基于MAPK/ERK通路探讨茯苓多糖对卵巢早衰模型大鼠的作用及其分子机制

Study on the effect and molecular mechanism of Poria cocos polysaccharide on premature ovarian failure model rats based on MAPK/ERK pathway

目的基于丝裂原活化蛋白激酶/细胞外调节蛋白激酶(MAPK/ERK)通路探讨茯苓多糖对卵巢早衰(POF)模型大鼠的作用及其分子机制。方法取50只SD雌性大鼠腹腔注射环磷酰胺诱导复制POF模型,随机分为5组:模型组、茯苓多糖低剂量组(100 mg/kg)、茯苓多糖高剂量组(200 mg/kg)、茴香霉素组(MAPK激活剂,10 mg/kg)、茯苓多糖高剂量+茴香霉素组,每组10只;另取10只SD雌性大鼠腹腔注射等剂量生理盐水作为对照组。分组处理后分别测定各组大鼠子宫指数及卵巢指数;酶联免疫吸附试验(ELISA)测定各组大鼠血清性激素[雌二醇(E2)、促黄体生成素(LH)、卵泡刺激素(FSH)]水平;HE染色观察各组大鼠卵巢形态;酶联免疫吸附试验检测各组大鼠血清及卵巢组织肿瘤坏死因子-α(TNF-α)、白细胞介素-4(IL-4)、白细胞介素-18(IL-18)水平;Western blotting检测各组大鼠卵巢组织MAPK/ERK通路相关蛋白表达。结果与对照组比较,模型组大鼠卵巢间质结构有一定程度的出血和纤维化损伤,卵泡发育失常,血清FSH和LH水平、血清和卵巢组织TNF-α、IL-4、IL-18水平、卵巢组织p-p38 MAPK/p38 MAPK及p-ERK1/2/ERK1/2相对表达量均升高(P<0.05),子宫指数和卵巢指数、E_(2)水平均降低(P<0.05)。与模型组比较,茯苓多糖低剂量组、茯苓多糖高剂量组大鼠卵巢形态损伤及卵泡发育失常均减轻,血清FSH及LH水平、血清和卵巢组织TNF-α、IL-4、IL-18水平、卵巢组织p-p38 MAPK/p38 MAPK及p-ERK1/2/ERK1/2相对表达量均降低(P<0.05),子宫指数和卵巢指数、E_(2)水平均升高(P<0.05),且茯苓多糖高剂量组效果更优(P<0.05)。茴香霉素组大鼠卵巢形态损伤及卵泡发育失常加重,血清FSH及LH水平、血清和卵巢组织TNF-α、IL-4、IL-18水平、卵巢组织p-p38 MAPK/p38 MAPK及p-ERK1/2/ERK1/2相对表达量均升高(P<0.05),子宫指数及卵巢指数、E_(2)水平均降低(P<0.05)。茯苓多糖高剂量+茴香霉素组逆转茯苓多糖作用,呈相反趋势(P<0.05)。结论茯苓多糖可通过抑制MAPK/ERK通路的激活减轻POF大鼠炎症,从而改善其卵巢功能和性激素分泌,缓解大鼠POF症状。

Objective To explore the effect and molecular mechanism of Poria cocos polysaccharide on premature ovarian failure(POF)model rats based on mitogen-activated protein kinase(MAPK)/extracellular regulated protein kinase(ERK)pathway.Methods The POF model was induced by intraperitoneal injection of cyclophosphamide in SD female rats,and was randomly divided into five groups:model group,low dose poria cocos polysaccharide(100 mg/kg)group,high dose poria cocos polysaccharide(200 mg/kg)group,anisomycin(MAPK activator,10 mg/kg)group,high dose poria cocos polysaccharide+anisomycin group,with 10 rats in each group,another 10 SD rats were injected intraperitoneally with the same dose of normal saline as the control group,the uterus index and ovary index of rats in each group were measured after treatment with Poria cocos polysaccharide and anisomycin;the levels of serum sex hormones[estradiol(E_(2)),luteinizing hormone(LH)and follicle-stimulating hormone(FSH)]were measured by enzyme-linked immunosorbent assay(ELISA)kit;the ovarian morphology of rats in each group was observed by HE staining;the levels of tumor necrosis factor-α(TNF-α),interleukin-4(IL-4)and IL-18 in serum and ovarian tissue of rats in each group were measured by ELISA kit;the expression of MAPK/ERK pathway related protein in ovarian tissue of rats in each group was detected by Western blotting.Results Compared with the control group,the interstitial structure of the ovary in the model group had some degree of hemorrhage,fibrosis damage,and follicular dysplasia,the serum FSH and LH levels,serum and ovarian tissue TNF-α,IL-4,IL-18 levels,ovarian tissue p-p38 MAPK/p38 MAPK and p-ERK1/2/ERK1/2 were increased(P<0.05),the uterine index,ovarian index and E_(2) level were decreased(P<0.05).Compared with the model group,the ovarian morphology damage and follicular dysplasia of rats in the low dose group and the high dose Poria cocos polysaccharide group were alleviated,the serum FSH and LH levels,serum and ovarian tissue TNF-α,IL-4,IL-18 levels,ovarian tissue p-p38 MAPK/p38 MAPK and p-ERK1/2/ERK1/2 were all decreased(P<0.05),uterine index,ovarian index and E_(2) level were increased(P<0.05),and the effect of pachyman high dose group was better(P<0.05);In the anisomycin group,the ovarian morphology damage and follicular dysplasia were aggravated,the levels of serum FSH and LH,serum and ovarian tissue TNF-α,IL-4,IL-18,and ovarian tissue p-p38 MAPK/p38 MAPK and p-ERK1/2/ERK1/2 were increased(P<0.05),uterine index,ovarian index and E_(2) level decreased(P<0.05).The high dose of pachyman+anisomycin group reversed the effect of pachyman,showing an opposite trend(P<0.05).Conclusion Poria cocos polysaccharide can alleviate the inflammation of POF rats by inhibiting the activation of MAPK/ERK pathway,thereby improving the ovarian function and sex hormone secretion,and alleviating the symptoms of premature ovarian failure in rats.