Zhou Qingqi, Li Xun, Hou Decai
Second Department of Orthopaedics, Dalian Hospital of Traditional Chinese Medicine, Dalian 116031, China.
Second Department of Orthopaedics, The First Affiliated Hospital of Liaoning University of Traditional Chinese Medicine, Shenyang 110000, China.
Int Immunopharmacol. 2025 Jun 17;158:114820. doi: 10.1016/j.intimp.2025.114820. Epub 2025 May 15.
Osteoarthritis (OA) is an age-related progressive joint disease characterized by loss of cartilage and subsequent inflammation. Perillaldehyde is a compound extracted from Perilla, which has multiple pharmacological activities including anti-inflammatory, and anti-apoptosis. However, it still remains unknown whether and how perillaldehyde could regulate chondrocyte apoptosis and inflammation in OA.
The interleukin-1beta (IL-1β)-treated chondrocytes and destabilized medial meniscus (DMM) -induced rats were used as in vitro and in vivo models of OA. Cell viability, proliferation, and apoptosis were investigated by cell counting kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU) and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays. The mitochondrial membrane potential was analyzed by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining. Light chain 3 (LC3) location, and expression of NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) and apoptosis-associated speck-like protein (ASC) were detected by immunofluorescence staining. Relative protein levels were measured via western blotting. Tumor necrosis factor-alpha (TNF-α), IL-6 and IL-8 levels were measured by enzyme-linked immunosorbent assay (ELISA). The cartilage injury in rats was investigated via hematoxylin-eosin (HE) staining.
Perillaldehyde attenuated IL-1β-induced inhibition of viability (from 44.45 % to 92.72 %, p < 0.05) and proliferative potential of chondrocytes (p < 0.05). Perillaldehyde mitigated mitophagy-associated apoptosis of chondrocytes by enhancing mitophagy (p < 0.05) and reducing cell apoptosis (from 32.36 % to 8.00 %, p < 0.05). Perillaldehyde attenuated NLRP3-mediated inflammation through reducing NLRP3, ASC, TNF-α, IL-6 and IL-8 levels (p < 0.05). ALOX5 expression was upregulated in OA (fold change: 2.61, p < 0.05), and decreased by perillaldehyde treatment (fold change: 1.34, p < 0.05). Perillaldehyde inhibits p65 nuclear factor kappa B (NF-κB) signaling activation (fold change: 3.19 to 1.33, p < 0.05) by regulating ALOX5 expression. ALOX5 overexpression reversed the effects of perillaldehyde on mitophagy-associated apoptosis and NLRP3-mediated inflammation (p < 0.05), and these roles were mitigated due to NF-κB inactivation (p < 0.05). Perillaldehyde attenuated cartilage injury in OA rats (p < 0.05).
Perillaldehyde attenuated IL-1β-induced mitophagy-associated apoptosis and NLRP3-mediated inflammation through decreasing ALOX5 and inactivating NF-κB signaling, indicating the potentially protective potential of perillaldehyde in osteoarthritis.
骨关节炎（OA）是一种与年龄相关的进行性关节疾病，其特征是软骨丢失及随后的炎症反应。紫苏醛是从紫苏中提取的一种化合物，具有多种药理活性，包括抗炎和抗凋亡作用。然而，紫苏醛是否以及如何调节OA中软骨细胞的凋亡和炎症反应仍不清楚。
将白细胞介素-1β（IL-1β）处理的软骨细胞和内侧半月板不稳定（DMM）诱导的大鼠分别作为OA的体外和体内模型。通过细胞计数试剂盒-8（CCK-8）、5-乙炔基-2'-脱氧尿苷（EdU）和末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）试验研究细胞活力、增殖和凋亡情况。采用5,5',6,6'-四氯-1,1',3,3'-四乙基苯并咪唑基羰花青碘化物（JC-1）染色分析线粒体膜电位。通过免疫荧光染色检测轻链3（LC3）定位以及NOD样受体热蛋白结构域相关蛋白3（NLRP3）和凋亡相关斑点样蛋白（ASC）的表达。通过蛋白质印迹法测定相关蛋白水平。采用酶联免疫吸附测定（ELISA）法检测肿瘤坏死因子-α（TNF-α）、IL-6和IL-8水平。通过苏木精-伊红（HE）染色研究大鼠的软骨损伤情况。
紫苏醛减轻了IL-1β诱导的软骨细胞活力抑制（从44.45%提高到92.72%，p<0.05）和增殖潜能抑制（p<0.05）作用。紫苏醛通过增强线粒体自噬（p<0.05）和减少细胞凋亡（从32.36%降至8.00%，p<0.05）减轻软骨细胞线粒体自噬相关凋亡。紫苏醛通过降低NLRP3、ASC、TNF-α、IL-6和IL-8水平减轻NLRP3介导的炎症反应（p<0.05）OA中5-脂氧合酶（ALOX5）表达上调（倍数变化：2.61，p<0.05），紫苏醛处理后表达降低（倍数变化：1.34，p<0.05）。紫苏醛通过调节ALOX5表达抑制p65核因子κB（NF-κB）信号激活（倍数变化：从3.19降至1.33，p<0.05）。ALOX5过表达逆转了紫苏醛对线粒体自噬相关凋亡和NLRP3介导的炎症反应的影响（p<0.05），并且由于NF-κB失活这些作用减弱（p<0.05）。紫苏醛减轻了OA大鼠的软骨损伤（p<0.05）。
紫苏醛通过降低ALOX5水平和使NF-κB信号失活减轻IL-1β诱导的线粒体自噬相关凋亡和NLRP3介导的炎症反应，表明紫苏醛在骨关节炎中具有潜在的保护作用。
