摘要
目的研究丁酸梭菌修复肠道屏障改善肠易激综合征(IBS)的作用机制。方法采用母婴分离联合三硝基苯磺酸建立IBS小鼠模型,以丁酸梭菌和丁酸钠(NaB)进行干预,并分为正常组、模型组、NaB组和丁酸梭菌组,每组9只。通过体重、腹泻及内脏高敏性(AWR)评估疗效;通过16S rRNA基因测序和高效液相色谱法分别检测肠道菌群及粪便丁酸水平;检测血异硫氰酸荧光素−葡聚糖(FITC-D)和二胺氧化酶(DAO)水平(肠上皮通透性)和紧密连接蛋白(ZO-1/Occludin)以评估肠道屏障功能;采用免疫组化与Western-blotting检测磷脂酰肌醇−3−激酶/丝苏氨酸蛋白激酶(PI3K/Akt)通路蛋白活性。体外机制验证采用脂多糖诱导Caco-2细胞屏障损伤模型,通过NaB干预、G蛋白耦联受体109A(GPR109A)沉默及PI3K激动剂/抑制剂正反验证,结合FITC-D渗透率、Western-blotting及免疫荧光评估屏障功能与PI3K/Akt通路活性。结果丁酸梭菌和NaB干预后IBS小鼠症状显著改善,体重增加、小鼠疼痛阈值上调、4 h排便数减少(均P<0.05)。丁酸梭菌干预后,肠道菌群失调显著恢复,产丁酸细菌丰度显著增加,粪便丁酸水平升高(均P<0.05)。丁酸梭菌和NaB干预后血FITC-D和DAO水平显著降低,Occludin和ZO-1表达显著升高,PI3K和Akt表达显著降低,PI3K/Akt通路活性抑制(均P<0.05)。体外实验证实NaB通过GPR109A抑制PI3K/Akt通路,促进紧密连接蛋白表达。结论丁酸梭菌可通过其代谢产物丁酸抑制PI3K/Akt通路活性,修复IBS小鼠肠道屏障功能。
Objective To explore the mechanisms underlying Clostridium butyricum-mediated restoration of intestinal barrier function and alleviation of irritable bowel syndrome(IBS).Methods The IBS mouse model was established using a dual approach of maternal separation and trinitrobenzene sulfonic acid(TNBS)induction.The mice were administered Clostridium butyricum and sodium butyrate(NaB)as the therapeutic intervention,and divided into normal group,modle group,NaB group and Clostridium butyricum group,with 9 cases in each group.Therapeutic effect was evaluated by monitoring body weight,diarrhea severity,and visceral hypersensitivity,quantified through the abdominal withdrawal reflex(AWR)scoring system.Gut flora composition was analyzed via 16S rRNA gene sequencing,while fecal butyrate concentrations were quantified using high-performance liquid chromatography(HPLC).Intestinal barrier integrity was assessed by measuring serum fluorescein isothiocyanate-dextran(FITC-D)and plasma diamine oxidase(DAO)concentrations,alongside tight junction protein expressions(ZO-1 and Occludin).PI3K/Akt signaling pathway activity was evaluated through immunohistochemistry(IHC)and Western-blotting.For in vitro validation,a lipopolysaccharide(LPS)-induced Caco-2 cell barrier injury model was established.Barrier function and PI3K/Akt pathway activity were assessed through FITC-D permeability assays,Western-blotting,and immunofluorescence following interventions with NaB,GPR109A silencing,and pharmacological modulation using PI3K agonists and inhibitors.Results Both Clostridium butyricum and NaB ameliorated IBS symptoms in model mice.Specifically,these interventions significantly promoted body weight gain during the treatment period,increased visceral pain thresholds,and reduced 4-hour fecal pellet output(all P<0.05).Clostridium butyricum reshaped gut flora composition,markedly elevating the abundance of butyrate-producing bacteria and fecal butyrate concentrations(all P<0.05).Furthermore,Clostridium butyricum and NaB interventions significantly reduced serum FITC-D and plasma DAO levels,and upregulated tight junction protein(Occludin and ZO-1)expressions(all P<0.05).Both treatments suppressed PI3K/Akt pathway activation,as evidenced by decreased PI3K and Akt phosphorylation levels(all P<0.05).In vitro validation confirmed that NaB inhibited PI3K/Akt signaling via the GPR109A receptor and enhanced tight junction protein expression.Conclusion Clostridium butyricum restores intestinal barrier integrity in IBS mice by suppressing PI3K/Akt pathway hyperactivation through its metabolite butyrate.
作者
王娇娇
杨良俊
方政
刘珊
朱佳杰
WANG Jiaojiao;YANG Liangjun;FANG Zheng;LIU Shan;ZHU Jiajie(Department of Gastroenterology,Tongde Hospital of Zhejiang Province,Hangzhou,Zhejiang 310012,China;不详)
出处
《中国微生态学杂志》
北大核心
2025年第11期1260-1268,共9页
Chinese Journal of Microecology
基金
浙江省自然科学基金(LQ21H290002)
国家自然科学基金(82004333)
浙江省中医药科技计划(2023ZR005)。
