Metabolic Distribution Of Mycobacteriophage D29in Mice And Effection On Airway Epithelial Cell

Objective:Facing the increasingly serious tuberculosis epidemic, new anti tuberculous drugs are in a dire needed of research and development in order to treat tuberculosis. Phage D29is a kind of lytic mycobacteriophage that infects and destroys both Mycobacterium smegmatis and Mycobacterium tuberculosis, so it has potential for diagnosis and treatment of tuberculosis. To investigate the metabolism of phage D29administered by different routes in mice and effection on morphology and function of airway epithelial cells, which can provide an appropriate administration route and safety for clinically pulmonary tuberculosis treatment.Methods:①Ninety-six male BALB/c mice weighing18g to22g were randomly divided into three groups:intranasal instillation group (n=48), subcutaneous injection group (S.C, n=24) and intramuscular injection group (I.M, n=24).7.5×108PFU phage D29were administered to each mouse in each group. Six mice from every group were killed by cervical dislocation after taking blood by ophthalmectomy, phage D29was enumerated in blood, lung, liver and spleen respectively at1h,2h,3h and4h (the intranasal instillation group enumerated in addition at5h,6h,8h,and12h according to the result).②Human airway epithelial9HTE cell line cultured in vitro was infected with high dose (109PFU) and low dose (107PFU) phage D29respectively. Proliferation of9HTE cells was detected by MTT assay; the secretion levels of IL-6, IL-8in supernatant fluid of9HTE cells were detected by ELIS A respectively; the expression of ICAM-1mRNA on9HTE cells was detected by RT-PCR; apoptosis was detected by flow cytometry (FCM). By all these approachs, we studied what effection on airway epithelial cells by phage D29.Results:①There were significant differences (P<0.05) in phage D29titers in lung among the three groups at different time points, the titers at1h in lung of the intranasal instillation group, the subcutaneous injection group and the intramuscular injection group were (5.7±3.2)×106PFU,(1.9±1.4)×104PFU,(1.7±1.1)×104PFU respectively and the complete inactivation time of mycobacteriophage D29in lung was12h,3h, and4h respectively. The titers in liver is significantly higher than (P<0.05) that in spleen in the subcutaneous injection group and the intramuscular injection group at1h, but no mycobacteriophage D29was detected in liver or spleen in the intranasal instillation group at all the time points.②Compared to the control group, there was no significant difference on cells proliferation between high and low dose group (P>0.05); both high and low dose phage D29induced no more greater IL-6release in supernatants fluid compared to phage buffer group, and there was no significant both the expressions of ICAM-1mRNA and apoptosis of9HTE cells among the groups (P>0.05) High dose phage D29induced greater IL-8release compared to normal group, but no difference compared to phage buffer group, but the level secretion of IL-8in all fours groups were very little.Conclusion:①The titers of phage D29given in intranasal way was the highest and the maintenance time was the longest in lung, compared with other two groups. Therefore the intranasal instillation is an appropriate administration route for pulmonary tuberculosis treatment, but it’s not an ideal administration route to treat extrapulmonary tuberculosis.②Phage D29had no significant effection on gowth and function of9HTE cells, so delivery through airway was relatively safe.③In conclusion, compared with subcutaneous and intramuscular injection, intranasal instillation was the best administration routes for phage D29delivery to treat pulmonary tuberculosis, and it had less effect on airway epithelial cells.