猪β防御素-2对猪源肠外致病性大肠杆菌的体内外抑菌活性研究

Antibacterial Activity of Porcine Beta Defensin-2 Against Porcine Extraintestinal Pathogenic Escherichia coli in vivo and in vitro

本研究旨在评价猪β防御素2(porcine beta defensin 2,PBD-2)在体内外对猪源肠外致病性大肠杆菌(ExPEC)的抗菌效果,为评估PBD-2在抗生素替代品中的应用前景提供参考。首先,在体外检测不同浓度PBD-2对猪源ExPEC PCN033的杀菌活性。随后,选取5周龄,体重在18~22 g之间的雌性昆明小鼠,检测PBD-2处理组和PBS对照组小鼠(n≥5)感染不同剂量的ExPEC PCN033后的存活率,脑、脾脏、肺脏组织和血液中的载菌量、炎性细胞因子白细胞介素-6(IL-6)、IL-12、IL-1β和肿瘤坏死因子-α(TNF-α)的水平及脑、脾脏、肺脏组织的病理变化程度。结果表明,PBD-2在25μg/mL时即可在体外极显著抑制猪源ExPEC PCN033的生长(P<0.01),且抑制作用随PBD-2的浓度升高而增强。在体内,与PBS对照组相比,PBD-2处理有效降低了小鼠感染不同剂量ExPEC PCN033后的死亡率。提高PBD-2的治疗剂量对降低小鼠死亡率的效果更加明显。腹腔注射和肌内注射的方式在PBD-2降低小鼠死亡率的效果方面优于口服途径。PBD-2治疗在降低小鼠死亡率的效果方面略低于氯霉素治疗。同时,PBD-2治疗极显著降低了小鼠感染ExPEC PCN03321 h后的脑、脾脏、肺脏组织和血液中的细菌载量(P<0.01),降低了血液中的IL-6、IL-12和IL-1β的含量(P<0.01),减轻了脑、脾脏和肺脏组织的病变程度。上述结果说明PBD-2在体外具有良好的抗猪源ExPEC的活性,同时在小鼠体内对猪源ExPEC感染具有治疗作用,表明PBD-2具有开发成为治疗性药物或者抗生素替代品的潜力。

The purpose of this study was to evaluate the antibacterial activity of porcine beta defensin 2(PBD-2)against porcine extraintestinal pathogenic Escherichia coli(ExPEC)in vivo and in vitro,and provide more insights for evaluating the value of PBD-2 as an antibiotic substitutes.Firstly,the bactericidal activity of different concentrations of PBD-2 against the porcine ExPEC PCN033 was tested in vitro.Subsequently,female Kunming mice aged 5 weeks and weighing between 18-22 g infected with different doses of ExPEC PCN033 were treated with PBD-2 or PBS(n≥5).The survival rate,the bacteria loads of brain,spleen,lung tissue and blood,the levels of inflammatory cytokines IL-6,IL-12,IL-1βand TNF-αand the pathological changes of brain,spleen and lung tissues were observed.The results showed that 25μg/mL of PBD-2 could extremely significantly inhibit the growth of ExPEC PCN033 in a concentration-dependent manner(P<0.01).The in vivo study revealed that PBD-2 treatment could effectively reduce the mortality rate of mice infected with ExPEC PCN033.The effects of PBD-2 were enhanced with increase of treatment dose of PBD-2.The effects of PBD-2 via intraperitoneal injection and intramuscular injection route were better than that of the oral route.PBD-2 treatment was less effective than chloramphenicol treatment in reducing the mortality of mice.At the same time,PBD-2 treatment extremely significantly reduced the bacteria loads in the brain,spleen,lung tissue and blood of mice at 21 h post infection with ExPEC PCN033(P<0.01).At the same time,PBD-2 treatment extremely significantly reduced the levels of IL-6,IL-12 and IL-1βof mice at 21 h post infection with PCN033(P<0.01).PBD-2 treatment also significantly reduced the degree of pathological damages of the brain,lung and spleen tissues of mice at 21 h post infection with ExPEC PCN033.These results indicated that PBD-2 had good activity against porcine ExPEC in vitro,and had therapeutic effect on swine derived ExPEC infection in mice,indicating that PBD-2 had the potential to develop into therapeutic drugs or antibiotic substitutes.