摘要
目的:探讨杜仲多糖对白细胞介素1β(interleukin-1β,IL-1β)诱导的软骨细胞损伤的影响及可能机制。方法:体外培养小鼠软骨细胞ATDC5,用含10μg/ml IL-1β处理ATDC5制作骨关节炎软骨细胞炎症模型,随机分为空白组、模型组、模型+杜仲多糖低浓度组、模型+杜仲多糖中浓度组和模型+杜仲多糖高浓度组。其中空白组细胞用常规培养基培养,模型组细胞用含10μg/ml IL-1β的培养基培养,模型+杜仲多糖低浓度组、模型+杜仲多糖中浓度组和模型+杜仲多糖高浓度组细胞分别用含100、200、400μg/ml杜仲多糖与10μg/ml IL-1β的培养基共同培养。分别培养24、48、72 h后,CCK-8法检测细胞活力。培养48 h后,流式细胞术和DAPI染色检测细胞凋亡,ELISA法检测细胞培养上清液中肿瘤坏死因子α(tumor necrosis factor-α,TNF-α),一氧化氮(netric oxide,NO),γ干扰素(interfero-γ,IFN-γ)和白细胞介素6(interleukin-6,IL-6)的表达,DCFH-DA法检测细胞中活性氧含量,Western-blot法检测金属蛋白酶组织抑制因子1 (tissue inhibrtor of metalloproteinase,TIMP-1),基质金属蛋白酶13 (mitochondrial membrane protential,MMP-13)及NF-κB信号通路相关P65,磷酸化P65(p-P65)的蛋白表达,免疫荧光染色观察NF-κB P65细胞定位。结果:与空白组比较,模型组ATDC5细胞活力及TIMP-1蛋白表达降低(P<0.05),细胞凋亡率,TNF-α、NO、IFN-γ和IL-6水平,活性氧(reactive oxygen species,ROS)含量,MMP-13和p-P65的蛋白表达及细胞核内P65+数量均升高(P<0.05)。与模型组比较,模型+杜仲多糖低浓度组、模型+杜仲多糖中浓度组和模型+杜仲多糖高浓度组ATDC5细胞活力及TIMP-1蛋白表达升高(P<0.05),而细胞凋亡率、TNF-α、NO、IFN-γ和IL-6水平、ROS含量、MMP-13和p-P65的蛋白表达及细胞核内P65+数量均降低(P<0.05)。结论:杜仲多糖可促进白细胞介素1β诱导的软骨细胞ATDC5增殖,并抑制其凋亡、炎症反应和基质降解,其作用机制可能与抑制NF-κB通路的激活有关。
Objective:To investigate the effects of Eucommia ulmoides Oliv polysaccharide on the injury of interleukin-1β(IL-1β)-induced chondrocyte and its possible mechanism.Methods:ATDC5 was treated with 10 μg/ml IL-1β to establish osteoarthritis chondrocyte inflammation model,mouse chondrocyte ATDC5 were cultured in vitro and randomly divided into blank group,model group,model+Eucommia ulmoides Oliv polysaccharide low concentration group,model+Eucommia ulmoides Oliv polysaccharide medium concentration group and model+Eucommia ulmoides Oliv polysaccharide high concentration group.The cells in the blank group were cultured with conventional medium;the cells in the model group cells were cultured with a medium containing 10 μg/ml IL-1β,and the cells in the model+Eucommia ulmoides Oliv polysaccharide low concentration group,model+Eucommia ulmoides Oliv polysaccharide medium concentration group and model+Eucommia ulmoides Oliv polysaccharide high concentration group were co-cultured with medium containing 100,200,400 μg/ml Eucommia ulmoides Oliv polysaccharide and 10 μg/ml IL-1β.After the cells of each group were cultured for 24 h,48 h and 72 h,CCK-8 method was used to detect cell viability.After the cells of each group were cultured for 48 h,flow cytometry and DAPI staining were used to detect cell apoptosis;ELISA method was used to detect the expression of TNF-α,NO,IFN-γ and IL-6 in cells;DCFH-DA method was used to detect the content of ROS in cells;Western blot was used to detect the protein expression of TIMP-1,MMP-13 and NF-κB signaling pathway-related P65 and p-P65;Immunofluorescence staining was used to observe the localization of NF-κB P65 cells.Results:Compared with the blank group,the ATDC5 cell viability and the protein expression of TIMP-1 in the model group reduced(P<0.05),while apoptosis rate,the levels of TNF-α,NO,IFN-γ and IL-6,the content of ROS,the protein expression of MMP-13 and p-P65,and the number of P65^(+) in the nucleus increased(P<0.05).Compared with the model group,the ATDC5 cell viability and the protein expression of TIMP-1 in the model+Eucommia ulmoides Oliv polysaccharide low concentration group,model+Eucommia ulmoides Oliv polysaccharide medium concentration group and model+Eucommia ulmoides Oliv polysaccharide high concentration group increased(P<0.05),while apoptosis rate,the levels of TNF-α,NO,IFN-γ and IL-6,the content of ROS,the protein expression of MMP-13 and p-P65,and the number of P65^(+)in the nucleus reduced(P<0.05).Conclusion:The results showed that Eucommia ulmoides Oliv polysaccharide could promote proliferation of IL-1β-induced chondrocyte ATDC5 and inhibit its apoptosis,inflammatory response and matrix degradation.Its mechanism may be related to the inhibition of the activation of NF-κB pathway.
作者
李宁博
骆晓飞
尹夏
魏瑄
LI Ning-bo;LUO Xiao-fei;YIN Xia;WEI Xuan(不详;Department of Orthopaedics,Zhengzhou Orthopaedics Hospital,Zhengzhou 450000,Henan,China)
出处
《中国骨伤》
CAS
CSCD
2022年第7期661-668,共8页
China Journal of Orthopaedics and Traumatology
基金
2018年河南省科技攻关项目(编号:182102310183)。
关键词
杜仲多糖
软骨细胞
细胞凋亡
炎症
NF-ΚB信号通路
Eucommia polysaccharides
Chondrocytes
Apoptosis
Inflammation
NF-κB signaling pathway
