Effects Of Compatibility Of Rhei Radix Et Rhizoma-Scutellariae Radix On Epilepsy After Traumatic Brain Injury

Objective:A closed brain injury model was established to investigate the effect of compatibility of Rhei Radix et Rhizoma-Scutellariae Radix on early stage epilepsy of traumatic brain injury and its mechanism of action,and provide a theoretical basis for clinical prevention of epilepsy after traumatic brain injury.Method:SPF-grade C57BL/6J male mice were randomly divided into 5 groups,blank control group,model group,Rhei Radix et Rhizoma-Scutellariae Radix low dose treatment group(1 g·kg-1),high dose group(4 g·kg-1),positive control group(2.6 g·kg-1 sodium valproate).A mouse model of closed brain injury was established by the weight-falling method in all test groups except for the blank control group,and the mice were given the corresponding drug gavage after mice brain injury for successively 7 days.Subacute dose of pentylenetetrazole(PTZ)was given to the mice after the last administration.1.HPLC analysis:HPLC was established to determine the content of baicalin,baicalein,aloe-emodin,and emodin in compatibility of Rhei Radix et Rhizoma-Scutellariae Radix,and preliminary quality control for the compatibility of Rhei Radix et Rhizoma-Scutellariae Radix.2.Behavioural methods:The epilepsy seizures were observed in each group of mice after the administration of epileptic agents.The number of mice with tonic-clonic seizures,seizure latency and average seizure intensity in each group were recorded.3.Electroencephalogram(EEG)analysis:The brain discharge pattern of epilepsy was recorded by cortical EEG.The Clutter interference was filtered out,and the sum of brain discharges from 0 to 20 Hz was calculated.4.Immunofluorescence(IF):The positive cells of glial fibrillary acidic protein(GFAP)in hippocampal DG area were counted by immunofluorescence staining of tissue sections,and the fluorescence density was compared.5.Real-time PCR:Quantitative PCR was used to detect the expression levels of IL-1? mRNA and TNF-a mRNA in hippocampus of mice in each group.6.Western blotting:detection of GFAP,inflammatory cytokines(TNF-a,IL-1?),apoptosis protein(Caspase-3,Bax,Bcl-2)and neuronal specific proteins Na+-K+-2Cl-cotransporter-1(NKCC1)expression levels in hippocampus.Result:1.HPLC:The liquid chromatographic pattern analysis showed that the content of baicalein was slightly higher than that of Scutellariae Radix after the compatibility of Rhei Radix et Rhizoma-Scutellariae Radix,and there was no significant change in the content of the other three components.The results of the assay showed that there was no obvious difference in the contents of emodin,aloe-emodin,baicalin,and baicalein in the same batch of samples after treated by the same extraction method.2.Behavior:The mice in the blank group had partial convulsive behavior and no obvious abnormalities.Most of the mice in the model group showed large-scale episodes of 4 to 5 grades,while the rest of the mice in the administered groups exhibited repeated episodes of 2 to 4 grades.As Compared with the model group,the latency of seizures in each drug treatment group was significantly prolonged(P<0.01),the number of mice with tonic-clonic seizures was significantly reduced within 30 min(P<0.01),and the degree of seizures was also significantly reduced.There was no significant difference between the high-dose group and the sodium valproate control group(P>0.05).3.Electroencephalogram:The electroencephalogram of the blank group showed normal,mainly based on the basic wave,no epileptic discharge;however,the electroencephalogram of the mouse in the model group showed a clear waveform of epileptic seizure discharge.The electrical signals showed spikes,sharp waves and slow waves with abnormally high frequency.As Compared with the blank group,the differences were significant(P<0.001).The compatibility of Rhei Radix et Rhizoma-Scutellariae Radix had different degrees of spikes in the low and high dose groups,and epileptic waves appeared in the high-dose group after a long incubation period.Compared with the model group,the epileptic wave frequency was significantly reduced(P<0.001).The mice treated with sodium valproate had occasional epileptic discharges,which was not statistically significant compared with the high-dose group(P>0.05).According to the statistics of the total brain discharge from 0 to 20 Hz,the results showed that the electroencephalogram discharges of the 5 groups ranged from large to small were model group>Rhei Radix et Rhizoma-Scutellariae Radix low dose group>Rhei Radix et Rhizoma-Scutellariae Radix high dose group>sodium valproate group>blank control group.4.Immunofluorescence:Compared with the model group,the GFAP immunoreactivity of the astrocytes in the hippocampal DG area of mice after intervention with Rhei Radix et Rhizoma-Scutellariae Radix was weakened,the number of positive cells was reduced,and the fluorescence density was also decreased.After statistical comparison,there was a significant difference between the drug treatment group and the model group(P<0.05).As Compared with the blank group,there was no significant difference in the number of GFAP positive cells between the Rhei Radix et Rhizoma-Scutellariae Radix high-dose group and the positive control group(P>0.05).5.Real-time PCR:The absolute quantification of TNF-a mRNA and IL-1? mRNA in the hippocampus of the 5 groups of mice was compared.As Compared with the blank control group,the expression of TNF-a mRNA and IL-1? mRNA in the hippocampus of the model group increased obviously(P<0.01);there was no significant change among the high-dose group,the positive control group and the blank control group(P>0.05).Compared with the model group,the expression of TNF-a mRNA and IL-1? mRNA in the drug treated group and the positive control group was decreased(P<0.05).6.Western blot:The expression of GFAP,IL-1?,TNF-a,Caspase-3,Bax and NKCC1 was detected by Western blotting.The protein expression of the model group was significantly higher than that of the blank group(P<0.01).The Rhei Radix et Rhizoma-Scutellariae Radix low-dose group,high-dose group and sodium valproate group were significantly lower than the model group(P<0.05).There was no significant difference between the high-dose group and the sodium valproate group(P>0.05).Conclusion:1.The compatibility of Rhei Radix et Rhizoma-Scutellariae Radix can prolong the seizure latency of mice with epilepsy in early stage of traumatic brain injury,reduce the level of seizures,inhibit the amplitude of epileptiform discharge,and decrease the frequency of discharges.2.The compatibility of Rhei Radix et Rhizoma-Scutellariae Radix can effectively inhibit the increase of GFAP positive cells in hippocampal DG area in mice with early stage epilepsy after traumatic brain injury,thus inhibiting the activation and proliferation of astrocytes.3.The compatibility of Rhei Radix et Rhizoma-Scutellariae Radix can inhibit the release of inflammatory mediators and apoptosis-related factors by regulating the corresponding factors,so as to improve the secondary brain dysfunction caused by epilepsy in the early stage of brain injury,In order to achieve the purpose of neuroprotection.