目的 基于网络药理学预测三七治疗幽门螺杆菌（Hp）相关疾病机制，研究三七中有效活性成分人参皂苷Rb₃对Hp造成的胃上皮细胞损伤的保护作用及机制。方法 使用Herb数据库收集“三七”的相关预测靶点，使用Gene Cards数据库收集Hp相关疾病的靶点；使用Draw Venn Diagram网站绘制Venn图，得到靶点交集；进行蛋白互作（PPI）网络分析、基因本体（GO）和京都基因与基因组百科全书（KEGG）富集分析。将GES-1细胞分为对照组、模型组及人参皂苷Rb₃低、中和高浓度（1、5、10 μ mol· L^-1）组，人参皂苷Rb₃组使用相应浓度的人参皂苷Rb₃预处理，培养过夜12 h至融合度为70%～80%。Hp悉尼株1 （SS1）按感染复数（MOI） 100加入细胞中制备损伤模型，人参皂苷Rb₃继续给药，共培养48 h。对照组不加SS1，对照组和模型组不加药。改良吉姆萨染色后通过光学显微镜观察细胞形态；结合Hoechst 33342荧光染色和Annexin V/PI双染流式细胞术检测细胞凋亡；试剂盒法检测活性氧（ROS）水平；采用实时荧光定量PCR（qRT-PCR）检测凋亡相关基因TP53、Bax、Bcl-2表达量；Western blotting法检测P53、p-Akt、cleaved/pro-Caspase 9、Bcl-2、Bax、cleaved/pro-Caspase 3的蛋白表达情况。结果 网络药理学结果表明三七治疗Hp相关疾病的靶点共16个，其PPI网络分析得到按度值大小排名前6位靶点为TP53、CASP3、PTGS2、IL6、TNF、IL1β。GO富集分析与KEGG富集分析结果均显示与凋亡相关。与模型组比较，经人参皂苷Rb₃处理后，GES-1细胞的细胞核染色质致密深染，破裂的细胞逐渐减少；Hoechst 33342荧光染色细胞核强荧光数目明显减少；细胞凋亡率显著降低（P＜0.05）；ROS水平显著降低（P＜0.05）；TP53与Bax的mRNA水平显著降低，Bcl-2 mRNA水平显著升高（P＜0.05）； p-Akt、Bcl-2蛋白表达显著升高（P＜0.05），P53、Bax、cleaved/pro-Caspase 9与cleaved/pro-Caspase 3蛋白表达显著降低（P＜0.05）。结论 三七可能通过包括炎症及凋亡在内的多种途径治疗Hp相关疾病，人参皂苷Rb₃对Hp诱导的胃上皮细胞凋亡发挥显著改善作用，其可能机制是降低氧化应激水平，并调节Akt的磷酸化和P53的表达。

Objective To explore the protective effect and mechanism of ginsenoside Rb₃ (G-Rb₃) on gastric epithelial cell injury caused by Helicobacter pylori (Hp). Methods Herb database was used to collect predicted targets of "Panax notoginseng", Gene Cards database was used to collect targets of Hp-related diseases. The Venn Diagram was drawn using the Draw Venn Diagram website to obtain the intersection of target points. Protein interaction (PPI) network analysis, gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis were performed. GES-1 cells were divided into control group, model group, and G-Rb₃ low, medium and high concentration groups (1, 5, 10 μmol·L^-1). G-Rb₃ group was pretreated with the corresponding concentration of G-Rb₃, and cultured overnight for 12 h until the degree of fusion was 70% — 80%. Hp Sydney strain 1 (SS1) was added into the cells according to the infection number (MOI) 100 to prepare the injury model, and G-Rb₃ was continued to be administered for 48 h. Control group did not add SS1, control group and model group did not add drugs. The effects of drugs on cell morphology were observed by optical microscope, the reversal effect of drugs on Hp-induced apoptosis was detected by Hoechst 33342 fluorescence staining and Annexin V/PI double staining. In mechanism research, on the one hand. qRT-PCR and Western blotting were used to detect the expression of apoptosis-related genes P53, Bax, Bcl-2 and related proteins P53, p-Akt, Cleaved/Pro-Caspase 9, Bcl-2, Bax, Cleaved/Pro-Caspase 3, and the difference of ROS levels between groups was detected. Results The results of network pharmacology showed that there were 16 targets for Panax notoginseng in the treatment of Hp-related diseases. The top six PPI network analysis were TP53, CASP3, PTGS2, IL6, TNF and IL1β. GO enrichment analysis and KEGG enrichment analysis showed apoptosis-related results. After pretreatment with G-Rb₃, the nuclear chromatin of gastric epithelial cells was dense and deeply stained, and the number of broken cells decreased gradually. Hoechst 33342 fluorescence staining was used to detect apoptotic cells. Compared with Hp group, the number of nuclear strong fluorescence in G-Rb₃ low, medium and high groups was significantly (P<0.05). Flow cytometry showed that compared with the normal group, the apoptosis rate of Hp group was significantly increased. Compared with Hp group, the apoptosis rate of G-Rb₃ group was significantly decreased (P<0.05). Reactive oxygen species detection showed that the fluorescence intensity of the three concentrations of G-Rb₃ was significantly lower than that of the model group (P<0.05). Compared with the Hp group, the expressions of TP53 and Bax genes were significantly decreased, and the expression of Bcl-2 gene was significantly increased (P<0.05). Western blotting results showed that compared with Hp group, p-Akt and Bcl-2 protein expression increased, P53, Bax, Cleaved-Caspase 9 and Cleaved-Caspase 3 protein expression decreased (P<0.05). Conclusion Panax notoginseng may treat Hp-related diseases through multiple pathways including inflammation and apoptosis. Its main component G-Rb₃ can effectively protect GES-1 and reduce SS1-induced apoptosis, which may be due to the promotion Akt phosphorylation and P53 degradation by regulating oxidative stress.

R285.5

江苏省中医药局科技项目(YB2015166)