马甲子对大鼠炎性肠病的干预及基于转录组学的机制研究

Intervention of Paliurus ramosissimus on Inflammatory Bowel Disease in Rats and Its Mechanism Based on Transcriptomics

目的:评价马甲子提取物对大鼠实验性炎性肠病(Inflammatory bowel disease, IBD)的干预作用,探讨其作用机制。方法:以2,4,6-三硝基苯磺酸(TNBS)为致炎剂复制大鼠IBD模型,灌胃给予马甲子,观察疾病活动指数(DAI)、结肠粘膜损伤指数(CMDI)、结肠长度、结肠组织病理、血清炎症相关因子白介素-6(Interleukin-6,IL-6)、IL-1β、肿瘤坏死因子(Tumour necrosis factor-α,TNF-α)、环氧合酶2(Cyclooxygenase 2,COX2)等指标;采用转录组测序(RNA-Seq)筛选差异基因,免疫蛋白印迹法(Western blot, WB)检测相关基因的蛋白表达。结果:马甲子820 mg/kg组可减轻IBD大鼠结肠组织炎症,显著增加结肠长度(P<0.01),明显降低血清中IL-6、IL-1β、TNF-α、COX2含量(P<0.05或P<0.01)。马甲子820 mg/kg组共筛选出514个差异基因(|lg2 Fold change|≥1.5,P<0.01),主要涉及IL-17信号通路(IL-17 signal pathway)、cAMP信号通路(cAMP signal pathway)等;其中与IBD密切相关的IL-17信号通路中,与模型对照组比较,马甲子820 mg/kg组可下调CCATT/增强子结合蛋白β(CCAAT/enhancer binding proteinβ,C/Ebpb)、丝裂原活化蛋白激酶家族(Mitogen active protein kinases,Mapks)以及下游炎症因子IL-1β、C-C趋化因子7(C-C motif chemokine 7,Ccl7)、S100钙结合蛋白A8(S100 Calcium Binding Protein A8,S100a8)、Cox2基因的表达;WB结果显示马甲子820 mg/kg组干预后IL-17信号通路中糖原合成酶激酶3β(Glycogen synthase kinase 3β,GSK3β)蛋白表达上调(P<0.01),C/EBPβ蛋白表达下调(P<0.01)。结论:马甲子能明显改善IBD大鼠的结肠炎性损伤,其作用机制可能与上调GSK3β蛋白表达,下调C/EBPβ的表达,从而影响下游炎症因子COX2、IL-6、IL-1β、TNF-α、CCL7、S100A8水平有关。

Objective:To evaluate the intervention effect of Paliurus ramosissimus extract on experimental inflammatory bowel disease(IBD)in rats and explore its mechanism of action.Methods:The rat model of IBD was induced using 2,4,6-trinitrobenzene sulfonic acid(TNBS).Rats were orally administered with P.ramosissimus extract,and the disease activity index(DAI),colonic mucosal damage index(CMDI),colon length,colonic tissue pathology,serum levels of inflammation-related factors interleukin-6(IL-6),IL-1β,tumor necrosis factor-α(TNF-α),and cyclooxygenase 2(COX2)were observed.Differential genes were screened using transcriptome sequencing(RNA-Seq),and protein expression of relevant genes was detected using Western blot.Results:The group treated with P.ramosissimus extract at a dose of 820 mg/kg alleviated colonic tissue inflammation in IBD rats,increased colon length(P<0.01),and significantly reduced serum levels of IL-6,IL-1β,TNF-α,and COX2(P<0.05 or P<0.01).A total of 514 differential genes(|log2 Fold change|≥1.5,P<0.01)were identified in the P.ramosissimus groups,mainly involving the IL-17 signaling pathway and cAMP signaling pathway.In the IL-17 signaling pathway,which was closely related to IBD,the P.ramosissimus extract at a dose of 820 mg/kg downregulated the expression of CCAAT/enhancer binding proteinβ(C/Ebpβ),mitogen-activated protein kinases(Mapk),as well as downstream inflammatory factors Il1β,C-C motif chemokine 7(Ccl7),S100 calcium-binding protein A8(S100A8),and Cox2 genes compared with the results in the model control group.Western blot results showed an upregulation of glycogen synthase kinase 3β(GSK3β)protein expression(P<0.01)and a downregulation of C/EBPβprotein expression(P<0.01)in the IL-17 signaling pathway after intervention with P.ramosissimus extract at a dose of 820 mg/kg.Conclusion:P.ramosissimus extract significantly improves colonic inflammatory damage in IBD rats,and its mechanism of action may be related to the promotion of GSK3βprotein expression and inhibition of C/EBPβactivity,thereby affecting the levels of downstream inflammatory factors COX2,IL-6,IL-1β,TNF-α,CCL7,and S100A8.