| Objective Previous studies revealed that rapamycin(RAPA) was effective in the treatment of systemic lupus erythematosus(SLE) patients and animal model. The bone marrow- mesenchymal stem cells(BM-MSCs) from SLE patients are senescent MSCs. This study was designed to further prove the effect of RAPA on MRL/lpr mice and its influence on the senescent phenotype of BM-MSCs and the immunoloregulation. We also investigated the role mTOR signaling pathway played in the senescence of BM-MSCs from SLE patients,and explored the possible mechanisms.Methods Twelve female BALB/c mice aged 16 weeks were enrolled in normal group. Meanwhile, twenty-four female MRL/lpr mice aged 16 weeks were enrolled and divided into control group and RAPA treatment group. We recorded the survival rate and the weight of mice and used coomassie brilliant blue staining to measure the 24-hour urinary protein excretion every two weeks.The mice were killed by cervical dislocation after 12 weeks. The level of anti-double-stranded DNA(ds-DNA) antibody in peripheral blood serum was detected by enzyme linked immunosorbent assay(ELISA). We used HE staining to observe the renal and pulmonary pathological changes. When the BM-MSCs were separated into three groups and then cultivated to passage 4 in vitro, cell morphology was observed and senescence associated β-galactosidase(SA-β-gal) staining was used to detect the activity of β-gal of cells. F-actinstaining was used to observe the changes of cytoskeletal structure. Cell Counting Kit(CCK)-8 method was used to detect the cell proliferation rate.Flow cytometry was used to detect the distribution of cell cycle. Western Blotting was used to detect the changes of cell cycle-related proteins. Three groups of BM-MSCs and purified CD4+ T cells were co-cultured indirectly.Flow cytometry was used to inspect the proportion of regulatory T(Treg) /T helper type 17(Th17). We used ELISA to observe the changes of transforming growth factor-β(TGF-β), interleukin-10(IL-10), IL-17 and IL-6 in cell culture supernatant. WB and immunofluorescence were used to analyze the changes of expression and localization of mTOR signaling pathway in three groups of BM-MSCs. BM-MSCs of SLE patients and normal individuals were isolated and cultivated in vitro. WB and immunofluorescence were used to distinguish the difference of expression and localization of m TOR signaling pathway between normal group and SLE group. The level of S6 phosphorylation triggered by RAPA, the mTOR inhibitor, was evaluated by WB under different concentrations or at different time. We used small interfering RNA(siRNA) to interfere the expression of mTOR, and detect the effects by reverse transcription polymerase chain reaction(RT-PCR), WB and immunofluorescence. After the intervention of RAPA and si RNA mTOR, we observed cell morphology and detected the activity of SA-β-gal, the changes of cytoskeletal structure, cell proliferation rate, the distribution of cell cycle, the expression of cell cycle-related proteins, the proportion of Treg/Th17 and the changes of cytokines. Finally, 1×106 of SLE BM-MSCs treated with RAPA were transplanted to cure the 8 MRL/lpr mice aged 16 weeks for 12 weeks, and then the survival rate, the weight, the 24-hour urinary protein excretion, the titer of ds-DNA, and the renal and pulmonary pathological changes of this treated group were compared with SLE BM-MSCs transplantation group and normal BM-MSCs transplantation group. The data were analyzed by SPSS 17.0.Results(1) Compared with the control group, the survival rate and the weight of the MRL/lpr mice treated with RAPA increased. We also found that the content of 24-hour urinary protein and the level of anti ds-DNA antibodiesdecreased. The results of HE staining showed that renal crescent formation and inflammatory cells infiltration also decreased distinctively. The damage index of tracheas and blood vessels and the infiltration degree of interstitial inflammatory cells were both reduced. Compared with normal group, the significant hypertrophic cell morphology, increase of SA-β-gal positive cells,disordered organization of cytoskeleton, slow proliferation rate of BM-MSCs,increased cell proportion of G0/G1 phase, and the high expression of cell cycle related proteins(p53, p21, p27) were found in the BM-MSCs from MRL/lpr mice. After RAPA treatment, the senescence phenotype in the BM-MSCs of MRL/lpr mice was reversed, but the cell proliferation remained unchanged.After the indirect co-cultivation with CD4+ T cells, the proportion of Treg cells in RAPA treatment group increased, and the proportion of Th17 cells decreased.Compared with the control group, the concentrations of anti-inflammatory factors TGF-β and IL-10 in the supernatant of the RAPA treatment group increased. Meanwhile, the concentrations of the inflammatory factors IL-17 and IL-6 decreased. The m TOR signaling pathway in the BM-MSCs of MRL/lpr mice was abnormally activated in comparison with BM-MSCs from normal group. However, the activation of mTOR signaling pathway in the BM-MSCs from RAPA treatment group was inhibited.(2) The mTOR signaling pathway in the SLE MSCs was abnormally activated in comparison with BM-MSCs from normal group. After the intervention of RAPA and m TOR siRNA, the cell volume and the number of SA-β-gal positive in SLE BM-MSCs was reduced,and the organization of cytoskeleton was neatly arranged. However, the rate of cell proliferation, the proportion of G0/G1 phase cells and the cell cycle related proteins remained unchanged. After the indirect co-cultivation with CD4+T cells, the proportion of Treg cells increased, while the proportion of Th17 cells decreased in the group interfered by RAPA and mTOR si RNA. The concentrations of TGF-β and IL-10 increased, but the concentrations of IL-17 and IL-6 decreased in the supernatant.(3) Compared with SLE BM-MSCs transplantation group, the survival rate and the weight of the MLR/lpr mice,which were transplanted with RAPA treated SLE MSCs, increased markedly,however, the content of 24-hour urinary protein and the level of anti ds-DNA antibodies decreased distinctively. The results of HE staining showed that renal and pulmonary pathological lesions were significantly reduced. However, this effect was inferior to that of the BM-MSCs transplantation group.Conclusions(1) RAPA treatment was effective on MRL/lpr mice.Through the inhibition of mTOR signaling pathway, the senescence phenotype of BM-MSCs from MRL/lpr mice was reversed, and the immunoloregulation was improved.(2) mTOR signaling pathway in BM-MSCs from SLE patients were in high expression. Through the intervention of RAPA and siRNA mTOR,the senescence phenotype of SLE BM-MSCs was inhibited and the immunoloregulation was improved.(3) SLE BM-MSCs transplantation treatment had no effect on MRL/lpr mice. However, the transplantation of SLE MSCs treated with RAPA was effective on MRL/lpr mice, but the therapeutic effect was inferior to that of the BM-MSCs transplantation group. |
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