| 杜仲多糖通过抑制NF-κB通路减轻IL-1β诱导的软骨细胞损伤 |
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投稿时间：2020-05-19
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| 作者 | Author | 单位 | Address | E-Mail |
| 李宁博 |
LI Ning-bo |
河南中医药大学第一附属医院骨科, 河南 郑州 450000 |
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| 骆晓飞 |
LUO Xiao-fei |
郑州市骨科医院骨科, 河南 郑州 450000 |
Department of Orthopaedics, Zhengzhou Orthopaedics Hospital, Zhengzhou 450000, Henan, China |
luoxiaofei152@163.com |
| 尹夏 |
YIN Xia |
河南中医药大学第一附属医院骨科, 河南 郑州 450000 |
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| 魏瑄 |
WEI Xuan |
郑州市骨科医院骨科, 河南 郑州 450000 |
Department of Orthopaedics, Zhengzhou Orthopaedics Hospital, Zhengzhou 450000, Henan, China |
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| 期刊信息：《中国骨伤》2022年，第35卷，第7期，第661-668页 |
| DOI：10.12200/j.issn.1003-0034.2022.07.013 |
| 基金项目:2018年河南省科技攻关项目（编号：182102310183） |
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中文摘要:
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、400 μg/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。 |
| 【关键词】杜仲多糖 软骨细胞 细胞凋亡 炎症 NF-κB信号通路 |
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| Eucommia ulmoides Oliv polysaccharide reduces the injury of IL-1β-induced chondrocyte by inhibiting NF-κB pathway |
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ABSTRACT
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 10μg/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. |
| KEY WORDS Eucommia polysaccharides Chondrocytes Apoptosis Inflammation NF-κB signaling pathway |
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| 引用本文，请按以下格式著录参考文献： |
| 中文格式: | 李宁博,骆晓飞,尹夏,魏瑄.杜仲多糖通过抑制NF-κB通路减轻IL-1β诱导的软骨细胞损伤[J].中国骨伤,2022,35(7):661~668 |
| 英文格式: | LI Ning-bo,LUO Xiao-fei,YIN Xia,WEI Xuan.Eucommia ulmoides Oliv polysaccharide reduces the injury of IL-1β-induced chondrocyte by inhibiting NF-κB pathway[J].zhongguo gu shang / China J Orthop Trauma ,2022,35(7):661~668 |
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