Li Xiaobin, Wu Dengying, Hu Zhichao, Xuan Jiangwei, Ding Xiaoxia, Zheng Gang, Feng Zhenhua, Ni Wenfei, Wu Aimin
Department of Orthopaedics, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, China.
Zhejiang Provincial Key Laboratory of Orthpaedics, Wenzhou, China.
Cell Physiol Biochem. 2018;48(6):2583-2595. doi: 10.1159/000492701. Epub 2018 Aug 17.
BACKGROUND/AIMS: Osteoarthritis is a degenerative joint disease characterized by cartilage degeneration and a chondrocyte inflammatory response that induces an inflammatory environment closely linked to extracellular matrix (ECM) degradation. Ligustilide (LIG) is a major component of the herb Radix Angelicae Sinensis, with demonstrated anti-inflammatory effects. To confirm whether LIG has an equally inhibitory effect on inflammation in human osteoarthritis chondrocytes, we performed in vivo and in vitro experiments to validate the above conjectures and determine the relevant mechanisms.
Quantitative realtime PCR and western blotting were performed to evaluate the expression of MMP-3, MMP-13, ADAMTS-5, iNOS, and COX-2 at both gene and protein levels. An enzyme-linked immunosorbent assay was used to evaluate the levels of other inflammatory factors (PGE2, TNF-α, and IL-6). The PI3K/AKT and nuclear factor kappa B (NF-κB) signaling pathways were also analyzed by western blotting, whereas immunofluorescence was used to assess the expression of collagen II and aggrecan. The in vitro effect of LIG was evaluated by intraperitoneal injection into a mouse osteoarthritis model induced by destabilization of the medial meniscus.
LIG lowered the phosphorylation levels of p65, IκBα, and IKKα/β and suppressed the IL-1β-induced expression of MMP-3, ADAMTS-5, iNOS, and COX-2 and the inflammatory factors PGE2, TNF-α, and IL-6. LIG markedly decreased IL-1β-induced degradation of collagen II and aggrecan. In vivo results showed that LIG-treated mouse cartilage showed less damage than the control group; the Osteoarthritis Research Society International (OARSI) score was also lower. LIG further reduced the thickness of the subchondral bone plate and alleviated the synovitis.
LIG may act as a promising therapeutic agent for osteoarthritis by attenuating IL-1β-induced inflammation in chondrocytes and ECM degradation via suppression of NF-κB activation by the PI3K/AKT pathway.
背景/目的：骨关节炎是一种退行性关节疾病，其特征在于软骨退变和软骨细胞炎症反应，该反应诱导了与细胞外基质（ECM）降解密切相关的炎症环境。藁本内酯（LIG）是中药当归的主要成分，具有抗炎作用。为了证实LIG对人骨关节炎软骨细胞炎症是否具有同样的抑制作用，我们进行了体内和体外实验以验证上述推测并确定相关机制。
采用定量实时PCR和蛋白质印迹法在基因和蛋白质水平评估基质金属蛋白酶-3（MMP-3）、基质金属蛋白酶-13（MMP-13）、含血小板反应蛋白基序的解聚素样金属蛋白酶-5（ADAMTS-5）、诱导型一氧化氮合酶（iNOS）和环氧化酶-2（COX-2）的表达。采用酶联免疫吸附测定法评估其他炎症因子（前列腺素E2（PGE2）、肿瘤坏死因子-α（TNF-α）和白细胞介素-6（IL-6））的水平。还通过蛋白质印迹法分析磷脂酰肌醇-3激酶（PI3K）/蛋白激酶B（AKT）和核因子κB（NF-κB）信号通路，而免疫荧光法用于评估Ⅱ型胶原和聚集蛋白聚糖的表达。通过腹腔注射到内侧半月板失稳诱导的小鼠骨关节炎模型中来评估LIG的体外作用。
LIG降低了p65、IκBα和IKKα/β的磷酸化水平，并抑制了白细胞介素-1β（IL-1β）诱导的MMP-3、ADAMTS-5、iNOS和COX-2以及炎症因子PGE2、TNF-α和IL-6的表达。LIG显著减少了IL-1β诱导的Ⅱ型胶原和聚集蛋白聚糖的降解。体内结果显示，LIG处理的小鼠软骨损伤比对照组少；国际骨关节炎研究学会（OARSI）评分也更低。LIG进一步降低了软骨下骨板厚度并减轻了滑膜炎。
LIG可能通过PI3K/AKT途径抑制NF-κB活化，减轻IL-1β诱导的软骨细胞炎症和ECM降解，从而成为一种有前景的骨关节炎治疗药物。
