| Research background:With the rapid development of society and changes in lifestyle and diet,high-fat diet(HFD)is enormously common in both developed and developing countries.HFD increases the risk of inflammatory bowel disease(IBD)and intestinal tumors,however,there are few reports on lipotoxicity in intestinal epithelial cells prior to HFD-induced intestinal lesions such as IBD and intestinal tumors and it still not has been received enough attention.The mechanisms underlying the direct lipotoxicity effects of HFD on intestinal epithelial cells are largely unknown.Purpose:To investigate the role of endoplasmic reticulum stress(ERS)and autophagy in the lipotoxicity of intestinal epithelial cells due to HFD.Methods:(1)8 weeks,12 weeks and 16 weeks of high-fat diet mice models were established,and changes in body weight and serum triglyceride(TG)were recorded.Hematoxylin & eosin(HE)staining was performed to observe the morphology ofmice intestinal epithelial cells.Western blot was used to detect ERS,autophagy and apoptosis-related protein expression in mice intestinal epithelial cells.(2)Human-like intestinal epithelial Caco2 cells were treated with different concentrations of palmitic acid(PA)(0-1.0 mM),a kind of saturated fatty acid,or oleic acid(OA)(0-1.0 mM),a kind of unsaturated fatty acid.CCK8 assay was performed to detect cells viability.Western blot was accessed to detect the effects of PA or OA on Caco2 cells ERS-associated protein,glucose-regulated protein 78(GRP78),inositol-requiring enzyme 1α(IRE1α),phosphorylated endoplasmic reticulum membrane protein kinase(p-PERK),phosphorylated eukaryotic initiation factor 2α(p-e IF2α)and activating transcription factor 6(ATF6),autophagy-related protein,Beclin1,autophagy-related genes 5(ATG5),microtubule-associated protein light chain 3(LC3)B/LC3 A and p62 and apoptosis-related protein,cleaved-caspase3 and B-cell lymphoma-2 associated X Protein(BAX)/Bcl2.RT-PCR was performed to detect effects of PA on X-box binding proteinⅠ(XBP1)and Beclin1 mRNA expressions.MRFP-GFP-LC3 plasmid was transfected into Caco2 cells to detect the effects of PA or OA on autophagy flux.The effects of PA or OA on apoptosis were detected by Annexin V-FITC/PI double staining.Combined with the ERS inhibitor 4-phenylbutyric acid(4-PBA),the IRE1αRNase inhibitor(4μ8C),the autophagy inhibitor 3-methyladenine(3-MA)or the autophagy agonist rapamycin,the role of ERS and autophagy in high-fat induced apoptosis in intestinal epithelial cells was detected.Results:(1)8 weeks,12 weeks and 16 weeks HFD induced the increase in body weight and serum TG and induced necrosis and detachment of intestinal epithelial cells in HFD mice(p < 0.05).Meanwhile,8 weeks,12 weeks and 16 weeks HFD induced the expression of ERS marker protein GRP78(p < 0.01),autophagy-related proteins including Beclin1,ATG5,LC3B/LC3 A and p62(p < 0.01),and apoptosis-related protein including cleaved-caspase3 and BAX/Bcl2(p < 0.05).(2)High concentrations of PA(0.5 mM and 1.0 mM)significantly inhibited the viability of Caco2 cells(p < 0.01);high concentrations of PA(0.5 mM and 1.0 mM)significantly increased the apoptosis-related protein expressions of cleaved-caspase3 and BAX/Bcl2 as well as the number of cells in early and late apoptosis(p < 0.05);high concentrations of PA(0.5 mM and 1.0 mM)increased protein expressions of GRP78 and IRE1α and activated XBP1s(p < 0.001);high concentrations of PA(0.5mM and 1.0 mM)increased expressions of autophagy-related proteins,including Beclin1,ATG5 and LC3B/LC3A(p < 0.001),as well as decreased expression of p62 and increased autophagy flux(p < 0.01).ERS inhibitor 4-PBA,IRE1α RNase inhibitor 4μ8C or autophagy inhibitor 3-MA could decrease the expressions of apoptosis-related protein including cleaved-caspase3 and BAX/Bcl2 induced by high concentrations of PA(1.0 mM)(p < 0.01);ERS inhibitor 4-PBA or IRE1α RNase inhibitor 4μ8C decrease the expressions of autophagy-related proteins including Beclin1,ATG5 and LC3B/LC3A(p < 0.001),as well as increased p62 protein induced by high concentrations of PA(1.0 mM)(p < 0.01);the autophagy agonist rapamycin reversed the effects of 4-PBA or 4μ8C on high concentrations of PA(1.0mM)induced protein expressions of autophagy(increased Beclin1,ATG5 and LC3B/LC3 A protein levels and decreased p62 protein levels,p < 0.01)and apoptosis(increased cleaved-caspase3 and BAX/Bcl2 protein levels,p < 0.001).Furthermore,IRE1α RNase inhibitor 4μ8C was found to inhibit the activation of XBP1 s and Beclin1 mRNA expression induced by high concentrations of PA(1.0 mM)(p <0.001).(3)High concentrations of OA(0.5 mM and 1.0 mM)significantly inhibited the viability of Caco2 cells(p < 0.01);high concentrations of OA(0.5 mM and 1.0 mM)induced the apoptosis-related protein expressions of cleaved-caspase3 and BAX/Bcl2 as well as the number of cells in late apoptosis(p < 0.05);high concentrations of OA(0.5 mM and 1.0 mM)induced protein expressions of GRP78,IRE1α and ATF6(p <0.001);high concentrations of OA(0.5 mM and 1.0 mM)induced expressions of autophagy-related proteins,including Beclin1,ATG5,LC3B/LC3 A and p62,and impaired autophagy flux(p < 0.05).Autophagy agonist rapamycin could decrease the increased expressions of apoptosis-related proteins including cleaved-caspase3 and BAX/Bcl2 induced by high concentrations of OA(1.0 mM)(p < 0.05);ERS inhibitor4-PBA decreased the expressions of autophagy-related proteins including Beclin1,ATG5,LC3B/LC3 A and p62 as well as apoptosis-related proteins including cleaved-caspase3 and BAX/Bcl2 induced by high concentrations of OA(1.0 mM)(p< 0.05).Conclusion:(1)High-fat diet induced endoplasmic reticulum stress,impaired autophagic flux and apoptosis in mice intestinal epithelial cells.(2)High concentrations of PA induced apoptosis in Caco2 cells via IRE1α-mediated endoplasmic reticulum stress-autophagy.(3)High concentrations of OA induced apoptosis in Caco2 cells through impaired endoplasmic reticulum stress-mediated autophagic flux. |
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