Chinese Academy of Medical Sciences and Peking Union Medical College Plastic Surgery Hospital and Institute, Beijing, China.
Biosci Rep. 2022 Mar 31;42(3). doi: 10.1042/BSR20212808.
Adipose-derived stem cells (ADSCs) showed decreased cell viability and increased cell death under oxygen-glucose deprivation (OGD). Meanwhile, vital necroptotic proteins, including receptor-interacting protein kinase (RIP) 3 (RIP3) and mixed lineage kinase domain-like pseudokinase (MLKL), were expressed in the early stage. The present study aims to explore the effect of necroptosis inhibition on ADSCs. ADSCs were obtained from normal human subcutaneous fat and verified by multidirectional differentiation and flow cytometry. By applying cell counting kit-8 (CCK-8), calcein/propidium iodide (PI) staining and immunostaining, we determined the OGD treatment time of 4 h, a timepoint when the cells showed a significant decrease in viability and increased protein expression of RIP3, phosphorylated RIP3 (pRIP3) and phosphorylated MLKL (pMLKL). After pretreatment with the inhibitor of RIP3, necroptotic protein expression decreased under OGD conditions, and cell necrosis decreased. Transwell assays proved that cell migration ability was retained. Furthermore, the expression of the adipogenic transcription factor peroxisome proliferator-activated receptor γ (PPARγ) and quantitative analysis of Oil Red O staining increased in the inhibitor group. The expression of vascular endothelial growth factor-A (VEGFA) and fibroblast growth factor 2 (FGF2) and the migration test suggest that OGD increases the secretion of vascular factors, promotes the migration of human umbilical vein endothelial cells (HUVECs), and forms unstable neovascularization. ELISA revealed that inhibition of RIP3 increased the secretion of the anti-inflammatory factor, interleukin (IL)-10 (IL-10) and reduced the expression of the proinflammatory factor IL-1β. Inhibition of RIP3 can reduce the death of ADSCs, retain their migration ability and adipogenic differentiation potential, reduce unstable neovascularization and inhibit the inflammatory response.
脂肪来源的干细胞（ADSCs）在氧葡萄糖剥夺（OGD）下表现出细胞活力降低和细胞死亡增加。同时，早期表达重要的坏死性蛋白，包括受体相互作用蛋白激酶（RIP）3（RIP3）和混合谱系激酶结构域样假激酶（MLKL）。本研究旨在探讨坏死性抑制对 ADSCs 的影响。ADSCs 从正常人皮下脂肪中获得，并通过多向分化和流式细胞术进行验证。通过应用细胞计数试剂盒-8（CCK-8）、钙黄绿素/碘化丙啶（PI）染色和免疫染色，我们确定了 OGD 处理时间为 4 小时，此时细胞活力明显降低，RIP3、磷酸化 RIP3（pRIP3）和磷酸化 MLKL（pMLKL）的蛋白表达增加。在 RIP3 抑制剂预处理后，OGD 条件下坏死性蛋白表达减少，细胞坏死减少。Transwell 测定证明细胞迁移能力得以保留。此外，在抑制剂组中，脂肪生成转录因子过氧化物酶体增殖物激活受体γ（PPARγ）的表达和油红 O 染色的定量分析增加。血管内皮生长因子-A（VEGFA）和成纤维细胞生长因子 2（FGF2）的表达和迁移试验表明，OGD 增加了血管因子的分泌，促进了人脐静脉内皮细胞（HUVECs）的迁移，并形成不稳定的新血管化。ELISA 显示 RIP3 抑制增加了抗炎因子白细胞介素（IL）-10（IL-10）的分泌，并降低了促炎因子 IL-1β的表达。RIP3 的抑制可以减少 ADSCs 的死亡，保留其迁移能力和脂肪生成分化潜力，减少不稳定的新血管形成并抑制炎症反应。
