标题民族药理学相关性
类风湿性关节炎（RA）是一种慢性全身性自身免疫性疾病。二妙散（EMS）已被证明具有良好的抗炎作用，被广泛用于RA的临床治疗。然而，确切的机制尚不完全清楚。
研究目的
本研究旨在探讨含EMS血清影响巨噬细胞M1/M2极化并可能通过微小RNA（miRNA）-33/NLRP3通路介导，从而阐明EMS治疗RA的分子机制。
材料和方法
我们通过使用 CCK-8 测量筛选了安全浓度的含 EMS 血清。RAW264.7 细胞与脂多糖 (LPS) (100 ng/mL) 和干扰素-γ (20 ng/mL) 一起培养 24 小时以诱导 M1 型巨噬细胞。在最后 30 分钟内加入三磷酸腺苷 (ATP) (5 mM) 以激活 NLRP3。转染 miRNA-33 模拟物后，通过 RT-qPCR 检测 miR-33 的含量。Western blot检测NLRP3、ASC、caspase-1、诱导型一氧化氮合酶（iNOS）和精氨酸酶-1（Arg-1）的蛋白表达水平。ELISA法测定血清和细胞上清液中IL-1β、IL-10、TNF-α、TGF-β和IL-18的含量。免疫荧光法检测CD86和CD206的荧光强度。
结果
结果表明，含EMS血清可促进Arg-1的蛋白表达水平和TGF-β、IL-10的分泌水平，抑制iNOS、IL-1β和TNF-α的水平，调节pro的平衡。 -炎症因子和抗炎因子。RT-qPCR 结果表明，含 EMS 的血清可以降低 miRNA-33 的水平。EMS含血清可降低NLRP3炎症小体相关蛋白的表达，下调IL-1β和IL-18的表达水平。这些结果表明 EMS 通过 miRNA-33/NLRP3 途径对巨噬细胞极化产生影响。
结论
含 EMS 的血清通过下调 miRNA-33 抑制 NLRP3 炎性体的激活，从而阻止 M1 型巨噬细胞的极化。
"点击查看英文标题和摘要"
Exploring the effect of Er miao San-containing serum on macrophage polarization through miR-33/NLRP3 pathway
Headings ethnopharmacological relevance
Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease. Er miao San (EMS) has been shown to have good anti-inflammatory effects and is widely used in the clinical treatment of RA. However, the exact mechanism is not completely understood.
Aim of the study
The aim of this study was to explore that EMS-containing serum affects M1/M2 polarization of macrophages and may be mediated through the microRNA (miRNA)-33/NLRP3 pathway, thereby elucidating the molecular mechanism of EMS treatment of RA.
Materials and methods
We screened for safe concentrations of EMS-containing serum by using CCK-8 measurement. RAW264.7 cells were cultured with lipopolysaccharide (LPS) (100 ng/mL) and interferon-γ (20 ng/mL) for 24 h to induce M1-type macrophages. Adenosine triphosphate (ATP) (5 mM) was added in the last 30 min to activate NLRP3. The content of miR-33 was detected by RT‒qPCR after transfection of the miRNA-33 mimic. The protein expression levels of NLRP3, ASC, caspase-1, Inducible Nitric Oxide Synthase (iNOS) and Arginase-1 (Arg-1) were detected by Western blot. The contents of IL-1β, IL-10, TNF-α, TGF-β and IL-18 in serum and cell supernatant were determined by ELISA. The fluorescence intensity of CD86 and CD206 was detected by immunofluorescence.
Results
The results showed that EMS-containing serum promoted the protein expression level of Arg-1 and the secretion levels of TGF-β and IL-10, inhibited the levels of iNOS, IL-1β and TNF-α, and regulated the balance of pro-inflammatory factors and anti-inflammatory factors. RT‒qPCR results showed that EMS-containing serum could reduce the level of miRNA-33. EMS-containing serum could reduce the expression of NLRP3 inflammasome-related proteins and downregulate the expression levels of IL-1β and IL-18. These results suggest that EMS exerts its effect on macrophage polarization through the miRNA-33/NLRP3 pathway.
Conclusion
EMS-containing serum inhibits the activation of the NLRP3 inflammasome by downregulating miRNA-33, thus preventing the polarization of M1-type macrophages.
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