| ObjectivesThe incidence and death rate of cerebrovascular disease has been increasing in recent years in China.Ischemic stroke accounts for 3/4 of stroke with high disability and mortality,affecting the quality of life.Lots of medical resources are consumed each year,and there is no particularly effective treatment for the disease.The researches of western medicine on ischemic stroke mainly focus on single target.However,due to the complicated pathological process and mechanism of stroke,the comprehensive efficacy is limited so development of western medicine has approaching a bottleneck.TCM has been used thousands of years in prevention and treatment of strok,and has played a very important role in the treatment of cardiovascular and cerebrovascular diseases.Searching a new efficacious drug for cerebral protection has become important for the treatment of stroke.TCM theory with multi-component collaboration and multiple targets has unique advantages.The rapid development of bioinformatics and metabolomics has provided a good opportunity for TCM development.The trends of metabolites are directly related to the physiological and pathological states of the disease through the overall study of metabolite maps,combined with multivariate statistical analysis and pathway analysis.Metabolomics provides technical support for the study of TCM.It can help us to systematically and profoundly reveal the scientific connotation of TCM syndromes and TCM prescriptions,and contributes to the inheritance and development of TCM theories.Terminalia Chebula Retz(TC)is one of the traditional drugs and is widely used for the treatment a variety of diseases.TC has antioxidant,anti-inflammatory and anti-neurotoxicity effects.Related study has found that polyphenolic compounds in natural medicines have an important role and potential medicinal value in the prevention and treatment of stroke.The main effective component of TC is polyphenolic compounds.However,its specific role and mechanism in brain protection have been rarely reported.Therefore,we study and validate its mechanism of action based on metabolomics methods.MethodsAt present,metabolomics has achieved good results in TCM substances and its mechanism of action,safety assessment of TCM and research on TCM and compound medicine.It has obtained relevant biomarkers and provided a certain amount of research and exploration in TCM.MCAO model of SD rats and OGD model of PC12 cells were used in this study.At firstl the pharmacodynamics of TCE was evaluated in vitro and in vivo.Then differential metabolites,networks and pathways in its main role were investigated.At last,the pathways of major potent substances were verified.The experimental procedure is mainly divided into four parts.1.The potency of TCE against cerebral ischemia reperfusion by traditional pharmacological methodIn vivo study male SD rats(250±30 g)were selected and randomly divided into Sham group,Model group,TCE group(100 mg/kg),TCE group(300 mg/kg)and TCE group(500 mg/kg)with eight rats in each group.After MCAO operation,the corresponding drugs were administered by gavage once a day for 7 consecutive times.After the end of the treatment,SD rats were evaluated for neurological behavior,brain water content,and TTC staining to calculate the percentage of cerebral infarct volume and the degree of brain injury.Then the morphological changes of the cells were observed by HE staining and TUNEL staining and the antioxidant and inflammatory factors in the serum were measured.In vitro study the OGD model of PC12 cells was used to simulate in vitro I/R process,and cell proliferation and apoptosis were observed by dynamic real-time imaging.The experiments were divided into 6 groups: normal group,OGD model group,TCE(12.5 ?g/ml)group,TCE(25 ?g/ml)group,TCE(50 ?g/ml)group,and TCE(100 ?g/ml)group.In order to observe the effect of TCE on cell migration,we used scratch and Transwell methods.2.The effect of TCE on metabolic network by metabolomicsMale SD rats(250±30 g)were selected and randomly divided into three groups: sham group,model group and TCE(500 mg/kg)group,with eight rats in one group.After MCAO operation,the corresponding dose of TCE was given by gavage once a day seven times.When the treatment was completed,the sample was left for measurement.Metabolomics study of TCE was made using UPLC-Q/TOF-MS technique.Data were pre-processed through Waters data acquisition software.SIMCA software was used to make multivariate statistical analysis of PCA,PLS-DA,and OPLS-DA to evaluate the TCE treatment of cerebral I/R injury in rats.Then the potential biomarkers were screened and the compounds were identified by HMDB,KEGG and Lipidmaps databases with the combination of VIP values and t-test results.Metabolic pathway analysis was made by Metabo Analyst 4.0 software.3.The cerebral protection mechanism of TCEMale SD rats(250±30 g)were selected and randomly divided into three groups: sham group,model group and TCE(500 mg/kg)group with eight rats in each group.After MCAO operation,the corresponding dose of TCE was given by gavage once a day seven times.At the end of the treatment,the head was decapitated and the brain tissue was taken.The expression of anti-oxidation pathway related proteins Nrf2,HO-1 and VEGF was detected by immunohistochemistry and Western blot.The expression of angiogenesis marker CD31 was determined by Western blot.The change of micro-vessel density in the infarct area after cerebral I/R was detected by FITC.4.Major pharmacodynamic substances of TCE and and its mechanism(1)The main polyphenols in TCE were investigated,including gallic acid,corilagin,capric acid,ellagic acid,and ellanic acid(CA)by HPLC analysis.The cell viability and LDH assay were used to investigate the expression of TCE and its polyphenols in the vascular endothelial cells after OGD injury and determine the main effective substances in TCE.(2)Male SD rats(250±30 g)were selected and randomly divided into three groups: sham group,model group and CA(20 mg/kg)group with eight rats in each group.After MCAO operation,administration was given by gavage once daily seven times.At the end of the treatment,the head was decapitated and the brain tissue was taken.The expression of anti-oxidation pathway related proteins Nrf2,HO-1 and VEGF was detected by immunohistochemistry and Western blot.(3)Male SD rats(200±30 g)were selected and randomly divided into three groups: sham group,model group and CA(20 mg/kg)group with eight rats in each group.After intragastric administration one hour after administration,anhydrous ethanol was intragastrically administered(5 ml/kg)and the rats were anesthetized four hours later.The pylorus and cardia were ligated in the stomach and the rats were sacrificed and decapitated.The expression of anti-oxidation pathway related proteins Nrf2,HO-1 and VEGF was detected by immunohistochemistry and Western blot.Results1.The findings of vivo study showed that TCE had enormous cerebral protection.It significantly improved neurological function damage,reduced brain water content in infarct area,reduced cerebral infarct size,and reduced pathological injury and apoptosis.The anti-oxidation parameters and cytokines were converted to normal after TCE administration.The mechanism of action may be related to anti-oxidation and anti-inflammatory effects.The optimal dose was 500 mg/kg.The results of vitro study showed that after OGD modeling,PC12 cells grew slowly,the cells became swollen,round and clustered,and the cell count was lower than that in normal group.After TCE administration,the cells were significantly improved,the cell membrane was intact,the processes were prominent,the proliferation was accelerated,and the apoptosis was reduced.There was a significant difference compared with model group.Our research in cell migration revealed that TCE promoted PC12 migration.TCE demonstrated its neuronal protective effect in promoting proliferation,promoting migration and inhibiting apoptosis in PC12.The optimal drug concentration was 25 ?g/ml.2.UPLC-Q/TOF-MS and other modern biological analysis methods were employed to make a systematic metabolic profiling of rat MCAO model and plasma after administration.With the combination of multivariate statistical analysis methods and pattern recognition techniques,potential biomarkers with strong correlations between modeling factors and drug effects were screened and 12 potential specific markers were screened out to mainly intervene in lipid metabolism of the body.The antioxidant effect of brain tissue and energy supply were increased through amino acid metabolism,glycolysis and TCA cycle.In addition,the consistency of metabolomics and pharmacodynamics for pharmacodynamic evaluation was confirmed.3.The study of TCE mechanism demonstrated that TCE played a role in anti-oxidation and angiogenesis by regulating the expression of Nrf2,HO-1 and VEGF proteins.4.The results of HPLC analysis showed that the main polyphenols in TCE were gallic acid,corilagin,quercetic acid,ellagic acid and ellanic acid(CA).CA significantly increased the survival rate of endothelial cells and reduced the level of cell supernatant LDH,which was one of the main effective substances of TCE.Our study of CA anti-oxidation and angiogenesis found that CA regulated the anti-oxidation of Nrf2,HO-1,VEGF and other proteins,promoted angiogenesis and healing,which is consistent with the results of TCE antioxidation and angiogenesis.Conclusion1.The pharmacodynamic evaluation of TCE in vivo and in vitro was performed through pharmacodynamic evaluation in this study.The results showed that TCE can significantly reduce the cerebral infarct size and the neurological impairment in rats.TCE has a strong antioxidant and pro-angiogenic effects in enhancing PC12 cell proliferation,reducing apoptosis and promoting migration.2.Through the metabonomics study,12 potential specific markers were screened out to mainly intervene in lipid metabolism,amino acid metabolism,glycolysis,and TCA cycle of the body increase the antioxidant capacity of brain tissue and increase energy supply.3.TCE can play a role in anti-oxidation and angiogenesis by regulating the expression of Nrf2,HO-1 and VEGF proteins.4.The primary effective substance of TCE was selected as citrilire acid and the multiple organ vascular protective effect of CA was studied through cell activity test and LDH level determination.The findings suggest that CA could exert its antioxidant and angiogenesis effects by regulating the expression of Nrf2,HO-1 and VEGF protein.In conclusion,this study has not only revealed the mechanism of TCE from a new perspective but also provided a new method for the evaluation of the efficacy of TCE. |
PDF Full Text Request