低频超声联合抗菌药物治疗耐药菌所致肺部感染的机制研究

Pulmonary infection caused by multidrug resistant bacteria is difficult to treat in clinic. Increasing the dose and combination of antimicrobials can hardly completely remove the pathogen and will bring the higher risk of adverse reactions because of antimicrobial resistance and lung-blood barrier. Low-frequency ultrasound (LFU), as a noninvasive targeted physical therapy, presents a unique advantage in promoting penetration and healing. In addition, our research group found that LFU can enhance the antimicrobial activity against pan-drug resistant bacteria. Therefore, based on the results of synergism effect with antibiotic, the possibility to accelerate the distribution of drug to lung tissue and the potential immune regulation and tissue repair capacity of LFU, we first proposed the idea of "combination therapy of LFU and antibiotic against pulmonary infection caused by drug-resistant bacteria". We designed three levels of experiments on the molecular, cellular and whole animal. Microarray method was applied to comprehensively analyze the molecular mechanism of the immunoregulation and tissue repair. The animal model of pulmonary infection suitable for live imaging system was constructed to achieve intuitive and real-time observation on the degree of infection. The feasibility and mechanism of LFU treatment on pulmonary infection were studied in detail from three aspects: synergism with antimicrobials, promoting drug distribution, immunoregulation and tissue repair. We hope to provide new ideas and methods for the clinical treatment of refractory pulmonary infections.

耐药菌所致肺部感染是临床面临的棘手问题，耐药性的攀升和肺-血屏障导致药物分布困难使得增大剂量和联合应用抗菌药物都难以完全清除肺部感染且会带来不良反应增加的风险。低频超声(LFU)作为一种无创靶向物理治疗方法，在促药渗透和创面修复方面展示出独特的优势，并且课题组前期研究还发现LFU能加强抗菌药物对泛耐药细菌的抗菌活性。因此，基于LFU抗菌增效的研究结果，并考虑其可靶向作用于肺部加速系统性用药肺组织的分布可能性以及潜在的免疫调节和组织修复能力，我们首次提出“LFU联合抗菌药物治疗耐药菌所致肺部感染”的设想，设计分子、细胞和整体动物三个层面的系统实验，采用microarray方法全面解析调节修复的分子机制，构建活体动物成像肺部感染模型直观实时评价，从抗菌增效、促药分布和调节修复三方面深入研究LFU联合抗菌药物治疗耐药菌所致肺部感染的可行性和作用机制，为临床治疗难治性肺部感染提供新的思路和方法。

肺部感染是临床最常见的感染性疾病，而医院内获得性肺部感染致病菌耐药性高。细菌的广泛耐药引发了抗菌药物应用的危机，虽然近些年不断有新型抗菌药物的研发，但耐药菌也紧随其后出现，因此，很多研究者提出了其他替代疗法来应对日益严重的细菌耐药性问题，低频超声（LFU）作为一种新型物理手段为肺炎的治疗提供了新的思路。本研究首先通过体外实验证明了LFU可协同抗菌药物有效抑制耐药菌及其生物被膜，摸索出了LFU的使用频率为40KHz、强度为92.36Mw/cm2，结果显示LFU可显著增强抗菌药物对耐药菌的杀菌效果，并且多次超声（M-LFU）与抗菌药物联合应用比单次超声（S-LFU）对细菌生物膜具有更好的协同作用。为了建立稳定的小鼠肺炎模型，通过摸索不同浓度的环磷酰胺剂量及对免疫抑制后的小鼠肺部荧光强度、载菌量、血液学检查和组织病理等指标评价，最终确定了术前第4天进行150 mg/kg、术前1天进行100 mg/kg环磷酰胺腹腔注射预处理的肺炎模型更稳定。为了进一步探索LFU联合抗菌药物对小鼠肺炎克雷伯菌肺炎的治疗效果，从小鼠行为学改变、肺部细菌清除率、肺组织病理学及细胞因子的结果看，LFU与美罗培南(MEM)或阿米卡星(AMK)联合使用可显著改善小鼠的行为状态。与单独使用抗菌药物组相比，LFU联合抗菌药物组的小鼠肺部荧光强度下降更显着，LFU联合抗菌药物组肺组织的平均菌落数(CFU)也较低。尽管未观察到血浆和BALF中细胞因子（IL-6 和 TNF-α）的显着变化，但与单独使用抗菌药物组相比，LFU联合抗菌药物组的炎症损伤较小。此外，通过小鼠尾静脉给药60min和30min后，LFU处理10min表现出分别促进了 MEM 和 AMK 在小鼠肺组织中的分布，特别是对于在肺组织分布较低的AMK，在60min时AMK的浓度在LFU联合抗菌药物组比单用药物组提高了432 ng/mL。终上所述，LFU的应用为肺炎治疗节约型用药和预防耐药菌提供了新方法，可协助抗菌药物尽快杀死致病菌，并促进炎症修复，值得在以后的工作中进一步研究，为其能够在临床转化应用提供基础。