To evaluate the anti-inflammatory effect of a number of drugs on microglia, I've used the Griess reagent to determine the levels of nitric oxide in the cell culture supernatant. Cells were pretreated with the compounds for two hours, then stimulated with LPS (100 ng) for 24 hours in the presence of the compounds. Results show a significant production of nitric oxide following LPS-mediated stimulation and reduction of such increased production i response to treatment. However, gene expression analysis for iNOS did not reflect these results. iNOS expression did not vary between the groups treated with LPS solely and those treated with LPS and the compounds, despite a noticeable difference in the Griess assay (LPS + drugs were incubated for 6 hours, not 24 h, before adding TRIzol). I was wondering what might be a possible explanation for this?
Thank you.
I think the most parsimonious explanation is that the compounds are acting on NO once it is formed rather than affecting is production by NOS. In other words, the compounds would be acting as antioxidants, scavenging NO, and not affecting NOS at all. This is actually very common for plant extracts, fractions, and isolated compounds.
In the case of the effects of LPS itself, remember that NOS need substrates to exert its activity (e.g, arginine) and, thus, the availability of these might affect NO production regardless of enzyme expression.
Thank you very much @Daniel.
Any ideas on how to validate this hypothesis?
The possible direct effect of compounds on NO is fairly easy to test. You can use sodium nitroprusside to generate NO in a test tube with or without compounds at several dilutions and perform the Griess assay as you would with cell supernatants. This will show if the compounds have the ability to directly scavenge NO.
The involvement of changes in substrate availability is more complex to test.
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