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重庆医科大学基础医学院细胞生物学与遗传学教研室,重庆 400016
刘恺文,Email:353194212@qq.com,研究方向:骨关节炎的进展与机制。
郭风劲,Email:guo.fengjin@cqmu.edu.cn。
收稿:2023-12-29,
修回:2024-03-22,
录用:2024-03-26,
纸质出版:2024-12-28
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刘恺文,叶元兰,郭风劲.香豆素调控SASP抑制细胞衰老缓解骨关节炎的试验研究[J].重庆医科大学学报,2024,49(12):1556-1565.
Liu Kaiwen,Ye Yuanlan,Guo Fengjin.Coumarin alleviates osteoarthritis by regulating senescence-associated secretory phenotype and inhibiting cell senescence: an experimental study[J].Journal of Chongqing Medical University,2024,49(12):1556-1565.
刘恺文,叶元兰,郭风劲.香豆素调控SASP抑制细胞衰老缓解骨关节炎的试验研究[J].重庆医科大学学报,2024,49(12):1556-1565. DOI: 10.13406/j.cnki.cyxb.003683.
Liu Kaiwen,Ye Yuanlan,Guo Fengjin.Coumarin alleviates osteoarthritis by regulating senescence-associated secretory phenotype and inhibiting cell senescence: an experimental study[J].Journal of Chongqing Medical University,2024,49(12):1556-1565. DOI: 10.13406/j.cnki.cyxb.003683.
目的
2
探究香豆素(coumarin)对骨关节炎(osteoarthritis,OA)疾病的影响及其具体作用机制。
方法
2
在Venny2.1.0网站将香豆素与OA的作用靶点交叉后,利用STRING数据库进行蛋白互作PPI分析,并利用Autodock vina1.1.2对筛选到的香豆素和OA疾病的靶点进行分子对接。接下来进行体外实验验证,实验分组设置为对照组、IL-1β组和IL-1β+coumarin组。CCK-8法检测香豆素对C28/I2细胞的细胞毒性,qPCR及免疫荧光检测各组OA相关标志物及SASP因子表达情况,并利用β-半乳糖苷酶染色检测细胞衰老水平。
结果
2
共筛选到香豆素作用靶点196个,其中香豆素与OA共同作用靶点127个。PPI分析筛选到了CCND1、EGFR、ERBB2、MAPK14等4个香豆素治疗OA的可能的核心靶点。分子对接结果显示,香豆素与核心靶点CCND1、EGFR、ERBB2、MAPK14能形成较稳定的复合物。CCK-8结果显示,5~15 μmol/L香豆素处理C28/I2细胞促进细胞增殖,而高于20 μmol/L的香豆素处理则会抑制细胞活性(
P
<
0.05);其中10 μmol/L香豆素在24 h、48 h、72 h均会促进细胞增殖(
P
<
0.05),且处理48 h增殖最为明显(
P
<
0.05)。qPCR结果显示,10 μmol/L的香豆素处理48 h能降低C28/I2细胞中细胞衰老标志基因p16、p21的mRNA水平,β-半乳糖苷酶染色的结果与qPCR的结果一致。香豆素处理后SASP因子IL-1β、IL-6、TNF-α、MMP9、MMP13的mRNA水平降低(
P
<
0.05)。同样地,免疫荧光结果也显示香豆素处理后部分SASP因子表达水平降低。
结论
2
香豆素可能通过作用于p38/MAPK14通路,抑制SASP导致的细胞衰老及软骨分解,从而治疗骨关节炎。
Objective
2
To investigate the effect of coumarin on osteoarthritis(OA) and its specific mechanism of action.
Methods
2
Venny2.1.0 website was used to obtain the intersecting targets of coumarin and OA,STRING database was used to perform a protein-protein interaction(PPI) analysis,and Autodock
vina1.1.2 was used to perform molecular docking between the targets of coumarin and the disease targets of OA. An
in vitro
experiment was conducted,and the cells were divided into control group,IL-1β group,and IL-1β+coumarin group. CCK-8 assay was used to observe the cytotoxicity of coumarin on C28/I2 cells,qPCR and immunofluorescence assay were used to measure the expression of OA-related markers and SASP factors in each group,and β-galactosidase staining was used to measure the level of cell senescence.
Results
2
A total of 196 action targets were obtained for coumarin,including 127 intersecting targets of coumarin and OA. PPI analysis obtained four possible core targets of coumarin in treating OA,i.e.,CCND1,EGFR,ERBB2,and MAPK14. Molecular docking showed that coumarin could form a relatively stable complex with the core targets
CCND1,EGFR
,ERBB2,and MAPK14. CCK-8 assay showed that treatment of C28/I2 cells with 5-15 μmol/L coumarin promoted cell proliferation,while treatment with coumarin at a concentration of
>
20 μmol/L inhibited viability(
P
<
0.05);in addition,10 μmol/L coumarin promoted cell proliferation at 24,48,and 72 hours(
P
<
0.05),with the most significant proliferation at 48 hours(
P
<
0.05). The results of qPCR showed that treatment with 10 μmol/L coumarin for 48 hours could reduce the mRNA levels of the cell senescence marker genes p16 and p21 in C28/I2 cells,and β-galactosidase staining obtained consistent results with qPCR. There were significant reductions in the mRNA levels of the SASP factors IL-1β,IL-6,TNF-α,MMP9,and MMP13 after coumarin treatment(
P
<
0.05). Similarly,immunofluorescence assay also showed reductions in the expression levels of some SASP factors after coumarin treatment.
Conclusion
2
Coumarin may exert a therapeutic effect on OA by acting on the p38/MAPK14 pathway and inhibiting cell aging and cartilage decomposition caused by SASP.
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