基于NLRP3炎癥小體活化探討“三色散”揮發油減輕膝骨關節炎滑膜炎癥的機制

廖太陽 張力 張皞晟 李曉辰 吳鵬 王培民

中圖分類號 R285 文獻標志碼 A 文章編號 1001-0408（2021）19-2335-07

DOI 10.6039/j.issn.1001-0408.2021.19.05

摘 要 目的：基于NOD樣受體家族3（NLRP3）炎癥小體的活化探討“三色散”揮發油減輕膝骨關節炎（KOA）滑膜炎癥的機制。方法：采取水蒸氣蒸餾法提取“三色散”揮發油，采用氣質聯用（GC-MS）技術分析其組成。提取雄性SD大鼠膝關節成纖維樣滑膜細胞（FLS），采用CCK-8法檢測10、25、50、100、250、500、1 000 μg/mL“三色散”揮發油對FLS存活率的影響;以脂多糖誘導12 h建立KOA炎癥細胞模型，檢測10、250 μg/mL“三色散”揮發油對炎癥模型細胞中NLRP3、胱天蛋白酶1（caspase-1）、凋亡相關斑點樣蛋白（ASC）的蛋白及其mRNA相對表達量的影響，檢測炎癥模型細胞上清液中白細胞介素1β（IL-1β）、IL-18水平。結果：提取得到黃色澄清且有獨特芳香氣味的“三色散”揮發油0.51～0.61 g，提取率為0.33%～0.41%（n=9）。從該揮發油中共分離得到化學成分41種，鑒定了其中的30種，其峰面積之和占總峰面積的90.073 6%;相對含量較高的成分依次為芳姜黃酮（17.573 9%）、δ-杜松烯（15.434 5%）、姜黃酮（11.509 5%）等。當“三色散”揮發油質量濃度為10～250 μg/mL時，其對FLS存活率均無顯著影響（P>0.05）。與空白組比較，模型組細胞中NLRP3、caspase-1、ASC的蛋白及其mRNA相對表達量以及細胞上清液中IL-1β、IL-18水平均顯著升高（P<0.05）;與模型組比較，“三色散”揮發油10、250 μg/mL組細胞及上清液中上述指標的相對表達量或水平均顯著降低（P<0.05）。結論：“三色散”揮發油可能通過抑制FLS中NLRP3炎癥小體的活化而減少下游炎癥級聯反應，從而發揮其改善KOA滑膜炎癥的作用。

關鍵詞 膝骨關節炎;滑膜炎癥;NOD樣受體家族3炎癥小體;三色散;揮發油;成纖維樣滑膜細胞

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.