TRPC对脂多糖诱导的星形胶质细胞TNF-α和NO分泌的影响

Effects of TRPC on LPS induced secretion of TNF-α and NO in cultured cortical astrocytes

目的探讨TRPC对脂多糖诱导的星形胶质细胞TNF-α和NO分泌的影响。方法通过摇床筛选法纯化大鼠大脑皮层星形胶质细胞,用免疫荧光法鉴定其纯度。细胞培养至80%左右融合时加入0.5μg/mL脂多糖(LPS)后用维持液分别培养0、2、6、12、24和48 h,检测TNF-α和NO分泌情况,观察TRPC阻断剂2-APB和SKF96365对LPS诱导的TNF-α和NO分泌的影响,并与不加LPS的对照组比较。结果 PCR结果显示,星形胶质细胞能够表达TRPC1、TRPC3～7 mRNA。LPS作用2 h后TNF-α显著升高,一直持续到48 h(P<0.01),而LPS作用24和48 h后,NO的分泌显著增加(P<0.01)。10μmol/L 2-APB和5μmol/L SKF96365均可抑制LPS引起的TNF-α和NO增加(P<0.01),但与对照组相比差异仍有统计学意义(P<0.01)。结论抑制TRPC通道能够减少LPS诱导的TNF-α和NO分泌,提示TRPC通道可能参与脑内炎症性疾病过程中星形胶质细胞的活化。

Objective To investigate the effects of the TRPC on LPS-induced secretion of TNF-α and NO in astrocytes. Methods The astrocytes were isolated and purified by shaking the flasks in a horizontal orbital shaker,and identified by immunofluorescence.When cell fusion ratio reach 80%,cultured cells were exposed to 0.5 μg/mL LPS for 0,2,6,12,24 and 48 hours,or treated with 10 μmol/L 2-APB and 5 μmol/L SKF96365 simultaneously.TNF-α and NO concentration in cell culture supernatants were measured. Results The production of TNF-α,which was induced by LPS for 2 to 48 hours,was significantly increased（P0.01）,so was the NO secretion induced by LPS for 24 to 48 horus（P0.01）.TRPC blockers,2-APB and SKF96365 both significantly inhibited the LPS induced secretion of TNF-α and NO of astrocytes（P0.01）. Conclusion The inhibition of TRPC channels reduces the LPS-induced secretion of TNF-α and NO,suggesting that TRPC channel regulates astrocytes activation in the pathophysiology of brain inflammation.