补肾活血方对肾虚血瘀型子宫内膜增生症大鼠的作用及机制研究

Study on the Effect and Mechanism of Bushen Huoxue Prescription on Rats with Endometrial Hyperplasia of Kidney Deficiency and Blood Stasis Type

目的探讨补肾活血方(巴戟天、菟丝子、沙苑子、党参、茯苓等)对肾虚血瘀型子宫内膜增生症(Endometrial hyperplasia,EH)大鼠的作用及机制。方法将90只SD大鼠随机分为正常组、模型组、米非司酮组(12 mg·kg^(-1))及补肾活血方低、中、高剂量组(1.2、2.4、4.8 g·kg^(-1)),每组15只。采用灌胃羟基脲及戊酸雌二醇,联合尾静脉注射高分子右旋糖苷法复制肾虚血瘀型EH大鼠模型。造模成功后,各组按照设定剂量灌胃给药,每日1次,连续21 d。实验结束后,采用生化法检测各组大鼠血清环磷酸腺苷(c AMP)、环磷酸鸟苷(c GMP)、皮质醇(CORT)、促肾上腺皮质激素(ACTH)、三碘甲状腺原氨酸(T_(3))、甲状腺素(T_(4))、促甲状腺激素(TSH)的水平以及全血黏度、血液流变学、凝血功能等指标;ELISA法检测血清雌激素(E2)、卵泡雌激素(FSH)、黄体生成素(LH)、一氧化氮(NO)、内皮素1(ET-1)的水平;HE染色法观察子宫内膜组织病理形态变化,并测量子宫内膜厚度;免疫组化法检测子宫内膜组织中基质金属蛋白酶9(MMP-9)、基质金属蛋白酶抑制物1(TIMP-1)蛋白表达水平;Western Blot法检测子宫内膜组织中MMP-9、TIMP-1、B细胞淋巴瘤2(Bcl-2)、Bcl-2相关X蛋白(Bax)、第10号染色体缺失性磷酸酶-张力蛋白同源物基因(PTEN)蛋白表达水平。结果与正常组比较,模型组大鼠血清CORT、ACTH、c AMP、T_(3)、T_(4)、TSH、ET-1水平及ET-1/NO比值均明显降低(P<0.05,P<0.01),c GMP、E2、FSH、LH、NO水平均明显升高(P<0.05,P<0.01);血液流变学各项指标及纤维蛋白原(FIB)含量均明显升高(P<0.05),凝血酶时间(TT)、凝血酶原时间(PT)、活化部分凝血活酶时间(APTT)均明显延长(P<0.05);子宫指数及子宫内膜厚度显著升高(P<0.01);子宫内膜组织MMP-9、Bcl-2蛋白表达显著上调(P<0.01),TIMP-1、PTEN、Bax蛋白表达显著下调(P<0.01)。与模型组比较,补肾活血方组大鼠血清CORT、ACTH、c AMP、T_(3)、T_(4)、TSH、ET-1水平及ET-1/NO比值均明显升高(P<0.05,P<0.01);c GMP、E2、FSH、LH、NO水平均明显降低(P<0.05,P<0.01);血液流变学各项指标及FIB含量均明显降低(P<0.05,P<0.01),TT、PT、APTT均明显缩短(P<0.05);子宫指数及子宫内膜厚度均明显降低(P<0.05,P<0.01);子宫内膜组织MMP-9、Bcl-2蛋白表达显著下调(P<0.05,P<0.01),TIMP-1、PTEN、Bax蛋白表达显著上调(P<0.05,P<0.01)。结论补肾活血方能够改善肾虚血瘀型EH大鼠的症状和体征,保护子宫内膜血管内皮功能,以缓解子宫内膜增生,其作用机制与恢复MMP-9/TIMP-1平衡,上调Bax、PTEN表达,抑制Bcl-2表达有关。

Objective To investigate the effect and mechanism of Bushen Huoxue Prescription(Morindae Officinalis Radix,Cuscutae Semen,Astragali Complanati Semen,Codonopsis Radix,Poria,etc.)on endometrial hyperplasia(EH)rats of kidney deficiency and blood stasis type.Methods A total of 90 SD rats were randomly divided into normal group,model group,Mifepristone group(12 mg·kg^(-1)),and low-,medium-,and high-dose Bushen Huoxue Prescription groups(1.2,2.4,4.8 g·kg^(-1)),with 15 rats in each group.The EH rat model of kidney deficiency and blood stasis type was replicated by intragastric administration of Hydroxyurea and Estradiol Valerate combined with tail vein injection of high molecular dextran.After successful modeling,each group was given intragastric administration according to the set dose,once a day for 21 consecutive days.At the end of the experiment,the levels of serum cyclic adenosine monophosphate(cAMP),cyclic guanosine monophosphate(cGMP),cortisol(CORT),adrenocorticotropic hormone(ACTH),triiodothyronine(T_(3)),thyroxine(T_(4)),thyroid stimulating hormone(TSH),whole blood viscosity,hemorheology and coagulation function were detected by biochemical method.The levels of serum estrogen(E2),follicular estrogen(FSH),luteinizing hormone(LH),nitric oxide(NO)and endothelin-1(ET-1)were detected by ELISA.The pathological changes of endometrial tissue were observed by HE staining,and the endometrial thickness was measured.The protein expression levels of matrix metalloproteinase-9(MMP-9)and tissue inhibitor of metalloproteinase-1(TIMP-1)in endometrial tissues were detected by immunohistochemistry.The protein expression levels of MMP-9,TIMP-1,B-cell lymphoma 2(Bcl-2),Bcl-2 related X protein(Bax)and phosphatase and tensin homologue deleted on chromosome 10(PTEN)in endometrial tissue were detected by Western Blot.Results Compared with the normal group,the levels of serum CORT,ACTH,cAMP,T_(3),T_(4),TSH,ET-1 and ET-1/NO ratio in the model group were significantly decreased(P<0.05,P<0.01),while the levels of cGMP,E2,FSH,LH and NO were significantly increased(P<0.05,P<0.01).The indexes of hemorheology and fibrinogen(FIB)content were significantly increased(P<0.05),and thrombin time(TT),prothrombin time(PT)and activated partial thromboplastin time(APTT)were significantly prolonged(P<0.05).The uterine index and endometrial thickness were significantly increased(P<0.01).The protein expressions of MMP-9 and Bcl-2 in endometrial tissue were significantly up-regulated(P<0.01),and the protein expressions of TIMP-1,PTEN and Bax were significantly down-regulated(P<0.01).Compared with the model group,the levels of serum CORT,ACTH,cAMP,T_(3),T_(4),TSH,ET-1 and ET-1/NO ratio in the Bushen Huoxue Prescription group were significantly increased(P<0.05,P<0.01).The levels of cGMP,E2,FSH,LH and NO were significantly decreased(P<0.05,P<0.01).The indexes of hemorheology and FIB content were significantly decreased(P<0.05,P<0.01),and TT,PT and APTT were significantly shortened(P<0.05).The uterine index and endometrial thickness were significantly decreased(P<0.05,P<0.01).The protein expressions of MMP-9 and Bcl-2 in endometrial tissue were significantly down-regulated(P<0.05,P<0.01),and the protein expressions of TIMP-1,PTEN and Bax were significantly upregulated(P<0.05,P<0.01).Conclusion Bushen Huoxue Prescription can improve the symptoms and signs of EH rats with kidney deficiency and blood stasis,protect endometrial vascular endothelial function,and alleviate endometrial hyperplasia.Its mechanism is related to the restoration of MMP-9/TIMP-1 balance,up-regulation of Bax and PTEN expressions,and inhibition of Bcl-2 expression.