| Background and ObjectiveEpilepsy is one of the most common chronic disease of the brain. Seizures seriously affected the quality of life of patients and their families. And also take the society a significant negative impact. But so far its pathogenesis is not fully understood. Animal models and clinical studies have shown that prolonged seizures and status epilepticus can cause multiple regions of the brain nerve cell apoptosis(neuronal apoptosis). Hippocampal neurons’ damage is most evident.Omi/HtrA2(high-temperature requirement serine protease A2) is a mitochondrial serine protease, which belongs to the pro-apoptotic factor.It can involve in apoptosis by cysteinyl aspartate specific protease caspase dependent pathway or a non-caspase-dependent pathway. Under normal physiological conditions, mature Omi/HtrA2 stored mainly in the mitochondria, membrane gap between the outer membrane. Studies have shown that when cells are stimulated to produce stress response, apoptosis-inducing factor increase the outer membrane permeability. Omi/HtrA2 is released into the cytoplasm, binding and breaking up IAPs(inhibitor of apoptosis protein). This will relieve the inhibition of caspase-3,7,9 by IAPs and bringthe activation of Caspase cascade reaction, which induces apoptosis. Meanwhile, when a variety of apoptotic stimulate cells, Omi/HtrA2 through its proteolytic activity, may degrade or clear the HAX-1(mitochondrial anti-apoptotic proteins), beginning a non-activated caspase-dependent apoptosis way.Omi/HtrA2 serine protease inhibitor(undetermined certain factor, Ucf-101) is Omi/HtrA2 specific inhibitors, which by inhibiting its activity, blocking Omi/HtrA2 of caspase caspase dependent apoptosis pathway and non-caspase dependent apoptosis pathway, thereby it can play a role in inhibiting apoptosis. Previous studies showed that, Ucf-101 have a protective effect on the heart, lung, kidney and other organ cells, and also reduce neuronal apoptosis by cerebral ischemia- reperfusion and neurodegenerative diseases caused,.but its role of the hippocampus in epilepsy rats at home and abroad has been reported rarely.In our study, through the establishment of lithium chloride-pilocarpine induced seizures model, we use HE, Nissl, TUNEL and immunohistochemistry staining to study the hippocampal tissue injury and apoptosis. We observe the influence of Ucf-101 on hippocampal neuron apoptosis, and detect Omi/HtrA2, caspase-9 and HAX-1 levels to explore the of the possible mechanisms by Western blot.Materials and methods160 healthy male rats were randomly divided into seven groups,which weight is 200-250 g and age 6 weeks. These groups include CON group, PILO, PILO+DMSO group, PILO+Ucf-101(0.7umol/kg) group and PILO+Ucf-101(1.5umol/kg) group. PILO group were divided into 2 h, 6h, 12 h, 24 h group among them. Each group included 20 SD rats. All rats were previously injected with lithium chloride(127mg/kg) In the abdominal cavity.After twenty hours later, We injected pilocarpine(30mg/kg) into the abdominal cavity of rats. Before 30 min of the injection of pilocarpine, all the rats were given the subcutaneous injection of the scopolamine, which were used to antagonize the peripheral chonlinegic response of pilocarpine. PILO+Ucf-101 Low dose group and the PILO+Ucf-101 high doses need to intraperitoneal injection of Ucf-101(0.7umol/kg, 1.5umol/kg) befor the injection of pilocarpine. PILO+DMSO group was injected with an equal volume of DMSO instead of Ucf-101.CON group was injected with an equal volume of saline instead of pilocarpine. According to Racine seizure classification standards to Ⅳ or Ⅴ level is successful modeling. After SE continued 60 min, we terminate seizures of the rats by intraperitoneal injection of diazepam(10mg/kg). We recorded the epileptic seizures success rate of each group of rats, survival rate and latency. We taken out brains of rats,which were Half of the total number of successful and viable model rats of CON group, PILO 24 h group, PILO+DMSO group,PILO+Ucf-101(0.7umol/kg) group and PILO+Ucf-101(1.5umol/kg) group.They were used for the HE, Nissl and TUNEL and Immunohistochemistry staining to detect the hippocampal injury and apoptosis after SE. The remaining rats were removed bilateral hippocampal tisse. It was stored at-80 degrees in the freezer. We used Western blot and Immunohistochemistry staining to detected the expression of the Omi/HtrA2、caspase9 and HAX-1. Results1 Behavioral observation : rats in CON don’t have any seizures. PILO group, PILO+Ucf-101(0.7umol/kg) group and PILO+Ucf-101(1.5umol/kg) group have seizures and the seizure grade of them is up to Ⅳ/ Ⅴgrade. And each group and subgroup of rats epileptic seizure latency has no significant difference(P> 0.05).2 HE staining results: Compared with the CON group, in PILO 24 h subgroup, PILO+Ucf-101 low-dose group and Ucf-101 high dose group, the number of hippocampal neurons were reduced(P<0.05). Compared with the PILO 24 h subgroup, the number of hippocampal neurons in PILO+DMSO group were reduced(P>0.05). Compared with the PILO 24 h subgroup, the number of hippocampal neurons in PILO+Ucf-101 low-dose group and Ucf-101 high dose group were increased(P<0.05). Compared with the PILO+Ucf-101 low-dose group, the number of hippocampal neurons in Ucf-101 high dose group were increased(P <0.05).3 Nissl staining results: Compared with the CON group, the number of hippocampal neurons in PILO 24 h subgroup, PILO+Ucf-101 low-dose group and Ucf-101 high dose group were reduced(P<0.05). Compared with the PILO 24 h subgroup, the number of hippocampal neurons in PILO+DMSO group were reduced(P>0.05). Compared with the PILO 24 h subgroup, the number of hippocampal neurons in PILO+Ucf-101 group were increased(P<0.05). Compared with the PILO+Ucf-101 low-dose group, the number of hippocampal neurons in Ucf-101 high dose group were increased(P<0.05).4 TUNEL staining results: Compared with the CON group, the number of TUNEL-positive cells in PILO 24 h subgroup, PILO+Ucf-101 low-dose group and Ucf-101 high dose group were increased(P<0.05). Compared with the PILO 24 h subgroup, the number of hippocampal neurons in PILO+DMSO group were increased(P>0.05). Compared with the PILO 24 h subgroup, the number of hippocampal neurons in PILO+Ucf-101 group were reduced(P<0.05). Compared with the PILO+Ucf-101 low-dose group, the number of hippocampal neurons in Ucf-101 high dose group were reduced(P<0.05).5 Immunohistochemistry staining results: For Omi/HtrA2 and caspase9, compared with the CON group, the expression in PILO 24 h subgroup, PILO+DMSO group, PILO + Ucf-101 low-dose group and Ucf-101 high dose group were reduced(P <0.05).; Compared with the PILO 24 h subgroup, tthe expression in PILO + Ucf-101 group were reduced(P<0.05); Compared with the PILO+Ucf-101 low-dose group, the expression in Ucf-101 high dose group were reduced(P<0.05). Compared with the PILO group, the expression in PILO+DMSO group were increased(P>0.05).For HAX-1, Compared with the CON group, the expression in PILO 24 h subgroup and PILO+DMSO group were reduced(P<0.05); Compared with the PILO 24 h subgroup, the expression in PILO+Ucf-101 group were increased(P<0.05). Compared with the PILO+Ucf-101 low-dose group, the expression in Ucf-101 high dose group were increased(P<0.05);Compared with the PILO group, the expression in PILO+DMSO group were reduced(P>0.05).6 Western blot analysis: Compared with CON group, in the expression of Omi/HtrA2 and caspase9 rise 2h after SE(P<0.05) and 24 hours reached the peak(P <0.05), while HAX-1 rise 2h(P<0.05) after SE, 8h reached the peak(P<0.05) and then decreased in the PILO 24 h subgroup. Compared with PILO 24 h subgroup, the expression of Omi/HtrA2 and caspase9 decreased(P<0.05) in PILO 24 h subgroup, and the expression of HAX-1 significantly increased(P<0.05). Compared with PILO+Ucf-101 low-dose group, the expression of HAX-1 in Ucf-101 high dose group significantly increased(P<0.05) and the expression of Omi/HtrA2 and caspase9 decreased(P<0.05). Compared with the PILO group, the expression of Omi/HtrA2 and caspase9 in PILO+DMSO group were increased(P>0.05), and the expression of HAX-1 were reduced(P>0.05). Conclusion1 Omi/HtrA2 and caspase9 increased continuously in 24 hours. HAX-1 inceresed in early time, decreased after 6 hours, instructing that epilepsy induce the endoplasmic reticulum stress.2 Ucf-101 can reduce the apoptosis of hippocampal neurons of epileptic rats., and it has a protective effect on brain injury caused by epilepsy. We thought the undelying mechanism were inhibiting the expression of Omi/HtrA2 and caspase9, increasing the expression of HAX-1.3 The neuroprotection of Ucf-101 was dose-dependent. |
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