| Objective:Arsenic is a relatively abundant element that is ubiquitous in nature.Arsenic and arsenic compounds are human Class I carcinogens recognized by the International Agency for Research on Cancer(IARC).The literature from epidemiological and experimental studies suggests that arsenic has immunomodulatory effects on the body,but the mechanism of arsenic-induced immune regulation is still incomplete.In recent years,the damage of arsenic to the immune system and immune cells has become a research hotspot.Dendritic cells(DC)are the strongest antigen presenting cells(APC)in the body's immune defense response.Play the role of immune surveillance.Studies have shown that inorganic arsenic can induce DC immune tolerance through a variety of mechanisms.Autophagy is a common stress response under normal and pathological conditions of cells,and it is also a unique catabolic pathway of eukaryotic cells.Studies have shown that autophagy in DC can affect its innate immune function as well as adaptive immune response.By reviewing relevant literatures at home and abroad,there are few reports on the effects of inorganic arsenic on DC autophagy.This study explored the possible effects of inorganic arsenic on the lysosomal-autophagy pathway of DC cells,and further explored the key transcription factor EB(TFEB)that regulates the cell lysosomal-autophagy pathway,and provided a richer theoretical basis for the possible mechanism of arsenic immunosuppressive effect on DC.Methods:In this study,C57BL/6 mice and bone marrow dendritic cells(BMDC)induced in vitro were used as research objects to observe and explore inorganic arsenic to BMDC through in vitro inorganic arsenic exposure and different interventions.The effect of autophagy function.Overexpression of TFEB by lentiviral infection technology was used to observe and explore the possible role of TFEB-mediated autophagy-lysosomal pathway in the induction of BMDC immunosuppressive effects by inorganic arsenic.The specific research methods are as follows:1.Observation of autophagosomes and lysosomes by transmission electron microscopyBMDC induction culture was collected until the sixth day,and after centrifugation,an appropriate amount of 2.5%glutaraldehyde(prepared in phosphate buffer)was added and fixed in a refrigerator at 4°C for 2 h or longer.After rinsing,fixing,re-rinsing,block dyeing,gradient dehydration,embedding gradient penetration,heating polymerization,etc.,the double-layer membrane autophagosomes and lysosomes were observed by transmission electron microscopy and the records were saved.2.Western blot immunoblot detection of protein expressionBMDC were resuspended in serum-free 1640 medium in 6-well cell culture plates.Treatment with specific concentrations of LPS and inorganic arsenic for 6 h according to the purpose of the experiment.After the treatment,the cells were gently pipetted and collected,the BMDC protein was extracted,and the protein concentration was determined.Preparation of SDS polyacrylamide gel according to the molecular weight of the target protein,followed by protein denaturation,protein loading,electrophoresis,membrane transfection,blocking,primary antibody incubation,secondary antibody incubation,washing,and finally ECL luminescence detection the levels of LC3,Atg5,Atg12,Atg16L1,p-m-TOR/m-TOR,p62/SQSTM1,TFEB,Lamin B,LAMP1,LAMP2,Cathepsin D,Cathepsin L,Cathepsin S,Cathepsin B,Legumain/AEP,ATP6V1A,ATP6V1E1,ATP6V0A1 and?-actin.3.BMDC immunofluorescence stainingThe BMDC induction culture was collected on the sixth day and subjected to LC3B immunofluorescence staining.4%paraformaldehyde was fixed for 15 min,and the immunostaining washing solution was washed for 1 min,3 times.The blocking solution was blocked and allowed to stand at room temperature for 1 h.Rabbit anti-LC3B monoclonal antibody was added at 4°C overnight.Wash the washing solution for 1 min,3 times.Green fluorescent anti-rabbit DyLight?488 was added dropwise.Wash the washing solution for 1 min,3 times.Anti-quenching tablets containing DAPI were added dropwise in the dark.Observations and photographs were taken under a Nikon 90I fluorescence microscope.4.Real-time PCR analysis of gene transcriptional activityThe BMDC induction culture was collected on the sixth day,and the cells were collected by gently pipetting.The supernatant was centrifuged at 1300 rpm/min,resuspended in pre-cooled PBS,and the supernatant was again centrifuged.Total RNA of BMDC was extracted by Trizol method,and the extracted RNA concentration was determined.And purity,reverse transcription of mRNA into cDNA using the GoScriptTM Reverse TranscriptionSystem reverse transcription kit.Real-time quantitative PCR analysis was performed using the GoTaq?qPCR Master Mix kit.The Q6 real-time quantitative PCR instrument was used for detection and analysis of the processed data.5.Lysotracker Red stainingOn the sixth day of BMDC induction culture,1?l of Lysotracker Red was added to20 ml of cell culture medium to prepare a working solution with a final concentration of50 nM.The cells were seeded on a 6-well plate slide at a suitable density,the cell culture medium was removed,and the Lysotracker Red working solution pre-warmed at 37°C was added,and the medium was changed after 30 min incubation.Laser confocal microscopy observation,photo analysis.6.TFEB lentivirus infectionOn the sixth day of cell-induced culture,a cell suspension of 1×10~5cells/ml was prepared in complete medium and inoculated into a 6-well plate.According to the cell MOI and virus titer,the corresponding lentivirus LV-TFEB and negative control virus LV-CON virus were added respectively,and cultured at 37°C for 10 h.Complete medium was added midway to maintain cell viability,and infection efficiency was observed after about 72 h of infection.7.Flow cytometry determination of BMDC surface moleculesCollect single-cell suspensions of BMDC in each experimental group,adjust the number of cells to about 2×10~5 cells/ml,discard the supernatant after centrifugation,and resuspend the BMDC in 100?l of the stain buffer according to the flow-through fluorescent antibody specification,according to the appropriate ratio.Labeling staining was carried out by adding MHC II-APC flow fluorescent antibodies.Incubate at 4°C for30 min in the dark,mix well after washing,and use BD flow cytometer for on-machine detection.8.ELISA determination of cytokines in supernatant of BMDCAccording to the ELISA kit instructions,antibodies TNF-?,IL-1?and IL-6 were first coated.The next day,the plate was discarded and washed three times.Add1×ELISAPOT dilution,incubate for 1 h at room temperature,and wash three times.The standard protein was diluted with 1×ELISAPOT dilution,the standard and the supernatant of BMDC were added,and the plate was incubated for 2 h at room temperature.After washing for three tests,Avidin-HRP was added and incubated for 1 h at room temperature.Discard the liquid and wash it six times.Add 1×TMB substrate solution and incubate for 15 min at room temperature in the dark.Finally,the ELISA stop solution was added and detected and analyzed using a microplate reader.9.Statistical analysisStatistical analysis was performed on the data using SPSS 13.0 software,and the results were expressed as meanąSD.Differences between groups were compared using one-way analysis of variance(ANOVA).If the variances were uniform,the SNK method was used for pairwise comparison.If the variances were not uniform,Dunnett's T3method was used for pairwise comparison.p<0.05 was considered statistically significant.Results:1.Inorganic arsenic exposure leads to an increase in the number of BMDC autophagosomes,an increase in LC3 protein levels,and an increase in the transcriptional activity of Map1lc3b gene.Compared with the Con group,a certain number of autophagosomes were observed after 1?M As exposure for 12 h.In LPS-induced mature DC,1?M As was induced to induce DC formation for 12 h compared with the Unt group.Autophagosome.Western blot analysis showed that the expression of BMDC LC3 I and LC3 II protein was significantly increased after exposure to As(0.25,0.5 and 1?M)for 6 h(p<0.05).After12 h of 1?M As,the fluorescence intensity of LC3 was significantly enhanced.In addition,1?M As induced a significant increase in the transcriptional activity of BMDC Map1lc3b and the difference was statistically significant(p<0.05).2.Influence of inorganic arsenic on the expression of ATG5,ATG5-ATG12,ATG16L1 and p-mTOR/mTOR proteins in BMDCWestern blot analysis showed that there was no difference in the expression of Atg5,Atg12-Atg5 and Atg16L1 in different concentrations of As.Different doses of As induced a decrease in the expression of p-mTOR in BMDC,the difference was statistically significant(p<0.05).The total protein expression level of mTOR remained basically unchanged.3.Inorganic arsenic induced BMDC autophagy with mTOR dependenceCompared with the untreated group,the Rap treatment group down-regulated the expression of p-mTOR and up-regulated the significant expression of LC3 II/LC3 I(p<0.05);compared with the As-treated group,the Rap-As co-treatment group induced LC3The difference in II/LC3 I was statistically significant(p<0.05).4.Inorganic arsenic upregulates p62/SQSTM1 protein expression and transcriptional activity of Sqstm1 in BMDCDifferent concentrations of As(0.25,0.5,and 1?M)dose-dependently induced an increase in p62 protein expression compared with the untreated group,and the difference was statistically significant at the As concentration of 1?M(p<0.05).Furthermore,inorganic arsenic induced a significant increase in p62 protein expression from 6 h,reaching a peak at 24 h,and the difference was statistically significant(p<0.05).In addition,1?M As significantly induced the transcriptional activity of BMDC Sqstm1,and there was a significant difference(p<0.05),which was consistent with the results of arsenic-induced LC3 and SQSTM1/p62 protein expression.5.Inorganic arsenic can inhibit BMDC autophagic fluxCompared with the As treatment group,the ratio of LC3 II/LC3 I in the CQ treatment group was significantly down-regulated(p<0.05),indicating that inorganic arsenic not only increased the synthesis of autophagosomes,but also inhibited the degradation of autophagosomes.In addition,compared with the untreated group,the autophagy-specific degradation substrate p62 protein in the As treatment group,the CQ treatment group and the CQ+As treatment group was significantly up-regulated(p<0.05),and the fold increase was basically the same.It is further indicated that inorganic arsenic inhibits the autophagic flow induced by LPS to promote mature BMDC,resulting in the accumulation of autophagy-degrading substrates such as p62.6.Inorganic arsenic affects TFEB nuclear translocationIn this study,BMDC nuclear protein,cytoplasmic protein and whole protein were collected after treatment of cells with 1?M As for 12 h.We used western blot to detect the protein expression level of TFEB.It was found that inorganic arsenic could down-regulate the expression of TFEB and nucleoprotein in mature BMDC induced by LPS.The difference was statistically significant(p<0.05),while TFEB in cytoplasmic protein.There was no significant change in the expression.The above results indicate that inorganic arsenic can inhibit nuclear translocation of BMDC TFEB.7.Inorganic arsenic inhibits BMDC lysosome production,membrane integrity and acidificationAs can induce the red fluorescence intensity of lysotracker red in LPS-induced mature BMDC to be significantly reduced,and the expression level is significantly reduced.The above results suggest that inorganic arsenic can reduce the number of acidic lysosomes induced by LPS induced by mature BMDC.Transmission electron microscopy showed that in the untreated group,the lysosome contained the food ingested and digested,and the lysosomal membrane was almost intact.In the As-treated LPS-promoted BMDC,lysosomes were observed.The phenomenon of incomplete film.In addition,western blot results showed that inorganic arsenic could down-regulate LAMP1-induced LAMP1 and LAMP2 in mature BMDC,and protein expression levels of lysosomal cathepsins CTSD,CTSL,CTSS and AEP.8.TFEB attenuates inorganic arsenic inhibition of BMDC lysosomal-associated protein expressionIn the untreated arsenic(Unt)group,the expression levels of LAMP2,CTSL,CTSB,CTSD and AEP in the LV-TFEB group were significantly higher than those in the LV-NC group,indicating that overexpression of TFEB induced lysosomes.The expression of the relevant membrane protein LAMP2 was significantly higher in the arsenic-treated(As)group than in the LV-NC group,and the expression levels of LAMP2,CTSL,CTSB,CTSD and AEP in the LV-TFEB group were also significantly increased.9.TFEB relieves inorganic arsenic inhibition of BMDC autophagic fluxThe protein levels of LC3 I,LC3 II,and p62 in the LV-TFEB group were significantly lower than those in the LV-TFEB group(p<0.05)in the arsenic-treated(Unt)group or the arsenic-treated(As)group.In addition,consistent with the protein expression results,in the arsenic-treated(As)group,LV-TFEB down-regulated the mRNA expression of Map1lc3b in BMDC compared with the LV-NC group,while the Sqstm1 gene expression level showed no significant change.10.Effect of TFEB on the inhibition of BMDC antigen presenting molecules,costimulatory molecules and chemokines by inorganic arsenicIn the arsenic-treated(As)group,LV-TFEB up-regulated the mRNA expression of Cd83 and Cd40 in BMDC compared with LV-NC group,but there was no statistical difference;however,there was no significant change in MHC II expression,as well as Cd86 and Cd80 gene expression.On the other hand,we also observed that in the arsenic-treated(As)group,LV-TFEB up-regulated the mRNA expression of Ccr5 and Ccr7 in BMDC compared with the LV-NC group,and the difference in gene expression of Ccr5 was up-regulated significantly(p<0.05).11.Effect of TFEB on the expression of BMDC chemokine inflammatory cytokines inhibited by inorganic arsenicELISA results showed that LV-TFEB significantly up-regulated the expression of TNF-?secreted by BMDC in the supernatant(p<0.05)in the arsenic-treated(As)group compared with the LV-NC group.Regardless of the arsenic-treated(Unt)group or the arsenic-treated(As)group,LV-TFEB up-regulated the expression of IL-1?secreted by BMDC in the supernatant compared with the LV-NC group.Realtime-PCR detection was found to be consistent with ELISA results.LV-TFEB up-regulated Tnf-?in BMDC compared with LV-NC group in both arsenic-treated(Unt)and arsenic-treated(As)groups.The gene transcriptional activity of Il-1?was statistically significant(p<0.05).12.Effect of TFEB on the expression of Th1 type cytokine IL-12 in BMDC inhibited by inorganic arsenicThe results of ELISA showed that LV-TFEB significantly up-regulated the secretion of BMDC in the supernatant of IL-12p70 in the arsenic-treated(Unt)group or the arsenic-treated(As)group compared with the LV-NC group.Expression(p<0.05).Realtime-PCR was performed on the other hand,which was consistent with the ELISA results.In the untreated arsenic(Unt)group,LV-TFEB up-regulated the genes of Il-12a and Il-12b in BMDC compared with the LV-NC group.Transcriptional activity,and the differences were statistically significant.In the arsenic-treated(As)group,LV-TFEB up-regulated the gene transcriptional activities of Il-12a and Il-12b in BMDC compared with the LV-NC group,but there was no statistical difference.13.Effect of TFEB on the expression of Th17 cytokines IL-6 and IL-23 in inorganic arsenic-inhibited BMDCELISA results showed that LV-TFEB up-regulated the expression of IL-6 secreted by BMDC in supernatants in the arsenic-treated(Unt)group or the arsenic-treated(As)group,compared with the LV-NC group.And the former has statistical differences.On the other hand,consistent with the ELISA results,realtime-PCR results showed that LV-TFEB can be up-regulated compared with LV-NC group in both the arsenic-treated(Unt)group and the arsenic-treated(As)group.The transcriptional activity of Il-6 gene in BMDC was statistically significant(p<0.05).In addition,LV-TFEB slightly up-regulated the transcriptional activity of Il-23a in BMDC,but the difference was not statistically significant.14.Effect of TFEB on the expression of Treg cytokines IL-10 and TGF-?in BMDC induced by inorganic arsenicELISA results showed that LV-TFEB up-regulated the expression of IL-10 secreted by BMDC in supernatants in the arsenic-treated(Unt)group or the arsenic-treated(As)group,compared with the LV-NC group.And the former has statistical differences.On the other hand,the mRNA expression level of Il-10 in the cells was detected by realtime-PCR method,regardless of whether the arsenic-treated(Unt)group or the arsenic-treated(As)group was administered,compared with the LV-NC group.LV-TFEB could down-regulate the transcriptional activity of Il-10 gene in BMDC,and the latter differences were statistically significant(p<0.05).In addition,LV-TFEB slightly up-regulated the transcriptional activity of Tgf-?in BMDC,but there was no statistical difference.Conclusion:1.Inorganic arsenic can affect the autophagy-lysosomal pathway of BMDC.On the one hand,mTOR-dependent induction of BMDC autophagosomes can also inhibit the autophagic flow of mature BMDC,and also have a certain inhibitory effect on lysosomal function.2.Overexpression of TFEB enhance the lysosomal function of BMDC and alleviate the arsenic-induced autophagic flow of BMDC induced by inorganic arsenic,and at the same time,it can antagonize the inhibitory effect of inorganic arsenic-induced BMDC. |
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