Effect Of “Yin Three Needles” On Insulin Resistance And Ovarian MTOR Protein Expression In PCOS-IR Rats

ObjectiveTo investigate the effects of“Yin three needles”on glucose metabolism,ovarian function and mTOR protein expression in ovarian tissues of rats with polycystic ovary syndrome-Insulin resistance（PCOS-IR）,to explore the regulatory effects of“Yin three needles”as an acupuncture point formula on rats with PCOS-IR,and to provide a scientific basis for the clinical treatment of PCOS-IR.This study will provide scientific basis for the selection of acupuncture points for the clinical treatment of PCOS-IR.MethodsThirty-two SPF female SD rats with regular motility cycles were randomly divided into 8control rats and 24 model preparation rats.The PCOS-IR model was prepared by gavage with letrozole and fed with high-fat diet for 28 days.(The rats were randomly divided into the model group,metformin group and“Yin three needles”treatment group（8 rats in each group）.The intervention was started on the day following the end of the modelling.The“Yin three needles”treatment group took Guan Yuan（RN4）,Guilai（ST29）and Sanyinjiao（SP6）acupoints,with Guan Yuan（RN4）acupuncture point being flat-pricked with a homemade double-pole needle,Guilai（ST29）acupuncture point downward on both sides and Sanyinjiao（SP6）straight-pricked on both sides.Guan Yuan（RN4）and Sanyinjiao（SP6）were connected to an electroacupuncture instrument with sparse and dense waves（10 S for sparse waves and 15 S for dense waves）at a frequency of 2/10 Hz and an intensity of 1m A,and the needles were left in place for 20 minutes each time.The intervention was performed once daily for 21 days.The metformin group was given metformin solution by gavage daily,and the rest of the operation was the same as that of the“Yin three needles”treatment group,except that no acupuncture was performed.The control and model groups were given the same hours of grasping,fixation and vaginal smear respectively,without any treatment.At the end of the intervention,the rats were collected uniformly;HE staining was performed to observe the morphological changes of the ovarian tissue in each group.The six sex hormones in serum and fasting insulin levels were measured by ELISA;the body weight and Lee’s index,fasting blood glucose,glucose tolerance test and ovarian wet weight were recorded in each group;the expression of target of Mammalian Target of Rapamycin（mTOR）protein in ovarian tissues of rats in each group was measured by Western Blot.Results（1）Ovarian function:Ovarian morphology:in the control group,follicles of different levels of development were seen,as well as a larger number of corpus luteum;compared with the control group,the ovaries of the model group showed a large number of dilated follicle-like follicles and a significant decrease in the number of corpus luteum;in the metformin group,a decrease in the number of dilated follicles and a small increase in the number of corpus luteum were seen;in the“Yin three needles”treatment group,a decrease in the number of follicles and an increase in the number of follicles of different levels of development were seen.The number of cystic follicles was reduced,the number of follicles at different levels of development was increased and the number of corpus luteum was increased in the“Yin three needles”treatment group.Compared to the control group,the levels of Luteinizing Hormone（LH）and Testosterone（T）were significantly higher in the model group（P<0.001）;Follicle Stimulating Hormone（FSH）,Estradiol（E₂）,Progesterone（P）and Prolactin（PRL）were significantly lower（P<0.05）.Ovarian organ indices increased with a statistically significant difference（P<0.05）.Compared with the model group,the LH and T levels of rats in the metformin and“Yin three needles”treatment group were significantly lower（P<0.001）;the FSH,E₂,P and PRL levels were significantly higher（P<0.05）;and the ovarian organ index decreased（P<0.05）.Compared with the metformin group,the elevated E₂and P levels were more significant in the“Yin three needles”treatment group（P<0.01）.（2）Insulin resistance:compared with the control group,body weight and Lee’s index were significantly increased in the model group（P<0.01,P<0.05）.Blood glucose values at the 30th min,60th min and 120th min of IPGTT in rats,and AUC values（area under the curve）were significantly higher,with statistically significant differences（all P<0.05）.Fasting blood glucose（FPG）,fasting insulin（FINS）,and HOMA-IR index were significantly higher,with statistically significant differences（all P<0.01）.Compared with the model group,body weight in the metformin and“Yin three needles”treatment group decreased significantly（P<0.01）,Lee’s index tended to decrease（P<0.05,P<0.05）,and blood glucose values in the rats in the metformin group at different times of IPGTT were not statistically significant（P>0.05）.AUC values decreased（P<0.05）.The difference was statistically significant（P<0.001）in the decrease of blood glucose value at the 30th min and 60th min in the rats of the“Yin three needles”treatment group.FINS and HOMA-IR indices decreased significantly in the metformin and“Yin three needles”treatment group,with a statistically significant difference（P<0.001）.There was a trend of decrease in body weight and Lee’s index in the“Yin three needles”treatment group compared with the metformin group,but the difference was not statistically significant（P>0.05）,and there was a statistically significant decrease in blood glucose value and AUC value in the rats at the 30th min of IPGTT test（P<0.05）.（3）Ovarian tissue mTOR protein expression:Compared with the control group,the ovarian tissue mTOR protein expression level in the model group was significantly lower than that in the control group（P<0.05）.Compared with the model group,the mTOR protein expression levels of rats in the metformin and the“Yin three needles”treatment group were not statistically significant（P>0.05）.Conclusions1.“Yin three needles”has a certain regulatory effect on insulin resistance and abnormal glucose metabolism in PCOS-IR model rats,which can reduce body weight,lower fasting insulin level,improve glucose tolerance and improve insulin resistance.2.“Yin three needles”can improve sex hormone disorders in PCOS-IR rats,improve ovarian tissue morphology and structure in PCOS-IR rats.It improves follicular development and regulates ovarian function.3.“Yin three needles”is superior to metformin in increasing estrogen and progesterone and in promoting ovulation in PCOS-IR rats.