A Study On The Protective Effection And Its Mechanism Of Baicalin On Hippocampal Neurons After Status Epilepticus In Mice

Part I Neuroprotective effect and its potential mechanism of Baicalin onhippocampal neurons after status epilepticus in mice（I）Objective This investigation was performed to explore the neuroprotective effectand its potential mechanism of Baicalin on hippocampal neurons after status epilepticusinduced by kainic acid in miceMethods132ICR male mice(weighing22–28g) were randomly divided intothree groups: sham control(n=44), status epilepticus(SE, n=44)and baicalin group(n=44).，to which baicalin was administered at doses of100mg/kg. To establish animalmodel of status epilepticus,0.1μg/5μl kainic acid(KA) was injected into theintracerebral ventricle of the mouse．The mice were then received intraperitonealinjection with baicalin (100mg/kg) or vehicle. Hematoxylin and Eosin(HE) staining wasused to observe the pathological changes and neuronal apoptosis was determined byTUNEL staining and the cleaved caspase-3protein expression in hippocampus area72hafter SE in mice. In addition, immunohistochemistry and western blot were used todetect the expression of XIAP protein expression6h、12h、24h after SE in mice,respectively.Results①To observe by HE staining: About72h after the onset of SE, mostneurons in the Hippocampal CA1and CA3subfields (without baicalin treatment),appeared shrunken with eosinophilic cytoplasm and triangulated pyknotic nuclei. Incontrol group, there was no neuronal damage. Baicalin (100mg/kg) reduced thenecrotic or seizure induced neuronal injury in the same areas.②TUNEL stainingshowed that the hippocampal CA3region of Sham group had a few tan TUNEL positivecells, but the tan TUNEL positive cells significantly increased in hippocampal CA3region of SE group. Compared with the SE group, Baicalin treatment group CA3regionTUNEL positive cells significantly attenuated（P＜0.05）.③Western blot showed thatthe expression of the protein1evel of cleaved caspase-3of Sham group were weak, butits increased significantly after status epilepticus. Compared with the SE group, Baicalin treatment group could obviously down-regulate the expression of cleaved caspase-3protein (P<0.05).④Baicalin significantly relieved the hippocampal injury after SE inmice；compared with the control group, the expression of CA3XIAP protein in the SEgroup was increased gradually since6h after SE，and reached the peak at12h，decreasedat24h.The difference was statistically significant(P<0.01). Compared with the SE group,the expression of XIAP protein was increased at6h,12h,24h in baicalin group(P<0.05).Conclusion Baicalin could significantly relieve neuronal apoptosis to protecthippocampal neurons after status epilepticus in mice； The protective effect ofBaicalin on the hippocampus after SE in mice,which might be associated with theupregulated expression of XIAP and down-regulated expression of cleaved caspase-3proteinPart II The neuroprotective mechanism of Baicalin on hippocampal neuronsafter status epilepticus in mice（II）Objective This investigation was performed to explore the neuroprotectivemechanism of Baicalin on hippocampal neurons after status epilepticus induced bykainic acid in mice.Methods Sixty ICR male mice were randomly divided into three groups: shamcontrol(n=20), status epilepticus(SE, n=20)and baicalin group (n=20).，to which baicalinwas administered at doses of100mg/kg. To establish animal model of status epilepticus,0.1μg/5μl kainic acid(KA) was injected into the intracerebral ventricle of themouse．The mice were then received intraperitoneal injection with baicalin (100mg/kg)or vehicle. Immunohistochemistry and western blot were used to detect the expressionof Bcl-2；quantitative reverse-transcription PCR（qRT-PCR）was used to quantify themiR-497expression in hippocampus.Results①Immunohistochemistry showed the Sham group had only a fewscattered yellow-brown positive cells of Bcl-2, while the SE group can be seennumerous and densely distributed positive cells. Compared with the SE group, thebrown granular positive cells of Baicalin treatment group increased significantly and their differences was obvious.②Western blot showed that the expression of the protein1evel of Bcl-2of Sham group were scant, but its increased significantly after statusepilepticus(P<0.01). Compared with the SE group, Baicalin could obviously up-regulatethe expression of Bcl-2protein (P<0.05).③qRT-PCR showed that the expression of themRNA1eveI of miR-497of Sham group were scant, but its increased significantly afterstatus epilepticus(P<0.05). Compared with the SE group, Baicalin could obviouslydown-regulate the expression of miR-497(P<0.05).Conclusion Our results suggest that baicalin possesses potent anti-apoptoticproperties, attenuates injury, and improves neurological outcomes in mice after SE,which might be associated with the down-regulated expression of miR-497, and theup-regulated expression of Bcl-2.