坤宁颗粒对痛经模型大鼠的抗炎镇痛作用研究

Study on anti-inflammatory and analgesic effects of Kunning Granules on dysmenorrhea model rats

关键词:  坤宁颗粒  PGF_2α  内分泌激素  氧化应激

Key words:  Kunning Granules  PGF_2α  endocrine hormone  oxidative stress

摘要:

[目的]研究坤宁颗粒对痛经模型大鼠COX-2/PGF_2α通路、内分泌激素、炎症因子和氧化应激水平的影响,探讨其保护作用及机制。[方法]取SD雌性大鼠,随机分为6组,正常对照组、模型组、益母草颗粒(3.5 g/kg)阳性对照组、坤宁颗粒低(1.4 g/kg)、中(2.8 g/kg)、高(5.6 g/kg)剂量组,除正常对照组外,其余组建立痛经模型,第1、10天灌胃戊酸雌二醇每只0.2 mg,第2~9天每只0.1 mg,从第5天各给药组灌胃给药,正常对照组和模型组灌胃蒸馏水,每日1次,第10天正常对照组大鼠腹腔注射生理盐水,其余各组大鼠腹腔注射缩宫素每只10 U。观察各组大鼠扭体反应次数;采用酶联免疫吸附实验(ELISA)检测大鼠血清前列腺素F_2α(PGF_2α)、前列腺素E₂(PGE₂)、β-内啡肽(β-EP)、雌二醇(E₂)、孕酮(PROG)、白介素-1β(IL-1β)、白介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)含量;比色法检测大鼠血清超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、丙二醛(MDA)水平;蛋白免疫印迹法(Western Blot)检测大鼠子宫组织环氧合酶-2(COX-2)、前列腺素E₂受体(PTGER₂)、雌激素受体α(ER_α)蛋白表达;苏木精-伊红(HE)染色观察子宫组织病理形态。[结果]与正常对照组相比,模型组大鼠扭体反应次数显著增加,大鼠血清中PGF_2α、E₂、IL-1β、IL-6、TNF-α、MDA水平和子宫组织COX-2、ER_α蛋白表达显著升高,而血清PGE₂、β-EP、PROG、SOD、GSH-Px水平显著降低,子宫PTGER₂蛋白表达显著降低,子宫组织可见明显充血、水肿。与模型组相比,坤宁颗粒可以显著降低大鼠扭体反应次数和血清中PGF_2α、E₂、IL-1β、IL-6、TNF-α、MDA水平,升高PGE₂、β-EP、PROG、SOD、GSH-Px水平,显著抑制子宫组织COX-2、ERα蛋白表达,升高PTGER₂蛋白表达,组织病理学明显改善。[结论]坤宁颗粒对痛经模型大鼠具有显著的改善作用,其机制可能与调节COX-2/PGF_2α通路和PGE₂、β-EP、E₂、PROG等内分泌激素、降低炎症因子水平、改善氧化应激水平有关。

Abstract:

[Objective] To study the effects of Kunning Granules on COX-2/PGF_2α pathway,endocrine hormones,levels of inflammatory factors and oxidative stress in rats with dysmenorrhea model,and to investigate their protective effects and mechanisms.[Methods] The female SD rats were randomly divided into 6 groups:normal control group,model group,leonurus granules (3.5 g/kg) positive control group,and low (1.4 g/kg),medium (2.8 g/kg) and high (5.6 g/kg) dose groups with Kunning Granules.The dysmenorrhea model was established in all groups except the normal control group.Estradiol valerate 0.2 mg/each rat was administered by gavage on the 1st and 10th day,and 0.1 mg/each rat on the 2nd to 9th day.From the 5th,each drug group was administered by gavage,and the normal control and model groups were given distilled water by gavage once a day.On the 10th day,the rats in the normal control group were injected intraperitoneally with saline,and the rats in the other groups were injected intraperitoneally with indocin 10 U/each rat.The numbers of writhing response in each group were observed.The levels of prostaglandin F_2α(PGF_2α),prostaglandin E₂(PGE₂),β-endorphin(β-EP),estradiol (E₂),progesterone (PROG),interleukin-1β (IL-1β),interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) in rat serum were measured by enzyme-linked immunoassay (ELISA).The levels of superoxide dismutase (SOD),glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) in rat serum were measured by colorimetric method.The expression of cyclooxygenase-2 (COX-2),prostaglandin E₂ receptor(PTGER₂) and estrogen receptor (ER_α) in rat uterine tissues was detected by Western blotting.HE staining was used to observe the histopathological pattern of the uterus.[Results] Compared with the normal control group,the numbers of writhing response were significantly increased in the model group.The serum levels of PGF_2α, E₂, IL-1β,IL-6,TNF-α,MDA,the expression of COX-2 and ERα protein in uterine tissues were significantly increased,while the serum levels of PGE₂, β-EP,PROG,SOD,GSH-Px and the expression of uterine PTGER₂ protein were significantly reduced.Uterine tissues were observed to be significantly congested and edematous.Compared with the model group,Kunning Granules significantly reduced the numbers of writhing response and serum levels of PGF_2α, E2,IL-1β,IL-6,TNF-α and MDA,while increased the levels of PGE₂, β-EP,PROG,SOD and GSH-Px.Kunning granules significantly inhibited the expression of COX-2 and ER_α protein in uterine tissues,while increased the expression of PTGER₂ protein.The histopathology was significantly improved.[Conclusion] Kunning Granules have significant improvement in dysmenorrhea model rats.The mechanism may be related to the regulation of the COX-2/PGF_2α pathway and endocrine hormones such as PGE₂, β-EP,E₂ and PROG,reduction of inflammatory factor levels and improvement of oxidative stress level.