| The challenge of treating drug resistant epilepsy is Drugs(Antiepileptic Drugs,AEDs)resistance of the patients,how to reduce or inhibit the drug resistance of patients to AEDs is an important problem to be solved urgently by domestic and foreign scholars.In long-term clinical practice,our team found that the addition of Chaibei Zhixian Decoction in the treatment of drug resistant epilepsy can improve the anti-epileptic efficacy of western medicine chemical drugs.Preliminary basic experimental results showed that its antiepileptic mechanism may be related to the inhibition of multi-drug Resistance Genel(Mdr1)and the encoded P-glycoprotein(P-gp),and the pathway of regulating P-gp is related to the inflammatory signaling pathway.On the basis of previous studies,this clinical study further expanded the sample size to observe the efficacy and safety of Chaibei Zhixian Decoction in the treatment of drug resistant epilepsy patients.Preliminary screening of changes in serum inflammatory factors.In order to further understand the mechanism of resistance and its contribution to the anti-drug resistance of Chaibei Zhixian Decoction and its main components in blood to epileptic rats induced by Kainic Acid(KA),the animal experimental study was conducted to investigate the anti-drug resistance mechanism of Chaibei Zhixian Decoction and its main components in blood[Objectives]Clinical trials were conducted to observe the efficacy and safety of Chaibei Zhixian Decoction in the treatment of drug resistant epilepsy,and the changes of serum inflammatory factors in patients with drug resistant epilepsy before and after treatment were observed by inflammatory antibody chip.Through animal experiments,the Chaibei Zhixian Decoction and its main blood components,α-asarone,β-asarone,peimine and peiminine were investigated for their anti-drug resistance mechanism to epileptic rats induced by KA.[Methods]Clinical researches:Clinical researches:Clinical researches consist of clinical observation and discussing mechanism,Eighty-four patients with drug-resistant seizures of focal disorder of consciousness were included,they were randomly divided into treatment group and control group.The treatment group was treated with Chaibei Zhixian Decoction on the basis of the original antiepileptic drugs,and the control group was treated with placebo on the basis of the original antiepileptic drugs,and the observation period was 3 months.The main efficacy indicators included monthly attack frequency,no attack Rate and 50%Response Rate(RR).Secondary efficacy indicators included changes in seizure degree,TCM syndromes,quality of life score(QOLIE-89),Neurological Disorders Depression Inventory For Epilepsy(NDDI-E),Self-rating depression scale(SDS),Hamilton Depression Scale(HAMD),Hamilton Anxiety Scale(HAMA)and safety evaluation.The serum of drug resistant epilepsy patients were taken before treatment,3 months after treatment and 6 months after treatment,and the changes of serum inflammatory factors before and after treatment were detected by inflammatory antibody chip,so as to explore the mechanism of action of the drug.Animal experiment:The models of epileptic rats were established by injecting KA into the lateral ventricle They were divided into model group,CBZ group,Chaibei Zhixian Decoction+CBZ group,α-asarone+CBZ group,β-asarone+CBZ group,peimine+CBZ group,peiminine+CBZ group,and set up sham operation group.After continuous gavage for 60 days,Western Blot and RT-PCR were used to detect the expression of P-gp and Mdr1a/b mRNA in hippocampus.Immunohistochemical was used to detect ionized calcium binding adapter molecule 1(Iba-1)and glial fibrillary acidic protein(GFAP),Liquid chromatography and mass spectrometry was used to detect the content of CBZ and CBZE,Nils staining was used to observe the pathology of hippocampus.[Results]Clinical research 1(1)Baseline data:A total of 84 patients with drug-resistant seizures of focal disorder of consciousness were included.The difference between the two groups in gender was statistically significant(P<0.05),while the differences in other basic datas were not statistically significant(P>0.05).The baseline data of the two groups were basically the same.(2)Main efficacy indicators:After 3 months treatment the difference of the monthly attack frequency and no attack rate between the two groups were statistically significant(P<0.01,P<0.05);After 2 and 3 months treatment,the difference of 50%response rate between the two groups was statistically significant(P<0.01,P<0.01)(3)Secondary efficacy indicators:comparison of attack severity between the two groups after 3 months of treatment showed statistical significance(P<0.05).After 3 months treatment,the difference of TCM syndromes between the two groups was statistically significant(P<0.05).(4)Evaluation of life quality and emotion scale:The total score of life quality of the two groups after 3 months treatment was statistically significant(P<0.05).Single items comparison between the two groups showed statistical significance in pain,energy/fatigue,attention,panic attacks,social support(P<0.01,P<0.01,P<0.05,P<0.01,P<0.01)SDS scores of the two groups were statistically significant after 3 months treatment(P<0.05).(5)Safety evaluation:during the observation period,no abnormal liver and kidney functions and blood routine were found related to the addition of Chaibei Zhixian Decoction,and no obvious adverse events were found.Clinical research 2(1)After the addition of Chaibei Zhixian Decoction for the treatment of patients with drug-resistant seizures of focal disorder of consciousness 3 months,compared with that before treatment,It showed a tendency to down-regulate serum IL-1β IL-4、IL-17、IL-1α,IL-15、IL-6、IL-11、I-309、MIG、GCSF、GM-CSF inflammatory factors.(2)The expression of I-309,IL-11 and MIG in serum of patients with drug-resistant seizures of focal disorder of consciousness decreased after the addition of Chaibei Zhixian Decoction 6 months compared with that before treatment,and the differences were statistically significant.Moreover,there was a tendency to down-regulate IL-1β、I-309、IL-1α、IL-15、IL-8、IL-17、IL-6、IL-11、IL-16、MIG、IL-1ra、GCSF、BCL、MIP-1β、MIP-1α、IL-12P40 inflammatory factors.(3)The addition of Chaibei Zhixian Decoction in the treatment of patients with drug-resistant seizures of focal disorder of consciousness after 6 months may have the effect of improving the serum inflammatory factors,and its KEGG pathway may be related to the toll-like receptor signaling pathway,cytokine receptor interaction and cytokine signaling pathway.Animal experiment(1)P-gp expression in hippocampal tissues of rats in each group:Compared with the sham operation group,the expressions of model group and CBZ group increased,and the differences were statistically significant(P<0.05,P<0.01).Compared with the model group,the expressions of sham operation group and Chaibei Zhixian Decoction+CBZ group decreased,and the difference was statistically significant(P<0.05,P<0.05).Compared with the CBZ group,the expressions of Chaibei Zhixian Decoction+CBZ group,α asarone+CBZ group,peimine+CBZ group and β asarone+CBZ group decreased,with statistically significant differences(P<0.01,P<0.05,P<0.05,P<0.05,P<0.05).(2)Expression of Mdrla/b mRNA in hippocampal tissues of rats in each group:Compared with the sham operation group,the expressions of model group and CBZ group increased,and the differences were statistically significant(P<0.05,P<0.01),Mdr1a mRNA expression was increased in peimnine+CBZ group,and the difference was statistically significant(P<0.05).Compared with the CBZ group,the expression levels of Mdrla/b mRNA in the hippocampus of Chaibei Zhixian Decoction,α asarone+CBZ group and peimine+CBZ group were significantly lower(P<0.01,P<0.05;P<0.01,P<0.05;P<0.01,P<0.01),The expression of MdrlamRNA decreased in β asarone+CBZ group,and the difference was statistically significant(P<0.01).(3)Expression of Iba-1 and GFAP in hippocampus:Compared with the sham operation group,the expressions of Iba-1 and GFAP in the model group and CBZ group increased,and the differences were statistically significant(P<0.01,P<0.05;P<0.05,P<0.05),Iba-1 expression was increased in β-asarone+CBZ group,and the difference was statistically significant(P<0.05).compared with the model group,the expression of Iba-1 in Chaibei Zhixian Decoction+CBZ group,α-asarone+CBZ group,peimine+CBZ group,peimnine+CBZ group decreased,and the differences were statistically significant(P<0.01,P<0.05,P<0.01,P<0.05).(4)CBZ and CBZE contents in right brain of rats in each group:Comparison of CBZ content in right brain:There was no significant statistical difference between the groups.Comparison of CBZE content in right brain:compared with the CBZ group,the content of CBZE in Chaibei Zhixian Decoction+CBZ group,α-asarone+CBZ group and peimine+CBZ group increased,with statistically significant differences(P<0.05,P<0.05,P<0.01)Compared with Chaibei Zhixian Decoction+CBZ group,the content of CBZ group decreased,and that of peimine+CBZ group increased,the differences were statistically significant(P<0.05,P<0.05).Compared with peimine+CBZ group,the contents of CBZ group,Chaibei Zhixian Decoction+CBZ group,α-asarone+CBZ group,β-asarone+CBZ group and peimnine+CBZ group all decreased,and the differences were statistically significant(P<0.01,P<0.05,P<0.05,P<0.01,P<0.01).(5)Nils staining of hippocampus in each groupIn the model group,hippocampal neurons decreased sharply,the arrangement was disordered,the proliferation of glial cells,and the degradation of nissl bodies was obvious.Compared with the model group,the number of neurons in the CBZ group increased,the glial cells proliferated more,and the number of nisi bodies was less.In Chaibei Zhixian Decoction+CBZ group,α-asarone+CBZ group,and peimine+CBZ group,neurons were abundant,nisin corpuscles were abundant,and a few glial cells were observed.In β-asarone+CBZ group and peimnine+CBZ group,neurons were abundant,nisin corpuscles were few,and vacuolar lesions were obvious.[Conclusions]1.Chaibei Zhixian Decoction is safe and effective in the treatment of patients with drug-resistant seizures of focal disorder of consciousness,which can reduce the number of seizures,reduce the severity of seizures,Improve patients’ quality of life and depression.2.The addition of Chaibei Zhixian Decoction after 3 months and 6 months of treatment has a trend of reducing serum inflammatory factors in patients with drug-resistant seizures of focal disorders of consciousness.After 6 months of the treatment,the serum levels of I-309,IL-11 and MIG in the patients were lower than those before treatment,and with the extension of treatment time,the effect of Chaibei Zhixian Decoction on reducing serum inflammatory factors in patients was gradually obvious.3.Chaibei Zhixian Decoction,α-asarone or peimine combined with CBZ may increase cerebral CBZE content in the brain of epileptic rats induced by KA and protect hippocampal neurons,and the mechanism may be related to the reduction of hippocampal P-gp and Mdr1a/b mRNA expression and the inhibition of hippocampal Iba-1 activation. |
