RESUMO
Chronic enteritis can produce an excess of reactive oxygen species resulting in cellular damage. Stanniocalcin-1(STC-1) reportedly possesses anti-oxidative activity, the aim of this study was to define more clearly the direct contribution of STC-1 to anti-oxidative stress in cattle. In this study, primary intestinal epithelial cells (IECs) were exposed to hydrogen peroxide (H2O2) for different time intervals to mimic chronic enteritis-induced cellular damage. Prior to treatment with 200 µM H2O2, the cells were transfected with a recombinant plasmid for 48 h to over-express STC-1. Acridine orange/ ethidium bromide (AO/EB) double staining and trypan blue exclusion assays were then performed to measure cell viability and apoptosis of the cells, respectively. The expression of STC-1 and apoptosis-related proteins in the cells was monitored by real-time PCR and Western blotting. The results indicated that both STC-1 mRNA and protein expression levels positively correlated with the duration of H2O2 treatment. H2O2 damaged the bovine IECs in a time-dependent manner, and this effect was attenuated by STC-1 over-expression. Furthermore, over- expression of STC-1 up-regulated Bcl-2 protein expression and slightly down-regulated caspase-3 production in the damaged cells. Findings from this study suggested that STC-1 plays a protective role in intestinal cells through an antioxidant mechanism.
Assuntos
Caspase 3/genética , Doenças dos Bovinos/genética , Enterite/veterinária , Regulação da Expressão Gênica , Glicoproteínas/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Animais , Animais Recém-Nascidos , Western Blotting/veterinária , Caspase 3/metabolismo , Bovinos , Doenças dos Bovinos/etiologia , Doenças dos Bovinos/metabolismo , Duodeno/metabolismo , Enterite/etiologia , Enterite/genética , Enterite/metabolismo , Células Epiteliais/metabolismo , Glicoproteínas/metabolismo , Peróxido de Hidrogênio/farmacologia , Masculino , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/veterináriaRESUMO
This study investigated the effect of exogenous insulin-like growth factor (IGF)-1 on the proliferation and differentiation of osteoblastic cells from Chinese Holstein cattle and the resultant bone nodule formation and mineralisation in vitro. The osteoblastic cells were isolated and cultured, then identified using Giemsa and alkaline phosphatase (ALP) staining methods. The effect of different concentrations of IGF-1 on cell growth was assessed by MTT assay. The ALP activity and osteocalcin (OC) concentration in the osteoblastic cells were measured by a colorimetric assay and a radioimmmunoassay, respectively. Calcium nodules were observed using alizarin red S stain, while the content of matrix calcium was determined by atomic absorption spectrophotometry. Cell proliferation in the cultures was stimulated by IGF-1 at concentrations ranging from 1 to 200ng/mL, with the maximum effect observed at 100ng/mL. This effect was observed from day 1 and peaked at day 5, but decreased at day 7. At concentrations of 10ng/mL and 100ng/mL, IGF-1 significantly induced ALP activity, OC level, matrix calcium content, and nodule formation of the osteoblastic cells by 20-180% (P<0.05 or P<0.01), compared to controls. The results suggested that IGF-1 is an anabolic agent for the proliferation, differentiation, mineralisation and calcium content of dairy cow osteoblasts, and could therefore act as a potential treatment for the metabolic bone diseases in these animals.
