甘草酸对巨噬细胞抗绵羊肺炎支原体感染的调控作用研究

为观察甘草酸在小鼠巨噬细胞系RAW264.7抗绵羊肺炎支原体(Mycoplasma ovipneumoniae,MO)感染中的作用,实验利用CCK-8细胞活性检测找到最佳甘草酸处理浓度;检测甘草酸对受MO感染的巨噬细胞活性的影响。流式细胞仪检测甘草酸对RAW264.7巨噬细胞生长周期的影响;ELISA检测甘草酸对受MO感染的巨噬细胞分泌TNF-α的影响;Western blot检测细胞凋亡因子Bax、Bad的表达情况。RT-PCR检测凋亡和自噬相关基因的表达情况。结果显示,浓度为12μmol/L的甘草酸显著升高RAW264.7的活性(P=0.012 9),且处于G1期的细胞数减少,G2期的细胞数增加。甘草酸(12μmol/L)可提高受MO感染的RAW264.7的增殖率(P=0.034 0),培养上清中TNF-α含量升高(P=0.015 2),巨噬细胞中促凋亡蛋白Bax表达量增加,但基因caspase 3和caspase 9的表达量显著下调(P<0.000 1),自噬相关基因Atg 7和Beclin 1表达量显著升高(P<0.000 1)。结果提示在MO感染巨噬细胞引起免疫抑制的情况下,甘草酸可通过促增殖、抑凋亡、促进TNF-α的表达、增加自噬来起到免疫调控作用。

The effects of glycyrrhizic acid on Mycoplasma ovipneuniae-infected marcrophages

This study was designed to investigate the role of glycyrrhizin acid in the infection of the mouse macrophage cell line RAW264.7 against Mycoplasma ovipneumoniae(MO).The optimal concentration of glycyrrhizic acid was determined by CCK-8 cell activity assay and the effects of glycyrrhizic acid on the activity of MO-infected macrophages were examined.The effect of glycyrrhizin acid on the growth cycle of RAW 264.7 macrophages was detected by flow cytometry.On the other hand,the effect of glycyrrhizin acid on the secretion of TNF-α by MO-infected macrophages was detected by ELISA.The expression of apoptosis factors Bax and Bad were detected by Western blot.RT-PCR was used to detect the expression of apoptosis and autophagy related genes.The results showed that glycyrrhizic acid at a concentration of 12 μmol/L significantly increased the activity of RAW264.7(P=0.012 9).In cell cycle experiment,it is found that the number of cells treated with glycyrrhizic acid in the G1 phase decreased while the number of cells in the G2 phase increased.Glycyrrhizic acid(12 μmol/L) increased the proliferation rate of MO-infected RAW264.7(P=0.034 0).The ELISA test resulted in an increase of TNF-α content in culture supernatant(P=0.015 2).Western Blot analysis to examine the expression level of proapoptotic protein was done and Bax protein was found increase in macrophages.For RT-PCR,it was found that the expression levels of Caspase 3 and Caspase 9 were significantly down-regulated(P<0.000 1),however the expression levels of autophagy-related genes Atg 7 and Beclin 1 were significantly increased(P<0.000 1).The results suggest that glycyrrhizic acid can play an immunoregulatory role by promoting proliferation,inhibiting apoptosis,promoting the expression of TNF-α and increasing autophagy in the case of MO-infected macrophages.