The Effects And Mechanism Of Exposure To PM_2.5 On The Clearance Of Klebsiella Pneumoniae Of Rats

Objective:Epidemiological studies reveal increased hospitalization forpneumonia when levels of particulate matter in the air are increased,especially in children and the olderly population. Klebsiella pneumoniae is anopportunistic Gram-negative bacillus, which is one of a primary pathogenamong the olderly population with pneumoniae. We developed a rat model inwhich the animals were exposed to PM_2.5,followed by infection withKlebsiella pneumoniae. We sought to determine the effects of PM_2.5exposureon susceptibility to infection and bacterial clearance by examining clinicalappearance score, bacterial loads in bronchoalveolar lavage fluid (BALF),histopathology assessment of lung, cell enumeration in BALF. We discussedmechanism of the increased susceptibility to infection by examining thecellulosity of tracheal membrane epithelium cells and condition of cilia byscanning electron microscope. In order to PM_2.5 expose lead to pulmonaryinfectious diseases provide the theoretical principle.Methods:86male SD rats of clean grade was divided into4groupsAccording to random number table:①c ontrol group;②normal saline+Klebsiella infection group;③PM_2.5+normal saline group;④PM_2.5+Klebsiella infection group；Klebsiella infection that tracheal instillation ofstandard strain of Klebsiella pneumoniae(1.0×105cfu/ml,1ml/kg),PM_2.5exposure that tracheal instillation of PM_2.5by30mg/kg.Sacrificed some rats ofeach group after1stday and7thday separately, sampled and determinedcorresponding indexes.Anesthetized with an intraperitoneal injection of chloral hydrate10%,bloodletting from femoral and collected the blood, centrifugal,collect theblood and centrifuge it, drew out the supernatant fluid, and detected thecontents of TNF-αand IL-6by ELASA. Sacrificed the animals by open-chest, taken the middle and inferior lobe of right lung, fixed by10%neutral formalin,conventional paraffin-embedded, sliced, HE staining, observed thepathomorphism changes of lung tissue and scored, induced bronchoalveolarlavage of left lung bronchial by tracheal intubation, took BALF.Precipitatedcell was suspended in200μl saline, WBC count. Took the upper trachea, andthen observed the cellulosity of tracheal membrane epithelium cells andcondition of cilia, by scanning electron microscope(SEM).Results:1Clinical score: The medians and quartile intervals of clinical manifestationscores of the first day of blank control group, NS+Klebsiella infection group,PM_2.5+NS group and PM_2.5+Klebsiella pneumoniae infection group were4(1),5(2),5(1),7(2) respectively; The medians and quartile intervals ofclinical manifestation scores of the seventh day of were1(1),5(1),4(2),8(4).namely the clinical manifestation scores of the first day of the animals ofPM_2.5+Klebsiella infection group were higher than that of blank controlgroup(P<0.01); and there were no statistics difference in the comparison ofthe clinical score change of PM_2.5+NS group,NS+Klebsiella infection groupand blank control group(P>0.05); there were no statistics difference in thecomparison of the clinical score change of PM_2.5+Klebsiella infection groupand NS+Klebsiella infection group,PM_2.5+NS group(P>0.05). The clinicalmanifestation scores of the seventh day of the animals of NS+Klebsiellainfection group, PM_2.5+NS group and PM_2.5+Klebsiella infection group wereall higher than that of blank control group(P<0.01), and the clinicalmanifestation scores of the animals of PM_2.5+Klebsiella infection group washigher than those of NS+Klebsiella pneumoniae infection group andPM_2.5+NS group(P<0.01). The decrease of the clinical scores of the seventhday of blank control group had statistical significance compared with those ofthe first day(P<0.05); And there were no significant improvement in theclinical scores of NS+Klebsiella infection group,PM_2.5+NS group andPM_2.5+Klebsiella pneumoniae infection group at different times(P>0.05).2Comparison of mortality rates: The total mortality rates of the seventh day of blank control group, NS+Klebsiella infection group, PM_2.5+NS group, andPM_2.5+Klebsiella infection group were0%,30%,20%,46.7%respectively.The mortality rates of the animals of NS+Klebsiella infection group,PM_2.5+NS group and PM_2.5+Klebsiella infection group were obviously higherthan that of blank control group, with statistical difference(P<0.0083). Andthe mortality rate of the animals of the seventh day of PM_2.5+Klebsiellainfection group was also obviously higher than those of NS+PM_2.5group andNS+Klebsiella infection group, and the difference had statistical significance(P<0.0083); The mortality rates of the seventh day of PM_2.5+Klebsiellainfection group was obviously higher than that of the first day(P<0.05), butThere were no significant differences of the mortality rates of the animals ofblank control group, NS+Klebsiella infection group and PM_2.5+NS group atdifferent times(P>0.05).3Lung histopathologic score: The histopathological grades of the first day andthe seventh day of NS+Klebsiella infection group, PM_2.5+NS group, andPM_2.5+Klebsiella infection group were higher than that of blank controlgroup, all had statistical differences(P<0.05). And the significant rise ofpathologic scores of PM_2.5+Klebsiella infection group compared toNS+Klebsiella infection group and PM_2.5+NS group had statisticalsignificance(P<0.05). All of the changes of pathological grades of the seventhday of all groups compared to those of the first day had no statisticalsignificance(P>0.05).4Changes in airway mucociliary of electron microscopy: Tracheal cilia of thePM_2.5+NS group showed the appearance of disorderly arrangement, adhesion,sparse, distributed in beam, ciliary beat bad, and visible to the microvilli etc.And the symptoms aggravated gradually with the exposure timelengthened.The cilia was short, irregular and some of ecclasis,even lack of thecilia in partial vision,and neonatal ciliated columnar epithelial cells could befound.Columnar epithelial cilia in trachea of the control group rats werestructured and arrangement rules.5The total cell count of BALF and cell classification: The results showed that the total cell count of BALF and the count and percentage of neutrophils ofthe first day and the seventh day of PM_2.5+NS group, PM_2.5+NS group andPM_2.5+Klebsiella infection group increased significantly compared with theblank control group(P<0.01), and the total cell count of BALF and the countand percentage of neutrophils of PM_2.5+Klebsiella infection group wereobviously higher than those of NS+PM_2.5group and NS+Klebsiella infectiongroup.(P<0.01) And the total cell count of BALF and the count andpercentage of neutrophils of the seventh day of NS+Klebsiella infectiongroup, PM_2.5+NS group and PM_2.5+Klebsiella infection group wereobviously higher than those of the first day(P<0.05). And there were nostatistical differences in the changes of the numbers of total cell count and cellclassification of blank control group at different times(P>0.05).6The changes of contents of IL-6and TNF-α in serum: The results showedthat concentrations of IL-6and TNF-α in serum of the first day and theseventh day of NS+PM_2.5group, PM_2.5+NS group and PM_2.5+Klebsiellainfection group increased significantly compared with the blank controlgroup(P<0.01), and concentrations of IL-6and TNF-α in serum ofPM_2.5+Klebsiella infection group were obviously higher than those ofNS+PM_2.5group and NS+Klebsiella infection group.(P<0.01).Andconcentrations of IL-6and TNF-α in serum of the seventh day ofNS+Klebsiella infection group and PM_2.5+Klebsiella infection group wereobviously higher than those of the first day(P<0.05). And there were nostatistical differences in the changes of concentrations of IL-6and TNF-α inserum of PM_2.5+NS group and blank control group at different times(P>0.05).7The germ tested rate of BALF: The positive rates of the germiculture of thefirst day in the NS+Klebsiella infection group and PM_2.5+Klebsiella infectiongroup were50%,75%respectively, differences of these two groups had nostatistical significance(P>0.05); The positive rates of the germiculture of theseventh day were25%,87.5%respectively,Those of the seventh day ofPM_2.5+Klebsiella infection group were obviously higher than those ofNS+Klebsiella infection group(P<0.05). There were no significant differences in the positive rates of the germiculture of these two groups at different timepoints in intragroup comparisons(P>0.05).8The colony count of Klebsiella pneumoniae of BALF: The medians andquartile intervals of the bacterial colony count(Iogl0cfu/ml) of the first day ofNS+Klebsiella infection group and PM_2.5+Klebsiella infection group were1.89(2.48),1.00(2.02) respectively; and those colony counts of germicultureof the seventh day were3.97(3.31),4.70(1.02) respectively. Those colonycounts of germiculture of the seventh day and the the first day ofPM_2.5+Klebsiella infection group were obviously higher than those ofNS+Klebsiella infection group(P<0.05). And there were no significantdifferences of colony counts of NS+Klebsiella infection group andPM_2.5+Klebsiella infection group compared at different time points(P>0.05).Conclusions:1The inflammatory response of the lungs and function impairment of thebronchus mucociliary system in the rats caused by exposure to PM_2.5.2The exposure to PM_2.5increased the susceptibility of the rats to Klebsiellapneumoniae infection; damaged the clearance of Klebsiella pneumoniae byrats' lung; aggravated the inflammatory response caused by the infection.3The mechanism of enhances the susceptibility of the rats to Klebsiellapneumoniae caused by exposure to PM_2.5involving in function impairment ofthe bronchus mucociliary system and interaction of inflammatory factors.