疏血通脉胶囊含药血清对大鼠骨髓间充质干细胞凋亡的影响

目的:探讨疏血通脉胶囊含药血清对大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)凋亡的影响以及作用机制。方法:体外培养大鼠BMSCs,以不同浓度双氧水(H2O2)诱导BMSCs凋亡,取最适宜浓度1.0 mmol·L~(-1)用于实验。疏血通脉胶囊低、中、高剂量(含生药1.81,3.62,7.24 g·kg~(-1))灌胃给药制备大鼠含药血清。选择第3代BMSCs,随机分正常组(不做处理,加入体积分数为15%正常大鼠血清培养液),模型组(H2O2诱导细胞凋亡,加入体积分数为15%正常大鼠血清培养液)和疏血通脉胶囊低、中、高剂量含药血清组(H2O2诱导细胞凋亡,分别加入对应剂量、体积分数为15%的疏血通脉胶囊含药血清培养液),共5组,作用24 h后噻唑蓝比色法(MTT)检测细胞相对存活率,Annexin V-FITC/PI流式细胞术检测细胞凋亡率,实时荧光定量PCR法(Real-time PCR)检测半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)和B细胞淋巴瘤/白血病-2(Bcl-2)mRNA的表达。结果:H2O2能体外模拟缺血缺氧环境诱导BMSCs凋亡。MTT结果显示疏血通脉胶囊低、中、高剂量含药血清组细胞存活率较模型组有明显提高(P0.01),且存活率随剂量增加而增高(P0.05);Annexin V-FITC/PI流式细胞术结果显示疏血通脉胶囊含药血清可以降低H2O2诱导的BMSCs凋亡率(P0.01)。Real-time PCR结果显示H2O2诱导可以增加BMSCs的Caspase-3 mRNA表达,降低Bcl-2 mRNA表达。疏血通脉胶囊含药血清处理下调了Caspase-3 mRNA的表达(P0.01),上调了Bcl-2 mRNA的表达(P0.01)。结论:疏血通脉胶囊含药血清可通过上调Bcl-2基因表达,负性调控Caspase-3基因,从而抑制H2O2导致的BMSCs凋亡。

Effect of Shuxue Tongmai Capsule-containing Serum on Apoptosis of Rat Bone Marrow Mesenchymal Stem Cells

Objective:To investigate the effect and mechanism of Shuxue Tongmai capsule-containing serum on apoptosis in rat bone marrow mesenchymal stem cells (BMSCs). Method:BMSCs were cultured with different concentrations of H₂O₂ in vitro to induce apoptosis, and then 1.0 mmol·L^-1 optimum concentration was used for the experiment. First, Shuxue Tongmai capsule-containing serum were prepared at low (1.81 g·kg^-1), middle (3.62 g·kg^-1) and high (7.24 g·kg^-1) doses. Then the third-generation BMSCs were divided into 5 groups in random. The first group was control group, which only added with normal rat serum with a volume fraction of 15% in culture medium. The second group was model group, which was added with normal rat serum with a volume fraction of 15% in culture medium and the apoptosis cells were induced with H₂O₂.The other three groups were drug-contained serum groups, which were respectively prepared at low (0.32 g·kg^-1), middle (0.64 g·kg^-1) and high (1.28 g·kg^-1) doses, and added with drug-contained serum with a volume fraction of 15%, and the apoptosis cells were induced with H₂O₂ in each group. After 24 hours, the cells'' relative survival rate was detected by 3-(4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2-H-tetrazolium bromide (MTT) method, and the apoptosis rate was evaluated by Annexin V-FITC/PI flow cytometry method. And cysteine aspartate protease-3 (Caspase-3) and B-cell lymphoma-2 (Bcl-2) mRNA expressions were assayed by Real-time PCR (Real-time PCR). Result:H₂O₂ could imitate the ischemic anoxic environment in vitro and induce the apoptosis of BMSCs. The MTT detection results showed that the cells'' survival rates of Shuxue Tongmai capsule-containing serum groups prepared at low, medium and high doses were significantly higher than that in the model group (P<0.01), and the survival rate increased with the dose concentration (P<0.05). And the AnnexinV-FITC/PI flow cytometry results showed that the drug-containing serum of Shuxue Tongmai capsule could decrease the apoptosis rates of BMSCs induced with H₂O₂ (P<0.01). The Real-time PCR results showed that the induction with H₂O₂ could increase BMSCs'' Caspase-3 mRNA expression and reduce their Bcl-2 mRNA expression. The treatment of Shuxue Tongmai capsule-containing serum could decrease Caspase-3 mRNA expression (P<0.01), and increase Bcl-2 mRNA expression (P<0.01). Conclusion:Shuxue Tongmai capsule-containing serum can negatively regulate Caspase-3 gene by increasing Bcl-2 gene expression, so as to inhibit the apoptosis of BMSCs caused with H₂O₂.