基于网络药理学探究血尿胶囊治疗急性肾盂肾炎的作用机制

Mechanism of Xueniao Capsule in Treatment of Acute Pyelonephritis Based on Network Pharmacology

目的:采用网络药理学和实验验证的方法探究血尿胶囊治疗急性肾盂肾炎(APN)的作用机制。方法:通过APN大鼠模型研究血尿胶囊对APN的作用效果。使用中药系统药理学数据库与分析平台(TCMSP),化学专业数据库,中医证候关联(SymMap)数据库检索菝葜、薏苡仁、棕榈子的化学成分,PharmMapper与SwissTargetPrediction数据库收集成分的靶点信息,使用治疗靶标数据库(TTD),DrugBank数据库,疾病相关的基因与突变位点数据库(DisGeNET),GeneCards数据库,在线人类孟德尔遗传数据库(OMIM)收集疾病靶点信息。使用基因注释工具(Metascap)对血尿胶囊治疗APN的关键基因进行基因本体(GO)富集分析及京都基因与基因组百科全书(KEGG)通路富集分析。使用实时荧光定量聚合酶链式反应(Real-time PCR)与蛋白免疫印迹法对预测结果进行验证。结果:与空白组和假手术组比较,模型组左右肾比值升高,脏器指数升高(P<0.05,P<0.01),白细胞(WBC),中性粒细胞(NEUT),单核细胞(MONO),淋巴细胞(LY)水平明显升高(P<0.05,P<0.01),核转录因子-κB(NF-κB),白细胞介素-6(IL-6),肿瘤坏死因子-α(TNF-α)水平明显升高(P<0.05,P<0.01)。与模型组比较,诺氟沙星组左右肾比值降低,脏器指数降低(P<0.05,P<0.01),WBC,NEUT,MONO,LY水平降低(P<0.05,P<0.01),NF-κB,IL-6,TNF-α水平降低(P<0.05,P<0.01);血尿胶囊低剂量组左右肾比值降低,脏器指数降低(P<0.05,P<0.01),WBC,NEUT,MONO,LY水平降低(P<0.05,P<0.01),NF-κB,IL-6,TNF-α水平降低(P<0.05,P<0.01);血尿胶囊中剂量组左右肾比值降低,脏器指数降低(P<0.05,P<0.01),WBC,NEUT,MONO,LY水平降低(P<0.05,P<0.01),IL-6,TNF-α水平降低(P<0.05,P<0.01);血尿胶囊高剂量左右肾比值降低,脏器指数降低(P<0.05,P<0.01),WBC,NEUT,MONO,LY水平降低(P<0.05,P<0.01),NF-κB,IL-6,TNF-α水平降低(P<0.05,P<0.01)。网络药理学分析得到菝葜活性化合物17种,薏苡仁活性化合物18种,棕榈子活性化学物6种,血尿胶囊治疗APN关键基因39个,GO分析富集704生物学过程,22个细胞组成,59个分子功能,KEGG信号通路共富集62条通路。实验验证结果表明与空白组比较,模型组的前列腺素-过氧化物合成酶2(PTGS2),丝裂原活化蛋白激酶1(MAPK1),磷脂酰肌醇3-激酶(PI3K),蛋白激酶B2(Akt2),Janus激酶2(JAK2),信号转导和转录激活因子3(STAT3)mRNA的表达量和PI3K,Akt2,JAK2,STAT3蛋白表达量显著升高(P<0.01);与模型组比较,血尿胶囊低剂量组MAPK1,PI3K,JAK2,STAT3 mRNA表达量和PI3K,JAK2,STAT3蛋白表达量显著降低(P<0.01);中剂量组MAPK1,PTGS2,PI3K,JAK2,STAT3 mRNA表达量和PI3K,JAK2,STAT3蛋白降低(P<0.05,P<0.01);高剂量组PTGS2,MAPK1,PI3K,Akt2,JAK2,STAT3 mRNA表达量和PI3K,Akt2,JAK2,STAT3蛋白表达量降低(P<0.05,P<0.01)。结论:血尿胶囊对APN具有一定的疗效,血尿胶囊发挥作用为多靶点,多途径的,其机制可能与抑制PI3K/Akt信号通路和JAK2/STAT3信号通路活性表达有关。

Objective:To explore the mechanism of Xueniao capsule in the treatment of acute pyelonephritis(APN)by network pharmacology and experimental verification.Method:The effect of Xueniao capsule on APN was investigated based on the APN model in rats.The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP),Chemistryl Database,and SymMap were searched for the chemical components of Smilacis Chinae Rhizoma,Coicis Semen,and Trachycarpi Petiolus.The target information of the components was collected from PharmMapper and SwissTargetPrediction,and disease target information from Therapeutic Target Database(TTD),DrugBank,DisGeNET,GeneCards,and Online Mendelian Inheritance in Man(OMIM).The key genes of Xueniao capsule for APN underwent Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses by Metascap.Real-time quantitative polymerase chain reaction(PCR)and Western blot were employed to verify the prediction results.Result:Compared with the blank group and the sham operation group,the model group showed an increased ratio of the left kidney to the right kidney and organ index(P<0.05,P<0.01),up-regulated white blood cells(WBC),neutrophils(NEUT),monocytes(MONO),and lymphocytes(LY)(P<0.05,P<0.01),and elevated levels of nuclear factor-κB(NF-κB),interleukin-6(IL-6),and tumor necrosis factor-α(TNF-α)(P<0.05,P<0.01).Compared with the model group,the norfloxacin group,the low-and high-dose Xueniao capsule groups showed a decreased ratio of the left kidney to the right kidney and organ index(P<0.05,P<0.01),dwindled levels of WBC,NEUT,MONO,and LY(P<0.05,P<0.01),and reduced levels of NF-κB,IL-6,and TNF-α(P<0.05,P<0.01).The medium-dose Xueniao capsule group showed a decreased ratio of the left kidney to the right kidney and organ index(P<0.05,P<0.01),reduced levels of WBC,NEUT,MONO,and LY(P<0.05,P<0.01),and dwindled levels of IL-6 and TNF-α(P<0.05,P<0.01).Network pharmacological analysis revealed 17 active compounds from Smilacis Chinae Rhizoma,18 active compounds from Coicis Semen,six active compounds from Trachycarpi Petiolus,and 39 key genes for the treatment of APN in Xueniao capsule.GO enrichment analysis demonstrated 704 biological processes,22 cellular components,and 59 molecular functions.Sixty-two pathways were enriched in KEGG enrichment analysis.The experimental verification results showed that compared with the blank group,the model group showed increased mRNA expression of prostaglandinendoperoxide synthase 2(PTGS2),mitogen-activated protein kinase 1(MAPK1)/extracellular signal-regulated protein kinase 2(ERK2),phosphoinositide 3 kinase(PI3K),protein kinase B2(Akt2),Janus kinase 2(JAK2),and signal transducer and activator of transcription 3(STAT3)and protein expression of PI3K,Akt2,JAK2,and STAT3(P<0.05,P<0.01).Compared with the model group,the low-dose Xueniao capsule group showed decreased mRNA expression of MAPK1,PI3K,JAK2,and STAT3 and protein expression of PI3K,JAK2,and STAT3(P<0.05,P<0.01).The medium-dose Xueniao capsule group showed decreased mRNA expression of MAPK1,PTGS2,PI3K,JAK2,and STAT3,and protein expression of PI3K,JAK2,and STAT3(P<0.05,P<0.01).The high-dose Xueniao capsule group showed reduced mRNA expression of PTGS2,MAPK1,PI3K,Akt2,JAK2,and STAT3 and protein expression of PI3K,Akt2,JAK2,and STAT3(P<0.05,P<0.01).Conclusion:Xueniao capsule has a certain curative effect on APN via multiple targets and multiple pathways.The mechanism may be related to the inhibition of the PI3K/Akt signaling pathway and the JAK2/STAT3 signaling pathway.