Expression Of HMGB1 In BM-MSCs Under Hypoxia And Effect Of HMGB1 On The Apoptosis And Adhesion Of BM-MSCs In Vitro

Objective Hypoxic preconditioning can regulate the survival ability of Mesenchymal Stem Cells(MSCs) and the mechanism is not clear. Under hypoxia, the levels of high mobility group box 1(HMGB1) will be increased by the cellular processes of secretion and apoptosis-associated release in many kinds of cells, it may be involved in the regulation of cell viability. The aim of this study is to prove that hypoxia can also induce the expression of HMGB1 in BM-MSCs and to investigate the role of HMGB1 in the apoptosis and adhesiveness of BM-MSCs. Both of these researches can offer new evidences and strategies to improve the survival ability of post-transplanted MSCs.Methods(1) MSCs were isolated and cultured from the femurs and tibias bone marrow of Sprague-Dawley rats, then the cells were identified by flow cytometric analysis.(2) Experiments were performed when cells of third passages became 80%-90% confluence. The cells were incubated in hypoxia incubator chambers in the presence of 1% O2 and normoxic controls were incubated in a humidified chamber containing 20% O2. To compare the difference of the expression of HMGB1 between hypoxic cultures and normoxic cultures, cells were harvested to accept the analysis of RT-PCR and Western blot after 24 hours culturing.(3) The serum-free DMEM was used to induce cell apoptosis. The third passages cells were seeded in 12-well plates, HMGB1 with different concentrations(0, 10, 50, 100 and 200ng/ml) were added when serum had withdrawed. After 24 hours of HMGB1 interfering, the cell apoptosis was evaluated by using the Annexin V-FITC/PI(AV-PI) apoptosis detection kit.(4) Fibronectin(FN) with a concentration of 1 μg/cm2 was used as a thin coating on 12-well plate surfaces overnight. MSCs were seeded in the 12-well plate, followed by 24 hours cultivation with HMGB1 at different concentrations. Then the nonadherent cells were removed by washing with PBS and the cells cohered to fibronectin were counted under microscopy.Results(1) Hypoxia resulted in a significant increase in the expression of HMGB1 m RNA and HMGB1 protein in MSCs after 24 hours of incubation when compared to cells under normoxia(P<0.05).(2) MSCs in the presence of different concentrations of HMGB1 were exposed to serum-free DMEM for 24 hours. Then, the apoptotic rates were examined by FACS analysis of AV/PI staining: Increased apoptosis rates of r BM-MSCs were observed after HMGB1 treatment. Compared with the serum-free DMEM culture group, the apoptotic rates of r BM-MSCs were significantly increased at the HMGB1 concentration of 50ng/ml and 100ng/ml(P<0.05). However, the apoptotic rates showed no significant difference at the HMGB1 concentration of 10ng/ml and 200ng/ml compared with the serum-free DMEM group.(3) With the increasing concentration of HMGB1, the adherent numbers of MSCs were increased. Compared with the single FN culture group(0ng/ml HMGB1), the umber of adhesive r BM-MSCs were significantly increased at the HMGB1 concentration of 50ng/ml、100ng/ml and 200ng/ml(P<0.05).Conclusions(1) Hypoxia can significantly increase the levels of HMGB1 in MSCs.(2) HMGB1 can accelerate apoptosis of r BM-MSCs, especially at the concentration of 50ng/ml and 100ng/ml. However, with the increase of the concentration of HMGB1, the effect of HMGB1 on cell apoptosis will be weaken.(3) HMGB1 can increase the adhesion ability of r BM-MSCs in a concentration-dependent manner.(4) Hypoxia can regulate the survival ability of MSCs, and its possible mechanism may be related with the up-regulation of HMGB1 and the change of biological effects on MSCs.