The Prophylactic Treatment Of Compound Essential Oil On Brain Injury Induced By Chronic PM_2.5 Exposure In Mice

Purpose: With the development of science and technology and the increase of industrialized cities,environmental pollution becomes more and more serious.People begin to pay more attention to the health problems caused by environmental pollution.In recent years,smog has emerged in most cities,so the impact of PM_2.5 on health has received more attention.However,at present,only limited studies have been conducted on the damage of PM_2.5 to cardiovascular system and respiratory system,and few studies have been conducted on the damage to nervous system.In this experiment,a model of PM_2.5 chronic exposure mice was constructed to study the damage of PM_2.5 to the body's brain,and at the same time,to study whether the compound essential oil can play a protective role in the anti-inflammation and anti-oxidation way.Methods: 1.PM_2.5 collection,suspension preparation and component analysis: all PM_2.5 samples in the experiment were collected from October 2015 to March 2016 in Langfang,Hebei province.The collected PM_2.5 filter film was cut into pieces and then placed in 70% ethanol for ultrasonic treatment for 2 hours.The ultrasonic liquid was filtered through 12 layers of gauze,evaporated and concentrated,and then vacuum freeze-dried to obtain PM_2.5 particles.Then,PM_2.5 suspension of the corresponding concentration was prepared with sterile normal saline and put into the dynamic poisoning cabinet for poisoning.The chemical composition of the extracted PM_2.5 particles was analyzed.2.Grouping and establishment of animal models: 36 BALB/c mice?half male and half female?were randomly divided into three groups,with 12 mice in each group,respectively the control group,PM_2.5 group and PM_2.5+ compound essential oil group.Mice in the PM_2.5 group and the PM_2.5+ compound essential oil group were placed in the dynamic exposure cabinet for poisoning at a concentration of 300 ?g/m3,5 hours a day,5 days a week,for 16 weeks.The mice in the PM_2.5+ compound essential oil group were inhaled to the compound essential oil one day after the start of the poisoning and until the day before the execution.The mice were inhaled with 1% compound essential oil for 30 minutes a day.3.Measurement of brain injury related indicators: Morris water maze experiment was used to detect behavioral changes after mice were poisoned.Brain tissue and serum were sacrificed and weighed and recorded.Brain tissue was stained with hematoxylin-eosin?H&E?to observe pathological changes.The expressions of iba-1 and GFAP were observed by immunofluorescence and immunohistochemical staining.The damage of neurons was observed by Nissl staining.MDA content in serum of mice was determined by MDA microassay kit.4.Measurement of inflammatory reaction and oxidative stress related indicators: MDA level in mouse brain tissues was determined using MDA microassay kit;m RNA expression levels of IKK?,TNF-?,IL-1? and HO-1 in hippocampal tissues were determined by q PCR.Western blot was used to detect the protein concentrations of NF-KB and HO-1 in the hippocampus.ROS expression was detected by ROS detection kit.5.Measurement of NRF2 and NOX2: the gene and protein expression levels of NRF2 and NOX2 in hippocampal tissues were detected by q PCR and Western blot,respectively.Results: 1.Body weight and organ coefficient results: Compared with the control group and the PM_2.5 group and the PM_2.5+ compound essential oil group,there were no statistically significant changes in body weight and organ coefficient;2.Pathological results: by observing the HE staining of mouse brain tissue,it was found in the PM_2.5 group that pyramidal cells in the hippocampal area were irregularly distributed and slightly widened,with loose and chaotic cell arrangement,swollen cells,and apical dendrites of pyramidal cells disappeared.In the PM_2.5+ compound essential oil group,pyramidal cells in the hippocampal area were regularly distributed,slightly widened,and some cells were edema.3.Immunofluorescence results: compared with the control group,the fluorescence signal of PM_2.5 group and PM_2.5+ compound essential oil group showed a trend of significantly enhanced to different degrees;4.Immunohistochemical results: compared with the control group,the levels of GPAP protein in PM_2.5 group and PM_2.5+ compound essential oil group increased,and the PM_2.5 group showed a more significant trend.5.The results of Nissl staining: compared with the control group,the Nissl staining showed slight cell vacuoles in the PM_2.5 group and the PM_2.5+ compound essential oil group after PM_2.5 exposure.6.Behavioral results: it was found in the water maze experiment that the incubation period of the mice was significantly prolonged after the exposure of chronic PM_2.5,and the number of crossing the platform was reduced.Compared with PM_2.5 group,the incubation period of PM_2.5+ compound essential oil group decreased,and the number of crossing the platform increased,but it was not statistically significant.7.Expressions of inflammatory reaction and oxidative stress: the expressions of IKK? m RNA,TNF-?m RNA,IL-1? m RNA and the protein of NF-KB in PM_2.5 group and PM_2.5+ compound essential oil group were increased,while the expressions of PM_2.5+ compound essential oil group were improved compared with PM_2.5 group.Both serum and tissue MDA levels were significantly increased in the PM_2.5 group compared with the control group,and decreased in the PM_2.5+ compound essential oil group compared with the PM_2.5 group.Compared with the control group,ROS expression in PM_2.5 group increased.Compared with PM_2.5 group,the ROS expression level of PM_2.5+ compound essential oil group decreased.On the contrary,the expression levels of HO-1 m RNA and protein decreased after the exposure of PM_2.5 chronic.Compared with PM_2.5 group,PM_2.5+ compound essential oil group showed an improved downward trend.8.Expression of NRF2 and NOX2: the expression level of NOX2 m RNA and protein in PM_2.5 group were significantly increased compared with the control group,while the expression level of NRF2 m RNA and protein were decreased.After inhalation of compound essential oil,compared with PM_2.5 group,the expression level of NOX2 m RNA and protein in PM_2.5+ compound essential oil group decreased,while the expression level of NRF2 m RNA and protein were significantly up-regulated.Conclusion: 1.PM_2.5 chronic exposure can lead to brain lesion,and the mechanism may be that the imbalance between NOX2 and NRF2 leads to inflammation and oxidative stress.The increase of reactive oxygen species?ROS?or inflammatory factors can cause brain damage.2.Compound essential oils made from peppermint,frankincense,eucalyptus,spruce simple essential oils can reduce brain damage caused induced by PM_2.5;The possible mechanism is to improve the inflammatory and oxidative stress response by inhibiting the increase of NOX2 and the up-regulation of NRF2,so as to reduce the reactive oxygen radical ROS or inflammatory factors and thus alleviate the brain damage.