研究目的

干细胞（Stem cell）具有自我更新并分化为多个细胞谱系的潜能，是机体内负责组织、器官发育和再生的最小组织单元，因此基于干细胞的再生医学技术具有极大治疗潜力。目前干细胞主要有四个来源，即胚胎组织、胎儿组织、成人组织以及诱导多能干细胞。虽然成人间充质干细胞的干性弱于其他三种细胞，但其不存在伦理或法律问题，且没有基因编辑相关风险。其中，脂肪来源干细胞（Adipose-derived stem cells, ADSCs）在人体含量丰富且易于微创获取，具有间充质、内胚层、和外胚层谱系的分化能力，是干细胞治疗和再生医学领域的热点种子细胞。在使用ADSCs治疗时，无论细胞疗法抑或基于ADSCs的组织工程疗法，移植后因缺血缺氧、机械损伤以及炎症反应等不利因素引起的氧化应激严重影响治疗效果。氧化应激微环境抑制干细胞增殖，加剧细胞凋亡，甚至诱导干细胞向不利于治疗的方向分化。虾青素（Astaxanthin, Axt）是一种在海洋中广泛存在的类胡萝卜素，因其具有抗氧化、抗炎以及抗肿瘤的作用而备受青睐。本研究通过构建氧化应激损伤模型，探究虾青素对ADSCs氧化应激损伤的调控作用及其潜在机制。

研究方法

       本研究第一部分利用5位在本科室行腹部吸脂手术女性患者的脂肪样本进行ADSCs提取与鉴定，随后探究虾青素对正常状态下ADSCs细胞活动的影响以及虾青素是否会激活ADSCs内核因子E2相关因子2（Nuclear factor erythroid2-related factor 2，Nrf2）信号通路。通过酶消化和离心提取血管基质组分（Stromal vascular fraction， SVF），然后使用贴壁培养法提纯得到ADSCs。通过成脂、成骨以及成软骨三系分化实验以及流式细胞表面标志分析对所使用的细胞进行鉴定。随后使用不同浓度的虾青素（0，1，2，5，10，20，40 μM）处理细胞24h或48h，使用CCK-8检测不同浓度虾青素对ADSCs细胞活性的影响，确定最佳作用浓度。在最佳浓度下探究虾青素对生理状态下ADSCs细胞增殖（Ki67以及Cyclin D1的免疫荧光染色）、细胞迁移（Transwell小室细胞迁移实验）以及细胞凋亡（Annexin V/PI染色+流式细胞分析）的影响。最后通过总Nrf2、胞质Nrf2、核Nrf2以及Kelch样ECH相关蛋白1（Kelch-like ECH associated protein 1，Keap1）蛋白水平检测（Western blot）以及Nrf2免疫荧光染色分析，判断Nrf2信号通路是否激活以及Nrf2是否有核转位现象。

       课题第二部分使用过氧化氢（H₂O₂）构建ADSCs氧化应激损伤模型，评估模型的有效性，并且利用该模型评估虾青素在氧化应激环境下对ADSCs的保护作用。使用不同浓度H₂O₂（0，50，100，200，400 μM）处理细胞24小时后检测细胞活性。选择效果较明显的作用浓度分析模型中ADSCs的细胞增殖（Cyclin D1）、细胞凋亡（cleaved Caspase 3）、细胞外基质合成（COL1A1及COL2A1）、成脂能力（PPAR-γ）相关蛋白的表达以及促炎细胞因子白细胞介素-6（Interleukin 6，IL-6）和肿瘤坏死因子-α（Tumor necrosis factor alpha，TNF-α）的分泌水平，确定H₂O₂造模的最佳浓度。使用虾青素（0，2，5，10 μM）预处理脂肪干细胞3 h再引入氧化应激损伤，评估虾青素对ADSCs的保护作用。除此之外，本部分实验利用活性氧簇（Reactive oxygen species，ROS）含量、丙二醛（Malondialdehyde，MDA）水平以及超氧化物歧化酶（Superoxidase dismutase，SOD）活性的检测评估ADSCs氧化应激水平。

       研究第三部分探究虾青素调控ADSCs氧化应激损伤的作用机制。收集经虾青素预处理（0，2，5，10 μM）的ADSCs并提取总蛋白，通过分析Nrf2及其下游抗氧化蛋白的表达情况，包括血红素加氧酶-1（Heme oxygenase-1，HO-1），NADPH醌氧化还原酶（NADPH quinone oxidoreductase，NQO1）以及谷胱甘肽过氧化物酶4（Glutathione peroxidase 4，Gpx4），判断Nrf2信号通路是否激活。ML385是Nrf2特异性抑制剂，首先确定ML385对本模型中ADSCs的最佳抑制浓度，再将细胞分为空白对照组（Control）、阴性对照组（Vehicle）、虾青素组（Axt）以及抑制剂组（Axt + ML385），验证Nrf2信号通路是否参与介导虾青素对ADSCs氧化应激损伤的调控作用。结果表明虾青素对ADSCs有较显著的抗凋亡作用，且这种作用不能被ML385完全阻断，通过检测经典凋亡通路Bcl-2/Bax/Caspase 3相关蛋白以及线粒体膜电位变化（JC-1），探索虾青素缓解氧化应激相关ADSCs凋亡的作用机制。

研究结果

       第一部分实验中，脂肪样本来源的细胞在光镜下具有成纤维细胞样形态，具有向软骨、骨以及脂肪谱系分化的能力，流式细胞分析提示其阳性细胞标志物为CD73、CD105以及CD90，阴性细胞标志物为CD1b、CD19、CD34以及CD45，符合脂肪干细胞特性。CCK8细胞活性检测提示，虾青素对体外培养的ADSCs无细胞毒性，2-20 μM 虾青素促进ADSCs细胞活性，其中10 μM作用效果最显著。此外，虾青素促进ADSCs细胞增殖以及细胞迁移，且不会诱导细胞凋亡。经虾青素处理后，ADSCs内总Nrf2表达上升，其下游抑制性蛋白Keap1表达下降，细胞核内Nrf2/细胞质Nrf2比例显著提高；同时，Nrf2免疫荧光染色提示，相比于空白对照组，虾青素组Nrf2进入细胞核内，表明虾青素能够引起Nrf2核转位并激活Nrf2信号通路。

       本研究第二部分成功构建ADSCs移植早期氧化应激损伤模型，明确虾青素对ADSCs氧化应激损伤的保护作用。200μM H₂O₂显著降低ADSCs细胞活性，抑制细胞增殖，促进细胞凋亡；细胞外基质中的重要组分COL1A1和COL2A1的合成显著降低，并且ADSCs脂肪分化潜能受损；细胞培养基中IL-6和TNF-α水平升高，提示存在炎症微环境。虾青素预处理能够保护ADSCs的细胞功能，恢复部分COL1A1和COL2A1的合成以及成脂分化能力。虾青素通过清除ROS，降低MDA水平以及提高总SOD活性，显著缓解细胞氧化应激。此外，虾青素抑制促炎细胞因子IL-6和TNF-α的分泌，改善炎症微环境。

       第三部分实验结果表明，虾青素在发挥抗氧化应激作用中激活了Nrf2/Keap1信号通路，细胞内总Nrf2以及其下游蛋白（包括HO-1、NQO1和GPX4）表达均上调。为了进一步验证Nrf2信号通路在虾青素细胞保护作用中的地位，我们利用ML385抑制Nrf2激活。结果表明，4 μM ML385 显著抑制Nrf2蛋白的表达，并能阻断虾青素对ADSCs的细胞保护作用。此外，JC-1线粒体膜电位染色揭示虾青素对ADSCs线粒体功能具有保护作用，Bcl-2/Bax/Caspase 3通路蛋白检测提示虾青素显著降低Bax以及cleaved Caspase 3的蛋白水平，提高Bcl-2/Bax比例，进而减轻氧化应激导致的细胞凋亡。

研究结论

       我们的研究结果表明：1）我们所提取的细胞符合脂肪来源干细胞的定义，虾青素能够促进正常培养条件下ADSCs的增殖、迁移活动，不诱导细胞凋亡，并能上调Nrf2并促进其核转位；2）氧化应激损伤降低ADSCs细胞活性，抑制细胞增殖，降低ADSCs成脂分化能力，减少细胞外基质的分泌以及诱发炎症微环境，进一步促进细胞凋亡。虾青素预处理能够保护ADSCs，显著缓解氧化应激，提高抗氧化能力，减轻氧化应激导致的细胞功能受损；3）Nrf2/Keap1信号通路介导虾青素对ADSCs氧化应激损伤的调控作用，此外虾青素可能通过Bcl-2/Bax/Caspase 3信号通路保护ADSCs线粒体功能，从而抑制ADSCs凋亡。

Background

With self-renewal and multi-lineage differentiation capacity, stem cells are the smallest units for development and regeneration of organ and tissue systems. Therefore, stem cell-based regenerative medicine has substantial therapeutic potential. At present, there are four main sources of stem cells, namely embryonic tissues, fetal tissues, adult tissues, and induced pluripotent stem cells. Clinical use of embryonic stem cells, fetal stem cells, or induced pluripotent stem cells is limited because of ethical or legal issues even though these cells are theoretically highly beneficial. Adult mesenchymal stem cells appear to be an ideal stem cell population for regenerative medicine. Among these cells, adipose-derived stem cells (ADSCs) with the potential to differentiate into the mesenchymal, endoderm, and ectoderm lineages, are abundant and easy to obtain through minimally invasive procedures. Thus, ADSCs are potential seed cells in stem cell therapy and regenerative medicine. During ADSCs treatment, oxidative stress caused by ischemia, hypoxia, mechanical injury, or inflammation affects the therapeutic effect. The oxidative stress microenvironment inhibits stem cell proliferation, aggravates cell apoptosis, and even induces stem cells to differentiate in a detrimental direction. Astaxanthin (Axt), a form of carotenoid referred to as xanthophyll, is widely distributed in the ocean and exhibits anti-oxidative, anti-inflammatory, and anti-tumor properties. In this study, by constructing an oxidative stress injury model, we explored the regulatory effect of Axt on oxidatively stressed ADSCs and the underlying mechanism.

Methods

In the first instance, ADSCs were isolated and characterized from five healthy adipose tissue samples obtained by selective abdominal liposuction in our department. Next, we examined the effects of Axt on ADSCs activities under physiological conditions and determined whether nuclear factor erythroid2-related factor 2 (Nrf2) signaling was activated. The stromal vascular fraction (SVF) was obtained by enzymatic digestion and centrifugation, and ADSCs were purified through adhesive cultivation of the SVF. Multilineage differentiation and a mesenchymal surface marker flow cytometry analysis were used to characterize ADSCs. Cell counting kit-8 (CCK-8) assays were conducted to evaluate the toxic effects of Axt at various concentrations (1, 2, 5, 10, 20, and 40 μM) on ADSCs for 24 h and 48 h and the optimal concentration was ascertained. Axt was evaluated for its effects on ADSCs proliferation, migration, and apoptosis respectively by immunofluorescence staining of Ki67 and Cyclin D1, a transwell migration assay, and Annexin V/PI staining. At last, Nrf2 activation and translocation were observed through semi-quantification of total Nrf2, total Kelch-like ECH associated protein 1 (Keap1), nuclear Nrf2, and cytoplasmic Nrf2 through Western blot and immunofluorescence staining of Nrf2.

Secondly, we developed and evaluated a cellular oxidative stress model induced by hydrogen peroxide to determine whether Axt protects ADSCs against oxidatively insult. To determine the ideal condition, we evaluated the effect of H₂O₂ on ADSCs at increasing concentrations (0, 50, 100, 200, 400 μM) by CCK-8 assay. The following western bolt and ELISA assay detected the expression of marker proteins in cell proliferation (Cyclin D1), cell apoptosis (cleaved Caspase 3), extracellular matrix synthesis (COL1A1, COL2A1), and adipogenic potential (PPAR-γ) and secretion of pro-inflammatory cytokines including Interleukin 6 (IL-6) and Tumor necrosis factor alpha (TNF-α). According to these results, 200 μM H₂O₂ was applied in the subsequent research to induce oxidative imbalance. Before introducing H₂O₂, we pretreated ADSCs with Axt (0, 2, 5, 10 μM) for 3 h to assess Axt's cytoprotective effects. In addition to the examinations mentioned before, we determined the content of reactive oxygen species (SOD), malondialdehyde (MDA), and superoxidase dismutase (SOD) to depict the oxidative stress context.

The third objective of this study is to investigate the mechanism by which Axt modulates oxidative stress in ADSCs. To determine whether the Nrf2/Keap1 signaling pathway is activated, we measured the expression of Nrf2 and downstream proteins such as heme oxygenase-1 (HO-1), NADPH quinone oxidoreductase (NQO1), and glutathione peroxidase 4 (Gpx4). In addition, we determined whether the Nrf2/Keap1 signaling pathway mediates Axt's cytoprotective effect, by using ML385, a specific inhibitor of Nrf2. Furthermore, we also performed JC-1 staining and detection of the Bcl-2/Bax/Caspase 3 pathway to explore Axt's potent anti-apoptosis property which cannot be totally blocked by ML385.

Results

In the first section, the fibroblast-like cells isolated from adipose samples were able to differentiate into adipose tissue, osseous tissue, and cartilage tissue with positive surface makers including CD73, CD105, and CD90 and negative makers including CD1b, CD19, CD34, and CD45, which were consistent with characteristics of ADSCs. CCK-8 assay revealed that Axt did not cytotoxically affect ADSCs. However, by contrast, 2-20 μM Axt significantly enhanced ADSCs’ cell viability and the concentration of 10 μM was optimal. Axt also promoted the proliferation and migration of ADSCs and did not induce apoptosis. Furthermore, compared with the control group, Axt remarkably upregulated total Nrf2, nuclear Nrf2, and cytoplasmic Nrf2 and downregulated Keap1. Immunofluorescence staining revealed that Axt exposure resulted in evident Nrf2 nuclear translocation. Based on the evidence, Axt elevates Nrf2 levels by inactivating its endogenous inhibitor, Keap1, and initiates Nrf2 translocation from the cytoplasm to the nucleus.

In the second section of this study, we established an oxidatively stressed ADSCs model and ascertained that Axt plays a cytoprotective role. The cell viability of ADSCs was significantly reduced by 200 μM H₂O₂ treatment. As a result of the oxidative insult, the proliferation of ADSCs was reduced, and prominent apoptosis was induced after 24 h of exposure to H₂O₂. In addition, the synthesis of COL1A1 and COL2A1 as well as the adipogenic capacity were impaired. Moreover, the increased secretion of IL-6 and TNF-α in culture media indicated an inflammation micro-environment. However, on account of Axt pretreatment, the cell activities of ADSCs were preserved in a dose-dependent manner under oxidative imbalance. With Axt, the synthesis of COL1A1 and COL2A1 was restored, as well as the adipogenic potential of ADSCs. By eliminating ROS content, reducing MDA levels, and increasing SOD activity, Axt greatly relieved oxidative stress. In addition, Axt mitigated inflammation by suppressing the secretion of IL-6 and TNF-α.

In the last section, we demonstrated that Axt exerts cytoprotective effects on ADSCs through activation of the Nrf2/Keap1 signaling pathway, in which the expression of total Nrf2 and downstream proteins including HO-1, NQO1, and Gpx4 was upregulated. Subsequently, to confirm that Axt's cytoprotective effects were mediated by Nrf2, we inhibited Nrf2 expression and function through ML385. 4 μM ML385 treatment led to a significant reduction in total Nrf2 expression and abolished the protective effects of Axt. Flow cytometry revealed that Axt also exerted a potent anti-apoptosis feature that cannot be fully reversed by ML385. We subsequently examined the Bcl-2/Bax/Caspase 3 pathway and observed that Axt remarkably decreased the expression level of Bax and cleaved Caspase 3 whereas the Bcl-2/Bax ratio was increased. JC-1 staining revealed that Axt restored the mitochondrial membrane potential in oxidatively stressed ADSCs.

Conclusion

The characteristics of cells isolated from adipose tissue agreed with the definition of adipose-derived stem cells. Axt promotes cell viability, proliferation, and migration without inducing apoptosis, and initiates activation and translocation of Nrf2 in physiologically cultured ADSCs. Oxidative injury inhibits cell viability, proliferation, adipogenic capacity, and synthesis of ECM and triggers an inflammatory microenvironment, which induces ADSCs to apoptosis. Axt safeguards ADSCs and preserved the cell functions and activities by significantly alleviating oxidative stress levels. Axt's cytoprotective effects on oxidatively stressed ADSCs are mediated by the Nrf2 signaling pathway. Moreover, Axt is capable of downregulating BAX/Caspase 3 signaling pathway to prevent mitochondrial injury and mitigate cell apoptosis.