槐定碱2种加药方式对LPS激活的RAW264.7巨噬细胞TLR4-JNK通路的影响

Effect of two Treatments of Sophoridine on TLR4-JNK Signaling Pathway of RAW264.7 Cells Induced by LPS

目的:观察槐定碱预处理和预混合2种给药方式对脂多糖(LPS)激活的RAW264.7巨噬细胞Toll样受体4(TLR4)及下游c-jun氨基末端激酶(JNK)、c-jun表达的影响,探讨槐定碱抗LPS的分子机制。方法:将RAW264.7细胞分为5组:RAW264.7细胞对照组加入未加血清的DMEM培养基;LPS组加含100μg/L LPS的DMEM培养基;槐定碱药物组加含31.25 mg/L槐定碱的DMEM培养基;槐定碱预处理组以含31.25 mg/L槐定碱的DMEM培养基孵育细胞24 h弃去槐定碱,而后加入100μg/L LPS的DMEM培养基继续孵育细胞;槐定碱预混合组以终浓度为31.25 mg/L槐定碱与100μg/L LPS预先混合1 h的DMEM培养基孵育细胞。上述5组处理完毕后于5、30、60、120 min收集细胞,以RT-PCR技术检测RAW264.7细胞TLR4、JNK、c-jun mRNA表达,免疫细胞化学和Western blot检测细胞c-jun的蛋白的表达。结果:RAW264.7细胞对照组与槐定碱药物组各项指标差异无统计学意义(P>0.05);LPS组RAW264.7细胞的TLR4、JNK、c-jun mRNA及c-jun蛋白表达均显著高于RAW264.7细胞对照组(P<0.01或P<0.05),但各指标显著升高的时间点有所不同;槐定碱预处理组与槐定碱预混合组RAW264.7细胞TLR4、JNK、c-jun mRNA及c-jun蛋白表达均显著低于LPS组(P<0.01或P<0.05),但各指标显著降低的时间点有所不同。结论:槐定碱可通过调控TLR4-JNK信号转导通路发挥抗LPS作用,其不同加药方式显示的效应表明其作用可能是多环节的。

Objective： To observe the effect of two treatments of sophoridine（ pretreating and premixing） on lipopolysaccharide（ LPS）-induced on TLR4,the expression of TLR4 and its downstream signaling molecule such as JNK and c-jun in RAW264. 7 cells were,and to detect its mechanism. Methods： RAW264. 7 cells were randomly divided into five groups,RAW264. 7 cells control group added absence serum DMEM to incubate cells; LPS group,with added 100 μg/L LPS DMEM to incubate cells; Sophoridine control group added 31. 25 mg/L sophoridine DMEM to incubate cells; sophoridine pretreating group,added 31. 25 mg/L sophoridine DMEM to incubate cells 24 h,then threw away sophoridine and added 100 μg/L LPS DMEM to incubate cells; sophoridine premixing group added the final concentration of 31. 25 mg/L sophoridine and 100 μg/L LPS DMEM to incubate cells 1 h. After above treatments,collected cells at 5,30,60,and 120 min,respectively. The mRNA expressions of TLR4,JNK and c-jun were determined by reverse transcription PCR（ RT-PCR）,the protein expression of c-jun in RAW264. 7 cells was measured by immunocytochemistry and Western blot. Results：Compared with RAW264. 7 cells control group,there were no statistical difference on each index in sophoridine control group（ P〈0. 05）,the mRNA expressions of TLR4,JNK,c-jun and c-jun protein expression were significantly increased in LPS group（ P〈0. 01 or P〈0. 05）,but each index increased were different at different time points; the mRNA expressions of TLR4,JNK,c-jun and c-jun protein expression in RAW264. 7 cells were lower in sophoridine pretreating and premixing group than LPS group,but each index decreased were different at different time points. Conclusion： Sophoridine induces the effect of antiendotoxin through regulating TLR4-JNK signal transduction pathway,which showing that the effect of different treatments of sophoridine may be multi-link adjustment.