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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 effects of sophoridine pretreatment and premixed administration on Toll-like receptor 4 (TLR4) and downstream c-jun N-terminal kinase (JNK) of RAW264.7 macrophages activated by lipopolysaccharide (LPS) c-jun expression of sophoridine anti-LPS molecular mechanism. Methods: RAW264.7 cells were divided into 5 groups: RAW264.7 cells control group was added to serum-free DMEM medium; LPS group DMEM medium containing 100μg / L LPS; sophoridine group plus 31.25 mg / L sophoridine; the sophoridine preconditioning group was incubated with DMEM medium containing 31.25 mg / L sophoridine for 24 h, then the sophoridine was discarded and then incubated with 100 μg / L LPS DMEM medium for further incubation Cells; the sophoridine pre-mixed group was incubated with DMEM medium pre-mixed with sophoridine at a concentration of 31.25 mg / L and 100 μg / L LPS for 1 h. The cells were harvested at 5, 30, 60 and 120 min after the above 5 treatments. The mRNA expression of TLR4, JNK and c-jun in RAW264.7 cells was detected by RT-PCR. The expression of c-jun was detected by immunocytochemistry and Western blot Protein expression. Results: There was no significant difference in the indexes between RAW264.7 cells and sophoridine group (P> 0.05). The expressions of TLR4, JNK, c-jun mRNA and c-jun protein in LPS RAW264.7 cells Was significantly higher than that of RAW264.7 cells (P <0.01 or P <0.05), but the time point of each index was significantly different; pretreatment with sophoridine pretreatment group and sophoridine RAW264.7 cells TLR4 , JNK, c-jun mRNA and c-jun protein expression were significantly lower than LPS group (P <0.01 or P <0.05), but the time point of each index decreased significantly. CONCLUSIONS: Sophoridine exerts anti-LPS effect by regulating TLR4-JNK signal transduction pathway. The effects of sophoridine on the expression of TLR4-JNK signal transduction pathway may be multi-link.