黄芪汤对12C6+离子辐射脑损伤模型鼠Bcl-2/NF-κB信号通路的调控机制研究

Study on Regulatory Mechanism of Huangqi Decoction on Bcl-2/NF-κB Signal Pathway in Rats with Brain Injury Induced by 12C6+ Ion Radiation

关键词:  黄芪汤|重离子辐射|细胞凋亡|B细胞淋巴瘤/白血病-2/核转录因子-κB信号通路

Key words:  Huangqi decoction|heavy ion radiation|apoptosis|Bcl-2/NF-κB signal pathway

摘要:

目的 观察黄芪汤对¹²C⁶⁺离子辐射脑损伤模型鼠脑组织B细胞淋巴瘤/白血病-2（B-cell lymphoma-2，Bcl-2）和核转录因子-κB（nuclear factor-κB，NF-κB）蛋白表达的影响，进一步揭示黄芪汤防辐射机制。方法 50只SPF级Wistar大鼠随机分为正常对照组，单纯辐射模型组，黄芪汤高、中、低剂量组（18，9，4.5 g·kg^-1·d^-1）。黄芪汤高、中、低剂量组预先给予黄芪汤灌胃2周，单纯辐射模型组和黄芪汤高、中、低剂量组大鼠脑组织于灌胃第8天给予4 Gy ¹²C⁶⁺离子束单次照射，正常对照组不予照射。辐射后第7天处死各组大鼠。HE染色法观察脑组织的病理形态变化，实时荧光定量PCR法和免疫组化法分别测定各组大鼠脑组织Bcl-2、Bax和caspase-3的基因表达和蛋白表达，Western blotting法检测各组大鼠脑组织NF-κB的蛋白表达。结果 与正常对照组比较，单纯辐射模型组体质量和脑系数均显著降低，脑组织Bcl-2的基因表达和蛋白表达均显著降低，Bax和caspase-3的基因表达和蛋白表达均显著升高，NF-κB的蛋白表达也显著升高（P<0.05），单纯辐射模型组大鼠脑组织出现不同程度的神经细胞数量减少，排列疏松，核固缩、深染及神经细胞周围间隙增宽。与单纯辐射模型组相比，黄芪汤中、高剂量组脑系数和脑组织Bcl-2的蛋白表达显著升高，黄芪汤高剂量组体质量和脑组织Bcl-2的基因表达均显著升高（P<0.05）；而黄芪汤各干预组的NF-κB蛋白表达均显著降低，中、高剂量组caspase-3的基因表达和蛋白表达均显著降低，高剂量组Bax的基因表达和蛋白表达也均显著降低（P<0.05），高剂量组神经细胞数量减少及脑组织排列疏松等情况较单纯辐射组明显好转，但未完全恢复正常。结论 黄芪汤对¹²C⁶⁺离子辐射所致大鼠脑损伤具有一定防护作用，其作用机制可能与促进脑组织Bcl-2/NF-κB信号通路的Bcl-2蛋白表达，抑制其Bax、caspase-3和NF-κB蛋白表达来减轻脑组织神经细胞凋亡有关。

Abstract:

OBJECTIVE To observe the effect of Huangqi decoction on expression of B-cell lymphoma-2(Bcl-2) and nuclear-factor-κB(NF-κB) of brain tissue in model rats with brain injury induced by ¹²C⁶⁺ ion radiation, and to further reveal its mechanism. METHODS Fifty SPF Wistar rats were randomly divided into five groups:normal control group, radiation alone model group, Huangqi decoction high-dose, middle-dose and low-dose group(18, 9, 4.5 g·kg^-1·d^-1). Rats in the Huangqi decoction high-dose, middle-dose and low-dose groups were given Huangqi decoction by gavage for two weeks. At day 8 of gavage, the brain of the rats of the radiation alone model group and the the Huangqi decoction high-dose, middle-dose and low-dose groups were radiated once by 4 Gy ¹²C⁶⁺ ion. The normal control group was not radiated. After radiation for 7 d, all groups of rats were executed. The pathomorphism changes of brain tissue were observed by HE, the gene and protein expression of Bcl-2, Bax and caspase-3 in brain tissue was assessed by RT-PCR and immunohistochemical stain, and expression of NF-κB protein in brain tissue was analyzed by Western blotting. RESULTS Compared with the normal control group, the body weight and brain coefficient significantly decreased, the gene and protein expression of Bcl-2 in brain tissue significantly decreased, the gene and protein expression of Bax, caspase-3 in brain tissue significantly raised, and the protein expression of NF-κB in the brain tissue significantly increased in the radiation alone model group(P<0.05), the number of nerve cells reduced at different degrees, loose arrangement, nuclear pyknosis and deep dyeing, widened gap around nerve cells in radiation alone model group. Compared with the radiation alone model group, the brain coefficient and the protein expression of Bcl-2 in brain tissue raised obviously in Huangqi decoction middle-dose, high-dose group, the body weight and the gene expression of Bcl-2 in brain tissue increased significantly in high-dose group(P<0.05). But, the protein expression of NF-κB declined obviously in Huangqi decoction intervention groups, the gene and protein expression of caspase-3 decreased significantly in Huangqi decoction middle-dose, high-dose group, the gene and protein expression of Bax decreased significantly in Huangqi decoction high-dose group(P<0.05), the nerve cells reduction and loose arrangement in Huangqi high-dose decoction group were relieved significantly. CONCLUSION Huangqi decoction has protective effect on ¹²C⁶⁺ ion radiation induced mice brain injuries, the mechanism may be related to the reduction of neuron apoptosis which was the up-regulation of Bcl-2 protein, down-regulation of Bax, caspase-3 and NF-κB by Bcl-2/NF-κB signal pathway.