几种临床常用抗生素诱导小鼠腹泻的研究

Study on diarrhea induced by several commonly used antibiotics in mice

目的建立稳定有效的抗生素相关性腹泻(antibiotic-associated diarrhea, AAD)小鼠模型,为抗生素相关腹泻的机制及药物的研究提供支持。方法各抗生素按约临床剂量的8倍,每日灌胃2次,连续5d;停止抗生素后,再观察8d;观察小鼠的腹泻情况;根据单用抗生素的结果,选取致腹泻较明显的进行组合:头孢拉定(1.00g/kg)+庆大霉素(0.16g/kg),头孢呋辛(0.26g/kg)+左氧氟沙星(0.20g/kg),头孢克肟(0.10g/kg)+罗红霉素(0.08g/kg),每日灌胃给药2次,连续5d,观察小鼠腹泻情况、肠道微生物菌群和结肠组织等。结果各抗生素单用,剂量达到约临床的8倍,小鼠腹泻率低,恢复快,不适于作为AAD模型继续研究。抗生素联用,小鼠腹泻出现时间提早、腹泻率高,其中头孢呋辛+左氧氟沙星于第3天出现明显腹泻症状,到第5天腹泻率达到100%,腹泻评分最高。停止抗生素6d后,即实验的第11天,该组小鼠全部可见腹泻症状,且无小鼠死亡。结肠组织观察发现,头孢呋辛+左氧氟沙星组小鼠结肠组织出现炎性细胞浸润;进一步发现结肠组织紧密连接蛋白表达下降。肠道菌群分析发现,该组小鼠与正常组比,肠道菌群结构发生显著变化,多样性显著降低,有益菌群显著降低。结论头孢呋辛+左氧氟沙星灌胃小鼠,每日2次,可复制稳定的抗生素相关腹泻模型,肠道菌群紊乱、抗生素对肠黏膜的直接损坏及二者共同作用导致了腹泻。

Objective To provide an effective antibiotic-associated diarrhea(AAD) animal model so as to advance the pathogenesis study and drug development. Methods Each of the antibiotics at a dose equivalent to 8 times of clinical dose was given intragastrically to mice twice daily for five consecutive days. After the 5 d antibiotic exposure, mice were observed for another 8 d. The diarrhea status of mice was recorded throughout the experiment. The combined use of antibiotics such as cefradine(1.00 g/kg)+gentamicin(0.16 g/kg), cefuroxime(0.26 g/kg)+levofloxacin(0.20 g/kg), and cefixime(0.10 g/kg)+roxithromycin(0.08 g/kg) selected according to the results of individual antibiotic exposure were used to treat mice as described in individual antibiotic treatment. In addition to diarrhea status, intestinal microbiota and expression of occludin and claudin-1 in colon tissue were investigated and the histological analysis of colon was performed. Results Although about 8 times of the clinical dose was used, the diarrhea status induced by each of the individual antibiotic were unsatisfactory as low diarrhea rate and fast recovery were observed. An improved diarrhea status such as an increased diarrhea rate, early onset and long duration of diarrhea were seen by combined use of those antibiotics. The combination of cefuroxime and levofloxacin initiated diarrhea on the third day and its diarrhea rate reached 100% on the fifth day. After the cancellation of antibiotics, the diarrhea status of this group lasted for another 6 d and no mice died. Histological analysis revealed colon tissue damage which was further proved by decreased expression of occludin and claudin-1 in colon tissue. Obvious intestinal microbiota imbalance in cefuroxime and levofloxacin treated group was observed. Conclusion The combination of cefuroxime and levofloxacin effectively induced AAD, and intestinal microbiota imbalance as well as toxicity of antibiotics individually or collectively play a role in the development of AAD.