DIDS protects against neuronal injury by blocking Toll-like receptor 2 activated-mechanisms.
Using an in vitro ischemia model (ischemic solution; IS model) that induces penumbral cell death, we examined the effect of 4,4'-diisothio-cyanostilbene-2,2'-disulfonic acid (DIDS) on cell injury/death and underlying molecular mechanisms. Propidium iodide (PI) uptake was used to quantify cell death in organotypic hippocampal slice cultures. A 24-h IS exposure caused a fivefold increase in mean PI fluorescence intensity. DIDS, dose-dependently (1-4000 microM), reduced the IS-induced PI uptake in hippocampal CA1 neurons with an IC(50) of 26 microM. This protective effect of DIDS was reversible and effective even 6 h following the onset of IS treatment. Gene expression profiling studies indicated that among approximately 46,000 transcripts tested, the most significantly up-regulated gene by IS was interleukin-1beta (IL-1beta) which was also the most significantly down-regulated gene when DIDS was added to the IS-treated slices. The addition of a recombinant IL-1 receptor antagonist (100 microg/mL) or neutralizing IL-1beta antibody significantly attenuated the IS-induced cell death, indicating that the up-regulation of IL-1beta with IS treatment contributed to the IS-induced cell death. Toll-like receptor 2 (TLR2), another gene that was significantly up-regulated by IS and suppressed by DIDS, was studied to determine whether it was related to the IL-1beta up-regulation. Indeed, this was the case as the IS-induced IL-1beta up-regulation was abolished in TLR2-/- mouse brain slices. Furthermore, the IS-induced cell death was significantly reduced in TLR2-/- when compared with that in wild-type slices, indicating that TLR2 is functionally upstream of IL-1beta in this IS model. We conclude that (i) IS up-regulates TLR2 expression and augments TLR2 signaling, causing over-expression of IL-1beta which leads to cell death and (ii) DIDS blocks IS-induced neuronal injury, at least partially, by suppressing the TLR2 pathway.
Yao
Hang
H
Department of Pediatrics (Section of Respiratory Medicine), University of California San Diego, La Jolla, California 92093-0735, USA.
Felfly
Hady
H
Wang
Juan
J
Zhou
Dan
D
Haddad
Gabriel G
GG
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology*; D000818:Animals; D015153:Blotting, Western; D016923:Cell Death / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D053583:Interleukin-1beta / Q000096:biosynthesis; D007511:Ischemia / Q000473:pathology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D018696:Neuroprotective Agents; D051193:Toll-Like Receptors / Q000037:antagonists & inhibitors* / Q000235:genetics; D015854:Up-Regulation / Q000187:drug effects
D053583:Interleukin-1beta; D018696:Neuroprotective Agents; D051193:Toll-Like Receptors; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid