电针通过抑制瘦素/亲肽素轴改善多囊卵巢综合征模型大鼠排卵功能障碍及性激素水平

Effect of Electro-acupuncture on Ovulatory Dysfunction and Sex Hormone Levels in Rats with Polycystic Ovary Syndrome through Inhibiting Leptin/Kisspeptin Axis

【目的】观察电针对多囊卵巢综合征(PCOS)模型大鼠的治疗作用及机制。【方法】从70只雌性Wistar大鼠中随机选择15只作为正常组,剩余55只连续20 d于颈背部皮下注射脱氢表雄酮(DHEA)60 mg/kg构建PCOS模型。选择造模成功的45只大鼠随机分为模型组、电针组及西药组,每组15只。其中:电针组大鼠给予电针刺激三阴交、足三里穴,西药组大鼠给予二甲双胍300 mg/kg灌胃,模型组、正常组给予灌胃等体积生理盐水,另西药组、模型组及正常组大鼠于非穴位处进行电针刺激,均每日1次,连续15 d。记录造模前、成模后及电针干预后大鼠体质量。干预结束后,测量大鼠卵巢直径及质量,苏木素-伊红(HE)染色法观察大鼠卵巢组织形态及卵泡发育情况,酶联免疫吸附分析(ELISA)检测大鼠血清瘦素(LEP)、雌二醇(E2)、睾酮(T)、促黄体生成素(LH)、促卵泡激素(FSH)水平,蛋白免疫印迹(Western Blot)法检测大鼠下丘脑弓状核瘦素受体(LEPR)、亲肽素(KP)、G蛋白偶联受体54(GPR54)、促性腺激素释放激素(GnRH)蛋白表达。【结果】DHEA可成功构建PCOS大鼠模型。与造模前比较,模型组、电针组及西药组成模大鼠体质量均显著增大(P<0.05);电针组和西药组大鼠体质量显著低于模型组(P<0.05)。治疗后,与正常组比较,模型组卵巢质量及直径显著增大,结构不完整,未见黄体,卵泡颗粒层变薄,囊性卵泡(CF)数目显著增多,发育中卵泡(DF)及黄体(CL)数目显著减少,血清LEP、E2、T及LH水平均显著升高,FSH水平显著降低,下丘脑弓状核组织内LEPR、KP、GPR54及GnRH蛋白表达水平均显著升高(均P<0.05);与模型组比较,电针组及西药组大鼠卵巢质量及直径显著减小,卵巢组织逐渐变得完整,卵泡颗粒层变厚,CF数目显著减少,DF及CL数目显著增多,血清LEP、E2、T及LH水平均显著降低,FSH水平显著升高,下丘脑弓状核组织内LEPR、KP、GPR54及GnRH蛋白表达水平均显著降低(均P<0.05);与电针组比较,西药组作用效果较强(P<0.05)。【结论】电针可调节PCOS大鼠性激素水平,改善排卵功能障碍,其作用机制可能与抑制LEP/KP轴,平衡内分泌功能紊乱有关。

Objective To observe the therapeutic effect and mechanism of electro-acupuncture in rats with polycystic ovary syndrome(PCOS)model. Methods Fifteen rats were randomly selected from 70 female Wistar rats as the normal group, and the remaining 55 rats were injected subcutaneously with dehydroepiandrosterone(DHEA)60 mg/kg on the back of the neck for consecutive 20 days to construct a PCOS model. The 45 rats with successful modeling were randomly divided into the model group,the electro-acupuncture group and the western medicine group, with 15 rats in each group. The rats in the electro-acupuncture group were given electroacupuncture stimulation at the Sanyinjiao(SP6)and Zusanli(ST36)acupoints,the rats in the western medicine group were given metformin(300 mg/kg)by gavage,the rats in the model group and the normal group were given the same volume of normal saline by gavage,and additionally,the rats in the western medicine group,the model group and the normal group were given electro-acupuncture stimulation at non-acupuncture points. The treaments in all groups were given once a day for consecutive 15 days. The body masses of the rats before modeling,after modeling and after the electro-acupuncture intervention were recorded. After ending intervention,the diameter and mass of rat ovarians were measured, the morphological features and follicular development of rat ovarian tissues were observed by hematoxylin-eosin(HE)staining,the levels of serum leptin(LEP),estradiol(E2),testosterone(T), luteinizing hormone(LH)and follicle stimulating hormone(FSH)were detected by enzymelinked immunosorbent assay(ELISA), the protein expression of leptin receptor(LEPR), kisspeptin(KP),G protein-coupled receptor 54(GPR54)and gonadotropin-releasing hormone(GnRH)in hypothalamic arcuate nucleus were detected by immunoblotting(Western Blot). Results DHEA could successfully construct the PCOS rat model. The body masses of the rats in the model group,the electro-acupuncture group and the western medicine group were significantly increased compared with those before modeling(P<0.05);the body masses of the rats in the electro-acupuncture group and the western medicine group were significantly lower than those of the model group(P<0.05). After treatment,compared with the normal group,the ovarian mass and diameter of the model group were significantly increased, the ovarian structure was incomplete, no corpus luteum was seen, the follicular granular layer was thinned,the number of cystic follicles(CF)was significantly increased,the number of developing follicles(DF)and corpus luteum(CL)was significantly decreased,the serum levels of LEP,E2,T and LH were significantly increased,the FSH level was significantly decreased,and the protein expression levels of LEPR,KP,GPR54 and GnRH in the hypothalamic arcuate nucleus tissues were significantly increased(all P<0.05);compared with the model group,the ovarian mass and diameter of the rats in the electro-acupuncture group and western medicine group were significantly reduced, the ovarian tissue gradually became intact, the follicular granular layer became thicker,the number of CF was significantly reduced,the number of DF and CL was significantly increased,the serum levels of LEP,E2,T and LH were significantly reduced,and the FSH level was significantly increased, and the protein expression levels of LEPR, KP, GPR54 and GnRH in the hypothalamic arcuate nucleus were significantly reduced(all P<0.05);compared with the electro-acupuncture group,the western medicine group had a stronger effect(P<0.05). Conclusion Electro-acupuncture is effective in regulating sex hormone levels and improving ovulatory dysfunction in PCOS rats,and its mechanism of action may be related to inhibiting the LEP/KP axis and balancing endocrine dysfunction.