脂多糖干预后的不同滑膜细胞来源炎性外泌体对软骨细胞的作用机制研究OACSTPCD
Study on the mechanism of action of different synovial cell-derived inflammatory exosomes on chondrocytes after lipopolysaccharide intervention
目的 观察脂多糖(LPS)诱导炎症后的不同滑膜细胞来源外泌体对软骨细胞的影响,探讨两种滑膜细胞外泌体在膝骨关节炎(KOA)疾病进展中引起软骨损伤的作用机制.方法 将两种滑膜细胞以1∶4共培养并用LPS诱导炎症,提取上清液中外泌体,再分别提取正常及LPS诱导炎症后的两种滑膜细胞外泌体.将术中取得的人软骨组织分离培养成软骨细胞,分为五组:第Ⅰ组加入FLS外泌体;第Ⅱ组加入正常的FLS外泌体;第Ⅲ组加入两种滑膜细胞共培养后的外泌体;第Ⅳ组加入炎性的MLS外泌体;第V组加入炎性的FLS外泌体.CCK-8检测各组软骨细胞活力.ELISA法检测各组软骨细胞上清液中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、IL-6水平.Western blot法检测各组软骨细胞中Toll样受体4(TLR4)、核因子κB(NF-κB)、核因子κB抑制因子激酶(IkK)、核因子κB抑制蛋白(IκB)、金属肽酶含血小板反应蛋白基元5(ADAMTS5)的蛋白表达量.结果 CCK-8显示,与正常滑膜细胞来源外泌体相比,3组炎性外泌体均可使软骨细胞活力降低(P＜0.05);ELISA检测显示Ⅲ、Ⅳ、Ⅴ组软骨细胞上清液中TNF-α、1L-1β、IL-6水平高于Ⅰ、Ⅱ组(P＜0.05),Ⅲ组软骨细胞上清液中TNF-α、IL-1 β、IL-6 水平高于 Ⅳ组而低于 Ⅴ 组(P＜0.05).Western blot 显示Ⅲ、Ⅳ、Ⅴ组软骨细胞中 TLR4、NF-κB、IkK、IκB、AD-AMTS5蛋白表达量高于Ⅰ、Ⅱ组(P＜0.05),Ⅲ组软骨细胞中TLR4、NF-κB、IkK、IκB、ADAMTS5蛋白表达量高于Ⅳ组而低于Ⅴ组(P＜0.05).结论 LPS诱导炎症后的两种滑膜细胞来源外泌体均可通过调控软骨TLRs/NF-κB信号通路,引起软骨炎症.MLS外泌体致炎效果强于FLS外泌体,但两种共培养情况下的外泌体致炎效果弱于单纯MLS外泌体.
Objective To observe the effect of different synovial cell secretions on chondrocytes after LPS-induced inflammation,and to explore the mechanism of two synovial cell secretions causing cartilage damage in the progres-sion of KOA disease.Methods Two kinds of synovial cells were co-cultured at 1∶4 and LPS-induced inflamma-tion.The supernatant and exocrine were extracted,and then the normal and LPS-induced inflammation were extrac-ted.The human cartilage tissue obtained during the operation was isolated and cultured into chondrocytes,which were divided into five groups:the first group was added with FLS secretion,the second group was added with nor-mal FLS secretion,the third group was added with secretion after co-culture of two kinds of synovial cells,the fourth group was added with inflammatory MLS secretion,and the fifth group was added with inflammatory FLS se-cretion.CCK-8 was used to detect the viability of chondrocytes in each group.TNF-α,IL-1β,IL-6 level in the su-pernatant of chondrocytes in each group was detected by ELISA.The protein expression of TLR4,NF-κB,IkK,IκB,ADAMTS5 in chondrocytes of each group was detected by Western blot method.Results CCK-8 showed that the activity of chondrocytes in the three groups of inflammatory secretions decreased compared with the secretions from normal synovial cells(P＜0.05);ELISA showed TNF-α,IL-1 β,IL-6 level in the supernatant of group Ⅲ,Ⅳ and V was higher than that of group Ⅰ and Ⅱ(P＜0.05),TNF-α,IL-1 β,IL-6 level in group Ⅲ was higher than that in group Ⅳ but lower than that in group Ⅴ(P＜0.05).Western blot showed the protein expression of TLR4,NF-κB,IkK,IκB,ADAMTS5 in chondrocytes of group Ⅲ,Ⅳ and Ⅴ was higher than that in group Ⅰ and Ⅱ(P＜0.05),the protein expression of TLR4,NF-κB,IkK,IκB,ADAMTS5 in group Ⅲ was higher than that in group Ⅳbut lower than that in group Ⅴ(P＜0.05).Conclusion Two kinds of synovial cell-derived secretions after LPS-induced inflammation can regulate cartilage TLRs/NF-κB signal pathway,causing cartilage inflammation.The in-flammatory effect of MLS secretion is stronger than that of FLS secretion,but the inflammatory effect of MLS secre-tion under two co-cultures is weaker than that of MLS secretion alone.
周俊;郭长青;王庆甫
北京中医药大学第三附属医院骨伤科,北京 100029北京中医药大学针灸推拿学院,北京 100029
临床医学
成纤维样滑膜细胞巨噬样滑膜细胞外泌体TLRs/NF-κB信号通路膝骨关节炎软骨细胞
fibroblast-like synoviocytesmacrophage-like synoviocytesexosomesTLRs/NF-κB signal pathwayknee osteoarthritischondrocytes
《安徽医科大学学报》 2024 (002)
243-248 / 6
国家自然科学基金面上项目(编号:81874475)
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