目的

分析多西环素联合左氧氟沙星调控高温需求因子A2（high temperature requirement factor A2，HtrA2）治疗耐碳青霉烯类鲍曼不动杆菌（Carbapenem-resistant Acinetobacter Baumannii，CRAB）肺炎的机制。

方法

选择60 只SD 大鼠，随机分为对照组、模型组、多西环素组、左氧氟沙星组、联合组、过表达（overexpressed，oe）-HtrA2 组，每组10 只。 模型组使用100 μl 1×10¹¹ cfu/L AB5075 菌液滴入大鼠鼻腔；多西环素组在模型组基础上使用20 mg/kg 多西环素灌胃给药；左氧氟沙星组在模型组基础上使用30 mg/kg 左氧氟沙星灌胃给药；联合组在模型组基础上使用20 mg/kg 多西环素＋30 mg/kg 左氧氟沙星混合灌胃给药；oe-HtrA2 组采用HtrA2 慢病毒经尾静脉注射SD 大鼠体内，每次200 μl 1×10⁹ IFU/L。 采用实时荧光定量聚合酶链反应检测HtrA2 mRNA 表达；采用菌落形成单位法检测肺组织细菌含量；采用试剂盒检测白细胞计数和中性粒细胞百分比、炎症因子及氧化应激水平；采用蛋白印迹法检测Toll 样受体（Toll-like receptor，TLR）信号通路相关蛋白水平。

结果

对照组、模型组、多西环素组、左氧氟沙星组、联合组和oe-HtrA2 组的HtrA2 mRNA 表达分别为（1.00±0.16）、（2.31±0.29）、（1.76±0.17）、（1.81±0.26）、（1.37±0.14）和（3.89±0.58），TLR2 蛋白水平分别为（1.00±0.20）、（3.65±0.60）、（2.88±0.43）、（2.74±0.65）、（1.87±0.21）和（2.92±0.49），TLR4 蛋白水平分别为（1.00±0.17）、（3.92±0.71）、（2.77±0.54）、（2.81±0.43）、（2.06±0.28）和（3.11±0.44），髓样分化因子88（myeloid differentiation factor 88，MyD88）蛋白水平分别为（1.00±0.15）、（2.94±0.45）、（2.21±0.39）、（2.25±0.23）、（1.44±0.16）和（2.41±0.27）（P＜0.05）。 多西环素组和左氧氟沙星组HtrA2 表达、白细胞计数及中性粒细胞百分比、炎症及氧化应激水平、TLR 信号通路蛋白水平低于模型组，高于联合组； oe-HtrA2 组与联合组结果相反。

结论

多西环素联合左氧氟沙星改善CRAB 肺炎大鼠肺部损伤，与调控HtrA2 抑制TLR2/4-MyD88 信号通路激活相关。

Objective

To study the mechanism of doxycycline combined with levofloxacin in regulating high temperature requirement factor A2 （HtrA2） for the treatment of rats with pneumonia caused by Carbapenem-resistant Acinetobacter Baumannii（CRAB）.

Methods

Sixty SD rats were randomly divided into control group，model group，doxycycline group，levofloxacin group，combination group and overexpressed（oe）-HtrA2 group，with 10 rats in each group.In the model group，100 μl 1×10¹¹ cfu/L AB5075 bacteria were dropped into the nasal cavity of rats； Doxycycline group was given 20 mg/kg doxycycline intragastric administration on the basis of model group.Levofloxacin group was given 30 mg/kg levofloxacin by gavage on the basis of model group.Based on the model group，the combined group was given 20 mg/kg doxycycline ＋30 mg/kg levofloxacin by intragastric administration；In group oe-HtrA2，HtrA2 lentivirus was injected into SD rats through the tail vein at 200 μl and 1×10⁹ IFU/L each time.Real-time fluorescence quantitative polymerase chain reaction was used to detect the expression of HtrA2 mRNA.The colony-forming unit method was employed to measure the bacterial content in lung tissues.Kits were utilized to detect white blood cell count，the percentage of neutrophils，the levels of inflammatory factors and oxidative stress.Western blotting was adopted to detect the levels of proteins related to the Toll-like receptor （TLR） signaling pathway.

Results

The expressions of HtrA2 mRNA in the control group，model group，doxycycline group，levofloxacin group，combination group and oe-HtrA2 group were （1.00±0.16），（2.31±0.29），（1.76±0.17），（1.81±0.26），（1.37±0.14） and （3.89±0.58） respectively.The protein levels of TLR2 in these groups were （1.00±0.20），（3.65±0.60），（2.88±0.43），（2.74±0.65），（1.87±0.21） and （2.92±0.49） respectively.The protein levels of TLR4 were （1.00±0.17），（3.92±0.71），（2.77±0.54），（2.81±0.43），（2.06±0.28） and （3.11±0.44）respectively.The protein levels of myeloid differentiation factor 88 （MyD88） were （1.00±0.15），（2.94±0.45），（2.21±0.39），（2.25±0.23），（1.44±0.16） and （2.41±0.27） respectively.There were statistically significant differences in the above indicators （all P＜0.05）.Compared with the model group，the expressions of HtrA2，white blood cell counts，percentages of neutrophils，levels of inflammation and oxidative stress，and protein levels of the TLR signaling pathway in the doxycycline group and the levofloxacin group were all decreased.The combination group showed further improvement.Compared with the combination group，the results in the oe-HtrA2 group were the opposite.

Conclusion

The combination of doxycycline and levofloxacin can improve lung injury in rats with CRAB pneumonia，which may be related to the regulation of HtrA2 and the inhibition of the activation of the TLR2/4-MyD88 signaling pathway.