The Liver Injury Induced By Pm_2.5.5 Short-term Exposure And The Preventive Effect Of Compound Essential Oils

Purpose:Air pollution is closely related to human health.PM_2.5.5 has attracted much attention as a hot spot in today's research.The inhalation of PM_2.5.5 can not only induce lung diseases,but also cause injury to the extrapulmonary organ.In addition,there is no proven method for countering PM_2.5-induced injury of extrapulmonary organ.The purpose of the study is exploring liver inflammation and oxidative stress-mediated liver injury induced by PM_2.5,and exploring the anti-inflammatory and antioxidant preventive effects of compound essential oils.Methods:1.Collection of PM_2.5.5 samples,preparation of suspension and preparation of compound essential oils:During the period February 2013 to April in Hebei,Langfang,China,a 68m³/h sampler was used to collect PM_2.5.5 particles.Ultra fine quartz fiber filters containing PM_2.5.5 particles were treated with 2 hours ultrasound in sterilized normal saline,and the obtained suspension was subjected to vacuum freeze drying.Finally,the obtained PM_2.5.5 particles were mixed with sterile saline to form a PM_2.5.5 suspension of 10mg/mL concentration,which was used to construct the lung inhalation mice model.According to aromatherapy,four kinds of essential oils of mint,eucalyptus,spruce and frankincense were prepared in a certain proportion to compound essential oils.2.Experimental design:All 96 Balb/c mice were randomly divided into four groups,with each group consisting of 24 animals.The animals were housed with free access to food and water.?1?Model control group:treated with 50?l sterile saline at day 0 and day 2 by tracheal perfusion;?2?PM_2.5injury model group:treated with 50?l PM_2.5.5 suspension at day 0 and day 2 by tracheal perfusion;?3?PM_2.5.5 injury model+saline group?Negative control group or treatment group?:treated with 50?l PM_2.5.5 suspension at day 0 and day 2 by tracheal perfusion,but these animals were also exposed to 200?l sterile saline by static inhalation for 30min per day one day before PM_2.5.5 perfusion and until they were sacrificed.;?4?PM_2.5.5 injury model+CEOs group?CEOs experimental group or treatment group?:the animals were treated as in Group III,static inhalation of 200?l sterile saline with 2 drops of CEOs for 30 min per day from the day before PM_2.5perfusion to the end of the day before sacrificed.3.Detection of liver inflammatory injury related markers:Mice were sacrificed at third,seventh,fourteenth days,and liver and serum samples were collected.Serum levels of ALT and AST were detected by automatic biochemical analyzer.Liver tissues were stained with hematoxylin and eosin?H&E?to observe the histopathological and morphological changes by inverted optical microscope.Real time fluorescence quantitative PCR?RT-PCR?was used to detect the gene expression levels of IL-6,TNF?,TLR4,MyD88,NF-?B1 in liver tissues.The protein expressions of TNF?and TLR4 in liver tissues were assessed using Western Blot.4.Expression changes of related indicators of oxidative stress injury in liver:RT-PCR was used to evaluate the gene expression levels of HO-1 and SOD-1 in liver tissues.The protein expression of HO-1 in liver tissues was assessed using Western Blot.Results:1.Serological correlation index test results:Serum ALT and AST levels increased in PM_2.5.5 injury model group compared with the model control group at three time-points.Serum ALT and AST levels decreased after pretreatment of CEOs compared with the negative control group at three time-points.2.Histopathological results:Histological changes displayed in livers of mice including hepatocytic edema and cytosolization in PM_2.5.5 injury model group compared with the model control group at three time-points,especially on day 7 and day14.Compared to the negative control group,hepatocytic edema and cytosolization in livers of mice relatively alleviated in CEOs experimental group at three time-points.3.Expression changes of related indicators of hepatic inflammatory injury.At three time points,the gene expressions of IL-6,TNF?,TLR4,MyD88,NF-?B1 in liver tissues increased in PM_2.5injury model group compared with the model control group.The protein expressions of TNF?and TLR4 were up-regulated as well.The gene expressions of IL-6,TNF?,TLR4,MyD88,NF-?B1 in liver tissues decreased in CEOs experimental group compared with the negative control group at three time points.Besides,CEOs also restrained the protein expressions of TNF?and TLR4.4.Expression changes of related indexes of oxidative stress injury in liver.Compared to the model control group,the m RNA and protein expressions of HO-1 in liver tissues increased in PM_2.5injury model group at three time-points.Correspondingly,the SOD-1?Superoxide dismutase-1?m RNA expression was temporary and significant increase as a protective reaction on day 3 firstly in PM_2.5.5 injury model group compared with the model control group,and then reduced with consumed by ROS,following dramatically increased with protective stress.Compared to the negative control group,the mRNA and protein expressions of HO-1 in liver tissues were down-regulated in CEOs experimental group at three time-points.Whereas,the SOD-1 mRNA expression was temporary and significant reduction on day 3 firstly in CEOs experimental group compared with the negative control group,and then increased on day 7,following continued to decline.Conclusion:Firstly,short-term exposure of PM_2.5.5 by systemic blood circulation could cause liver inflammatory injury and oxidative stress-mediated liver injury in Balb/c mice.Moreover,the activated TLR4/MyD88 signaling pathway might participate in PM_2.5-induced liver inflammatory injury response.Secondly,CEOs could attenuate liver inflammatory injury and oxidative stress-mediated liver injury induced by PM_2.5exposure.Similarly,CEOs might alleviate PM_2.5-induced liver inflammatory injury through inhibiting the activation of TLR4/MyD88 signaling pathway.