前言:目的:观察苦参石油醚提取物(PEEP)对类风湿关节炎(RA)的治疗作用。方法:体外:采用细胞计数试剂盒-8(CCK-8)检测细胞活性,筛选最佳提取物浓度;采用一氧化氮(NO)比色法和Q-PCR法筛选有效位点;采用Westernblot检测RAW264.7细胞中p38、p-p38、p-MK2、TTP(TTP)的表达。体内:采用完全弗氏佐剂(CFA)建立大鼠模型。观察不同剂量PEEP对CFA大鼠的生活状态,爪子肿胀,脾脏指数,X光片,苏木精伊红(HE)染色;肿瘤坏死因子α(TNF-α)的分泌,采用酶联免疫吸附试验(ELISA)检测白细胞介素-6(IL-6)和前列腺素E2(PGE2);采用Westernblot检测CFA大鼠踝关节p38、p-p38、p-MK2和TTP的表达。结果:体外:PEEP,aculeate米勒的乙酸乙酯提取物(EEEP),敏化丙酸杆菌的正丁醇提取物(BEEP)对RAW264.7巨噬细胞没有毒性作用。PEEP,EEEP,和BEEP减少脂多糖(LPS)诱导的RAW264.7细胞中NO的分泌,只有PEEP能明显抑制炎症因子TNF-α和IL-6的mRNA表达水平;PEEP依赖性地减少LPS诱导的RAW264.7细胞中TNF-α和IL-6的分泌,降低p-p38和p-MK2的表达以及TTP磷酸化水平。体内:PEEP改善CFA大鼠的生活条件,减少足部肿胀,脾脏指数,骨表面侵蚀和关节间隙变窄;减少滑膜细胞的形成,足部和踝关节中的炎症细胞和血管造影。PEEP减少TNF-α的分泌,大鼠血清IL-6、PGE2,下调踝关节中p-p38和p-MK2的表达,减少TTP的磷酸化。结论:PEEP能改善CFA大鼠的生活状态,减少足部肿胀的程度,保护免疫器官,减少炎症细胞浸润,软骨损伤,血管nus形成,减少炎症和RA损伤。通过调控p38丝裂原活化蛋白激酶(p38/MAPK)的信号通路,减少TNF-α的释放,血清中的IL-6和PGE2。
Objective: To investigate the therapeutic effect of petroleum ether extract of P. aculeate Miller (PEEP) on rheumatoid arthritis (RA). Methods: In vitro: The Cell Counting Kit-8 (CCK-8) was used to detect cell activity and select the optimal concentration of the extract; the effective site was screened by nitric oxide (NO) colorimetric method and Q-PCR method; the expression of p38, p-p38, p-MK2, and Tristetraprolin (TTP) in RAW 264.7 cells were detected by Western blot. In vivo: The rat model was established by complete Freund\'s adjuvant (CFA). The different doses of PEEP on CFA rats were observed with life status, paw swelling, spleen index, X-ray, Hematoxylin eosin (HE) staining; the secretion of Tumor necrosis factor α (TNF-α), interleukin-6 (IL-6) and Prostaglandin E2 (PGE2) were detected by Enzyme linked immunosorbent assay (ELISA); the expressions of p38, p-p38, p-MK2, and TTP in the ankle joints of CFA rats were detected by Western blot. Result: In vitro: PEEP, Ethyl Acetate Extract of P. aculeate Miller (EEEP), N-butanol Extract of P. aculeate Miller (BEEP) have no toxic effects on RAW264.7 macrophages. PEEP, EEEP, and BEEP reduce the secretion of NO in RAW264.7 cells induced by lipopolysaccharide (LPS), only PEEP significantly inhibited the mRNA expression levels of inflammatory factors TNF-α and IL-6; PEEP-dependently reduce the secretion of TNF-α and IL-6, decrease the expression of p-p38 and p-MK2, and the level of TTP phosphorylation in LPS-induced RAW264.7 cells. In vivo: PEEP improve the living conditions of CFA rats, reduce foot swelling, spleen index, bone surface erosion and joint space narrowing; reduce the formation of synovial cells, inflammatory cells and pannus in the foot and ankle joints. PEEP reduce the secretion of TNF-α, IL-6, PGE2 in rat serum, downregulate the expression of p-p38 and p-MK2 in the ankle joint, and reduce the phosphorylation of TTP. Conclusion: PEEP improve the living conditions of CFA rats, reduce the degree of foot swelling, protect immune organs, reduce inflammatory cell infiltration, cartilage damage, pannus formation, reduce inflammation and RA damage. The mechanism through regulating the signal pathway of p38 mitogen-activated protein kinase (p38/MAPK), which reduces the release of TNF-α, IL-6, and PGE2 in the serum.