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

Antagonistic effect of Polygonum cillinerve against methicillin-resistant Staphylococcus aureus and its immunoregulatory mechanism：An experimental study

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。

Abstract:

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 in－fluence 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 significant－ly 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 sig－nificantly 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 fur－ther regulates the pattern recognition receptors TLRs/NOD-like receptors and the downstream inflammatory factors TNF-α and IL-6.