基于“治未病”理论探讨电针对DOR大鼠Th1/Th2免疫平衡的影响

目的 基于“治未病”理论，探讨电针对雷公藤多苷诱导的卵巢储备功能减退(Diminished ovarian reserve, DOR)模型大鼠卵巢功能和Th1/Th2免疫平衡的影响。方法 将27只动情周期正常的雌性SPF级SD大鼠随机分为空白组、模型组和电针组，每组9只。模型组和电针组采用雷公藤多苷片混悬液按50 mg·kg^-1·d^-1连续灌胃14 d,每日1次，空白组每日给予1次等体积的生理盐水灌胃，连续14 d。电针组大鼠在灌胃1 h后，进行电针干预，选取“中脘”“关元”或双侧“肾俞”穴，2组穴位隔日交替，每日1次，每次10 min,连续14 d。电针参数设置为2/100 Hz疏密波(疏波与密波交替持续的时间各约1.5 s),电流强度为1.0 mA。每日采用阴道脱落细胞涂片观察大鼠动情周期；干预结束后，采用HE染色观察卵巢组织学形态；ELISA检测大鼠血清FSH、E₂、IL-4、IFN-γ含量；免疫组化、Western blot和qPCR分别检测大鼠卵巢组织中T-bet、GATA3蛋白及mRNA的表达情况；流式细胞术...

Effect of Electroacupuncture on the Immune Balance of Th1/Th2 in Diminished Ovarian Reserve Rats Based on the Preventive Treatment Theory

  OBJECTIVE  To explore the effect of electroacupuncture on ovarian reserve function and Th1/Th2 immune balance in DOR rats based on the preventive treatment theory.  METHODS  A total of 27 female SPF Sprague-Dawley rats with normal estrus cycle were randomly divided into control group, DOR group, and electroacupuncture group, with 9 rats in each group. The DOR group and the electroacupuncture group were treated with intragastric administration of tripterygium glycosides(TGs) tablet suspension at a dose of 50 mg·kg-1·d-1 for 14 days to establish the DOR models. An equal volume of normal saline was given to the control group once a day for 14 days. One hour after intragastric administration, rats in the electroacupuncture group had a daily 10-minute electroacupuncture treatment at bilateral "Shenshu" (BL23) or "Guanyuan" (CV4) and "Zhongwan" (CV12). The two groups of acupoints were alternated every other day. The electroacupuncture parameters were as follows: 2/100 Hz, 1.0 mA. Vaginal smears were obtained every day starting the first day of intervention to monitor the estrus cycle of the rats. After the intervention, HE staining was used to observe the histological morphology of ovaries; the level of estradiol (E₂), follicle-stimulating hormone (FSH), interleukin-4 (IL-4) and interferon-γ (IFN-γ) were measured through ELISA kits; the expression of T-bet and GATA3 mRNA was detected by qPCR; the protein levels of T-bet and GATA3 in ovarian tissue were detected by immunohistochemistry and Western blot; the ratio of Th1 and Th2 cells in the spleen was detected by flow cytometry.  RESULTS  Compared with the control group, the number of primordial follicles in the DOR group decreased (P < 0.05), and atretic follicles increased (P < 0.01); both the ovarian index and spleen index were reduced in the DOR group (P < 0.01); serum E₂ and IL-4 levels decreased (P < 0.05), while the levels of FSH and IFN-γ increased in the DOR group (P < 0.05); GATA3 protein and mRNA expression lessened (P < 0.05, P < 0.01), while T-bet protein and mRNA expression were upregulated in ovarian tissues (P < 0.05); the T-bet/GATA3 ratio was elevated (P < 0.05); the proportion of Th1 cells as well as Th1/Th2 ratio increased (P < 0.01), and the proportion of Th2 cells decreased (P < 0.05). In comparison with DOR group, the number of primordial follicles in the electroacupuncture group increased notably, which was accompanied by decreased atretic follicles (P < 0.01); the ovarian index and spleen index increased (P < 0.01); the levels of E₂ and IL-4 both increased(P < 0.05, P < 0.01), while FSH and IFN-γ concentrations decreased in DOR group (P < 0.01); GATA3 protein and mRNA were expressed higher in ovarian tissues (P < 0.05), while the expression of T-bet protein and mRNA was downregulated (P < 0.05, P < 0.01); the T-bet/GATA3 ratio is reduced (P < 0.01); the proportion of Th1 cells alone with Th1/Th2 ratio decreased (P < 0.05, P < 0.01), and the proportion of Th2 cells was higher than that in DOR group (P < 0.05).  CONCLUSION  Electroacupuncture has a protective effect on ovarian reserve in DOR rats, and its mechanism may be related to the regulation of Th1/Th2 immune balance.