Effect Of Exposure To PM_2.5on Pulmonary Inflammation After Klebsiella Pneumoniae Infection In Rats

Objective: Epidemiological studies reveal increased hospitalization forpneumonia when levels of particulate matter in the air are increased,especiallyin children and the olderly population.Klebsiella pneumoniae is one of themost frequent bacterial pathogen causing pulmonary infection among elderlyand immunocompromised individuals.In this study,we developed a rat modelin which the animals were infected with Klebsiella pneumoniae followed byexposure to PM_2.5。We sought to determine the effects of PM_2.5exposure onlung inflammation caused by klebsiella infection by examining clinicalmarkers,classification of pulmonary pathology, the cell count inBALF,colonies count,rats mortality.We discussed potential mechanisms byexamining the cellulosity of tracheal membrane epithelium cells and conditionof cilia by scanning electron microscope and the contents of TNF-α and IL-6.Methods:92male SD rats of clean grade was divided into4groupsaccording to random number table:①control group;②Klebsiella+normalsaline group(For short:Klebsiella group);③PM_2.5+normal saline group(Forshort: PM_2.5group);④Klebsiella+PM_2.5group(For short: Klebsiella+PM_2.5group).Klebsiella pneumoniae infection:Mice were instilled suspension liquidof standard strain of Klebsiella pneumoniae via the trachea1ml/kgwith1.0×106cfu/ml; PM_2.5:Mice were instilled suspension liquid of particulatematter via the trachea1ml/kg with30mg/ml.Part rats were killed randomly atthe1stday and the7thday in different groups. Observe the clinical presentationof the rats,and give a mark;Calculating the mortality rate of different groups;The level of TNF-α and IL-6in blood serum were detected by the method ofELISA;The pathomorphism changes of lung tissue was observed and gradedby the method ofHE staining;Bronchoalveolar lavage fluid(BALF)cells wascounted and classified.The number of colonies of BALF was counted. The composition of cells and the change of cilia in trachea epithelium wereobserved by scanning electron microscope(SEM).Results:1.Clinical presentation score: The median and interquartile of clinicalpresentation score at the1stday and7thday in each group were: control group4(1),1(1),Klebsiella group6(2),6(1), PM_2.5group5(1),4(2), Klebsiella+PM_2.5group5(2),8(4),There were no significant differences in different groups at the1stday.The clinical presentation score of the Klebsiella group,PM_2.5group andKlebsiella+PM_2.5group were all significantly higher than control group on the7thday(P<0.05).The clinical presentation score inKlebsiella+PM_2.5group washigher than in Klebsiella group and PM_2.5group(P<0.05); Compared withthe1stday, the clinical presentation score were decraesed at7thday in controlgroup and PM_2.5group(P<0.05).NO significant differences were found on the1stday and7thday in Klebsiella group and Klebsiella+PM_2.5group.2. Rats mortality:There was no significant differences of the rats mortality indifferent groups at the1stday. The rats mortality of the Klebsiella group, PM_2.5group and Klebsiella+PM_2.5group are all significantly higher than in thecontrol group on the7thday.Klebsiella+PM_2.5group was higher than Klebsiellagroup and PM_2.5group(P<0.05).Compared with the1stday, the rats mortalitywere significantly incraesed at7thday in Klebsiella group and Klebsiella+PM_2.5group (P<0.05). The rats mortality of other groups were no significantdifferences between the1stday and the7thday.3.The level of IL-6and TNF-α in blood serum:The level of IL-6in bloodserum at the1stday in each group(control group, Klebsiella group, PM_2.5group,Klebsiella+PM_2.5group)were0.16±0.04,pg/ml,0.88±0.10pg/ml,0.40±0.10pg/ml,1.32±0.54pg/ml; The levels of IL-6in blood serum at the7thday in each group concluding control group, Klebsiella group, PM_2.5groupand Klebsiella+PM_2.5group were0.18±0.07pg/ml,0.59±0.09pg/ml,0.55±0.17pg/ml and1.24±0.29pg/ml,and the levels of TNF-α in blood serumat the1stday in each group were168.34±48.68ng/L,670.01±83.01ng/L,400.33±67.48ng/L,647.23±41.12ng/L; The levels of TNF-α in blood serum at the7thday in each group were162.48±25.97ng/L,710.49±32.43ng/L,374.70±73.15ng/L,927.54±83.95ng/L. The level of IL-6and TNF-α in thegroups (Klebsiella group,PM_2.5group and Klebsiella+PM_2.5group)weresignificantly higher than the control group at the1stday and the7thday. Thelevel of IL-6and TNF-α in the Klebsiella+PM_2.5group was increasedcompared with Klebsiella group and PM_2.5group(P<0.05).The level of IL-6and TNF-α had no difference between the1stday and the7thday.4.The grades of pulmonary histopathology: The Pathological damage inKlebsiella group,PM_2.5group,and Klebsiella+PM_2.5group were more seriousthan in control group at different time (P<0.05); The grades of pulmonaryhistopathology of Klebsiella+PM_2.5group was more serious than in Klebsiellagroup and PM_2.5group(P<0.05); Compared with the1stday, the grades ofpulmonary histopathology were significantly incraesed at the7thday inKlebsiella+PM_2.5group(P<0.05). NO differences were found at the1stday andthe7thday in other groups(P>0.05).5. Cell count and classification in BALF:The numbers of cells and theperecent of neutrophil (%) had a significant increase in Klebsiella group,PM_2.5group and Klebsiella+PM_2.5group compared with the control group atthe different time(P<0.05); The numbers of cells and the perecent ofneutrophil(%) in Klebsiella+PM_2.5group was higher than in Klebsiella groupand PM_2.5group(P<0.05);Compared with the control group,the perecent ofmacrophage(%) in PM_2.5group and Klebsiella+PM_2.5group was significantdecreased on each time point(P<0.05);Compared with the1stday, Thenumbers of cells and the perecent of neutrophil (%)were significantlyincraesed at the7thday in Klebsiella group,PM_2.5group and K lebsiella+PM_2.5group (P<0.05). Compared with the1stday, the perecent of macrophage(%)was significantly decraesed at the7thday in PM_2.5group and Klebsiella+PM_2.5group (P<0.05).6. Bacterial colony counts of BALF: The median and interquartile of bacterialcolony counts in Klebsiella group and Klebsiella+PM_2.5group were6×103cfu/ml (103cfu/ml),104cfu/ml(103cfu/ml) respectively on the1stday. The median and interquartile of bacterial colony counts in Klebsiella group andKlebsiella+PM_2.5group were8×103cfu/ml (4×103cfu/ml),105cfu/ml (104cfu/ml) respectively on the7thday;There were no statistical significancebetween the Klebsiella group and Klebsiella+PM_2.5group(P>0.05).Thebacterial colony counts in Klebsiella+PM_2.5group was higher than inKlebsiella group on the7thday(P<0.05). There was no statistical significanceon the1stday and the7thday (P>0.05).7. The changes of trachea mucous cilia observed by SEM:Columnar epithelialcilia in trachea of the control group rats were intensive and structured.With theexposure time lengthened, tracheal cilia of the Klebsiella group, PM_2.5groupand Klebsiella+PM_2.5group showed the appearance of disorderly arrangemen,adhesion,sparse,distributed in beam,ciliary beat bad and goblet cellhypersecretion squamous metaplasia and neonatal ciliated columnar epithelialcells could be found. A large part of the Cilia was deletion, fracture at the7thday in Klebsiella+PM_2.5group.Conclusions:1. Exposure to PM_2.5can induce lung inflammation.2. Exposure to PM_2.5could enhances pulmonary Inflammation effect ofKlebsiella pneumoniae Infection, diminish bacterial clearance and increasemortalityof the rats.3. The mechanisms of PM_2.5exacerbate lung inflammation involves thedamage of mucocil-liary barrier and interaction of various Chemokines.