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温经汤加味对EM肾虚血瘀证大鼠局部微环境Caspase-8,MMP-9, E-cadherin, N-cadherin的影响
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山西中医药大学,山西 晋中 030619

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通讯作者:

崔轶凡,博士,副教授,从事中医妇科痛证的临床与基础研究,Tel:0351-3179792,E-mail:cuiyf1120@163.com

中图分类号:

R2-0;R22;R285.5;R289;R33

基金项目:

国家自然科学基金面上项目(81774356);山西省科技厅面上项目(201901D111341);山西省卫生健康委员会科研项目(2017128);山西中医药大学中医妇科优势病种证治研究创新团队项目(2018TD-017)


Modified Wenjingtang Affects Caspase-8 MMP-9 N-cadherin and E-cadherin in Local Microenvironment of EM Rats with Kidney Deficiency and Blood Stasis
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Shanxi University of Chinese Medicine,Jinzhong 030619,China

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    摘要:

    目的 探讨温经汤加味对子宫内膜异位症(EM)肾虚血瘀证大鼠的干预作用及其机制研究。方法 SPF级雌性未孕大鼠随机选出正常组,其余大鼠采用“冰水浸泡+皮下注射盐酸肾上腺素法”进行肾虚血瘀证造模,造模结束后,仅开腹不进行内膜移植大鼠,设为假手术组,其余大鼠以自体内膜移植法进行EM造模,EM肾虚血瘀证造模成功大鼠随机分为阳性药组及温经汤加味(中药)低、中、高剂量组。中药低、中、高剂量组分别以5,10,20 g·kg-1灌胃,每日给药1次,连续4周;阳性药选取达那唑63 mg·kg-1灌胃,每日给药1次,连续4周。4周后,取各组大鼠内膜组织(异位、在位)进行苏木素-伊红(HE)染色,观察其组织病理学变化;免疫组化(IHC)法、蛋白质免疫印迹(Western blot)法检测半胱氨酸天冬氨酸蛋白酶-8(Caspase-8),基质金属蛋白酶-9(MMP-9),上皮细胞钙黏蛋白(E-cadherin)及神经钙黏蛋白(N-cadherin)蛋白表达;实时荧光定量聚合酶链式反应(Real-time PCR)检测Caspase-8,MMP-9,E-cadherin,N-cadherin mRNA表达。结果 经IHC法和Western blot法,与假手术组比较,EM肾虚血瘀证模型组大鼠在位、异位内膜组织中MMP-9,N-cadherin蛋白表达显著升高(P<0.01),Caspase-8,E-cadherin蛋白表达显著降低(P<0.01);与模型组比较,达那唑组、中药低、中、高剂量组大鼠在位、异位内膜组织中MMP-9,N-cadherin蛋白明显降低(P<0.05,P<0.01),Caspase-8,E-cadherin蛋白表达明显升高(P<0.05,P<0.01)。经Real-time PCR法检测,与假手术组大鼠比较,模型组大鼠在位、异位内膜组织中MMP-9,N-cadherin mRNA表达显著升高(P<0.01),Caspase-8,E-cadherin mRNA表达显著降低(P<0.01);与模型组大鼠在位内膜比较,达那唑组、中药高、中剂量组在位、异位内膜组织MMP-9,N-cadherin mRNA表达明显降低,Caspase-8,E-cadherin mRNA表达明显升高(P<0.05,P<0.01)。结论 温经汤加味能够通过调节Caspase-8,MMP-9,E-cadherin,N-cadherin等因子水平异常表达,改善EM肾虚血瘀证大鼠免疫抑制、阻断微血管新生,从而起到治疗EM的作用;以上微观指标的变化有望成为体现疾病(EM),证候(肾虚血瘀证)及病理机制(免疫抑制、微血管新生)的重要标志物。

    Abstract:

    Objective To investigate the therapeutic effect and mechanism of modified Wenjingtang on endometriosis (EM) rats with kidney deficiency and blood stasis.Method Healthy non-pregnant female Sprague-Dawley (SD) rats of SPF grade were randomly divided into the blank group and experimental group. After being modeled via soaking in ice water and subcutaneous injection of epinephrine hydrochloride, the ones in the experimental group were further divided into the sham operation group and EM model group, with the former only undergoing laparotomy and the latter further receiving autologous endometrial transplant for triggering EM. The successfully modeled rats with EM due to kidney deficiency and blood stasis were randomized into the positive drug (danazol, 63 mg·kg-1) group and low- (5 g·kg-1), medium- (10 g·kg-1), and high-dose (20 g·kg-1) modified Wenjingtang groups. The corresponding drugs were administered by gavage, once per day, for four weeks. Then the ectopic and eutopic endometrial tissues were stained with hematoxylin-eosin (HE) to observe the histopathological changes. The protein and mRNA expression levels of cysteinyl aspartate-specific proteinase-8 (Caspase-8), matrix metalloproteinase-9 (MMP-9), N-cadherin, and E-cadherin were detected by immunohistochemistry (IHC), Western blotting, and real-time polymerase chain reaction (Real-time PCR), respectively.Result The IHC and Western blot revealed that the protein expression levels of MMP-9 and N-cadherin in eutopic and ectopic endometrial tissues of the model group were significantly increased as compared with those in the sham operation group (P<0.01), while the levels of Caspase-8 and E-cadherin was significantly decreased (P<0.01). Compared with the model group, the danazol and low-, medium-, and high-dose modified Wenjingtang groups exhibited obviously down-regulated MMP-9 and N-cadherin protein expression in eutopic and ectopic endometrial tissues (P<0.05,P<0.01), but up-regulated Caspase-8 and E-cadherin (P<0.05, P<0.01). Real-time PCR uncovered that the mRNA expression levels of MMP-9 and N-cadherin in eutopic and ectopic endometrial tissues of the model group were significantly elevated as compared with those in the sham operation group (P<0.01), whereas the levels of Caspase-8 and E-cadherin significantly declined (P<0.01). The comparison with the eutopic endometrial tissue in the model group showed that the mRNA expression levels of MMP-9 and N-cadherin in the danazol group and high- and medium-dose modified Wenjingtang groups were significantly down-regulated, while those of Caspase-8 and E-cadherin were significantly up-regulated (P<0.05,P<0.01).Conclusion Modified Wenjingtang alleviates the immunosuppression and blocks the angiogenesis in EM rats with kidney deficiency and blood stasis syndrome by regulating the expression of such cytokines as Caspase-8, MMP-9, N-cadherin, and E-cadherin, thus exerting the therapeutic effect against EM. The above-mentioned micro-indicators are potential markers reflecting the disease (EM), syndrome (kidney deficiency and blood stasis), and pathological mechanisms (immunosuppression and angiogenesis).

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崔轶凡,孙瑞英,王志平,曹娟.温经汤加味对EM肾虚血瘀证大鼠局部微环境Caspase-8,MMP-9, E-cadherin, N-cadherin的影响[J].中国实验方剂学杂志,2021,27(11):42~51

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  • 收稿日期:2021-02-23
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  • 在线发布日期: 2021-05-06
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