连翘叶茶对大鼠肝硬化的影响及其机理研究

doi:10.13560/j.cnki.biotech.bull.1985.2024-0286

摘要/Abstract

摘要：

【目的】 研究连翘叶茶粗提物及其茶水对四氯化碳（CCl₄）诱导大鼠肝硬化的影响，阐明不同剂量连翘叶茶粗提物及茶水对肠道菌群多样性的影响及其机理。【方法】 30只雄性SD大鼠按照平均体重相近的原则随机分为对照组、模型组、低、中、高剂量连翘叶茶灌胃组和茶水组，采用皮下注射CCl₄+灌胃CCl₄橄榄油混合液的方法构建肝硬化大鼠模型，建模成功后给予不同剂量连翘叶茶粗提物灌胃及茶水组干预10 d后，收取血清进行ELISA检测，肝脏组织通过HE染色和提取RNA进行RT-PCR检测，粪便样本进行16S rRNA测序。【结果】 连翘叶茶粗提物及茶水组均能显著改善大鼠肝硬化形态，尤其茶水组可明显逆转肝硬化状态，肝细胞脂肪变性、中央静脉周围炎性细胞浸润和汇管区纤维结缔组织增生较模型组均明显改善；连翘叶茶粗提物及茶水组可显著提高益生菌Rothia、Parabacteroides和Lactobacillus丰度，促进不饱和脂肪酸合成和酸呼吸通路的富集；连翘叶茶粗提物及茶水组可抑制血液中DAO、TLR-4、LPS内毒素因子的水平，促进抑炎因子1L-4及肠道屏障功能因子ZO-1表达，各组之间肠道微生物组成与DAO、LPS、1L-4和TLR4相关，其中与肠道损伤指标DAO相关性最大。【结论】 连翘叶茶粗提物及茶水组可能通过改善大鼠肝硬化形态、减弱炎症反应、改善肠道微生态功能失调及保护肠屏障功能抑制肝硬化发生。

关键词: 连翘叶茶, 肝硬化, 微生物多样性, 大鼠, 炎症因子, 机理

Abstract:

【Objective】 To study the effects of crude extracts of Forsythia suspensa leaves and their tea solution on carbon tetrachloride（CCl₄）induced liver cirrhosis in rats, and to elucidate the effects and mechanisms of different doses of the crude extracts and tea solution of F. suspensa leaves on gut microbiota diversity. 【Method】 The 30 male SD rats were randomly divided into control group, model group, low, medium, and high-dose F. suspensa leaf tea orally administered group, and tea water group according to the principle of similar average body weight. Liver cirrhosis rat model was constructed by subcutaneous injection of CCl₄+CCl₄ olive oil mixture orally administered group. At the same time, different doses of F. suspensa leaf tea crude extract orally administered, and tea water group were intervened for 10 d. Serum was collected for ELISA detection, liver tissue was stained with HE, the liver tissue RNA was extracted for RT-PCR detection, and fecal samples were subjected to 16S rRNA sequencing. 【Result】 Both the crude extract of F. suspensa leaves and the tea group significantly improved the morphology of liver cirrhosis in rats, especially the tea group significantly reversed the status of liver cirrhosis. Liver cell steatosis, inflammatory cell infiltration around the central vein, and fibrous connective tissue proliferation in the portal area were significantly improved compared to the model group. The crude extract of F. suspensa leaves and the tea group significantly increased the abundance of probiotics Rothia, Paraacteroides, and Lactobacillus, promoting the synthesis of unsaturated fatty acids and the enrichment of acid respiration pathways. The crude extract of F. suspensa leaf tea and the tea group inhibited the levels of DAO, TLR-4, and LPS endotoxin factors in the blood, promoted the expression of anti-inflammatory factor 1L-4 and intestinal barrier function factor ZO-1. The composition of intestinal microbiota among the groups was correlated with DAO, LPS, 1L-4, and TLR4, with the highest correlation with intestinal injury index DAO. 【Conclusion】 The crude extract of F. suspensa leaf tea and tea group inhibit in the pathogenesis of liver cirrhosis by improving the pathological morphology of liver cirrhosis in rats, reducing inflammatory response, improving intestinal microecological dysfunction, and protecting intestinal barrier function.

Key words: Forsythia suspensa leaf tea, liver cirrhosis, diversity of microbes, rat, inflammatory factors, mechanism

吴永娜, 滕文龙, 张磊, 王德富, 牛颜冰. 连翘叶茶对大鼠肝硬化的影响及其机理研究[J]. 生物技术通报, 2024, 40(11): 285-295.

WU Yong-na, TENG Wen-long, ZHANG Lei, WANG De-fu, NIU Yan-bing. Effect of Forsythia suspensa Leaf Tea on with Cirrhosis and Its Mechanism in Rats[J]. Biotechnology Bulletin, 2024, 40(11): 285-295.

图/表 12

图1 肝硬化和正常大鼠肝脏Masson染色 A：对照组大鼠，肝小叶正常，中央静脉无炎症浸润，汇管区无结缔组织； B：模型组大鼠，纤维组织增生明显，假小叶形成

Fig. 1 Masson staining of liver between cirrhosis and normal rats A: The rats in the control group had normal liver lobules, no inflammatory infiltration of the central vein, and no connective tissue in the manifold area. B: Rats in the model group had obvious fibrous tissue hyperplasia and pseudolobule formation

图2 肝硬化大鼠肝脏形态特征 Control：对照组；Model：肝硬化模型组；Tea：连翘茶水组；Low、Medium和High：分别为连翘叶茶粗提物0.5、0.7和1 g/mL。下同

Fig. 2 Morphological characteristics of livers in cirrhotic rats Control: Control group. Model: Cirrhosis model group. Tea: Forsythia tea group. Low, Medium and High: 0.5, 0.7 and 1 g/mL crude extract of Forsythia leaf tea, respectively. The same below

图3 不同处理组间细菌群落的α多样性特征

Fig. 3 α diversity characteristics of bacterial community in different treatments

图4 不同处理组间 β多样性特征 A：β多样性PLSDA模型聚类分析；B：维恩图特征分析

Fig. 4 β diversity characteristics among different treatments A: Cluster analysis of β diversity via PLSDA mode. B: Characteristic analysis via the Venn diagram

图5 不同处理组间TOP 10的肠道细菌门（A）和属（B）水平上的组成及差异

Fig. 5 Composition and difference of TOP 10 gut bacteria among different treatments at phylum（A）and genus（B）level

图6 不同处理组间肠道细菌属水平上差异菌群

Fig. 6 Differential microbiota of gut bacteria among different treatments at genus level

图7 不同处理组间肠道细菌LefSe分析图

Fig. 7 LefSe analysis of gut bacteria in different treatments

图8 不同处理组间肠道细菌LAD图

Fig. 8 LAD analysis of gut bacteria in different treatments

图9 不同处理组间差异功能通路分析 A：不饱和脂肪酸代谢通路；B：阿拉伯糖转运结合蛋白通路；C：延胡索酸呼吸通路；D：β-木糖苷水解酶GH43；不同字母代表P<0.05水平上差异显著，下同

Fig. 9 Analysis of differential functional pathways in different treatments A: Metabolic pathways of unsaturated fatty acids. B: Pathways of arabinosaccharide transport system substrate-binding protein. C: Respiratory pathway of fumaric acid. D: β-xylosidase GH43. Different letters indicate significant differences at the P<0.05 level. The same below

图10 不同处理组间血清中炎症因子的分析 1L-4：白介素4；DAO：二胺氧化酶；LPS：脂多糖；TLR4：Toll样受体（TLR）家族成员

Fig. 10 Analysis of inflammatory factor in serum in different treatments 1L-4: Interleukin 4. DAO: Diamine oxidase. LPS: Lipopolysaccharide. TLR4: A member of the toll-like receptor（TLR）family

图11 不同处理组间肝脏屏障功能蛋白和炎症因子的表达

Fig. 11 Analysis of barrier functional proteins and inflammatory factor in liver tissue in different treatments

图12 不同处理组间肠道微生物与临床因子相关性分析 A：差异菌群与差异炎症因子斯皮尔曼相关性分析；B：差异菌群与差异炎症因子CCA分析

Fig. 12 Correlation analysis between intestinal microbes and clinical factors among different treatments A: Spearman correlation analysis between differential microbiota and differential inflammatory factors. B: CCA analysis of differential microbiota and differential inflammatory cytokines

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