摘要
目的:探究丹皮酚是否通过调节真杆菌属介导的G蛋白胆汁酸偶联受体5(TGR5)/蛋白激酶A(PKA)/环腺苷单磷酸反应结合元件(CREB)信号通路对酒精性肝损伤小鼠发挥保护作用。方法:将C57BL/6小鼠随机分成5组:正常组、模型组、丹皮酚组(480 mg·kg^(-1))、抗生素组(Abs组)和抗生素+丹皮酚组。Lieber-De Carli液体饲料喂养C57BL/6小鼠造模第2天起灌胃给药,连续10 d。测定小鼠血脂、肝功能、炎症因子及氧化应激水平,通过苏木素-伊红(HE)染色法和油红O染色观察肝脏组织形态变化和脂肪堆积情况,采用16S r DNA测序检测空白组、模型组和丹皮酚组小鼠肠道菌群多样性变化,蛋白免疫印迹法(Western blot)检测丹皮酚对小鼠回肠组织闭锁小带蛋白-1(ZO-1)、紧密连接蛋白-1(Claudin-1)和TGR5/PKA/CREB信号通路相关蛋白表达水平的影响。结果:相较于空白组,模型组小鼠的血脂、肝功能、氧化应激水平,炎症因子的表达升高(P<0.01),肝脏脂肪空泡明显,回肠黏膜损伤严重,且回肠组织ZO-1、Claudin-1和TGR5/PKA/CREB蛋白含量显著下降(P<0.01),肠道菌群发生改变,变形菌门明显增多,拟杆菌门与厚壁菌门的比值降低;杜氏杆菌属、乳杆菌属、双歧杆菌属等菌属相对丰富度降低,埃希氏-志贺氏菌属、摩根氏菌属、普罗维登斯菌属、变形杆菌属的相对丰度显著增加。与模型组比较,丹皮酚明显降低了乙醇饲料诱导的肝损伤小鼠的血脂、肝功能、氧化应激水平,炎症因子的表达(P<0.05),减少肝脏脂肪空泡,改善并恢复了回肠肠道屏障,恢复了肝细胞和回肠细胞正常结构。改善了由乙醇引起的肠道菌群紊乱的情况,使真杆菌属等有益菌属相对丰富度增加。回肠组织中ZO-1、Claudin-1、TGR5/PKA/CREB蛋白表达水平升高(P<0.05)。结论:丹皮酚对小鼠酒精性肝损伤具有保护作用,作用机制是通过调节真杆菌属介导的TGR5/PKA/CREB信号通路达到抗炎效果和改善肠道屏障来实现的。
Objective:To investigate whether paeonol exerts a protective effect on mice with alcoholic liver injury by regulating the Takeda G-protein-coupled Receptor 5(TGR5)/protein Kinase A(PKA)/cAMP response binding element(CREB)signaling pathway mediated by Eubacterium.Methods C57BL/6 mice were randomly divided into 5 groups:normal group,model group,paeonol group(480 mg·kg-1),antibiotic group(Abs group),and antibiotic+paeonol group.Lieber-DeCarli liquid feed fed C57BL/6 mice on the second day of modeling and administration for 10 days.Measuring blood lipids,liver function,inflammatory factors and oxidative stress levels in mice.Hematoxylin-eosin staining(HE)and oil red O staining were used to observe the morphological changes and fat accumulation of liver tissues.16S rDNA sequencing was used to detect the diversity of intestinal microbiota in the blank,model and paeanol groups.Western blot was used to detect the effect of paeonol on the expression levels of protein related to the signaling pathway of atresia band protein 1(ZO-1),claudin-1(Claudin-1)and G protein bile acid-coupled receptor 5(TGR5)/protein kinase A(PKA)/cAMP response binding element(CREB)in mouse ileal tissue.Result:Compared with the blank group,the blood lipids,liver function,oxidative stress levels,and the expression of inflammatory factors in the model group increased(P<0.01),the liver fat vacuoles were obvious,the ileal mucosa was seriously damaged,and the protein contents of ZO-1,Claudin-1 and TGR5/PKA/CREB in ileal tissue decreased significantly(P<0.01),the intestinal flora changed,the proteobacteria phylum increased significantly,and the ratio of Bacteroidetes to Firmicutes decreased.The relative abundance of Bifidobacterium and other genera decreased,while the relative abundance of Escherichia-Shigella,Morganella,Providence and Proteus increased significantly.Compared with the model group,paeonol significantly reduced the blood lipids,liver function,oxidative stress levels,and expression of inflammatory factors in mice with alcohol diet-induced liver injury(P<0.05),reduced liver fat vacuoles,improved and restored the ileal intestinal barrier,and restored the normal structure of hepatocytes and ileal cells.The intestinal microbiota disorder caused by alcohol was improved,and the relative abundance of beneficial bacteria such as Eubacterium spp.was increased.The protein expression levels of ZO-1,Claudin-1 and TGR5/PKA/CREB in ileal tissue were increased(P<0.05).Conclusion:Paeonol has a protective effect on alcoholic liver injury in mice,and the mechanism of action is achieved by modulating the eubacterium-mediated TGR5/PKA/CREB signaling pathway to achieve anti-inflammatory effect and improve the intestinal barrier.
作者
张鲁宁
刘玲玲
姜胜男
吴琦峰
颜贵明
ZHANG Luning;LIU Lingling;JIANG Shengnan;WU Qifeng;YAN Guiming(School of Pharmacy,Anhui University of Traditional Chinese Medicine,Hefei 230012,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2024年第12期78-86,共9页
Chinese Journal of Experimental Traditional Medical Formulae
基金
安徽省自然科学基金项目(2022AH050521,2008085MH300)。
