State Key Laboratory of Southwestern Chinese Medicine Resources, School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China.
State Key Laboratory of Southwestern Chinese Medicine Resources, School of Ethnic Medicine, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China.
Phytomedicine. 2022 Dec;107:154462. doi: 10.1016/j.phymed.2022.154462. Epub 2022 Sep 17.
BACKGROUND: Rheumatoid arthritis (RA) is a systemic autoimmune disease that often results in joint destruction. Ershiwuwei Lvxue Pill (ELP), a prescription of Tibetan medicine, has been used for centuries for the clinical treatment of RA in Tibet, China. In a previous study, we reported that ELP could ameliorate RA symptoms in CIA rats by inhibiting the inflammatory response and inducing apoptosis in synovial tissues. It is still needed further to clarify the mechanisms of action of ELP in mitigating RA. PURPOSE: In this study, we aim to elucidate the mechanism of action of ELP to improve RA joint damage and explore the changes in the intestinal flora and host metabolites. METHODS: Firstly, we analyzed the main absorbed constituents of ELP in the serum of rats by ultra-performance liquid chromatography quadrupole-time-flight mass spectrometry (UPLC-Q-TOF/MS). Then, we verified the alleviating effects of ELP on cartilage injury and bone erosion as well as the inflammatory response in CIA rats by microCT, H&E staining, safranin-O staining, and ELISA. Moreover, we investigated the main factors that mediate joint damage, including the production of matrix metalloproteinases (MMPs) and osteoclast activity in the ankle of rats by immunohistochemistry and tartrate-resistant acid phosphatase (TRAP) staining. Further, we explored the molecular mechanisms of the MMPs production and osteoclast activity in CIA rats treated with ELP through various experiments such as ELISA, qRT-PCR, western blotting, and immunofluorescence assay. Besides, we investigated gut microbiota composition by 16S rDNA sequencing and serum metabolites through untargeted metabolomics. In addition, we analyzed the correlation between gut microbiota and metabolites by Spearman correlation analysis. RESULTS: In this study, we identified 20 compounds from rat serum samples, which could be the ELP components that improve RA. Moreover, we found that ELP could alleviate cartilage and bone injury by reducing MMP-1, MMP-3, and MMP-13 expression and osteoclast activity in CIA rats. Further studies demonstrated that ELP could reduce joint damage by inhibiting osteoprotegerin (OPG)/receptor activator for nuclear factor-κB ligand (RANKL) /nuclear factor-κB (NF-κB) and extracellular signal-regulated kinase (ERK)/c-Jun N-terminal kinases (JNK) signal pathways. The 16S rDNA sequencing analysis indicated that there was a significant difference in the gut microbiota composition between the normal and CIA rats, and these differences were changed after ELP administration. ELP could alter the gut microbiota by increasing the abundance of the genus Lactobacillus and decreasing the abundance of Dorea, [Eubacterium]_ventriosum_group, Anaerostipes, Collinsella, Coprococcus_1, Ruminiclostridium_5, Ruminococcus_1, Family_XIII_UCG-001, Butyricicoccus, Erysipelotrichaceae_UCG-003, Lachnoclostridium, Faecalibacterium, Lachnospiraceae_UCG-010, Roseburia, Rs-E47_termite_group_norank, Treponema_2 genera. Non-targeted metabolomics analysis showed that ELP reduced arachidonic acid levels. The serum arachidonic acid level was significantly correlated with the abundance of 41 genera, particularly Collinsella and Lactobacillus. CONCLUSION: Our study shows that ELP can improve RA joint damage by inhibiting MMPs production and osteoclast activity, and regulating intestinal flora and host metabolites, which provides a novel insight into the ELP in alleviating RA.
背景：类风湿性关节炎（RA）是一种系统性自身免疫性疾病，常导致关节破坏。二十味乳鹏丸（ELP）是一种藏药方剂，在中国西藏地区已使用数百年治疗 RA。在之前的研究中，我们报道 ELP 可以通过抑制炎症反应和诱导滑膜组织细胞凋亡来改善 CIA 大鼠的 RA 症状。仍需要进一步阐明 ELP 减轻 RA 的作用机制。
目的：本研究旨在阐明 ELP 改善 RA 关节损伤的作用机制，并探讨肠道菌群和宿主代谢物的变化。
方法：首先，我们通过超高效液相色谱-四极杆飞行时间质谱联用技术（UPLC-Q-TOF/MS）分析 ELP 在大鼠血清中的主要吸收成分。然后，我们通过 microCT、H&E 染色、番红 O 染色和 ELISA 验证了 ELP 对 CIA 大鼠软骨损伤和骨侵蚀以及炎症反应的缓解作用。此外，我们通过免疫组化和抗酒石酸酸性磷酸酶（TRAP）染色研究了介导关节损伤的主要因素，包括基质金属蛋白酶（MMPs）的产生和破骨细胞活性。进一步，我们通过 ELISA、qRT-PCR、western blotting 和免疫荧光测定等多种实验研究了 ELP 治疗 CIA 大鼠 MMPs 产生和破骨细胞活性的分子机制。此外，我们通过 16S rDNA 测序和非靶向代谢组学研究了肠道微生物群落组成和血清代谢物。此外，我们通过 Spearman 相关性分析分析了肠道微生物群和代谢物之间的相关性。
结果：本研究从大鼠血清样本中鉴定出 20 种化合物，这些化合物可能是改善 RA 的 ELP 成分。此外，我们发现 ELP 可以通过降低 CIA 大鼠中 MMP-1、MMP-3 和 MMP-13 的表达和破骨细胞活性来减轻软骨和骨损伤。进一步的研究表明，ELP 通过抑制核因子-κB（NF-κB）信号通路中的骨保护素（OPG）/核因子κB 受体激活剂配体（RANKL）/核因子-κB（NF-κB）和细胞外信号调节激酶（ERK）/c-Jun N-末端激酶（JNK）信号通路来减轻关节损伤。16S rDNA 测序分析表明，正常和 CIA 大鼠之间的肠道微生物群落组成存在显著差异，而 ELP 给药后这些差异发生了变化。ELP 通过增加乳杆菌属的丰度和降低 Dorea、[Eubacterium]ventriosum_group、Anaerostipes、Collinsella、Coprococcus_1、Ruminiclostridium_5、Ruminococcus_1、Family_XIII_UCG-001、Butyricicoccus、Erysipelotrichaceae_UCG-003、Lachnoclostridium、Faecalibacterium、Lachnospiraceae_UCG-010、Roseburia、Rs-E47 termite_group_norank、Treponema_2 属的丰度来改变肠道微生物群。非靶向代谢组学分析表明，ELP 降低了花生四烯酸水平。血清花生四烯酸水平与 41 个属的丰度显著相关，特别是 Collinsella 和 Lactobacillus。
结论：本研究表明，ELP 通过抑制 MMPs 的产生和破骨细胞活性以及调节肠道菌群和宿主代谢物来改善 RA 关节损伤，为 ELP 缓解 RA 提供了新的见解。
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