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益生菌通过调控 Wnt/β- 连环蛋白信号通路对肝硬化大鼠肠道菌群、炎症及肝纤维化程度的影响。

Effects of probiotics on intestinal flora, inflammation and degree of liver cirrhosis in rats with liver cirrhosis by regulating Wnt/β-catenin signaling pathway.

机构信息

Department of Hepatology, Qingdao No.6 People's Hospital, Qingdao, Shandong, China.

出版信息

J Biol Regul Homeost Agents. 2021 Jan-Feb;35(1):25-33. doi: 10.23812/20-242-A.


DOI:10.23812/20-242-A
PMID:33472728
Abstract

We aimed to explore the effects of probiotics on intestinal flora, inflammation and degree of liver cirrhosis in rats with liver cirrhosis, and to verify the Wnt/β-catenin signaling pathway that regulates this process. A total of 30 SD rats were randomly divided into 3 groups, namely, control group (n=10), model group (n=10) and probiotic group (n=10). Rats in the model group were used to construct liver cirrhosis models using carbon tetrachloride (CCL4) method, and those in the probiotic group were administered with probiotic preparations by gavage for 8 weeks. Then the feces of rats in each group were taken to detect the composition of intestinal flora, and changes in the content of inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), monocyte chemotactic protein 1 (MCP-1) and interferon-gamma (IFN-γ), in peripheral blood collected were examined by enzyme-linked immunosorbent assay (ELISA). Next, changes in the degree of liver cirrhosis were analyzed by hematoxylin and eosin (H&E) staining, and the expression levels of the Wnt/β-catenin signaling pathway-related molecules, including β-catenin, glycogen synthase kinase (GSK)-3β and Frizzled-2, in liver tissues in each group were detected via polymerase chain reaction (PCR) and Western blotting (WB). Compared with rats in the control group, those in the model group had a disordered structure of hepatic lobule and hyperplasia of a large number of fibrous tissues. In contrast to those in the model group, the liver lobule structure was greatly improved, the edema cells were obviously reduced, and the hyperplasia of collagen fibers was remarkably alleviated in the probiotic group. Moreover, the degree of liver cirrhosis in the probiotic group was significantly reduced compared with that in the model group. Moreover, the rats in the model group exhibited a higher Bifidobacterium level in the intestinal tract, while those in the probiotic group displayed higher levels of microorganisms in the intestinal tract, such as Lachnospiraceae, Ruminococcaceae, Actinbacteria, Slackia and Pasteurellaceae. In comparison with that in the control group, the level of salt-tolerant Lactobacillus in the intestinal tract of rats in the model group was significantly decreased, while that in the probiotic group was partially increased (P=0.023). Meanwhile, some intestinal flora of rats in the control group, model group and probiotic group were closely correlated. Specifically, highly positive correlations were found between Bacteroidetes and Paraeggerthella (r=0.423, P=0.034) and between Firmicutes and Lactobacillus (r=0.318, P=0.027), but strongly negative associations were detected between Firmicutes and Paraeggerthella (r=-0.691, p=0.004) and between Paraeggerthella and Lactobacillus (r=-0.384, P=0.047). In addition, the levels of inflammatory cytokines TNF-α IL-6, MCP-1 and IFN-γ in the plasma of rats in the model group were markedly higher than those in the control group (P<0.05), whereas such levels in the probiotic group were decreased compared with those in the model group (P<0.05). PCR results revealed that the expression levels of β-catenin and Frizzled-2 in the model group were higher than those in the control group, whereas they were lower in the probiotic group than those in the model group (P<0.05). Furthermore, the model group had a decreased level of GSK-3β in comparison with the control group, but the probiotic group had a higher level of GSK-3β than the model group (P<0.05). WB results were consistent with PCR results. Probiotics can affect intestinal flora, inflammation and degree of liver cirrhosis in rats with liver cirrhosis, and its mechanism may be related to the Wnt/β-catenin signaling pathway.

摘要

本研究旨在探讨益生菌对肝硬化大鼠肠道菌群、炎症反应和肝纤维化程度的影响,并验证调节该过程的 Wnt/β-连环蛋白信号通路。将 30 只 SD 大鼠随机分为 3 组,即对照组(n=10)、模型组(n=10)和益生菌组(n=10)。模型组大鼠采用四氯化碳(CCL4)法构建肝硬化模型,益生菌组大鼠给予益生菌制剂灌胃 8 周。然后取各组大鼠粪便检测肠道菌群组成,采用酶联免疫吸附试验(ELISA)检测外周血中肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、单核细胞趋化蛋白 1(MCP-1)和干扰素-γ(IFN-γ)等炎症因子的含量。接下来,通过苏木精和伊红(H&E)染色分析肝硬化程度,并通过聚合酶链反应(PCR)和蛋白质印迹(WB)检测各组大鼠肝组织中 Wnt/β-连环蛋白信号通路相关分子β-连环蛋白、糖原合成酶激酶(GSK)-3β和 Frizzled-2 的表达水平。与对照组大鼠相比,模型组大鼠肝小叶结构紊乱,大量纤维组织增生。与模型组相比,益生菌组大鼠肝小叶结构得到明显改善,水肿细胞明显减少,胶原纤维增生明显缓解。此外,益生菌组大鼠的肝硬化程度明显低于模型组。此外,模型组大鼠肠道内双歧杆菌水平较高,而益生菌组大鼠肠道内微生物如lachnospiraceae、ruminococcaceae、actinbacteria、slackia 和 pasteurellaceae 水平较高。与对照组相比,模型组大鼠肠道内耐盐乳杆菌水平明显降低,而益生菌组大鼠肠道内部分增加(P=0.023)。同时,对照组、模型组和益生菌组大鼠的部分肠道菌群密切相关。具体来说,拟杆菌门和 Paraeggerthella 之间存在高度正相关(r=0.423,P=0.034),厚壁菌门和乳杆菌之间存在高度正相关(r=0.318,P=0.027),但厚壁菌门和 Paraeggerthella 之间存在强烈的负相关(r=-0.691,p=0.004),Paraeggerthella 和乳杆菌之间也存在强烈的负相关(r=-0.384,P=0.047)。此外,模型组大鼠血浆中 TNF-α、IL-6、MCP-1 和 IFN-γ 等炎症因子的水平明显高于对照组(P<0.05),而益生菌组大鼠的这些水平则低于模型组(P<0.05)。PCR 结果显示,模型组大鼠β-连环蛋白和 Frizzled-2 的表达水平高于对照组,而益生菌组大鼠的表达水平低于模型组(P<0.05)。此外,模型组大鼠 GSK-3β 水平低于对照组,而益生菌组大鼠 GSK-3β 水平高于模型组(P<0.05)。WB 结果与 PCR 结果一致。益生菌可影响肝硬化大鼠的肠道菌群、炎症反应和肝硬化程度,其机制可能与 Wnt/β-连环蛋白信号通路有关。

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