葛根素对支气管上皮细胞和嗜中性粒细胞接触共培养诱导白细胞介素-6合成的抑制作用

Synthesis of IL-6 in co-cultured bronchial epithelial and neutrophil cells and the inhibitory effect of puerarin

目的葛根素可清除炎性介质,但具体机制尚不明确。文中旨在观察支气管上皮细胞(Beas-2B细胞)和嗜中性粒细胞(Neutrophils细胞)接触共培养体系中白细胞介素-6(IL-6)合成,探讨不同剂量葛根素对共培养体系IL-6的抑制作用。方法提取支气管上皮细胞(Beas-2B细胞)和嗜中性粒细胞(Neutrophils细胞),并分为6组:粒细胞组(Neutrophils细胞单独培养)、上皮细胞组(Beas-2B细胞单独培养)、共培养组(Beas-2B+Neutrophils细胞接触共培养)以及共培养+葛根素_(200)组、共培养+葛根素_(100)组、共培养+葛根素_(50)组(Beas-2B+Neutrophils+葛根素,调节葛根素终浓度分别为200、100、50μg/mL)。接种密度为1×10~6/孔,进行上清液IL-6浓度分析。将各联合培养组中的Beas-2B和Neutrophils细胞分别分离提取,进行不同细胞IL-6基因表达分析。结果共培养18 h,共培养组IL-6浓度[(280.409±21.340)pg/mL]较上皮细胞组[(240.002±23.727)pg/mL]、粒细胞组[(2.771±1.146)pg/mL]、共培养+葛根素_(50)组[(244.205±9.335)pg/mL]、共培养+葛根素_(100)组[(218.168±18.635)pg/mL]、共培养+葛根素_(200)组[(179.539±7.340)pg/mL]明显增加(P<0.05);共培养+葛根素_(200)组IL-6浓度较共培养+葛根素_(50)组、共培养+葛根素_(100)组明显降低(P<0.05)。共培养组Beas-2B细胞IL-6基因表达(1.151±0.013)较上皮细胞组(1.000±0.215)、共培养+葛根素_(50)组(0.398±0.024)、共培养+葛根素_(100)组(0.332±0.016)、共培养+葛根素_(200)组(0.306±0.015)明显升高(P<0.05);共培养+葛根素_(200)组Beas-2B细胞IL-6基因表达较上皮细胞组、共培养+葛根素_(50)组明显降低(P<0.05)。结论支气管上皮细胞和嗜中性粒细胞接触共培养可以促进炎性介质IL-6的合成与释放,并随着葛根素浓度升高,其合成的抑制逐渐增强。

Objective Puerarin can remove inflammatory mediators,but the underlying mechanisms are not yet clear. The purpose of this study is to observe the synthesis of IL-6 in co-cultured bronchial epithelial（ Beas-2B） and neutrophil cells,and to investigate the inhibitory effect of puerarin on IL-6. Methods Beas-2B and neutrophil cells were extracted and divided into six groups： neutrophil cells cultured alone（ NC）, Beas-2B cells cultured alone（ BC）,Beas-2B and neutrophil cells co-cultured（ BC＋NC）,and Beas-2B and neutrophil cells co-cultured with puerarin at 50 μg/mL（ BC＋NC＋P50）,100 μg/mL（ BC＋NC＋P100） and 200 μg/mL（ BC＋NC＋P200）. The concentrations of IL-6 in different groups were quantitatively measured by ELISA and the expression of the IL-6 gene detected by real-time PCR. Results After 18 hours of culturing,the concentration of IL-6 was significantly increased in the BC＋NC group（ [280.409±21.340] pg/mL） as compared with those in BC（ [240.002±23.727] pg/mL）,NC（ [22.771± 1.146] pg/mL）,BC ＋NC ＋P50（ [244. 205 ± 9. 335] pg/mL）,BC ＋NC ＋P100（ [218.168±18.635]pg/mL）,and BC＋NC＋P200（ [179.539±7.340]pg/mL）（ P〈0.05）,but lower in the BC＋NC＋P200 than in the BC＋NC＋P50 and BC＋NC＋ P100 groups（ P〈0. 05）. The expression of the IL-6 gene was markedly up-regulated in the BC ＋ NC group（ 1.515± 0. 013） in comparison with those in BC（ 1. 000 ± 0. 215）,BC ＋ NC ＋ P50（ 0. 398 ± 0. 024）,BC ＋ NC ＋ P100（ 0. 332 ±0.016）,and BC＋NC＋P200（ 0.306±0.015）（ P〈0.05）,but lower in the BC＋NC＋P200 than in the BC and BC＋NC＋P50 groups（ P〈0.05）. Conclusion Direct-contact co-culture of Beas-2B and neutrophil cells significantly enhances the synthesis and release of the inflammatory mediator IL-6,which can be inhibited by puerarin.