Study On The Effects Of PM2.5 On The Blood Glucose Regulated By LncRNA NEAT1 In Mice

Objective:The real-time PM2.5?particle size<2.50?m?exposure mice model was established to observe the effect of PM2.5 exposure on blood glucose.To explor the effect of lnc RNA NEAT1-regulated apoptosis and inflammation of islet cells on the blood glucose changes of mice after PM2.5exposured.Methods:1. Establishment of animal model?1?PM2.5 exposureThirty 6-week-old male C57 mice were randomly divided into three groups with 10 mice in each group:concentrated air group?CA?,unfiltered air group?UA?and filtered air group?FA?.The mice in the CA group were placed in the chamber of the PM2.5 concentration enrichment system.The mice in the UA group were exposed to the outdoor atmosphere,and the mice in the FA group were placed in the chamber of outdoor atmospheric equipped with HEPA.The mice in each group were exposed to PM2.5 for 6 hours a day and other time were exposed to clean air.The mice were exposed for a total of8 weeks.?2?Particle size distribution analysis and concentration detection of PM2.5The aerodynamic particle size spectrometer was used to detect the distribution of particle size in the exposure chambers.The PM2.5concentration was monitored by aerosol particle detector in real time.2. Effect of PM2.5 exposure on blood glucose in miceOral glucose tolerance test?OGTT?and Intra-peritoneal insulin tolerance test?ip ITT?were used to evaluate the effect of PM2.5 exposure on blood glucose in mice.3.Effect of PM2.5 exposure on systemic inflammationThe effects of PM2.5 exposure on white blood cell count and lymphocyte subsets distribution were evaluated by peripheral blood routine test in mice.4.Effect of PM2.5 on the pathological changes and apoptosis in pancreasHematoxylin-eosin?HE?staining was used to observe the pathological changes of pancreatic tissue in mice.The e apoptosis in pancreas of mice was detected by terminal deoxynucleotidyl transferase mediated d UTP Nick end labeling?TUNEL?.5.Effects of PM2.5 exposure on the expression of lnc RNA NEAT1 and the levels of inflammation and apoptosis related proteins in pancreatic tissueReal-time fluorescence quantitative polymerase chain reaction?qRT-PCR?and Western blotting were used to detect the expression of lnc RNA NEAT1,programmed death ligand-1?PD-L1?,STAT3,p-STAT3,IL-6,TNF-?and Caspase-3 in pancreatic tissue.6.Establishment of cell modelThe mouse islet?cells?Beta-TC-6?were divided into three groups:control group?Con,0?g/m L?,low dose group(PM2.5^low,50?g/m L),medium dose group(PM2.5^medium,100?g/m L)and high dose group(PM2.5^high,200?g/m L).Beta-TC-6 cell line were exposred to PM2.5 at different doses for24 hours.Stably low expression of NEAT1(PM2.5^sh-NEAT1)Beta-TC-6 cell line was constructed through RNA interference technique.NEAT1(PM2.5^sh-NEAT1)Beta-TC-6 cells were treated by 200?g/m L PM2.5 for 24 hours.7.Effects of PM2.5 exposure on the expression of lnc RNA NEAT1 and the levels of inflammation and apoptosis related proteins in Beta-TC-6 cellsqRT-PCR and Western blotting were used to detect the expression of NEAT1,PD-L1,STAT3,p-STAT3 and Caspase-3 in Beta-TC-6 cells.Results:1.Particle size distribution and concentration of PM2.5PM2.5 in the CA group accounts for 99.49%of the total number of particles and UA group accounts for 92.14%.During the whole exposure period,the average concentrations of PM2.5 in the UA group,the CA group 0.00?g/m³,respectively.2.Effect of PM2.5 on blood glucose in miceThe results of glucose tolerance test showed that the blood glucose of UA and CA groups was significantly higher than that of FA group?P<0.05?.Insulin tolerance test showed that after intraperitoneal injection of insulin,the decreasing rate of blood glucose in UA and CA groups was significantly slower than that in FA group?P<0.05?.Taken together,PM2.5 exposure could increase the blood glucose and cause insulin resistance in mice.3. Effect of PM2.5 exposure on systemic inflammationThe results of blood routine test showed that the peripheral blood leukocyte count,monocyte count,lymphocyte count and neutrophil count significantly increased after PM2.5 exposure?P<0.05?.This result suggested that PM2.5 exposure could promote the occurrence of inflammation in mice.4. Effect of PM2.5 on pathological changes and apoptosis in pancreasThe results of HE staining showed that destroyed islet structure,degenerated islet cell vacuole,adipocyte infiltrated and pancreatic fibrosis was observed in pancreas of mice after PM2.5 exposure.The results of TUNEL assay showed that the number of apoptotic cells in pancreatic tissue in UA and CA groups was significantly higher than that in FA group?P<0.05?.5. Effects of PM2.5 exposure on the expression of lnc RNA and the levels the proteins related with inflammation and apoptosis in pancreatic tissueThe results of qRT-PCR showed that the expression of NEAT1 in pancreatic tissue significantly increased after PM2.5 exposure?P<0.05?.The results of Western blotting showed that the expression of PD-L1 was decreased?P<0.05?and the expression of p-STAT3/STAT3,IL-6,TNF-?and Caspase-3 was increased in pancreatic tissue of mice after PM2.5exposure?P<0.05?.The above results further suggest that PM2.5 exposure could promote the activation of inflammatory signal pathway and the occurrence of apoptosis in pancreatic tissue.6. Effects of PM2.5 exposure on the expression of lnc RNA and the levels the proteins related with inflammation and apoptosis in islet?cellsThe results of qRT-PCR showed that the expression of NEAT1 in Beta-TC-6 cells significantly increased after PM2.5 exposure?P<0.05?,and the expression of NEAT1 decreased significantly after sh-NEAT1 was transfected by liposome?P<0.05?.Western blotting results showed that the expression of PD-L1 decreased?P<0.05?and the expression of p-STAT3/STAT3 and Caspase-3 increased in Beta-TC-6 cells exposed to PM2.5?P<0.05?.Knocking down the expression of NEAT1 could reverse the effect of PM2.5 on the expression of the above proteins in Beta-TC-6 cells?P<0.05?.Our results suggested that PM2.5 exposure could promote the activation of STAT3 related inflammatory signaling pathway and induces apoptosis by upregulating the expression of NEAT1 in islet?cells.Conclusions:1.PM2.5 exposure could increase blood glucose and cause insulin resistance in mice.2.PM2.5 exposure could upregulate the expression of NEAT1 and promote the ability of inflammation and apoptosis in pancreatic tissue of mice.3.PM2.5 exposure could promote the activation of STAT3 related inflammatory signaling pathway and apoptosis by upregulating the expression of NEAT1 in islet?cells.