白藜芦醇通过重塑肠道菌群改善类风湿性关节炎的作用机制研究

Mechanism of Resveratrol Improving Rheumatoid Arthritis by Remodeling Intestinal Flora

目的:探讨白藜芦醇对类风湿性关节炎的影响,研究肠道菌群的重塑及其作用机制。方法:选取雄性DBA/1小鼠作为实验对象,采用尾根部皮下注射Ⅱ型胶原与弗氏不完全佐剂混合物的方法诱导小鼠(CIA)模型。将诱导成功的小鼠随机分为CIA模型组、CIA+甲氨蝶呤组、CIA+白藜芦醇低剂量干预组及CIA+白藜芦醇高剂量干预组,每组10只。造模第21天灌胃给予不同剂量白藜芦醇[2、0.5 g/(kg·d)],对照组给予等量0.9%氯化钠溶液,甲氨蝶呤组给予0.5 g/(kg·d),连续21 d。采用关节炎分数法对小鼠关节炎症程度进行评分,通过苏木精-伊红染色法(HE)染色评价关节破坏程度,观察各组小鼠关节组织病理变化情况;提取小鼠血清采用酶联免疫吸附法(ELISA)检测肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)及白细胞介素-1β(IL-1β);使用流式细胞术检测免疫小鼠第42天脾脏Treg细胞(Treg),辅助性T细胞17(Th17)和CD8+T细胞的变化情况;提取免疫第42天小鼠粪便样品中微生物基因组DNA,采用实时PCR(RT-PCR)定量分析拟杆菌、乳杆菌、双歧杆菌以及放线菌的丰度;分析各组小鼠肠道菌群结构差异与T细胞的相关性,阐明肠道菌群对Th17/Treg平衡关系的调控。结果:与CIA模型组比较,CIA+白藜芦醇高、低剂量组均可以降低CIA小鼠血清中炎症因子TNF-α、IL-6及IL-1β的表达(P<0.01),高剂量组更显著。病理检测表明,高剂量白藜芦醇可以显著改善CIA小鼠的关节病变。RT-PCR检测表明,白藜芦醇可促进拟杆菌和双歧杆菌的生长。流式细胞术及相关性分析等方法明确了重塑肠道菌群可恢复CIA小鼠Th17/Treg细胞之间平衡。结论:白藜芦醇可通过增加拟杆菌、乳杆菌和双歧杆菌的丰度有效改善CIA模型小鼠相关症状。

Objective:To investigate the effect of resveratrol on rheumatoid arthritis and study the remodeling of intestinal flora and its mechanism.Methods:DBA/1 male mice were selected as the experimental object.The mouse model of rheumatoid arthritis(collagen induced arthritis,CIA)was induced by subcutaneous injection of the mixture of type II collagen and Freund’s incomplete adjuvant at the tail root.The successfully induced mice were randomly divided into CIA model group,CIA+methotrexate group,CIA+resveratrol low-dose intervention group and CIA+resveratrol high-dose intervention group,with 10 mice in each group.On the 21 stday of modeling,different doses of resveratrol[2,0.5 g/(kg·d)]were given by gavage,the control group was given the same amount of 0.9%sodium chloride solution,and the methotrexate group was given 0.5 g/(kg·d)for 21 days.The degree of joint inflammation in mice was scored by arthritis index scoring method,the degree of joint damage was evaluated by hematoxylin eosin staining(HE),and the pathological changes of joint tissues in each group were observed.Mouse serum was extracted and tumor necrosis factor-α(TNF-α)was detected by enzyme-linked immunosorbent assay(ELISA)、interleukin-6(IL-6)and interleukin-1β(IL-1β).Flow cytometry was used to detect spleen Treg cells(Treg)and helper T cells 17(Th17)on day 42 of immunized mice and CD8+T cells;the microbial genomic DNA was extracted from the fecal samples of mice on the 42;day of immunization,and the abundance of Bacteroides,Lactobacillus,Bifidobacterium and Actinomycetes was quantitatively analyzed by real time PCR(RT-PCR);the correlation between the structural difference of intestinal flora and T cells was analyzed,and the regulation of intestinal flora on Th17/Treg balance was clarified.Results:Compared with CIA model group,CIA+resveratrol high and low dose groups can reduce the expression of serum inflammatory factor TNF-α、IL-6 and IL-1βin CIA mice(P<0.01),the high-dose group was more significant.Pathological examination showed that high-dose resveratrol could significantly improve the joint lesions of CIA mice.RT-PCR showed that resveratrol could promote the growth of Bacteroides and Bifidobacteria.Flow cytometry and correlation analysis confirmed that resveratrol could reshape the intestinal flora and restore the balance between Th17/Treg cells of CIA mice.Conclusion:Resveratrol can increase the growth of Bacteroides,Lactobacillus and Bifidobacterium can effectively improve the related symptoms of CIA model mice.