Study On The Negative Effects Of Pm2.5 On Lung Tissue Of Rats And The Expression Of MiR-155.

Atmospheric fine particles,namely PM2.5,refers to the suspended particles in the atmosphere in the aerodynamic diameter less than or equal to 2.5 ? m,with small particle size,large surface area characteristics,propagation distance,the stagnation time is long,easy access to the alveolar terminal,and easily dissolved in the blood and respiratory system.Epidemiological studies found that the main components of PM2.5 are complex and have a carrier role.Fine particles can be loaded with sulfate,nitrate,ammonium salt,carbon containing particles,heavy metals,minerals,bacteria and viruses.When PM2.5 inhaled in the lungs,it can cause acute pulmonary inflammatory response and release a variety of inflammatory factors.At the same time,PM2.5 also contains organic compounds such as PAHs and lipopolysaccharides,which can produce free radicals in the lungs,break the fast oxidation and antioxidant balance,and then cause lung function damage,resulting in negative effects of lung.Although there are many theories on the pathogenesis of PM2.5,the mechanism and regularity of the specific negative effects of the lung still need to be further explored.miR-155(microRNA-155)is one of the important members of the miRNAs family,is a short chain of NLE RNA consists of 23 nucleotides.Studies have found that miR-155 plays an important role in pulmonary inflammation in mice,and has confirmed the important role of miR-155 in regulating gene expression and inflammatory response related to T cell function.It was also found that the expression of miR-155 increased in two cases of pneumonia in rats,and increased in the macrophages of the two infected patients.The negative effect of PM2.5 is closely related to the inflammatory response of the lung,and miR-155 plays an important role in the inflammatory response.Therefore,through the experiments of PM2.5 exposed rats,PM2.5 exposure model,by detecting the expression of miR-155 in lung tissues of rats and inflammatory factors,correlation analysis of miR-155 and inflammatory factors,and then explain the miR-155 and PM2.5 in rat induced pulmonary inflammatory response in significance.Objective1.to investigate the negative effects and mechanisms of atmospheric fine particles(PM2.5)on the lung tissue of rats.2.to investigate the expression and significance of miR-155 in the lung tisse of rats exposed to PM2.5.MethodsExperimental MaterialsExperimental animalsThe SD rats were purchased in Liaoning Changsheng Biotechnology Co.,Ltd.,the license number is SCXK(Liao)2015-0001],body weight 200-220g.The collection of PM2.5 and the preparation of suspensionThe Shenyang municipal environmental monitoring center station is continuously sampled for 4 months(2016.11-2017.2).Before the experiment,PM2.5 was used to make the particulate matter of 2mg/ml in the saline solution,and the ultrasonic oscillation 5min was used to mix and sterilize the particles.Experiment ProcedureGrouping:according to the body weight of rats reduced rank,were randomly divided into PM2.5 rats once treated group(PI group),three PM2.5 treated group(P3 group)and control group(O group),the treated group after treatment were sacrificed according to the time of 3H,12h,24h.Each group was divided into 3 subgroups.Group P1 was given the PM2.5 of each rat 30mg/kg(PM2.5 suspension with a concentration of 2mg/ml).Group P3 was anesthetized at first,third,sixth days,and the dose of PM2.5 was the same every time.The O group did not do anything.Sample collection1.Bronchoalveolar lavage fluid(BALF):the rats were sacrificed after anesthesia,tracheal intubation,clipping the right main bronchus,slow injection of 0 ? 3 ml nonnal saline perfusion of left lung,repeated 3 times,after centrifugation,cell pellets were smear,Wright staining.Jimusai,the proportion of counting inflammatory cells under microscope.2 The light lung tissues were inflated with 1 0%paraformaldehyde fixed for 4 h in formalin,embedded with paraffin,sectioned in the sagital plane,and stained with hematoxylin and eosin(H&E).In H&E stained lung sections,Airway inflammation pathological scores,average interalveolar septal wall distance(mean linear intercept MLI)and Mean alveoli number(MAN)were measured.3 Serum:exposed to abdominal cavity of rats,the abdominal aorta of rats was found,and 10ml of arterial blood was extracted by 20ml syringe.Centrifuge centrifugation was used to keep the supernatant fluid stored at-70 C for detecting TNF-,IL-6,IL-1? and other inflammatory indexes.Detection1 Detection of TNF-alpha,IL-6 and IL-1 beta in the serum of each group:the double antibody sandwich ELISA method was used to complete the operation according to the explanation of the kit.2 The expression of SOD,MDA and GSH-Px in the lung tissue of each group was detected,and the operation was carried out according to the kit.3 The expression of NF-KB in lung tissue of each group was detected by immunohistochemistry(IHC)staining method.In each experimental group,1 stained sections were selected from each animal.7 visual fields were randomly selected on each slice.The average optical density of NF-KB was measured by image analyser.4 The expression of miR-155 in lung tissue by reverse transcription polymerase chain reaction(RT-PCR).(1)the extraction of total RNA of rat lung tissue.(2)reverse transcriptional reaction(3)PCR reactionResults1.In the general blank control group,the rats were active and active,the fur was bright,the body was fat,strong,and the breathing was smooth.PM2.5 a coughing and sneezing after a drug exposure group.The three PM2.5 rats in addition to cough,sneeze,also appeared arch bending,and symptoms such as shortness of breath deeply.There was no significant difference in weight between the rats in each group(P>0.05).2.Pathological changes and histological observation of small airway inflammation:the airway inflammation score of the exposure group was significantly higher than that of the control group,and the degree of inflammatory reaction increased with the increase of the number and amount of infection.The average diameter of alveoli was widened and the density of alveoli became smaller and some alveolar fusion appeared in the infected group.In the control group,the alveolar size was homogeneous,the alveolar wall did not become thinner and broken,no alveolar fusion was found,and there was no inflammatory infiltration and congestion and edema in the wall and alveolar wall.The inflammatory infiltration,edema and even fine particles of the tube wall,alveolar wall and interstitial tissue were visible in the lung tissue of the infected rats.In the lung tissue of the rats,the lung tissue was broken and alveolar wall was broken and the alveoli were fused,the alveolar septum became thinner and the alveolar cavity became larger.3.Bronchoalveolar lavage fluid(BALF)inflammatory cells in PM2.5 exposed groups of lymphocytes and neutrophils were significantly higher than the control group,and with the increase of PM2.5 number and exposure dose,percentage of myeloid cells and lymphocytes are also increasing,and gradually reduce the proportion of phagocytic cells(P<0.01)in P1 and P3.The two group,there was no significant difference between the 3 groups(P>0.05).4.The expression of SOD,MDA and GSH-Px in lung tissue in rats:lung tissue superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)expression than the control group in the lung tissue of rats in low volume,and with the increase of exposure dose and frequency decreased(P<0.01).The expression of malondialdehyde(MDA)in lung tissue of the exposed group was significantly higher than that of the control group,and the expression level of MDA increased with the increase of dose and times(P<0.01).5.The expression of NF-KB in lung tissue:the expression of NF-KB in PM2.5 exposed rats was significantly higher than that in the control group,and the expression of NF-KB increased gradually with the increase of dose and times of exposure.The difference was statistically significant(F=45.06,P<0.01).6.Expression of inflammatory factors TNF-?,IL-6 and IL-1 ? in serum:The expression of inflammatory factors in the PM2.5 group was more than that in the control group,and with the increase of the number of PM2.5,the increase of the expression was more obvious.Among the subgroups of group P1 and group P3,the expression of group 24h was the highest,and the difference was statistically significant.7.Expression and correlation analysis of miR-155 in lung tissue:The expression of miR-155 in PM2.5 group was more than that in control group,and the expression level increased with the increase of exposure times(F:20.19,P<0.01).Pearson correlation analysis showed that miR-155 expression in lung tissue was positively correlated with the expression level of TNF-?,IL-6 and IL-1 ? in serum(r=0.768,0.752,0.729,P<0.01).Conclusions1.PM2.5 can directly damage the structure of lung tissue induced by lung tissue,and can also further cause lung injury by activating the inflammatory reaction and oxidative stress of the lungs and the body.2.The expression of miR-155 increased with the increase of PM2.5 exposure times and dosages.MiR-155 was associated with PM2.5 induced pulmonary inflammatory reaction.Therefore,miR-155 might be involved in PM2.5 induced pulmonary inflammatory response.