Clinical And Mechanistic Study On Electroacupuncture Liver Meridian Acupoints Treatment Of DOR Based On The Theory Of "Cong Gan Lun Zhi"

Objective1.A randomized controlled study was used in this study.To evaluate the clinical effectiveness based on the theory of "cong gan lun zhi"acupuncture in the treatment of Diminished ovarian reserve(DOR),female patients with Diminished ovarian reserve who met the criteria were screened by setting up a liver meridian treatment group and non-meridian non-acupoints group,We observed the changes of anti-Mullerian hormoe(AMH),basic follicle-stimulating hormon(bFSH),basic luteinizing hormone(bLH),basic estradiol,the number of antral follicles(AFC),the modified Kupperman score,MENqol score,SAS score and SDS score were assessed before and after treatment by two groups.At the same time,the pregnancy rates were observed three months after the end of treatment.2.Based on the previous clinical study,we established a cyclophosphamide-induced DOR model in rats through experimental study,and observed the effects of each group on the ovarian function and apoptotic proteins Bax and Bcl-2 in the DOR model rats,and initially explored the mechanism of each group to regulate the changes of ovarian hormones,delay follicular atresia,and improve the function of the ovarian reserve;meanwhile we investigate the mechanism of DOR treatment by electroacupuncture of liver meridian acupoints from Keap1-Nrf2 pathway further more,so as to provide a scientific basis for the treatment about"cong gan lun zhi"acupuncture.Methods1.Clinical StudiesIn this study,a randomized controlled study was conducted to include 60 female patients with DOR who met the criteria,and the 60 subjects were divided into a liver meridian group and a non-meridian non-acupoints group in a 1:1 ratio.The liver meridian group was treated with electro-acupuncture using the "cong gan lun zhi" protocol:Taichong(LR3),Ligou(LR5),Ququan(LR8),and Jimai(LR12).The control group was treated with electro-acupuncture at four non-meridian non-acupoints on the lateral thigh,the lateral calf,and the lateral heel bone,respectively.Two groups of acupuncture points at the proximal end of the 2mm shallow stab an auxiliary needle,the main point connected to the negative pole,the auxiliary needle connected to the positive pole,then connected to the electro-acupuncture instrument,the parameters of the electroacupuncture instrument in liver meridian group were showing continuous wave,frequency of 2Hz,current intensity of 0.5-1.0mA,increased the current intensity gradually to the patient’s local skin slightly twitching as degree.The needles were kept for 30 minutes each time,once every other day,3 times a week,Saturday and Sunday off,for a total of 36 treatments.The changes in serum sex hormones(FSH,LH,E2),AMH and the number of antral follicles,modified Kupperman score,MENqol score SAS score and SDS score were assessed before and after treatment,while the pregnancy rates were observed three months after the end of treatment.2.Experiment Research:Literature evaluationIn this study,SD female rats with normal motility cycles were selected as the subjects and randomly divided into normal group(8)and model preparation group(24).Constructing SD rat DOR model by single intraperitoneal injection of 75mg/kg CTX once(1 ml of CTX solution was prepared by adding 0.9%saline at a dose of CTX 75 mg/kg),and continuously for 7 days.After successful modeling,they were randomly divided into model group,liver meridian group and non-meridian non-acupoints group.Normal group and model group:fixed daily but no electroacupuncture intervention treatment;liver meridian group and non-meridian non-acupoints group:fixed rats after successful modeling,and the treatment protocol refer to the clinical trial,once every other day,3 times a week,Saturday and Sunday off,a total of 3 weeks of treatment.The estrous cycle of rats were observed daily,and the rats were executed after all the intervention.The changes of ovarian histology were observed by HE staining,and the serum folliculopoietin(FSH),luteinizing hormone(LH)and estradiol(E2)in rat’s Serum were measured by EILSA.The expression levels of keapl,Nrf2,Bax and Bcl-2 mRNA and protein in rat ovarian tissues were detected by QPCR and Western blot.Results1.Clinical Studies1.1 Baseline general dataThere was no significant difference in age and course of disease between the two groups(P>0.05),which were comparable.1.2 Ovarian function(1)Serum AMHThe level of AMH decreased in the non-meridian non-acupoints group(P<0.01)and increased in the liver meridian group(P<0.01)after treatment.And the level of AMH in the liver meridian group were higher than in the non-meridian non-acupoints group(P<0.01).(2)Serum FSH content、Serum LH content、Serum E2 contentThe level of FSH,the level of E2 and the level of LH increased(P<0.01,P<0.05)in the non-meridian non-acupoints group after treatment,the level of FSH and LH decreased(P<0.05)and the level of E2 level increased(P<0.01)in the liver meridian group after treatment.Compared with the non-meridian non-acupoints group,the level of FSH and LH were lower(P<0.01)and the level of E2 was higher than in the liver meridian group(P<0.05).(3)The number of antral folliclesThe number of AFC decreased(P<0.05)in the non-meridian non-acupoints group and the number of AFC increased(P<0.01)in the liver meridian group after treatment.The number of AFC in the liver meridian group was higher than in the non-meridian non-acupoints group after the treatment(P<0.01).(4)The Pregnancy ratesCompared the pregnancy of the two groups at 3 months after the end of treatment,it was found that the pregnancy rate of the liver meridian group was 13.33%,and the clinical pregnancy rate of the non-meridian non-acupoints group was 0.1.3 Emotional symptoms scale(1)The modified Kupperman scoreThe modified Kupperman score was decreased(P<0.05)in the non-meridian non-acupoints group and the modified Kupperman score was also decreased(P<0.01)in the liver meridian group.Compared with the non-meridian non-acupoints group,the modified Kupperman score in the liver meridian group decreased better than in the non-meridian non-acupoints after treatment(P<0.05).(2)The MENqol scoreThe MENqol score decreased(P<0.01)in both groups after treatment.Compared with the non-meridian non-acupoints group,the MENqol score in the liver meridian groupdecreased better than in the non-meridian non-acupoints after treatment(P<0.05).(3)The SAS scoreThe SAS score decreased(P<0.01)in both groups after treatment.Compared with the non-meridian non-acupoints group,the SAS score in the liver meridian group decreased better than in the non-meridian non-acupoints after treatment(P<0.05).(4)The SDS scoreThe SDS score decreased(P<0.01)in both groups after treatment.Compared with the non-meridian non-acupoints group,the SDS score in the liver meridian group decreased better than in the non-meridian non-acupoints after treatment(P<0.01).2.Experiment studies2.1 Morphological observation of ovarian tissue in each groupAfter HE staining,the ovarian tissue was intact,the number of primordial follicles,primary follicles and secondary follicles in the cortical area of the blank group was normal,and the number of atretic follicles was normal.Compared with the blank group,the number of primordial follicles,primary follicles and secondary ovaries decreased(P<0.05,P<0.01)and the number of atretic follicles increased(P<0.05,P<0.01)in other groups of the rats,among which the number of atretic follicles in the model group increased significantly(P<0.01).Compared with the model group,the number of follicles at all levels increased(P<0.05,P<0.01)and the number of atretic follicles decreased(P<0.05,P<0.01)in the liver meridian group and non-meridian non-acupoints group(P<0.05,P<0.01).Compared with the non-meridian non-acupoints group,the number of follicles at all levels increased(P<0.05,P<0.01)and the number of atretic follicles decreased(P<0.05).2.2 The effects of electroacupuncture on serum FSH,LH,E2 and AMH in rats of each groupsCompared with the blank group,the FSH and LH in the serum of rats in model group were significantly increased(P<0.01),while the AMH and E2 were significantly decreased(P<0.01).Compared with the model group,the FSH and LH in serum of rats in non-meridian non-acupoints group and liver meridian group were decreased(P<0.05,P<0.01),while the AMH and E2 were increased(P<0.05,P<0.01).Compared with the non-meridian non-acupoints group,the FSH and LH in serum of rats in the liver meridian group were decreased(P<0.05),while the AMH and E2 were increased(P<0.05).2.3 Effects of electroacupuncture on Bax,Bcl-2 mRNA and protein in rat ovarian tissues of each groupCompared with the blank group,the expression of mRNA and protein of Bax increased(P<0.01)in the ovarian tissues of rats and and the expression of mRNA and protein of Bcl-2 decreased in the model group(P<0.01).Compared with the model group,the expression of mRNA and protein of Bax decreased(P<0.01,P<0.05)and the expression of mRNA and protein of Bcl2 increased in both the liver meridian group and the non-meridian non-acupoints group(P<0.01,P<0.05).Compared with the non-meridian non-acupoints group,the expression of mRNA and protein of Bax decreased(P<0.05)and the expression of mRNA and protein of Bcl-2 increased(P<0.05)in the liver meridian group.2.4 Effects of electroacupuncture on keapl,Nrf2 mRNA and protein in rat ovarian tissues of each groupCompared with the blank group,the expression of mRNA and protein of Keap-1、Nrf2 decreased(P<0.01)in the ovarian tissues of rats in the model group.Compared with the model group,the expression of mRNA and protein of Keap-1、Nrf2 increased(P<0.05,P<0.01)in both the liver meridian group and the non-meridian non-acupoints group.Compared with the non-meridian non-acupoints group,the expression of mRNA and protein of Keap-1、Nrf2 increased(P<0.05)in the liver meridian group.Conclusion1.Electroacupuncture liver acupoints can improve the ovarian reserve function,relieve the anxiety of patients with DOR and improve the quality of life,and the effect is better than electroacupuncture of non-meridian non-acupoints.2.Electroacupuncture liver acupoints may improve ovarian function in DOR model rats.The mechanism may be through up-regulating Keap1/Nrf2 pathway by regulating serum hormone levels and the expression of apoptosis-related proteins of Bax and Bcl-2,thus Regulating follicular development and reducing follicular atresia.