| ObjectiveTo explore the inhibitory effect of Wuling capsule combined with carbamazepine on rats with kainic acid(KA)-induced epilepsy,the effect of Morris water maze on the learning and memory function of rats,and the effect of hematoxylin-eosin staining(HE)on rats.The protective effect of hippocampal neurons on the pharmacokinetics of CBZ in refractory epilepsy rats by immunohistochemistry,Western blot and RT-PCR for detection of brain tissue and hippocampus in rats with epilepsy The effects of HMGB1,TLR4,NF-κB/p65 and P-gp/MDR1 expression provide a theoretical basis for further investigation of the mechanism of Wuling capsule combined with CBZ in reversing drug resistance to refractory epilepsy.Methods1.Clean-grade healthy adult male Wistar rats were divided into blank group,model group,CBZ group,CBZ+ low dose(0.3g/kg)Wuling capsule group(CBZ+L),CBZ+ medium dose(0.6g/kg)Wuling Capsule group(CBZ+M),CBZ+ high dose(1.2g/kg)Wuling capsule group(CBZ+H),8 rats in each group,a total of 6 groups.In the blank group,the sham operation model group was established by microinjection of normal saline into the lateral ventricle,and the rats in the other groups were injected with KA-induced RE rat model.Rats in the blank group and the model group were given normal saline every day.The remaining groups were 80mg/kg CBZ,80mg/kg CBZ+0.3g/kg Wuling capsule,80mg/kg CBZ+0.6g/kg.Ling capsule,80mg/kg CBZ+1.2g/kg Wuling capsule was administered by intragastric administration once a day for 6 days/week for 10 weeks.Changes in symptoms of rats in each group were monitored,and the level of seizures and frequency of seizures were recorded.2.Rats with successful model establishment were randomly selected from 8 rats in each group.A total of 6 groups were treated with Morris water maze after 60 days of intragastric administration.Record the time of the rat found the platform and the frequency of crossed the original set platform.3.After the Morris water maze test,18 Wistar rats were randomly selected,3 in each group,6 groups in total,anesthetized by intraperitoneal injection of 10% chloral hydrate(0.35 ml / kg),and normal saline and 4% paraformaldehyde were perfused along the apex.Broken head taken rat brain tissue,paraffin-embedded sections,and HE staining for pathological examination.4.124 rats after successful modeling were randomly divided into blood concentration group and brain medicine concentration group.The plasma concentration components were CBZ group,CBZ+L group,CBZ+M group,CBZ+H group,6 groups in each group,a total of 4 groups;the brain medicine concentration grouping method was the same as above,25 groups in each group,a total of 4 groups.The CBZ group was given normal saline by intragastric administration.The CBZ+L group,CBZ+M group and CBZ+H group were given 0.3g/kg,0.6g/kg and 1.2g/kg Wuling capsules once a day for 7 consecutive days.On the 8th day after intragastric administration for 30 minutes,CBZ(80 mg/kg)was administered by intragastric administration.Blood and brain tissues were collected through the internal iliac vein at a pre-designed time point.The concentration of CBZ in plasma and brain tissue was determined by HPLC.5.Rats were intragastrically administered for 60 days,3 rats in each group,a total of 6 groups.10% chloral hydrate(0.35 ml / kg)was intraperitoneally injected with anesthesia,normal saline and 4% paraformaldehyde perfusion,decapitated brain,paraffin-embedded sections,immunohistochemical method to observe the brain tissue of each group of rats HMGB1,TLR4,NF-κB/p65 and P-gp proteins were analyzed by Image Pro Plus software to analyze the expression of corresponding proteins in each group.6.Rats were administered with gastric administration for 60 days,3 rats in each group,a total of 6 groups.After the intact hippocampus tissues were removed as described above,the expressions of HMGB1,TLR4,NF-κB/p65 and MDR1 mRNA were detected by real-time quantitative PCR.7.Rats 60 days after gavage were randomly selected,3 rats in each group,6 groups in total,10% chloral hydrate(0.35ml/kg)was intraperitoneally injected,and the hippocampus was decapitated and determined by Western Blot.HMGB1,TLR4,NF-κB/p65 and P-gp proteins were detected in hippocampus of rats,and the expression of corresponding proteins in each group was statistically analyzed by Quantity one software.Results1.Long-term use of CBZ alone can reduce the level of seizures induced by KA in rats,and has no significant inhibitory effect on the frequency of attacks.Combined with low,medium and high doses of Wuling capsule can inhibit the level of seizures and reduce the frequency of seizures.Among them,the combination of medium and high dose Wuling capsules was better than CBZ alone(P<0.05).2.With the prolongation of the experimental days,the time of the navigation of the low-,medium-,and high-dose Wuling capsules in the rats with the combination of CBZ and CBZ was significantly lower than that of the model group(P<0.01),and the combination group The effect was better than that of the CBZ group alone(P<0.01).The frequency of the CBZ combined with the high-dose Wuling capsule group crossing the effective platform was significantly higher than that of the model group and the CBZ group(P<0.05).3.The results of HE staining showed that compared with CBZ alone,the nucleus pyknosis,lysis and lysis of hippocampal neurons in CBZ+M group and CBZ+H group were improved,and the number of neurons was relatively high.4.Compared with the CBZ group,the area under the curve of CBZ in the plasma of CBZ+M group and CBZ+H group was significantly increased(A<0h>h)and the maximum blood concentration Cmax(P<0.05).The clearance rate of CLz/F was significantly decreased(P<0.01).The concentration of CBZ in the brain tissue of CBZ+M group and CBZ+H group was also significantly increased(P<0.05).5.The results of IHC showed that the expression of HMGB1,TLR4,NF-κB/p65 and P-gp protein in brain tissue and hippocampus of KA-induced epileptic rats was significantly increased(P<0.05).Compared with the model group,HMGB1,TLR4,NF-κB/p65,and P-gp proteins were significantly decreased in the brain and hippocampus of CBZ+M group and CBZ+H group(P<0.05),and HMGB1 in hippocampus of CBZ group rats.There was no significant difference in the expression of TLR4 and NF-κB/p65,but the expression of P-gp protein was increased.The HMGB1 and TLR4 proteins in the brain of CBZ+L group were significantly decreased(P<0.01),and the NF-κB/p65 protein was decreased.There was no significant difference in the expression of P-gp protein between the trend(P<0.05).Compared with CBZ group,HMGB1,TLR4 and P-gp proteins in brain and hippocampus of CBZ+M group and CBZ+H group were significantly decreased(P<0.01),and NF-κB/p65 protein expression was decreased(P<0.05).),the four proteins in the CBZ+L group were not significantly reduced.6.The results of RT-PCR showed that HMGB1,TLR4,NF-κB/p65 and MDR1 mRNA in hippocampus of KA-induced epileptic rats were significantly increased.Compared with CBZ group,HMGB1,TLR4,NF-κB/p65 and MDR1 mRNA in hippocampus of CBZ+M group and CBZ+H group were significantly lower(P<0.05).7.The results of WB showed that compared with the blank group,the HMGB1,TLR4 and P-gp proteins in the hippocampus of the model group,CBZ group,CBZ+L group,CBZ+M group and CBZ+H group were significantly increased(P< 0.05),NF-κB/p65 protein was significantly increased in model group,CBZ group,CBZ+L group and CBZ+H group(P<0.05).Compared with the model group,the HMGB1,TLR4,NF-κB/p65 and P-gp proteins in the hippocampus of CBZ+M group and CBZ+H group were significantly decreased(P<0.01).Compared with CBZ group,HMGB1,TLR4,NF-κB/p65 and P-gp protein in hippocampus of CBZ+M group and CBZ+H group were significantly decreased(P<0.01).ConclusionWuling Capsule combined with CBZ can inhibit the seizure of KA-induced epileptic rats,improve the learning and memory ability of rats caused by epilepsy,alleviate the damage of hippocampal neurons,and improve the blood and brain tissue of CBZ in epileptic rats.The distribution has the effect of reversing the drug resistance of refractory epilepsy.The mechanism may be to alleviate the brain damage caused by KA-induced epileptic seizures,inhibit the high expression of HMGB1 protein in hippocampus,decrease the activation of TLR4,and inhibit NF-κB/p65.Thereby,the production of the drug resistance gene P-gp is down-regulated to play a role. |
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