Study On The Effect Of Ambient PM_2.5 On Respiratory System Under Different Cold Stresses And The Related Mechanism In Rats

Objectives Ambient particulate matters and low temperature stress were reported to have effects on the respiratory disease hospital admissions and deaths, interaction effect was found between the two factors. The purpose of this study is to discuss the effect of fine particulate matter(PM2.5) on respiratory system within different cold stresses and the related mechanism by estimating inflammation and oxidative stress responses.Methods 1.Experiment in vivo 48 Wistar male rats, matched by weight, were randomly assigned to six groups, Every group contains 8 rats, each rat was treated with cold stress alone(0°C, 10°C, and 20°C(normal control) and cold stresses plus PM2.5 exposures respectively. Cold stress alone groups were treated with intratracheal instillation of 0.25 ml normal saline, while cold stress plus PM2.5 exposure groups were treated with intratracheal instillation of 8 mg/0.25 ml PM2.5. These procedures were carried out for three times with an interval of 48 hours for each treatment. All rats were sacrificed after 48 hours of the third treatment. 2. Experiment in vitro Alveolar macrophages(AMs) were isolated and cultured by using the modified techniques of primary culture. These AMs were divided into different groups,cultured and exposed in 18°C(strong hypothermia), 24°C(moderate hypothermia), 30°C(weak hypothermia), 37°C(normal temperature) groups and each temperature group was divided into different groups with the final ambient PM2.5 dosages of 0μg/ml(cold stress alone), 25μg/ml(low dose), 50μg/ml(middle dose) and 100μg/ml(high dose)groups. The morphological changes of AMs were observed under the inverted microscope. Neutral red absorption test and MTT assay were used to observe phagocytosis function and the relative survival rates of AMs 3.Biomarker estimations The bronchoalveolar lavage fluid(BALF) was collected for counting inflammatory cells by using hematocytometers. The TNF-α, IL-6, IL-8, CRP, MIP-1α, MSR1 and GM-CSF were detected with ELISA kit. The SOD, GSH-Px and MDA were determined with assay kit.Results 1. Experiment in vivo Results showed higher level of total cells and neutrophil in the BALF of PM2.5 exposed groups(P<0.05). All PM2.5 exposure groups showed significantly higher level of four inflammatory cytokines than their corresponding cold stress alone groups(P<0.05). The levels of SOD, GSH-Px and MDA in groups with PM2.5 instillation were significantly different from their corresponding cold stress exposure alone groups(P<0.05). Significantly higher levels of IL-6 and CRP in lung and IL-6, IL-8 and CRP in the blood were found in all PM2.5 exposed groups with different cold stresses(P<0.05). The level of IL-6 and CRP in lung and IL-6 and CRP in blood were obviously related(P<0.05). Interactive effect of PM2.5 exposure and cold stress was found in the neutrophil, IL-6 and IL-8 level of BALF and in the SOD and IL-6 level of lung(P<0.05). 2. Experiment in vitro(1) Morphological changes of AMs The morphological and number changes of AMs were observed under different cold stress, PM2.5 and combined effect of PM2.5 exposure and cold stress.(2) Phagocytosis function and survival rates of AMs Results showed that the increase of PM2.5 dose and decrease of temperature levels led to the decrease of the phagocytosis function and the relative survival rates of AMs(3) Cytokine level in supernatant Results indicated that all PM2.5 exposure groups showed significantly higher level of inflammatory cytokines and MDA, lower level of MSR1 and SOD than their corresponding cold stress alone groups(P<0.05), and dose-dependence with PM2.5 were found in these changes. The SOD of 18°C and 24°C cold stress alone groups, 18°C high dose group showed significantly lower level than their corresponding normal temperature control groups(P<0.05). MDA showed a significantly higher level in 18°C cold stress alone group and 18°C every-dosage groups than their corresponding control groups(P<0.05). The MDA of 30°C middle dose group showed a significantly lower level than its corresponding normal temperature control group(P<0.05).Conclusions 1. These data demonstrated that both PM2.5 exposure and cold stress had effects on respiratory system and the extremely cold stress may enhance the toxic effect of ambient PM2.5 exposure. The lower temperature of cold stress lead the more significant effect. The interactive toxic mechanism could be through proving oxidative stress and higher production of pro-inflammatory cytokines in lung and even in blood. 2. The inflammatory and oxidative stress injury of AMs by PM2.5 exposure and cold stress may be the major cause of respiratory system damage.