基于生物信息学探讨血竭散对克罗恩病大鼠结肠组织Notch1、Nrf2、GPX4和PTGS2表达影响

Exploration of the Effect of Xuejie San(血竭散)on Expressions of Notch1,Nrf2,GPX4 and PTGS2 in Colon Tissues of Crohn's Disease Rats Based on A Bioinformatic Analysis

目的基于生物信息学探析血竭散缓解克罗恩病(Crohn's disease,CD)的作用靶点和机制。方法运用BATMAN-TCM数据库挖掘血竭散治疗CD的潜在靶点和分子机制,并采用2,4,6-三硝基苯磺酸诱导建立CD大鼠模型行体内实验验证。造模后,将30只大鼠随机分为空白组,模型组,血竭散低、高剂量组和N-[N-(3,5-二氟苯乙酰基)-L-丙氨酸基]-S-苯甘氨酸叔丁酯(N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester,DAPT)组,每组各6只。苏木精-伊红染色法观察大鼠结肠组织病理学改变,酶联免疫吸附法检测结肠组织中炎症细胞因子表达,免疫组化法检测4-羟基壬烯醛(4-hydroxynonenal,4-HNE)表达,实时荧光定量PCR法检测Notch1和核因子E2相关因子2(nuclear factor E2-related factor 2,Nrf2)mRNA表达,蛋白免疫印迹法检测Notch1细胞内结构域(Notch1 intracellular domain,NICD1)、Nrf2、谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)和前列腺素内过氧化物合酶2(prostaglandin-endoperoxide synthase 2,PTGS2)蛋白表达。结果生物信息学分析结果显示,Notch1和PTGS2是血竭散潜在靶点;调控氧化还原酶活性和脂代谢是血竭散潜在作用机制。血竭散组和DAPT组干预后大鼠结肠组织病理学评分显著低于模型组(P<0.05)。与模型组相比,血竭散组和DAPT组能显著降低大鼠结肠组织中白介素6(interleukin-6,IL-6)、IL-17、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、4-HNE表达(P<0.05);抑制Notch1 mRNA表达,上调Nrf2 mRNA表达(P<0.05);减少NICD1和PTGS2蛋白表达,增加Nrf2和GPX4蛋白表达(P<0.05)。结论血竭散可能通过抑制Notch1信号,增强Nrf2/GPX4活性,抑制肠黏膜细胞脂质过氧化,减少促炎细胞因子分泌,缓解CD肠炎。

Objective To explore therapeutic targets and mechanisms of Xuejie San(血竭散,XJS)in the treatment of Crohn's disease(CD)based on a bioinformatic analysis.Methods BATMAN-TCM database was used to explore the therapeutic targets and mechanisms of XJS in the treatment of CD,which were verified in a CD rat model induced by 2,4,6-trinitrobenzene sulfonic acid.After establishment of CD rat models,30 rats were randomly divided into control group,model group,XJS low-dose group,XJS high-dose group,and N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester(DAPT)group with 6 rats in each group.Histopathological changes in the colon of rats were observed by hematoxylin-eosin staining.Expressions of inflammatory cytokines and 4-hydroxynonenal(4-HNE)were respectively investigated by enzyme-linked immunosorbent assay and immunohistochemical staining.The mRNA expressions of Notch1 and nuclear factor E2-related factor 2(Nrf2)were examined by quantitative real-time PCR.Western blot was used to detect the protein expressions of Notch1 intracellular domain(NICD1),Nrf2,glutathione peroxidase 4(GPX4),and prostaglandin-endoperoxide synthase 2(PTGS2).Results Data from the BATMAN-TCM database showed that Notch1 and PTGS2 were the potential targets of XJS,and XJS could regulate oxidoreductase activities and lipid metabolism.The histopathological scores and expressions of interleukin-6(IL-6),IL-17,tumor necrosis factor-α(TNF-α),and 4-HNE were significantly lower in XJS groups and DAPT group than those in model group(P<0.05).In addition,XJS groups and DAPT group were able to inhibit the mRNA expression of Notch1 and the protein expressions of NICD1 and PTGS2,while increase the mRNA expression of Nrf2 and the protein expressions of Nrf2 and GPX4(P<0.05).Conclusion XJS might suppress lipid peroxidation in intestinal mucosal cells to reduce the secretion of proinflammatory cytokines and ameliorate CD colitis via enhancing the activity of Nrf2/GPX4 signaling pathway negatively regulated by Notch1.