文献类型：期刊文献

中文题名：PI3K/AKT信号通路在党参干预溃疡性结肠炎中的差异基因表达

英文题名：Differential Gene Expression of PI3K/AKT Signaling Pathway in the Treatment of Ulcerative Colitis by Dangshen(党参)

作者：李芳[1];陈正君[1];葛俊李[1];王春霞[1];杨扶德[1]

第一作者：李芳

机构：[1]甘肃中医药大学,兰州730000

第一机构：甘肃中医药大学

年份：2024

卷号：40

期号：1

起止页码：61

中文期刊名：中药药理与临床

外文期刊名：Pharmacology and Clinics of Chinese Materia Medica

收录：;北大核心:【北大核心2023】；CSCD:【CSCD2023_2024】；

基金：科技部国家重点研发计划(编号:2018YFC1706305);甘肃省自然科学基金项目(编号:22JR5RA590、23JRRA1369);甘肃省教育科技创新项目(编号:2022A-064、2023A-297)。

语种：中文

中文关键词：党参;溃疡性结肠炎;转录组学;差异基因;PI3K/AKT

外文关键词：Dangshen(觉参);Ulcerative colitis;Transcriptomics;Dfferential gene;PI3K/Akt

摘要：目的:基于转录组学分析党参对UC大鼠结肠组织基因差异表达的影响,确证PI3K/AKT信号通路在党参干预UC中通路富集基因的差异化表达。方法:SD大鼠随机分为正常对照组和造模组,采用2,4,6-三硝基苯磺酸(TNBS)-乙醇复合方法制备UC模型;造模成功大鼠随机分为模型对照组、柳氮磺吡啶0.3 g/kg组、党参4.5、9、18 g/kg组,药物连续干预7 d后收集大鼠血清、结肠组织,计算疾病活动指数(DAI),HE染色观察大鼠结肠黏膜损伤评分并计算结肠指数;ELISA法检测血清白介素-1β(IL-1β)、IL-6、IL-8、肿瘤坏死因子-α(TNF-α)、降钙素(PCT)、C-反应蛋白(CRP)含量;转录测序分析正常对照组、模型对照组、党参18 g/kg组大鼠结肠组织差异表达基因,进行基因本体(GO)分析及基因组百科全书(KEGG)富集分析可能的信号通路;Werstern blot法检测结肠组织AKT、PI3K蛋白表达;qRT-PCR法检测结肠组织蛋白激酶B(Akt)、磷脂酰肌醇-3激酶(Pi3k)、IV型胶原a6蛋白(Col4a6)、层粘连蛋白α2(Lama2)、神经生长因子(Ngfr)、层粘连蛋白γ3亚基(Lamc3)、层粘蛋白β(Lamb2)、氨基酸整合素亚基α7(Itga7)、溶血磷脂酸受体3(Lpar3)、酪氨酸激酶受体Eph亚族(Efna1)、催乳素受体(Prlr)、3’-磷酸肌醇依赖性蛋白激酶1(Pdpk1)和细胞周期蛋白D1(Ccnd2)mRNA表达。结果:与正常对照组比较,模型对照组大鼠体质量显著降低,结肠长度显著缩短,疾病活动指数、结肠指数、结肠组织病理评分显著升高(P<0.01);与模型对照组比较,各药物组大鼠体质量显著升高(P<0.01),结肠长度明显增加,疾病活动指数、结肠指数、结肠组织病理评分明显降低(P<0.05或P<0.01)。模型对照组与正常对照组的4727个差异基因及受试药物组与模型对照组的1058个差异表达基因均显著富集于PI3K/AKT信号通路,其中Col4a6、Lama2、Ngfr、Lamc3、Lamb2、Itga7、Lpar3、Efna1、Prlr、Pdpk1、Ccnd2是富集于PI3K/AKT信号通路的显著差异表达基因。与正常对照组比较,模型对照组血清IL-1β、IL-6、IL-8、TNF-α、PCT、CRP含量显著升高(P<0.01),结肠组织PI3K、AKT蛋白表达显著上调(P<0.01),Pi3k、Akt、Pdpk1、Ccnd2基因表达显著上调(P<0.01),Col4a6、Lama2、Ngfr、Lamc3、Lamb2、Itga7、Lpar3、Efna1、Prlr基因表达显著下调(P<0.01);与模型对照组比较,各药物组血清IL-1β、IL-6、IL-8、TNF-α、PCT、CRP含量显著降低(P<0.01),结肠组织PI3K、AKT蛋白表达明显下调(P<0.05或P<0.01),Pi3k、Akt、Pdpk1、Ccnd2基因表达明显下调(P<0.05或P<0.01),Col4a6、Lama2、Ngfr、Lamc3、Lamb2、Itga7、Lpar3、Prlr基因表达明显上调(P<0.05或P<0.01)。结论:党参可能通过抑制PI3K/AKT信号通路,调控Col4a6、Lama2、Ngfr、Lamc3、Lamb2、Itga7、Lpar3、Efna1、Prlr、Pdpk1、Ccnd2基因发挥治疗UC的作用,运用转录组学技术发现差异基因为揭示中药党参治疗UC的机制提供了新的研究视角。
Objective:To confirm the differential expression of genes in the phosphatidylinositol 3 kinase(PI3K)/protein kinase B(Akt)signaling pathway in the mouse model of ulcerative colitis(UC)treated by Dangshen(觉参)based on the transcriptomic data.Method:SD rats were randomized into a blank group and a modeling group.The mouse model of UC was established by the 2,4,6-trinitrobenzenesulfonic acid(TNBS)-ethanol method.The successfully modeled rats were randomized into model,positive drug(salazosulfapyridine,SASP,o.3 g/kg),and Dangshen(18,9,and 4.5 g/kg)groups.After 7 consecutive days of drug intervention,the serum and colon tissue samples of each group were collected.The disease activity index(DAI)was calculated.The pathological sections were stained with hematoxylin-eosin(HE),and the tissue damage index(TDI)of the colon mucosa and the colon index were calculated.Enzyme-linked immunosorbent assay was employed to measure the levels of interleukin-1β(IL-1β),IL-6,IL-8,tumor necrosis factor-α(TNF-α),calcitonin(PCT),and C-reactive protein(CRP)in the serum.Transcriptional sequencing was carried out to analyze the differentially expressed genes(DEGs)in the colon tissue among the blank,model,and Dangshen(18 g/kg)groups.Gene Ontology(CO)annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment were performed to analyze the signaling pathway in DEGs.Western blotting(WB)was employed to determine the protein levels of Akt and PI3K in the colon tissue.Real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)was employed to measure the mRNA levels of Akt,Pi3k,collagen type IVα-6(Col4a6),lamininα2(Lama2),nerve growth factor receptor(Ngf),laminin subunit 3(Lamc3),lamininβ2(Lamb2),integrin subunitα7(Iga7),lysophosphatidic acid receptor 3(Lpar3),ephrin A1(Efnal),prolactin receptor(Prlr),3-phosphoinositide-dependent protein kinase 1(Pdpk1),and cyclin d2(Ccnd2)in the colon tissue.Results:Compared with the blank group,the modeling decreased the body weight(P<0.01),shortened the colon(P<0.01),and increased the DAI,colon index,and colon TDI(P<0.01).Compared with the model group,the drug interventions increased the body weight(P<0.01),extended the colon(P<0.01 or P<0.05),and decreased the DAI,colon index,and colon TDI(P<0.01 or P<0.05).The 4727 DEGs between the model group and the blank group,as well as the 1058 DEGs between the drug intervention group and the model group,were significantly enriched in the PI3K/Akt signaling pathway.Among them,Col4a6,Lama2,Ngfr,Lamc3,Lamb2,Itga7,Lpar3,Efnal,Prlr,Pdpkl,and Ccnd2 showed significantly differential expression.Compared with the blank group,the modeling elevated the levels of IL-1β,IL-6,IL-8,TNF-α,PCT,and CRP in the serum(P<0.O1),up-regulated the protein levels of PI3K and Akt and the mRNA levels of Pi3k,Akt,Pdpk1,and Ccnd2 in the colon tissue(P<0.01),and down-regulated the mRNA levels of Col4a6,Lama2,Ngfr,Lamc3,Lamb2,Itga7,Lpar3,Efnal,and Prlr in the colon tissue(P<0.O1).Compared with the model group,the drug intervention lowered the levels of IL-1β,IL-6,IL-8,TNF-α,PCT,and CRP in the serum(P<0.O1),down-regulated the protein levels of PI3K and Akt and the mRNA levels of Pi3k,Akt,Pdpk1,and Ccnd2 in the colon tissue(P<0.01 or P<0.05),and up-regulated the mRNA levels of Col4a6,Lama2,Ngfr,Lamc3,Lamb2,Itga7,Lpar3,and Prlr in the colon tissue(P<0.01 or P<0.05).Conclusion:Dangshen may exert therapeutic effects on UC by inhibiting the PI3K/Akt signaling pathway and regulating the expression of Col4a6,Lama2,Ngfr,Lamc3,Lamb2,Itga7,Lpar3,Efnal,Prlr,Pdpk1,and Ccnd2.Using transcriptomic to explore the significant DEGs provides a new research perspective for deciphering the mechanism of Dangshen in treating UC.