脂多糖刺激小胶质细胞分泌Pro-cathepsin L及对神经元凋亡的影响

The release of lysosomal Pro-cathepsin L of BV-2 cells upon lipopolysaccharide stimulation and its toxicity to neurons

目的观察脂多糖刺激对小胶质细胞系BV-2(BV-2细胞)分泌Pro-cathepsin L和神经元凋亡的影响。方法通过脂多糖刺激BV-2细胞产生炎性反应建立炎性细胞模型,Western blotting法分别观察脂多糖刺激前后BV-2细胞条件培养液中Pro-cathepsin L表达水平,以及经Cathepsin L抑制剂预处理前后多巴胺能神经元细胞系PC-12(PC-12细胞)活化Caspase-3表达变化。结果脂多糖刺激BV-2细胞1和3 h后,Pro-cathepsin L表达水平显著增加,与刺激前比较差异均有统计学意义(P=0.015,0.021)。与BV-2细胞共培养并经脂多糖刺激1和3 h后,PC-12细胞活化(Caspase-3表达水平升高,且显著高于刺激前水平(均P=0.000);经Cathepsin L抑制剂预处理及脂多糖刺激1和3 h后,PC-12细胞活化Caspase-3表达水平下降,且显著低于单纯脂多糖刺激组(P=0.005,0.001)。结论小胶质细胞在炎性反应条件下可向细胞外释放Pro-cathepsin L,促进神经元表达活化型Caspase-3,在神经变性疾病发生发展中发挥重要作用。

Objective To investigate the secretion of lysosomal Pro- eathepsin L and its contribution to neuronal apoptosis upon lipopolysaecharide （LPS） stimulation. Methods The effect on the release of Pro-cathepsin L in BV-2 cells upon LPS stimulation was determined by Western blotting. Dopaminergic cell line PC-12 cells were cultured with conditioned medium collected from BV-2 cells upon LPS stimulation, then the activated Caspase-3 expression in PC- 12 ceils was determined by Western blotting. After combination with Cathepsin L inhibitor （iCL） and LPS, the activated Caspase-3 expression in PC-12 cells was observed. Results The secretion of Pro-cathepsin L was increased significantly at 1 and 3 h upon LPS stimulation in BV-2 cells （P = 0.015, 0.021）. Expression of activated Caspase-3 in PC-12 cells co-cultured with conditioned medium collected from BV-2 cells upon LPS stimulation at 1 and 3 h was increased （P = 0.000, for all）. However, pretreatment with iCL in BV-2 cells could partially reverse Caspase- 3 activation （P = 0.005, 0.001）. Conclusion Inflammation increases secretion of Pro-cathepsin L in microglia, which can induce the Caspase-3 activation of neurons, which may participate in the pathogenesis of neurodegenerative diseases.