目的 基于網(wǎng)絡(luò)藥理學(xué)預(yù)測三七治療幽門螺桿菌（Hp）相關(guān)疾病機制，研究三七中有效活性成分人參皂苷Rb₃對Hp造成的胃上皮細(xì)胞損傷的保護作用及機制。方法 使用Herb數(shù)據(jù)庫收集“三七”的相關(guān)預(yù)測靶點，使用Gene Cards數(shù)據(jù)庫收集Hp相關(guān)疾病的靶點；使用Draw Venn Diagram網(wǎng)站繪制Venn圖，得到靶點交集；進行蛋白互作（PPI）網(wǎng)絡(luò)分析、基因本體（GO）和京都基因與基因組百科全書（KEGG）富集分析。將GES-1細(xì)胞分為對照組、模型組及人參皂苷Rb₃低、中和高濃度（1、5、10 μ mol· L^-1）組，人參皂苷Rb₃組使用相應(yīng)濃度的人參皂苷Rb₃預(yù)處理，培養(yǎng)過夜12 h至融合度為70%～80%。Hp悉尼株1 （SS1）按感染復(fù)數(shù)（MOI） 100加入細(xì)胞中制備損傷模型，人參皂苷Rb₃繼續(xù)給藥，共培養(yǎng)48 h。對照組不加SS1，對照組和模型組不加藥。改良吉姆薩染色后通過光學(xué)顯微鏡觀察細(xì)胞形態(tài)；結(jié)合Hoechst 33342熒光染色和Annexin V/PI雙染流式細(xì)胞術(shù)檢測細(xì)胞凋亡；試劑盒法檢測活性氧（ROS）水平；采用實時熒光定量PCR（qRT-PCR）檢測凋亡相關(guān)基因TP53、Bax、Bcl-2表達量；Western blotting法檢測P53、p-Akt、cleaved/pro-Caspase 9、Bcl-2、Bax、cleaved/pro-Caspase 3的蛋白表達情況。結(jié)果 網(wǎng)絡(luò)藥理學(xué)結(jié)果表明三七治療Hp相關(guān)疾病的靶點共16個，其PPI網(wǎng)絡(luò)分析得到按度值大小排名前6位靶點為TP53、CASP3、PTGS2、IL6、TNF、IL1β。GO富集分析與KEGG富集分析結(jié)果均顯示與凋亡相關(guān)。與模型組比較，經(jīng)人參皂苷Rb₃處理后，GES-1細(xì)胞的細(xì)胞核染色質(zhì)致密深染，破裂的細(xì)胞逐漸減少；Hoechst 33342熒光染色細(xì)胞核強熒光數(shù)目明顯減少；細(xì)胞凋亡率顯著降低（P＜0.05）；ROS水平顯著降低（P＜0.05）；TP53與Bax的mRNA水平顯著降低，Bcl-2 mRNA水平顯著升高（P＜0.05）； p-Akt、Bcl-2蛋白表達顯著升高（P＜0.05），P53、Bax、cleaved/pro-Caspase 9與cleaved/pro-Caspase 3蛋白表達顯著降低（P＜0.05）。結(jié)論 三七可能通過包括炎癥及凋亡在內(nèi)的多種途徑治療Hp相關(guān)疾病，人參皂苷Rb₃對Hp誘導(dǎo)的胃上皮細(xì)胞凋亡發(fā)揮顯著改善作用，其可能機制是降低氧化應(yīng)激水平，并調(diào)節(jié)Akt的磷酸化和P53的表達。

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

江蘇省中醫(yī)藥局科技項目(YB2015166)