中国组织工程研究

中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (14): 2161-2166.doi: 10.12307/2022.477

• 软骨组织构建 cartilage tissue construction • 上一篇    下一篇

姜黄素对H2O2诱导软骨细胞自噬的调控

曹舒兴1，宋永周1，李  明1，张洪亮1，赵立辉2，马  维1   

  1. 1河北医科大学第二医院骨科， 河北省石家庄市   050000；2河北省深泽县医院放射科，河北省深泽县   052560
  • 收稿日期:2021-03-08 修回日期:2021-03-09 接受日期:2021-04-28 出版日期:2022-05-18 发布日期:2021-12-21
  • 通讯作者: 马维，博士，主任医师，河北医科大学第二医院骨科， 河北省石家庄市 050000
  • 作者简介:曹舒兴，男，1984年生，河北省正定县人，汉族，2012年河北医科大学研究生学院毕业，硕士，主治医师，主要从事骨关节炎、骨质疏松症方面的研究。
  • 基金资助:
    河北省卫生厅科研基金(20190068) ，项目负责人：曹舒兴 ；河北省财政厅老年病课题 (361004)，项目负责人：李明

Regulatory role of curcumin in hydrogen peroxide-induced autophagy in chondrocytes

Cao Shuxing1, Song Yongzhou1, Li Ming1, Zhang Hongliang1, Zhao Lihui2, Ma Wei1   

  1. 1Department of Orthopedics, Second Hospital of Hebei Medical University, Shijiazhuang 050000, Hebei Province, China; 2Department of Radiology, Shenze County Hospital, Shenze 052560, Hebei Province, China
  • Received:2021-03-08 Revised:2021-03-09 Accepted:2021-04-28 Online:2022-05-18 Published:2021-12-21
  • Contact: Ma Wei, MD, Chief physician, Department of Orthopedics, Second Hospital of Hebei Medical University, Shijiazhuang 050000, Hebei Province, China
  • About author:Cao Shuxing, Master, Attending physician, Department of Orthopedics, Second Hospital of Hebei Medical University, Shijiazhuang 050000, Hebei Province, China
  • Supported by:
    the Scientific Research Fund of Hebei Provincial Health Department, No. 20190068 (to CSX); the Subject of Geriatric Diseases of Hebei Provincial Finance Department, No. 361004 (to LM)

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摘要：

文题释义：
姜黄素：是一种从姜黄中提取的多酚，作为添加剂广泛用于日常食物中，它具有抗炎和抗氧化的功能，有望治疗一系列与慢性炎症相关的疾病，包括癌症、心血管疾病、代谢综合征、阿尔茨海默病、骨关节炎以及其他常见病和衰老疾病。
自噬：是一种保守的分解代谢过程，在人类退行性疾病中起着重要的作用。自噬通过对受损蛋白和功能失调细胞器的降解，实现能量循环，以维持某些应激条件下细胞代谢的稳态。

背景：姜黄素对骨关节炎具有良好的治疗作用，但具体机制还不清楚；研究表明自噬的激活可降低骨关节炎的严重程度。
目的：探讨姜黄素对H2O2诱导软骨细胞自噬的调控作用。
方法：体外培养C57小鼠关节软骨细胞，分为对照组、模型组以及姜黄素不同剂量组(10，20，40，80 μmol/L)。模型组给予H2O2(1 mmol/L)处理，模拟骨关节炎体内氧化应激状态；姜黄素组中不同浓度姜黄素与软骨细胞培养48 h后，加入H2O2，24 h后收集细胞进行检测。CCK-8法检测各组细胞活力；酶联免疫吸附法检测炎症因子白细胞介素6和肿瘤坏死因子α水平； TUNEL染色检测凋亡状况；流式细胞法检测各组细胞凋亡率；Western-blot法检测凋亡相关蛋白(活化半胱天冬酶3、Bax/Bcl-2)、细胞自噬相关蛋白(Beclin-1、LC3-Ⅱ/Ⅰ蛋白)及自噬相关信号通路蛋白(蛋白激酶B、哺乳动物雷帕霉素靶蛋白)的表达情况；实时荧光定量PCR检测软骨基质降解相关基因(基质金属蛋白酶13、血小板结合蛋白基序的解聚蛋白样金属蛋白酶5、Ⅱ型胶原蛋白α1、软骨蛋白聚糖)的表达水平。
结果与结论：①与模型组相比，姜黄素干预后软骨细胞的活力明显升高(P < 0.05)；炎症因子表达明显下降(P < 0.05)，凋亡率明显下降(P < 0.05)，凋亡相关标志物活化半胱天冬酶3以及Bax/Bcl-2比值显著下降(P < 0.05)，自噬相关标志物LC3-Ⅱ/Ⅰ和Beclin-1表达水平显著升高(P < 0.05)，蛋白激酶B、哺乳动物雷帕霉素靶蛋白的表达水平显著降低(P < 0.05)；②实时荧光定量PCR显示，基质金属蛋白酶13和血小板结合蛋白基序的解聚蛋白样金属蛋白酶5 mRNA表达降低，Ⅱ型胶原蛋白α1和软骨蛋白聚糖mRNA表达升高(P < 0.05)；③提示姜黄素通过促进自噬降低H2O2诱导的软骨细胞凋亡，这种作用可能与蛋白激酶B/哺乳动物雷帕霉素靶蛋白信号通路有关。
缩略语：磷脂酰肌醇3-激酶：phosphatidylinositol-3-kinase，PI3K；蛋白激酶B：protein kinase B，Akt；哺乳动物雷帕霉素靶蛋白：the mammalian target of Rapamycin，mTOR

https://orcid.org/0000-0003-3354-1877 (曹舒兴) 

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

关键词: 姜黄素, 骨关节炎, 软骨细胞, 自噬, H2O2

Abstract: BACKGROUND: Curcumin has a good therapeutic effect on osteoarthritis, but the specific mechanism is still unclear. Activation of autophagy can reduce the severity of osteoarthritis. 
OBJECTIVE: To investigate the regulatory effect of curcumin on hydrogen peroxide (H2O2)-induced autophagy in chondrocytes. 
METHODS: C57 mouse articular chondrocytes were cultured in vitro and randomly divided into control group, model group (treated with H2O2) and curcumin low-, medium- and high-dose groups (10, 20, 40, 80 μmol/L). Model group was treated with H2O2 (1 mmol/L) to simulate oxidative stress in osteoarthritis. In the different curcumin groups, chondrocytes were treated with different concentrations of curcumin for 48 hours, followed by addition of H2O2. Twenty-four hours later, the cells were collected for detection. Cell counting kit-8 assay was used to detect cell viability in each group. Interleukin-6 and tumor necrosis factor-α levels were detected by ELISA. TUNEL staining was used to detect cell apoptosis. The apoptosis rate of each group was detected by flow cytometry. The expression levels of cleaved caspase-3, Bax/Bcl2, Beclin1, LC3-II/I, protein kinase B (Akt) and mammalian target of rapamycin (mTOR) were detected by western blot. The expression of cartilage matrix degradation related genes, including matrix metalloproteinase 13, ADAM metallopeptidase with thrombospondin type 1 motif 5, type II collagen α1, and Aggrecan, were detected by qRT-PCR.
RESULTS AND CONCLUSION: Compared with the model group, the activity of chondrocytes was significantly increased after curcumin intervention (P < 0.05). The expression of inflammatory cytokines was significantly decreased (P < 0.05). Apoptosis was significantly decreased (P < 0.05). Apoptosis-related markers, cleaved caspase-3 and Bax/Bcl2 ratio, were significantly decreased (P < 0.05). The expression of autophagy-related markers, LC3-I/II and Beclin1, was significantly increased (P < 0.05). The expression levels of Akt and mTOR were significantly decreased (P < 0.05). qRT-PCR showed that the mRNA expressions of matrix metalloproteinase 13 and ADAM metallopeptidase with thrombospondin type 1 motif 5 were decreased, while the expressions of type II collagen α1 and Aggrecan were increased (P < 0.05). It is suggested that curcumin can reduce the H2O2-induced apoptosis in chondrocytes by promoting autophagy, and this effect may be related to the Akt/mTOR signaling pathway.

Key words: curcumin, osteoarthritis, chondrocyte, autophagy, hydrogen peroxide

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