基于网络药理学预测达原饮治疗新型冠状病毒肺炎作用机制及实验验证

Prediction and Experimental Verification of the Mechanism of Dayuan Decoction in Treating COVID-19 Based on Network Pharmacology

目的:研究达原饮治疗新型冠状病毒肺炎(corona virus disease 2019,COVID-19,简称新冠肺炎)的配伍内涵,并验证网络药理学预测的作用机制。方法:运用相关数据库资源,在获取达原饮代表性化学成分后,利用反向分子对接技术筛选得到关键性作用靶点,并构建各拆方组和全方组的蛋白相互作用网络、化合物-靶点-通路网络。通过腹腔注射脂多糖建立急性肺炎大鼠模型,随机分为正常组、模型组、破结逐邪组、清热滋阴组、甘草组和全方组,除空白组外,其余5组大鼠分别腹腔注射5 mg·kg^(-1)脂多糖溶液,建立急性肺炎模型,正常组和模型组按10 mL·kg^(-1)灌胃生理盐水,其余4组分别灌胃给药达原饮提取液2 mL,连续3 d。于末次给药2 h后,分离大鼠淋巴细胞,采用酶联免疫吸附测定(enzyme linked immunosorbent assay,ELISA)法检测脾淋巴细胞中白细胞介素-2(Interleukin-2,IL-2)和肿瘤坏死因子(tumor necrosis factor,TNF)-α含量,蛋白免疫印迹法(western blot,WB)检测磷脂酰肌醇3-激酶(phosphatidylinositol3-kinase,PI3-K)、蛋白激酶B(protein kinase B,Akt)蛋白及其磷酸化蛋白表达。结果:25个化合物主要作用于156个相关靶点和30条相关潜在通路,通过代谢途径、PI3K-Akt、IL-17、低氧诱导因子-1(hypoxia-inducible factor-1,HIF-1)等信号通路协同发挥调节代谢、抗炎和增强机体免疫等作用。与空白组比较,模型组大鼠IL-2、TNF-α含量显著增加,差异有统计学意义(P<0.05);与模型组比较,破结逐邪组、清热滋阴组、甘草组和全方组大鼠IL-2含量显著降低,差异有统计学意义(P<0.05);破结逐邪组、清热滋阴组、甘草组IL-2、TNF-α水平与全方组比较,差异有统计学意义(P<0.05)。与正常组比较,模型组大鼠PI3K、Akt、p-PI3K、p-Akt蛋白表达均显著升高,差异有统计学意义(P<0.05)。与模型组比较,全方组、清热滋阴组和破结逐邪组大鼠p-PI3K、p-Akt的表达均显著下降,差异有统计学意义(P<0.05),甘草组仅显著降低p-Akt含量,清热滋阴组显著降低PI3K蛋白表达水平,差异有统计学意义(P<0.05)。全方组PI3K、Akt、p-PI3K、p-Akt蛋白表达与破结逐邪组、清热滋阴组、甘草组比较,差异有统计学意义(P<0.05)。结论:达原饮中各药味通过协同配合,从炎症因子IL-2、TNF和PI3K、Akt及其磷酸化蛋白表达方面起到治疗COVID-19作用。

Objective:To study the compatibility connotation of Dayuan Decoction(达原饮,DYD)in the treatment of novel coronavirus disease 2019(COVID-19)and to verify the mechanism of network pharmacological prediction.Methods:Using relevant database resources,after obtaining the representative chemical components of DYD,the key action targets were screened by reverse molecular docking technology;the protein interaction network of each disassembled formula group and the whole formula group,and compound-target-pathway network were constructed.The rat model of acute pneumonia was established by intraperitoneal injection of lipopolysaccharide(LPS).The rats were randomly divided into the normal group,the model group,the group of breaking knot and expelling pathogens,the group of clearing heat and nourishing yin,the licorice group and the whole formula group.Except for the blank group,the rats in the other five groups were injected with 5 mg·kg^(-1) LPS(1 g·L^(-1))solution intraperitoneally to establish the acute pneumonia model.The normal group and model group were both perfused with 10 mL·kg^(-1) normal saline,while the other four groups were all given 2 mL of DYD extract by gavage for 3 days continuously.Two hours after the last administration,rat lymphocytes were isolated.The contents of interleukin-2(IL-2)and tumor necrosis factor(TNF)-αin spleen lymphocytes were detected by enzyme linked immunosorbent assay(ELISA);Western blot(WB)was used to detect the expression of phosphatidylinositol 3-kinase(PI3-K),protein kinase B(Akt)and their phosphorylated proteins.Results:The 25 compounds mainly acted on 156 related targets and 30 related potential pathways,and played a synergistic role in regulating metabolism,anti-inflammatory and enhancing immunity through metabolic pathways,PI3 K-Akt,IL-17,hypoxia inducible factor-1(HIF-1)and other signal pathways.Compared with the blank group,the contents of IL-2 and TNF-αin the model group increased significantly,and the difference was statistically significant(P<0.05).Compared with the model group,the content of IL-2 in each disassembled formula group and the whole formula group decreased significantly,and the difference was statistically significant(P<0.05).Compared with the whole formula group,there were statistically significant differences in IL-2 and TNF-αin each disassembly group(P<0.05).Compared with the normal group,the protein expressions of PI3 K,Akt,p-pi3 k and p-Akt in the model group increased significantly,and the difference was statistically significant(P<0.05).Compared with the model group,the expressions of p-PI3 k and p-Akt in the whole formula group,the group of clearing heat and nourishing yin,the group of breaking knot and expelling pathogens,decreased significantly,and all the differences were statistically significant(P<0.05),while only the content of p-Akt in licorice group decreased significantly,and the expression level of PI3 K protein in the group of clearing heat and nourishing yin,and all the differences were statistically significant(P<0.05).The protein expressions of PI3 K,Akt,p-PI3 k and p-Akt in the whole formula group were significantly different from those in each disassembled formula group(P<0.05).Conclusion:Through synergistic cooperation,the medicinal flavors in DYD increase the efficacy on the expressions of inflammatory factors IL-2,TNF,PI3 K,Akt and their phosphorylated proteins.