The Responses Of Antioxidant Signaling Pathway Nrf2/Are To Spring Viraemia Of Carp Virus In Epithelioma Papulosum Cyprini Cells

An increasing number of studies have documented that oxidative stress induced by viral infection is one of the major pathogenic mechanisms (s) and may contribute to inflammatory responses and tissue injury. Nuclear factor-E2related factor2(Nrf2) is the key regulator of cellular defence systems in the counteraction with oxidative stress. The downstream effective genes, mediated by the Nrf2/ARE signal transduction pathway, perform a fundamental role in cellular detoxication, anti-oxidative, ant-inflammatory and anti-apoptotic capacities, and may also participate in the absorption, entry, replication, assembly and release of virus. Thus, an detailed investigation on the interaction between Nrf2and virus is in needed, which may provide new targets for the prevention and therapeutic of virus-induced diseases.In the present study, the full-length Nrf2of fathead minnow (Pimephales promelas) was cloned. Then, the transcription and expression profile of Nrf2upon Spring Viraemia of Carp Virus (SVCV) infection and the impact of activation of Nrf2on the replication of SVCV were studied in Epithelioma Papulosum Cyprini (EPC) cells. The major results are summarized as follow:1. Molecular cloning and homologous analysis of the Pimephales promelas Nrf2The full-length cDNA of Nrf2was cloned from EPC cells by RT-PCR and3’/5’-RACE. According to sequence analysis, the Nrf2cDNA was2115bp long, with159bp and183bp in the3’/5’untranslated region, respectively. A typical poly A signal (AATAAA) could also be found in the3’non-coding region. The ORF of Nrf2is1773bp in length, predicting a protein of590amino acids residues with a highly conserved CNC-bZip domain (AA441-AA552). Pairwise comparisons revealed that the Nrf2originated from Pimephales promelas was similar to those from mammals, birds and other fish. A higher homology to fish (52.5%-82.1%) was found, and the homology to Homo Sapiens, Mus musculus, Gallus gallus was relatively low (46.7%,45.9%and46.3%, respectively).2. The transcriptional and expression profiles of Nrf2upon SVCV infectionReal-time PCR and western blotting analyses were performed to study the impact of SVCV infection on the expression of Pimephales promelas Nrf2in vitro. The results demonstrated that, the transcriptional level of Nrf2increased upon SVCV infection and plateaued at12h post-infection following by a small decrease. For the protein profile of Nrf2, an obviously increased level could be detected at the early phase of viral infection, and a time-dependent upward tendency was also showed. The Nrf2expression level was peaked at12h post-infection and maintained at a relatively high level even at the later infection period. These results indicated that the SVCV infection activated the nuclear translocation of Nrf2, thus increasing the transcriptional and expression levels of Nrf2.3. The impact of Nrf2activation on the infection of SVCVTwo compounds, CDDO-Me and SFN, were to used to investigate the impacts of Nrf2activation on the infection of SVCV. Based on MTT assays, the LD50of CDDO-Me and SFN for EPC cells in48h were2.5μM,22μM, respectively. Then, real-time PCR and western blotting analyses were performed to verify the drug’s potential capacity to trigger the nuclear translocation of Nrf2. The results illustrated that10μM of SFN could significantly facilitate the nuclear accumulation of Nrf2, enhance the expressions of Nrf2-responsive genes and endow the EPC cells with higher antioxidant ability. Meanwhile, after pretreatment with SFN for16h, the transcriptional profile of SVCV-G gene in infected EPC cells appeared to decrease, though showed no statistically significant compared with that of the DMSO-pretreatment. In addition, CDDO-Me was not likely to induce the Nrf2-dependent cell defenses in EPC cells.