| 摘要: |
| 目的 探讨低强度脉冲聚焦超声（FLIPUS）对脂多糖（LPS）诱导的小鼠膝关节软骨细胞损伤中炎症因子和细胞焦亡相关蛋白表达的影响及其保护软骨的作用机制。方法 提取C57BL/6J小鼠膝关节原代软骨细胞进行体外实验，用1 μg/mL LPS处理软骨细胞12 h模拟骨关节炎样软骨细胞损伤。细胞分为对照组（仅用含10% FBS的培养基培养，不予任何处理）、LPS组（LPS处理12 h）和LPS+FLIPUS组（LPS处理12 h后再用FLIPUS干预20 min）。采用蛋白质印迹法检测各组软骨细胞中Ⅱ型胶原蛋白α1（COL2α1）、炎症细胞因子（IL-18和IL-1β）、焦亡相关蛋白[核苷酸结合寡聚化结构域样受体3（NLRP3）、cleaved caspase 1、消皮素D N端片段（GSDMD-N）]、软骨基质降解因子[基质金属蛋白酶（MMP）13、MMP3和血小板结合蛋白基序的解聚蛋白样金属蛋白酶5（ADAMTS5）]的表达。采用钙黄绿素乙酰氧基甲酯（calcein AM）/PI荧光染色和乳酸脱氢酶（LDH）释放实验检测细胞活性，采用扫描电子显微镜观察细胞的形态变化，采用ELISA法检测细胞上清液中IL-1β、IL-18水平。结果 成功提取小鼠膝关节原代软骨细胞并模拟骨关节炎样软骨细胞损伤。与对照组相比，LPS组小鼠膝关节软骨细胞中COL2α1蛋白表达降低（P<0.01），MMP13、MMP3、ADAMTS5、NLRP3、GSDMD-N、cleaved caspase 1、IL-18和IL-1β蛋白表达增加（均P<0.01），细胞上清液中IL-18、IL-1β水平升高和LDH释放增加（均P<0.01），细胞肿胀变形，细胞膜上出现较多孔隙，丧失完整性，细胞死亡率增加（P<0.01）。与LPS组相比，LPS+FLIPUS组小鼠膝关节软骨细胞中COL2α1蛋白表达上升（P<0.01），MMP13、MMP3、ADAMTS5、NLRP3、GSDMD-N、cleaved caspase 1、IL-18和IL-1β蛋白表达下降（P<0.05，P<0.01），细胞上清液中IL-18、IL-1β水平下降和LDH释放减少（P<0.05，P<0.01），细胞肿胀程度减轻，细胞膜上孔隙减少，细胞死亡率下降（P<0.05）。结论 FLIPUS可能通过抑制炎症因子和细胞焦亡相关蛋白表达减轻LPS诱导的小鼠膝关节软骨细胞损伤 |
| 关键词: 低强度脉冲聚焦超声 骨关节炎 炎症 软骨细胞 细胞焦亡 |
| DOI：10.16781/j.CN31-2187/R.20230516 |
| 投稿时间：2023-09-11修订日期：2023-10-10 |
| 基金项目:国家自然科学基金青年科学基金（81802234），重庆市自然科学基金面上项目（cstc2020jcyj-msxmX0134），重庆医科大学未来医学青年创新团队发展支持计划项目（W0196），重庆医科大学附属第二医院“宽仁英才”项目（kryc-gg-2116），重庆市中青年医学高端人才项目，重庆市科卫联合医学科研项目（2024GDRC002）. |
|
| Effects of focused low-intensity pulsed ultrasound on inflammatory cytokines and pyroptosis-related proteins in mouse knee joint chondrocyte injury |
| WEI Xia,LI Dongqian,LUO Shiqi,JIA Lang* |
(Department of Rehabilitation Medicine, The Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, China
*Corresponding author) |
| Abstract: |
| Objective To explore the effects of focused low-intensity pulsed ultrasound (FLIPUS) on the expression of inflammatory cytokines and pyroptosis-related proteins in mouse knee joint chondrocyte injury induced by lipopolysaccharide (LPS) and the mechanism of chondroprotection. Methods Primary chondrocytes were isolated from knee cartilage of C57BL/6J mice and stimulated with LPS for 12 h to mimic osteoarthritic chondrocyte injury in vitro. The chondrocytes were divided into control group (only cultured in medium containing 10% FBS without any treatment), LPS group (LPS treatment for 12 h), and LPS+FLIPUS group (LPS treatment for 12 h followed by FLIPUS intervention for 20 min). The expression of collagen Ⅱ α1 (COL2α1), inflammatory cytokines (interleukin[IL]-18 and IL-1β), pyroptosis-related proteins (nucleotide-binding oligomerization domain-like receptor 3 [NLRP3], cleaved cysteine aspartic acid specific protease 1 (caspase 1), and N-terminal fragment of gasdermin D[GSDMD-N]), and cartilage matrix degradation factors (matrix metalloproteinase [MMP] 13, MMP3, and a disintegrin and metalloproteinase with thrombospondin 5[ADAMTS5]) were detected by Western blotting. The viability of chondrocytes was detected by calcein acetoxymethyl ester/propidium iodide (calcein AM/PI) fluorescent staining and lactate dehydrogenase (LDH) release assay, the morphology of chondrocytes was observed by scanning electron microscopy, and the levels of IL-1β and IL-18 in cell supernatant were detected by enzyme-linked immunosorbent assay. Results The primary chondrocytes were isolated from knee cartilage of the mice and osteoarthritic chondrocyte injury in vitro were successfully simulated. Compared with the control group, the expression of COL2α1 was significantly decreased in the LPS group (P<0.01), while the expression levels of MMP13, MMP3, ADAMTS5, NLRP3, GSDMD-N, cleaved caspase 1, IL-18, and IL-1β were significantly increased (all P<0.01); the levels of IL-18, IL-1β, and LDH in the cell supernatant were significantly increased (all P<0.01), with cell swelling and deformation, more pores on the membrane, loss of integrity, and increased cell mortality (P<0.01). Compared with the LPS group, the expression of COL2α1 was significantly increased in the knee chondrocytes of mice in the LPS+FLIPUS group (P<0.01), while the expression levels of MMP13, MMP3, ADAMTS5, NLRP3, GSDMD-N, cleaved caspase 1, IL-18, and IL-1β were significantly decreased (P<0.05, P<0.01); the levels of IL-18, IL-1β, and LDH in the cell supernatant were significantly decreased (P<0.05, P<0.01), with relieved cell swelling and membrane pores and decreased cell mortality (P<0.05). Conclusion FLIPUS may reduce chondrocytes injury induced by LPS in mice via inhibiting the expression of inflammatory cytokines and pyroptosis-related proteins. |
| Key words: focused low-intensity pulsed ultrasound osteoarthritis inflammation chondrocyte pyroptosis |
