| Previous study suggested that Spring Viraemia of Carp Virus(SVCV) infection EPC cells can induce oxidative stress, down-regulate the expression of heme oxygen-1(HO-1), disturb the balance of intracellular redox-cycling and further cause proteins and DNA oxidative damage, which is one of the major pathogenic mechanisms(s) of SVCV. Nrf2, an important transcription factor, plays a pivotal role in maintaining intracellular homoeostasis as well as in anti-oxidative, anti-inflammatory and anti-apoptotic.Nrf2 also participates in viral infection. Thus, investigation on the interaction between Nrf2-ARE signaling pathways and SVCV is necessary, which may help to elucidate the relationship between the Nrf2-ARE pathway and SVCV infection and facilitate the development of new therapeutic strategies against SVCV. The major results are summarized as follows: 1. The effects of SFN and CDDO-Me on the activation of Nrf2 and the replication of SVCV.Two compounds, SFN and CDDO-Me, were used to assess the role of Nrf2 activation on SVCV replication. Using the MTT method, the LD50 of two compounds in FHM cells were determined as 37.65 μM and 6.823 μM, respectively. Then, real- time PCR and western- blot were performed to verify the drug’s potential capacity to trigger the nuclear translocation of Nrf2. The results illustrated that the level of Nrf2 transcription was significantly increased at 6 h and peaked at 12 h after treated with SFN. HO-1, the downstream gene of Nrf2, was slightly up-regulated at early stages and significantly higher at 12 h. Nevertheless, both of Nrf2 and HO-1 returned to basal levels at 24 h. But the level of SOD1 transcription were significantly up-regulated from 12 h to 24 h. For CDDO-Me treatment, the transcriptional levels of Nrf2, HO-1 and SOD1 were all reached maximum level at 24 h. However, HO-1 levels were decreased mildly during 3 to 6 h and then followed with a continual increase. SOD1 show a time-dependent increase from 3h to 24 h in FHM cells after treated with CDDO-Me. In according with the dynamic variation of m RNA, the protein levels of Nrf2 and HO-1 were significantly increased in FHM cells after treated with SFN or CDDO-Me. Moreover, the TAC of FHM cells pretreated with SFN or CDDO-Me were promptly increased and maintained a relatively high level. Then the influence of Nrf2 on the replication of SVCV were estimated. FHM cells were pretreated with SFN and CDDO-Me for 12 h and then infected with SVCV at an MOI of 0.1. The m RNA levels of the SVCV-G gene were significantly decreased at 12 h and 24 h post SVCV infection. In accordance with the inhibition of replication of SVCV-G gene, the viral titer of SVCV was also decreased. The similar results were also observed in EPC cells. These results demonstrate that up-regulation of Nrf2 dampens SVCV replication. 2. Knocking down the expression of Nrf2 or HO-1 sensitizes cells to SVCV.To further verify the protective role of the Nrf2 against SVCV, Nrf2 was knockdown by si RNA transfection. Comparing with the negative control groups transfected with nontargeting si RNA, transfection of si RNA targeted to Nrf2 decreased the expression of Nrf2 up to 50 percent. And no obvious enhancement of Nrf2 were observed when these cells were incubated with SFN or CDDO-Me after transfected with si RNA. Furthermore, the up-regulation of the downstream genes of Nrf2(HO-1, SOD1) were significantly inhibited in the Nrf2-si RNA transfected-cells after treatment with SFN or CDDO-Me. Similarly, the protein levels of Nrf2 and HO-1 were also significantly decrease when Nrf2-si RNA were transfected into FHM cells. In addition, the total antioxidant capacity of FHM cells were significantly decrease after transfection with Nrf2-si RNA. Knocking down the expression of Nrf2 and HO-1 alone increase the expression of SVCV-G genes about 2.5-fold. Treatment with SFN and CDDO-Me showed no significant impact on the replication of SVCV-G gene when Nrf2 or HO-1 were knocked down by si RNA transfection. |
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