| Prescriptions are the primary forms in clinic of Chinese Medicine,with the characteristics of multiple ingredients,multiple targets and multiple ways.The theory of compound compatibility is the essence in the prescriptions of traditional Chinese medicine(TCM).Prescriptions are not a simple superposition of drug efficacy,but for the guideline of efficacy enhancing and toxicity reducing.Studying the compatibility principles can not only reveal the scientific essence of prescriptions,but also point out the direction for the reasonable application of TCM.At present,exploring the pharmacokinetic interactions of prescriptions,that is,the interaction of drugs in the absorption,distribution,metabolism and excretion(ADME),is still an important idea and method to explore the law of drug compatibility.However,the mechanism of compatibility of prescriptions with perspective of pharmacokinetic interaction is mostly focused on the single component or single target evaluation system,which is obviously not suitable for the pharmacokinetic interaction of Chinese medicine prescription with the multi component and multi target characteristic.Also,the normal animal is mostly used in present research,while the animal model in the pathological state is rarely used for explaining mechanism of compatibility.Drug transporter is a membrane protein,widely distributed in the gut,blood-brain barrier,liver and kidney.Transporters play an important role in drug absorption,distribution and excretion in vivo.Transporters are the key factors influencing the in vivo process of drugs.The relationship between the functional changes of transporters in physiological/pathological conditions and diseases has caused for concern gradually.Additionally,the inhibition or induction effect of the substrates makes an important mechanism resulting in pharmacokinetic interactions in co-administration of drugs.In recent years,researches on the compatibility mechanism of TCM based on transporters have made some progress,mainly concerned the effect of compatibility on bioavailability,distribution and metabolic pathways.However,there is less research on the interactions of excretion,which needs to be paid more attention."Syndrome" refers to the specific disease for specific prescriptions as the core and basis of treatment by differentiation of syndromes.The components of the prescription are complex and have the integrity characteristics of multi-target,multi-link and multi-level,which is complementary to ’overall concept’ in TCM.It can eliminate or ameliorate the "syndrome" of the disease by systematic regulating in vivo.Although the etiology and pathogenesis of insomnia is complex,the "heart-kidney imbalance"is the most common type of insomnia.It is understood that the dysfunction of the heart and kidney,which is related to the imbalance between the two organs,can be regarded as amanifestation of the disorder of the function caused by the dysfunction exchange of substance in the heart and kidney.At present,the well-known mechanism about the insomnia disorder is that the HPA axis of neuroendocrine is disordered by inhibition and excitatory neurotransmitter imbalance in brain or blood.The interpretation of insomnia mechanism still lacks more systematic and.Jiao-tai-wan,recorded in the book of "Han Tong Medicine" in Ming Dynasty,composed of Rhizoma Coptidis(RC)and Cortex Cinnamon(CC)(10:1,g/g),has been used to treat insomnia in China for centuries.RC has the effects of clearing heart fire and tranquilizing the mind as sovereign drug,which property and flavor were painstakingly and cold.CC,with the property of pungent,sweet and hot,has the effect of warming kidney and enhancing yang,letting the fire back to its origin as adjuvant drug.In recent years,there is obvious pharmacokinetic interaction between RC and CC,which may be an important reason for its compatibility.While the compatibility mechanism of Jiao-tai-wan from the view of prescription combining syndrome remains to be investigated.Proteomics,as an important part of the new systems biology,can reflect the function of all protein in a certain state at the biological holography perspective.It provides a powerful technical support for exploring the essence of the syndrome and revealing the target of drug action.Meanwhile,it is great significance to reveal the scientific principles of syndrome differentiation and the action mechanism of prescriptions.Membrane protein,as an important target of drugs,plays an important role in tumor and systema nervosum disease.The functional changes of membrane protein is closely related to disease happening,and the mechanism of prescriptions action can be revealed through the systematic regulation on the variation of membrane protein under the pathological state.At present,research on the combination of multi tissue membrane proteins with proteomics technology,explaining the essence of syndrome and the mechanism of action of drugs,is rarely reported.Therefore,the following new ideas and methods are proposed.Firstly,our study is to investigate the differences of coptis alkaloids in the dynamic distribution and excretion characteristics in healthy and insomniac rats after the combination of RC with CC.Secondly,the causes of these differences were discussed from the aspect of drug transporters function,and then preliminarily revealed the mechanism pharmacokinetics interaction between RC and CC.Finally,we analyzed the expression of the heart and kidney membrane protein in"heart-kidney imbalance" insomniac rats,and studied the regulation of Jiao-tai-wan.A new research model of combining prescription with syndrome will be established,which could be used to explore the essence of the syndrome of "heart-kidney imbalance" and the mechanism of Jiao-tai-wan action.For this purpose,the following researches have been carried out:Part one:Tissue distribution of coptis alkaloids in normal/insomniac rats before and after compatibility with RC and CC1.The method of determining the content of berberine(BBR),epiberberine(EBBR),coptisine(COP),jatrorrhizine(JAT),palmatine(PAL),berberrubine(BBRB)and magnoflorine(MAG)was determined by HPLC-MS/MS.The differences in tissue distribution characteristics of coptis alkaloids of RC combined with CC were investigated in normal and insomniac rats.According to the previous plasma pharmacokinetic experiments,the normal and insomniac model rats were orally administrated with RC and JTW powder(10:1)at the dose of 3.0 g·kg-1·d,3.3 g·kg-1·d,once per day for 7 days.The tissue samples were collected at 15 min,30 min(absorption phase),1 h,4 h(distribution phase)and 6 h(elimination phase),and the contents of coptis alkaloids in the tissue samples at each time point were detected.The distribution changes and accumulation of coptis alkaloids in different tissues were analyzed when RC combined with CC.The results showed that the coptis alkaloids are mainly distributed in heart,liver and kidney.The Cmax and AUC0-t of BBR were the largest in different tissues among the all components,which might be related to the highest content in RC.The accumulation content of MAG in kidney is the highest and the content of BBRB is the highest in liver,indicating that these two components have obvious liver-kidney targeting.2.In normal and pathological conditions,there were significant differences in the tissue distribution characteristics of coptis alkaloids.In the normal condition,all compounds reached the highest concentration within 30 min after intragastric administration of RC.Then,the content of each compounds was rapidly eliminated in the tissue within 1 h,the content of 6 h in the tissues was the lowest.After combined with CC,the AUC0-t of coptis alkaloids decreased significantly in the heart and kidney,while the Tmax was prolonged and the absorption was slow.In the pathological condition,the distribution of the coptis alkaloids in the heart was descended and then increased.The distribution content of coptis alkaloids were increased(P<0.05)at 6 h and appeared double peaks.It may be related to the absorption of double peak.In addition,the AUC0-t of coptis alkaloids in the group increased significantly in tissues,while the Tmax was significantly prolonged in the kidney to eliminate slowly.It is concluded that in normal rats,combining with CC can reduce the accumulation of coptis alkaloid in the heart,kidney and brain,while in the insomniac model rats,the accumulation of coptis alkaloids was increased in the same tissues,and the clearance of coptis alkaloids was postponed.Additionally,the coptis alkaloids also showed strong tissue targeting after compatibility.The scientific connotation of the compatibility principle of RC-CC was explained from the view of dynamic interaction of tissue distribution.Part two:the excretion characteristics of coptis alkaloids in normal/insomniac rats when RC combined with CC1.A new HPLC-MS/MS method was developed for the simultaneous determination of BBR,EBBR,JAT,COP,PAL,EBBR and MAG in rat urine and feces.The urine and feces samples in normal and insomniac rats were collected at 0-3,3-6,6-9,9-12,12-24 h after administration of RC-CC(1:0,10:1)at a dose of 3.0 g·kg-1·d,3.3 g·kg-1·d,once per day for 7 days.The results showed that 6 kinds of coptis alkaloids were detected in urine and fecal samples,but the content of MAG in urine was lower than the quantitative limit(LLOQ).No quantitative analysis of MAG was made in urine samples.2.In the urinary and fecal excretion test of normal rats,the cumulative excretion amounts of coptis alkaloids in compatibility group were significantly higher than that of RC group(P<0.05),especially the PAL and COP,which were increased by 28.08 and 13.68 times in urine sample,respectively.In insomniac rats,the cumulative excretion amounts of coptis alkaloids in compatibility group decreased significantly compared with those in RC group(P<0.05).For example,the amounts of PAL and COP were decreased by 18.48 and 12.16 times,respectively.And the other components were almost decreased by 1.3-2.2 times.The results showed that there was a significant difference in the urinary and fecal excretion amounts of coptis alkaloids in normal and insomniac rats.Combining with CC promoted the excretion amounts of coptis alkaloids in normal rats.However,it inhibited the excretion amounts in insomniac model rats,and prolonged the retention time of drugs in vivo.It was speculated that RC and CC exhibited synergistic effect under the pathological condition.Part three:Study on the mechanism of pharmacokinetic Interaction between RC and CC based on transportersBased on the interaction of distribution and excretion characteristics has been proved between RC and CC in vivo under the pathological condition.We compared the differences in OCTs and P-gp activity and mRNA expression in the normal and insomnia rats,and analyzed the regulation on the differences after compatibility.The mechanism of interaction of coptis alkaloids in RC and CC in vivo was preliminarily investigated.1.Changes of OCTs activity in normal and insomniac rats and the regulatory effects of RC-CC compatibilityMetformin is a recognized specific substrate for OCTs.The changes of plasma pharmacokinetic parameters of metformin in normal and pathological state were determined to reflect the change of OCTs activity by LC-MS/MS.The results showed that the Cmax,AUC(0-t),and Tmax of metformin in insomnia group were significantly increased compared with that in the normal group,while t1/2,Vz/F is significantly reduced.It was deduced that OCT1,OCT3 in the gastrointestinal tract,OCT1 in the liver and OCT2 in the kidney activity might be activated in insomnia pathological conditions.After the regulation of the OCTs activity by RC,CC and RC-CC compatibility in insomnia model rats,the AUC(0-t),Cmax and MRT(0-t)in the compatibility group were significantly higher than those in the insomniac group,t1/2,Vz/F were significantly increased,and CLz/F was significantly decreased.This deduced that OCT1,OCT3 in the gastrointestinal tract,OCT1 in the liver,and OCT3 in the brain might be activated,and OCT2 activity in the kidney might be inhibited;The RC group showed the same trend,but the change of pharmacokinetic parameters was not as remarkably as the compatibility group.The pharmacokinetic parameters of metformin in the CC group remained almost unchanged.To a certain extent,this result explained the principle of"increasing efficiency" after compatibility.2.Changes of P-gp activity in normal and insomniac rats and the regulatory effects of RC-CC compatibilityFexofenadine is recognized as a specific substrate for P-gp.The changes of plasma pharmacokinetic parameters of fexofenadine under normal and pathological conditions were determined to reflect the change of P-gp activity by LC-MS/MS.The results showed that compared with in normal rats,MRT(0-t),Tmax,t1/2 of fexofenadine increased significantly,and Cmax decreased significantly in insomniac model rats.This indicated that the rate of absorption of fexofenadine by the gastrointestinal tract slowed down,and the residence time of the drug in the tissue was prolonged.It was conducted that the P-gp activity in the gastrointestinal tract was activated in pathological conditions,and might be inhibited in the brain,kidney,and liver.After regulation of the compatibility group,the Cmax of fexofenadine increased significantly,MRT(0-t),t1/2 and Vz increased significantly,and CLz/F significantly decreased.The results showed that fexofenadine increased in vivo accumulation time and the clearance rate decreased.It is inferred that the activity of P-gp in the gastrointestinal tract and other tissues after administration of RC combined with CC might be inhibited,and the of coptis alkaloids might be increased.3.Changes of OCT and P-gp mRNA expression in normal and insomniac rats and effects of RC-CC compatibilityThis section of the experiment examined the insomnia model and the regulatory effect of RC-CC compatibility from the gene level.We used RT-PCR method and randomly divided them into normal group,model group,RC group,CC group and RC-CC compatibility group.The differential expression levels of OCTs and P-gp mRNA in heart,liver,kidney and brain tissue were examined.The results suggested that OCTs and P-gp mRNA expression in heart,kidney,liver and brain tissue under pathological conditions have different changes,but the variation is not obvious.It had some effects on the expression of OCTs and P-gp genes in brain,heart,liver and kidney tissues in RC,CC and RC-CC compatibility groups.The expression levels of OCTs and P-gp mRNA in the compatibility group were closer to normal levels.The pharmacokinetic interactions of RC-CC may be achieved through the comprehensive regulation of OCTs and P-gp gene expression in multiple tissues.However,its definite regulation still needs further verification in vitro.Part four:to investigate the mechanism of "heart-kidney imbalance" insomnia and explore the targets of Jiao-tai-wan by membrane proteomics combined with prescription-syndrome relationBased on the corresponding theory of TCM syndrome,this chapter explores the essence of insomnia and the targets of the treatment of the disease in terms of protein level.The experimental rats were randomly divided into normal group,model group and Jiao-tai-wan group.After 7 days of continuous gavage,the heart and kidney tissues were taken and membrane protein was extracted.The expression analysis of differential membrane protein was found by iTRAQ labeling membrane proteomics technology,according to multiple>1.2 or<0.8 as the basis.Compared with the normal group,the difference protein number 176 and 311 differential membrane proteins were identified in the cardiac and renal tissues of the insomnia model group.And the differential membrane proteins in the heart were mainly enriched in 250 biological processes and 11 KEGG pathways,such as such as urea metabolism and viral myocarditis.And the differential membrane proteins in the kidneys were mainly enriched in 199 biological processes and 4 KEGG pathways,including physical process and systemic lupus erythematosus.It indicates that the membrane proteins of brain and kidney have changed significantly under the condition of"heart-kidney imbalance"insomnia.And they are related to many biological processes and pathways.44 and 57 differential membrane proteins were transferred to normal expression levels in the heart and kidney,including OCT2 transporter,gamma aminobutyric acid transporter,adenylate cyclase,cytochrome C oxidase,GULT1 and GULT5,which have important biological functions.The membrane protein of the normalization of Jiao-tai-wan pill in the heart is enriched in 61 biological processes,such as fatty acid metabolism and propionic acid metabolism,and other 5 KEGG pathways;the normal membrane proteins in the kidneys of the kidneys are enriched in 7 biological processes,such as cation transport,and 4 KEGG pathways,such as the oxidative phosphorylation,and the ribosome.That is to say,Jiao-tai-wan pill may treat"heart-kidney imbalance" insomnia by regulating these proteins and pathways.Finally,OCT2 was selected as the representative for Western-Blot verification,and the results were consistent with the results of membrane proteomics analysis.To sum up,the following conclusions are as followed:(1)In the pathological state of insomnia,the accumulations of coptidis alkaloids in most tissues increased significantly,the urinal and fecal excretion accumulations decreased,and thus prolonging the retention time of the coptidis alkaloids in the body after the combined with CC.The synergistic effect of RC and CC compatibility is indicated by pharmacokinetic interaction.(2)the interaction mechanism between RC and CC was preliminarily studied at the level of the transporter function.The results showed that the activity and mRNA expression of the OCTs and P-gp were changed in the pathological state.The changes the activity and mRNA expression were regulated by the JTW and other drugs.The mRNA expression of OCTs and P-gp in most tissues recovered to the normal level by the regulation of JTW.The mechanism of pharmacokinetic interaction between RC and CC was preliminarily revealed,in order to provide some references for rational drug use in the clinic.(3)for the first time,with iTRAQ membrane proteomics technology and the prescription and syndrome,we explored the connotation of "heart and kidney discord" in TCM and the mechanism of "restoring normal coordination between heart and kidney" by JTW.The results showed that the membrane proteins in the heart and kidney tissues were changed significantly under the insomniac state,and were related to many biological processes and pathways.In addition,the action mechanism of JTW may probably correct these differential proteins and regulate related pathways to treat the "heart and kidney discord" insomnia.By the combination between the system biological method and the prescriptions and syndromes,it reflects the system regulation and integrity characteristics of TCM theories,and the multiple targets,multiple pathways and multiple targers of prescription.It will provide a new way of thinking for the corresponding research of other TCM syndromes. |
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