Pei Wenbin, Huang Xiaojian, Ni Bowei, Zhang Rui, Niu Guangyi, You Hongbo
Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Rhode Island School of Design, Providence, RI, United States.
Front Pharmacol. 2021 Sep 28;12:730312. doi: 10.3389/fphar.2021.730312. eCollection 2021.
Osteoarthritis (OA), which is identified by chronic pain, impacts the quality of life. Cartilage degradation and inflammation are the most relevant aspects involved in its development. Signal transducer and activator of transcription 3(STAT3), a member of the STATs protein family, is associated with inflammation. Alantolactone (ALT), a sesquiterpene lactone compound, can selectively suppress the phosphorylation of STAT3. However, the pharmacological effect of ALT on OA is still imprecise. In this study, IL-1β (10 ng/ml) was applied to cartilage chondrocytes, which were treated with different concentrations of Alantolactone for 24 h. The expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2(COX2), matrix metalloproteinases (MMPs) and thrombospondin motifs-5 (ADAMTS5) were detected by western blot. Protein expression of Collagen Ⅱ was observed by western blot, safranin O staining and immunofluorescence. Manifestation of autophagy related proteins such as autophagy-related gene-5 (ATG5), P62, LC3Ⅱ/Ⅰ and PI3K/AKT/mTOR-related signaling molecules were measured by western blot and autophagic flux monitored by confocal microscopy. Expression of STAT3 and NF-κB-related signaling molecules were evaluated by western blot and immunofluorescence. , 2 mg/kg ALT or equal bulk of vehicle was engaged in the destabilization of medial meniscus (DMM) mouse models by intra-articular injection, the degree of cartilage destruction was classified by Safranin O/Fast green staining. Our findings reported that the enhance of inflammatory factors containing iNOS, COX2, MMPs and ADAMTS5 induced by IL-1β could be ameliorated by ALT. Additionally, the diminish of Collagen Ⅱ and autophagy which was stimulated by IL-1β could be alleviated by ALT. Mechanistically, STAT3, NF-κB and PI3K/AKT/mTOR signal pathways might be involved in the effect of ALT on IL-1β-induced mouse chondrocytes. ALT protected cartilage in the DMM mouse model. Overall, this study illustrated that ALT attenuated IL-1β-induced inflammatory responses, relieved cartilage degeneration and promoted impaired autophagy restraining of STAT3 and NF-κB signal pathways, implying its auspicious therapeutical effect for OA.
骨关节炎（OA）以慢性疼痛为特征，会影响生活质量。软骨降解和炎症是其发展过程中最相关的方面。信号转导和转录激活因子3（STAT3）是信号转导和转录激活因子（STATs）蛋白家族的成员，与炎症相关。土木香内酯（ALT）是一种倍半萜内酯化合物，可选择性抑制STAT3的磷酸化。然而，ALT对OA的药理作用仍不明确。在本研究中，将白细胞介素-1β（IL-1β，10 ng/ml）应用于软骨细胞，并使用不同浓度的土木香内酯对其进行24小时处理。通过蛋白质免疫印迹法检测诱导型一氧化氮合酶（iNOS）、环氧化酶-2（COX2）、基质金属蛋白酶（MMPs）和含血小板反应蛋白基序的解聚素-5（ADAMTS5）的表达。通过蛋白质免疫印迹法、番红O染色和免疫荧光观察Ⅱ型胶原蛋白的蛋白表达。通过蛋白质免疫印迹法检测自噬相关蛋白如自噬相关基因5（ATG5）、P62、微管相关蛋白1轻链3Ⅱ/Ⅰ（LC3Ⅱ/Ⅰ）的表达以及磷脂酰肌醇-3激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白（PI3K/AKT/mTOR）相关信号分子的表达，并通过共聚焦显微镜监测自噬通量。通过蛋白质免疫印迹法和免疫荧光评估STAT3和核因子κB（NF-κB）相关信号分子的表达。通过关节内注射，将剂量为2 mg/kg的ALT或等量的赋形剂应用于内侧半月板不稳定（DMM）小鼠模型，通过番红O/固绿染色对软骨破坏程度进行分级。我们的研究结果表明，ALT可改善IL-1β诱导的炎症因子iNOS、COX2、MMPs和ADAMTS5的增加。此外，ALT还可缓解IL-1β刺激引起的Ⅱ型胶原蛋白减少和自噬减弱。从机制上讲，STAT3、NF-κB和PI3K/AKT/mTOR信号通路可能参与了ALT对IL-1β诱导的小鼠软骨细胞的作用。ALT对DMM小鼠模型中的软骨具有保护作用。总体而言，本研究表明，ALT可减轻IL-1β诱导的炎症反应，缓解软骨退变，并通过抑制STAT3和NF-κB信号通路促进受损自噬，这意味着其对OA具有良好的治疗效果。
