卡博替尼抑制产单核细胞李斯特菌侵袭Caco-2细胞的机制

Inhibitory effect of cabozantinib against Listeria monocytogenes invasion in Caco-2 cells in vitro

目的探讨c-Met受体阻断剂卡博替尼(Cabozantinib,XL-184)能否抑制产单核细胞李斯特菌(Listeria monocytogenes,LM)进入Caco-2细胞并降低对细胞的损伤。方法通过侵袭实验研究XL-184在抑制LM进入Caco-2细胞中的作用,并研究剂量及时间与侵袭率的关系;Caco-2细胞种植于Transwell上室,LM感染单层Caco-2细胞,然后在上室中加入辣根过氧化物酶(HRP),通过测定跨上皮细胞电阻(TEER)值及计数从上室渗透到下室的LM数量和HRP浓度,预测X-184对单层细胞通透性的保护作用;检测乳酸脱氢酶(LDH)释放水平推测细胞膜的渗透性;荧光染色鉴定细胞活性。结果相比对照组,侵袭实验显示,XL-184处理组的LM侵袭率明显降低(P=0.000),且侵袭率随XL-184浓度和作用时间的增加而显著降低,联合用药效果优于单独用药(P<0.05)。细胞通透性实验表明,XL-184可明显抑制LM诱导的跨上皮细胞电阻(TEER)的降低(P<0.05),并可降低辣根过氧化物酶和细菌介导的细胞通透性(P=0.000);细胞活性实验表明,XL-184可提高细胞存活率(P<0.01);同时,可显著降低LM诱导的LDH释放(P<0.05)。结论 XL-184可能在抑制LM侵袭Caco-2及降低LM诱导的Caco-2损伤中起重要作用。因此,XL-184有望成为潜在的治疗和预防李斯特菌感染的有效药物。

Objective To investigate the effect of c-Met inhibitor cabozantinib（XL-184） in inhibiting Listeria monocytogenes（LM）from invading Caco- 2 cells to reduce the cell injury. Methods The cell invasion capacity of LM was assayed in Caco- 2 cells incubated with different doses of XL-184 for different durations. Caco-2 cells incubated with XL-184 were seeded on the upper room of the transwell chamber, and the cell monolayer was exposed to LM infection followed by addition of horseradish peroxidase（HRP）. The trans-epithelial electric resistance（TEER）, HRP concentration and LM colony-forming unit（CFU） were measured in the cell monolayer. Fluorescent staining was used to evaluate the cell viability, and LDH release from the cells was examined to assess the changes in cell membrane permeability. Results XL- 184 significantly decreased LM invasion rate in Caco-2 cells in a dose- and time-dependent manner（P=0.000）, and this effect was enhanced by co-incubation of the cells with ampicillin（P〈0.05）. In the cell membrane permeability assay in the monolayer cells, XL- 184 markedly inhibited LM- induced reduction of TEER（P〈0.05） and significantly suppressed LM- induced enhancement of cell membrane permeability shown by reduced HRP concentration and LM count in the lower chamber（P=0.000）. The cells infected with LM showed significantly lowered cell viability, which was rescued by XL- 184（P〈0.01）; XL- 184 also dose- dependently reduced LDH release from the cells（P〈0.05）. Conclusion XL-184 can suppress LM invasion in Caco-2 cells to reduce the cell injury, suggesting its value as a promising candidate agent for prevention and treatment of LM infections.