基于NLRP3炎症小体活化探讨“三色散”挥发油减轻膝骨关节炎滑膜炎症的机制

廖太阳 张力 张皞晟 李晓辰 吴鹏 王培民

中圖分类号 R285 文献标志码 A 文章编号 1001-0408（2021）19-2335-07

DOI 10.6039/j.issn.1001-0408.2021.19.05

摘 要 目的：基于NOD样受体家族3（NLRP3）炎症小体的活化探讨“三色散”挥发油减轻膝骨关节炎（KOA）滑膜炎症的机制。方法：采取水蒸气蒸馏法提取“三色散”挥发油，采用气质联用（GC-MS）技术分析其组成。提取雄性SD大鼠膝关节成纤维样滑膜细胞（FLS），采用CCK-8法检测10、25、50、100、250、500、1 000 μg/mL“三色散”挥发油对FLS存活率的影响;以脂多糖诱导12 h建立KOA炎症细胞模型，检测10、250 μg/mL“三色散”挥发油对炎症模型细胞中NLRP3、胱天蛋白酶1（caspase-1）、凋亡相关斑点样蛋白（ASC）的蛋白及其mRNA相对表达量的影响，检测炎症模型细胞上清液中白细胞介素1β（IL-1β）、IL-18水平。结果：提取得到黄色澄清且有独特芳香气味的“三色散”挥发油0.51～0.61 g，提取率为0.33%～0.41%（n=9）。从该挥发油中共分离得到化学成分41种，鉴定了其中的30种，其峰面积之和占总峰面积的90.073 6%;相对含量较高的成分依次为芳姜黄酮（17.573 9%）、δ-杜松烯（15.434 5%）、姜黄酮（11.509 5%）等。当“三色散”挥发油质量浓度为10～250 μg/mL时，其对FLS存活率均无显著影响（P>0.05）。与空白组比较，模型组细胞中NLRP3、caspase-1、ASC的蛋白及其mRNA相对表达量以及细胞上清液中IL-1β、IL-18水平均显著升高（P<0.05）;与模型组比较，“三色散”挥发油10、250 μg/mL组细胞及上清液中上述指标的相对表达量或水平均显著降低（P<0.05）。结论：“三色散”挥发油可能通过抑制FLS中NLRP3炎症小体的活化而减少下游炎症级联反应，从而发挥其改善KOA滑膜炎症的作用。

关键词 膝骨关节炎;滑膜炎症;NOD样受体家族3炎症小体;三色散;挥发油;成纤维样滑膜细胞

Discussion on the Mechanism of Synovitis of KOA Relieved by “Sanse Powder” Volatile Oil Based on the Activation of NLRP3 Inflammasome

LIAO Taiyang，ZHANG Li，ZHANG Haosheng，LI Xiaochen，WU Peng，WANG Peimin（Dept. of Orthopedics， the Affilliated Hospital of Nanjing University of Chinese Medicine/Jiangsu Province Hospital of Chinese Medicine，Nanjing 210029， China）

ABSTRACT   OBJECTIVE： To discuss the mechanism of synovitis of knee osteoarthritis （KOA） relieved by “Sanse powder” volatile oil based on the activation of Nod-like receptor family 3 （NLRP3） inflammasome. METHODS： “Sanse powder” volatile oil was extracted by distillation-condensation process and the components of it were analyzed by GC-MS. Fibroblast-like synovial cells （FLS） were extracted from the knee joint of male SD rats. The effects of 10， 25， 50， 100， 250， 500， 1 000 μg/mL “Sanse powder” volatile oil on the viability of FLS were examined by CCK-8 assay. KOA inflammatory cell model was induced by lipopolysaccharide for 12 h. The effects of 10， 250 μg/mL “Sanse powder” volatile oil on the protein and mRNA expression of NLRP3， caspase-1 and apoptosis-associated speck-like protein （ASC） were detected. The levels of IL-1β and IL-18 in supernatants of FLS were determined. RESULTS： Totally 0.51-0.61 g volatile oil with yellow clear and unique aromatic odor were extracted from “Sanse powder”， and the extraction rate was 0.33%-0.41% （n=9）. A total of 41 chemical components were isolated， and 30 of them were identified and their peak area accounted for 90.073 6% of the total peak area. The components with high relative content were gingerol flavonoids （17.573 9%）， δ-junionene （15.434 5%）， gingerol （11.509 5%）， etc. When the concentration of volatile oil was 10-250 μg/mL， there was no significant effect on survival rate of FLS （P>0.05）. Compared with blank group， the relative protein and mRNA expression of NLRP3， caspase-1 and ASC and the levels of IL-1β and IL-18 in supernatant were significantly increased in model group （P<0.05）.  Compared with model group， relative expression or levels of above indexes were all decreased significantly in FLS and supernatant of “Sanse powder” volatile oil 10 and 250 μg/mL groups （P<0.05）. CONCLUSIONS： “Sanse powder” volatile oil can inhibit NLRP3 inflammasome activation in FLS and reduce the downstream inflammatory cascade response， thus exerting its efficacy in ameliorating synovial inflammation of KOA.