甘草养阴汤联合甲氨蝶呤干预老年类风湿关节炎小鼠价值及基于DIA蛋白组学分析的效应机制

Value of Gancao Nurish-yin decoction combined with MTX for the intervention of aging collagen-induced arthritis mouse models and its effect mechanism based on DIA proteomic analysis

目的 探讨老年类风湿关节炎(ERA)小鼠的分泌表型特点及甘草养阴汤联合甲氨蝶呤对ERA小鼠的干预作用和整体效应机制。方法 DBA/1雄性小鼠共72只,随机取10只作为正常组;另取12只建立胶原诱导关节炎(CIA)模型,作为RA组;剩余50只小鼠在CIA模型基础上加D-半乳糖刺激建立ERA模型,将造模成功小鼠随机分为ERA组、甲氨蝶呤组、甘草养阴汤组、甲氨蝶呤+甘草养阴汤组。正常组、RA组、ERA组小鼠给予生理盐水灌胃,每日1次;甲氨蝶呤组给予甲氨蝶呤1 mg/kg灌胃,每周2次;甘草养阴汤组给予甘草养阴汤3.0 g/kg灌胃,每日1次;甲氨蝶呤+甘草养阴汤组给予甲氨蝶呤和甘草养阴汤灌胃,药物用量及方法同甲氨蝶呤组和甘草养阴汤组。各组均灌胃4周。对各组小鼠后肢关节炎病变进行评分,HE染色观察各组踝关节组织水肿、滑膜增生、软骨/骨侵蚀、血管翳形成、脂肪沉积情况并进行评分,采用非依赖采集(DIA)蛋白组学技术分析各组小鼠的分泌表型特点。结果 ERA组后肢肿胀更明显,关节炎评分及肢关节水肿、滑膜增生、软骨/骨侵蚀评分均明显高于RA组(P均<0.05),血管翳形成及脂肪沉积评分与RA组比较差异均无统计学意义(P均>0.05);甘草养阴汤组、甲氨蝶呤+甘草养阴汤组关节炎评分及关节水肿、软骨/骨侵蚀、脂肪沉积评分均明显低于ERA组(P均<0.05),甲氨蝶呤组关节炎评分及关节水肿、滑膜增生、软骨/骨侵蚀、血管翳形成、脂肪沉积评分与ERA组比较差异均无统计学意义(P均>0.05)。ERA组与RA组比较,上调71个蛋白,下调70个蛋白,主要富集于中性粒细胞胞外陷阱形成、脂质和动脉粥样硬化、鞘脂信号通路等。甲氨蝶呤+甘草养阴汤除了对多种炎症因子的抑制作用,还倾向于对脂质代谢等通路靶点的调节,尤其是上调叶酸合成通路相关靶点。结论 ERA小鼠的代谢改变可能导致更为严重的关节损伤,甘草养阴汤对甲氨蝶呤治疗ERA具有减毒增效作用。

Objective It is to investigate the secretory phenotypic characteristics of elderly rheumatoid arthritis(ERA)mice and the interventional effects and overall effect mechanism of Gancao nurishing-yin decoction(GCNY)combined with methotrexate(MTX)on them.Methods A total of 72 DBA/1 male mice were selected,in which 10 ones were randomly selected as the normal group,another 12 mice were selected to establish the collagen-induced arthritis(CIA)models as the RA group,the remaining 50 mice were stimulated with D-galactose on the basis of the CIA model to establish the ERA models,and the successfully modeled mice were randomly divided into the ERA group,MTX group,GCNY group,and MTX+GCNY group.The mice in the normal group,RA group and ERA group were given normal saline by gavage once per day,the MTX group was given MTX 1 mg/kg by gavage twice per week,the GCNY group was given GCNY 3.0 g/kg by gavage once per day,the MTX+GCNY group was given MTX combined with GCNY by gavage,and the dosage and administration were the same as those of the MTX group and GCNY group.All the groups were gavaged for 4 weeks.Arthritic lesions in the hind limbs of the mice in each group were scored,and their tissue edema,synovial hyperplasia,cartilage/bone erosion,pannus formation,and fat deposition of the ankle joint were observed by HE staining and scored,and their secretory phenotypic characteristics were analyzed by data independent acquisition(DIA)proteomics techniques.Results In the ERA group,the swelling of the hind limbs was more obvious,and the arthritis scores and scores of joint edema,synovial hyperplasia,and cartilage/bone erosion were significantly higher than those in the RA group(all P<0.05),while the scores of pannus formation and fat deposition were not statistically significant compared with those in the RA group(all P>0.05);the arthritis scores,and scores of joint edema,cartilage/bone erosion,and fat deposition in the GCNY group and MTX+GCNY group were significantly lower than those in the ERA group(all P<0.05),and the arthritis scores and scores of joint edema,synovial hyperplasia,cartilage/bone erosion and fat deposition in the MTX group were not statistically significant compared with those in the ERA group(all P>0.05).71 proteins were up-regulated and 70 proteins were down-regulated in the ERA group compared with the RA group,mainly enriched in neutrophil extracellular trap formation,lipid and atherosclerosis,and sphingolipid signaling pathways.The combination of MTX and GCNY not only inhibited various inflammatory factors,but also tended to regulate targets of lipid metabolism and other pathways,especially upregulated the targets related to folate synthesis pathway.Conclusion Metabolic changes in ERA mice may lead to more severe joint damage,and GCNY has a detoxifying and synergistic effect in the treatment of ERA with MTX.