有氧运动联合水飞蓟宾对阻塞性黄疸小鼠模型肠黏膜屏障的影响

Effect of aerobic exercise combined with silybin on intestinal mucosal barrier in mice with obstructive jaundice

目的探讨有氧运动联合水飞蓟宾激活阻塞性黄疸小鼠肠组织microRNA-21/TLR4/NF-κB通路轴改善肠黏膜屏障损伤的效果。方法选取健康雄性昆明小鼠采用手术线直角悬挂胆总管法构建阻塞性黄疸模型,随机分成模型组(M组)、运动组(E组)、水飞蓟宾组(S组)、运动联合水飞蓟宾组(ES组),另设假手术组(GO组),每组10只。有氧运动和水飞蓟宾灌胃干预7周后,采用一般行为学观察、ELISA酶联免疫、回肠组织HE染色、免疫组化染色,肠组织相关因子蛋白、mRNA检测及肝组织mRNA表达谱测序分析,探讨其干预效果。计量资料多组间比较采用单因素方差分析,进一步两组间比较采用LSD-t检验。采用多因素方差分析有氧运动与水飞蓟宾间交互效应,并进行简单效应分析。结果 M组腹部、尾部皮肤黄染严重,出现黄尿及粪便呈浅灰色,开腹可见胆管悬挂处呈囊性扩张。相比M组,E、S、ES组肝功能指标ALT、AST和TBil水平下降(F值分别为567. 56、1376. 09、512. 81,P值均<0. 001),血浆二胺氧化酶(DAO)、D-乳酸及内毒素水平降低(F值分别为650. 29、1130. 05、396. 04,P值均<0. 001),上述指标除DAO水平以外,有氧运动和水飞蓟宾间均存在协同效应(P值均<0. 05)。镜下显示,M组肠黏膜结构破坏严重,绒毛稀疏且高度不一,隐窝深度变浅,部分腺体萎缩、脱落,局部有炎症细胞浸润及肌层萎缩。相比M组,E、S组病理症状明显改善,ES组干预效果最佳。与M组相比,E、S、ES组TLR4、NF-κB、TNFαmRNA表达降低(F值分别为104. 69、153. 55、262. 38,P值均<0. 01)和蛋白表达下降(F值分别为2683. 83、419. 73、572. 41,P值均<0. 01),microRNA-21基因水平上调(F=194. 58,P <0. 01),且除microRNA-21基因水平外,有氧运动和水飞蓟宾间存在协同效应(P值均<0. 05)。Illumina高通量测序筛选肝组织炎症相关因子及关联分析显示,其因子的差异变化主要富集于NF-κB、TGFβ、TNF、TLR等炎性通路,涉及组织细胞信号转导、凋亡抑制、免疫反应、毒性等,且有氧运动和水飞蓟宾间可显著降低炎症和免疫相关信号通路的富集。结论 7周有氧运动或水飞蓟宾灌胃可通过上调阻塞性黄疸小鼠肠组织中microRNA-21表达,抑制TLR4/NF-κB信号通路及其下游炎性因子释放,发挥对肠黏膜屏障的保护作用,有氧运动与水飞蓟宾联合应用效果最佳。

Objective To investigate the effect of aerobic exercise combined with silybin in improving intestinal mucosal barrier injury in mice with obstructive jaundice by activating the microRNA-21/TLR4/NF-κB pathway axis in intestinal tissue. Methods Healthy male Kunming mice were selected,and a model of obstructive jaundice was established by the method of hanging the common bile duct using an operating suture line at a right angle.The mice were randomly divided into model group(M group),exercise group(E group),silybin group(S group),and exercise+silybin group(ES group),and a sham-operation group was also established,with 10 mice in each group.After the intervention of aerobic exercise combined with intragastric administration of silybin for 7 weeks,general behavioral observation,ELISA,HE staining of ileal tissue,immunohistochemical staining,measurement of the protein and mRNA expression of intestinal tissue-related factors,and a sequencing analysis of mRNA expression profiles in liver tissue were performed to observe intervention effect.A one-way analysis of variance was used for continuous data,and the least significant difference t-test was used for comparison between groups.A multivariate analysis of variance was used to investigate the interaction effect between aerobic exercise and silybin,and a simple effect analysis was performed. Results The mice in the M group had severe jaundice on the skin of the abdomen and tail,yellow urine,and light gray feces,and laparotomy showed cystic dilatation at the bile duct suspension.Compared with the M group,the E,S,and ES groups had significant reductions in the liver function parameters alanine aminotransferase,aspartate aminotransferase,and total bilirubin(F=567.56,1376.09,and 512.81,all P<0.001),as well as significant reductions in the plasma levels of diamine oxidase(DAO),D-lactic acid,and endotoxin(F=650.29,1130.05,and 396.04,all P<0.001),and there was a synergistic effect between aerobic exercise and silybin in all the above indices except DAO(all P<0.05).Microscopic observation showed that the M group had severe damage of the structure of the intestinal mucosa,sparse villi with different heights,a reduction in the depth of intestinal crypts,atrophy and detachment of some glands,and local infiltration of inflammatory cells and muscular atrophy.Compared with the M group,the E and S groups had significant improvements in the above pathological symptoms,and the ES group had the best intervention effect.Compared with the M group,the E,S,and ES groups had significant reductions in the mRNA and protein expression of TLR4,NF-κB,and TNFα(mRNA:F=104.69,153.55,and 262.38,all P<0.01;protein:F=2683.83,419.73,and 572.41,all P<0.01),as well as a significant increase in the expression of microRNA-21(F=194.58,P<0.01),and there was a synergistic effect between aerobic exercise and silybin in all the above indices except the expression of microRNA-21(all P<0.05).According to Illumina high-throughput sequencing for the screening of liver inflammation-related factors and correlation analysis,the differentially expressed factors were mainly enriched in the inflammatory pathways such as NF-κB,TGFβ,TNF,and TLR,which involved tissue cell signal transduction,apoptosis inhibition,immune response,and toxicity,and aerobic exercise and silybin significantly reduced the enrichment of inflammation and immune-related signaling pathways. Conclusion In mice with obstructive jaundice,aerobic exercise or intragastric administration of silybin for 7 weeks can exert a protective effect on intestinal mucosal barrier by upregulating the expression of microRNA-21 in intestinal tissue and inhibiting the TLR4/NF-κB signaling pathway and the release of its downstream inflammatory factors,and aerobic exercise combined with silybin has the optimal effect.