五加生化胶囊治疗原发性痛经的作用及机制研究

Effect and Mechanism of Wujia Shenghua Capsule Against Primary Dysmenorrhea

目的:通过制备原发性痛经大鼠模型,探讨五加生化胶囊对原发性痛经的治疗作用,并深入分析其作用机制。方法:60只SPF级大鼠,随机分为正常对照组、模型对照组、五加生化胶囊110、220、440 g/kg组、吲哚美辛6.75 mg/kg组,每组10只。采用皮下注射苯甲酸雌二醇联合腹腔注射缩宫素法制备痛经大鼠模型。造模第5 d开始灌胃给药,连续给药7 d,1次/d。末次给药后,腹腔注射缩宫素,记录30 min内扭体次数及扭体潜伏期;Elisa法测定血清中雌二醇(E2)、孕酮(PROG)、白介素-1β(IL-1β)、白介素-18(IL-18)含量,子宫组织中前列腺素E_(2)(PGE_(2))、前列腺素2α(PGF;)含量;子宫、卵巢组织称质量,计算脏器指数;HE染色观察子宫组织病理变化情况;免疫组化法检测子宫组织NOD样受体蛋白3(NLRP3)和IL-1β蛋白表达情况;Western-blot法检测子宫组织NLRP3、含半胱氨酸的天冬氨酸蛋白水解酶1(Caspase-1)、凋亡相关的斑点样蛋白(ASC)、核因子κB(NF-κB p65)蛋白表达情况。结果:与正常对照组比较,模型对照组大鼠扭体次数显著升高,潜伏期显著缩短(P<0.01);血清E2、IL-1β、IL-18含量显著升高(P<0.01),PROG含量显著降低(P<0.01);子宫组织PGF;含量显著升高,PGE_(2)含量显著降低(P<0.01);子宫指数、卵巢质量及指数显著升高(P<0.01);子宫组织内壁柱状褶皱参差不齐,内膜变薄、空泡样变,外膜局部增厚,肌层有充血现象;子宫组织中NLRP3、IL-1β蛋白表达平均光密度值显著增加(P<0.01);NLRP3、ASC、Cleaved caspase-1、NF-κB p65(核)蛋白表达显著上调(P<0.01)。与模型对照组比较,给药7 d后,五加生化胶囊440 g/kg组大鼠扭体次数显著降低,潜伏期显著延长(P<0.01);血清E2、IL-1β、IL-18含量显著降低,PROG含量显著升高(P<0.01);子宫组织PGF;含量显著降低,PGE_(2)含量显著升高(P<0.01);子宫指数、卵巢质量及指数均显著降低(P<0.01);子宫内膜相对光滑平整、空泡变化和充血现象明显改善;子宫组织中NLRP3、IL-1β蛋白表达平均光密度值显著降低(P<0.01);NLRP3、ASC、Cleaved caspase-1、NF-κB p65(核)蛋白表达均显著下调(P<0.01)。结论:五加生化胶囊对原发性痛经有预防作用,机制可能与调节血清激素水平、抑制子宫炎症因子反应有关。

Objective:To explore the therapeutic effects of Wujia Shenghua Capsule on primary dysmenorrhea in model rats and investigate its underlying mechanism.Methods:Sixty rats of SPF grade were randomly divided into the normal group,model group,440,220,and 110 g/kg Wujia Shenghua Capsule groups,and 6.75 mg/kg indomethacin group,with 10 rats in each group.The rat model of dysmenorrhea was duplicated by subcutaneous injection of estradiol benzoate combined with intraperitoneal injection of oxytocin.The rats were administered with the corresponding drugs since the 5 th day of modeling,once per day,for seven consecutive days.After the last administration,oxytocin was intraperitoneally injected,and the number of twisting within 30 min and twisting latency were recorded.The contents of serum estradiol(E2),progesterone(PROG),interleukin-1β(IL-1β),interleukin-18(IL-18)and uterine prostaglandin E2(PGE_(2)),prostaglandin 2α(PGF;)were determined by Elisa.The uterine and ovarian tissues were weighed and the viscera indexes were calculated.Pathological changes in uterine tissues were observed by HE staining.The protein expression levels of NOD-like receptor protein 3(NLRP3)and IL-1βin uterine tissues were detected by immunohistochemistry,and the protein expression levels of cysteine dependent aspartate directed protease-1(Caspase-1),apoptotic speck protein(ASC),and nuclear factorκB(NF-κB p65)in uterine tissues by Western blotting.Results:Compared with the normal group,the model group displayed significantly increased number of twisting,shortened twisting latency(P<0.01),elevated serum E2,IL-1β,and IL-18(P<0.01),uterine PGF;,uterine index,and ovarian weight and index(P<0.01),and reduced PROG and uterine PGE_(2)(P<0.01).The cylindrical folds in inner uterine wall were irregular.The endometrium became thinner with vacuolar degeneration,while the perimetrium was thickened locally with hyperemia observed in the muscle layer.Both the average optical densities of NLRP3 and IL-1βprotein expression(P<0.01)and the protein expression levels of NLRP3,ASC,cleaved caspase-1,and NF-κB p65 were significantly up regulated(P<0.01).Compared with the model group,after seven days of administration,Wujia Shenghua Capsule at 440 g/kg remarkably lowered the number of twisting,prolonged the twisting latency(P<0.01),decreased the levels of serum E2,IL-1β,and IL-18(P<0.01)and uterine PGF;,uterine index,and ovarian weight and index(P<0.01),and raised PROG(P<0.01)and uterine PGE_(2)(P<0.01).The endometrium was relatively smooth and flat,and the vacuolization and hyperemia were improved.The average optical densities of NLRP3 and IL-1βprotein expression(P<0.01)and the protein expression levels of NLRP3,ASC,cleaved caspase-1,and NF-κB p65 were obviously down regulated(P<0.01).Conclusion:Wujia Shenghua Capsule has a preventive effect on primary dysmenorrhea,which may be related to its regulation of serum hormone levels and inhibition of uterine inflammation.