基于网络药理学和实验验证的六君子汤治疗慢性萎缩性胃炎的作用机制研究

Mechanism of Six Gentlemen Decoction in Treating Chronic Atrophic Gastritis Based on Network Pharmacology and Experimental Verification

目的:通过网络药理学方法预测六君子汤治疗慢性萎缩性胃炎(CAG)的潜在分子机制,并结合动物实验进行疗效和机制验证。方法:在TCMSP数据库中收集六君子汤的活性成分和药物靶点;通过PharmGkb、OMIM、GeneCards和DrugBank数据库筛选CAG的相关靶点。药物靶点与疾病靶点取交集得出共同靶点,使用Cytoscape3.8.0软件制作活性成分-交集靶点网络图,利用R软件对其进行GO和KEGG富集分析。采用复合因素造模法建立慢性萎缩性胃炎大鼠模型,造模16周后进行模型评价,随后用六君子汤干预4周。观察大鼠胃黏膜病理形态变化,通过酶联免疫法检测大鼠血清中白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)含量;蛋白免疫印迹法、实时荧光定量聚合酶链反应检测Janus激酶1(JAK1)和转录激活因子3(STAT3)、P53、B淋巴细胞瘤-2(BCL-2)表达水平来验证六君子汤对CAG的影响。结果:筛出六君子汤活性成分125个,潜在靶点241个,CAG靶点377个,交集靶点48个,关键靶点涉及IL-1β、IL-6、TP53、BCL-2等,KEGG富集分析得出六君子汤治疗CAG涉及到了JAK/STAT、PI3K/Akt、HIF-1、IL-17等信号通路。实验结果表明,与模型组相比,六君子汤组大鼠胃黏膜外观和病理形态均有所改善,大鼠血清中IL-6含量降低(P<0.05),IL-1β含量显著降低(P<0.01);胃黏膜组织中JAK1、STAT3、BCL-2 mRNA表达降低(P<0.05),P53 mRNA表达显著降低(P<0.01);JAK1、STAT3、P53蛋白显著降低(P<0.01),BCL-2蛋白含量降低(P<0.05)。结论:六君子汤可以改善慢性萎缩性胃炎,其机制可能是通过抑制IL-1β、IL-6从而进一步抑制下游JAK1/STAT3信号通路的表达,随着该通路激活而抑制P53、Bcl-2因子表达,从而有效调控胃黏膜细胞凋亡,达到修复胃黏膜的作用有关。

Objective:To predict the potential molecular mechanism of six gentlemen decoction in the treatment of chronic atrophic gastritis(CAG)by network pharmacological method,and to verify its efficacy and mechanism in combination with animal experiments.Methods:The active components and drug targets of six gentlemen decoction were collected in TCMSP database;the relevant targets of CAG are filtered through databases PharmGkb,OMIM,GeneCards and DrugBank.The common target is obtained by intersection of drug target and disease target.The active ingredient intersection target network diagram is made by using the software Cytoscape 3.8.0,and the enrichment analysis of GO and KEGG is carried out by using R software.The rat model of chronic atrophic gastritis(CAG)was established by compound factor modeling method.The model was evaluated 16 weeks after modeling,and then the rats were intervened with six gentlemen decoction for 4 weeks.The pathological changes of rat gastric mucosa were observed,and the levels of interleukin-1β(IL-1β)and interleukin-6(IL-6)in serum of rats were detected by enzyme-linked immunoassay.The expression levels of Janus kinase 1(JAK1),Signal transducer and activator of transcription3(STAT3),P53,and B-cell lymphoma-2(BCL-2)were detected by Western blot and real-time fluorescence quantitative polymerase chain reaction to verify the effect of six gentlemen decoction on CAG.Results:125 active ingredients,241 potential targets,377 CAG targets,48 intersection targets were screened out,and the key targets involved IL-1β,IL-6,TP53,BCL-2,etc.The KEGG enrichment analysis shows that the treatment of CAG with six gentlemen decoction involves JAK/STAT,PI3K/Akt,HIF-1,IL-17 and other signal pathways.The experimental results showed that compared with the model group,the appearance and pathological morphology of gastric mucosa of rats in six gentlemen decoction group were improved,the content of IL-6 in serum of rats decreased(P<0.05),and IL-1βcontent decreased significantly(P<0.01).The expression of JAK1,STAT3,and BCL-2 mRNA decreased(P<0.05)in gastric mucosa and P53 mRNA decreased significantly(P<0.01);JAK1,STAT3,P53 protein decreased significantly(P<0.01),and BCL-2 protein content decreased(P<0.05).Conclusion:Six gentlemen decoction can improve chronic atrophic gastritis,its mechanism may be through inhibiting IL-1βand IL-6.It can subsequently inhibit the expression of downstream JAK1/STAT3 signal pathway,and inhibit the expression of P53 and BCL-2 factors with the activation of this pathway,so as to effectively regulate the apoptosis of gastric mucosa cells and achieve the role of repairing the mucosa.