CD14抑制肽对内毒素诱导U937细胞表达TNF-α的影响

Effects of CD14 inhibitory peptide on TNF-α expression of U937 induced by lipopolysaccharides

目的观察CD14抑制肽(CD14inhibitory peptide,CD14-IP)对内毒素诱导的U937细胞表达TNF-α的影响。方法U937细胞用佛波脂(PMA)诱导成熟后分5组:正常对照组、LPS组、高剂量抑制肽组、中剂量抑制肽组和低剂量抑制肽组。LPS组给予终浓度为100ng/mL的LPS和100ng/mL的LBP,高、中和低剂量抑制肽组除给予LPS和LBP外,分别给予终浓度为10μg/mL、1.0μg/mL和0.1μg/mL的CD14-IP。ELISA测定细胞培养上清TNF-α的浓度。进一步观察不同时间应用CD14-IP(1.0μg/mL)对LPS诱导U937细胞TNF-α和TNF-αmRNA表达的影响。用RT-PCR测定细胞TNF-αmRNA的表达。结果LPS组和抑制肽各组TNF-α浓度较正常组明显增高(P<0.05),高、中剂量抑制肽组TNF-α水平比LPS组明显降低(P<0.05),高、中剂量抑制肽组之间TNF-α浓度无明显差别(P>0.05),低剂量抑制肽组TNF-α浓度与LPS组无统计学差异(P>0.05)。不同时间应用CD14-IP时,CD14-IP早期应用对TNF-α和TNF-αmRNA的表达抑制作用显著。结论CD14-IP能显著减少LPS诱导的U937细胞TNF-α和TNF-αmRNA的表达,早期应用效果较好,可能对LPS所致急性肺损伤有保护作用。

Objective To investigate the effects of CD14 inhibitory peptide （CD14-IP） on TNF-α expression of U937 induced by lipopolysaccharides （LPS）. Methods U937 cells were stimulated by Phorbol myfistate acetate （PMA）, and then divided into five groups： control group, LPS group, high dose inhibitory peptide （HIP） group, moderate dose inhibitory peptide （MIP） group, and low dose inhibitory peptide （LIP） group. The cells in LPS group were incubated with 100 ng/mL （final concentration） LPS and 100 ng/mL （final concentration） LBP. The cells in HIP, MIP, and LIP groups were incubated with 10, 1.0, and 0.1 μg/mL CD14-IP respectively, besides LPS and LBP. The concentration of TNF-α released from cultured cells was detected by ELISA. Subsequently, U937 cells were incubated with 1.0 μg/mL CD14-IP. After treatment for different times, the mRNA and protein expression levels of TNF-α in each treatment point were determined by RT-PCR and ELISA, respectively. Results The concentrations of TNF-α in LPS group and every CD14-IP group were significantly higher than that in control group （ P 〈 0.05）. Compared with LPS group, the TNF-α protein expressions in HIP and MIP groups were significantly reduced （ P 〈 0.05）. There was not significant difference in the concentration of TNF-α between HIP and MIP groups （ P 〉 0.05）. The concentration of TNF-α in LPS group was not significantly higher than that in LIP group （ P 〉0.05）. After treatment with CD14-IP, the expressions of TNF-α and TNF-α mRNA decreased in a time-dependent manner, compared with LPS group. Conclusion CD14-IP can reduce the TNF-α mRNA and protein expression of U937 induced by hpopolysaccharide in a time-dependent manner, which suggests a potential protective effects of CD14-IP against LPS-induced inflammatory disorders such as acute hmg injury.