The Effects Of Toll-like Receptor (TLR)-4 Signaling Pathway On The Proliferation And Oxidative Stress-induced Apoptosis Of Mesenchymal Stem Cells (MSCs)

ObjectiveTo study the effects of Toll-like receptor (TLR)-4 signaling pathway on the proliferation as well as oxidative stress-induced apoptosis of mesenchymal stem cells (MSCs), and to explore the mechanisms underling the effects of TLR4 signaling pathway.MethodsMSCs were harvested from femurs of C57BL/10J(wild-type), C57BL/10ScNJ (TLR4lps-del), C57BL/6J(wild-type) as well as MyD88-knock out(-/-) mice respectively, and then were cultured by passing on. Phenotype and TLR4 expressed by the MSCs were assayed using flow cytometry analysis. To study the effect of TLR4 on proliferation of MSCs, the MSCs were treated with or without different concentration (0.1,1.0, and 10.0μg/ml, respectively) of lipopolysaccharides(LPS) for 48 hours or 72 hours, and then the number of MSCs were counted by Cell counting Kit-8 (CCK-8). To study the effect of TLR4 on survival of MSCs, MSCs were incubated with or without different concentration of LPS and then the MSCs underwent H2O2/serum-deprevation (H2O2/SD). The apoptosis of MSCs were analyzed on a FACScan flow cytometer and were further confirmed by detecting apoptotic nuclear morphology using fluorescence microscopy upon Hoechst 33258 staining. To explore the mechanisms underling the effects of TLR4, the phosphorylation of Akt Ser 473 and NF-κB p65 Ser 536 were detected by Western bloting with specific antibodies after MSCs had been treated with LPS for 30 minutes. IL-6 concentration in culture media of MSCs treated with LPS was determined by enzyme-linked immunosorbent assay (ELISA) for IL-6. To explore the effect of statin associating with LPS on the survival of MSCs, MSCs were administered with different dose of simvastatin in advance and then treated with LPS, and then the proliferation and apoptosis of MSCs were measured by the above-mentioned means.ResultsThe adherent, fibroblast-like cells that derived from mice bone marrow expressed phenotype of MSCs. The cultured MSCs incubated with 5-azacytidine (10μmol/L) for 24 hours and then cultured without 5-azacytidine for four weeks were able to express cardiac troponin I andα-sarcomeric actin when detected by immunocytochemistry. MSCs isolated from wild-type mice expressed TLR4, either at mRNA or receptor level. The proliferation of MSCs incubated with LPS 0.1μg/ml or 1.0μg/ml for 48 hours and LPS 1.0μg/ml for 72 hours were significantly higher than that of each control group. When LPS rose up to 10.0μg/ml, the proliferation of MSCs decreased adversely. However, LPS1.0μg/ml could not significantly promote the proliferation of MSCs derived from TLR4lps-del or MyD88-/- mice. Compared with control group, LPS 1.0μg/ml could significantly reduce apoptosis induced by H2O2/SD (P<0.001) while LPS 10μg/ml increased the apoptosis of MSCs adversely. Western bloting showed LPS 0.1 or 1.0μg/ml could enhance the phosphorylation both of Akt at Ser 473 and NF-κB p65 at Ser 536. When LPS rose up to 10μg/ml, the phosphorylation of Akt at Ser 473 or NF-κB p65 at Ser 536 lowered adversely. However, LPS could not enhanced the phosphorylation of Akt Ser 473 in TLR4lps-del MSCs.Low-dose of simvastatin (0.1,1.0μmol/L) associating with LPS could enhance the proliferation while could not significantly reduce the apoptosis of MSCs induced by H2O2/SD. ELISA showed that LPS (0.1,1.0,10.0μg/ml) could enhance MSCs to secret IL-6, which did not depend upon the concentration of LPS.ConclusionMSCs could be isolated and purified from the cells of bone marrow by passing on, and the MSCs were able to differentiate into myocardia-like cells. MSCs derived from bone marrow of mice expressed functional TLR4, both at mRNA and protein level. LPS could modulate the proliferation and oxidative stress-induced apoptosis of MSCs in vitro via TLR4 signaling pathway. The effects of LPS on the survival of MSCs were concentration-dependent and depended on TLR4/MyD88 signaling pathway. Low dose of LPS was helpful while high dose of LPS was adverse for the survival of MSCs. Appropriate dose of simvastatin pretreatment associating with low concentration of LPS could further enhance the proliferation and may be helpful for the survival of MSCs.