Melatonin Regulates Nanoscale Silicon Dioxide Induced Autophagy,Apoptosis And Inflammatory Reaction In RAW264.7 Cell

Background The alveolar macrophage is one of the main effect of immune cells,effectively identify and combined with the removal of pathogens,and release a variety of bioactive substances involved in immune defense responses.Pm2.5 refers to the material is less than 2.5 microns in diameter,including inorganic composition,organic compounds,trace metal elements,elemental carbon(EC),biological material(such as bacteria,bacteria,mold,etc.).Grain diameter greater than 2.5 micron material harm to the human body is relatively small;grain diameter less in the 2.5 micron particles,it can cause a variety of diseases.When silicon dioxide is sucked into the body,the immune system starts with natural immunity,and macrophages play an important role.SiO2 is mainly caused by the dust in the air,autophagy and apoptosis of macrophage stimulated with the silica in a series of changes of immune molecules,especially apoptotic changes in the concentration changes of the relevant point in time has not been clarified clearly.Melatonin is produced by mammals and human pineal an amine hormone.Melatonin in macrophages in the process of silica to gobble up if there is a regulation,especially the changes of macrophage apoptosis will have a regulatory role has yet to clarify clearly.Objectives1.Study activated level of autophagy and apoptosis signaling protein expression in RAW264.7 cells stimulated with SiO2.2.Investigate the role of melatonin in autophagy and apoptosis pathways in SiO2 stimulated RAW264.7 at a cellular level,in order to clear melatonin on macrophage function in the process of immune response.Methods1.Determined by MTT detection of nano silicon dioxide on RAW264.7 cell survival The survival rate of RAW264.7 cells stimulated with SiO2 was detected by MTT crystallization of ELISA tests.2.LDH value of the activity at the different time points of RAW264.7 cells stimulated by nano silicon was detected The activity value of LDH enzyme in RAW264.7 cells was obtained by the ELISA detection of LDH in the culture fluid.3.Treatment group and intervention group of RAW264.7 cells Treatment group:Use 100μg /ml of suspension in DMEM culture of 10 nm SiO2 in 0,2,4,8,12,24 hours to stimulate RAW264.7 respectively,collect all time points supernatant and cell protein components,or for subsequent experiments.Intervention group: Do the following four groups,three holes each group,each hole 1ml DMEM cultures,0 h group: without 10-20 nano SiO2 suspension and Melatonin,DMSO group:only add 1μl DMSO;SiO2 group:10-20 of nano SiO2 suspension makes the hole concentration reached 100μg /ml,SiO2 + Melatonin groups: to join 10-20 of nano SiO2 suspension makes hole concentration reached 100μg /ml,at the same time to join 1μl200 mol/L melatonin to make its final concentration reached 200μmol/ml.After 12 h after three sets of role,collect 0 h group and DMSO group,the cultivation of SiO2 and SiO2 + Melatonin groups of cells supernatant and protein composition,or for subsequent experiments.4.ELISA detection of supernatant fluid TNF alpha,IL-1β and IL-18 of treatment group and intervention group supernatant fluid.The TNF alpha of 0 h,2 h,4 h,8 h,12 h,24 h were dectected by ELISA kits.TNF alpha,IL–18 and IL-1β value of 0 h group and DMSO group,SiO2 and SiO2 +Melatonin groups were also dectected with ELISA kits.TNF alpha,IL-1β,and the level of IL – 18 were calculated as reference to OD value of the standard.5.Western Blot and half quantitative analysis of the expression of autophagy and apoptosis signal protein Using Western Blot method inspection 0,2,4,8,12,24 hours of beclin1,bax,bcl,bax/bcl,LC3 and the change trend of caspase-3;moreover detecting 0 h group and DMSO group,SiO2 and SiO2 + Melatonin groups of beclin1,bax,bcl,bax/bcl,LC3 and the change trend of caspase-3.6.Treatment group and intervention group of laser confocal microscopy imaging With GFP-LC3 plasmid RAW264.7 cells grown in processing chip,according to the treatment group and intervention group processing steps.Put on the wafer seal,imaging laser confocal microscopy of treatment group and intervention group.7.The RAW264.7 cell apoptosis rate was detected by flow cytometry instrument Through the Annexin V-EGFP FITC channels(FL1)green fluorescence wavelength and PI PE-Cy5 channels(FL4)red fluorescence under 488 nm to detect the treatment group and intervention group cell apoptosis rate by Q2 + Q3.8.The results of transmission electron microscope scanning of RAW264.7stimulated with nano silicon dioxide and melatonin.Through the preparation of 0 h group and DMSO group,SiO2 and SiO2 + Melatonin groups of electron microscope slides,the JEM-1230 type transmission electron microscope scan RAW264.7 cells for phagosome and bubble.9.Statistical methods SPSS20.0 software was used for statistical analysis.Data were expressed as mean±standard error;Using single factor analysis of variance,the P value < 0.05 showed significant difference.Results1.MTT had no effect on the survival rate of the RAW264.7 cells of nano-silica stimulation Through the experiment we found that compared with the control group(0 mu g/ml silica)RAW264.7 cells stimulated with 100μg/ml nano silicon dioxide at different time points have no effect on survival.2.LDH value of RAW264.7 cells of induced by nano-silica increased at the different time points compared with 0h The detection showed that the value of the LDH on RAW264.7 cells of induced by nano-silica was higher and higher.2h,4h,8h,12 h and 24 h time point values have significant statistical significance compared with 0h time point.3.The tnf-alpha in the SiO2-stimualated liquid was gradually increased for 2 h,4h,8 h,12 h,24 h,while the level of TNF-alpha in the intervention group with melatonin was decreased and the level of IL-1 beta and IL-18 were increased When the 100 μg/ml nano SiO2 stimulated RAW264.7 cells for 2 h,4 h,8 h,12 h,24 h.TNF alpha at each time point was gradually increased for 2 h,4 h,8 h,12 h,24 h compared with 0h controls with significant statistical significance.Compared with SiO2 group,the IL-1β and IL-18 levels in SiO2 + Melatonin group were significantly increased and with significant statistical significance.Compared with SiO2 group,the TNF-alpha decreased significantly and with significant statistical significance.DMSO group,SiO2 and SiO2 + Melatonin groups compared with 0 h control group have a significant statistical significance.4.LC3 and Bax signaling pathways was activated in SiO2-stimulated Raw264.7cells,LC3 in the intervention group with melatonin was increased and Bax decreased When the 100 μg/ml nano SiO2 stimulate RAW264.7 cells for 2 h,4 h,8 h,12 h,24 h.beclin1,bax,bax/ bcl-2,caspase 3,LC3 showed a trend of increasing with time,the Bcl-2 showed a trend of decreasing over time.LC3 have the most obvious changes in 12 h and 24 h,compared with 0 h the differences were statistically significant.Bax and caspase 3 differences in 24 h time point compared with 0 h were statistically significant.Beclin1 at 2 h,4 h,8 h,12 h,24 h point has increased,but compared with 0 h there is no statistically significant difference.The Bcl-2 point decreases with time and 24 h minimum,no statistically significant difference compared with 0 h.0 h group and DMSO group,SiO2 and SiO2 + Melatonin groups were detected,beclin1,LC3 point showed a trend of increasing with time,LC3 increased obviously at SiO2 + Melatonin group than SiO2 group,and the differences were statistically significant.Bax at SiO2 +Melatonin group significantly reduced than at SiO2 group,and the differences were statistically significant.bax/ bcl-2,Bcl-2 and caspase-3 at SiO2 + Melatonin group significantly reduced than at SiO2 group,but the differences were no significant statistical.5.GFP-LC3 point particles of laser confocal microscope imaging showed a significant increase in treatment group and a significant decrease in intervention group The cytoplasm GFP-LC3 pointlike particles of RAW264.7 at 12 h and 24 h increased obviously than other time points.The cytoplasm GFP-LC3 pointlike particles of RAW264.7 at SiO2 + Melatonin group increased obviously than SiO2 group,the differences were statistically significant.6.Flow cytometry instrument detection showed that the number of apoptosis RAW264.7 of the SiO2 stimulation was increasing with time,and the decreased by Melatonin With 100 μg/ml of suspension in DMEM culture of 10 nm SiO2 in 0,2,4,8,12,24 hours to stimulate RAW264.7 respectively,the rates of apoptosis increased with time,and have the significant statistical significance.The rates of apoptosis in SiO2 +Melatonin group reduced than SiO2 group,and have significant statistical significance.7.Transmission electron microscope scanning showed that the number of phagosomes of the RAW264.7 was increased with the stimulation of nano-silica stimulated by Melatonin Consuming vesicles was obviously formed at SiO2 and SiO2 + Melatonin groups which contained number of phagosome.SiO2 + Melatonin groups had many phagosome than SiO2 group with significant statistical significance.Compared SiO2 + Melatonin group and SiO2 group with 0 h group and DMSO group,the former phagosome number increased obviously,and the differences has significant statistical significance.Conclusions1.SiO2 leads to RAW264.7 autophagy and apoptosis growing.2.Melatonin has a promoting nano SiO2 stimulation of RAW264.7 cells autophagy,decrease apoptosis.3.The cytokines can reduce the content of tnf-alpha and increase the content of IL-1beta and IL-18 in the inflammatory aspects of nano-silica and melatonin stimulated RAW264.7.