The Expression Of XIAP,Smac,HtrA2 And XAF1 In The Rat Hippocampus And Quercetin Treament Following Status Epilepticus

Background and Purpose: Status epilepticus(SE) is one of themedical emergency which can cause acute and permanent central neuralsystem damage, especially neuronal damage of limbic system such ashippocampus. It can promote the formation and development of seizures,and at last result in brain dysfunction. Apoptotic neuronal cell death isone of the forms of neuronal cell death after SE. The activation of caspasecascade plays a key role in apoptosis. X-linked inhibitor of apoptosisprotein(XIAP)is the most potent and versatile inhibitor of caspases in thelAP family, playing anti-apoptotic function, which is also regulatednegatively by Smac, HtrA2 and XAF1. It has been reported that XIAPand its negative regulators implicate in the pathophysiologicalmechanisms of cerebral ischemic injury and traumatic brain injury in rats.However, little is known about the regulation of XIAP,Smac,HtrA2 andXAF1 following SE in rats. It was reported that oxidative stress isassociated with XIAP pathway, and it involve in the mechanism ofneuronal injury following SE. The antioxidation of quercetin have beenshown to protect the tissue and cells against oxidative stress, but, there is no report about quercetin treatment following SE in rats. So wehypothesize that quercetin can inhibit apoptosis and protect neurons byinfluencing the regulation of XIAP pathway. The present study wasundertaken to investigate expression of XIAP,Smac,HtrA2 and XAF1 inthe rat hippocampus and quercetin treatment following SE, to explorepathophysiological mechanisms of expression of XIAP and its negativeregulators after SE and the possible neuroprotective mechanism ofquercetin on SE-induced neuronal injury, so as to provide a new methodor way for the protection of brain damage after SE.Methods: Adult male Sprague-Dawley rats were randomlyassigned to control group, SE model group and quercetin treatment group.The lithium-pilocarpine model of status epilepticus was established in rat.The dorsal hippocampus was chosen for study at 2h, 4h, 8h, 24h and 72hafter SE. XIAP, Smac, HtrA2, XAF1 and activated caspase-3 proteinwere examined using immunohistochemistry; Western blot was used todetect the protein levels of XIAP,Smac,HtrA2 and activated caspase-3;XIAP mRNA were examined using semiquantitative RT-PCR; Apoptoticneurons were examined using TUNEL staining; Nissl staining wasperformed for the evaluation of hippocampal neuronal loss.Results:1. The lithium-pilocarpine model of SE:After injections of lithium-pilocarpine, 86.6% of the rats developed SE, 10.0% of the rats died. The achievement ratio of pilocarpine-inducedrat SE model was 76.6%.2. Expressions of XIAP mRNA and protein: XIAP immunoreactivitywas localized in the perinuclear region of cytoplasm within hippocampalneuron in control group. XIAP immunoreactivity exhibited a diffusedistribution within the neuron after SE. Compared with the control group,the expression of CA1/CA3 XIAP protein in the SE group was increasedgradually since 2h, and reached a peak at 8h (P＜0.01), then decreasedobviously at 72h. The expression of CA1/CA3 XIAP protein at 2h and 4hhad no significant differences between the quercetin group and the SEgroup (P＞0.05), but increase in the expression of CA1/CA3 XIAPprotein was found in quercetin group at 8h and 24h (P＜0.05, P＜0.01).Western blot showed that there were no significant differences in XIAPprotein levels between the control group and the SE group. Comparedwith the control group, the XIAP mRNA levels revealed increase at 2h,4h and 8h (P＜0.01), and then decreased at 24h in the SE group. Therewere no significant differences in the XIAP mRNA levels at 2h and 4hbetween the quercetin group and the SE group (P＞0.05), but increase inthe XIAP mRNA levels was found in quercetin group at 8h and 24h (P＜0.05, P＜0.01).3. Expressions of Smac protein: The immunohistochemical analysisshowed the amount of Smac within hippocampal neurons was very lower in control group. Smac immunoreactivity exhibited a diffuse distributionwithin the neuron after SE. The expression of CA1/CA3 Smac proteinwas increased generally from 2h to 24h, and decreased at 72h in the SEgroup. Western blot analysis showed a significant increase in Smacprotein levels from 2h after SE.4. Expressions of HtrA2 protein: The expression of HtrA2 proteinwas very lower within hippocampal neurons in control group, whereasdiffuse pattern of HtrA2 expression was observed in the neuronsfollowing SE. There was increased HtrA2 expression from 2h to 24h, andreduced at 72h in the CA1and CA3 of the SE group. The same result wasshowed by Western blot analysis of HtrA2 protein levels.5. Expression of XAF1 protein: The expression of XAF1 proteinwas very lower within hippocampal neurons in control group, whereasthere was increase of CA1/CA3 XAF1 protein expression from 2h to 24h,and reduction at 72h in the SE group.6. Expressions of activated caspase-3 protein: There was lack ofimmunoreactivity for activated caspase-3 in control group. Theexpression of CA1/CA3 activated caspase-3 protein revealed significantincrease from 4h to 24h, and reduction at 72h in the SE group. Therewere no significant differences in the expression of activated caspase-3protein in the CA1 and CA3 at 2h and 4h between the quercetin groupand the SE group(P＞0.05), but decrease in the expression of activated caspase-3 protein was found in the quercetin group at 8h, 24h and 72h (P＜0.05 and P＜0.01). Western blot analysis of the activated caspase-3protein levels showed significant increase from 4h to 72h in SE group (P＜0.01).7. In situ apoptotic cells detection: No TUNEL positive cell wasfound in CA1 and CA3 of the control group. The number of CA1/CA3TUNEL positive cells increased from 4h after SE, reached a peak at 24h,and decreased at 72h after SE. No significant differences were found inthe number of CA1/CA3 TUNEL positive cells at 2h and 4h between thequercetin group and the SE group(P＞0.05). but the number of CA1/CA3TUNEL positive cells in the quercetin group was fewer than in the SEgroup at 8h, 24h and 72h (P＜0.05, P＜0.01).8. Analysis of hippocampal neurons loss: Compared with the controlgroup, the number of CA1/CA3 surviving neurons was decreasedgradually in the SE group from 4h to 72h (P＜0.01). The number of CA1/CA3 surviving neurons was reduced in the quercetin group from 8h to72h compared with the control group(P＜0.01), but was more than in theSE group from 8h to 24h (P＜0.01).Conclusions:1. XIAP, Smac, HtrA2 and XAF1 are involved in the regulation ofneuronal apoptosis and implicated in pathophysiological mechanisms ofneuronal damage after SE. 2. Quercetin have a protective effects on SE-induced neuronalinjury.3. Quercetin may exert its neuroprotective effects by up-regulatingthe expression of XIAP.4. XIAP may be a potential therapeutic target for the neuroprotectivetreatment of brain injury induced by SE.