University of Rijeka. Faculty of Medicine. Department of Anatomy.
Abstract
CILJ ISTRAŽIVANJA. Ispitati da li tijekom ishemijsko-reperfuzijske (IR) ozljede
kralježnične moždine dolazi do ekspresije i aktivacije Nrf2 transkripcijskog faktora, u
kojim stanicama se eksprimira te da li aktivnost Nrf2 signalnog puta ovisi o dužini
trajanja reperfuzije. Ispitati da li mangan porfirin (MnP) djeluje neuroprotektivno
putem Nrf2 i NFkB transkripcijskih faktora.
MATERIJAL I METODE. Korišten je eksperimentalni model IR ozljede kralježnične
moždine klemanjem abdominalne aorte u trajanju 45 minuta u štakora. MnP je davan
prije operativnog zahvata i svakih 12 sati nakon zahvata. Životinje su žrtvovane
nakon 1, 6, 48 ili 168 sati nakon reperfuzije. Neurološki status je ocjenjivan svih 7
dana. Spektrofotometrijom su mjerenje razine oštećenja lipida i proteina te razina
antioksidativnih enzima. Western blotom mjerili smo razine ekspresije SOD1, SOD2,
GSH-Px, Nrf2, HO-1, NF-kB. Metodom PCR-a analizirana je ekspresija mRNA HO-1,
Gclc i CAT. Imunofluorescentnom metodom prikazana je lokalizacija i intenzitet
ekspresije Nrf2, NF-kB, NeuN, ChaT, GFAP, Iba1 i PLP1.
REZULTATI. Neurološki status je bio značajno bolji nakon 48 i 168 sati davanjem
MnP. MnP je značajno snizio razinu produkata lipidne peroksidacije i sadržaja
karboniliranih proteina nakon 48 i 168 sati od reperfuzije. Također značajno je
povisio aktivnost SOD enzima u prvih 6 sati dok je aktivnost GPx enzima bila
povećana i do 48 sati. IR ozljeda značajno povećava ekspresiju Nrf2 proteina u
nuklearnoj frakciji u prvih 6 sati dok MnP ne mijenja njegovu ekspresiju. Ciljni protein
Nrf2 puta, HO-1 je značajno povećan i nakon 48 sati. Povećan je nivo ekspresije
mRNA HO-1, Gclc i CAT molekula. MnP značajno smanjuje ekspresiju NFkB
proteina u nuklearnoj frakciji u prvih 6 sati. Histomorfološka analiza je pokazala
ekspresiju Nrf2 proteina u CHaT pozitivnim neuronima prednjeg roga sive tvari.
Prosječni intenzitet Nrf2 ekspresije raste s dužinom trajanja reperfuzije i direktno je
ovisan o IR ozljedi. MnP snižava broj astrocita posebno u prednjem funikulu bijele
tvari 6 i 48 sati od reperfuzije. Analizom NF-kB ekspresije i aktivacije pokazali smo da
se NFkB eksprimira u astrocitima u ovisnosti o IR ozljedi kao i o vremenu trajanja
reperfuzije. MnP je značajno snizio broj stanica s NFkB pozitivnom jezgrom.
ZAKLJUČAK. IR ozljeda potiče ekspresiju i aktivaciju Nrf2 proteina ovisno o trajanju
reperfuzije. MnP djeluje protektivno smanjujući ekspresiju NF-kB proteina u
nuklearnoj frakciji.AIM. To investigate whether Nrf2 expression and activation occurs during ischemia –
reperfusion injury of spinal cord, in which cells the Nrf2 expression is observed and
does the Nrf2 signalling pathway activity depends on the time of reperfusion. To
investigate whether manganese porphyrin (MnP) presents neuroprotective effects
through transcription factors Nrf2 and NF-kB.
MATERIAL AND METHODS. The study was conducted on experimental model of
spinal cord IR injury by clamping abdominal aorta during 45 min in rats. MnP was
administered just before the surgery and every 12 h post surgery. The animals were
sacrificed after 1, 6, 48 and 168 h of reperfusion. Neurologic score was evaluated
during 7 days. Spectrophotometric measurement of lipid peroxidation products,
protein carbonyl content and levels of antioxidant enzyme activity were performed.
Using Western blot levels of expression of SOD1, SOD2, GSH-Px, Nrf2, HO-1 and
NF-kB were measured. The expressions of mRNA HO-1, Gclc and CAT were
analyzed by PCR method. Immunofluorescent analysis was used to show localization
and intensity of expression of Nrf2, NF-kB, NeuN, ChaT, GFAP, Iba1 and PLP1.
RESULTS. The neurologic score was significantly better during 48 and 168 h of
reperfusion in group with MnP treatment. MnP significantly decreased the levels of
lipid peroxidation products and the protein carbonyl content during 48 and 168 h of
reperfusion. Also MnP significantly increased the activity of SOD enzymes during 6 h
of reperfusion, while the activity of GPx enzyme was increased even during 48 h of
reperfusion. IR injury significantly increased expression of Nrf2 protein in nuclear
fraction during 6 h of reperfusion while MnP does not effect Nrf2 expression. Nrf2
signalling pathway target protein, HO-1, was significantly increased even during 48 h
of reperfusion. Also the level of mRNA HO-1, Gclc and CAT expression was
increased. MnP significantly decreased expression of NF-kB protein in nuclear
fraction during 6 h of reperfusion. Histological analysis revealed Nrf2 expression in
CHaT positive neurons localized in anterior horn of grey matter. Mean intensity of
Nrf2 expression increased with the longer reperfusion time and was directly
dependant on IR injury. MnP decreased astrocyte number in anterior funiculus of
white matter during 6 and 48 h of reperfusion. By analysing NF-kB expression and
activation we showed that NF-kB was expressed in astrocytes depending on IR injury as well as on time of reperfusion. MnP significantly decreased number of cells with
nuclear positive NF-kB expression.
CONCLUSION. IR injury induces expression and activation of Nrf2 protein depending
on time of reperfusion. MnP acts protective by decreasing expression of NF-kB
protein in nuclear fraction
