The Effect Of ROS On F-actin Cytoskeleton And Migration Capacity Of MSCs From SLE Patients And Its Mechanisms

Objective:To investigate the impact of intracellular ROS (reactive oxygen species) level on F-actin cytoskeleton and migration capacity of MSCs (mesenchymal stem cells) in SLE (systemic lupus erythematosus) patients and the possible mechanisms as well as the effect of oral administration of NAC (N-acetylcysteine) on MRL/lpr mice.Methods:The F-actin cytoskeleton was observed by fluorescence microscopy after staining with Alexa Fluor594phalloidin. Transwell system was performed to determine the migration ratio of MSCs. RT-PCR (real time polymerase chain reaction) and western blot were carried out to detect changes of Rho signaling pathway. Serum level of SOD (superoxide dismutase) was detected by ELISA (enzyme-linked immunosorbent assay) and intracellular ROS level of MSCs was tested by flow cytometry. In order to find out the effect of ROS on F-actin cytoskeleton of MSCs, H2O2or NAC was added into the growth medium of MSCs to change their intracellular ROS level before testing. The osteogenesis ability was observed by staining with alizarin red. MSCs were co-cultured with PBMCs (peripheral blood mononuclear cells) for3days and the IFN-y and IL-4levels in the culture supernatant were subsequently detected by ELISA to analyze the immunomodulatory function of MSCs. The in vivo experiment was performed by oral administration of NAC in MRL/lpr mice for8weeks. The serum levels of anti-ds-DNA antibody and ANA (antinuclear antibody) as well as proteinuria were detected by ELISA and BCA (bicinhoninic acid) assay. The pathology of the kidney was analysed by HE staining. The bone marrow-derived MSCs were isolated and cultured. CFSE was used to stain the cells and tissues were collected after the infusion of MSCs into the MRL/lpr mice to analyse the in vivo homing.Results:F-actin cytoskeleton of MSCs was rearranged and highly expressed in SLE patients. The migration capacity was impaired in SLE MSCs as they were passaged. Both of the mRNA level and protein expression of RhoA and Racl were down-regulated in SLE MSCs while Cdc42was up-regulated. Reduced serum SOD level and higher intracellular ROS level were observed in SLE patients. The F-actin cytoskeleton of normal MSCs was rearranged after up-regulation of ROS level by H2O2and the migration ratio was subsequently reduced. The addition of NAC reversed the rearrangement of F-actin cytoskeleton and the impairment of migration capacity of SLE MSCs by down-regulating ROS level. H2O2inhibited^psteogenic ability of normal MSCs and NAC enhanced that of SLE MSCs. However, ROS had no effect on the immunomodulatory function of MSCs. Cdc42expression was up-regulated by H2O2while down-regulated by NAC. Oral administration of NAC reduced serum level of anti-ds-DNA antibody in MRL/lpr mice. Serum ANA level was also decreased with no significant difference. The proteinuria was decreased with an amelioration of kidney pathology. Bone marrow-derived MSCs from NAC treated MRL/lpr mice showed long straight F-actin, increased migration ratio and reduced ROS level compared with that from untreated mice. The in vivo homing to different organs of MSCs from treated mice was also enhanced.Conclusion:These experimental findings suggest that the rearrangement of F-actin cytoskeleton of MSCs in SLE patients leads to the impairment of their migration capacity. The high ROS level may contribute to it via up-regulation of Cdc42. Oral administration of NAC has therapeutic effect on MRL/lpr mice and partially reversed the abnormalities of their BM-MSCs.