Inhibition of RIP3 increased ADSC viability under OGD and modified the competency of adipogenesis, angiogenesis, and inflammation regulation

Abstract

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 and mixed lineage kinase domain-like pseudokinase (MLKL), were expressed in the early stage. This 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 CCK8, calcein/PI staining and immunostaining, we determined the OGD treatment time of 4 hours, 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 gamma (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 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.

The necroptosis-inducing pseudokinase mixed lineage kinase domain-like regulates the adipogenic differentiation of pre-adipocytes

“…In this study, PPARγ was also negatively correlated with hepatic RIPK3 in patients with NAFLD ( Afonso et al., 2021 ). Moreover, the inhibition of RIPK3 using a Glaxo Smith Kline inhibitor increased the expression of PPARγ as well as triglyceride accumulation in ASC ( Yang et al., 2022 ). Thus, RIPK3 appears as a lipid metabolism regulator, which activity might be targeted ( e.g.…”

The Influence of Intervertebral Disc Microenvironment on the Biological Behavior of Engrafted Mesenchymal Stem Cells

“…These results suggest that IVD-like low-glucose conditions may be a positive factor for MSC transplantation in the treatment of IDD, while too low concentrations of glucose can have significant negative effects. Recently, Yang et al [109] further studied and found that oxygen-glucose deprivation AD-MSCs: adipose-derived mesenchymal stem cells; AF-MSCs: amniotic fluid-derived mesenchymal stem cells; BMSCs: bone marrow-derived mesenchymal stem cells; ECM: extracellular matrix; IL-1β: interleukin-1β; IVD: intervertebral disc; NP: nucleus pulposus; NP-MSCs: nucleus pulposus-derived mesenchymal stem cells; PB-MSCs: peripheral blood-derived mesenchymal stem cells; PMSCs: placenta-derived mesenchymal stem cells; SMSCs: synovialderived mesenchymal stem cells; TGF: transforming growth factor; UC-MSCs: umbilical cord-derived mesenchymal stem cells.…”

“…These results suggest that IVD-like low-glucose conditions may be a positive factor for MSC transplantation in the treatment of IDD, while too low concentrations of glucose can have significant negative effects. Recently, Yang et al [ 109 ] further studied and found that oxygen-glucose deprivation not only significantly inhibited the viability and adipogenesis of AD-MSCs but also inhibited cell proliferation and migration. Inhibition of receptor-interacting protein kinase-3 (RIP3) increased the viability, proliferation, and migration of AD-MSCs under oxygen-glucose deprivation and reduced unstable neovascularization and inhibited inflammatory responses [ 109 ].…”

“…Recently, Yang et al [ 109 ] further studied and found that oxygen-glucose deprivation not only significantly inhibited the viability and adipogenesis of AD-MSCs but also inhibited cell proliferation and migration. Inhibition of receptor-interacting protein kinase-3 (RIP3) increased the viability, proliferation, and migration of AD-MSCs under oxygen-glucose deprivation and reduced unstable neovascularization and inhibited inflammatory responses [ 109 ].…”

Identification of hub genes regulating the cell activity and function of adipose-derived stem cells under oxygen-glucose deprivation

“…When ADSCs are deprived of oxygen and glucose, some studies have demonstrated that ADSCs might regulate survival potential and stemness through autophagy ( Li et al, 2017 ; Yang et al, 2021 ). In our previous study, a preexperiment was carried out, and we developed an OGD model to imitate the hypoxic and glucose-free environment in vivo ( Yang et al, 2022 ). We also regulated RIP3, a key target in necroptosis, to relieve the effect of OGD treatment.…”