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Halofuginone (HF) is an extract from the widely used traditional Chinese medicine (TCM) Dichroa febrifugathat facilitates the recovery of wounds and attenuates hepatic fibrosis. However, the role of HF in theepithelial-mesenchymal transition (EMT) of IPEC-J2 cells remains unclear. The current study explored theanti-EMT effect of HF in IPEC-J2 cells and illustrates its molecular mechanism. Transforming growth factorb1 (TGF-b1), as a recognized profibrogenic cytokine, decreased the level of the epithelial marker E-cadherinand increased the level of the mesenchymal markers, such as N-cadherin, fibronectin (FN), vimentin (Vim),and a-smooth muscle actin (a-SMA), in IPEC-J2 cells depending on the exposure time and dose. HF markedlyprevented the EMT induced by TGF-b1. Dissection of the mechanism revealed that HF inhibited IPEC-J2 cellEMT via modulating the phosphorylation of SMAD2/3 and the SMAD2/3-SMAD4 complexnuclear translocation. Furthermore, HF could promote the phosphorylation of eukaryotic translation initiationfactor-2a (eIF2a), which modulates the SMAD signaling pathway. These results suggested that HF inhibitsTGF-b1-induced EMT in IPEC-J2 cells through the eIF2a/SMAD signaling pathway. Our findings suggest thatHF can serve as a potential anti-EMT agent in intestinal fibrosis therapy.
RESUMEN
Objective By establishing multivariate necrotizing enterocolitis (NEC) model in newborn rats to study the expression of the related molecules protein kinase R-like ER kinase(PERK),pho-protein kinase R-like ER kinase(p-PERK),eukaryotic translation initiation factor 2 alpha(eIF2a),pho-eukaryotic translation initiation factor 2 alpha (p-eIF2a),CCAAT/enhancer-binding protein-homologous protein (CHOP) in the PERK/eIF2a/ CHOP signaling pathway to explore the pathogenesis of NEC,and in order to provide a theoretical basis for clinical pre-vention and treatment of NEC.Methods One hundred and fifty neonatal Sprague-Dawley (SD) rats were divided into 3 groups according to random number table.NEC group:fed with formula,experienced hypoxia and cold stress,and lipopolysaccharide(LPS) fed via orogastric gavage to induce NEC model.Salubrinal(SAL) group:estabhshing NEC model and intraperitoneal injection of SAL (1 mg/kg).Control group:the same amount of 9 g/L saline was injected intraperitoneally.Ten rats selected from each group randomly were decapitated at 0,12,24,48 and 72 h,respectively,and intestinal tissues were obtained,intestinal general situation and made pathology scores observed.Expressions of PERK,p-PERK,eIF2a,p-eIF2a,CHOP proteins were detected by Western blot technique and the expression levels of CHOP mRNA gene were detected by real-time polymerase chain reaction.Results The control group had no NEC.The incidence of NEC in NEC group was 90％ (9/10 cases).The incidence of NEC in SAL group was 40％ (4/10 cases).The p-PERK and p-eIF2a protein expressions in NEC and SAL groups were increased with the modeling time.The p-PERK and p-eIF2a protein expressions of NEC and SAL groups were up-regulated compared with the control group (p-PERK:1.528 ± 0.264,1.402 ± 0.233 vs.0.303 ± 0.036;p-eIF2a:0.969 ± 0.076,1.173 ± 0.066 vs.0.209 ±0.045;P ＜0.05).The p-eIF2a protein expressions of SAL group were higher than those of the NEC group (P ＜ 0.05).The CHOP protein and CHOP mRNA expressions in the NEC and the SAL groups were increased with the modeling time.The CHOP protein and CHOP mRNA expressions of NEC and SAL groups were up-regulated than those of the control group (CHOP protein:1.456 ± 0.223,0.929 ± 0.064 vs.0.165 ± 0.026;CHOP mRNA:0.343 ±0.035,0.198 ± 0.044 vs.0.017 ± 0.010;P ＜ 0.05).The CHOP protein and CHOP mRNA expressions of SAL group were lower than that in the NEC group (P ＜ O.05).Conclusions The PERK/eIF2a/CHOP signal pathway may participate in NEC,and salubrinal probably by inhibiting p-eIF2a dephosphorylation,and inhibiting CHOP gene and protein expression to work.
RESUMEN
Hepatitis C virus (HCV) is a major cause of liver disease throughout the world. The NS5A and E2 proteins of HCV genotype 1 were reported to inhibit the double-stranded (ds) RNA-dependent protein kinase (PKR), which is involved in the cellular antiviral response induced by interferon (IFN). The response to IFN therapy is quite different between genotypes, with response rates among patients infected with types 2 and 3 that are two-three-fold higher than in patients infected with type 1. Interestingly, a significant percentage of HCV genotype 3-infected patients do not respond to treatment at all. The aim of this paper was to analyse the sequences of fragments of the E2 and NS5A regions from 33 outpatients infected with genotype 3a, including patients that have responded (SVR) or not responded (NR) to treatment. HCV RNA was extracted and amplified with specific primers for the NS5A and E2 regions and the PCR products were then sequenced. The sequences obtained covered amino acids (aa) 636-708 in E2 and in NS5A [including the IFN sensitivity determining region (ISDR), PKR-binding domain and extended V3 region)]. In the E2 and NS5A regions, we did observe aa changes among patients, but these changes were not statistically significant between the SVR and NR groups. In conclusion, our results suggest that the ISDR domain is not predictive of treatment success in patients infected with HCV genotype 3a.