益母草水煎液对原发性痛经大鼠的治疗作用及其代谢组学分析

Therapeutic effect of Leonuri Herba aqueous decoction on primary dysmenorrhea in rats and its metabolomic analysis

研究益母草水煎液对原发性痛经(primary dysmenorrhea,PD)的治疗作用,并结合非靶向代谢组学分析其潜在作用机制。将40只雌性成年大鼠,随机分为正常组、模型组、布洛芬(0.12 g·kg^(-1))组和益母草水煎液高、低剂量(5、2.5 g·kg^(-1))组,每组8只。采用肌肉注射苯甲酸雌二醇联合腹腔注射缩宫素法制备PD大鼠模型。自造模第4天起灌胃给药,连续7 d。末次给药后,腹腔注射缩宫素,记录扭体潜伏期和30 min内扭体次数;测定子宫与卵巢脏器系数;通过ELISA和生化试剂盒测定子宫组织中雌二醇(estradiol,E_(2))、孕酮(progesterone,Prog)、催产素(oxytocin,OT)、环氧合酶2(cyclooxygenase 2,COX-2)、前列腺素E_(2)(prostaglandin E_(2),PGE_(2))、前列腺素F_(2α)(prostaglandin F_(2α),PGF_(2α))和Ca^(2+)含量;苏木素-伊红(hematoxylin-eosin,HE)染色观察子宫和卵巢组织形态学变化;免疫组化法检测子宫组织催产素受体(oxytocin receptor,OTR)、前列腺素E_(2)受体3(prostaglandin E_(2) receptor 3,EP3)和雌激素受体α(estrogen receptor alpha,ERα)的蛋白表达情况;荧光定量聚合酶链反应(quantitative real-time polymerase chain reaction,qPCR)法检测子宫组织OTR、PGE_(2)受体1~4(EP1、EP2、EP3、EP4)和PGF_(2α)受体(prostaglandin F_(2α) receptor,FP)的mRNA表达情况;采用超高效液相色谱-高分辨质谱仪联用(liquid chromatography quadrupole-time-of-flight mass spectrometry,LC-QTOF-MS)技术进行非靶向代谢组学分析,筛选潜在生物标志物并富集代谢通路。结果显示,益母草能够显著减少PD大鼠扭体次数(P<0.05或P<0.01);显著降低子宫和卵巢系数(P<0.01),并使其组织形态得到改善;益母草处理后,PD大鼠子宫组织中的PGE_(2)含量显著提高(P<0.05),COX-2、PGF_(2α)、Ca^(2+)含量及PGF_(2α)/PGE_(2)显著降低(P<0.05或P<0.01),OT含量降低,而E_(2)、Prog含量有进一步提高趋势;子宫组织中OTR和EP3蛋白表达显著下调(P<0.05或P<0.01),ERα蛋白表达显著上调(P<0.05);子宫组织中FP、EP4的mRNA表达均显著下调(P<0.01),EP1、EP3和OTR的mRNA表达有下调趋势。非靶向代谢组学分析发现益母草处理后的PD大鼠血浆中有10个差异代谢物得到恢复。以上结果表明,益母草能够有效防治PD,调控前列腺素(prostaglandins,PGs)合成及对应受体结合可能是其潜在的作用机制。

This study aimed to investigate the therapeutic effect of Leonuri Herba aqueous decoction on primary dysmenorrhea(PD) and explore the underlying mechanism in conjunction with untargeted metabolomics.Forty adult female rats were randomly divi-ded into a normal group,a model control group,ibuprofen(0.12 g·kg^(-1)) group,and high-and low-dose Leonuri Herba aqueous decoction(5 and 2.5 g·kg^(-1)) groups,with eight rats in each group.The PD rat model was prepared using intramuscular injection of estradiol benzoate combined with intraperitoneal injection of pitocin.Drugs were administered by gavage from the 4th day of modeling for 7 d.After the last administration,pitocin was injected intraperitoneally,and the writhing latency and writhing times within 30 min were recorded.The uterine and ovarian coefficients were determined.Estradiol(E_2),progesterone(Prog),oxytocin(OT),cyclooxyge-nase 2(COX-2),prostaglandin E_2(PGE_2),prostaglandin F_(2α)(PGF_(2α)),and Ca^(2+) levels in uterine tissues were measured by ELISA and biochemical kits.Morphological changes in uterine and ovarian tissues were observed by hematoxylin-eosin(HE) staining.The protein expression of oxytocin receptor(OTR),prostaglandin E_(2) receptor 3(EP3),and estrogen receptor alpha(ERα) in uterine tissues was detected by immunohistochemistry.The mRNA expression of OTR,PGE_(2) receptors 1-4(EP1,EP2,EP3,and EP4),and PGF_(2α) receptor(FP) in uterine tissues was detected by quantitative real-time PCR.Untargeted metabolomics analysis was performed by ultra-high-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(LC-QTOF-MS) technology to screen potential biomarkers and enrich metabolic pathways.The results showed that Leonuri Herba was able to significantly reduce the writhing times in PD rats(P<0.05 or P<0.01),significantly reduce the uterine and ovarian coefficients(P<0.01),and improve their histomorphology.After treatment with Leonuri Herba,PGE_(2) content was significantly increased(P<0.05),COX-2,PGF_(2α) and Ca^(2+) content,and PGF_(2α)/PGE_(2) was significantly decreased(P<0.05 or P<0.01),and OT content was decreased,while E_(2) and Prog content tended to further increase in uterine tissues of PD rats.Correspondingly,OTR and EP3 protein expression was significantly downregulated(P<0.05 or P<0.01) and ERα protein expression was upregulated(P<0.05) in uterine tissues.The mRNA expression of FP and EP4 in uterine tissues was significantly downregulated(P<0.01),and the mRNA expression of EP1,EP3,and OTR showed a decreasing trend.The untargeted metabolomics results showed that 10 differential metabolites were restored in the plasma of PD rats after Leonuri Herba treatment.The results indicate that Leonuri Herba is effective in the prevention and treatment of PD,and the underlying mechanism may be attributed to the regulation of PGs synthesis and corresponding receptor binding.