly increased,and the difference was statistically significant（P<0.001）.Compared with the non-acupoint electrothermal acupuncture group,the serum E₂content in the Sanyinjiao electrothermal acupuncture group was significantly increased,and the difference was statistically significant（P<0.001）.3.Serum INHB hormone level:Compared with the blank group,the serum INHB content in the model group,the non-acupoint electrothermal acupuncture group and the Sanyinjiao electrothermal acupuncture group were significantly decreased,and the difference were statistically significant（P<0.001,P<0.001,P<0.05）.Compared with the model group,the serum INHB content in the non-acupoint electrothermal acupuncture group showed an increasing trend,and the difference was not statistically significant（P>0.05）.The serum INHB content in the Sanyinjiao electrothermal acupuncture group was significantly increased,and the difference was statistically significant（P<0.001）.Compared with the non-acupoint electrothermal acupuncture group,the serum INHB content in the Sanyinjiao electrothermal acupuncture group was significantly increased,and the difference was statistically significant（P<0.001）.4.Morphological changes of ovarian tissue:Optical microscope showed that the ovaries of rats in the blank group were full,the follicles at all levels were normal,and the number of atresia follicles was less.The ovarian volume of the model group decreased,the number of follicles and corpus luteum at different developmental stages decreased,and the number of atresia follicles increased significantly.There was no significant difference in ovarian volume between non-acupoint electrothermal acupuncture group and model group.The number of growth follicles and corpus luteum decreased,and the number of atresia follicles increased.In the electrothermal acupuncture Sanyinjiao group,the ovarian volume increased,all levels of follicles developed well,the number of growing follicles and corpus luteum increased,and the number of atresia follicles decreased.5.Relative expression of GDF9 in ovary:Compared with the blank group,the relative expression levels of GDF9 protein and m RNA in the ovary of the model group and the non-acupoint electroacupuncture group were significantly decreased,and the differences were statistically significant（P<0.001）.The relative expression of GDF9 protein in ovary of the Sanyinjiao electrothermal acupuncture group was decreased,and the difference was statistically significant（P<0.05）.Compared with the model group,the relative expression levels of GDF9protein and m RNA in the ovary of the non-acupoint electrothermal acupuncture group showed an increasing trend,and the difference were not statistically significant（P>0.05）.The relative expression levels of GDF9 protein and m RNA in the ovary of the Sanyinjiao electrothermal acupuncture group were significantly increased,and the difference were statistically significant（P<0.001）.Compared with the non-acupoint electrothermal acupuncture group,the relative expression levels of GDF9 protein and m RNA in the ovary of the Sanyinjiao electrothermal acupuncture group were significantly increased,and the difference were statistically significant（P<0.001,P<0.01）.Conclusions1.Electrothermal acupuncture Sanyinjiao has a certain regulatory effect on POI model rats,which can increase the ovarian organ index of POI model rats and improve the morphological structure of ovarian tissue.2.Electrothermal acupuncture Sanyinjiao can reduce serum FSH,increase E₂ and INHB content,up-regulate ovarian GDF9 expression,improve follicular development and regulate ovarian function.3.The regulation effect of electrothermal acupuncture Sanyinjiao point on POI model rats was significantly better than that of electrothermal acupuncture non-acupoints,indicating that Sanyinjiao point has specific regulation on ovarian function.