Fig 1.
Effect of NO donors on cell viability and NO release in the presence or absence of benzo[a]pyrene in VSMCs.
(A) VSMCs were cultured in serum-free DMEM in the presence or absence of sodium nitroprusside (SNP, 1 mmol/L) for 12 or 24 h. (B) VSMCs were treated with SNP (1 mmol/L) in the presence or absence of benzo[a]pyrene (10 μmol/L) for 24 h. (C) VSMCs were treated with SNP (1–30 mmol/L) for 24 h. (D) VSMCs were treated with streptozotocin (STZ, 30 mmol/L) in the presence or absence of benzo[a]pyrene (10 μmol/L) for 24 h. (E) VSMCs were treated with streptozotocin (STZ, 1–30 mmol/L) for 24 h. Cell viability was determined by MTT assay. Cell survival was expressed as % of untreated control. The NO (nitrite/nitrate) levels were determined using the nitrite/nitrate colorimetric assay kit. All data are represented as mean ± SEM from three independent experiments. *P < 0.05 as compared with the control. #P < 0.05 as compared with SNP alone (B) or STZ alone (D).
Fig 2.
Effect of NO donor on subdiploid DNA content and cell apoptosis in the presence or absence of benzo[a]pyrene in VSMCs.
VSMCs were cultured in serum-free DMEM. VSMCs were treated with SNP (1 mmol/L) in the presence or absence of benzo[a]pyrene (10 μmol/L) for 24 h (A and B) or 12 h (C and D). (A) and (B) Cells were fixed with 70% ethanol and stained with propidium iodide (PI), and the DNA content was analyzed by flow cytometry. Representative images are shown (A). The percentage of subdiploid DNA content in cells was calculated (B). (C) and (D) The cells were collected, and stained with Annexin V-FITC and PI. The percentage of annexin V-positive (annexin V (+)), PI-negative (PI (-)) or PI (+) cells was calculated from fluorescence-1 (FL1-H) / fluorescence-2 (FL-2-H) dot plots, and is shown in the respective upper right and lower right hand corner. Data are represented as mean ± SEM from six independent experiments. *P < 0.05 as compared with control. #P < 0.05 as compared with SNP alone. (E) VSMCs were treated with SNP (1 mmol/L) in the presence or absence of benzo[a]pyrene (10 μmol/L) for 6 or 12 h. The protein expression of bcl-2 and α-tubulin (internal control) was determined by Western blotting. One representative experiment of three is shown.
Fig 3.
Inhibition of cell viability by NO donor was partially suppressed by conditioned medium of benzo[a]pyrene-treated cells.
VSMCs were cultured in serum-free DMEM in the presence or absence of benzo[a]pyrene (10 μmol/L) for 24 h and then the condition medium was collected. Cells were refreshed with DMEM containing SNP (1 mmol/L) as “w”. The condition medium was added to another cell culture dish as “cm”. Cells were co-incubated with SNP and benzo[a]pyrene as “co”. After 24 h, the cell viability was determined by MTT assay (A) and the subdiploid DNA content was determined by flow cytometry (B). Data are represented as mean ± SEM from three independent experiments. *P < 0.05 as compared with control. #P < 0.05 as compared with SNP alone.
Fig 4.
Role of interleukin-6 (IL-6) in the anti-apoptotic effect of benzo[a]pyrene on SNP-treated VSMCs.
(A) VSMCs were cultured in serum-free DMEM in the presence or absence of benzo[a]pyrene for 24 h. The IL-6 production was measured by ELISA. Data are represented as mean ± SEM from three independent experiments. *P < 0.05 as compared with the control. (B) VSMCs were treated with SNP (1 mmol/L) in the presence or absence of benzo[a]pyrene (10 μmol/L) for 24 h. The subdiploid DNA content was determined by flow cytometry. Representative images are shown. (C) VSMCs were treated with SNP (1 mmol/L) in the presence or absence of benzo[a]pyrene (10 μmol/L) for 12 h. The annexin V-FITC and PI staining was analyzed by flow cytometry. Data are represented as mean ± SEM from three independent experiments. #P < 0.05 as compared with SNP alone. $P < 0.05 as compared with SNP+benzo[a]pyrene.
Fig 5.
Benzo[a]pyrene induced the activation of both NF-κB and p38 MAPK, which were involved in the benzo[a]pyrene-increased IL-6 production in VSMCs.
VSMCs were cultured in serum-free DMEM in the presence or absence of benzo[a]pyrene (10 μmol/L) for 0.25–4 h (A) or 5–30 min (B). The protein expressions of NF-κB-p65 (nuclear protein) and IκBα (A) and phospho-p38/p38 MAPK (B) were determined by Western blotting. The protein fold changes, which were normalized to C23, α-tubulin, or p38, were shown below each blot. Experiments were repeated three times. One representative experiment is shown. (C) VSMCs were cultured in serum-free DMEM in the presence or absence of benzo[a]pyrene for 24 h. The IL-6 production was measured by ELISA. Data are represented as mean ± SEM from three independent experiments. *P < 0.05 as compared with control. #P < 0.05 as compared with benzo[a]pyrene alone.
Fig 6.
Role of NF-κB and p38 MAPK in the anti-apoptotic effect of benzo[a]pyrene on SNP-treated VSMCs.
VSMCs were pretreated with SB203580 (3 μmol/L) or PDTC (10 μmol/L) followed by treatments of benzo[a]pyrene (10 μmol/L) and SNP (1 mmol/L) for 12 h. The annexin V-FITC and PI staining was analyzed by flow cytometry (A). Cell viability was determined by MTT assay (B). Data are represented as mean ± SEM from three independent experiments. *P < 0.05 as compared with the control. #P < 0.05 as compared with SNP alone. $P < 0.05 as compared with SNP+benzo[a]pyrene.
Fig 7.
A proposed model showed the ability of benzo[a]pyrene to suppress a death signal in VSMCs triggered by NO through an IL-6 signaling pathway.