Dunaliella salina alga extract inhibits the production of interleukin-6, nitric oxide, and reactive oxygen species by regulating nuclear factor-κB/Janus kinase/signal transducer and activator of transcription in virus-infected RAW264.7 cells

Abstract

Recent investigations have demonstrated that carotenoid extract of Dunaliella salina alga (Alga) contains abundant β-carotene and has good anti-inflammatory activities. Murine macrophage (RAW264.7 cells) was used to establish as an in vitro model of pseudorabies virus-induced reactive oxygen species (ROS) response. In this study, antioxidant activities of Alga were measured based on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, trolox equivalent antioxidant capacity assays, reducing power, and virus-induced ROS formation in RAW264.7 cells. Anti-inflammatory activities of Alga were assessed by its ability to inhibit the production of interleukin-6 and nitric oxide (NO) using enzyme-linked immunosorbent assay, then the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway was investigated by measuring the inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear factor-κB (p50 and p65), JAK, STAT-1/3, and suppressor of cytokine signaling 3 (SOCS3) by Western blotting. In addition, Alga inhibited virus replication by plaque assay. Our results showed that the Alga had high antioxidant activity, significantly reduced the virus-induced accumulation of ROS, and inhibited the levels of nitric oxide and interleukin-6. Further studies revealed that Alga also downregulated the gene and protein expressions of iNOS, COX-2, nuclear factor-κB (p50 and p65), and the JAK/STAT pathway. The inhibitory effects of Alga were similar to pretreatment with specific inhibitors of JAK and STAT-3 in pseudorabies virus -infected RAW264.7 cells. Alga enhanced the expression of SOCS3 to suppress the activity of the JAK/STAT signaling pathway in pseudorabies virus-infected RAW264.7 cells. In addition, Alga has decreased viral replication (p < 0.005) at an early stage. Therefore, our results demonstrate that Alga inhibits ROS, interleukin6, and nitric oxide production via suppression of the JAK/STAT pathways and enhanced the expression of SOCS3 in virus-infected RAW264.7 cells.

Keywords: Dunaliella salina; anti-inflammatory; pseudorabies virus; reactive oxygen species.

Figure 1

Reducing power of Alga, β-carotene and α-tocopherol. Each value is expressed as mean ± SD (n = 3). Alga = D. salina carotenoid extract; SD = standard deviation.

Figure 2

Alga inhibits the production of ROS in PRV-infected RAW264.7 cells. RAW264.7 cells were untreated (DMSO and basal) or pretreated with 100 μM, 50 μM, and 25 μM of Alga for 1.5 hours, and then infected with PRV or left uninfected (basal). After 24 hours, the cells were stained with 10 μM of DCFH-DA for 30 minutes, and ROS production was determined using a fluorescence microplate reader. All measurements were performed in triplicate. Data are presented as mean ± SD (n = 3). ^#p < 0.01, significant compared with PRV-infected RAW264.7 cells (DMSO). *p < 0.01, significant compared with basal. Alga = D. salina carotenoid extract; DMSO = dimethyl sulfoxide; DCFH-DA = dichloro-dihydro-fluorescein diacetate; MOI = multiplicity of infection; PRV = pseudorabies virus; ROS = reactive oxygen species; SD = standard deviation.

Figure 3

Alga inhibits the production of (A) IL-6 and (B) NO in PRV-infected RAW264.7 cells. RAW264.7 cells were untreated (DMSO and basal) or pretreated with 100 μM, 50 μM, and 25 μM of Alga for 1.5 hours, and then infected with PRV or left uninfected (basal). After 6–24 hours, IL-6 and NO production was measured by ELISA. All measurements were performed in triplicate. Data are presented as mean ± SD (n = 3). *p < 0.01, significant compared with PRV-infected RAW264.7 cells (DMSO). Alga = D. salina carotenoid extract; DMSO = dimethyl sulfoxide; ELISA = enzyme-linked immunosorbent assay; IL = interleukin; PRV = pseudorabies virus; SD = standard deviation.

Figure 4

Effect of JAK/STAT inhibitors on PRV-induced ROS and inflammatory response in RAW264.7 cells. (A) IL-6 and (B) NO production was determined by ELISA and Griess reagent. RAW264.7 cells were pretreated with different concentrations of STAT-3 inhibitor (Line 1: 25 μM; Line 3: 50 μM) or JAK2 inhibitor AG490 (Line 2: 10 μM, Line 4: 20 μM), and DMSO (Line 5), or received no treatment (mock, Line 6) for 1.5 hours; the cells were then infected with PRV or left uninfected (basal, Line 7) for 24 hours. (C) Cell lysates were prepared and subjected to Western blot analysis using antibodies against iNOS, COX-2, and NF-κB (p50 and p65); the signals were normalized against the GAPDH. (D) RAW264.7 cells were untreated (DMSO) or pretreated with Alga (100 μM), AG490 (20 μM), and STAT-3 inhibitor (50 μM) for 1.5 hours, and then infected with PRV or left uninfected (basal). After 24 hours, the cells were stained with 10 μM of DCFH-DA for 30 minutes, and the ROS production was determined using a fluorescence microplate reader. All measurements were performed in triplicate. The results are presented as means ± SD of three independent experiments. *p < 0.01, significant compared with PRV-infected RAW264.7 cells (DMSO). Alga = D. salina carotenoid extract; DMSO = dimethyl sulfoxide; ELISA = enzyme-linked immunosorbent assay; IL = interleukin; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; iNOS = inducible nitric oxide synthase; COX-2 = cyclooxygenase; JAK = Janus kinase; NF-κB = nuclear factor-κB; PRV = pseudorabies virus; SD = standard deviation; ROS = reactive oxygen species; STAT = signal transducer and activator of transcription.

Figure 5

Alga inhibits virus-induced activation of iNOS, COX-2, and NF-κB in RAW264.7 cells. RAW264.7 cells were untreated (DMSO) or pretreated with 100 μM of Alga for 1.5 hours, and then infected with PRV (DMSO) or left uninfected (basal). After 6–24 hours, protein levels of (A) iNOS and COX-2, and (B) NF-κB p50 and p65 in the RAW264.7 cells were assayed via Western blot analysis. All measurements were performed in triplicate. Alga = D. salina carotenoid extract; DMSO = dimethyl sulfoxide; iNOS = inducible nitric oxide synthase; COX-2 = cyclooxygenase; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; NF-κB = nuclear factor-κB; PRV = pseudorabies virus.

Figure 6

Alga inhibits the expressions of JAK, pJAK, STAT-1, STAT-3, pSTAT-1, and pSTAT-3 in PRV-infected RAW264.7 cells. RAW264.7 cells were untreated (DMSO) or pretreated with 100 μM of Alga for 1.5 hours, and then infected with PRV or left uninfected (basal) for 6–24 hours. Total protein was subjected to 10% SDS–PAGE followed by Western blotting using JAK, pJAK, STAT-1, pSTAT-1, STAT-3, pSTAT-3, and GAPDH antibodies. All measurements were performed in triplicate. Alga = D. salina carotenoid extract; DMSO = dimethyl sulfoxide; JAK = Janus kinase; PRV = pseudorabies virus; SDS-PAGE = sodium dodecyl sulfate polyacrylamide gel electrophoresis; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; pSTAT = tyrosine-phosphorylated signal transducer and activator of transcription; STAT = signal transducer and activator of transcription.

Figure 7

Alga regulates the expression of SOCS3 in PRV-infected RAW264.7 cells. RAW264.7 cells were untreated (DMSO) or pretreated with 100 μM of Alga for 1.5 hours, and then infected with PRV or left uninfected (basal) for 6–24 hours. Total protein was subjected to 10% SDS–PAGE followed by Western blotting using SOCS3 and GAPDH antibodies. All measurements were performed in triplicate. Data are presented as mean ± SD (n = 3). Alga = D. salina carotenoid extract; DMSO = dimethyl sulfoxide; PRV = pseudorabies virus; SDS-PAGE = sodium dodecyl sulfate polyacrylamide gel electrophoresis; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; SD = standard deviation; SOCS3 = suppressor of cytokine signaling 3.

Figure 8

Antiviral activity of RAW264.7 cells treated with Alga. RAW264.7 cells were pretreated with the Alga (100 μM) or DMSO for 1.5 hours. The cells were infected with the PRV of 0.1 MOI for 12 hours. Supernatants were collected and frozen at −70°C. Afterward, the supernatants were used to determine the level of virus titer by plaque assay. The data represent the average number of PFU/mL. The results are presented as means ± SD (n = 3); p < 0.01, significant compared with PRV-infected RAW264.7 cells (DMSO). Alga = D. salina carotenoid extract; DMSO = dimethyl sulfoxide; MOI = multiplicity of infection; PFU = plaque forming unit; PRV = pseudorabies virus; SD = standard deviation.