Effects Of FK506 On Oxidative Stress And Expression Of Active Caspase-3 In The Hippocampus Of Rats With Status Epilepticus

Background and Objective: Epilepsy is one of the most common neurological disorders that affect people of all ages worldwide. Morbidity rate for epilepsy is 5%o-6%o according epidemiologic survey. It is harm on people's healthy and life and has high fatality rate. Status epilepticus (SE) generally refers to the occurrence of a single unremitting seizure with a duration longer than 30 minutes or frequent clinical seizures without an interictal return to the baseline clinical state. SE is a medical emergency and results in subcellular changes that lead to neuronal damage and death in the hippocampus. Notably, oxidative stress and hippocampal neuronal cell death were observed during SE in many studies.Oxidative stress is defined as an imbalance between higher cellular levels and reactive oxygen species (ROS), such as superoxide radical, hydrogen peroxide and nitric oxide (NO) [5], and cellular antioxidant defense including superoxide dismutase (SOD) [6]. Brain tissue is particularly vulnerable to oxidative damage because of its high consumption of oxygen and the consequent generation of high quantities of free radical. Growing datas suggest that injury resulting from oxidative stress may play an important role in pathophysiology following acute neurological insults such as stroke and seizure [7-9].An immunosuppressive agent, FK506, has been used in clinical settings for the prevention of allograft rejection, which orchestrates its immunosuppressive action by binding to FK506 binding protein 12 (FKBP12). Recent studies in vitro and vivo have demonstrated that FK506 not only induces some neurological diseases such as focal cerebral ischemia, but also plays a role in neuroprotection and neurotrophy. In kainate-induced seizure, FK506 was shown to protect neuron through preventing the sprouting of mossy fibres in the hippocampus. In middle cerebral artery occlusion model, cortical damage was remarkably reduced by intravenous injection of FK506. Despite the protective effect of FK506 on neuron was observed in experimental cerebral ischemia and seizure model, the mechanism by which FK506 targets neuron remains unclear.Our current study was designed to establish lithium chloride-pilocarpin-induced SE (LPCSE) rat models to investigate whether and how oxidative stress participates in this process and the effects of tacrolimus on expression of nitric oxide (NO), nitric oxide synthase (NOS), malondialdehyde (MDA), superoxide dismutase (SOD) and active caspase-3, and also to reseach on anti-epilepsy mechanisms of FK506.Methods:1. Pilocarpine was intraperitoneally injected to make modles of SE. The rats were randomly divided into 4 groups (54 rats in each group, n=216):control group, pilocarpine group (pilo group), saline+pilo group and FK506 group. In FK506 group, the animals were further treated with FK506 (Fujisawa, Japan) (2mg/kg,i.p) 24h and 1h before pilocarpine administration. In addition, rats in saline+pilocarpine group were treated as FK506 group except that FK506 was replaced by the same dose of 0.9% saline. Pilocarpine group animals were treated with pilocarpine with no pretreament. Control group animals were treated as pilocarpine group with the same dose of 0.9% saline instead of pilocarpine. The animals of each group, which consisted of 7 subgroups (6 rats in each subgroups) with 0.5h,2h,6h,12h,1 day,3 days, and 7 days after treatment, were sacrificed for estimation of NO, NOS, MDA, SOD and immunoblotting.6 rats were killed at 6 h and 6 rats at 7 days for immunohistochemical techniques.2. The seizure activity of rats induced by KA was scored using a modified Racine scale.3. To evaluate pathology features of hippocampal neurons of each group by H&E staining with hematoxylin and eosin.4. To evaluate NO, NOS activity, MDA and SOD activity by using commercially available kits. As the activity of nNOS has not been measured directly, and it is reported that nNOS is the mainly isoform resulting in an increased of cNOS (constitutive nitric oxide synthase, including the isoforms of nNOS and eNOS) activity by immunoblotting, so cNOS activity was measured to reflect the activity of nNOS indirectly.5. To detect nNOS, iNOS and active caspase-3 immunoreactive positive cells in hippocampus by immunohistochemistry.6. Western blotting was used to measure the protein expressions of nNOS, iNOS and active caspase-3 at different time point and effect of FK506 on them.7. The results were expressed asχ±s, The SPSS13.0 statistic software was used to analyze the resultsResults:1. Behavioral episodes induced by pilocarpine injections showed typical increases in their intensity and duration, gradually progressing towards status epilepticus. The rats treated with FK506 showed highly significant differences in their latency period reaching to stage IV-V (P＜0.05) and the profile of proportions of rats suffering from seizures of different intensities. The latency period of the group with FK506 was significant longer and the percentage of animals reaching stage V was considerably lower compared with pilo or saline+pilo group (P＜0.05). Control animals did not exhibit any behavioral seizure activity.2. Pilocarpine injection intraperitoneally resulted in a loss of neuron in pyramidal cells of hippocampus in CA3 region. It has been observed in some neurons that cytoplasm appears heavy staining and nucleolus disappears with pyknotic nucleus. The surviving neuron numbers after SE were significantly decreased compared with that of control (P<0.05). FK506 significantly attenuated the neuron loss induced by seizures (P<0.05).3. NO level started to increase at 30min after SE and remained significantly elevated until 7d with two peaks at 6h and 7d (P<0.01, P<0.01, respectively). After pre-treatment with FK506, NO level at 6h and 7d induced by seizures decreased remarkably (all P<0.05).4. cNOS activation started to increase at 30min after SE, reached a maximum level at 6h (P<0.05, P<0.01, respectively). Differently, the iNOS activation did not increase until 12h, and attained the peak at 7d after SE (P<0.05, P<0.01, respectively). After pre-treatment with FK506, cNOS activation at 6h and iNOS activation at 7d decreased remarkably (all P<0.05).5. The MDA level started to increase at 30min after SE and reached a maximum level at 7d (P<0.05, P<0.01, respectively). Pre-treatment with FK506 could remarkably decrease MDA level at 7d (P<0.05).6. SOD activation started to increase at early stage (P< 0.05) with a peak at 2h after SE (P<0.01), and returned to basal level at 24h after SE (P>0.05). However, SOD activation started to increase again at 7d after SE (P<0.05). Pre-treatment with FK506 could increase SOD activation at 6h and 7d after SE (P<0.05).7. nNOS, iNOS and active caspase-3 positive cells was measured by immunohistochemistry. The results showed that nNOS positive cells appeared remarkably at 6h and iNOS and caspase-3 positive cells appeared significantly at 7d after SE. FK506 could inhibit expressiones of positive cells of nNOS, iNOS and active caspase-3.8. Western blotting was performed to the determine protein expression in nNOS, iNOS and caspase-3 at 2,6,72h and 7d. Compared with control group, protein expression of nNOS increased at 2h and attained to peak at 6h after SE (P<0.05, P< 0.01, respectively), but protein expression of iNOS reached to maximum at 7d after SE (P<0.01). Meanwhile, protein expression of active caspase-3 gradually increased from 72h to 7d after SE (P<0.05, P<0.01, respectively). FK506 could significantly inhibit protein expression of nNOS at 6h after SE (P<0.05), and protein expression of iNOS (P<0.05) and caspase-3 at 7d after SE(P<0.05).Conclusions: 1.NO meditated oxidative stress may have dual neurotoxic effects following SE: an early damage likely resulting from mitochondrial dysfunction and a prolonged apoptotic neuronal cell death involving in the activation of caspase cascade.2. Oxidative stress mediated hippocampal neuron death occurs prior to caspase 3 activation after LPCSE. The finding potentially explains the mechanism of early cell death after SE and suggests antioxidant based neuroprotective strategies for SE.3. The results suggest that FK506 protects hippocampal neurons from damage via suppressing oxidative stess and caspase-3 cascade at later stages after SE. The findings have implications for potential clinical use of FK506 against SE.