减味寿胎丸对流产模型大鼠子宫蜕膜脂质过氧化及铁死亡蛋白表达的影响

Effect of Jianwei Shoutai Pill(减味寿胎丸)on Lipid Peroxidation and Ferroptosis Protein Expression in Decidua of Abortion Rats

目的探讨减味寿胎丸治疗流产的潜在分子机制。方法SD雌性及雄性大鼠各48只,按雌、雄1∶1合笼持续配种2周,以观察到阴道栓作为妊娠第0天,共获得妊娠大鼠40只,随机分为正常组、模型组、地屈孕酮组、减味寿胎丸组,每组10只。自妊娠第0天开始,正常组和模型组雌鼠予10 ml/(kg·d)纯水灌胃,减味寿胎丸组予减味寿胎丸药液1.8 g/(kg·d)灌胃,地屈孕酮组予地屈孕酮片5.4 mg/(kg·d)灌胃,每日1次,连续13天。在妊娠第13天上午,模型组、地屈孕酮和减味寿胎丸组予浓度为1.2 mg/ml米非司酮药液10 ml/kg灌胃建立流产大鼠模型。妊娠第14天取材,HE染色观察各组大鼠子宫蜕膜组织的病理形态改变,检测蜕膜组织脂质过氧化相关指标[还原型谷胱甘肽(GSH)、谷胱甘肽过氧化物酶(GPX)、丙二醛(MDA)]水平及总铁离子[Fe^((Ⅱ+Ⅲ))]含量,普鲁士蓝(Perls)染色检测大鼠蜕膜组织铁离子分布,免疫组化及Western Blot法检测蜕膜组织铁死亡关键蛋白[谷胱甘肽过氧化物酶4(GPX4)、溶质载体家族7成员11(SLC7A11)、转铁蛋白受体1(TFR1)]蛋白分布及相对表达量。结果HE染色结果显示,正常组大鼠蜕膜组织血管丰富,蜕膜细胞排列紧密;模型组大鼠蜕膜组织血管分布少,胞浆水肿,部分细胞核消失;地屈孕酮组及减味寿胎丸组大鼠蜕膜组织病理较模型组明显改善。模型组大鼠较正常组蜕膜组织中GSH、GPX水平及GPX4、SLC7A11蛋白表达降低,Fe^((Ⅱ+Ⅲ))含量、MDA水平及TFR1蛋白表达升高(P<0.05或P<0.01)。与模型组比较,减味寿胎丸组大鼠蜕膜组织中GSH、GPX水平及GPX4、SLC7A11蛋白表达升高,MDA水平、Fe^((Ⅱ+Ⅲ))含量、TFR1蛋白表达降低(P<0.05或P<0.01);地屈孕酮组GPX水平升高,Fe^((Ⅱ+Ⅲ))含量降低(P<0.01)。地屈孕酮组与减味寿胎丸组各指标差异均无统计学意义(P>0.05)。结论减味寿胎丸可减轻流产大鼠脂质过氧化及过度铁累积,抑制子宫蜕膜组织铁死亡,从而防止流产的发生。

Objective To explore the underlying mechanism of Jianwei Shoutai Pill(减味寿胎丸,JSP)in treating abortion.Methods Forty-eight male rats were bred in cages with 48 female rats at the ratio of 1∶1 for two weeks;and day 0 of pregnancy was when the vaginal suppository was found.Forty rats with successful pregnancy were randomized into normal group,model group,dydrogesterone group and JSP group with ten rats in each group.From day 0 of pregnancy,rats in the normal group and model group were given 10 ml/(kg·d)pure water by gavage;the JSP group was administered with 1.8 g/(kg·d)JSP by gavage,and the dydrogesterone group was given 5.4 mg/(kg·d)didroxyprogesterone by gavage;all the administrations were given once daily for 13 days.The abortion rat model was established in the model group,dydrogesterone group and JSP group by gavage with 10 ml/kg mifepristone solution at a concentration of 1.2 mg/ml on day 13 of pregnancy,and decidual tissues were collected on day 14 of pregnancy.HE staining was used to observe the pathological changes of decidual tissue,and to detect the levels of lipid peroxidation-related indexes including reduced glutathione(GSH),glutathione peroxidase(GPX),malondialdehyde(MDA)),as well as the content of total iron(Fe^((Ⅱ+Ⅲ))).Prussian blue(Perls)staining was used to detect the distribution of iron ion in the decidua tissue,and the immunohistochemistry and Western Blot method were used to detect the protein distribution and relative expressions of glutathione peroxidase 4(GPX4),solute carrier family 7 member 11(SLC7A11)and transferrin receptor 1(TFR1)which could reflect ferroptosis.Results The HE staining results indicated that the decidual tissue of the rats in the normal group was rich in blood vessels,and the decidual cells were tightly arranged;the decidual tissue in the model group had less blood vessels,with cytoplasmic edema and partially missing nucleus;the dydrogesterone group and the JSP group had improved pathological results of decidual tissue than the model group.Compared to the normal group,the model group had significantly lower GSH and GPX levels,and GPX4 and SLC7A11 protein expression,as well as higher Fe^((Ⅱ+Ⅲ))content,MDA level and TFR1 protein expression in decidual tissue(P<0.05 or P<0.01).Compared to the model group,the JSP group significantly increased the GSH,GPX levels,GPX4 and SLC7A11 protein expression,and reduced the MDA level,Fe^((Ⅱ+Ⅲ))content and TFR1 protein expression(P<0.05 or P<0.01);the dydrogesterone group had higher GPX level and lower Fe^((Ⅱ+Ⅲ))content(P<0.01).There were no significant differences between dydrogesterone group and JSP group in all indexes.Conclusion JSP may reduce lipid peroxidation and excessive iron accumulation to inhibit ferroptosis in the decidual tissue,thereby preventing abortion ultimately.