湖南农业大学伍树松教授课题组Gut Microbes发文：原儿茶酸在代谢相关脂肪肝疾病中的保护作用

湖南农业大学动物科学技术学院Jijun Tan（第一作者）、伍树松教授（通讯作者）在国际期刊《Gut Microbes》（Q1，IF=12.2）上发表题为“Protection against Metabolic Associated Fatty Liver Disease by Protocatechuic Acid”的研究性论文。

研究背景

代谢相关脂肪肝疾病（MAFLD）越来越被认为是由肝脏脂肪过度堆积和代谢综合征发展而来最普遍的慢性肝病。最近的荟萃分析显示，根据 Medline和Embase数据库，代谢相关脂肪肝占欧洲、亚洲和北美的39.22%，患病率较高。MAFLD的发生和发展机制复杂，但主要基于遗传和营养因素之间的相关性，包括脂质代谢紊乱、胰岛素抵抗和肠道微生态不平衡。肠道菌群、免疫系统和肝脏之间的相互作用是代谢相关疾病的首发性物质，胰岛素作用被认为是脂肪沉积和代谢的关键调节因子，因此，基于肠肝轴理论的MAFLD研究引起了广泛关注。而一些传统有益细菌的负面影响往往被忽视，例如产生短链脂肪酸（SCFAs）的细菌或属于乳酸杆菌属的微生物群。研究表明，粪肠球菌（E. faecalis）是一种属于乳酸杆菌属的机会性病原体，具有通过以下方式诱发酒精性肝脂肪变性的潜力分泌溶细胞素，通过将三甲基赖氨酸代谢为N，N，N-三甲基-5-氨基戊酸，具有促进MAFLD的能力，可减少肉碱合成，减少脂肪酸的氧化。因此，潜在肠道菌群的变化成为了解MAFLD发病机制和预防的首要问题。

由于具有抗氧化剂、抗炎和抗菌能力，多酚具有抑制MAFLD的潜力。研究表明，花青素3-葡萄糖苷（C3G）是植物中最常见的花青素之一，具有改善MAFLD小鼠炎症和氧化应激的潜力，通过调节肠道微生物群。然而，摄入C3G后迅速降解为酚酸，酚酸可能发挥主要生物学功能，而原儿茶酸（PCA）、间苯三甲醛（PGA）、香草酸（VA）、阿魏酸（FA）及其衍生物已被证明是C3G在血液循环中的主要酚类代谢产物。基于抗氧化和抗炎活性，PCA、PGA、VA和FA被认为是C3G的潜在生物活性酚类代谢产物。因此，本研究旨在探讨C3G、PCA、PGA、VA和FA对MAFLD的保护作用，并探讨高脂饮食诱导小鼠模型中肠道菌群的调控，阐明典型生物活性代谢产物PCA的保护机制。

 

结论与展望

膳食补充PCA，一种多酚C3G的生物活性酚类代谢物，可减轻胰岛素抵抗，减少HFD-肝脂质积累，炎症和肝损伤诱导MAFLD模型。微生物测序和脂质组学分析表明，肠球菌与脂质代谢产物的变化有显著相关性，FMT实验表明，粪肠球菌可以通过下调胰岛素活性相关基因特别是CPT1α来加速肝脂肪变性，而PCA可以通过增强Fgf1、Igfbp2、Irs1和Irs2的表达来逆转肝脂肪变性。

研究亮点

• 原儿茶酸（PCA）对高脂饮食诱导的C57BL/6J小鼠MAFLD有积极作用
• PCA对脂质代谢和肠道菌群的调控作用
• PCA通过降低肠球菌属的相对丰度来降低厚壁菌门/拟杆菌门比值，与脂质组学分析中LDL-c、AST、ALT和大部分上调肝脂质的水平呈正相关
• 粪肠球菌引起肝脏炎症、脂肪沉积和胰岛素抵抗，肉碱棕榈酰转移酶-1α（CPT1α）表达降低，PCA可通过抑制粪肠球菌逆转
• 高比例的肠道粪肠球菌会加速 MAFLD，CPT1α 和 Fgf1 的表达降低，这可以通过膳食补充 PCA 来预防。

 

图文赏析

 

Figure 1. C3G, PCA and PGA showed the protective effect against MAFLD in mice. (a) Growth curves of body weight. (b) Representative sections of liver by hematoxylin – eosin (H&E) staining (original magnification × 100). Levels of (c) ALT, (d) AST, (e) glucose and (f) HDL-c/LDL-c ratio in serum were measured using an automatic biochemical analyzer. Cytokines including (g) IL-1β, (H) IL-2, (I) IL-6, (J) TNF-α and (K) MCP-1 in serum were measured with their respective ELISA kit. Data represent as mean ± SD (n = 4), and bars with different letters differ significantly (P < 0.05). Hepatic histopathological scores were evaluated based on NAFLD Activity Score (NAS). The score ranging from 0 to 8 is defined as the unweighted sum of the scores for steatosis (0–3), lobular inflammation (0–3) and ballooning (0–2). ALT, alanine aminotransferase; AST, aspartate aminotransferase; C3G, cyanidin-3-glucoside; FA, ferulic acid; HDL-c, high density lipoprotein cholesterol; HFD, high-fat diet; IL, interleukin; LDL-c, low density lipoprotein cholesterol; LFD, low-fat diet; MCP-1, monocyte chemoattractant protein-1; PCA, protocatechuic acid; PGA, phloroglucinaldehyde; TNF-α, tumor necrosis factor-α; VA, vanillic acid.

 

 

Figure 2. PCA showed a dose-dependent effect against MAFLD by attenuating insulin resistance in mice. (a) Growth curves of body weight. (b) Intraperitoneal fat weight. (c) Liver weight. (d) Representative sections of intraperitoneal fat by hematoxylin – eosin (H&E)staining with original magnification × 100 (left) and × 400 (right). (e) Representative liver sections by hematoxylin – eosin (H&E)staining with original magnification × 100 (left) and × 400 (right). (f) Representative liver sections by oil red staining (original magnification × 100). (G1) Serum level of AST. (G2) Serum level of ALT. (h) the content of MDA in liver. (i) the HDL-c/LDL-c ratio in serum. (j) Serum level of TNF. (K) HOMA-IR index based on serum levels of glucose and insulin. Data represent as mean ± SD (n = 6).Bars with different letters differ significantly (P < 0.05). Hepatic histopathological scores were evaluated based on NAFLD Activity Score(NAS). The score ranging from 0 to 8 is defined as the unweighted sum of the scores for steatosis (0–3), lobular inflammation (0–3) and ballooning (0–2). ALT, alanine aminotransferase; AST, aspartate aminotransferase; HDL-c, high density lipoprotein cholesterol; HFD, high-fat diet; HOMA-IR, homeostasis model assessment-estimated insulin resistance; LDL-c, low density lipoprotein cholesterol; LFD, low-fat diet; MDA, malondialdehyde; PCA, protocatechuic acid; TNF, tumor necrosis factor.

 

 

Figure 3. PCA alleviated MAFLD by inhibiting Enterococcus. (a) Ratio of Firmicutes to Bacteroidetes. (b) the relative abundance of Enterococcus. (c) Correlation of gut microbiota and serum indicators. (d) Correlation of gut microbiota and hepatic lipid metabolites.Feces were collected at the first day (Feces0W) and final day (Feces12W) of experiment, and cecal contents (Cecal contents12W) were collected after sacrifice. The intensity of the colors represented the degree of association (red, positive correlation; blue, negative correlation). Data represent as mean ± SD (n = 6), and significant correlations were marked by *P < 0.05, **P < 0.01. ALT, alanine aminotransferase; AST, aspartate aminotransferase; Cerm, ceramide; Glu, glucose; HDL-c, high density lipoprotein cholesterol; LDL-c, low density lipoprotein cholesterol; PS, phosphatidylserine; TG, triglyceride.

 

 

Figure 4. Effects of E. faecalis and PCA on MAFLD by FMT experiments. The first FMT experiment (A-K) aimed to investigate the effect of E.faecalis on MAFLD in mice (n = 6). (a) Body weight of mice. (b) Representative anatomical drawing. (c) Liver weight of mice. (f) Representative liver sections by hematoxylin – eosin (H&E) staining with original magnification × 100 (left) and × 400 (right). (e) TNF-α, (f) IL-1β, and (g) CPT1α in serum. (h) Representative CPT1α blots in liver. (i) NEFA, (j) TG, and (K) carnitine in 10% liver homogenate. The second FMT experiment (L-W) aimed to know the effect of PCA on E. faecalis associated MAFLD (n = 8). (l) Growth curves of mice body weight. (m)Intraperitoneal fat rate. (n) Liver weight. (o) HOMA-IR index based on serum levels of glucose and insulin. (p) Representative sections of intraperitoneal fat by hematoxylin-eosin (H&E) staining with original magnification × 100 (left) and × 400 (right). (q) Representative liver sections by hematoxylin-eosin (h and e) staining with original magnification × 100 (left) and × 400 (right). (r) Representative liver sections by oil red staining (original magnification × 100). (S) IL-1β and (t) CPT1α in serum. (u) Representative CPT1α blots in liver. (v) NEFA and (W) TG in 10% liver homogenate. Data represent as mean ± SD, and significant correlations were marked by *P < 0.05, **P < 0.01, ***P < 0.001, or bars with different letters differ significantly (P < 0.05). Hepatic histopathological scores were evaluated based on NAFLD Activity Score (NAS). The score ranging from 0 to 8 is defined as the unweighted sum of the scores for steatosis (0–3), lobular inflammation (0–3) and ballooning (0–2). CPT1α, carnitine palmitoyltransferase-1 alpha; E. faecalis, Enterococcus faecalis; FMT, fecal microbiota transplantation; HOMA-IR, homeostasis model assessment-estimated insulin resistance; LFD, low-fat diet; NEFA, non-esterified free fatty acids; PCA, protocatechuic acid; rE.faecalis, recipient mice of E. faecalis; rLFD, recipient mice of LFD; TG, triglyceride.

 

 

Figure 5. PCA ameliorated insulin resistance induced by E. faecalis in MAFLD mice. (a) Differential genes expressed between CTL and E.faecalis group. (b) Differential genes expressed between E. faecalis group and E. faecalis + PCA group. (c) Heatmap involving in CTL, PCA group, E. faecalis group and E. faecalis + PCA group. (d) Expression of Fgf1. (E) Expression of Igfbp2. (f) Expression of Irs1. (g) Expression of Irs2. Data represent as mean ± SD (n = 3), and significant correlations were marked by *P < 0.05. CTL, control group; E, E. faecalis group; E_PCA, E. faecalis + PCA group; Fgf1, fibroblast growth factor 1; Igfbp2, insulin-like growth factor binding protein 2; Irs1, insulin receptor substrate 1; Irs2, insulin receptor substrate 2; PCA, protocatechuic acid.

 

原文链接

 

https://doi.org/10.1080/19490976.2023.2238959

通讯作者简介

伍树松，博士，教授，博士生导师，动物营养与饲料科学方向，湖南省“百人计划”青年学者，湖湘高层次人才聚集工程创新人才。主要研究方向为动物营养代谢调控及天然活性产物的应用，在植物多酚的生物活性尤其是抗氧化、抗炎、抑菌及调控脂肪代谢等方面的作用及调控机制有一定的研究基础，以第一或通讯作者发表相关学术论文20余篇，先后兼职日本国立鹿儿岛大学特别研究员、国际食品与健康学会组委会成员、日本农芸化学学会会员、日本食品因子学会会员、美国生物化学与分子协会会员，以及《Molecular Nutrition & Food Research》、《Food & Function》、《Animal Nutrition》等杂志审稿人。