风轮菜总提取物、总皂苷和总黄酮对子宫异常出血大鼠抗炎止血作用机制研究

Anti-inflammatory and hemostatic effects of total extract,saponins,and flavonoids of Clinopodium chinense in female rats with abnormal uterine bleeding and mechanism

研究风轮菜总提取物(total extraction of Clinopodium chinense,TEC)、总皂苷(total saponin of C.chinense,TSC)及总黄酮(total flavonoids of C.chinense,TFC)对子宫异常出血(abnormal uterine bleeding,AUB)大鼠抗炎止血作用及其机制。取同1 d受孕SD雌鼠,灌胃米非司酮(12.4 mg·kg^(-1))联合米索前列醇(130μg·kg^(-1)),建立AUB大鼠模型。造模成功大鼠随机分为模型组、TEC组、TSC组、TFC组、益母颗粒(leonurus granules,LG)组和戊酸雌二醇(estradiol valerate,EV)组,每组8只;另取8只未受孕的雌鼠作为正常组。各组灌胃给予相应受试药物,每日1次,连续给药7 d。给药结束后取血、取材。比色法测定大鼠子宫出血量;HE染色法子宫内膜病理变化;免疫组化法测定子宫内膜微血管密度;ELISA法测定血浆中TXB2、6-keto-PGF_(1α)、IL-6、TNF-α含量、血清中LH、FSH、E_(2)及孕酮水平;Western blot法测定子宫组织中ERα、PR、MMP-2、MMP-9、VEGF蛋白表达。与模型组相比,TEC、TSC、TFC均能明显减少子宫出血量,减轻子宫内膜病理损伤及升高子宫内膜组织中微血管密度;TEC、TSC可显著增加TXB2水平及TXB2/6-keto-PGF_(1α)比值;TEC、TFC可显著降低IL-6和TNF-α水平;TEC可显著增加孕酮水平,TFC可显著增加E_(2)、FSH和LH水平,TSC则可显著增加孕酮和FSH水平。此外,TEC可下调子宫组织中PR、MMP-2、VEGF蛋白表达,TSC可明显下调MMP-9蛋白表达,而TFC则可明显下调PR、MMP-9、VEGF蛋白表达,上调ERα蛋白表达。综上,TEC、TSC、TFC均可治疗大鼠功能性子宫出血,但TSC侧重于增加凝血功能和促进子宫内膜修复;TFC侧重于调节雌性激素水平和减轻炎症反应。总体而言,以TEC治疗作用最佳,该文从一定程度上揭示了风轮菜治疗AUB作用机制,也说明了中药药效作用整体性的特点。

This study aims to explore the anti-inflammatory and hemostatic effects of the total extract of Clinopodium chinense(TEC),total saponins of C.chinense(TSC),and total flavonoids of C.chinense(TFC)in female rats with abnormal uterine bleeding(AUB),and the possible mechanism.Mifepristone(i.g.,12.4 mg·kg^(-1))and misoprostol(i.g.,130μg·kg^(-1))were used to induce AUB in SD female rats conceiving on the same day.Then the AUB rats were randomized into model group,TEC group,TSC group,TFC group,Yimucao Granules(LG)group,and estradiol valerate(EV)group,with 8 rats in each group.Another 8 non-pregnant female rats were selected as normal group.During the experiment,each group was given the corresponding drug by gavage once a day for 7 days.After the administration,blood and uterine tissue were collected.The uterine bleeding volume was measured by ultraviolet spectrophotometry and the pathological changes of endometrium were observed based on hematoxylin-eosin(HE)staining.In addition,the microvessel density of endometrium was determined by immunohistochemistry,and the content of thromboxane B2(TXB2),6-keto-PGF_(1α),interleukin-6(IL-6),and tumor necrosis factor-α(TNF-α)in plasma and levels of lutenizing hormone(LH),follicle stimulating hormone(FSH),estradiol(E_(2)),and progesterone in serum were detected by enzyme-linked immunosorbent assay(ELISA).The mRNA and protein expression of estrogenreceptorα(ERα),progesterone receptor(PR),matrix metalloproteinase(MMP)-2,MMP-9,and vascular endothelial growth factor(VEGF)in uterine tissue was determined by Western blot.Compared with the model group,TEC,TSC,and TFC can reduce uterine bleeding volume,alleviate the pathological damage of endometrium,and increase the microvessel density in endometrium.Moreover,TEC and TSC can significantly raise plasma TXB2 level and ratio of TXB2 to 6-keto-PGF_(1α),and TEC and TFC can significantly reduce the levels of IL-6 and TNF-α.In addition,TEC significantly elevated serum progesterone level and TFC significantly increased serum levels of E_(2),FSH,and LH.TSC can significantly raise serum progesterone and FSH levels.In addition,TEC can significantly down-regulate the protein expression of PR,MMP-2,and VEGF and TSC significantly reduced the expression of MMP-9.TFC significantly decreased the expression of PR,MMP-9,and VEGF,and up-regulated the expression of ERα.In conclusion,TEC,TSC,and TFC all show therapeutic effects on AUB,particularly TEC.TSC exerts the effects by enhancing the coagulation function and promoting endometrial repair,and TFC by regulating estrogen levels and reducing inflammatory response.This study reveals the mechanism of C.chinense against AUB and also explains the holistic characteristics of Chinese medicine.