The Effect Of Green Tea Polyphenols On The Expression Of XIAP And Caspase-3in Hippocampus Of Epileptic Rats

Objective: Epilepsy is a common chronic neurological disorder,characterized by cerebral temporary dysfunction resulting from abnormaldischarge of cerebral neurons. The neurobiological changes of epilepsy areneuronal death, glial cell proliferation, and synaptic plasticity. Neuronal deathis the most prominent feature of the early stages of epilepsy. Seizures cancause neuron damage has been widely confirmed; therefore exploring newneuroprotective agent has important clinical value. Apoptosis, also known asprogrammed cell death, is controlled by a very fine regulation, which is one ofthe important ways of neuronal cell death after epilepsy. Epilepsy can increasethe release of excitatory amino acids and activate the postsynaptic membraneN-methyl-D-aspartate (N-methyl-D-asparate, NMDA) receptor, promoting theopening of calcium channel and the calcium massive influx and ultimatelyleading to intracellular calcium overload. In addition, the massive influx ofcalcium ions can activate nitric oxide synthase (NOS) and promote thegeneration of reactive oxygen species, ultimately leading to programmed celldeath.X-Linked Inhibitor of Apoptosis Protein (XIAP) is the most potentendogenous inhibitor of apoptosis in the family of Inhibitor of ApoptosisProteins (IAPs). It can play a neuroprotective role in the negative regulation ofapoptosis by inhibiting the enzymatic activity of caspase-3in a variety ofbrain injury diseases. Green tea polyphenols (GTPs) is kind of polyphenoliccompounds extracted from green tea, which is easy to reach the centralnervous system through the Blood-brain barrier. Recently, green teapolyphenols have been found with functions in ant-ioxidant, anti-apoptotic,anti-inflammatory and it has been an important nerve cell protective agent.Researches have also demonstrated that green tea polyphenols could showneuroprotective effects on Parkinson’s disease and Alzheimer disease, but whether it can play the role in the impaired hippocampal neurons in epilepsyhas not been reported. In view of it, chronic epileptic rats model kindled bypentylenetetrazol (PTZ) were established and GTPs was administrated. Weobserved the alteration of behavior and the hippocampal neuronal injury inrats, and the expression of XIAP and caspase-3protein in hippocampusthrough the methods of Western Blot and Immunohistochemistry, forexploring the neuroprotective effects of green tea polyphenols in epilepsy andthe potential mechanisms.Methods:36clean grade adult male Wistar rats (7-8weeks) wererandomly divided into three groups consisting of12animals each.They are thenormal control group (NS group), epilepsy group (PTZ group)，and green teapolyphenols group(GTPs group). PTZ and GTPs were dissolved inphysiological saline. The administration work was conducted between08:30-09:30AM (15injections total).The PTZ group: PTZ was injectedintraperitoneally every day in a dose of35mg/kg; The NS group: The ratswere received0.9%saline (3.5ml/kg) every day; The GTPs group: The ratswere Received GTPs pretreatment in doses of50mg/kg in addition to PTZ(35mg/kg). GTPs were given30min before PTZ. Animals were observed for1hour after each injection, seizure activity was observed for scored accordingto Racine.24h after the last administion,6rats in each group were perfusedand the brains were removed. HE staining was performed to observe theNeuron damage in hippocampus in rats. The expression of XIAP andcaspase-3protein in the CA1region of the hippocampus was determinedthrough the methods of Immunohistochemistry.6rats in each group weresacrificed and the hippocampi were quickly separated. The expression ofXIAP and caspase-3protein in hippocampus was determined through themethods of Western Blot.Results:1.Results of behavior: After repeated administration of PTZ(35mg/kg) by intraperitoneal injection, the seizure grade of the rats in the PTZgroup and GTPs group gradually increased and ultimately achieved grade Ⅳ-Ⅴ. No seizures were found in the normal group.Compared with the PTZ group, the latency to generalized tonic-clonic seizure (GTCS) in the GTPsgroup was prolonged significantly; The duration of GTCS was shortenedsignificantly (p<0.05).2. Results of HE staining: The NS group: Neurons in hippocampus werearranged in neat rows with more levels, normal morphology, transparentcytoplasm, uniformly distributed chromatin and clearly nucleolus. There wasno significantly neuron loss; The PTZ group: Neurons in hippocampus werearranged scattered with unclear boundaries, increased gap and obvious neuronloss.There was showed acidophilic neurons manifested as cell shrinkage,chromatin condensation and nuclear pyknosis; The GPTs group: Neurons inhippocampus tended to be with normal morphology and still neat arrangement.Compared with the PTZ group, the number of surviving cells increasedsignificantly with only a small amount of acidophilic neurons.3. Result of Western blot: The expression of XIAP protein in PTZ groupwas decreased obviously compared to NS group (P<0.05). The expression ofcaspase-3protein in PTZ group was increased obviously compared to NSgroup (P<0.05). Compared to PTZ group，the protein level of XIAP in theGTPs group was increased significantly; The protein levels of caspase-3weredecreased significantly (p<0.05).4. Result of Immunohistochemistry: The mean optical density of positiveXIAP protein in the CA1region of the hippocampus in PTZ group wasdecreased obviously compared to NS group (P<0.05). The mean opticaldensity of positive caspase-3protein in the CA1region of the hippocampus inPTZ group was increased obviously compared to NS group (P<0.05).Compared to PTZ group，the mean optical density of positive XIAP protein inthe CA1region of the hippocampus in the GTPs group was increasedsignificantly; The mean optical density of positive caspase-3protein weredecreased significantly (p<0.05).Conclusions:1. This study successfully established the chronic epilepticrat model kindled by PTZ. GTPs could effectively inhibit PTZ-inducedseizures by increasing the latency to GTCS and decreasing the duration of GTCS.2. Epilepsy could make anti-apoptotic protein XIAP reduced andapoptosis-executed protein caspase-3increased.3. GTPs could increase the level of XIAP protein and decrease the levelof caspase-3protein. Thus, we speculate that GTPs probably play aneuroprotective role in epilepsy by inhibiting apoptosis.