基于16S rDNA测序研究当归拈痛汤对风湿热痹佐剂性关节炎大鼠肠道菌群的影响

Effect of Danggui Niantongtang on Intestinal Flora in Adjuvant-induced Arthritis Rats of Wind-dampness-heat Arthralgia:Based on 16S rDNA Sequencing

目的:从肠道菌群变化研究当归拈痛汤治疗风湿热痹佐剂性关节炎(AA)大鼠的作用机制。方法:将60只SD大鼠随机分为6组:正常组、风湿热痹模型组(FSR组)、当归拈痛汤(DGNTT)低、中、高剂量组(5.67、11.34、22.68 g·kg^(-1)),甲氨蝶呤组(MTX,1.35 mg·kg^(-1)),每组10只,除正常组外,其余各组均注射灭活结核分支杆菌(Mtb)佐剂进行免疫,置于风湿热环境的人工气候箱中进行64 h干预造模。初次免疫当天起,给予大鼠灌胃给药,连续灌胃28 d。对大鼠进行关节炎指数(AI)检测,使用足趾容积测量仪检测大鼠足趾容积变化及苏木素-伊红(HE)染色观察膝关节滑膜组织形态改变,并且采用16S rDNA高通量基因测序法分析给药治疗前后大鼠的肠道菌群变化。结果:当归拈痛汤能够减轻风湿热痹证AA大鼠滑膜组织增生和炎症程度及血管翳形成,并且16S rDNA测序结果显示,与正常组比较,FSR组厚壁菌门、乳酸杆菌属、普雷沃氏菌属9、拟普雷沃氏菌属的相对丰度明显降低(P<0.05,P<0.01),拟杆菌门、拟杆菌属的相对丰度显著升高(P<0.01);与FSR组比较,当归拈痛汤各剂量组和MTX组中乳酸杆菌属均明显升高(P<0.05,P<0.01),拟杆菌门显著降低(P<0.01),当归拈痛汤中、高剂量组和MTX组中厚壁菌门、普雷沃氏菌属9、拟普雷沃氏菌属显著增加,拟杆菌属明显降低,差异均具有统计学意义(P<0.05,P<0.01)。Spearman相关性分析显示,拟杆菌属、螺杆菌属与AI呈正相关(P<0.05),普雷沃氏菌属9、候选单胞生糖菌属与AI呈负相关(P<0.05,P<0.01);普雷沃氏菌属9与足趾容积呈负相关(P<0.01);瘤胃球菌科NK4A214群、克里斯滕森菌科R-7、拟杆菌属与脾脏指数呈正相关(P<0.05),普雷沃氏菌属9与脾脏指数呈负相关(P<0.01)。结论:当归拈痛汤治疗风湿热痹类风湿关节炎具有明显疗效,能够改善AA大鼠菌群结构,提升益生菌的丰度,抑制致病菌生长,促使肠道菌群恢复至正常水平,其作用机制可能与改善肠道免疫代谢功能,维持肠道稳态相关。

Objective:To explore the mechanism of Danggui Niantongtang(DGNTT)against adjuvant-induced arthritis(AA)in rats with wind-dampness-heat arthralgia(FSR)based on the variation of intestinal flora.Method:A total of 60 SD rats were randomized into normal(control)group,FSR group,low-,medium-,and high-dose DGNTT(5.67,11.34,22.68 g·kg^(-1))groups,and methotrexate(MTX)group(1.35 mg·kg^(-1)),with 10 rats in each group.The rats,except the control group,were injected with Mtb adjuvant and then exposed to artificial climatic chamber(hot and humid with wind)for 64 h for modeling.The rats were treated with water,DGNTT or MTX for 28 days from the day of injection.Arthritis index(AI)of rats was measured and paw volume was determined with a volume meter.The morphology of synovial tissues of the knees was observed based on hematoxylin-eosin(HE)staining and the changes of intestinal flora were analyzed based on 16S rRNA sequencing.Result:DGNTT can alleviate the hyperplasia of synovial tissue and inflammation of AA rats with FSR and inhibit the formation of pannus.The results of 16S rRNA sequencing showed that the relative abundance of Firmicutes,Lactobacillus,Prevotella 9,and Alloprevotella decreased(P<0.05,P<0.01)and the relative abundance of Bacteroidetes and Bacteroides increased(P<0.01)in FSR group compared those in the control group.Compared with the FSR group,all DGNTT groups and MTX group had high relative abundance of Lactobacillus(P<0.05,P<0.01)and low relative abundance of Bacteroidetes(P<0.01)and medium-dose and high-dose DGNTT groups and MTX group showed high abundance of Firmicutes,Prevotella 9,and Alloprevotella and low abundance of Bacteroides(P<0.05,P<0.01).Spearman's correlation analysis suggested that the abundance of Bacteroides and Helicobacter was in positive correlation with AI(P<0.05),while the abundance of Prevotella 9 and Candidatus Saccharimonas was in negative correlation with AI(P<0.01,P<0.05).There was a negative correlation between the abundance of Prevotella 9 and paw volume(P<0.01),and the abundance of Ruminococcaceae NK4A214 group,Christensenellaceae R-7 group,and Bacteroides was in negative correlation with spleen index(P<0.05).The abundance of Prevotella 9 was in negative correlation with spleen index(P<0.01).Conclusion:DGNTT is effective for arthritis with FSR,as it can regulate the composition of intestinal flora in AA rats by increasing the abundance of probiotics and inhibiting the growth of pathogenic bacteria.The mechanism is the likelihood that it improves intestinal immune metabolism to ensure intestinal homeostasis.