益气阴祛瘀浊方剂联合全反式维甲酸减轻大鼠子宫内膜纤维化的机制

Mechanism of yiqiyinquyuzhuo recipe combined with all-trans retinoic acid for relieving endometrial fibrosis in rats

目的探讨益气阴祛瘀浊方剂联合全反式维甲酸(ATRA)减轻大鼠子宫内膜纤维化的机制。方法选取20只雌性大鼠,采用刮勺反复轻刮子宫内膜至粗糙构建大鼠子宫内膜纤维化模型,随机均分为手术未给药组(仅造模)、方剂组(造模后每日灌肠20 mL/kg益气阴祛瘀浊方剂)、ATRA组(造模后每日灌肠2.5 mg/kg ATRA)及联合组(造模后每日灌肠20 mL/kg益气阴祛瘀浊方剂+2.5 mg/kg ATRA),另选5只健康雌性大鼠作为正常对照组(常规饲养,不作任何处理);各组大鼠灌肠给药1次/d、连续5 d,第6天处死取子宫,采用苏木素伊红染色(HE)法检测各组大鼠子宫内膜纤维化、实时荧光定量聚合酶链式反应(PCR)检测子宫内膜转化生长因子-β(TGF-β)和β-连环蛋白(β-catenin)的蛋白及mRNA表达。结果正常组大鼠子宫内膜呈粉红色、血管正常分布、无出血或淤血、间质区结缔组织纤维规律排列,手术未给药组大鼠子宫内膜第6天可见出血区、结缔组织富集、间质内可见少量血管,方剂组和ATRA组大鼠子宫内膜较手术未给药组有不同程度的修复、间质纤维化程度明显减轻、且联合组大鼠子宫内膜修复效果尤为明显;正常对照组大鼠子宫内膜TGF-β和β-catenin蛋白表达均低于手术未给药组,方剂组、ATRA组及联合组大鼠子宫内膜TGF-β和β-catenin蛋白表达均高于正常对照组,且低于手术未给药组(P<0.05);方剂组、ATRA组大鼠子宫内膜TGF-β和β-catenin mRNA表达分别较正常对照组和手术未给药组升高(P<0.05);方剂组、ATRA组大鼠子宫内膜腺体数量分别较正常对照组和联合组均减少,较手术未给药组均增多(P<0.05);方剂组、ATRA组、联合组大鼠子宫内膜纤维化面积较正常对照组均增多,较手术未给药组均减少,且联合组大鼠较正常对照组增多(P<0.05)。结论益气阴祛瘀浊方剂联合ATRA减轻大鼠子宫内膜纤维化的机制可能与影响TGF-β和β-catenin的表达有关。

Objective To study the mechanism of Yiqiyinquyuzhuo recipe combined with all-trans retinoic acid(ATRA)to alleviate the fibrosis of endometrium in rats.Methods Twenty female rats were selected and a rat model of endometrial fibrosis was constructed by repeatedly and gently scraping endometrium with a spatula until the endometrium was rough.The rats were randomly divided into a non-administered group(modeling only),a prescription group(daily enema of 20 mL/kg yiqiyinquyuzhuo recipe after modeling),an ATRA group(daily enema of 2.5 mg/kg ATRA after modeling),a combination group(daily enema of 20 mL/kg yiqiyinquyuzhuo recipe after modeling plus 2.5 mg/kg ATRA),and the normal control group consisting of another 5 randomly selected healthy rats(conventional feeding and without any treatment).Rats in each group were given enema 1 time/d for 5 consecutive days.On the 6 th day,they were killed and uteruses were taken out.The endometrial fibrosis in each group was detected by hematoxylin and eosin staining(HE),and protein and mRNA expressions of transforming growth factor-β(TGF-β)andβ-catenin protein(β-catenin)were detected by real-time fluorescence quantitative polymerase chain reaction(PCR).Results The endometrium of the rats in the normal group was pink,with normal distribution of blood vessels,no bleeding or congestion,and connective tissue fibers in the interstitial area were regularly arranged.The endometrium of the rats in the non-administered group showed bleeding areas and enriched connective tissue,and a small number of blood vessels within the interstitium were visible on the 6 th day.Compared with the non-administered group,the endometrium of the rats in the prescription group and the ATRA group was repaired to different degrees,the interstitial fibrosis was significantly reduced,and the effect of endometrial repair was particularly obvious in the combination group.The expression of TGF-βandβ-catenin protein in the endometrium of the normal control group was lower than that of the non-administered group,while the expression of TGF-βandβ-catenin protein in the endometrium of the prescription group,ATRA group,and combination group was higher than that of normal control group and lower than that of non-administered group(P<0.05).The expression of TGF-βandβ-catenin mRNA in the endometrium of rats in the prescription group and ATRA group increased compared with that of the normal control group and the non-administered group(P<0.05).The number of endometrial glands in rats in the prescription group and ATRA groups decreased compared with the normal control group and the combination group,and it increased compared with the non-administered group(P<0.05).The endometrial fibrosis area of the rats in the prescription group,ATRA group,and combination group increased compared with that in the normal control group,and decreased compared with the non-administered group;and that of the combination group increased compared with the normal control group(P<0.05).Conclusion Yiqiyinquyuzhuo recipe combined with ATRA in alleviating endometrial fibrosis in rats may be related to the expression affecting TGF-βandβ-catenin.