透骨消痛胶囊抑制脂多糖诱导软骨细胞炎症反应的机制研究

摘要

Objective To explore the mechanism of Tougu Xiaotong capsule (TXC)inhibiting lipopolysaccharide (LPS)-stimulated inflammation in chondrocytes.Methods The chondrocytes harvested from the knee joints of 4-week-old SD rats were cultured in vitro and divided into 3 groups:the sham group,model group and TXC group.The sham group was cultured in 2 mL low-glucose Dulbecco Modified Eagle Medium (DMEM/LOW)containing 10% fetal bovine serum (FBS);the model group was cultured in 2 mL DMEM/LOW containing 10% FBS and 10 ng/mL LPS;the TXC group was cultured in 2 mL DMEM/LOW containing 10%FBS,10 ng/mL LPS and 300μg/mL TXC. After chondrocytes being treated for 8 h,the microRNA (miRNA)-140 expression was detected by quantitative real-time polymerase chain reaction (qPCR ), and the expressions of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS )4 and matrix metalloproteinase (MMP)-3 were observed by immunofluorescence staining,the average optical density of each group was analyzed by Image J. Results Compared with the sham group,the expression of MMP-3 (P<0.05 )and ADAMTS4 (P<0.01 )in the model group were significantly increased.Compared with the model group,the expression of MMP-3 (P<0.05)and ADAMTS4 (P<0.01)in the TXC group were significantly decreased.The expression of miRNA-140 in the model group was lower than that in the control group (P<0.01)and the TXC group (P<0.01).Conclusion TXC may delay the cartilage degeneration in osteoarthritis (OA)by promoting the expression of miRNA-140 to reduce the secretion of inflammatory cytokines such as ADAMTS4 and MMP-3 and thus inhibit the inflammation-mediated cartilage matrix degradation.%目的探讨透骨消痛胶囊抑制脂多糖（LPS）诱导的软骨细胞炎症反应作用机制。方法对4周龄雄性SD大鼠采用机械-Ⅱ型胶原酶消化法获取膝关节软骨细胞并进行体外培养，将软骨细胞分为空白组、模型组以及透骨消痛胶囊组，空白组加入含10％胎牛血清（FBS）的低糖型Dulbecco 改良 Eagle 培养基（DMEM／LOW）2 mL，模型组加入含10 ng／mL LPS、10％FBS的DMEM／LOW培养基2 mL，透骨消痛胶囊组加入含10 ng／mL LPS、透骨消痛胶囊（300μg／mL）、10％FBS 的DMEM／LOW培养基2 mL。干预8 h后，实时荧光定量聚合酶链式反应（qPCR）检测各组微小 RNA（miRNA）-140表达变化，免疫荧光观察各组带有血小板凝血酶敏感蛋白样模体的解整链蛋白金属蛋白酶（ADAMTS）4、基质金属蛋白酶（MMP）-3表达变化，Image J 分析各组平均光密度。结果与空白组相比，模型组 MMP-3表达升高（P＜0．05）、ADAMTS4表达明显升高（P＜0．01）；与模型组相比，透骨消痛胶囊组 MMP-3表达下降（P＜0．05）、ADAMTS4表达明显下降（P＜0．01）。与空白组相比，模型组软骨细胞miRNA-140表达明显降低（P＜0．05）；与模型组相比，透骨消痛胶囊组软骨细胞miRNA表达明显升高（P＜0．05）。结论透骨消痛胶囊能促进miRNA-140表达，降低ADAMTS4、MMP-3分泌，抑制炎症反应介导的软骨基质降解，从而延缓骨关节炎软骨退变。