基于MAPK/ERK/NF-κB通路探讨祛瘀温经汤治疗寒凝血瘀证原发性痛经的作用机制

Exploring the mechanism of Quyu Wenjing Decoction in treating primary dysmenorrhea with Cold Coagulation and Blood Stasis Syndrome based on MAPK/ERK/NF-κB pathway

目的探究祛瘀温经汤调控MAPK/ERK/NF-κB信号通路治疗寒凝血瘀证原发性痛经(PD)的机制。方法将60只SPF级雌性未孕SD大鼠分为空白(Control)组10只和造模组50只。造模组大鼠采用苯甲酸雌二醇联合缩宫素+冰水浴法制备寒凝血瘀证PD大鼠模型,再将造模组大鼠随机分为模型(Model)组、少腹逐瘀颗粒(S-F)组、祛瘀温经汤低(Q-L)、中(Q-M)、高(Q-H)剂量组。药物灌胃干预11d后观察大鼠的一般情况、扭体反应;血液黏度仪检测血液流变学指标;HE染色观察子宫组织病理形态;ELISA法检测白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)含量;Western Blot法检测细胞外信号调节激酶(ERK1/2)、磷酸化-ERK1/2(p-ERK1/2)、细胞核内转录因子(NF-κB)蛋白表达水平;qRT-PCR法检测ERK1、ERK2、NF-κB mRNA转录水平。结果与空白组比较,各组大鼠血液流变学指标、ERK1/2、p-ERK1/2、NF-κB蛋白表达及mRNA转录水平、IL-6、TNF-α含量均显著升高(P<0.01);与模型组比较,各治疗组大鼠血液流变学指标、ERK1/2、p-ERK1/2、NF-κB蛋白表达及mRNA转录水平、IL-6、TNF-α含量均降低(P<0.05,P<0.01),且呈剂量相关性。结论祛瘀温经汤治疗寒凝血瘀证PD的作用机制可能与其下调MAPK/ERK/NF-κB信号通路中ERK1/2、p-ERK1/2、NF-κB的高表达、降低血液流变学指标及IL-6、TNF-α的含量有关,通过抑制炎性因子过度刺激,减缓PD发生发展。

Objective To explore the mechanism of Quyu Wenjing Decoction regulating MAPK/ERK/NF-κB signaling pathway in the treatment of primary dysmenorrhea(PD)with cold coagulation and blood stasis syndrome.Methods Sixty SPF female non-pregnant SD rats were divided into blank(Control)group(10 rats)and model group(50 rats).The rats in the model group were treated with estra⁃diol benzoate combined with oxytocin+ice water bath to prepare the PD rat model of cold coagulation and blood stasis syndrome,and then the rats in the model group were randomly divided into model(Model)group,Shaofu Zhuyu Granule(S-F)group,Quyu Wenjing Decoction low(Q-L),medium(Q-M)and high(Q-H)dose groups.The general condition and writhing reaction of rats were ob⁃served after 11 days of drug intervention.Hemorheology indexes were detected by blood viscosity meter.The pathological morphology of uterine tissue was observed by HE staining.The contents of interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)were de⁃tected by ELISA.The protein expression levels of extracellular signal-regulated kinase(ERK1/2),phosphorylated-ERK1/2(p-ERK1/2)and nuclear transcription factor(NF-κB)were detected by Western Blotting.The transcription levels of ERK1,ERK2 and NF-κB mRNA were detected by qRT-PCR.Results Compared with the control group,the hemorheological indexes,the expression of ERK1/2,p-ERK1/2 and NF-κB protein and mRNA transcription levels,and the contents of inflammatory factors(IL-6 and TNF-α)in each group were significantly increased(P<0.01).Compared with the model group,the hemorheology indexes,the ex⁃pression of ERK1/2,p-ERK1/2,NF-κB protein and mRNA transcription levels,the contents of inflammatory factors(IL-6,TNF-α)in each treatment group were decreased(P<0.05,P<0.01),and showed a dose-dependent manner.Conclusion The mechanism of Quyu Wenjing Decoction in the treatment of PD with cold coagulation and blood stasis syndrome may be related to down-regulating the high expression of ERK1/2,p-ERK1/2 and NF-κB in MAPK/ERK/NF-κB signaling pathway,diminishing the hemo⁃rheological indexes and the contents of IL-6 and TNF-α,and slowing down the occurrence and development of PD by inhibiting the ex⁃cessive stimulation of inflammatory factors.