Downregulation of inducible nitric oxide synthase (iNOS) expression is implicated in the antiviral activity of acetylsalicylic acid in HCV-expressing cells

Abstract

Previously, we described that acetylsalicylic acid (ASA) decreases HCV expression, but the mechanisms involved have not been clearly established. We evaluated the participation of inducible nitric oxide synthase (iNOS) in the regulation of HCV-RNA induced by ASA. Huh7 cells expressing non-structural HCV proteins were exposed to 4 mM ASA and incubated at the same times we reported HCV downregulation (24-72 h), and iNOS mRNA and protein levels were then measured by real-time PCR and Western blot, respectively. Nitric oxide levels were measured at the same time. Inhibition of iNOS mRNA by small interfering RNAs (siRNA) and activation of the iNOS gene promoter by ASA treatment were evaluated. In Huh7 replicon cells treated with ASA, we found decreased levels of iNOS mRNA, iNOS protein and nitrosylated protein levels at 48-72 h. ASA exposure also reduced the transactivation of the iNOS promoter in HCV replicon cells at 48 h, and this was partly due to the decrease in the affinity of transcription factor C/EBP-β for its binding site in the iNOS promoter. siRNA silencing of iNOS decreased HCV-RNA expression (65 %) and potentiated the antiviral effect (80 %) of ASA compared with control cells. ASA reduces iNOS expression by downregulating promoter activity, mRNA and protein levels at the same time that it decreases HCV expression. These findings suggest that the antiviral activity of ASA is mediated partially through the modulation of iNOS.

Antiviral activity of aspirin against RNA viruses of the respiratory tract—an in vitro study

“…However, this does not appear to be the sole effect of aspirin against HCV as others have described an additional effect on the inducible nitric oxide synthase (iNOS), which seems to be implicated in the antiviral activity of aspirin in HCV-expressing cells. 34 The effect of aspirin on the iNOS expression by downregulating the promoter activity, mRNA and protein expression levels also appears to be only part of the story, but one which correlates with the known COX-2 inhibiting 34 Possible speculation about the mode of action can be made about the fact that, in our experiment, only RNA viruses were the target of the antiviral activity of aspirin. This suggests at least some level of specificity.…”

“…In the case of hepatitis C virus (HCV), an effect on the cellular receptor claudin‐1 is postulated leading to an effect on virus entry. However, this does not appear to be the sole effect of aspirin against HCV as others have described an additional effect on the inducible nitric oxide synthase (iNOS), which seems to be implicated in the antiviral activity of aspirin in HCV‐expressing cells . The effect of aspirin on the iNOS expression by downregulating the promoter activity, mRNA and protein expression levels also appears to be only part of the story, but one which correlates with the known COX‐2 inhibiting activity of aspirin .…”

Antimicrobial Effects of Antipyretics

“…Fungi (unlike bacteria and viruses) produce prostaglandins, and although their exact function is uncertain, it is thought that they influence virulence, in particular controlling the yeast-to-hypha transition and biofilm production (32,34,81). Aside from prostaglandin inhibition, as detailed in this review, other mechanisms by which antipyretic drugs influence pathogens include inhibiting virus replication (75)(76)(77)(78)(79), inhibiting or promoting bacterial and fungal growth (1-3, 5-9, 16-21, 34, 45, 52, 82-85), altering the expression of virulence factors (5,6,9,29,30,37,39,(43)(44)(45), changing the surface hydrophobicity of microbes (9), influencing biofilm production (16,21,30,32,33,35,(37)(38)(39)(40)(41), affecting motility (22,23), adherence (9,16,(25)(26)(27)(28)(29)(30)(31), and metabolism (16,18,46), interacting with the transport and release of antibiotics by PMNL …”

“…ASA/SAL inhibit the cell entry and replication of hepatitis C virus (75)(76)(77), as well as the replication of flavivirus and influenza virus (78,79). The inhibition of replication mostly occurs at therapeutic levels (78,79).…”

Quantification of nitric oxide by high‑performance liquid chromatography‑fluorometric method in subgenomic hepatitis C virus‑replicon expressing Huh7 cells upon treatment with acetylsalicylic acid

“…An increase in the levels of NO was observed in cells exposed to arginine, equivalent to two orders of magnitude, whereas L-NIL reduced NO production and iNOS expression at the transcriptional level. NO was previously quantified using the Griess method (27), but the detection range of the calibration curve using Griess method was 1.56-100 µM, whereas for the HPLC-fluorescence method the range was 125-1,000 pM. Therefore, HPLC may be more useful for the detection of low NO concentrations.…”