Regulation Of High-mobility Group Box-1on P-glyco-Protein Expression In The Epileptic Seizure Rats Or Mice

Background:Over-expression of P-glycoprotein (P-gp) in brain encoding by multidrug resistance-1(mdrl) is one of the major formation mechanism of Drug-Resistant Epilepsy (DRE). Recent studies showed that inflammation as a common risk factors may be associated with the occurrence and development of various epilepsy, and induced the expression of P-gp by relavant inflammatory epilepsy. High-mobility group box-1(HMGB1) as a major inflammatory activation factor acting on the toll like receptor4(TLR4), one of the pattern recognition receptor, mediated immune inflammation, reduced the threshold of epileptic seizures, and aggravated seizures, which may be associated with the formation of DRE. Research in other diseases confirmed that the TLR receptor activation play an important role in the classic nuclear factor kappa B(NF-icB)signaling pathways; the activation of NF-κB mediated the formation of acute sezures or chronic epilepsy, and associated with the over-expression of P-gp in epilepsy; HMGB1also can promote the over-expression of P-gp in gastric adenocarcinoma. However, the relationship of high HMGB1expression between P-gp over-expression, and the role of HMGB1/TLR4/NF-κB Signaling pathway in the epileptic brain regulating seizure-induced P-gp expression has not been fully elucidated. Part I Effect of high-mobility group box-1and the antagonistof BoxA on P-glycoprotein expression in thehippocampus of epileptic seizure ratsObjective:To investigate the regulation of HMGB1and BoxA on P-gp expression in the hippocampus of epileptic seizure rat.Methods:Male SD rats were randomly divided into sham operation group, epilepsy group, low, medium and high doses of HMGB1pretreated groups with12rats in each group, and low, medium and high doses of BoxA pretreated groups with6rats in each group. Various doses of recombinant HMGB1protein (10,100,1000ng) and BoxA (14,140,1400ng) were injected intracerebroventricularly15min before micro-injection of kainic acid(kainic acid, KA,3μl,0.5μg/μl) into the hippocampus induced seizure rats respectively. The latency period to the onset of stage Ⅲ seizure time was observed to assess the epileptic susceptibility and all the rats were sacrificed after24hours of the operation. The damage of the hippocampal tissue, the expression levels of mdrl mRNA and P-gp were detected by immunohistochemical method, real-time PCR and Western blot respectively.Results:The epileptic seizure latency in medium and high doses of HMGB1pretreated groups were lowed than that of the epilepsy group (P<0.05). Compared with epilepsy group, the damage of the hippocampal tissue and neuron loss in hippocampus CA3area were increased (P<0.05), and the expression levels of mdrl mRNA and P-gp were up-regulated in medium and high doses of HMGB1pretreated groups(P<0.05), but there is no significant differences between the low-does HMGB1pretreated groups and epilepsy group. Compared with epilepsy group, the expression levels of mdr1α/b mRNA and P-gp were down-regulated in medium and high doses of BoxA pretreated groups(P<0.05), and mdr1α mRNA were also down-regulated in low-does BoxA pretreated groups(P<0.05), but the expression levels of mdr1b mRNA and P-gp in low-does BoxA pretreated groups have no significant differences between epilepsy group.Conclusions:HMGB1could increase the seizure susceptibility, the damage of hippocampal tissue and the expression levels of P-gp in the hippocampus of seizure rats, and regulate the P-gp over-expression in the brain of epileptic seizure rats.Part Ⅱ Regulation of HMGB1/TLR4/NF-κB Signaling pathways on P-glycoprotein expression in the hippocampus of epileptic seizure miceObjective:To investigate the regulation of HMGB1/TLR4/NF-κB Signaling pathway on seizures, brain damage, and P-gp expression in the hippocampus of epileptic mice.Methods:1. Acute seizure mice:Male C57BL/6mice were randomly divided into sham operation group, epilepsy group, HMGB1pretreated groups, BoxA pretreated groups with12mice in each group, and HMGB1pretreated wild type, TLR4gene knock out mice with6mice in each group. Recombinant HMGB1protein (1μl,5.5μg/μl) or BoxA (1μl,2.5μg/μl) were injected into the hippocampus15min before micro-injection of KA (0.5μl,0.014μg/μl) into the same places induced acute seizure mice respectively. Mice were sacrificed after24hours of the operation. The damage of the hippocampal tissue was detected by immunohistochemical method, the expression levels of HMGB1, TLR4, IKKβ, p-65, p-p-65, P-gp were detected by Western blot respectively.2. Chronic epileptic mice:Male C57BL/6mice were randomly divided into sham operation group, epilepsy group, HMGB1pretreated groups, BoxA pretreated groups with6mice in each group. Micro-injection of KA (0.05μl,4μg/μl) into the hippocampus induced chronic epileptic mice. after14d, Recombinant HMGB1protein (1μl,5.5μg/μl) or BoxA (1μl,2.5μg/μl) were injected into the hippocampus respectively. Mice were sacrificed after24hours of the operation, the expression levels of p-p-65, P-gp were detected by Western blot respectively.Results:1. In acute seizure mice compared with epilepsy group, the damage of the hippocampal tissue and neuron loss in hippocampus CA1area were increased (P<0.05), and the expression levels of HMGB1, TLR4, IKK β, p-65, p-p-65, P-gp were up-regulated in HMGB1pretreated groups respectively (P<0.05). Compared with epilepsy group, the hippocampal tissue and neuron loss in hippocampus CA1area were not increased, and the expression levels of HMGB1, TLR4, p-p-65, P-gp were down-regulated in BoxA pretreated groups respectively (P<0.05). Compared with wild type mice, the expression levels of p-p-65, P-gp was down-regulated in TLR4gene knock out mice2. In chronic epileptic mice compared with epilepsy group, the expression levels p-p-65, P-gp were up-regulated in HMGB1pretreated groups(P<0.05), p-p-65was down-regulated in the BoxA pretreated groups(P<0.05), but the expression of P-gp has no significant differences P-gp down-regulated between the BoxA pretreated groups and epilepsy group.Conclusions:HMGB1could trigger the TLR4activation, and induce the NF-κB signaling pathway to regulate the P-gp over-expression in the brain of epilepsy mouse.