（一）文献基本信息
文献题目：Lycium barbarum polysaccharide modulates gut microbiota to alleviate rheumatoid arthritis in a rat model
研究人员：Lai W, Wang C, Lai R, et al.
研究单位：The First Clinical Medical College of Jinan University, Jinan University, Guangzhou, 510630, Guangdong, China.
发表时间：2022.06
期刊名称：npj Science of Food
影响因子：7.00
期刊分区：Q1
（二）核心亮点
1. LBP relieved the typical features of RA in rats, including the reduction of paw swell, arthritis scores and the contents of pro-inflammatory cytokines such as IL-1α, IL-1β, IL-12 and IL-17, and the recovery of the content of anti-inflammatory cytokine IL-10.
LBP 减轻了大鼠 RA 的典型特征，包括爪肿胀、关节炎评分和促炎细胞因子如 IL-1α、IL-1β、IL-12 和 IL-17 的含量减少，以及恢复了 抗炎细胞因子IL-10的含量。
2. LBP 改善了 RA 大鼠关节和软骨的病理表现。
LBP improved the pathological manifestations of joint and cartilage in RA rats.
3. 多组学分析还表明，LBP 重塑了肠道微生物结构，特别是调节了 Romboutsia、Lactobacillus、Dubosiella、Faecalibaculum、Lachnospiraceae_NK4A136_group 和 uncultured_bacterium_f_Ruminococcaceae 的相对丰度，并调节了结肠上皮中的基因表达，尤其是 Dpep3 的抑制表达 , Gstm6, Slc27a2, Col11a2, Sycp2, SNORA22, Tnni1, Gpnmb, Mypn 和 Acsl6 由于 DNA 超甲基化。
Multi-omics analysis also manifested that LBP reshaped the gut microbial structure, specifically the modulation of the relative abundance of Romboutsia, Lactobacillus, Dubosiella, Faecalibaculum, Lachnospiraceae_NK4A136_group and uncultured_bacterium_f_Ruminococcaceae, and regulated the gene expression in colonic epithelium, especially for the suppressed expression of Dpep3, Gstm6, Slc27a2, Col11a2, Sycp2, SNORA22, Tnni1, Gpnmb, Mypn and Acsl6 due to the DNA hypermethylation.
（三）思路与方法
1. II型胶原诱导的大鼠模型；
2. HE染色及病理学评价；
3. 血清中细胞因子含量的测定；
4. 16S rDNA测序和生物信息学分析；
5. 结肠上皮细胞转录组分析；
6. 结肠上皮细胞甲基化组分析；
7.结肠上皮组织和肠内容物中S-腺苷甲硫氨酸（SAM）含量的检测。
1. Type II collagen-induced rat model;
2. HE staining and pathological evaluation;
3. Determination of cytokine content in serum;
4. 16S rDNA sequencing and bioinformatics analysis;
5. Transcriptome analysis of colonic epithelial cells;
6. Methylome analysis of colonic epithelial cells;
7. Detection of S-adenosylmethionine (SAM) content in colonic epithelial tissue and intestinal contents.
（四）摘要
Abstract
Rheumatoid arthritis (RA) seriously impairs the quality of life of sufferers. It has been shown that Lycium barbarum polysaccharide (LBP), a natural active indigestible ingredient with medicinal and edible functions, can effectively relieve RA, however, whether this effect is related to gut microbiota is not known. This study aimed to explore the RA alleviating mechanism of LBP mediated by gut microbiota using a collagen-induced arthritis rat model. The results showed that LBP significantly changed the gut microflora structure accompanied with the RA alleviation. Specifically, a LBP intervention reduced the relative abundance of Lachnospiraceae_NK4A136_group and uncultured_bacterium_f_Ruminococcaceae and significantly increased the abundance of Romboutsia, Lactobacillus, Dubosiella and Faecalibaculum. The mRNA contents of several colonic epithelial genes including Dpep3, Gstm6, Slc27a2, Col11a2, Sycp2, SNORA22, Tnni1, Gpnmb, Mypn and Acsl6, which are potentially associated to RA, were down-regulated due to the DNA hypermethylation, possibly caused by the elevating content of a bacterial metabolite S-adenosyl methionine (SAM). In conclusion, our current study suggests that LBP alleviated RA by reshaping the composition of intestinal microflora which may generate SAM, inducing DNA hypermethylation of RA-related genes in the host intestinal epithelium and subsequently reducing their expression.
摘要
类风湿性关节炎 (RA) 严重损害患者的生活质量。研究表明，枸杞多糖（LBP）是一种具有药用和食用功能的天然难消化活性成分，可有效缓解 RA，但这种作用是否与肠道菌群有关尚不清楚。本研究旨在使用胶原诱导的关节炎大鼠模型探索肠道微生物群介导的 LBP 的 RA 缓解机制。结果表明，LBP 显著改变了肠道菌群结构，伴随着 RA 的缓解。具体来说，LBP 干预降低了Lachnospiraceae_NK4A136_group和 uncultured_bacterium_f_ Ruminococcaceae的相对丰度，并显着增加了Romboutsia、Lactobacillus、Dubosiella和Faecalibaculum。Dpep3 , Gstm6 , Slc27a2 , Col11a2 , Sycp2 , SNORA22 , Tnni1 , Gpnmb , Mypn和Acsl6几个结肠上皮基因的mRNA含量可能与 RA 相关，由于 DNA 高甲基化而下调，这可能是由细菌代谢物 S-腺苷甲硫氨酸 (SAM) 含量升高引起的。总之，我们目前的研究表明，LBP 通过重塑可能产生 SAM 的肠道微生物群落的组成、诱导宿主肠上皮细胞中 RA 相关基因的 DNA 高甲基化并随后降低其表达来减轻 RA。
（五）图表
Fig. 1: The severity of rheumatoid arthritis.a Joint swell of the left hind foot. b The diameter of paw swell in each group (mm). c The arthritis score of rats in each group. The error bars on the bar charts represented the standard deviations. The “*” indicated the P value was less than 0.05, “**” indicated the P value was less than 0.01, and “***” indicated the P value was less than 0.001. d Pathological sections of joint after HE staining in each group. Black arrow: diffusion or infiltration of inflammatory cells. Red circle: synovial hyperplasia and hypertrophy and formation of pannus. The magnification was 100×. The length of the scale bar was 250 μm.
图 1：类风湿性关节炎的严重程度。a 左后脚关节肿胀。b 每组爪肿胀的直径 (mm)。c 每组大鼠的关节炎评分。条形图上的误差条表示标准偏差。“*”表示 P 值小于 0.05，“**”表示 P 值小于 0.01，“***”表示 P 值小于 0.001。d 各组HE染色后关节病理切片。黑色箭头：炎症细胞扩散或浸润。红圈：滑膜增生肥大并形成血管翳。放大倍数为 100 倍。比例尺的长度为 250 μm。
Fig. 2: The contents of cytokines in the serum. a IL-1α, b IL-1β, c IL-12, d IL-17, and e IL-10. The error bars on the bar charts represented the standard deviations. The “*” indicated the P value was less than 0.05 while “**” indicated the P value was less than 0.01.
图2：血清中细胞因子的含量。a IL-1α、b IL-1β、c IL-12、d IL-17 和 e IL-10。条形图上的误差条表示标准偏差。“*”表示 P 值小于 0.05，而“**”表示 P 值小于 0.01。
Fig. 3: Metagenomics information of intestinal microflora among the Control, Model and Lyc group. a The Venn diagram on the OTU level among groups. b PLS-DA plot among groups. c The relative abundance of microflora on the phylum level and d on the genus level (n = 8 for Control and Model group, n = 7 for Lyc group).
图3：对照组、模型组和Lyc组肠道菌群宏基因组学信息。a 组间 OTU 级别的维恩图。b 各组之间的 PLS-DA 图。c 微生物群落在门水平上和 d 在属水平上的相对丰度（对照组和模型组的 n⟩=⟩8，Lyc 组的 n⟩=⟩7）。
Fig. 4: Correlation heatmap among the joint status, the contents of SAM and cytokines, the abundances of specific gut bacteria and the FPKM value of differentially expressed genes. Noted that, Gut bacterium 1 represents the uncultured_bacterium_f_Rumino-coccaceae; Gut bacterium 2 represents the Lachnospiraceae_NK4A136_group.
图4：关节状态、SAM和细胞因子含量、特定肠道细菌丰度和差异表达基因FPKM值之间的相关热图。注意，Gut bacterium 1代表uncultured_bacterium_f_Rumino-coccaceae；肠道细菌 2 代表 Lachnospiraceae_NK4A136_group。
Fig. 5: Transcriptomics and methylomics information between the Model and Lyc group. a Volcano plot of differentially expressed genes (DEGs) between groups. b The top 30 KEGG classes which DEGs were classified. Noted that, CP: Cellular Processes, EIP: Environmental Information Processing, GIP: Genetic Information Processing, OS: Organismal Systems. c The KEGG pathways which DEGs significantly enriched. d Distribution of differentially methylated regions (DMR) and e differentially methylation sites (DMS) on different gene elements. Noted that, different colors and numbers in circle represented different gene elements.
图 5：Model 和 Lyc 组之间的转录组学和甲基化组学信息。a 组间差异表达基因 (DEG) 的火山图。b DEGs 被分类的前 30 个 KEGG 类。注意到，CP：细胞过程，EIP：环境信息处理，GIP：遗传信息处理，OS：有机系统。c DEG 显着富集的 KEGG 通路。d 差异甲基化区域 (DMR) 和 e 差异甲基化位点 (DMS) 在不同基因元件上的分布。注意，圆圈中的不同颜色和数字代表不同的基因元件。
Fig. 6: The contents of S-adenosyl methionine (SAM) of each group. a SAM contents in colonic epithelial tissue and b intestinal contents. The error bars on the bar charts represented the standard deviations. The “*” indicated the P value was less than 0.05, “**” indicated the P value was less than 0.01, and “***” indicated the P value was less than 0.001.
图6：各组S-腺苷甲硫氨酸（SAM）含量。a 结肠上皮组织中的 SAM 含量和 b 肠内容物。条形图上的误差条表示标准偏差。“*”表示 P 值小于 0.05，“**”表示 P 值小于 0.01，“***”表示 P 值小于 0.001。
（六）不足
目前的研究缺乏对那些相对丰度增加的细菌产生 SAM 能力的直接证实，以及对几个基因在 RA 缓解过程中未定义功能的阐明。
The current study lacked the direct confirmation on the capability of SAM production by those bacteria with increased relative abundance, as well as the elucidation about the undefined function during RA alleviation of several genes.
（七）启发
多组学分析结合动物实验的研究方法对提高研究结果的可靠性具有重要意义。
【参考文献】
Lai W, Wang C, Lai R, et al. Lycium barbarum polysaccharide modulates gut microbiota to alleviate rheumatoid arthritis in a rat model[J]. npj Science of Food, 2022, 6(1): 1-10.
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