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Objective It is not yet clear whether 1,25-(OH)2D3acts on endoplasmic reticulum stress(ERS)and autoph-agy in pulmonary fibrosis(PF).This study aimed to investigate the roles of ERS and autophagy in the development and progression of pulmonary fibrosis in rats and the effects of 1,25-(OH)2D3on the expressions of ERS-related molecules and autophagy-related gene 12 (ATG12). Methods Ninety male SD rats were randomly divided into a control, a PF model and a treatment group of equal number.Bleomycin was injected into the tracheas of the latter two groups of rats to induce PF.On the second day after modeling, the rats of the treatment group were injected intraperitoneally with 1,25-(OH)2D3at 2 μg/kg,those of the PF model group with 1,25-(OH)2D3solvent at 200 μL,and those of the control group with iso-tonic saline at 200 μL,all once 2 days.Then the mRNA expressions of PERK,ATF4 and ATG12 were measured by real-time PCR and the protein expressions of PERK and ATF4 detected by immunohistochemistry. Results At 14, 21 and 28 days after treat-ment,the expression levels of PERK were significantly higher in the PF model group(2.30±0.19, 3.59±0.27, and 4.63±0.19) and treatment group(1.44±0.34,1.92±0.17,and 2.52±0.15)than in the control(1.01±0.23,1.05±0.09,and 1.04±0.08)(P<0.05), and so were the expression levels of ATF4 in the PF models(2.10±0.12, 3.91±0.14, and 6.20±0.28)and treated rats (1.49±0.27,2.52±0.42, and 4.02±0.31)than in the controls(1.04±0.07,1.05±0.08,and 1.03±0.10)(P<0.05).Compared with the control group,the PF model and treatment groups showed markedly increased expression levels of ATG 12 mRNA at 14 days (P<0.05), but decreased at 21 and 28 days(P<0.05).Both the expressions of PERK and ATF4 proteins were remarkably higher in the model and treatment groups than in the control at 14,21 and 28 days(P<0.05), increasing in a time-dependent manner. Conclusion By suppressing the PERK -eIF2α-ATF4 signaling pathway,1,25-(OH)2D3inhibits the development and progression of pulmonary fibrosis.
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Objective To investigate the regulatory roles of rno-miR-30b-5p in the expression of Atg5,Atg12 and Becn1 autophagy genes and their expressions in rats with experimental autoimmune uveitis (EAU).Methods Application of dual luciferase report system was conducted to detect the regulatory roles of rno-miR-30b-5p in Atg5,Atg12 and Becn1 gene expression.Lewis rats were randomly divided into control group and EAU group,with 6 rats in each group.Next,rats in EAU group were immunized to establish EAU model.After treatments,Genesis-D fundus camera was used to observe the fundus inflammation every day.After immunization for 12 days,the pathological features of rat ciliary body and retina were detected,and meanwhile,the spleen and lymph nodes in both groups were isolated to detect the expressions of rno-miR-30b-5p,Atg5,Atg12 and Becn1 genes by quantitative PCR (Q-PCR);the levels of autophagy related proteins were determined by ELISA.Results Dual luciferase report gene expression assay confirmed that Atg5,Atg12 and Becn1 were target genes of rno-miR-30b-5p.Twelve days after immunization,compared with the control group,rats in the EAU group had severe iris adhesions and severe blood vessel swelling,and pathological examination revealed massive infiltration of inflammatory cells in the ciliary body and retina.Furthermore,rno-miR-30b-5p mRNA was 0.46 ±0.01,0.29 ±0.17in the spleen and lymph nodes in EAU group,respectively,which was down-regulated when compared with the control group (P ＜0.01);whereas the expressions of Atg5,Atg12 and Becn1 were significantly upregulated,and the differences were statistically significant (all P ＜ 0.05).ELISA results showed that Atg5,Atg 12 and Becn 1 protein expression levels in the spleen and lymph nodes in EAU rats were significantly higher than those in the control group (all P ＜ 0.05).Conclusion rno-miR-30b-5p can regulate the expressions of Atg5,Atg12 and Becn1 autophagy-related genes.The down-regulation of rno-miR-30b-5p expression in the spleen and lymph nodes in EAU rats can significantly up-regulate the expressions of Atg5,Atg12 and Becn1 genes,thereby regulating the pathogenesis of uveitis.
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Autophagy is a self-degradation system of cellular components through an autophagosomal-lysosomal pathway. Over the last 15 yr, yeast genetic screens led to the identification of a number of genes involved in the autophagic pathway. Most of these autophagy genes are present in higher eukaryotes and regulate autophagy process for cell survival and homeostasis. Significant progress has recently been made to better understand the molecular mechanisms of the autophagy machinery. Especially, autophagy process, including the regulation of autophagy induction through mTOR and the nucleation and elongation in autophagosome formation through class III phosphatidylinositol 3-kinase complex and ubiquitin-like conjugation systems, became evident. While many unanswered questions remain to be answered, here, we summarize the recent process of autophagy with emphasis on molecules and their protein complexes along with advanced molecular mechanisms that regulate the autophagy machinery.