紫杉醇对海马神经元γ-氨基丁酸B型受体表达的影响及核因子-κB通路的调控作用

Effects of paclitaxel on γ-aminobutyric acid B receptors expression and nuclear factor-κB pathway regulation in hippocampal neurons

目的 通过给予核因子-κB（NF-κB）抑制剂（SN50）及其上游通路p38丝裂原活化蛋白激酶（p38MAPKs）抑制剂（SB203580）,观察紫杉醇对海马神经元γ-氨基丁酸B型受体（GABAB受体）表达影响及p38MAPKs/NF-κB通路在其中的调控作用.方法 选取原代培养5d、浓度为1 ×109/L的海马神经元随机分为6组：对照组（C组）、10 μmol/L SB203580处理组（SB组）、53 mg/LSN50处理组（SN组）、1μmol/L紫杉醇处理组（N组）、10 μmol/L SB203580＋1μmol/L紫杉醇处理组（SB＋N组）、53 mg/L SN50＋1μmol/L紫杉醇处理组（SN ＋N组）,培养时间为24 h,观察6组海马神经元形态学变化、早期凋亡率、NF-κB及GABAB受体蛋白表达变化.结果 与C组比较,N、SB ＋N和SN＋N组海马神经元的轴突和树突分枝均有所减少,而SB组及SN组的神经元形态结构基本正常.N、SB ＋N和SN＋N组的海马神经元GABAB受体表达与NF-κB蛋白表达及早期凋亡率变化一致：与C组比较,N、SB＋N和SN ＋N组的NF-κB蛋白（N组：3.452±0.654;SB ＋N组：1.729±0.461;SN＋N组：1.604±0.361）、早期凋亡率[N组：（49.16±3.12）％;SB ＋N组：（31.18±3.02）％;SN ＋N组：（28.47±3.75）％]及GABAB受体表达（N组：0.381 ±0.014;SB ＋N组：0.243 ±0.013;SN＋ N：0.268 ±0.027）均明显增高（P＜0.05）,SN组的NF-κB蛋白、早期凋亡率及GABAB受体表达则均显著降低（P＜0.05）,而SB组的GABAB受体表达及早期凋亡率则无显著变化（P＞0.05）;与N组比较,其余5组海马神经元NF-κB蛋白表达、早期凋亡率及GABAB受体均显著降低（P＜0.05）,但与SB组或SN组比较,SB ＋N组与SN ＋N组NF-κB蛋白、早期凋亡率及GABAB受体表达的增高幅度明显减少（P＜0.05）.而SB组与SN组,SB ＋N组与SN ＋N组比较,上述3项指标差异无统计学意义（P＞0.05）.结论 紫杉醇可通过上调NF-κB蛋白诱发海马神经元凋亡及GABAB受体表达增多,阻断NF-κB通路可下调GABAB受体表达,以减缓紫杉醇引起的细胞凋亡等中枢神经毒性,NF-κB在其中发挥关键调控作用.

Objective To investigate the effects of paclitaxel on γ-aminobutyric acid B （GABAs） receptors expression and p38 mitogen-activated protein kinase （p38MAPK）/nuclear factor-κB （NF-κB） pathway in the hippocampal neurons regulated by giving NF-κB inhibitor （SN50） and p38MAPK inhibitor （SB203580）.Methods The primary cultured hippocampal neurons which had been cultivated for 5 days in vitro were randomly selected,and the density was about 1 × 109/L.These neurons were randomly divided into six groups：control group （C group）,10 μmol/L SB203580 group （SB group）,53 mg/L SN50 group （SN group）,1 μmol/L paclitaxel group （N group）,10 μmol/L SB203580 ＋ l0 μmol/L paclitaxel group （SB ＋ N group）,and 53 mg/L SN50 ＋ 10 μmol/L paclitaxel group （SN ＋ N group）.All of the hippocampal neurons were cultured for 24 h.Morphologic changes,the early apoptosis rate,and expression of GABAB receptors and NF-κB in hippocampal neurons were observed in six groups.Results As compared with C group,the axons and dendrites of hippocampal neurons in N group,SB ＋ N group and SN ＋ N group were decreased,while the morphology and structure of neurons in SB group and SN group were normal.The expression of GABAB receptors and NF-κB protein and early poptnsis rate in hippocampal neurons of N group,SB ＋ N group and SN ＋ N group were consistent.As compared with C group,the expression of GABAB receptors （N：0.381 ± 0.014;SB ＋ N：0.243 ± 0.013;SN ＋ N：0.268 ± 0.027）,early apoptosis rate [N：（49.16 ± 3.12） ％;SB ＋ N：（31.18 ± 3.02） ％;SN ＋ N：（28.47 ± 3.75）％] and NF-κB protein （N：3.452 ±0.654;SB ＋N：1.729 ±0.461;SN ＋N：1.604 ±0.361） were significantly up-regulated in N group,SB ＋ N group and SN ＋ N group （P ＜ 0.05）,while the expression of GABAB receptors,early apoptosis rate and NF-κB protein were down-regulated in SN group （P ＜ 0.05）,and the expression of GABAB receptors and early apoptosis rate had no significant changes in SB group （P ＞ 0.05）.As compared with N group,the expression of GABAB receptors,early apoptosis rate and NF-κB protein were down-regulated in the other five groups of hippocampus neurons （P ＜ 0.05）,but as compared with SB group or SN group,the increases in the expression of GABAB receptors,early apoptosis rate and NF-κB protein were significantly reduced （P ＜ 0.05）.There was no significant difference between SB group and SN group,as well as between SB ＋ N group and SN ＋ N group （P ＞ 0.05）.Conclusion Paclitaxel can induce apoptosis of hippocampal neurons and increase GABAB receptors expression by up-regulating NF-κB protein,and the inhibition of NF-κB pathway can down-regulate the expression of GABAB receptors to slow the central neurotoxicity induced by paclitaxel,in which NF-κB may play a key regulatory role.