乌司他丁下调LPS诱导的小鼠巨噬细胞MiR-21表达及初步机制研究

Ulinastatin down regulates MiR-21 expression in murine macrophages induced by LPS and its preliminary mechanism

目的:观察在脂多糖(lipopolysaccharide,LPS)诱导的小鼠巨噬细胞株RAW264.7产生炎症反应中微小RNA-21(Micro RNA-21,mi R-21)的表达情况,以及乌司他丁(Ulinastatin,UTI)在炎症反应中对mi R-21的干预作用。方法:设置正常组、模型组(LPS 500 ng/m L剌激组)、实验组(LPS 500 ng/m L+UTI 1 000 U/m L组)和阴性实验对照组(UTI 1 000 U/m L),观察RAW264.7细胞生长状态;四甲基偶氮唑(methyl thiazolyl tetrazolium,MTT)法检测不同浓度UTI对RAW264.7细胞活性的影响;用不同浓度UTI预处理2 h后,LPS刺激小鼠RAW264.7细胞诱导炎症模型,RT-PCR检测肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)m RNA、白介素-6(interleukin-6,IL-6)m RNA、mi R-21的表达。结果:(1)UTI对RAW264.7细胞的毒性作用:UTI浓度小于1 000 U/m L时对RAW264.7细胞无毒性作用,比较差异无统计学意义(P=0.117)。(2)与正常组相比,在不同浓度(100,250,500 ng/m L)LPS刺激RAW264.7细胞后,TNF-αm RNA及IL-6 m RNA的分泌(1.311±0.037,1.549±0.039,1.667±0.059;1.167±0.019,1.518±0.058,1.740±0.071)与基础分泌量(1.000±0.000)相比明显升高(P=0.000),且mi R-21表达(1.082±0.114,1.671±0.087,2.789±0.107)明显升高,并随LPS浓度升高而增加(P=0.000);(3)不同浓度UTI(10,100,1 000 U/m L)能有效降低TNF-αm RNA及IL-6 m RNA(1.000±0.000,1.998±0.212,1.421±0.100,1.122±0.154,0.991±0.139;1.000±0.000,1.880±0.045,1.179±0.108,1.053±0.070,0.960±0.088)的表达,并降低mi R-21(1.000±0.000,2.789±0.107,2.675±0.135,2.258±0.170,1.369±0.279)的表达,差异有统计学意义(P1=0.000,P2=0.000,P3=0.000)。结论:LPS刺激小鼠RAW264.7细胞可促进mi R-21表达升高,其表达水平在一定程度上反映了细胞炎症反应状态;UTI可通过下调mi R-21抑制相关信号通路从而在免疫炎症反应中发挥保护作用。

Objective：To detect the expression of micro RNA-21 in lipopolysaccharide（LPS） induced injured models of mouse RAW264.7 macrophages and the influence of Ulinastatin（UTI）on the expression of mi R-21. Methods：The growth status of RAW264.7cells was observed in the normal group,the model group（LPS）,the experimental group（LPS ＋ UTI） and the negative control group（drug-non-stimulation group）. Methyl thiazolyl tetrazolium（MTT）assay was used to determine the effect of different concentration of UTI on the activity of RAW264.7 cells. After pre-treatment with different concentrations of UTI for 2 h,the expressions of TNF-αm RNA,IL-6 m RNA and mi R-21 were detected by RT-PCR in RAW264.7 cells induced by LPS. Results：（1）The toxic effects of UTI on RAW264.7 cell：it was non-toxic of UTI on RAW264.7 cells while the concentrations were less than 1 000 U/m L;there was no statistically significant difference（P=0.117）.（2）After RAW264.7cells being treated by LPS with different concentrations（100,250,500 ng/m L）,the expressions of mi R-21（1.000 ±0.000,1.082±0.114,1.671±0.087,2.789±0.107）and TNF-α and IL-6（1.311±0.037,1.549±0.039,1.667±0.059;1.167±0.019,1.518±0.058,1.740±0.071）were up-regulated respectively in a dose-dependent manner（P1=0.000,P2=0.000,P3=0.000）.（3）And the effect could be attenuated by UTI in different concentrations（10,100,1 000 U/m L）（mi R-21：1.000±0.000,2.789±0.107,2.675±0.135,2.258±0.170,1.369±0.279,P=0.000;TNF-α：1.000±0.000,1.998±0.212,1.421±0.100,1.122±0.154,0.991±0.139,P=0.000;IL-6：1.000±0.000,1.880±0.045,1.179±0.108,1.053±0.070,0.960±0.088,P=0.000）. Conclusion：The expression of mi R-21 is significantly changed after LPS stimulating RAW264.7macrophages,indicated that it is related to the degree of inflammation. UTI could down-regulate the mi R-21-related signaling pathway to play a protective role in immune and inflammatory response.