Interference Effects Of Methylparaben On ?-cell Failure And Its Molecular Mechanism

Methylparaben(MeP)is one of the most commonly used preservatives widely employed in foodstuffs,cosmetics and pharmaceutical products.With such large amounts of exposure to MeP,it was observed in the human and companion animal samples in high co ncentration.A crude positive association MeP and diabetes was found in a case-control study,but limited data was available on the interference effects of MeP on ?-cell dysfunction in vivo.To examine the ?-cell disrupting potential of MeP and its molecular mechanism,?-cell function in CD-1 mice was assessed after short-term exposure to MeP.RNA-seq was employed to determine the candidate genes mediated by MeP in the pancreatic and ?TC-6 cells.MeP mediated thePI3K/Akt-NF-?B2 signal pathway,and the secr etion of CCL5,CCL19 and CXCL12 through the ERR?in ?TC-6 cell was examined.This research laid the foundation for further management of MeP and may provide an environmental toxicological explanation for the high incidence of diabetes.Details are as follo ws:(1)Effects of short-term exposure to MeP on ?-cell function in mice.A sensitive liquid chromatography tandem mass spectrometry(LC/MSMS)method was developed and validated for quantification of parabens in mice daily diet.The validated method was us ed to analyze mice daily diet and to ensure no unknown parabens were present during the test involvement.Male CD-1 mice were intraperitoneally injected with 3mg/kg,10mg/kg and30mg/kg of MeP for 7 days respectively to study the effect on the function of?-cells.We found MeP could significantly reduce blood glucose tolerance,insulin tolerance,concentration of serum insulin,and glucose-stimulated insulin secretion in mice after 7 days of treatment at doses of 10mg/kg.The effect of MeP on the distributi on of proinsulin in mice was observed by histopathological methods.The results showed that increased proinsulin area in pancreatic islets was observed in mice exposed to 30mg/kg MeP.(2)Effects of MeP on gene expression profiling in pancreatic and ?TC-6cells.RNA-seq was conducted to get the gene expression profiling changes in the pancreatic and ?TC-6 cells after MeP treatment.In the pancreas,1396 genes were identified as differentially expressed(DEGs)after 10mg/kg MeP treatment for 7 days,which wa s confirmed as reliable evidence of MeP exposure.Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis demonstrated that the DEGs were mainly enriched in “Metabolic pathways,”“Pathways in cancer” and the “PI3K-Akt signaling pathway.” In the ?TC-6 cells,3818,3167 and 2058 genes were identified as DEGs after0.1?M,1?M and 10?M MeP treatment,respectively.Gene expression trend analysis(Series Test of Cluster,STC)found that theP value in the inverted U-shaped dose-effect curve was extremely s ignificant(P < 0.01),and the number of enriched genes was the most(1796).Results of protein-protein interaction(PPI)showed estrogen receptor 1(ESR1),nuclear receptor subfamily 4 group A member 3(NR4A3)and estrogen-related receptor gamma(ERR?)were hub genes.KEGG analysis showed that 150 co-regulated genes by MeP in pancreatic tissue and ?TC-6 cells were mainly focused on “Cell adhesion molecules”,“Pathways in cancer”and the “PI3K-Akt signaling pathway”.(GO)analysis revealed that those genes were mainly enriched in“Immune system process,”“Membrane” and “Protein binding.” Verification of the DEGs of the transcriptome data was conducted by q PCR.Results showed consistent expression trends in q PCR and RNA-seq,indicating that the RNA-seq results ar e reliable.The above results indicate that the interference effect of MeP on the function of ?-cells may be related to the expression changes of genes in pancreatic ?-cells.It also suggests that MeP can participate in the regulation of the inflammatory p rocess of mice pancreatic?-cells and that thePI3K-Akt signaling pathway is involved.The hub gene ERR? is closely related to the MeP and ?-cells.(3)Effects of MeP-mediated PI3K/Akt-NF-?B2 signal pathway through the ERR?.We applied the automated dockin g of the flexible MeP into known active sites of ERR?,which showed MeP could bind itself to a test protein via more than one hydrogen bond.The binding amino acids of MeP in ERR? were GLU275 and ARG316.The binding energy value of MeP was calculated as-6.2kcal/mol with ERR?.The p RP-EGFP-ERR? plasmid was transfected into?TC-6 cells to overexpression of ERR? in the cells.The results indicated ERR? was successfully overexpressed in ?TC-6 cells(?TC-6-ERR?).We found that overexpression of ERR? in ?TC-6 cells enables increased protein expression of PI3 K p85,PI3 K p110,Akt Thr308 and Akt Ser473.Furthermore,10?M MeP could also significantly increase the protein or phosphorylated protein expression of PI3 K p85,PI3 K p110,Akt Thr308,Nf-?B2 P52 and Nf-?B2 p100 in ?TC-6-ERR? cells.Moreover,10?M MeP could significantly increase secretion of CCL5 and CCL19 in ?TC-6-ERR? cells.In conclusion,we demonstrated that short-term exposure to MeP declined insulin levels,glucose tolerance,insulin tolerance and insul in sensitivity leads to ?-cell failure in mice in doses of 10mg/kg.Furthermore,analysis of pancreatic and ?TC-6 cells RNA-seq data reveals 150 genes co-regulated by MeP,which enriched in “Cell adhesion molecules”,“Pathways in cancer”and the “PI3K-Akt signaling pathway”.In addition,MeP inducing the secretion of CCL5 and CCL19 in ?TC-6-ERR? cells involed in PI3K/Akt-NF-?B2signaling pathway,and this process is partially induced by ERR?.Overall,our results add to the growing body of literature indicat ing the potential negative health outcomes associated with MeP exposure.