榆栀止血颗粒调控PI3K-Akt通路对子宫出血大鼠的保护作用

Protective effect of Yuzhi Zhixue Granule regulating PI3K Akt pathway on uterine bleeding model rats

目的探讨榆栀止血颗粒(YZZXKL)对子宫出血大鼠磷脂酰肌醇-3-激酶(PI3K)/蛋白激酶B(Akt)通路的调控及对子宫的保护作用。方法将SD雌性孕鼠灌胃给予米非司酮(13.8 mg/kg)和米索前列醇片(135μg/kg)复制大鼠子宫出血模型,随机分为模型组(Model组)、YZZXKL低(4 g/kg)、高(16 g/kg)剂量组、PI3K/Akt通路抑制剂LY294002组(LY组,0.3 mg/kg)、YZZXKL+LY组(16 g/kg+0.3 mg/kg),各组10只。另取10只灌胃给予等量生理盐水为正常对照组(Normal组)。造模成功后,Normal组和Model组灌胃和腹腔注射给予生理盐水10 mL/kg,各药物处理组分别灌胃给予相应剂量YZZXKL或腹腔注射给予LY,1次/d,共7 d。给药期间于大鼠阴道置入棉球检测子宫出血量(UBQ);末次给药24 h后,处死大鼠,取血清和子宫组织,酶联免疫吸附法(ELISA)检测大鼠血清雌二醇(E2)、孕酮(P);半自动凝血分析仪检测凝血酶时间(TT)、凝血酶原时间(PT)、活化部分凝血活酶时间(APTT)、血浆纤维蛋白原(FIB)含量;苏木精-伊红试剂(HE)染色观察子宫组织形态;蛋白免疫印迹法(Western blot)检测子宫组织PI3K、p-Akt、内皮型一氧化氮合酶(eNOS)、血管内皮生长因子(VEGF)、基质金属蛋白酶(MMP-1)蛋白相对表达水平。结果与Normal组相比,Model组和YZZXKL+LY组大鼠子宫组织可见间质纤维化及炎性细胞浸润等病理损伤,血清E2、P、FIB含量、子宫组织PI3K、p-Akt、eNOS、VEGF蛋白表达水平均降低(P<0.05),UBQ、凝血指标TT、PT及APTT、MMP-2蛋白表达均升高(P<0.05)。与Model组和YZZXKL+LY组相比,YZZXKL低、高剂量组大鼠子宫组织病理损伤减轻,E2、P、FIB、PI3K、p-Akt、eNOS、VEGF蛋白表达水平均升高(P<0.05),UBQ、TT、PT、APTT、MMP-2蛋白表达均降低(P<0.05),且YZZXKL各剂量组上述指标呈剂量依赖性;而LY组大鼠子宫组织病理损伤加重,E2、P、FIB、PI3K、p-Akt、eNOS、VEGF水平均降低(P<0.05),UBQ、TT、PT、APTT、MMP-2水平均升高(P<0.05)。Model组和YZZXKL+LY组相比,上述指标差异无统计学意义(P>0.05)。结论YZZXKL可提高子宫异常出血大鼠凝血功能和激素水平,改善出血情况,其作用机制可能通过激活PI3K-Akt-eNOS通路,促进血管生成实现。

Objective To explore the regulatory effect of Yuzhi Zhixue Granule(YZZXKL)on phosphatidylinositol-3-kinase(PI3K)/protein kinase B(Akt)pathway in rats with uterine bleeding and its protective effect on uterus.Methods SD female pregnant rats were given mifepristone(13.8 mg/kg)and misoprostol tablets(135 mg/kg)by gavageμG/kg)rat uterine bleeding model was copied and randomly divided into model group(model group),YZZXKL low(4 g/kg),high(16 g/kg)dose group,PI3K/Akt pathway inhibitor LY294002 group(LY group,0.3 mg/kg),YZZXKL+LY group(16 g/kg+0.3 mg/kg),with 10 rats in each group.Another 10 rats were given the same amount of normal saline by gavage as the normal control group(normal group).After successful modeling,normal group and model group were given 10 ml/kg of normal saline by gavage and intraperitoneal injection,and each drug treatment group was given the corresponding dose of YZZXKL by gavage or LY by intraperitoneal injection,once a day,for 7 days.During the administration period,cotton balls were placed into the vagina of rats to detect the amount of uterine bleeding(UBQ).24 hours after the last administration,the rats were killed,the serum and uterine tissue were taken,and the serum estradiol(E2)and progesterone(P)were detected by enzyme-linked immunosorbent assay(ELISA).The contents of thrombin time(TT),prothrombin time(PT),activated partial thromboplastin time(APTT)and plasma fibrinogen(FIB)were measured by semi-automatic coagulation analyzer.Hematoxylin eosin reagent(he)staining was used to observe the morphology of uterus.The relative expression levels of PI3K,P Akt,endothelial nitric oxide synthase(eNOS),vascular endothelial growth factor(VEGF)and matrix metalloproteinase 1(MMP 1)in uterine tissues were detected by Western blot.Results Compared with normal group,interstitial fibrosis and inflammatory cell infiltration were observed in uterine tissue of model group and YZZXKL+LY group.The contents of serum E2,P and FIB,the expression levels of PI3K,P-Akt,eNOS and VEGF in uterine tissue were decreased(P<0.05),and the expression levels of UBQ,coagulation indexes TT,Pt,APTT and MMP 2 were increased(P<0.05).Compared with model group and YZZXKL+LY group,the pathological damage of uterine tissue in YZZXKL low and high dose groups was reduced,the expression levels of E2,P,FIB,PI3K,P Akt,eNOS and VEGF protein were increased(P<0.05),and the expression levels of UBQ,TT,Pt,APTT and MMP 2 protein were decreased(P<0.05),and the above indexes in YZZXKL dose groups were dose-dependent.In LY group,the pathological injury of uterine tissue was aggravated,the levels of E2,P,FIB,PI3K,P Akt,eNOS and VEGF were decreased(P<0.05),and the levels of UBQ,TT,Pt,APTT and MMP 2 were increased(P<0.05).There was no significant difference in the above indexes between model group and YZZXKL+LY group(P>0.05).Conclusion YZZXKL can improve the coagulation function and hormone level in rats with abnormal uterine bleeding and improve the bleeding situation,which may be achieved by activating PI3K-Akt-eNOS pathway and promoting angiogenesis.