Clinical And Experimental Research On The Therapy Based On Yin And Yang Differentiation On Frequent Epilepsy Attack

The therapy based on yin and yang differentiation is a kind of traditional therapy in Chinese medicine (CM) which treats epilepsy attacking frequently under two conditions that are yinxian and yangxian, one of approaches of classification of epilepsy from the respect of CM on the basis of different pathogenesis between cold and heat property in terms of CM. This therapy originated from the suggestion of Chao Yuanfang in the Yuan era. Then Wushengying was put forward to treating yinxian in Shi Yi De Xiao Fang writed by Wei Yilin in the Yuan period. And Dingxianwan was recommended to treat yangxian in Yi Xue Xin Wu authored by Cheng Guopeng in the qing dynasty. It is advisable to treat yinxian in wushengyin (WSY) and Erchentang (ECT), while yangxian in huanglianjiedutang (HLJDT) and dingxianwan (DXW) in the teaching material of Internal of Medicine of CM. However, clinical appliance for these decoctions is little, the reason for which may result from their inclusion of certain toxic Chinese medicines like Rhizoma Arisaematis Erubescentis, Rhizoma Pinelliae, Rhizoma Typhonii Gigantei, all of which have not been processed, and Cinnabaris. Besides, domestic researches on anti-epileptic effects and mechanisms of Chinese medicine are carried on by pathological animal models of western medicine (WM) nowadays, and animal models characterized by CM differentiation of symptoms and signs, which is named zheng hou in traditional Chinese medicine, have not been established. From what had been discussed above, we designed and undertook this program which was consisted of two parts that were clinical trial and empirical study which included the animal experiment and the cell culture experiment. And the main aim was to explore the effects, safety, and mechanisms of the therapy based on yin and yang differentiation, and set up epileptic animal models which were yinxian and yangxian characterized by zheng hou of CM. Part1Clinic Trial of The Therapy Based on Yin and Yang DifferentiationObjectiveTo observe clinical effects and safety of the therapy based on yin and yang differentiation in epilepsy attacking frequently.MethodsPatients who suffered from epileptic seizure more than three times were randomly divided into the treatment group and the control group. The control group only received conventional AEDs, while the treatment group received conventional AEDs, Chinese herbal drugs that were JWWSY and ECT, or HLJDT and DXW on the basis of the therapy based on yin and yang differentiation, and intravenous drip of xingnaojing parenteral solution as well. Therapeutic effects were measured by the decrease in seizure rate between prior and post-treatment, zheng hou evaluation, average total dosages of different AEDs, NHS3, MBI, and MRS respectively.ResultsThere were12cases who participated in this trial, and11of them completed the program, of which5were in the control group, and6were in the treatment group, while1lost due to sudden death. Although most of parameters like the decrease in seizure rate between prior and post-treatment, average total dosages of different AEDs, NHS3, MBI, MRS, and etc we measured were no statistical difference between the two groups. In terms of evaluation of zheng hou, the control group adepted in improving asthenia(P<0.05), while the treatment group was effective at ameliorating wind, asthenia, renal yin deficiency, phlegm, and spleen energy deficiency(P<0.05), especially at phlegm and spleen energy deficiency (P<0.01). There was no side effect in all the cases in the treatment group.ConclusionIt is superior for the therapy based on yin and yang differentiation in terms of improvement in relative symptoms and signs of zheng hou of patients suffering from epilepsy attacking frequently, especially in zhenghou of phlegm and spleen energy deficiency, and no side effect was observed.Part2Experimental Research of The Therapy Based on Yin and Yang Differentiation Section1Animal ResearchExperiment1Research on Effects of JWSY and ECT on Yinxian Epileptic RatsObjectiveTo explore anti-epileptic effects and mechanisms of JWWSY and ECT, and establish yinxian epileptic rat model.MethodsThe yinxian epileptic rat model was made by the method of feeding on high fat diet and CM in cold property for a long time, and then the being induced seizure by PILO. Yinxian epileptic rats induced by PILO were treated with JWWSY and ECT, the representative recipe for treatment of yinxian, while HLJDT and DXW, the representative recipe for treatment of yangxian, was used as the control formula. Time spots of seizure latency period and seizure status were record, and concentrations of amino acid neurotransmitters were measured by HPLC method, and the expression of caspase-3was determined by immunohistochemisty technology, and pathological changes were observed by hematoxylin and eosin stain. All of indexes mentioned above were adopted to evaluate the anti-epileptic effects and mechanisms of JWWSY and ECT, and assess the quality of epileptic animal model characterized by zheng hou.ResultsAll kinds of concentrations of amino acid neurotransmitters measured in this experiment were lower, and the expression of caspase-3was higher in all the epileptic rats, in all of which the variation of yinxian CM group was the least, and the yinxian CM control group was the second, when compared with normal control group. However, there was no statistical difference in concentrations of amino acid neurotransmitters of Glu, Asp, GABA, Gly, and Ala, and in the expression of caspase-3between epileptic rats and normal control ones. Also, There was no recognizable difference in time spots of seizure latency period and seizure status among epileptic groups. And the tissue of the cerebral cortex and hippocampus, lung, liver, and kidney of all epileptic rats was injured severely, but there was no significant difference in all epileptic groups.ConclusionJWWSY and ECT, as well as HLJDT and DXW can inhibit the declination of different sorts of amino acid neurotransmitters, including Glu, Asp, GABA, Gly, and Ala, and apoptosis in the central nervous system, and effects of JWWSY and ECT on which are better, which indicates that such decoctions may have certain effects in treating the yinxian epileptic rats induced by PILO, and JWWSY and ECT seems to be superior. Yinxian epileptic rat model was set up in this experiment initially.Experiment2Research on Effects of HLJDT and DXW on Yangxian Epileptic Rats ObjectiveTo explore anti-epileptic effects and mechanisms of HLJDT and DXW, and establish yangxian epileptic rat model.MethodsThe yangxian epileptic rat model was made by the method of feeding on high fat diet and CM in heat property for a long time, and then being induced seizure by PILO. Yangxian epileptic rats induced by PILO were treated with HLJDT and DXW, the representative recipe for treatment of yangxian, while JWWSY and ECT, the representative recipe for treatment of yinxian, was used as the control formula. Time spots of seizure latency period and seizure status were record, and concentrations of amino acid neurotransmitters were measured by HPLC method, and the expression of caspase-3was determined by immunohistochemisty technology, and pathological changes were observed by hematoxylin and eosin stain. All of indexes mentioned above were adopted to evaluate the anti-epileptic effects and mechanisms of HLJDT and DXW, and assess the quality of epileptic animal model characterized by zheng hou.ResultsAll kinds of concentrations of amino acid neurotransmitters measured in this experiment were lower, and the expression of caspase-3was higher in all the epileptic rats, when compared with normal control group. In all of epileptic groups, the variation of amino acid neurotransmitters including Asp and Ala and the expression of caspase-3of yangxian CM group was the least. In contrast, the variation of the indexes concerned of yangxian CM control group was the biggest. However, there was no statistical difference in concentrations of amino acid neurotransmitters of Glu, Asp, GABA, Gly, and Ala, and in the expression of caspase-3between epileptic rats and normal control ones. And there was no meaningful difference in time spots of seizure latency period and seizure status among epileptic groups. The tissue of the cerebral cortex and hippocampus, lung, liver, and kidney of all epileptic rats was injured severely, but there was also no significant difference in all of the epileptic groups.ConclusionHLJDT and DXW can inhibit the declination of different sorts of amino acid neurotransmitters, including Glu, Asp, GABA, Gly, and Ala, and apoptosis in the central nervous system, which shows that such decoctions may have certain effects in treating the yangxian epileptic rats induced by PILO. The yangxian epileptic rats model was established initially in this experiment.Section2Cell Culture ResearchExperiment1Research on Establishment of PC12Cell Trauma Model Induced by Glutamate ObjectiveTo explore a proper methods to establish PC12cell trauma model for the following experiments.MethodsPC12cells were treated with various concentrations of Glu, and cell morphology and viability were measured by MTT assay at24h,48h, and72h respectively so that the concentration-effect and concentration-time relationship between the effects of Glu neurotoxicity and the viability of PC12cells was investigated.ResultsThe results of the observation of cell morphology indicated that the cell reflection, dimensionality, sketch, and the growth condition become bad to worse step by step as the increasing concentrations of Glu at the same time spot from5mM Glu group, while there was on obvious difference in cell morphology at different time spots comparison. The outcomes of the vitality of cells manifested that the cell vitality declined gradually as the increase of the concentration of Glu from5mM at24h,48h, and72h time spots. However, the vitality of cells in normal control group and trauma model groups induced by Glu in different concentrations. The relationship between effects of Glu neurotoxicity and viability of PC12cells was in manner of concentration and time-dependence:the higher the concentration of Glu is, the lower the viability of PC12cells becomes;the longer the incubation time is, the higher the viability of PC12cells becomes. There was significant difference in the cell vitality between PC12cells administered by Glu in the concentration of10mmol/L and in the normal control group.ConclusionThe relationship between effects of Glu neurotoxicity and viability of PC12cells was in manner of concentration and time-dependence:the higher the concentration of Glu is, the lower the viability of PC12cells becomes;the longer the incubation time is, the higher the viability of PC12cells becomes. PC12cells administered by Glu neurotoxicity in the concentration of10mmol/L for24h maybe the ideal conditions for the following experiment.Experiment2Therapeutic effects of JWSY and ECT drug-containing serum on PC12cell trauma model induced by Glu neurotoxicityObjectiveTo move forward a single step to explore the therapeutic action and mechanisms of anti-epileptic effects of JWWSY and ECT at the cellular level.MethodsThe experimental cells were randomly devided into six groups, which were the normal control group, the Glu trauma model group, the normal rat serum group, the cold phlegm rat group, the yinxian CM control group, and the yinxian CM group. The PC12cell trauma models induced by Glu neurotoxicity were treated in JWWSY and ECT drug-containing serum made by rats with body constitution in cold phlegm, which were made by the method of feeding on high fat diet and CM in cold property for a long time, while HLJDT and DXW drug-containing serum was used as the control formula. The MTT assay was utilized to detect the viability of cells, and flow cytometry was adopted to assess the concentration of intra-cellular calcium.ResultsThe cell viability of all groups administrated by Glu declined, in all of which the yinxian CM serum group was the least, and the yinxian CM control serum group was the second, when compared with the normal control group. There was a significant difference between groups administrated by Glu and the normal control group (P<0.01), except the yinxian CM serum group. And there was a meaningful difference between the yinxian CM serum group and the yinxian CM control serum group as well (P<0.01).Except the Glu trauma model group, the concentrations of intra-cellular calcium of all groups administrated by Glu decreased, in all of which the yinxian CM serum group was the most, and the yinxian CM control serum group was the second, when compared with the正常对照group. Both the yinxian CM control serum group and the yinxian CM serum group had a recognizable difference in contrast to the正常对照group(P<0.01). And there also was a noticeable difference between the yinxian CM control serum group and the yinxian CM serum group(P<0.01).ConclusionJWWSY and ECT, as well as HLJDT and DXW drug-containing serum made by rats with body constitution in cold phlegm can increase the vitality and inhabit internal flow of calcium of the PC12cell trauma models induced by Glu neurotoxicity, which indicates that the drug-containing serum of such decoctions, especially JWWSY and ECT drug-containing serum, may have certain therapeutic effects in treating the PC12cell trauma models.Experiment3Therapeutic effects of HLJDT and DXW drug-containing serum on PC12cell trauma model induced by Glu neurotoxicity ObjectiveTo further explore the therapeutic action and mechanisms of anti-epileptic effects of HLJDT and DXW at the cellular level.MethodsThe experimental cells were randomly devided into six groups, which were the normal control group, the Glu trauma model group, the normal rat serum group, the heat phlegm rat serum group, the yangxian CM control serum group, and the yangxian CM serum group. The PC12cell trauma models induced by Glu neurotoxicity were treated in HLJDT and DXW drug-containing serum made by rats with body constitution in heat phlegm, which were made by the method of feeding on high fat diet and CM in heat property for a long time while JWWSY and ECT drug-containing serum was used as the control formula. The MTT assay was utilized to detect the viability of cells, and flow cytometry was adopted to assess the concentration of intra-cellular calcium.ResultsThe cell viability of all groups administrated by Glu declined, in all of which the range in declination of the yangxian CM control serum group was the least, and the yangxian CM serum group was the second, when compared with the normal control group. There was a significant difference between groups administrated by Glu and the normal control group(P<0.01). And there was a meaningful difference between yangxian CM serum group and the yangxian CM control serum group as well (P<0.01).Except the Glu trauma model group, the concentrations of intra-cellular calcium of all groups administrated by Glu decreased, in all of which, the range of the heat phlegm rat serum group was the most, the yangxian CM serum group was the second, and the yangxian CM control serum group was the last, when compared with the normal control group. And except the yangxian CM control serum group, both the heat phlegm rat serum group and the yangxian CM serum group had a statistical difference in contrast to the normal control group(P <0.05), while there was no noticeable difference between the yangxian CM control serum group and the yangxian CM serum group.ConclusionHLJDT and DXW, as well as JWWSY and ECT drug-containing serum made by rats with body constitution in heat phlegm can increase the vitality of the PC12cell trauma models induced by Glu neurotoxicity, and the HLJDT and DXW drug-containing serum can also inhabit the internal flow of calcium, which manifests that there seems to be certain the therapeutic effects for the drug-containing serum of such decoctions in treating the PC12cell trauma models, but which decoction, the HLJDT and DXW, or JWWSY and ECT drug-containing serum of yangxian pre-seizure rats,is better needs further exploration.