I am working on microglial cells (macrophages from the Central Nervous System). It's really difficult to do transfection (transient, siRNA) in these cells due to their phagocytic capacities. I want to inhibit the Nrf2 pathway and mainly Nrf2 translocation. The use of siRNA is possible but really hard to perform. Thus, has anyone used Nrf2 inhibitors to block Nrf2 activation?
What transfection protocol you are using? Maybe you can advise, I transfected with siRNA every week.
Didn't use it myself but it looks very good in this paper:
http://www.pnas.org/content/early/2011/01/04/1014275108
let us know if it worked for you.
We deliver peptides and proteins into cells (interfering with protein protein interactions could be a way to inhibit the pathway or block Nrf2 translocation). We have a method that works in cells that are hard to transfect. I don't know if it would work in microglial cells but let me know if you're interested...
Thanks for your answers and your help !
@ José : our transfection experiments are performed with lipofectamine according to the manufacturer’s protocol but the output is very low. Have you got another protocol? You say that you transfected with siRNA every week, but is it on microglia or other macrophagic cells?
@ Barak and Jean-Philippe : effectively, I have seen that different compounds against Nrf2 signaling have been published. Concerning, the Brusatol, it’s really expensive (due to its synthesis). Some compounds, like Luteolin and Trigonelline, also seem to be active. I am currently checking the litterature …..
@ Jean-Philippe : what sort of peptides do you deliver? What is/are the mechanism(s) of these compounds?
I know nothing about microglial cells but we have used siLentFect from biorad for primary human macrophages (which are also very difficult) and works pretty well although we have to administer it twice, 0 and 48h and then harvest at 72h. might work for u...
Goodluck
... I will try with the alkaloid trigonelline which has been shown to have inhibitory effects on the Nrf2 signalling pathway. Once again, thanks for all your comments !
I deliver peptides and proteins by going through the endocytic pathway. We have a compound that make endosomes leaky and peptides/proteins that are endocytosed can escape into cytosol and nucleus. It's very efficient. the surprise is also that the cell is ok with it as far as we can tell (microarray analysis shows no response on transcription level, cells are healthy...etc)
if you know of a peptide/protein that would block Nrf2, this could be a way to go..just an idea ( and delivery could fail in microglial cells, I don't know!)
Hi, Tony, Do you have the plasmid about Nrf2 high and low expression. Can you send it to me if it is possible. I also do the same work in the neuron cell. Thank you very much . My email is. I am looking forward to receiving your reply.
Thanks for your answers !
Chiara, I am doing some dose-response experiments with Trigonelline on microglial cells. What is the concentration that you use for this compound ? for what type of cells ?
Tony Heurtaux what concentration of Trigonelline you are using? I am planning to do some dose curve 48 hrs..But any idea from where to start with?
Hi Suman, I currently use Trigonelline at 5 nM for a 6h exposure. It works fine.
Thanks a lot Tony. What type of cell line you are using? I am going to use pancreatic cancer cell lines. Any idea about concentrations in cancer cells for longer time like 48 hrs?
Suman, I work on brain primary cells (microglia). I have no idea concerning the effects on cancer cell lines. The best to do is to perform a dose ranging on your cells. Trigonelline will block the Nrf2 pathway. Have you got a positive control/treatment showing an "increase" of the Nrf2 pathway ? A Trigonelline exposure could thus decrease this "increase" of the Nrf2 pathway ...
Suman, where sis you buy the TRigonelline from . can you please send me teh catalogue number. Thank you.
Hi, tony. I am interested in the trigonelline effects, have you seen some inhibitory effect on Nrf2 expression or downstream target changes with tirgonelline? Thanks!
Hi Zhangting,
I have not checked for the expression of Nrf2 (by WB with nuclear proteins for example). However, HO-1 expression (qPCR) appears to be down-regulated by the presence of Trig. after an Nrf2-increased treatment. NQO1 expression is also down-regulated but at a lower level (qPCR).
https://www.researchgate.net/publication/49728490_Brusatol_enhances_the_efficacy_of_chemotherapy_by_inhibiting_the_Nrf2-mediated_defense_mechanism
This article seems to explain some potential treatment.
Article Brusatol enhances the efficacy of chemotherapy by inhibiting...
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