基于网络药理学研究妇科调气解毒方对慢性子宫内膜炎的治疗作用机制

Therapeutic Mechanism of Gynecological Tiaoqi Jiedu Formula on Chronic Endometritis Based on Network Pharmacology

目的:探讨妇科调气解毒方对慢性子宫内膜炎的作用机制。方法:通过网络药理学,确定妇科调气解毒方作用于子宫内膜炎可能的关键靶点和关键通路。在该理论基础上,建立子宫内膜炎小鼠模型,分为空白组、模型组、西药组(盐酸左氧氟沙星),以及低、中及高剂量组(给予低、中及高剂量的妇科调气解毒方),通过酶联免疫吸附试验(ELISA)法、实时荧光定量聚合酶链反应(PCR)及蛋白质印迹法验证网络药理学结果。结果:在妇科调气解毒方中筛选出219个化合物和相应靶点698个;与子宫内膜炎的靶点取交集获得36个关键靶点。京都基因与基因组百科全书通路富集得到77条代谢通路,主要包括核因子κB(NF-κB)信号通路、晚期糖基化终末产物(AGE)-AGE受体信号通路、肿瘤坏死因子信号通路和白细胞介素(IL)17信号通路等。ELISA法结果显示,与空白组相比,模型组髓过氧化物酶(MPO)活性,炎症因子IL-6及IL-1β含量显著升高,差异均有统计学意义(P<0.01);与模型组相比,妇科调气解毒方低剂量组IL-6含量显著降低,差异有统计学意义(P<0.05),但组间MPO活性、IL-1β含量的差异无统计学意义(P>0.05)。与模型组相比,中剂量组、高剂量组及西药组MPO活性、IL-6及IL-1β含量明显降低,差异均有统计学意义(P<0.01)。实时荧光定量PCR及蛋白质印迹法结果显示,与空白组比较,模型组子宫组织中磷酸化蛋白65(P-P65)、Toll样受体4(TLR4)的基因及蛋白表达量显著升高,差异有统计学意义(P<0.01或P<0.001)。与模型组比较,低剂量组不能明显抑制P-P65及TLR4 mRNA及蛋白的表达量(P>0.05),P-P65 mRNA、TLR4 mRNA表达量在中、高剂量组及西药组中明显降低(P<0.05或P<0.01,P<0.001),P-P65蛋白、TLR4蛋白表达量在高剂量组及西药组中明显降低(P<0.01或P<0.05),差异均有统计学意义。各组P65蛋白mRNA及蛋白表达量的差异均无统计学意义(P>0.05)。结论:在脂多糖诱导的子宫内膜炎小鼠模型中,妇科调气解毒方可以明显下调炎症因子水平,抑制TLR4/NF-κB信号通路中关键基因蛋白的表达,从而达到抗炎、保护子宫内膜的目的。

OBJECTIVE:To probe into the mechanism of gynecological Tiaoqi Jiedu formula on chronic endometritis.METHODS:Through network pharmacology,the possible key targets and key pathways of gynecological Tiaoqi Jiedu formula on endometritis were identified.On the basis of this theory,the mouse model of endometritis was established and divided into the blank group,model group,western medicine group(levofloxacin hydrochloride),and low-dose group,medium-dose group and high-dose group(low-dose,medium-dose and high-dose gynecological Tiaoqi Jiedu formula).The network pharmacology results were verified by enzyme-linked immunosorbent assay(ELISA),real-time fluorescent quantitative polymerase chain reaction(PCR)and Western blotting.RESULTS:A total of 219 compounds and 698 corresponding targets were screened from gynecological Tiaoqi Jiedu formula.A total of 36 key targets were obtained by intersections with endometritis targets.Seventy-seven metabolic pathways were obtained by Kyoto encyclopedia of genes and genomes enrichment,including nuclear factorκB(NF-κB)signaling pathway,advanced glycation end products(AGE)-AGE receptor signaling pathway,tumor necrosis factor signaling pathway and interleukin(IL)-17 signaling pathway.ELISA results showed that the levels of MPO,IL-6 and IL-1βin the model group were significantly higher than those in the blank group(P<0.01).Compared with the model group,the low-dose gynecological Tiaoqi Jiedu formula group had a significant reduction in IL-6,with statistically significant difference(P<0.05),but there was no significant difference in MPO and IL-1βbetween two groups(P>0.05).Compared with the model group,MPO,IL-6 and IL-1βin the medium-dose group,high-dose group and western medicine group decreased significantly,with statistically significant differences(P<0.01).The results of real-time fluorescence quantitative PCR and Western blotting showed that the gene and protein expression of P-P65 and TLR4 in the uterine tissue of the model group were significantly higher than those of the normal group,with statistically significant differences(P<0.01 or P<0.001).Compared with the model group,the low-dose group could not significantly inhibit the expression of P-P65 and TLR4 mRNA and protein(P>0.05),while the expression of P-P65 mRNA and TLR4 mRNA in the medium-dose group,high-dose group and western medicine group decreased significantly(P<0.05 or P<0.01,P<0.001),the expression of P-P65 protein and TLR4 protein in the high-dose group and western medicine group decreased significantly(P<0.01 or P<0.05),the differences were statistically significant.There was no significant difference in the expression of P65 mRNA and protein in each group(P>0.05).CONCLUSIONS:Gynecological Tiaoqi Jiedu formula can significantly down-regulate the levels of inflammatory factors and inhibit the expression of key genes in TLR4/NF-κB signaling pathway in the mouse model of lipopolysaccharide(LPS)-induced endometritis,so as to achieve the purpose of anti-inflammatory and endometrial protection.