基于肠道微生态探讨运脾解毒通络祛湿方对胶原诱导性关节炎小鼠的影响

Effects of Yunpi Jiedu Tongluo Qushi Decoction by regulating gut microbiome in collagen-induced arthritis mice

目的:探讨运脾解毒通络祛湿方通过调控肠道菌群对胶原诱导性关节炎(CIA)小鼠的治疗作用。方法:32只雄性DBA/1小鼠,随机分为空白对照组、CIA模型组、运脾解毒通络祛湿方组、甲氨蝶呤组,CIA模型组及药物组均注射牛Ⅱ型胶原制备CIA模型。药物组连续7周接受相应药物治疗,检测各组小鼠踝关节厚度、关节炎指数及血清抗CCP抗体含量,HE染色评估关节病理改变,采用悬浮芯片系统检测血清白细胞介素(IL)-6、IL-17、肿瘤坏死因子α(TNF-α)的含量,运用Illumina MiSeq高通量测序技术检测肠道菌群的改变。结果:与CIA模型组比较,运脾解毒通络祛湿方组小鼠踝关节厚度、关节炎指数评分、血清抗CCP抗体及炎症因子IL-6、IL-7含量显著降低(P<0.05,P<0.01),HE染色结果显示小鼠关节炎性细胞浸润、骨质破坏显著改善,菌群测序结果显示中药组的肠道微生物多样性明显恢复,且在属水平下调了Lactobacillus的丰度并上调了Lachnospiraceae_NK4A136_group的丰度,相关性分析显示此二种菌属与CIA疾病活动性存在高度关联。结论:运脾解毒通络祛湿方可以有效缓解CIA小鼠免疫炎症反应,改善关节病理损伤,作用机制可能与调节肠道菌群,恢复肠道微环境稳态有关。

Objective:To explore the curative effect of Yunpi Jiedu Tongluo Qushi Decoction on collagen-induced arthritis(CIA)mice by regulating the intestinal microbiome.Methods:A total of 32 DBA/1 male mice were randomly divided into four groups:control group,CIA group,Yunpi Jiedu Tongluo Qushi decoction group,and methotrexate group.CIA model was prepared by injection of bovine type II collagen.Each group was received corresponding drugs for 7 weeks.The thickness of ankle,the arthritis index and serum anti-CCP antibody were evaluated.Hematoxylin-eosin staining was performed to observe the joint inflammation of the mice.The levels of serum IL-6,IL-17,TNF-αin each group were measured by Bio-Plex system.Illumina MiSeq high-throughput sequencing technology was used to investigate the alterations of gut microbiota.Results:Compared with the CIA group,the thickness of ankle,the arthritis index and the content of serum anti-CCP and inflammatory cytokines IL-6,IL-7 in the Yunpi Jiedu Tongluo Qushi decoction group were significantly reduced(P<0.05,P<0.01).Hematoxylin-eosin staining analysis showed that the joint pathological damage were significantly reduced.Illumina MiSeq high-throughput gene sequencing revealed that the diversity of the intestinal flora of the Yunpi Jiedu Tongluo Qushi decoction group was effectively restored and the relative abundance of Lactobacillus was suppressed while the abundance of Lachnospiraceae_NK4A136_group was increased.Spearman correlation analysis demonstrated that Lactobacillus and Lachnospiraceae_NK4A136_group were highly correlated with CIA activity.Conclusion:Yunpi Jiedu Tongluo Qushi decoction can effectively alleviate the immune inflammatory response of CIA mice and reduce the pathological damage of joints by regulating the structure and function of the intestinal flora and restoring homeostasisoftheintestinal microenvironment.