Protective effect and related mechanism of tanshinol borneol ester on homocysteine induced rat bone marrow mesenchymal stem cells damage

Objective To investigate the protective effect and potential mechanism of tanshinol borneol ester (TBE) on homocysteine (Hey) induced rat bone marrow mesenchymal stem cells (BMSCs) damage. Methods BMSCs were isolated and cultured in vitro by density gradient centrifugation and adherent culture method. BMSCs were divided into the control (normal isolation and culture), TBE-1 (10 μmol/L TBE-1 solution with 100 μl),TBE-2 (10 μmol/L TBE-2 solution with 100 μl),Hcy (0.5 mmol/L Hey solution with 100 μl), Hey + TBE-1 (0. 5 mmol/L Hey solution with 100 pi,and 10 pmol/L TBE-1 solution with 100 μl) ,Hcy + TBE-2 (0. 5 mmol/L Hey solution with 100 μl,and 10 μmol/L TBE-2 solution with 100 μl), Hey + TBE-1 + inhibitor group(0. 5 mmol/L Hey solution with 100 pi, 10 μmol/L TBE-1 solution with 100 μl, and 25 μmol/L LY294002 (specific blocker of phosphatidylinositol 3 kinase) solution with 100 pi), Hey + TBE-2 + inhibitor group (0. 5 mmol/L Hey solution with 100 pi, 10 μmol/L TBE-2 solution with 100 μl, and 25 μmol/L LY294002 solution with 100 pi). Cell proliferation activity was detected by MTT assay. The T-SOD activity and malonaldehyde level of cells were measured by anthineoxidase method and TBA method, respectively, to evaluate cell oxidative and antioxidative activities. The ultrastructure of cells was observed under transmission electron microscope. The expression level of PKB and NF-kB of cells in various groups were detected with the immunocytochemical method. Results (1) Cell proliferation activity in TBE-1 group and TBE-2 group was significantly increased compared with the control group (both P < 0. 01), and was similar between TBE-1 group and TBE-2 groups after 1 ,12 ,24 and 48 hours treatment. (2)The T-SOD activity in TBE-1 group and TBE-2 group was significantly higher than in control group (both P < 0. 01), while it was significantly lower in Hey group, Hey + TBE-1 group, and Hey + TBE-2 group than in control group(all P <0. 01), and was similar between control group, Hey + TBE-1 + inhibitor group, and Hey + TBE-2 + inhibitor group (all P > 0. 05). The T-SOD activity was higher in Hey + TBE-1 group and Hey + TBE-2 group than in Hey group (both P < 0. 01), and was higher in Hey + TBE-1 + inhibitor group than in Hey + TBE-1 group (P < 0. 05) and was higher in Hey + TBE-2 + inhibitor group than in Hey + TBE-2 group (P < 0. 05). The malonaldehyde level was lower in TBE-1 group and TBE-2 group than in control group (both P < 0. 01), was higher in Hey group, Hey + TBE-1 group, Hey + TBE-2 group, Hey + TBE-1 + inhibitor group, and Hey + TBE-2 + inhibitor group than in control group (all P < 0. 01), was lower in Hey + TBE-1 group and Hey + TBE-2 group than in Hey group (both P < 0. 01), was higher in Hey + TBE-1 + inhibitor group than in Hey + TBE-1 group (P < 0. 05), was higher in Hey + TBE-2 + inhibitor group than in Hey + TBE-2 group (P < 0. 05). (3) Under electron microscope, BMSCs showed profound swelling, senescence and apoptosis of cells increased significantly in Hey group, Hey + TBE-1 + inhibitor group, and Hey + TBE-2 + inhibitor group when compared with control group. BMSCs in the TBE-1 and TBE-2 groups presented with abundant rough endoplasmic reticulum, and very active cell metabolism signs. Compared with Hey group, BMSCs edema, the number of aging and apoptotic cells, and cell injury severity were significantly less in TBE-1 + Hey group and TBE-2 + Hey. (4) The PKB level was higher in TBE-1 group and TBE-2 group than in control group (both P < 0. 01), was lower in Hey group, Hey + TBE-2 group, Hey + TBE-1 + inhibitor group, and Hey + TBE-2 + inhibitor group than in control group (all P < 0. 01), was similar between control group and Hey + TBE-1 group(P > 0. 05), was higher in Hey + TBE-1 group and Hey + TBE-2 group than in Hey group (both P < 0. 05), was lower in Hey + TBE-1 + inhibitor group than in Hey + TBE-1 group(P < 0. 05), and was lower in Hey + TBE-2 + inhibitor group than in Hey + TBE-2 group(P < 0. 05). The NF-kB level was higher in TBE-1 group and TBE-2 group than in control group(both P < 0. 01), was lower in Hey group, Hey + TBE-1 group, Hey + TBE-2 group, Hey + TBE-1 + inhibitor group, and Hey + TBE-2 + inhibitor group than in control group(P < 0. 01 or 0. 05), was higher in Hey + TBE-1 group and Hey + TBE-2 group than in Hey group (both P < 0. 05), was lower in Hey + TBE-1 + inhibitor group than in Hey + TBE-1 group (P < 0. 05) and was lower in Hey + TBE-2 + inhibitor group than in Hey + TBE-2 group (P < 0. 05). Conclusion Tanshinol borneol ester can promote the proliferation of BMSC, and attenuate the homocysteine induced rat BMSCs damage possibly through activation of phosphatidylinositol 3 kinase/PKB signal transduction and its downstream signal pathway protein NF-κB.