The Preliminary Study On The Relations Between Pneumonia Induced By RSV Combined With Klebsiella Pneumonia Infectionand TLR4-NF-κB Signal Transduction Pathway

ObjectiveThis study employs respiratory syncytial virus(RSV) combined with klebsiella pneumonia(Kp) infection induced pneumonia in SD rats, observed the histopathological changes in lung tissues of SD Rat, detected the expression of TLR4 pathways relevant factors, explored its relationship with TLR4-NF-κB signal transduction pathway.MethodsA total of 60 SD rats were divided into four groups randomly:Control group, RSV group, Kp group and RSV+Kp group. Each group had 15 animals. All animals were infected with the method of intranasal. 0,1,3,5,7 days after RSV or Kp innoculation, we observed the weights, lung index, histopathological changes in the lung tissues by H&E staining, expression of TLR4 and NF-kB m RNA in the lung tissues which were detected by RT-PCR, expression of NF-kB protein in the lung tissues which were measured by Western blot, concentration of TNF-a,IL-1b in BALF which were detected by ELISA.Results(1) Lung index: Compared with control group, in RSV group,Kp group and RSV+Kp group, LI showed increasing trend gradually.RSV group and Kp group increased at 3,5 d.p.i, the difference had statistically significant(P < 0.05), increased obviously at 7 d.p.i,the difference had statistically significant(P < 0.01);Compared with RSV group and Kp group, LI SD rats after infection showed a trend of increase with the extension of time, the difference had statistically significant at 5,7 d.p.i.(F=7.027, P < 0.05)(2) Lung tissue pathology: Lung tissue changed in RSV group and Kp group at 3 d.p.i., lung tissue changed in RSV+Kp group at 1 d.p.i.; Compared with RSV group and Kp group, RSV+Kp group lung tissue inflammation is aggravated. there were diffuse thickening of alveolar interval and the alveolar walls are destroyed, and with the extension of time, the infection alveolar structure increase gradually. Compared with RSV group and Kp group, lung tissue pathology significantly increased in RSV+Kp group in the same time.(3) The transcription level of m RNA: In control group, the expression of TLR4 m RNA, NF-kB m RNA were low, the expression of RSV group, Kp group and RSV+Kp group TLR4 m RNA,NF-kB m RNA increased gradually with the extension of the infection time. The expression of TLR4 m RNA in RSV group increased at 5,7 d.p.i, the difference had statistically significant(P < 0.01); NF-kB m RNA increased at 5 d.p.i, the difference had statistically significant(P < 0.05); increased obviously at 7 d.p.i, the difference had statistically significant(P < 0.01); The expression of TLR4 m RNA in Kp group increased at 3 d.p.i, the difference had statistically significant(P < 0.05), increased obviously at 5,7 d.p.i,the difference had statistically significant(P < 0.01); NF-kB m RNA increased at 3 d.p.i, the difference had statistically significant(P < 0.05), increased obviously at 5,7 d.p.i, the difference had statistically significant(P < 0.01);Compared with RSV group, the expression of RSV+Kp group TLR4 m RNA, NF-kB m RNA increased at 5,7 d.p.i, the difference had statistically significant(P < 0.05); Compared with RSV group, the expression of RSV+Kp group TLR4 m RNA, increased obviously at 7 d.p.i, the difference had statistically significant(P < 0.05), the expression of NF-kB m RNA increased at 5,7 d.p.i, the difference had statistically significant(P < 0.05).(4) The expression of NF-?B protein: SD rats in control group the NF-κB protein levels in the lung tissue expression quantity is low, the expression of RSV group, Kp group and RSV+Kp group NF-?B protein increased gradually with the extension of the infection time. RSV group increased obviously at 5 d.p.i, the difference had statistically significant(P < 0.05); increased obviously at 7 d.p.i, the difference had statistically significant(P < 0.01); the expression of NF-κB protein in Kp group increased obviously at 3 d.p.i, the difference had statistically significant(P < 0.05); increased obviously at 5,7 d.p.i, the difference had statistically significant(P < 0.01); Compared with RSV group, the expression of RSV+Kp group NF-?B protein increased obviously at 3,5,7 d.p.i, the difference had statistically significant(P < 0.05).Compared with Kp goup, the expression of NF-?B protein in RSV+Kp group increased obviously at 5,7 d.p.i., the difference had statistically significant(P < 0.05).(5) TNF-??IL-1? contents: In control group, TNF-α and IL-1??were low, in RSV group, Kp group and RSV+Kp group, IL-1?,TNF-? contents increased gradually with the extension of the infection time. TNF-α increased obviously at 5,7 d.p.i in RSV group and Kp group, the difference had statistically significant(F=9.137,P < 0.05). compared with RSV group, TNF-? in RSV+Kp group increased at 5 d.p.i., the difference had statistically significant(P < 0.05), increased obviously at 7 d.p.i, the difference had statistically significant(P < 0.01); compared with Kp group, increased obviously. at 5,7 d.p.i, the difference had statistically significant(P < 0.05). IL-1? in RSV group and Kp group increased obviously at 5,7 d.p.i, the difference had statistically significant(F=8.737,P < 0.01); compared with RSV group and Kp group, IL-1? in RSV+Kp group increased obviously at 5,7 d.p.i, the difference had statistically significant(F=8.871,P < 0.01).Conclusion(1)Established the pneumonia animal model of SD rats by intranasal method with RSV combined with Kp infection is feasible.(2)RSV combined with Kp infection has the synergistic effects, aggravates pulmonary inflammation.(3)RSV combined with Kp infection can induce pulmonary defence cells release cytokines TNF-??and IL-1?, etc. by TLR4-NF-κB signal transduction pathway.