基于炎症相关信号通路探讨活血化瘀通络汤治疗输卵管炎性不孕作用机制

Based on inflammation-related signaling pathways to explore the mechanism of action of Huoxue Huayu Tongluo Decoction in the treatment of salpingitis infertility

目的从TLR4/NF-κB,JAK/STAT及MAPK信号通路探讨活血化瘀通络法治疗输卵管炎性不孕的作用机制,并对比活血化瘀通络法与活血化瘀、通络单用及应用通路抑制剂疗效。方法以输卵管上皮细胞为对象,采用LPS干预建立输卵管上皮细胞炎性模型。实验分为正常组、模型组、活血化瘀组、通络组、活血化瘀通络组、JAK/STAT抑制剂组及MAPK抑制剂组。应用ELISA及RT-PCR法检测细胞上清中IL-1β、IL-6及TNF-α表达量;Western blot法检测输卵管炎症上皮细胞中TLR4、p65、p-p65、IκBα、p-IκBα、ERK、p-ERK、JNK、p-JNK、p38、p-p38、JAK1~3、p-JAK1~3、Tyk2、p-Tyk2、STAT3及p-STAT3蛋白的表达。结果LPS建立输卵管上皮细胞炎性模型最佳时间为24h,最佳剂量为200ng/ml;各组药物均可显著降低细胞上清中IL-1β、IL-6及TNF-α表达量(P<0.05);均可抑制TLR4、JAK/STAT及MAPK信号通路关键蛋白磷酸化(P<0.05),且组间比较均以活血化瘀通络组效果最佳(P<0.05)。结论中药组与通路抑制剂有类似功效,均可降低炎症因子表达、抑制炎症相关信号通路传导,从而发挥抗炎功效,达到延缓输卵管炎进程作用,且组间比较表明活血化瘀通络组药效最佳,提示活血化瘀通络法联用有协同增效作用。

Objective To investigate the mechanism of TLR4/NF-κB, JAK/STAT and MAPK signaling and fallopian infertility, and compare the effect of blood circulation, blood stasis and application inhibitors. Methods Tubal epithelial cells were established by LPS intervention, divided into normal group, model group, blood stasis group, collaterals, A group, JAK/STAT inhibitor and MAPK inhibitor group.IL-1, IL-6 and TNF-expression were measured in cell supernatant by ELISA and RT-PCR;Western blot for expression of TLR4, p65, p-p65, I B, p-I B, ERK, p-ERK, p-E R K, JNK, p-JNK, p38, p-p38, J A K 1-3, p-JAK1~3, Tyk2, p-Tyk2, STAT3 and p-STAT3 proteins.Results The optimal dose and time of action of LPS were 200 ng/mL and 24 h, respectively;Each group significantly reduced IL-1, IL-6 and TNF-α expression in the cell supernatant(P<0.05);all can inhibit the phosphorylation of key proteins in TLR4/NF-κB, JAK/STAT and MAPK signaling pathways(P<0.05), and showed the best effect by promoting blood circulation(P<0.05). Conclusion shows that traditional Chinese medicine group and pathway inhibitors have similar effect, can reduce the expression of inflammatory factors, inhibit the inflammation-related signaling, thus play anti-inflammatory effect, to delay the process of saltuitis, and the group showed to promote blood circulation of blood stasis, with synergistic effect.