平肝止痫复方联合卡马西平干预HMGB1/TLR4信号炎症通路的相关作用研究

Effect of Pinggan Zhixian Compound(平肝止痫复方)Combined with Caramazepine on Intervention of HMGB1/TLR4 Signaling Inflammation Pathway

目的研究平肝止痫复方联合卡马西平对难治性癫痫作用及机制。方法海人酸注射于Wistar大鼠海马CA3区的造模方法制作癫痫模型,予0.03 g/(kg·d)苯妥英钠连续灌胃14 d后结合Racine分级及脑电图筛选成功制作难治性癫痫模型78只。将制作完成的癫痫大鼠随机分为模型组、平肝止痫复方组、CBZ组、CBZ+平肝止痫复方低、中、高剂量组(L、M、H),每组13只,再加假手术组7只,共7组。分组后给予假手术组、模型组灌胃生理盐水,剩余大鼠分别以CBZ 0.03 g/(kg·d),平肝止痫复方1.32 g/(kg·d),CBZ+平肝止痫复方低剂量(CBZ+L)、CBZ+平肝止痫复方中剂(CBZ+M)、CBZ+平肝止痫复方高剂量(CBZ+H)组分别给予:0.03+0.66 g/(kg·d),0.03+1.32 g/(kg·d),0.03+2.64 g/(kg·d),灌胃给药8周;观察各组大鼠治疗前后体质量、各致痫组大鼠治疗后发作级别变化、各致痫组大鼠治疗后防卫反应分级变化、各致痫组发作平均持续时间变化,并对各组大鼠治疗后进行脑电图监测;8周药物灌胃结束后股动脉取血,ELISA分别检测各组难治性癫痫大鼠血清TNF-α、IL-1β、IL-6的表达;RT-PCR、Western Blot检测大鼠海马HMGB1、TLR4基因和蛋白表达。结果治疗后大鼠体质量变化:与卡马西平组比较,CBZ+M、CBZ+H组体质量明显增加,差异具有显著统计学意义(P<0.01);CBZ+L组体质量增加,差异具有统计学意义(P<0.05)。各致痫组大鼠治疗后发作级别变化:CBZ+M、CBZ+H组与CBZ组比较,治疗后发作分级等级减少,差异有显著统计学意义(P<0.01)。各致痫组大鼠治疗后防卫反应分级:CBZ+M、CBZ+H组与CBZ组比较,药物灌胃后防卫反应等级减少,差异有显著统计学意义(P<0.01)。各致痫组大鼠治疗后癫痫发作平均持续时间:与卡马西平组比较,CBZ+M、CBZ+H组平均持续发作时间明显减少,差异有显著统计学意义(P<0.01);CBZ+L组平均发作持续时间减少,差异有统计学意义(P<0.05)。脑电图结果示CBZ+M、CBZ+H组均可显著抑制海人酸难治性癫痫大鼠的痫性放电;酶联免疫吸附实验(Enzyme-linked immunosorbent assay,ELISA)检测TNF-α表达:与卡马西平组比较,CBZ+M、CBZ+H大鼠血清TNF-α含量降低,差异有显著统计学意义(P<0.01),CBZ+L组大鼠血清TNF-α含量降低,差异有统计学意义(P<0.05)。IL-1β表达:同卡马西平组比较,CBZ+M、CBZ+H组大鼠血清IL-1β含量显著降低,差异有显著统计学意义(P<0.01)。IL-6表达:与卡马西平组比较,CBZ+M、CBZ+H组大鼠血清IL-6含量降低,差异有显著统计学意义(P<0.01);实时荧光定量PCR(Real-time fluorescent quantitative PCR,RT-PCR)检测各组大鼠海马组织中HMGB1mRNA相对表达量:同卡马西平组比较,CBZ+L、CBZ+M、CBZ+H组大鼠海马HMGB1mRNA相对表达量均下降,差异有显著统计学意义(P<0.01)。各组大鼠海马组织中TLR4mRNA相对表达量:同卡马西平组比较,CBZ+M、CBZ+H组大鼠海马TLR4mRNA相对表达量均下降,差异具有显著统计学意义(P<0.01),CBZ+L组TLR4mRNA相对表达量下降,差异有统计学意义(P<0.05);蛋白免疫印迹(Western Blot,WB)检测各组大鼠海马组织中HMGB1蛋白表达:同卡马西平组相比,CBZ+L、CBZ+M、CBZ+H组大鼠海马HMGB1的蛋白表达量均显著下降,差异具有显著统计学意义(P<0.01)。各组大鼠海马组织中TLR4蛋白表达:与卡马西平组比较,CBZ+M、CBZ+H组大鼠海马TLR4的蛋白表达量下降,差异具有显著统计学意义(P<0.01),CBZ+L组大鼠海马TLR4的蛋白表达量下降,差异具有统计学意义(P<0.05)。结论平肝止痫复方联合卡马西平能降低海人酸难治性癫痫大鼠海马组织HMGB1、TLR4mRNA的相对表达量,显著下调海马组织异常升高的HMGB1、TLR4蛋白表达量;平肝止痫复方联合卡马西平能降低难治性癫痫大鼠血清TNF-α、IL-1β、IL-6表达水平。提示平肝止痫复方联合卡马西平用药后可通过减少炎症因子释放,抑制胶质细胞活化,减少HMGB1释放,抑制HMGB1/TLR4炎症信号通路的传递,从而减少炎症因子释放,抑制神经元异常兴奋,达到保护神经元,发挥抗癫痫作用。

Objective To study the effect and mechanism of Pinggan Zhixian Compound(平肝止痫复方)combined with carbamazepine(CBZ)on refractory epilepsy.Methods The model of hippocampal CA3 region in Wistar rats was set up by injecting kainic acid.After 14 days of continuous intragastric administration of 0.03 g/(kg·d)phenytoin sodium,combined with Racine classification and EEG screening,78 refractory epileptic models were successfully established.The completed epilepsy rats were randomly divided into model group,Pinggan Zhixian Compound group,CBZ group,CBZ+Pinggan Zhixian Compound low-dose,medium-dose and high-dose groups(L,M,H),with 13 rats in each group and 7 rats in sham operation group,a total of 7 groups.After grouping,the sham operation group and model group were given normal saline intragastrically.The remaining rats were given CBZ[0.03 g/(kg·d)],Pinggan Zhixian Compound[1.32 g/(kg·d)],CBZ+Pinggan Zhixian Compound low-dose(CBZ+L)[0.03 g/(kg·d)+0.66 g/(kg·d)],CBZ+Pinggan Zhixian Compound medium-dose(CBZ+M)[0.03 g/(kg·d)+1.32 g/(kg·d)],and CBZ+Pinggan Zhixian Compound high-dose(CBZ+H)[0.03 g/(kg·d)+2.64 g/(kg·d)]for 8 weeks.The changes of body weight,seizure grade,defense response grade and average duration of seizure in each group were observed before and after treatment.EEG monitoring was performed after treatment.Blood samples were collected from the femoral artery after 8 weeks of drug intervention,and the expressions of TNF-α,IL-1βand IL-6 in serum of refractory epilepsy rats were detected by ELISA.The expressions of HMGB1 and TLR4 genes and proteins in rat hippocampus were detected by RT-PCR and Western Blot.Results Compared with those of the CBZ group,the body weights of CBZ+M and CBZ+H groups were increased significantly,and the difference was statistically significant(P<0.01).The body weight of CBZ+L group was increased,and the difference was statistically significant(P<0.05).Compared with those of the CBZ group,the grade of defense response after treatment in CBZ+M group and CBZ+H group after drug intervention was decreased,and the difference was statistically significant(P<0.01).Compared with that of the CBZ group,the mean duration of epileptic seizure after treatment in CBZ+M and CBZ+H groups was significantly decreased(P<0.01).The mean duration of attack was decreased in CBZ+L group,and the difference was statistically significant(P<0.05).EEG results showed that CBZ+M and CBZ+H groups could significantly inhibit the epileptic discharge in rats with refractory epilepsy.Compared with that of the CBZ group,serum TNF-αcontent in CBZ+M and CBZ+H group was decreased,and the difference was statistically significant(P<0.01).The serum TNF-αcontent in CBZ+L group was decreased,and the difference was statistically significant(P<0.05).Compared with that of CBZ group,the serum IL-1βcontent in CBZ+M and CBZ+H groups was significantly decreased(P<0.01).Compared with that of the CBZ group,the serum IL-6 content in CBZ+M and CBZ+H groups was decreased(P<0.01).Compared with that in CBZ group,the HMGB1 mRNA relative expression levels in the hippocampus of rats in CBZ+L,CBZ+M and CBZ+H groups were decreased(P<0.01).The relative expression level of TLR4 mRNA in the hippocampus of rats in each group:Compared with that of CBZ group,the relative expression levels of TLR4 mRNA in the hippocampus of rats in CBZ+M and CBZ+H groups were decreased with statistically significant differences(P<0.01).The relative expression level of TLR4 mRNA in the CBZ+L group was decreased with statistically significant differences(P<0.05).Compared with that of CBZ group,the protein expressions of HMGB1 in the hippocampus of rats in CBZ+L,CBZ+M and CBZ+H groups were significantly decreased with statistical significance(P<0.01).Compared with that of CBZ group,the protein expressions of TLR4 in the hippocampus of rats in CBZ+M and CBZ+H groups were decreased with statistically significant difference(P<0.01).The protein expression of TLR4 in the hippocampus of rats in CBZ+L group was decreased with statistically significant difference(P<0.05).Conclusion Pinggan Zhixian Compound combined with carbamazepine can reduce the relative mRNA expression levels of HMGB1 and TLR4 in hippocampus of rats with refractory epilepsy,and significantly down-regulate the abnormally elevated protein expression levels of HMGB1 and TLR4 in hippocampus.Pinggan Zhixian compound combined with carbamazepine can decrease the expression levels of TNF-α,IL-1βand IL-6 in serum of intractable epilepsy rats.It is suggested that Pinggan Zhixian compound combined with CBZ can reduce the release of inflammatory factors,inhibit the activation of glial cells,reduce the release of HMGB1 and inhibit the transmission of the HMGB1/TLR4 inflammatory signal pathway,thereby reducing the release of inflammatory factors and inhibiting the abnormal excitability of neurons,so as to protect neurons and play an anti-epileptic effect.