| Background:Systemic lupus erythematosus(SLE)is an inflammatory autoimmune disease,involving in many tissues and organs dysfunctions.It is manifested by over expression of several pro-inflammatory cytokines and immune dysregulation.Our previous studies demonstrated that there were many dysfunctions of bone marrow MSCs derived from SLE patients,including decreased proliferation,enhanced senescence and apoptosis,abnormal cell skeleton and suppressed migration,dysregulated immune response.Additionally the efficacies of allogenic MSCs,from either bone marrow or umbilical cord were better than that of autologous MSCs for lupus.Both B6-MSCs and lupus-MSCs from young mice efficiently ameliorated SLE-like disease.However lupus-MSCs from older mice did not alleviate lupus,which suggested genetic factors did not result in MSCs dysfunction.Therefore the causes of MSCs dysfunction might be mainly attributed to systemic inflammatory microenvironment.Objective:The aim of the present study is to investigate whether inflammatory serum affects the senescence of MSCs,and which signaling pathway is critical in this senescent process.In consideration of complex ingredients in SLE serum,we need to make it clear that which inflammatory factor(s)in SLE serum and whether the key pathways above play an important role in MSCs senescence.Method:Human sera were collected from sex-matched,age-matched active SLE patients(n=8)and healthy volunteers(n=9)in Drum Tower Hospital,and then the complement was inactivated at 56℃ for 30min,eventually saved in the-80℃ freezer.UC-MSCs were cultured in DMEM/F12 containing 10%SLE or normal serum.The mRNA levels of p53 and p21 were detected by Real time PCR two days later.Three days later MSCs were stained with SA-β-gal.The percentages of SA-β-gal positive cells were evaluated.At the same time total protein was extracted from MSCs,The protein levels of p53/p21 and cell senescence related pathways,including Jak/stat,Ras/MEK/Erk,PI3K/Akt,NF-κB pathways,were detected by Western Blot.MSCs were pretreated with the specific inhibitors of activated pathways.The senescent effects of MSCs-induced by SLE serum were compared.Next 80 ceytokines and chemokines were detected in SLE serum and normal serum by protein microarray.Top 2 increased cytokines were demonstrated in more patients and healthy volunteers by ELISA.After blocking cell senescence related cytokines(IFNγ,IFNα,leptin and NAP2),the senescence of MSCs induced by SLE serum were further compared.Then MSCs were stimulated with the key factors alone or together in different concentrations,and the senescent effects were further evaluated.Several C57BL/6 mice,ob/ob mice and MRL/lpr mice were randomly divided into two groups(6 mice per group),respectively.One group was injected intraperitoneally(i.p.)with 0.2 ml recombinant mouse leptin(1μg/g,twice a day).The other group was treated with 0.2ml PBS for 3 days.Body weight was measured before and after leptin administration,Coomassie brilliant blue method was used to test the concentration of urine protein.Then the levels of serum leptin,autoantibodies(such as anti-nuclear antibody and anti-dsDNA antibody),total IgG were examined by ELISA.The subsets of CD4+CD25+Foxp3+regulatory T cells and CD4+IL-17+T cells were tested in peripheral blood and spleen by flow cytometry.Spleen mononuclear cells were cultured with 0 and 100ng/ml recombinant mouse leptin for 3 days.The subsets of Treg and Thl7 cells in vitro were also examined by flow cytometry.Next,the senescence of bone marrow MSCs was evaluated by SA-β-gal staining,p53/p21 mRNA by Real time PCR and the protein expression by Western blot.Results:Compared with healthy controls,increased percentage of SA-β-gal positive cells and elevated levels of p53/p21 were observed in MSCs from SLE patients.Moreover,MSCs cultured in SLE serum showed more SA-β-gal positive cells and higher levels of p53/p21.The expressions of p-statl/5,p-MEK,p-Erk,p-Akt were also significantly upregulated in senescent MSCs induced by SLE serum,which indicated that Jak/statl/5,MEK/Erk and PI3K/Akt pathways were all over-activated in this senescence process.Only LY294002(the specific inhibitor of PI3K/Akt)decreased the frequency of SA-p-gal positive MSCs and suppressed the levels of p53 and p21.The result of protein microarray and ELISA revealed that leptin and NAP2 were top 2 increased cytokines in SLE serum(>2.5 times).Blockade of leptin or NAP2 in SLE serum,but not IFNα or IFNγ,decreased the percentage of SA-β-gal positive MSCs,and the levels of p53/p21.Blockade both leptin and NAP2 could further decrease the frequeneies of seneseent eells.Leptin R and CXCR2 were detectable on MSCs cultured in FBS,normal serum or SLE serum.However,there is no statistically significant difference among these three groups.Leptin or NAP2 treatment also increased the number of senescent MSCs in a dose dependent manner,respectively.The senescent effect was further accelerated by stimulation with leptin and NAP2 together.However LY294002 administration almost completely abrogated MSCs senescence induced by leptin and NAP2.Compared with those PBS treated mice,leptin administration reduced the body weight of B6 mice,ob/ob mice and MRL/lpr mice,and elevated the serum leptin levels.In vivo,leptin remarkably increased urine protein,unregulated the production of autoantibodies,including anti-nuclear antibody(ANA),anti-dsDNA antibody(anti-dsDNA)and total IgG.In addition,leptin also upregulated the percentage of Th 17 cells,and downregulated the percentage of Treg cells in vivo and in vitro.Moreover,leptin also enhanced MSCs senescence in vivo,which was characterized by increased percentage of SA-p-gal positive cells and elevated levels of p53/p21.Conclusion:SLE serum accelerates the senescent effect of MSCs.The PI3K/Akt pathway is involved in this process.Leptin and NAP2 synergistically accelerate MSCs senescence in SLE serum through the activation of PI3K/Akt pathway.In vivo leptin treatment also enhances the senescence of bone marrow MSCs,and exacerbates lupus disease in MRL/lpr mouse model.Therefore leptin may accelerate SLE disease through indirectly promoting MSCs dysfunction or directly activating immune cells. |
