丹白颗粒对宫腔粘连大鼠TGF-β/Smad信号通路及内膜纤维化的影响

Effects of Danbai Granule on TGF-β/Smad Signaling Pathway and Endometrial Fibrosis in Rats with Intrauterine Adhesions

目的:探讨丹白颗粒对宫腔粘连(IUA)大鼠子宫内膜纤维化的影响及其可能机制。方法:将SD大鼠随机分为假手术组(15只)和模型组(65只),通过机械性刮除及感染性损伤手术进行大鼠IUA模型构建,假手术组仅进行开腹及子宫缝合手术,将构建成功的60只大鼠分为模型组、丹白颗粒低剂量组(2.5g/kg)、丹白颗粒中剂量组(5.0g/kg)、丹白颗粒高剂量组(10.0g/kg),每组15只,造模成功后24h开始给药,丹白颗粒低中高剂量组按剂量灌胃给药,假手术组与模型组以等量生理盐水灌胃,灌胃时间为上午7∶00至10∶00,持续灌胃24d;末次给药24h后,采集大鼠子宫组织,测定大鼠子宫通透性及AFS评分,以苏木精-伊红染色(HE)检测各组大鼠子宫组织病理变化、内膜厚度、腺体数量;以Masson染色检测大鼠子宫内膜纤维化程度;以酶联免疫吸附(ELISA)试剂盒检测大鼠子宫组织胶原含量;以蛋白免疫印迹法(Western blot)检测子宫内膜转化生长因子1(TGF-β1)/Smad通路蛋白表达。结果:与假手术组相比,模型组大鼠宫腔消失、腔壁组织呈现粘连状态,大鼠子宫内膜厚度及腺体数量显著降低,子宫通畅性评分、AFS评分、子宫不可溶性胶原含量、子宫内膜纤维化面积显著升高,子宫内膜TGF-β1、Smad3蛋白表达水平明显升高,Smad7蛋白表达水平明显降低(P<0.05);与模型组相比,丹白颗粒低、中、高剂量组大鼠子宫内膜厚度、腺体数量有一定的回升,子宫通畅性、子宫内膜纤维化程度得到缓解,不可溶性胶原含量显著降低,TGF-β1、Smad3蛋白表达水平呈降低趋势,Smad7蛋白表达水平呈升高趋势(P<0.05),且呈现一定的剂量效应关系。结论:丹白颗粒可有效缓解IUA大鼠子宫内膜纤维化程度,可能与抑制TGF-β/Smad通路有关。

Objective:To investigate the effect of Danbai Granule on endometrial fibrosis in rats with intrauterine adhesion(IUA)and its possible mechanism.Methods:SD rats were randomly divided into sham operation group(15 rats)and model group(65 rats),the IUA model was established by mechanical curettage and infective injury,and the sham operation only underwent laparotomy and uterine suture.The successful models were divided into model group,Danbai Granule low-dose group(2.5 g/kg),Danbai Granule middle-dose group(5.0 g/kg)and Danbai Granule high-dose group(10.0 g/kg),with 15 in each group.The drug was administered 24 hours after successful modeling,Danbai Granule low,medium and high dose groups was given by gavage according to the dosage,while the sham operation group and the model group were given the same amount of normal saline,the time of intragastric administration was from 7∶00 to 10∶00 a.m,continuous gavage for 24 days.At 24 hours after the last administration,the uterine tissues were collected,the uterine permeability and AFS score were measured,hematoxylin eosin staining(HE)was used to detect the pathological changes,endometrial thickness and the number of glands in each group.Masson staining was used to detect the degree of endometrial fibrosis;enzyme linked immunosorbent assay(ELISA)was used to detect the collagen content in rat uterus;and Western blot was used to detect the expression of transforming growth factor-β1(TGF-β1)/Smad pathway protein in endometrium.Results:Compared with those in the sham operation group,the uterine cavity disappeared;the cavity wall tissue showed adhesion state,the endometrial thickness and the number of glands were significantly reduced in the model group;the uterine patency score,AFS score,content of insoluble collagen and area of endometrial fibrosis were significantly higher;the protein expression levels of TGF-β1 and Smad3 in endometrium were significantly higher,and the expression level of Smad7 protein were significantly lower(P<0.05);compared with those in the model group,the endometrial thickness and the number of glands in the Danbai Granule low,medium and high dose groups were higher to a certain extent,the patency of uterus and the degree of endometrial fibrosis were relieved,and the content of insoluble collagen was significantly lower;the protein expression levels of TGF-β1 and Smad3 showed an decreasing trend;the expression of Smad7 protein showed an increasing trend(P<0.05);there was a certain dose effect relationship.Conclusion:Danbai granule can effectively alleviate the degree of endometrial fibrosis in IUA rats,which may be related to the inhibition of TGF-β/Smad pathway.