杨梅素减轻 H2O2 诱导的大鼠髓核间充质干细胞衰老和凋亡。

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

机构信息

Department of Orthopedics, Wuhan Hospital of Traditional Chinese Medicine, Wuhan, China.

出版信息

Folia Histochem Cytobiol. 2023;61(2):98-108. doi: 10.5603/FHC.a2023.0007.

DOI:10.5603/FHC.a2023.0007

PMID:37435897

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.

摘要

简介

间充质干细胞（MSCs）的移植已被报道为退化的椎间盘（IVD）再生的一种有前途的新靶点。然而，MSC 的培养和存活限制仍然是基于 MSC 的生物治疗的挑战。杨梅素是一种常见的天然类黄酮，已被证明具有抗衰老和抗氧化能力。因此，我们研究了杨梅素的生物学功能及其与椎间盘退变（IDD）中细胞衰老相关的机制。

材料和方法

从 4 月龄 Sprague-Dawley（SD）大鼠的髓核中分离出间充质干细胞（NPMSCs），并通过表面标志物和多能分化进行鉴定。将大鼠 NPMSCs 在 MSC 培养基或不同浓度 H2O2 的培养基中培养。将杨梅素或杨梅素与 EX527 的组合添加到培养基中以研究杨梅素的作用。通过细胞计数试剂盒-8 测定（CCK-8）评估细胞活力。通过 Annexin V/PI 双重染色测定细胞凋亡率。用 JC-1 染色后通过荧光显微镜分析线粒体膜电位（MMP）。通过 SA-β-Gal 染色确定细胞衰老。使用 MitoSOX 绿选择性估计线粒体活性氧（ROS）。通过蛋白质印迹法评估凋亡相关蛋白（Bax、Bcl2 和 cleaved caspase-3）、衰老标志物（p16、p21 和 p53）和 SIRT1/PGC-1α 信号通路相关蛋白（SIRT1 和 PGC-1α）。

结果

从髓核组织中分离的细胞符合 MSC 的标准。在培养 24 小时的大鼠 NPMSCs 中，浓度高达 100 μM 的杨梅素无细胞毒性。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 产生和降低 MMP。此外，杨梅素预处理可延迟大鼠 NPMSC 衰老，表现在衰老标志物表达减少。在用 100 μM H2O2 处理之前，用 10 μM EX527（一种选择性 SIRT1 抑制剂）预处理 NPMSCs，可逆转杨梅素对细胞凋亡的抑制作用。