| Backgrounds:Inorganic arsenic(i As)is one of the most common environmental chemical contaminants widely found in groundwater,coal mines,and industrial wastewater.Drinking arsenic-contaminated groundwater is the most crucial route of chronic arsenic exposure in humans.Numerous epidemiological studies have demonstrated that the liver,the main metabolic site of i As in the human body,is the most important organ for arsenicosis.Long-term arsenic exposure can trigger hepatic insulin resistance(hepatic IR),type 2 diabetes(T2D),nonalcoholic steatohepatitis(NASH),and liver fibrosis(LF).Hepatic IR is an essential pathological change and a critical initiating event in T2D and NASH,but the molecular mechanism of i As-induced hepatic IR needs to be further investigated.Our previous studies have shown that i As can induce the activation of NOD-like receptor protein 3(NLRP3)inflammasome in hepatocytes,triggering cellular inflammatory responses and participating in the development of hepatic IR.However,the mechanism by which i As induces upregulation of NLRP3 transcript level and protein level remains unclear,and its regulatory role in inflammasome activation still needs to be further explored.Post-transcriptional modifications of ribonucleic acid(RNA)are important for regulating RNA metabolism,protein expression,and cellular physiological processes.More than one hundred post-transcriptional modifications of RNA were identified,among which N6-methyladenosine(m~6A)modification,the most common and essential post-transcriptional modification of RNA,regulates the splicing,translation and degradation processes of RNA,and plays a crucial role in the development of diseases.Studies have shown that altered levels of intracellular m~6A methylation modifications may be closely associated with the development of T2D and NASH.However,it is not clear whether m~6A methylation modification level is altered in i As-induced hepatic IR,and whether m~6A methylation plays an essential regulatory role in arsenic-induced hepatic IR.The methyl metabolic pathway is the most important way of i As metabolism in vivo.Arsenite methyltransferase(AS3MT)is the key regulatory enzyme of i As methyl metabolism and the is first step rate-limiting enzyme that regulates the conversion of i As to monomethylarsonic and dimethylarsinic.S-adenosylmethionine(SAM)is almost the only methyl donor in cells.The process of i As metabolism will inevitably affect the level of m~6A methylation modification in cells,which affects the cellular physiological processes and the development of diseases.Therefore,the role mechanism of AS3MT in arsenic-induced hepatic IR and the relationship between AS3MT-mediated i As methyl metabolism and m~6A methylation modification deserve to be explored in depth.Objective:Based on the above research background,the main research objectives of this study are as follows.1.The role of m~6A methylation in i As-induced hepatic IRTo establish a mouse model of the early onset of arsenic-induced hepatic IR,investigate the changes of m~6A methylation modification in hepatocytes after i As exposure,and investigate the role of m~6A methylation in arsenic-induced hepatic IR after the intervention of key regulatory enzymes.2.Study on the regulatory role of m~6A methylation on i As-induced NLRP3inflammasome activationThe m~6A-seq was performed on mouse liver and the sequencing results were enriched and analyzed to find possible targets or pathways for the effect of m~6A methylation on arsenic-induced hepatic IR.The activation of several inflammasomes in mouse liver was examined to explore the role of NLRP3 in the early onset of arsenic-induced hepatic IR and its uniqueness.The m~6A methylation of NLRP3 was detected to explore the mechanism of m~6A methylation in the i As-induced upregulation of NLRP3 transcription and protein levels.3.Study on the role of arsenic methyltransferase in NLRP3 m~6A methylation and hepatic IRThe gene and protein levels of AS3MT were examined in mouse liver,and its role in i As-induced NLRP3 inflammasome activation and hepatic IR was explored after its expression interfered.The level of intracellular m~6A methylation modification and changes in NLRP3 m~6A methylation were investigated after the intervention of its expression.Methods:1.Establish a mouse model of early onset of i As-induced hepatic IRMale C57BL/6 mice aged 4-6 weeks were selected and randomly divided into a control group and an arsenic exposure group,with ten in each group.The control group was given normal drinking water and standard feed.The mice in the arsenic-exposed group were given standard feed and arsenic-containing water(4 mg/L).Other feeding conditions such as light time,ambient temperature and humidity,and water change time were kept the same.Monitor the fasting blood glucose(FBG)level of mice every week,and perform oral glucose tolerance test(OGTT)and insulin tolerance test(ITT)on mice at the end of the sixth week.The fasting serum insulin(FSI)level of mice was used to calculate the insulin resistance index(Homeostasis model assessment-IR,HOMA-IR).After the mice were sacrificed,liver and serum samples were collected.The q RT-PCR method was used to detect the expression of gluconeogenic genes Pck and G6pc in mouse liver tissues,and the expression of insulin signaling pathway-related proteins in mouse liver tissues was detected by Western blot.Hematoxylin and eosin(HE)staining,Masson staining,and oil red O staining were performed on liver tissue sections to detect pathology,fibrosis and lipid aggregation in mice.2.Detection of m~6A methylation in the mice liver of early onset of i As-induced hepatic IRWestern blot was used to detect the cytoplasmic and nuclear expression levels of m~6A methylation-related enzymes in mouse liver.ELISA was used to detect the m~6A methylation content of total RNA and m RNA in mouse liver tissues.m~6A-seq was performed on mouse liver to detect the difference in m~6A methylation modification of the transcriptome,and perform GO and KEGG enrichment analysis of the differential genes.3.Detection of the role of m~6A methylation in i As-induced hepatic IRThe expression of m~6A methylation key enzymes METTL14 and IGF2BP2 in normal human hepatocytes L-02 cells was interfered with by small interfering RNA(si RNA)technique,followed by i As(4μM)treatment for 24 h and insulin(100 n M)stimulation for 10 min.Western blot was performed to detect the expression of insulin signaling pathway-related proteins,and the insulin-stimulated glucose uptake level was detected by the colorimetric method.4.Detection of NLRP3 inflammasome in i As-induced hepatic IRThe expression levels of NLRP3 inflammasome in the mice liver of arsenic-induced early hepatic IR were measured by Western blot,and the levels of interleukin-1β(IL-1β)and IL-18 in the serum of mice were measured by ELISA.The NLRP3 expression was measured by immunofluorescence in L-02 cells,and the protein expression levels of NLRC4 and AIM2 were also measured by Western blot in mouse liver.The cells were pretreated with MCC950(5μM),a specific inhibitor of NLRP3inflammasome activation,for 2 h and then treated with i As(4μM)for 24 h and stimulated with insulin(100 n M)for 10 min.Western blot was used to examine the expression of insulin signaling pathway-related proteins,and the level of insulin-stimulated glucose uptake was examined by the colorimetric method.5.Detection of the role of m~6A methylation in i As-induced NLRP3 inflammasome activationThe expression of m~6A methylation key enzymes METTL14 and IGF2BP2 in L-02cells was interfered with by si RNA,followed by i As(4μM)treatment for 24 h.The expression of NLRP inflammasome-related proteins was examined by Western blot,and IL-1βand IL-18 in culture supernatants were examined by ELISA.The interactions between METTL14/IGF2BP2 and NLRP3 were detected by the co-immunoprecipitation(Co-IP).Additionally,L-02 cells were treated with actinomycin D(Act D)for 0,3 and 6h after i As treatment,RNA was extracted,and the gene expression levels of NLRP3 were examined by q RT-PCR.6.Detection of the role of AS3MT in i As-induced hepatic IR and its effect on m~6A methylation modificationThe gene expression levels of AS3MT in the liver of i As-induced early hepatic IR mice and i As-treated L-02 cells were examined by q RT-PCR,and the protein expression levels of AS3MT were examined by Western blot and immunohistochemistry(IHC).The expression of AS3MT in L-02 cells was knocked down by si RNA,followed by treatment with i As(4μM)for 24 h and then stimulation with insulin(100 n M)for 10 min.The expression of insulin signaling pathway-related proteins in the cells was measured by Western blot,and the insulin-stimulated glucose uptake level was measured by the colorimetric method.In addition,m~6A methylation-related enzyme expression level in cytoplasmic and nucleus was detected in L-02 cells by Western blot,and m~6A methylation content changes in total RNA and m RNA were detected by ELISA.7.Detection of the interaction between AS3MT and NLRP3 and its effect on NLRP3m~6A methylationThe AS3MT expression in L-02 cells was knocked down by si RNA,or the cells were pretreated with MCC950 for 2 h,followed by i As(4μM)treatment for 24 h.The expression levels of AS3MT and NLRP3 inflammasome-associated proteins in the L-02cells were detected by Western blot.The interactions between endogenously expressed AS3MT and NLRP3 in mouse liver tissues as well as in L-02 were examined by the Co-IP method,and a series of truncated plasmids were designed based on the amino acid sequences and functional domains of human-derived AS3MT and NLRP3,and the HEK293T cells were used for transfection.Then the interactions regions were examined by the Co-IP method.In addition,the expression of AS3MT was knocked down by si RNA,and Co-IP was performed to detect the interaction between the m~6A methylation key enzyme with NLRP3.After i As treatment,the cells were treated with Act D for 0,3 and 6h,and the gene of NLRP3 was detected by the q RT-PCR method.The expression level of NLRP3 was examined by q RT-PCR.8.Statistical analysisData were presented as the means ±standard error of the mean(SEM).Statistically significant differences between groups were identified by the unpaired Student’s t-test or one-way ANOVA followed by an LSD post hoc test in SPSS 26(IBM,Inc.,NY,USA).Statistical information is outlined in figure legends;differences were significant at P<?0.05.Results:1.The role of m~6A methylation in i As-induced insulin resistance in liver1.1 Compared with control mice,FBG and FSI levels were significantly upregulated,and the HOMA-IR index was increased in exposed mice.In addition,GTT and ITT were significantly impaired,the expression levels of liver gluconeogenic genes Pck and G6pc were upregulated,and the phosphorylation levels of insulin signaling-related proteins were decreased in the exposed mice compared with the control mice.However,there were no significant differences in body weight and organ coefficients between the two groups of mice,and no apparent lesions were found in the liver of the exposed mice,indicating that the arsenic-induced early hepatic IR model was successfully established.1.2 Western blot results showed that the expression levels of METTL14 and IGF2BP2,the key enzymes of m~6A methylation,were significantly higher in the nuclei of the exposed mice liver compared with the control mice.m~6A-seq results showed that the overall level of m~6A methylation was upregulated in the livers of the exposed mice compared with the control mice,and ELISA results further confirmed that the m~6A methylation contents of total RNA and m RNA in the exposed mice liver were significantly higher than those in the control group.1.3 Consistent with the in vivo results,the phosphorylation level of insulin signaling pathway-related proteins in L-02 cells was reduced after i As exposure,and the insulin-stimulated glucose uptake capacity was decreased.The i As-induced reduction of insulin sensitivity in L-02 cells was significantly improved after the interference of METTL14and IGF2BP2.2.Study on the regulatory role of m~6A methylation on i As-induced NLRP3inflammasome activation2.1 The activation of NLRP3 inflammasome was also observed in arsenic-induced early hepatic IR.Inhibiting NLRP3 inflammasome using MCC950 significantly improved the i As-induced decrease in L-02 cell sensitivity,and i As specifically regulated NLRP3inflammasome rather than other inflammasomes.2.2 Enrichment analysis of m~6A-seq results revealed that m~6A methylation could regulate NLRP3-related signaling pathways.Co-IP results revealed significant interactions between METTL14/IGF2BP2 and NLRP3 in exposed mice liver compared with the control group.In addition,RNA stability assay revealed that the half-life of NLRP3m RNA was significantly shorter,and NLRP3 m RNA stability was reduced compared with arsenic exposure.2.3 Compared with the control group,the NLRP3 inflammasome was activated in L-02cells after i As treatment,and the levels of inflammatory factors IL-1βand IL-18 in cell culture supernatants were significantly increased.In contrast,knockdown of METTL14and IGF2BP2 significantly inhibited i As-induced NLRP3P inflammasome activation,and the levels of inflammatory factors IL-1βand IL-18 in cell culture supernatants were also significantly reduced.3.Study on the role of arsenic methyltransferase in NLRP3 m~6A methylation and hepatic IR3.1 Western blot results showed that the protein expression levels of AS3MT were significantly upregulated in mice liver and L-02 cells after i As exposure,and IHC results found that the levels of AS3MT were elevated in i As-exposed mice liver.The q RT-PCR results also showed that the expression level of the AS3MT gene was significantly upregulated in the i As-exposed mice liver.In addition,knockdown of AS3MT significantly ameliorated the i As-induced insulin sensitivity impairment.3.2 Interference of AS3MT significantly inhibited i As-induced NLRP3 inflammasome activation,while inhibiting NLRP3 inflammasome using MCC950 did not reduce i As-induced AS3MT expression.The Co-IP results showed that i As exposure promoted the interaction of AS3MT with NLRP3,and truncated Co-IP results showed that the AS3MT201-375 segment and the NLRP3 211-550 segment are the most likely interaction region.3.3 Consistent with the in vivo results,the expression levels of METTL14 and IGF2BP2,critical enzymes for m~6A methylation,were significantly upregulated in the nucleus after i As exposure.Knockdown of AS3MT significantly inhibited the expression of METTL14and IGF2BP2 in the nucleus.In addition,ELISA results revealed that inorganic arsenic exposure upregulated m~6A methylation contents of total RNA and m RNA in L-02 cells,while knockdown of AS3MT significantly decreased m~6A methylation contents of total RNA and m RNA in L-02 cells.3.4 Co-IP results showed that knockdown of AS3MT significantly inhibited the level of interaction between METTL14/IGF2BP2 and NLRP3.In addition,RNA stability assay revealed that the half-life of NLRP3 m RNA was significantly shortened after the knockdown of AS3MT.Conclusion:1.m~6A methylation level is up-regulated and promotes the early onset of i As-induced hepatic IR.2.The early onset of i As-induced hepatic IR is dependent on NLRP3 inflammasome,and m~6A methylation is involved in i As-induced NLRP3 expression and inflammasome activation.3.AS3MT promotes i As-induced hepatic IR and may promote i As-induced NLRP3inflammasome activation by regulating NLRP3 m~6A methylation modification. |
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