Nuclear factor-kappaB p65 and upregulation of interleukin-6 in retinal ischemia/reperfusion injury in rats.
We previously demonstrated that endogenous interleukin-6 (IL-6) is upregulated and may be neuroprotective after retinal ischemia. The purpose of this study is to investigate the role of nuclear factor kappa-B (NF-kappaB) in regulating IL-6 expression after ischemia. NF-kappaB p65 mRNA levels were significantly elevated between 2 and 12 h after the insult. A high number of NF-kappaB p65 positive cells were detected in the inner retina at 12 h after ischemia. Activated nuclear NF-kappaB p65 and IL-6 were colocalized in cells, which were also marked by a microglial/phagocytic cell marker (ED1) in the inner retina. Carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG-132, a proteasome inhibitor, which inhibits IkappaB degradation and hence prevents the activation and translocation of NF-kappaB into the nucleus) abolished the increase in NF-kappaB p65 mRNA levels after the insult, while there was no effect by helenalin (an inhibitor which inhibits NF-kappaB activity by alkylation of the p65 subunit, thereby blocking its binding to the target DNA). However, MG-132 and/or helenalin significantly diminished the increase in IL-6 mRNA levels after the insult. Small interfering RNAs (siRNAs, inhibit target gene expression through the sequence-specific destruction of the target messenger RNA) against NF-kappaB p65 significantly reduced the increase in NF-kappaB p65 mRNA levels as well as IL-6 mRNA levels after ischemia. The number of retinal ganglion cells (RGCs) was also significantly decreased using the inhibitors of NF-kappaB compared with those of the controls after ischemia. These findings support the hypothesis that upregulation of endogenous retinal IL-6 in retinal I/R injury in microglial/phagocytic cells is controlled predominantly by NF-kappaB p65.
Wang
Jiantao
J
Department of Ophthalmology, Doheny Eye Institute, University of Southern California Keck School of Medicine, Los Angeles, CA 90033, USA.
Jiang
Shaoyun
S
Kwong
Jacky M K
JM
Sanchez
Ruben N
RN
Sadun
Alfredo A
AA
Lam
Tim T
TT
D000704:Analysis of Variance; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D002452:Cell Count / Q000379:methods; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004347:Drug Interactions; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D007150:Immunohistochemistry / Q000379:methods; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism*; D007976:Leupeptins / Q000494:pharmacology; D008297:Male; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D011917:Rats, Inbred Lew; D015427:Reperfusion Injury / Q000378:metabolism* / Q000503:physiopathology; D012160:Retina / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology*; D012165:Retinal Ganglion Cells / Q000187:drug effects / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012717:Sesquiterpenes / Q000494:pharmacology; D045789:Sesquiterpenes, Guaiane; D013997:Time Factors
D000894:Anti-Inflammatory Agents, Non-Steroidal; D015853:Cysteine Proteinase Inhibitors; D015850:Interleukin-6; D007976:Leupeptins; D016328:NF-kappa B; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012717:Sesquiterpenes; D045789:Sesquiterpenes, Guaiane; C001329:helenalin; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde