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
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摘要
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。
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.
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.
引文
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14 Zhao RJ,Li YQ,Wang H,et al.Relationship of caspase family and apoptosis[J].Chin J Anim Sci(中国畜牧杂志),2010,46(17):73-78.
15 Zhao D,He LF,Liu H,et al.Mitochondria,cytochrome c,caspase and apoptosis[J].JMPC(医学动物防制),2012,28:1337-1340.
16 Benedetti F,Davinelli S,Krishnan S,et al.Sulfur compounds block mcp-1 production by mycoplasma fermentans-infected macrophages through nf-κb inhibition[J].J Transl Med,2014,12:145.
17 Ibuki Y,Goto R.Contribution of inflammatory cytokine release to activation of resident peritoneal macrophages after in vivo low-dose γ-irradiation[J].J Radiat Res,2018,40:253.
18 Zin?cker S,Wang MY,Gaustad P,et al.Mycoplasma contamination revisited:mesenchymal stromal cells harboring mycoplasma hyorhinis potently inhibit lymphocyte proliferation in vitro[J].PLoS One,2011,6:e16005.
19 Mawarti H,Rajin M,Asumta Z.The effects of aloe vera on tnf-a levels,the percentage of nk cells and th 17 cells in rat that received izoniazid and rifampycin[J].Med Arch,2017,71:308-311.
20 Krayem I,Bazzi S,Karam M.The combination of crp isoforms with oxldl decreases tnf-α and il-6 release by u937-derived macrophages[J].Biomed Rep,2017,7:272.
21 Huang JH,Li L,Qiu X,et al.Stimulating effects on alveolar type Ⅱ epithelial a549 cells by tnf-α and lps[J].Immunol J(免疫学杂志),2013,29:211-215.
2 Parrott GL,Takeshi K,Jiro F.A compendium for mycoplasma pneumoniae[J].Front Microbiol,2016,7:513.
3 Liu X,Fang XM,Zou XL.Alterations of toll-like receptors tlr2 and tlr4 and gene expression of proinflammatory factors tnf-α and il-1β in lungs of swine after mycoplasma pneumoniae infection[J].Jiangsu J Agr Sci(江苏农业学报),2011,27:305-309.
4 Zhao LY.Changes of immune function and inflammation factors of mycoplasma pneumoniae pneumonia infants in acute and recovery stage[J].MCHCC(中国妇幼保健),2014,29(1):86-88.
5 Li XH.Role of macrophage autophagy in m.ovipneumoniae infection resistance[D].Yinchuan:Ningxia University(宁夏大学),2017.
6 Yang J,Change XQ,Wang X,et al.Extraction and antimicrobial activity of active components from glycyrrhiza[J].Henan Sci(河南科学),2017,35:1587-1591.
7 Gao XJ,Wu XL,Wu YL,et al.Separation of licorice flavonoids-producing salt-resistant endophytic fungi from wild glycyrrhiza uralensis fisch living in ningxia district[J].Nat Prod Res Dev(天然产物研究与开发),2016,28:1549-1556.
8 Wang BK,Mao YL.Glycyrrhizic acid activates chicken macrophages and enhances their salmonella-killing capacity in vitro[J].J Zhejiang Univ-Sci B(Biomed &Biotechnol),2018,19:785-795.
9 Xiang Q.Clinical observation of yunzhijun capsules combined with diammonium glycyrrhizinate capsules in trea[J].Drugs & Clinic(药物与临床),2017,32:1118-1121.
10 Chakotiya A S,Tanwar A,Srivastava P,et al.Effect of aquo-alchoholic extract of glycyrrhiza glabra against pseudomonas aeruginosa in mice lung infection model[J].Biomed & Pharmacother,2017,90:171-178.
11 Hussain,Iqbal Z,Abbas RZ,et al.Immunomodulatory activity of glycyrrhiza glabra extract against mixed eimeria infection in chickens[J].Int J Agric Biolk(国际农业与生物工程学报),2017,19:928-932.
12 Liang R,Dong ZY,Shi ZY,et al.Effect of single chinese herb on the adhesion of mycoplasma suis to erythrocytes[J].J Trad Chin Vet Med(中兽医医药杂志),2017,36(4):13-16.
13 Bewley MA,Naughton M,Preston J,et al.Pneumolysin activates macrophage lysosomal membrane permeabilization and executes apoptosis by distinct mechanisms without membrane pore formation[J].Mbio,2014,5(5):10-14.
14 Zhao RJ,Li YQ,Wang H,et al.Relationship of caspase family and apoptosis[J].Chin J Anim Sci(中国畜牧杂志),2010,46(17):73-78.
15 Zhao D,He LF,Liu H,et al.Mitochondria,cytochrome c,caspase and apoptosis[J].JMPC(医学动物防制),2012,28:1337-1340.
16 Benedetti F,Davinelli S,Krishnan S,et al.Sulfur compounds block mcp-1 production by mycoplasma fermentans-infected macrophages through nf-κb inhibition[J].J Transl Med,2014,12:145.
17 Ibuki Y,Goto R.Contribution of inflammatory cytokine release to activation of resident peritoneal macrophages after in vivo low-dose γ-irradiation[J].J Radiat Res,2018,40:253.
18 Zin?cker S,Wang MY,Gaustad P,et al.Mycoplasma contamination revisited:mesenchymal stromal cells harboring mycoplasma hyorhinis potently inhibit lymphocyte proliferation in vitro[J].PLoS One,2011,6:e16005.
19 Mawarti H,Rajin M,Asumta Z.The effects of aloe vera on tnf-a levels,the percentage of nk cells and th 17 cells in rat that received izoniazid and rifampycin[J].Med Arch,2017,71:308-311.
20 Krayem I,Bazzi S,Karam M.The combination of crp isoforms with oxldl decreases tnf-α and il-6 release by u937-derived macrophages[J].Biomed Rep,2017,7:272.
21 Huang JH,Li L,Qiu X,et al.Stimulating effects on alveolar type Ⅱ epithelial a549 cells by tnf-α and lps[J].Immunol J(免疫学杂志),2013,29:211-215.