Objective To explore the role of intracellular reactive oxygen species (ROS) in the senescence of bone marrow mesenchymal stem cells(BMSCs)in patients with systemic lupus erythematosus(SLE) and the underlying mechanisms that controls the intracellular ROS levels in vitro. Methods Human bone marrow aspirates were collected from iliac of eight donors and eight SLE patients and cultured in vitro.Morphological appearance of BMSCs at different passages was examined by inverted microscope. Nuclear size was measured by fluorescence microscope. BMSCs were monitored using the senescence associated β-galacto-sidase (SAβ-gal) assay to characterize senescence in vitro. The quantification of intracellular ROS production was detected by flow cytometry. Real-time PCR technique was used to determine the gene expressions of PI3K, KRas, NRas and FoxO3 at transcription level. The expression of FoxO3, phospho-FoxO3 (p-FoxO3),AKT and phospho-AKT (p-AKT) protein were determined by Western blot analysis. Statistical analysis was conducted with t-test and Mann-Whitney rank test.Results There were no differences in morphology and nuclear size[(31.4±4.5) vs (28.2±4.8) μm, P=0.628] of BMSCs between SLE patients and normal controls.The percentage of SA β-gal positive BMSCs from SLE patients was higher than that from healthy controls [(31.8±9.0)% vs (12.4±0.7)%, P＜0.05]. Intracellular ROS levels of BMSCs from SLE patients increased more significantly than healthy donors in vitro (34600±9600 vs 17 958±5400, P＜0.05). No significant differences in the expression of PI3K, NRas, KRas and FoxO3 from SLE subjects were observed at mRNA levels compared with normal controls, though all showed a similar upward trend. The expression of p-FoxO3 and p-AKT of BMSCs from SLE patients increased significantly compared with healthy controls at protein levels.Conclusion These data suggest that BMSCs from SLE patients aged more quickly, with high SA β-gal activity and up-regulation of intracellular ROS, which is associated with up-regulation of p-FoxO3 and pAKT at protein levels. These results indicate that bone marrow mesenchymal cell senescence may be associated with the pathogcnesis of SLE by maintaining the lifespan of BMSCs.

Objective To investigate the apoptosis of bone marrow mesenchymal stem cells(BMSCs)from systemic lupus erythematosus(SLE)patients and the expression of apoptotic molecules.Methods BMSCs were isolated from bone marrow of SLE patients and normal controls by density eentrifugation and adhesive culture in vitro.The apoptosis of BMSCs were evaluated by TUNEL assay.The expressions of Fas.Bcl-2 and the activity of Caspase 8 were detected by flow cytometry.Real-time PCR technique was used to determine the gene expressions of Fas,Bcl-2,Bax,Bcl-w,Caspase 8 and Apaf-1.Meanwhile,cytochrome C was detected by immunocytochemistry.Statistical analysis was conducted with t-test and Mann-Whitney rank test.Results The percentage of apoptotic BMSCs increased in SLE patients compared with healthy donors[(64±10)%vs [14±9)%,U=0,P<0.05 ].The expression of Bcl-2 in BMSCs of SLE patients was lower than the normal controls at protein leve[(11±9)%vs(56±18)%,U=0,P<0.05 ],and mRNA level(0.2±0.2vs 2.4±0.7,U=24,P<0.05).More cytochrome C positive pellets in the cytosolic fraction could be detected in BMSCs from SLE patients compared with healthy controls [(56±21)%vs (16±16)%,U=1,P<0.05].The activity of Caspase 8 was enhanced[(49±14)%vs(16±12)%,U=0.P<0.05 ],although with no significant difierence at mRNA Ievel.Both groups expressed Fas but with no significant difference (U=19,P>0.05).Conclusion BMSCs from SLE patients undergo more apoptosis,the mechanisms may be associated with the down regulation of Bcl-2,up-regulation of Cytoehrome C in cytoplasm and the activation of Caspase 8,which directs the intrinsic and extrinsic apoptosis pathways.

ObjectiveTo investigate the effect of IκB kinase (IKK-β) on migration and proliferation of bone marrow mesenchymal stem cells(BMSCs) from patients with systemic lupus erythematosus (SLE).MethodsHuman bone marrow aspirates were collected from iliac of six donors and six SLE patients and cultured in vitro.Migration of BMSCs were observed by wound healing and transwell migration assays.Proliferation of BMSCs was quantified by cell counting kit-8 assay.Total RNA was extracted and reverse transcribed into complementary DNA.Real-time PCR technique was used to determine the gene expression of IKK-β at transcription level.The expression of IKK-β and phospho-IKK-β(p-IKK-β) protein were determined by Western blotting analysis.Statistical analysis was conducted with or Mann-Whitney rank test.Results① The migration rate of BMSCs from SLE patients(5.2±3.8)‰ were significantly reduced as compared with normal controls (7.0±2.9)‰(P＜0.05 ).The proliferation of BMSCs of SLE patients (0.21±0.49)was lower than that from healthy controls ( 1.00±0.35 )(P＜0.05 ).②) No difference in IKK-β3 mRNA expression between SLE ( 1.9± 1.4) subjects and normal controls (1.9±2.4) (P＞0.05).IKK-β protein expression of BMSCs from SLE patients (1.41 ±0.19) increased significantly compared with healthy controls (0.93±1.24) (P＜0.05).③ Inhibitor of IKK-β caused a significant increase in cell migration (3.3±1.6)‰ and proliferation (1.13±0.26) of BMSCs from SLE patients compared with untreated cells (2.3±1.1)‰ and (0.81±0.17),respectively (P＜0.05).ConclusionMigration and proliferation of BMSCs are significantly decreased in SLE patients.IKK-β may be involved in migration and proliferation of BMSCs.