Myricetin alleviates H2O2-induced senescence and apoptosis in rat nucleus pulposus-derived mesenchymal stem cells.

IF 1.7 4区生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY Folia histochemica et cytobiologica Pub Date : 2023-01-01 DOI:10.5603/FHC.a2023.0007

Tian Xie, Ruijie Pan, Wenzhuo Huang, Sheng Dong, Shizhen Wu, Yuhui Ye

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引用次数: 1

Abstract

Introduction: Transplantation of mesenchymal stem cells (MSCs) has been reported to be a novel promising target for the regeneration of degenerated intervertebral discs (IVDs). However, the culture and survival limitations of MSCs remain challenging for MSC-based biological therapy. Myricetin, a common natural flavonoid, has been suggested to possess antiaging and antioxidant abilities. Therefore, we investigated the biological function of myricetin, and its related mechanisms involving cell senescence in intervertebral disc degeneration (IDD).

Material and methods: The nucleus pulposus-derived mesenchymal stem cells (NPMSCs) were isolated from 4-month-old Sprague-Dawley (SD) rats and identified by examining surface markers and multipotent differentiation. Rat NPMSCs were cultured in an MSC culture medium or culture medium with different concentrations of H2O2. Myricetin or the combination of myricetin and EX527 were added to the culture medium to investigate the effects of myricetin. Cell viability was evaluated by cell counting kit-8 assays (CCK-8). The apoptosis rate was determined using Annexin V/PI dual staining. The mitochondrial membrane potential (MMP) was analyzed by a fluorescence microscope after JC-1 staining. The cell senescence was determined by SA-β-Gal staining. MitoSOX green was used to selectively estimate mitochondrial reactive oxygen species (ROS) Apoptosis-associated proteins (Bax, Bcl2, and cleaved caspase-3), senescence markers (p16, p21, and p53), and SIRT1/PGC-1α signaling pathway-related proteins (SIRT1 and PGC-1α) were evaluated by western blotting.

Results: The cells isolated from nucleus pulposus (NP) tissues met the criteria for MSCs. Myricetin showed no cytotoxicity up to a concentration of 100 μM in rat NPMSCs cultured for 24 h. Myricetin pretreatment exhibited protective effects against H₂O₂-induced apoptosis. Myricetin could also alleviate H₂O₂-induced mitochondrial dysfunctions of increased mitochondrial ROS production and reduced MMP. Moreover, myricetin pretreatment delayed rat NPMSC senescence, as evidenced by decreased exppression of senescence indicators. Pretreatment of NPMSCs with 10 μM EX527, a selective inhibitor of SIRT1, prior to exposure to 100 μM H2O2, reversed the inhibitory effects of myricetin on cell apoptosis.

Conclusions: Myricetin could affect the SIRT1/PGC-1α pathway to protect mitochondrial functions and alleviate cell senescence in H₂O₂-treated NPMSCs.

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杨梅素减轻h2o2诱导的大鼠髓核源间充质干细胞的衰老和凋亡。

导语:间充质干细胞(MSCs)移植已被报道为退变椎间盘(IVDs)再生的一个新的有希望的目标。然而，间充质干细胞的培养和生存限制仍然是基于间充质干细胞的生物治疗的挑战。杨梅素是一种常见的天然类黄酮，已被认为具有抗衰老和抗氧化能力。因此，我们研究了杨梅素在椎间盘退变(IDD)中参与细胞衰老的生物学功能及其相关机制。材料与方法:从4月龄的SD大鼠中分离得到髓核源间充质干细胞(NPMSCs)，并通过表面标记和多能分化进行鉴定。大鼠NPMSCs分别在MSC培养基和不同浓度H2O2培养基中培养。在培养基中加入杨梅素或杨梅素与EX527的组合，考察杨梅素的作用。采用细胞计数试剂盒-8 (CCK-8)测定细胞活力。Annexin V/PI双染色法测定细胞凋亡率。JC-1染色后，荧光显微镜下分析线粒体膜电位(MMP)。采用SA-β-Gal染色法测定细胞衰老情况。使用MitoSOX绿色选择性评估线粒体活性氧(ROS)凋亡相关蛋白(Bax, Bcl2和cleaved caspase-3)，衰老标志物(p16, p21和p53)， SIRT1/PGC-1α信号通路相关蛋白(SIRT1和PGC-1α)通过western blotting进行评估。结果:从髓核(NP)组织中分离的细胞符合MSCs的标准。杨梅素对培养24 h的大鼠NPMSCs在100 μM浓度下无细胞毒性。杨梅素预处理对h₂O₂诱导的细胞凋亡具有保护作用。杨梅素还可以减轻h2o2诱导的线粒体功能障碍，增加线粒体ROS的产生和降低MMP。此外，杨梅素预处理可延缓大鼠NPMSC衰老，衰老指标表达降低。在暴露于100 μM H2O2之前，用10 μM EX527 (SIRT1的选择性抑制剂)预处理NPMSCs，逆转了杨梅素对细胞凋亡的抑制作用。结论:杨梅素可通过影响SIRT1/PGC-1α通路，保护线粒体功能，缓解H₂O₂处理的NPMSCs细胞衰老。

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来源期刊

Folia histochemica et cytobiologica 生物-生化与分子生物学

CiteScore

2.80

自引率

6.70%

发文量

审稿时长

6-12 weeks

期刊介绍： "Folia Histochemica et Cytobiologica" is an international, English-language journal publishing articles in the areas of histochemistry, cytochemistry and cell & tissue biology. "Folia Histochemica et Cytobiologica" was established in 1963 under the title: ‘Folia Histochemica et Cytochemica’ by the Polish Histochemical and Cytochemical Society as a journal devoted to the rapidly developing fields of histochemistry and cytochemistry. In 1984, the profile of the journal was broadened to accommodate papers dealing with cell and tissue biology, and the title was accordingly changed to "Folia Histochemica et Cytobiologica". "Folia Histochemica et Cytobiologica" is published quarterly, one volume a year, by the Polish Histochemical and Cytochemical Society.