桂枝茯苓胶囊对子宫肌瘤大鼠子宫系数和激素水平的影响相关机制研究

Study on the mechanism of Guizhi Fuling capsule’s effect on uterine coefficient and hormone level in rats with uterine fibroids

目的探讨桂枝茯苓胶囊对子宫肌瘤大鼠的保护作用及其机制是否与腺苷酸活化蛋白激酶(AMPK)-核因子E2相关因子2 (Nrf2)通路有关。方法选择50只无特定病原体(SPF)级健康雌性未孕SD大鼠为研究对象,由西安交通大学提供,体重180~220 g。按照随机数字表法均分为5组:对照组、模型组、桂枝茯苓胶囊低、中、高剂量组,各10只。对照组不建模,前肢腋下皮下注射0.9%氯化钠溶液1 mL 30 d,再灌胃0.9%氯化钠溶液1 mL 42 d。模型组、桂枝茯苓胶囊低、中、高剂量组通过前肢腋下皮下注射己烯雌酚0.2 mg·kg^(-1)·d^(-1),30 d,建立子宫肌瘤模型。模型组再灌胃0.9%氯化钠溶液1 mL 42 d,桂枝茯苓胶囊低、中、高剂量组分别再灌胃桂枝茯苓胶囊0.8、1.6、3.2 g·kg^(-1)·d^(-1),42 d。观察各组大鼠子宫系数的变化,苏木精和伊红染色观察子宫肌瘤大鼠子宫平滑肌形态,酶联免疫分析测定血清雌二醇和孕酮水平,实时荧光定量PCR检测AMPK、Nrf2、Bcl-2和Bax的mRNA表达情况。结果与对照组比较,模型组大鼠的子宫系数、子宫体径均显著增加,血清雌二醇浓度、血清孕酮水平显著升高,模型组大鼠子宫中AMPK、Nrf2、Bcl-2的mRNA表达显著增加,Bax的mRNA表达显著降低,差异均有统计学意义(P <0.05)。与模型组比较,低、中、高剂量组大鼠的子宫系数、子宫体径显著减少,血清雌激素水平显著降低,并改善子宫组织的病理状况,子宫中AMPK、Nrf2、Bcl-2的mRNA表达显著降低,Bax的mRNA表达显著增加,差异均有统计学意义(P <0.05),作用以桂枝茯苓胶囊高剂量组作用最为明显。结论桂枝茯苓胶囊对大鼠子宫肌瘤具有明显的抑制作用。其机制可能是通过AMPK-Nrf2通路提高Bax基因表达、下调Bcl-2表达,并减少血浆雌二醇和孕酮来促进子宫肌瘤细胞凋亡。

Objective To investigate the protective effect of Guizhi Fuling capsule on uterine fibroids in rats and whether the mechanism was related to adenylate activated protein kinase( AMPK)-nuclear factor E2 related factor 2( Nrf2) pathway. Methods Fifty SPF-free healthy female non-pregnant SD rats were selected as the research objects,provided by Xi’an Jiaotong University,weighing 180-220 g. According to the random number table method,they were divided into 5 groups: the control group,the model group,and the Guizhi Fuling capsule low,medium and high dose groups,each with 10 rats. In the control group,no modeling was performed. Forelimbs were subcutaneously injected with 1 mL of 0. 9% sodium chloride solution for 30 days,followed by intragastric administration of 1 mL of 0. 9% sodium chloride solution for 42 days. Model group and Guizhi Fuling capsule low,medium and high dose groups were injected subcutaneously with diethylstilbestrol 0. 2 mg·kg^(-1)·d^(-1)under the armpit of the forelimbs for 30 days to establish a uterine fibroids model. The model group was re-administered with 0. 9% sodium chloride solution 1 mL for 42 days,and the Guizhi Fuling capsule low,medium and high dose groups were re-administered with Guizhi Fuling capsule0. 8,1. 6,3. 2 g·kg^(-1)·d^(-1),respectively,42 d. The changes in the uterine coefficient of rats in each group were observed. The morphology of uterine smooth muscle in rats with uterine fibroids was observed and stained with hematoxylin and eosin. The levels of serum estradiol and progesterone were measured by enzyme-linked immunoassay test. The mRNA expression of AMPK,Nrf2,Bcl-2 and Bax was detected by real-time fluorescent quantitative PCR. Results Compared with the control group,uterine coefficient,and uterine body diameter of the model group were increased significantly,and the level of serum estradiol concentration and serum progesterone were significantly increased,the expression of AMPK,Nrf2,Bcl-2 mRNA in the uterus of the model group were increased significantly,and the expression of Bax mRNA were decreased significantly,the differences were statistically significant( P < 0. 05). Compared with the model group,uterine coefficient,and uterine body diameter of rats in the low,medium,and high dose groups were significantly reduced,and the level of serum estrogen was significantly reduced,and the pathological condition of the uterine tissue was improved,the expression of AMPK,Nrf2,Bcl-2 mRNA were significantly reduced,Bax mRNA expression was significantly increased,and the differences were statistically significant( P < 0. 05). The effect was the most obvious in the Guizhi Fuling capsule high-dose group. Conclusion This study showed that Guizhi Fuling capsule had a significant inhibitory effect on uterine fibroids in rats.The mechanism might be that the AMPK-Nrf2 pathway enhanced the expression of Bax gene,down-regulated the expression of Bcl-2 in uterine from the rats,and reduced serum levels of estradiol and progesterone,and subsequently promoted the apoptosis of uterine fibroid cells,suggesting that Guizhi Fuling capsule could be an alternative to current uterine fibroid treatments.