Image_1_Endoplasmic Reticulum Stress Mediated MDRV p10.8 Protein-Induced Cell Cycle Arrest and Apoptosis Through the PERK/eIF2α Pathway.JPEG

<p>In this study, the mechanism of Muscovy duck reovirus (MDRV) p10.8 protein-induced pathogenesis was investigated, with a focus on endoplasmic reticulum (ER) stress. In chicken embryo fibroblasts cell lines (DF1), pCI-neo-flg-p10.8 protein transfection increased the phosphorylation (p-) levels of PERK and eIF2α as shown by Western blotting analysis and led to the dissociation of BiP from PERK as shown by co-immunoprecipitation (Co-IP) analysis. Results of treatment with both ER stress activator and inhibitor further confirmed that p10.8 protein induced ER stress. Subsequently, using flow cytometry analysis, it was also found that p10.8 protein induced cell cycle arrest during the G0/G1 phase. Furthermore, p10.8 transfection increased the phosphorylation levels of PERK and eIF2α, and reduced the expression levels of CDK2, CDK4, and Cyclin E according to Western blotting analysis. Treatment with ER stress activator and ER stress inhibitor after p10.8 protein transfection in DF1 cells further indicated that p10.8 protein induced ER stress, which resulted in cell cycle arrest. The results of knockdown of either PERK or eIF2α genes further confirmed that p10.8 protein-induced ER stress led to cell cycle arrest through the PERK/eIF2α pathway. Further results showed that p10.8 protein induced ER stress and apoptosis in DF1 cells. The expression levels of p-PERK, p-eIF2α, CHOP, cleaved-Caspase12, and cleaved-Caspase3 were increased by p10.8 protein. Test results of treatment with each of Tunicamycin, TUDCA and knockdown of PERK, and eIF2α, confirmed that p10.8 protein induced ER stress involving apoptosis via the PERK/eIF2α pathway. In conclusion, MDRV p10.8 protein induced ER stress that caused cell cycle arrest and apoptosis through the PERK/eIF2α pathway.</p

1,25(OH)₂D₃ Deficiency Induces Colon Inflammation via Secretion of Senescence-Associated Inflammatory Cytokines

<div><p>Epidemiological studies showed that 1,25-Dihydroxyvitamin D[1,25(OH)₂D₃] insufficiency appears to be associated with aging and colon cancer while underlying biological mechanisms remain largely unknown. Inflammatory bowel disease is one of the risk factors for colon cancer. In this study, we investigated whether 1,25(OH)₂D₃ deficiency has an impact on the colon of 25-hydroxyvitamin D 1α-hydroxylase knockout [Cyp27b1^−/−] mice fed on a rescue diet (high calcium, phosphate, and lactose) from weaning to 10 months of age. We found that 1,25(OH)₂D₃ deficient mice displayed significant colon inflammation phenotypes including shortened colon length, thinned and disordered mucosal structure, and inflammatory cell infiltration. DNA damage, cellular senescence and the production of senescence-associated inflammatory cytokines were also increased significantly in the colon of Cyp27b1^−/−mice. Furthermore, the levels of ROS in the colon were increased significantly, whereas the expression levels of antioxidative genes were down-regulated dramatically in the colon of Cyp27b1^−/−mice. Taken together, our results demonstrated that 1,25(OH)₂D₃ deficiency could induce colon inflammation, which may result from increased oxidative stress and DNA damage, subsequently, induced cell senescence and overproduction of senescence-associated secretory factors. Therefore, our findings suggest that 1,25(OH)₂D₃ may play an important role in preventing the development and progression of colon inflammation and colon cancer.</p></div

1,25(OH)₂D₃ deficiency induced the ROS production and decreased antioxidants.

<p>(A) Flow cytometry analysis of ROS in colons from the three groups of 10-month-old mice. (B) H₂O₂ and (C) Total SOD level of colons measured by spectrophotometry. (D)Real-time RT-PCR was performed on extracts of colon from WT and Cyp27b1^-/- mice for the gene expression of antioxidants. (E) Western blot analyses of colonic SOD2 and Prdx2 expression in the WT and Cyp27b1^-/- mice, β-actin was used as a loading control.</p

1,25(OH)₂D₃ deficiency induced the genes expression of the senescence-associated inflammatory cytokines.

<p>Immunohistochemical analyses of IL-1ɑ (A) IL-6 (B) IL-8 (C) HGF1 (D) MMP3 (E) expressions in the colon from the WT and Cyp27b1^-/- mice. Positive cells in lamina propria were labeled by arrow. The histogram shows the mean percentage of the positive area determined from five randomly selected fields. Magnification, ×400. Bar, 25μm. Values are mean ± SEM, n = 5. *P < 0.05, ** P < 0.01. (F) Real-time RT-PCR was performed on extracts of colon from WT and Cyp27b1^-/- mice for the gene expression of IL-1α, IL-1β, IL-6, IL-8, HGF1, MMP-3 and MMP-13. (G) Western blot analyses of colonic NF-κB (p105 and p50) expression in the WT and Cyp27b1^-/- mice, β-actin was used as a loading control.</p

1,25(OH)₂D₃ deficiency induced DNA damage and cellular senescence.

<p>Immunohistochemical analyses of 8-OHdG (A) γH2AX (B) expression and analysis of SA-β-gal positive cells (C) in the colon from the WT and Cyp27b1^-/- mice. The positive cells in lamina proppria are marked by arrow. The histogram shows the mean percentage of the 8-OHdG or γH2AX, SA-β-gal positive cells determined from five randomly selected fields. Magnification, ×400. Bar, 25μm. Values are mean ± SEM, n = 5. *P < 0.05. (D): Western blot analyses of colonic γH2AX expression in the WT and Cyp27b1^-/- mice, β-actin was used as a loading control.</p

Effect of 1,25(OH)₂D₃ deficiency on serum biochemistry.

<p>(A) Deletion efficiency of the Cyp27b1 allele was analyzed using genomic DNA from the tail. (B) Quantitative PCR assay for Cyp27b1 mRNA in colons. (C) Serum calcium, (D) serum phosphorus and (E) serum 1,25(OH)₂D₃ from 10-month-old Cyp27b1^-/- and WT mice (n = 5; mean±SEM). **: p< 0.01.</p