朱砂七拮抗耐药金黄色葡萄球菌的实验研究及免疫调节机制初探
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摘要
目的:本研究通过多水平评价体系明确朱砂七是否可以拮抗耐药金黄色葡萄球菌(methicillin resistant Staphylococcus aureus,MRSA),并探讨其发挥拮抗作用的免疫调节机制。方法:采用试管二倍稀释法进行药物敏感试验,观察药物对MRSA生长的抑制作用,再检测朱砂七是否影响金MRSA对宿主细胞的侵入,纤粘连蛋白粘附试验评价朱砂七对细菌粘附作用的影响。荧光定量PCR(real time-PCR,RT-PCR)法检测朱砂七对AT-Ⅱ细胞模式识别受体Toll样受体(Toll like receptor,TLRs)和胞质受体家族核苷结合寡聚结构域(nucleotide-binding oligomerization domain,NODs)及其下游炎性因子肿瘤坏死因子-α(tumor necrosis factor,TNF-α)/白细胞介素-6(interleukin-6,IL-6)基因表达的影响。通过Western blot检测朱砂七对AT-Ⅱ细胞模式识别受体MAPK和核因子-κB(nuclear factor-κB,NF-κB)蛋白表达水平的影响。结果:朱砂七组AT-Ⅱ细胞的MRSA侵入数量(471.57±34.32)明显低于模型组(1014.54±127.51,P<0.05)和阳性对照组(810.05±101.33,P<0.05)。朱砂七组MRSA感染AT-II细胞纤粘连蛋白吸附比例(0.25±0.01)明显低于模型组(1.04±0.12,P<0.05)和阳性对照组(0.74±0.07,P<0.05)。RT-PCR结果显示,MRSA可明显升高AT-Ⅱ细胞TLR2、NOD2及其下游炎性因子IL-6、TNF-α(P<0.05),朱砂七可降低其升高水平(P<0.05)。Western blot检测结果表明,朱砂七可以明显下调MAPK和NF-κB信号通路蛋白表达(P<0.05)。结论:朱砂七可以拮抗耐药金黄色葡萄球菌,该作用可能涉及MAPK和NF-κB信号通路,进而调控TLRs/NLRs模式识别受体及其下游炎性因子TNF-α/IL-6。
Objective:To investigate whether Polygonum cillinerve can antagonize methicillin-resistant Staphylococcus aureus(MRSA using a multilevel evaluation system and the immunoregulatory mechanism of its antagonistic effect. Methods:Tube double dilution was used to perform drug sensitivity test to observe the inhibitory effect of Polygonum cillinerve on the growth of Staphylococcus aureus and whether Polygonum cillinerve can affect the invasion of MRSA in host cells. Fibronectin adhesion test was used to evaluate the influence of Polygonum cillinerve on bacterial adhesion. Real-time PCR was used to analyze the effect of Polygonum cillinerve on the mRNA expression of Toll-like receptors(TLRs),nucleotide-binding oligomerization domains(NODs),and downstream inflammatory factors tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6) in AT-Ⅱ cells. Western blot was used to investigate the effect of Polygonum cillinerve on the protein expression of mitogen-activated protein kinase(MAPK) and nuclear factor-κB(NF-κB) in AT-Ⅱ cells. Results:The Polygonum cillinerve group had a significantly lower number of AT-Ⅱ cells with MRSA invasion than the model group and the positive control group(471.57±34.32 vs. 1 014.54±127.51/810.05±101.33,P<0.05),as well as a significantly lower fibronectin adsorption ratio in AT-Ⅱ cells infected with MRSA(0.25±0.01 vs. 1.04±0.12/0.74±0.07,P<0.05). Real-time PCR showed that MRSA significantly increased the levels of TLR2,NOD2,and downstream inflammatory factors TNF-α and IL-6(P<0.05),while Polygonum cillinerve significantly reduced their levels(P<0.05). Western blot showed that Polygonum cillinerve significantly downregulated the expression of the proteins involved in the MAPK and NF-κB signaling pathways(P<0.05). Conclusion:Polygonum cillinerve can antagonize MRSA,and the MAPK and NF-κB signaling pathways might be involved in this process. It further regulates the pattern recognition receptors TLRs/NOD-like receptors and the downstream inflammatory factors TNF-α and IL-6.
Objective:To investigate whether Polygonum cillinerve can antagonize methicillin-resistant Staphylococcus aureus(MRSA using a multilevel evaluation system and the immunoregulatory mechanism of its antagonistic effect. Methods:Tube double dilution was used to perform drug sensitivity test to observe the inhibitory effect of Polygonum cillinerve on the growth of Staphylococcus aureus and whether Polygonum cillinerve can affect the invasion of MRSA in host cells. Fibronectin adhesion test was used to evaluate the influence of Polygonum cillinerve on bacterial adhesion. Real-time PCR was used to analyze the effect of Polygonum cillinerve on the mRNA expression of Toll-like receptors(TLRs),nucleotide-binding oligomerization domains(NODs),and downstream inflammatory factors tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6) in AT-Ⅱ cells. Western blot was used to investigate the effect of Polygonum cillinerve on the protein expression of mitogen-activated protein kinase(MAPK) and nuclear factor-κB(NF-κB) in AT-Ⅱ cells. Results:The Polygonum cillinerve group had a significantly lower number of AT-Ⅱ cells with MRSA invasion than the model group and the positive control group(471.57±34.32 vs. 1 014.54±127.51/810.05±101.33,P<0.05),as well as a significantly lower fibronectin adsorption ratio in AT-Ⅱ cells infected with MRSA(0.25±0.01 vs. 1.04±0.12/0.74±0.07,P<0.05). Real-time PCR showed that MRSA significantly increased the levels of TLR2,NOD2,and downstream inflammatory factors TNF-α and IL-6(P<0.05),while Polygonum cillinerve significantly reduced their levels(P<0.05). Western blot showed that Polygonum cillinerve significantly downregulated the expression of the proteins involved in the MAPK and NF-κB signaling pathways(P<0.05). Conclusion:Polygonum cillinerve can antagonize MRSA,and the MAPK and NF-κB signaling pathways might be involved in this process. It further regulates the pattern recognition receptors TLRs/NOD-like receptors and the downstream inflammatory factors TNF-α and IL-6.
引文
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[15] Cui JJ,Yuan JF,Zhang ZQ. Anti-oxidation activity of the crude polysaccharides isolated from Polygonum cillinerve(Nakai)Ohwi in immunosuppressed mice[J]. J Ethnopharmacol,2010,132(2):512-517.
[16] Zhang TZ,Yang SH,Yao JF,et al. Sangxingtang inhibits the inflammation of LPS-induced acute lung injury in mice by down-regulating the MAPK/NF-κB pathway[J]. Chin J Nat Med,2015,13(12):889-895.
[17] Ye J,Piao H,Jiang J,et al. Polydatin inhibits mast cell-mediated allergic inflammation by targeting PI3K/Akt,MAPK,NF-κB and Nrf2/HO-1 pathways[J]. Sci Rep,2017,7(1):11895.
[2] Pauchard LA,Blot M,Bruyere R,et al. Linezolid and atorvastatin impact on pneumonia caused by Staphyloccocus aureus in rabbits with or without mechanical ventilation[J]. PLoS One,2017,12(11):e0187187.
[3] Kerremans JJ,Goessens WH,Verbrugh HA,et al. Rapid identification of Staphyloccocus aureus in positive-testing blood cultures by slidex staph plus agglutination test[J]. J Clin Microbiol,2008,46(1):395.
[4]沈林海,赵岚,谢利军,等.金黄色葡萄球菌的耐药性分析与基因分型研究[J].中华医院感染学杂志,2015,25(11):2408-2410.
[5]袁文常.金黄色葡萄球菌适应性耐药及MRSA耐药调控机制的研究[D].重庆:第三军医大学,2013.
[6]王晓梅,袁菊丽.朱砂七提取物对α-葡萄糖苷酶的抑制作用[J].化学与生物工程,2011,28(10):32-34.
[7]姬丽娜.朱砂七多糖的体外抑菌作用及对小鼠免疫功能的影响[D].咸阳:西北农林科技大学,2013.
[8] Wu Y,Wang X,Liu P,et al. Quantitative determination of anthraquinones and resveratrol in Polygonum cillinerve(Nakai)Ohwi by HPLC-PAD[J]. J AOAC Int,2017,100(1):25-29.
[9]王晓梅,袁菊莉,李健,等.朱砂七及其提取物的抗炎作用研究[J].现代中药研究与实践,2011,25(5):48-50.
[10]赵勤,郭惠玲,郭玲,等.朱砂七总蒽醌抗肿瘤的实验研究[J].辽宁中医杂志,2008,35(8):1222-1224.
[11]王晓梅,李宗孝,任莉君.朱砂七多糖的抗氧化作用研究[J].安徽农业科学,2010,38(34):19346-19347.
[12]王晓梅,赵立芳,支娟娟,等.朱砂七总鞣质的提取及抗氧化活性研究[J].化工时刊,2011,25(1):25-28.
[13]赵勤,冯宝平,胡锐,等.朱砂七抗Ⅱ型单纯疱疹病毒的体外研究[J].中药药理与临床,2012,28(3):79-81.
[14]王振苗.苦苣菜水提物的抗炎作用及其作用机制研究[D].石家庄:河北医科大学,2015.
[15] Cui JJ,Yuan JF,Zhang ZQ. Anti-oxidation activity of the crude polysaccharides isolated from Polygonum cillinerve(Nakai)Ohwi in immunosuppressed mice[J]. J Ethnopharmacol,2010,132(2):512-517.
[16] Zhang TZ,Yang SH,Yao JF,et al. Sangxingtang inhibits the inflammation of LPS-induced acute lung injury in mice by down-regulating the MAPK/NF-κB pathway[J]. Chin J Nat Med,2015,13(12):889-895.
[17] Ye J,Piao H,Jiang J,et al. Polydatin inhibits mast cell-mediated allergic inflammation by targeting PI3K/Akt,MAPK,NF-κB and Nrf2/HO-1 pathways[J]. Sci Rep,2017,7(1):11895.
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