防粘汤对宫腔粘连模型大鼠的治疗作用研究

The Effect of Fangzhan Decoction on Intrauterine Adhesion of Model Rats

目的:观察防粘汤对宫腔粘连模型大鼠炎症因子、T细胞亚群及磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/Akt)信号通路关键分子蛋白表达的影响,探讨防粘汤对宫腔粘连的治疗作用。方法:首先使用HPLC法分析防粘汤各药物主要有效成分,将76只SD大鼠随机分为正常组和造模组,采用机械刮宫及感染双重损伤建立宫腔粘连大鼠模型,造模成功后,将造模组动物随机分为模型组、雌激素组及防粘汤低、中、高剂量(4.14 g·kg^(-1)·d^(-1)、8.28 g·kg^(-1)·d^(-1)、16.56 g·kg^(-1)·d^(-1))组。每天1次,连续灌胃给药21 d后处死大鼠,收集其血清、脾脏、肝脏及双侧子宫组织,采用HE及Masson染色观察其组织病理学变化;HE染色观察肝脏组织病理变化;ELISA法检测血清中TNF-α、IL-6及TGF-β1的表达;Western Blot法检测子宫组织中PI3K、p-Akt及Akt蛋白的表达;流式细胞仪检测大鼠脾淋巴细胞CD4^(+)、CD8^(+)亚群的变化。结果:防粘汤中含有金丝桃苷、甘草苷、落新妇苷、阿魏酸、丹酚酸B及淫羊藿苷。与正常组相比,模型组大鼠子宫组织明显肿胀、短缩,质硬,无弹性,子宫内膜菲薄,结构被破坏,腺体数量少,有大量炎性细胞浸润,胶原纤维增生明显;血清TNF-α、IL-6及TGF-β1表达显著升高(P<0.05,P<0.01);子宫组织中PI3K、Akt、p-Akt蛋白表达明显增多(P<0.01);CD4^(+)细胞比例下降而CD8^(+)细胞比例上升,CD4^(+)/CD8^(+)比值降低(P<0.05,P<0.01)。与模型组相比,防粘汤中、高剂量组大鼠子宫形态改善明显,炎症渗出明显减少,子宫内膜腺体数量增多,纤维化面积减少;血清TNF-α、IL-6及TGF-β1水平明显降低(P<0.05,P<0.01);子宫组织中PI3K、Akt、p-Akt蛋白表达显著减少(P<0.05,P<0.01);CD4^(+)/CD8^(+)比值升高(P<0.01)。结论:防粘汤能促进宫腔粘连大鼠子宫内膜组织修复,减少炎性浸润,改善免疫调节,抑制PI3K/Akt信号通路中关键蛋白的表达,延缓子宫内膜纤维化。

Objective:To observe the effect of Fangzhan decoction(FZD)on the expression of inflammatory factors,T lymphocyte subsets and key molecular proteins of phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt)signaling pathway in intrauterine adhesions model rats,and to explore the therapeutic effect of FZD on intrauterine adhesions.Methods:The main effective ingredients of each medicinal in FZD were analyzed by HPLC.Seventy-six SD rats were randomly divided into normal group and model group.The rat model of uterine adhesion was established by mechanical curettage and infection.After successful modeling,the model group was randomly divided into model group,estrogen group and low,medium and high dose of FZD(4.14 g·kg^(-1)·d^(-1),8.28 g·kg^(-1)·d^(-1),16.56 g·kg^(-1)·d^(-1))groups.All rats were gavaged once a day for 21 consecutive days,and then euthanized.Their serums,spleens,livers and bilateral uterine tissues were collected.Histopathological changes were detected using HE and Masson staining;HE staining was used to observe the pathological changes of liver tissues;ELISA was used to detect the expression of TNF-α,IL-6 and TGF-β1 in serum;Western blot was used to detect the expression of PI3K,p-Akt and Akt protein in uterine tissues;and flow cytometry was used to detect the changes of CD4^(+)and CD8^(+)subsets in rat spleen lymphocytes.Results:FZD contained hyperoside,glycyrrhizin,astilbin,ferulic acid,salvianolic acid B and icariin.Compared with the normal group,the uterine tissues of rats in the model group were significantly swollen,shortened,hard,and inelastic,the endometrium was thin,the structure was destroyed,and the number of glands was reduced,there were a large number of inflammatory and infiltrated cells,and collagen fiber proliferation was significant.Serum TNF-α,IL-6 and TGF-β1 expression was significantly increased(P<0.05,P<0.01).PI3K,Akt,and p-Akt protein expression was significantly increased in the uterine tissues(P<0.01).The proportion of CD4^(+)cells decreased while the proportion of CD8^(+)cells increased,and the CD4^(+)/CD8^(+)ratio decreased(P<0.05,P<0.01).Compared with the model group,the uterine morphology of rats in the medium and high dose FZD groups was significantly improved,the inflammatory exudation was significantly reduced,the number of endometrial glands increased,and the fibrosis area was reduced.The serum TNF-α,IL-6 and TGF-β1 levels were significantly reduced(P<0.05,P<0.01);the PI3K,Akt,and p-Akt protein expression in the uterine tissue was significantly reduced(P<0.01);and the CD4^(+)/CD8^(+)ratio was increased(P<0.01).Conclusion:Fangzhan Decoction can promote the repair of endometrial tissue,reduce inflammatory infiltration,improve immune disorders,inhibit the expression of key proteins in PI3K/Akt signaling pathway,and delay endometrial fibrosis in rats with intrauterine adhesions.