黄芩素对TBHP 诱导小鼠髓核细胞衰老的保护作用
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作者单位:

1.广州中医药大学,广州 510405;2.广州中医药大学第一附属医院,广州 510405;3.广州中医药大学岭南医学研究中心,广州 510405

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R-33


Baicalein protects mouse nucleus pulposus cells from TBHP-induced senescence
Author:
Affiliation:

1. Guangzhou University of Chinese Medicine, Guangzhou 510405, China. 2. First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510405. 3. Lingnan Medical Research Center, Guangzhou University of Chinese Medicine, Guangzhou 510405

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    摘要:

    目的 研究黄芩素(Baicalein,BAI)对叔丁基过氧化氢(tert-Butyl hydroperoxide, TBHP)诱导的小鼠髓核细胞(nucleus pulposus cells,NPCs)的影响。 方法 取8 周龄小鼠,体外分离培养髓核细胞,传代至P2~P3 代。将实验分为对照组、模型组及黄芩素组3 组。β 半乳糖苷酶染色检测髓核细胞衰老变化。实时荧光定量PCR(Real-time quantitative PCR,RT-qPCR)检测聚集蛋白聚糖(aggrecan)、Ⅱ型胶原蛋白(collagen Ⅱ)、基质金属蛋白酶-3(matrix metalloproteinase-3,MMP-3)、基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)、肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(IL-1β)、p53、p16 mRNA 表达。免疫荧光法(immunofluorescence,IF)检测TNF-α 表达水平。免疫印迹试验(Western blot)检测白细胞介素-6(IL-6)、aggrecan、p53、p16、IL-1β、TNF-α蛋白表达。 结果 TBHP 最佳干预浓度为100 μmol/ L,干预时间4 h;黄芩素最佳干预浓度为1. 0 μg/ mL,干预时间为24 h。与对照组比较,模型组β 半乳糖染色阳性率较对照组显著升高(P<0. 05),p53、p16、IL-1β、TNF-α mRNA及蛋白表达显著升高(P<0. 05),MMP-3 mRNA 表达显著升高(P<0. 05),MMP-9 mRNA 表达升高,差异无统计学意义(P>0. 05),aggrecan mRNA 及蛋白表达显著降低(P<0. 05),collagen Ⅱ mRNA 表达降低,差异无统计学意义(P>0. 05),IL-6 蛋白表达升高,差异无统计学意义(P>0. 05);与模型组比较,黄芩素组β 半乳糖染色阳性率较模型组明显降低(P<0. 05),p53、p16、IL-1β、TNF-α mRNA 及蛋白表达显著降低(P<0. 05),MMP-3、MMP-9 mRNA 表达显著降低(P<0. 05),aggrecan mRNA 及蛋白表达显著升高(P<0. 05),collagen Ⅱ mRNA 表达显著升高(P<0. 05),IL-6蛋白表达显著降低(P<0. 05)。 结论 黄芩素能延缓TBHP 诱导的小鼠髓核细胞衰老的进程。

    Abstract:

    Objective To investigate whether Baicalein (BAI) protects nucleus pulposus cells (NPCs) exposed to tert-Butyl hydroperoxide (TBHP). Methods NPCs were extracted from 8-week-old mice and cultured to passages 2 and 3 for experiments in control, model, and baicalein groups. SA-β-galactosidase staining was used to detect senescence of NPCs. Real-time quantitative PCR was used to measure aggrecan, collagen Ⅱ, matrix metalloproteinase-3 (MMP-3), matrix metalloproteinase-9 (MMP-9), tumor necrosis factor α (TNF-α), interleukin (IL)-1β, p53 and p16 mRNA expression. TNF-α was detected by immunofluorescence. Protein expression of aggrecan, p53, p16, IL-1β and TNF-α was detected by Western blot. Results The concentration of TBHP was 100 μmol/ L, and the treatment time was 4 h. The optimal concentration of baicalein was 1 μg/ mL with a treatment time of 24 h. Compared with the control group, the positive rate of SA-β-galactose staining in the TBHP group was significantly increased (P<0. 05), p53, p16, IL-1β and TNF-α mRNA and protein expression was significantly increased ( P<0. 05), and MMP-3 mRNA expression was significantly increased (P<0. 05). MMP-9 mRNA expression was increased, but the difference was not statistically significant (P>0. 05), aggrecan mRNA and protein expression was significantly decreased (P<0. 05), collagen Ⅱ mRNA expression was decreased, but the difference was not statistically significant (P>0. 05), and IL-6 protein expression was increased, but the difference was not statistically significant (P>0. 05). Compared with the model group, the positive rate of SA-β-galactose staining in the baicalein group was significantly lower (P<0. 05). p53, p16, IL-1β, and TNF-α mRNA and protein expression was significantly lower (P<0. 05), and MMP-3 and MMP-9 mRNA expression was significantly lower (P<0. 05). Aggrecan mRNA and protein expression was significantly increased (P<0. 05), collagen Ⅱ mRNA expression was significantly increased (P<0. 05), and IL-6 protein expression was significantly decreased (P<0. 05). Conclusions Baicalein delays senescence of NPCs induced by TBHP.

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甘延池,何嘉辉,尚 奇,陈桂锋,陈弘林,张学来,张 友,颜先伟,刘慧雯,梁 德,任 辉,江晓兵.黄芩素对TBHP 诱导小鼠髓核细胞衰老的保护作用[J].中国比较医学杂志,2023,33(5):61~68.

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  • 收稿日期:2022-09-27
  • 在线发布日期: 2023-06-15
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