基于网络药理学、分子对接及动物实验探讨芍药汤治疗溃疡性结肠炎的作用机制

Action Mechanism of Shaoyao Decoction in Treatment of Ulcerative Colitis Based on Network Pharmacology,Molecular Docking and Animal Experiment

目的:采用网络药理学、分子对接及动物实验探索芍药汤治疗溃疡性结肠炎(ulcerative colitis,UC)的作用机制。方法:检索芍药汤与UC的作用靶点,从而获取两者的交集靶点。基于蛋白质-蛋白质相互作用(protein-protein interaction,PPI)网络,采用CytoNCA插件进行拓扑分析,筛选关键靶点。针对交集靶点进行京都基因和基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)富集分析。构建“活性成分-靶点-通路”网络以筛选核心靶点,最后进行分子对接验证。从40只大鼠中随机选取10只作为空白组,其余大鼠使用葡聚糖硫酸钠(dextran sulfate sodium,DSS)诱导UC模型。造模成功的大鼠随机分为模型组、芍药汤组(32 g·kg-1)、美沙拉秦组(0.5 g·kg-1),连续灌胃10 d。末次灌胃后,计算大鼠的疾病活动指数(disease activity index,DAI)评分及结肠血液流变学指标。采用HE染色观察结肠病理形态,ELISA法检测血清白细胞介素(interleukin,IL)-6、IL-1β、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)含量,Western blot法检测结肠核因子-κB(nuclear factor-κB,NF-κB)、过氧化物酶体增殖物激活受体-γ(peroxisome proliferator activated receptor-γ,PPAR-γ或PPARG)表达水平。结果:芍药汤靶点262个,UC靶点2801个,两者的交集靶点169个。关键靶点包括基质金属蛋白酶9(matrix metalloproteinase 9,MMP9)、PPARG、IL-6、信号转导和转录激活因子3(signal transducers and activators of transduction 3,STAT3)等。KEGG通路主要包括NF-κB信号通路、IL-17信号通路、趋化因子信号通路等。分子对接显示,活性成分与关键/核心靶点的结合能小于-5 kcal·mol^(-1)。动物实验显示,模型组大鼠DAI评分较空白组增加(P<0.05),经芍药汤组与美沙拉秦治疗后减少(P<0.05)。与空白组比较,模型组大鼠结肠血流灌注量、血细胞移动速度下降,移动血细胞浓度升高(P<0.05);芍药汤治疗后上述指标的趋势得到逆转(P<0.05)。HE染色显示,空白组大鼠结肠组织结构正常;模型组大鼠结肠黏膜溃疡,腺体缺损,伴有炎症细胞浸润;美沙拉秦组和芍药汤组大鼠结肠病理形态较模型组改善。模型组大鼠血清TNF-α、IL-6、IL-1β含量较空白组升高(P<0.05),芍药汤和美沙拉秦干预后降低(P<0.05)。与空白组比较,模型组大鼠结肠NF-κB蛋白表达水平升高,PPAR-γ蛋白表达水平降低(P<0.05);经芍药汤与美沙拉秦干预后上述蛋白的趋势被逆转(P<0.05)。结论:芍药汤可能通过调控NF-κB、IL-17等信号通路,作用于PPARG、IL-6、IL-1β等靶点,有效改善UC大鼠的症状。

Objective:To explore the mechanism of action of Paeonia lactiflora broth in the treatment of ulcerative colitis(UC)using network pharmacology,molecular docking and animal experiments.Methods:The targets of action of Paeonia lactiflora broth and UC were searched,to obtain the intersecting targets of the two.Based on protein-protein interaction(PPI)network,topology analysis was performed using CytoNCA plug-in to screen the core targets.Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis was performed for the intersected targets.An"active ingredient-target-pathway"network was constructed to screen the key ingredients,and finally,molecular docking validation was performed.Ten rats were randomly selected from 40 rats as a blank group,and the remaining rats were induced with dextran sulfate sodium(DSS)to model UC.Successful rats were randomly divided into the model group,the peony soup group(32 g·kg-1),and the mesalazine group(0.5 g·kg-1),and were gavaged continuously for 10 d.After the last gavage,the disease activity index(DAI)scores of the rats and the hemorheological indexes of the colon were calculated.The pathological morphology of colon was observed by HE staining,serum interleukin-6(IL-6),IL-1βand tumor necrosis factor-α(TNF-α)were detected by ELISA,and nuclear factor-κB(NF-κB)was detected by Western blot.factor-κB(NF-κB),peroxisome proliferator activated receptor-γ(PPAR-γor PPARG)expression level by Western blotting.Results:There were 262 paeoniflora broth targets,2801 UC targets,and 169 targets at the intersection of the two.The core targets included matrix metalloproteinase 9(MMP9),PPARG,IL-6,signal transducers and activators of transduction 3(STAT3),etc.KEGG Pathway Mainly include NF-κB signaling pathway,IL-17 signaling pathway,chemokine signaling pathway,etc.Molecular docking showed that the binding energy of the key components to the core target was less than-5 kcal·mol^(-1).Animal experiments showed that the DAI scores of rats in the model group were increased compared with those in the blank group(P<0.05)and were decreased after treatment in the Paeonia lactiflora tonics group with mesalazine(P<0.05).Compared with the blank group,rats in the model group showed a decrease in colonic perfusion volume and blood cell movement velocity and an increase in mobile blood cell concentration(P<0.05);the trend of the above indexes was reversed after treatment with paeoniflora broth(P<0.05).HE staining showed that rats in the blank group showed a normal colonic histology;rats in the model group showed colonic mucosal ulceration with glandular defects accompanied by infiltration of inflammatory cells;rats in the mesalazine group and the peony broth group showed colonic pathology that was normal.The pathological morphology of colon of rats in Mesalazine group and Paeonia lactiflora group was improved compared with that of the model group.The serum TNF-α,IL-6 and IL-1βlevels of rats in the model group were elevated compared with those in the blank group(P<0.05),and decreased after the intervention of Paeoniae Radix and Mesalazine(P<0.05).Compared with the blank group,the expression level of NF-κB protein in the colon of rats in the model group was elevated,and the expression level of PPAR-γprotein was reduced(P<0.05);the trend of the above proteins was reversed after the intervention of Paeoniae Radix Tang and Mesalazine(P<0.05).Conclusion:Paeonia lactiflora broth may effectively improve the symptoms of UC rats by regulating the signaling pathways such as NF-κB and IL-17,and acting on the targets of PPARG,IL-6 and IL-1β.