[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(Hcy) 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 Hcy solution with 100 μl), Hcy + TBE-1(0.5 mmol/L Hcy solution with 100 μl, and 10 μmol/L TBE-1 solution with 100 μl), Hcy + TBE-2 (0.5 mmol/L Hcy solution with 100 μl, and 10 μmol/L TBE-2 solution with 100 μl), Hcy+ TBE-1+ inhibitor group(0.5 mmol/L Hcy solution with 100 μl, 10 μmol/L TBE-1 solution with 100 μl, and 25 μmol/L LY294002(specific blocker of phosphatidylinositol 3 kinase) solution with 100 μl), Hcy+ TBE-2+ inhibitor group(0.5 mmol/L Hcy solution with 100 μl, 10 μmol/L TBE-2 solution with 100 μl, and 25 μmol/L LY294002 solution with 100 μl). 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-κB 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 Hcy group, Hcy+ TBE-1 group, and Hcy+ TBE-2 group than in control group(all P<0.01), and was similar between control group, Hcy+ TBE-1+ inhibitor group, and Hcy+ TBE-2+ inhibitor group(all P>0.05). The T-SOD activity was higher in Hcy+ TBE-1 group and Hcy+ TBE-2 group than in Hcy group(both P<0.01), and was higher in Hcy+ TBE-1+ inhibitor group than in Hcy+ TBE-1 group(P<0.05) and was higher in Hcy+ TBE-2+ inhibitor group than in Hcy+ 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 Hcy group, Hcy+ TBE-1 group, Hcy+ TBE-2 group, Hcy+ TBE-1+ inhibitor group, and Hcy+ TBE-2+ inhibitor group than in control group(all P<0.01), was lower in Hcy+ TBE-1 group and Hcy+ TBE-2 group than in Hcy group(both P<0.01), was higher in Hcy+ TBE-1+ inhibitor group than in Hcy+ TBE-1 group(P<0.05), was higher in Hcy+ TBE-2+ inhibitor group than in Hcy+ TBE-2 group(P<0.05). (3)Under electron microscope, BMSCs showed profound swelling, senescence and apoptosis of cells increased significantly in Hcy group, Hcy+ TBE-1+ inhibitor group, and Hcy+ 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 Hcy group, BMSCs edema, the number of aging and apoptotic cells, and cell injury severity were significantly less in TBE-1+ Hcy group and TBE-2+ Hcy. (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 Hcy group, Hcy+ TBE-2 group, Hcy+ TBE-1+ inhibitor group, and Hcy+ TBE-2+ inhibitor group than in control group(all P<0.01), was similar between control group and Hcy+ TBE-1 group(P>0.05), was higher in Hcy+ TBE-1 group and Hcy+ TBE-2 group than in Hcy group(both P<0.05), was lower in Hcy+ TBE-1+ inhibitor group than in Hcy+ TBE-1 group(P<0.05), and was lower in Hcy+ TBE-2+ inhibitor group than in Hcy+ TBE-2 group(P<0.05). The NF-κB level was higher in TBE-1 group and TBE-2 group than in control group(both P<0.01), was lower in Hcy group, Hcy+ TBE-1 group, Hcy+ TBE-2 group, Hcy+ TBE-1+ inhibitor group, and Hcy+ TBE-2+ inhibitor group than in control group(P<0.01 or 0.05), was higher in Hcy+ TBE-1 group and Hcy+ TBE-2 group than in Hcy group(both P<0.05), was lower in Hcy+ TBE-1+ inhibitor group than in Hcy+ TBE-1 group(P<0.05) and was lower in Hcy+ TBE-2+ inhibitor group than in Hcy+ 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.