Study On The Effect And Mechanism Of Guanxin Shutong On Cerebral Ischemia Injury Based On “Dispelling Stasis To Promote Regeneration”

BackgroundIschemic stroke has the characteristics of high morbidity,disability,fatality,and recurrence rates,which seriously threaten human health and quality of life.The pathogenesis of stroke is complex,involving multiple pathological processes such as oxidative stress,calcium overload,excitatory amino acid toxicity,inflammatory response,apoptosis,energy metabolism disorders,and angiogenesis disorders.Traditional Chinese medicine(TCM)has the advantages of multi-component,multi-target,and overall regulation,and can act on multiple pathological processes after stroke.Therefore,TCM is an important breakthrough point for developing new drugs in the prevention and treatment of stroke.TCM believes that the symptoms of stroke can be divided into the wind,fire,phlegm,blood stasis,and deficiency.However,blood stasis is the pathological core throughout the entire stroke.Therefore,TCM with the effect of “dispelling stasis to promote regeneration” is often used in treating stroke.The purpose is to dissipate blood stasis in the body,so that new blood can be regenerated and blood vessels can be unblocked.It is similar to the “recanalization and angiogenesis of blood vessels” in modern medicine.Therefore,finding TCM with the effect of “dispelling stasis to promote regeneration” may be the key to developing new drugs for the prevention and treatment of stroke.The research group has devoted to the studies on the pharmacodynamic substances of TCMs on cardio-cerebral diseases and the Mechanisms of Action.With the support of several National Science Foundation of China,we have completed the protective mechanism and compatibility of Danhong injection,Safflower-Frankincense on cardiocerebral diseases.In the recent excavation of TCM prescriptions,we noticed that Guanxin Shutong Capsule(GXSTC),composed of Choerospondiatis Fructus,Salviae miltiorrhizae Radix et Rhizoma,Caryophylli Flos,Borneolum Syntheticum,and Concretio Silicea Bambusae,has the effect of promoting blood circulation and removing blood stasis,clearing meridians and collaterals,promoting qi and relieving pain,which is mainly used in the clinical treatment of coronary heart disease and angina pectoris.But from the perspective of efficacy,we speculate that GXSTC also has the effect of “dispelling stasis to promote regeneration”.In recent years,several clinical trials have further demonstrated that GXSTC has a significant clinical effect in treating ischemic stroke,and could markedly improve the nerve function of stroke patients.However,the pharmacodynamic components and potential mechanisms of action of GXSTC against CBVDs remain unclear.However,most of the current studies on the pharmacodynamic substances and their pharmacological mechanism of TCM are based on the research idea of a single component,a single target,and a single pathway.It is difficult to scientifically interpret pharmacodynamic substances and their mechanisms of action of TCM against complexdiseases,and it is also impossible to reflect the “holistic view” of traditional Chinese medicine in treating diseases.In recent years,systems pharmacology has emerged internationally.It uses a variety of public databases to collect,predict and screen drug and disease-related targets,construct a "drug-gene-target-disease" network,and reveal the relationship between drug and disease from a holistic perspective,which is in accordance with the therapeutic strategies of TCMs.Therefore,it has been widely used to investigate the complex pharmacological mechanisms of TCMs.In this study,we intend to systematically study the pharmacodynamic substances of GXSTC and its mechanism of action on cerebrovascular diseases by using systems pharmacology.Objectives1.To clarify the effect of GXSTC on cerebral ischemia injury,screen its pharmacodynamic substances,and predict its possible molecular mechanism based on systematic pharmacology and pharmacological experiments;2.To screen the chemical components of GXSTC entering the blood and brain by using UPLC-MS/MS and GC-MS methods,and to determine its possible pharmacodynamic substances and compatibility components;3.To clarify the effect and compatibility of GXSTC components against cerebral ischemia injury in vivo and in vitro,and determine the optimal compatibility ratio;4.To evaluate the effect of promoting angiogenesis and compatibility of GXSTC components on cerebral ischemia injury in vivo and in vitro,and pay attention to the regulatory mechanism of the p-ERK/HIF-1α/VEGFA signal axis.MethodsChapter 1 Study on the effect and mechanism of GXSTC on cerebral ischemia injuryFirst,the main components of GXSTC were identified by using HPLC-DAD and literature research.Then,the targets and related mechanisms of these components against cerebral ischemia were predicted by systematic pharmacology.Finally,the protective effect and molecular mechanism of GXSTC against cerebral ischemia injury were further verifiedin vivo experiment.The SD rats were randomly divided into sham,MCAO,MCAO+GXSTC(0.5,1.0,2.0 g/kg)groups.Rats in the MCAO+GXSTC groups were pretreated with GXSTC(0.5,1.0,and 2.0 g/kg per day,respectively)via intragastric administration for 7 days,and rats in the Sham and MCAO groups were pretreated with the same amount of 0.9% saline solution.Then the rats were anesthetized,and a MCAO was established after administration on the 7th day.After 24 h of reperfusion,SD rats in each group were evaluated for neurological function score,TTC staining was carried out to detect cerebral infarction volume,HE staining was performed to detect pathological damage,and Western blotting was performed to detect the protein expression levels of core targets VEGFA,e NOS,COX-2,and c-Jun.Chapter 2 Screening the pharmacodynamic substances of GXSTC on cerebral ischemia injuryFifty SD rats were randomly divided into sham group,Sham+GXSTC group(2.0 g/kg),MCAO model group,and MCAO+GXSTC-2.0 g/kg treatment group.After 5 days of adaptive feeding,the MCAO and MCAO+GXSTC groups underwent the standard MCAO model.Subsequently,each group was given GXSTC or normal saline by intragastric administration,twice a day,for 5 consecutive days.Then,the blood was collected from the abdominal aorta at 1 h after administration on the 5th day,and the cerebral cortex of rats was collected and homogenized.The corresponding solvent was added to extract the plasma and brain tissue homogenate.UPLC-MS/MS method was used to detect gallic acid,5-hydroxymethyl furfural,danshensu,protocatechualdehyde,protocatechuic acid,ellagic acid,rosmarinic acid,salvianolic acid A,salvianolic acid B,dihydrotanshinone I,tanshinone I,cryptotanshinone,tanshinone IIA and eugenol in plasma and brain tissue of each group.The distribution of eugenol and borneol in plasma and brain tissue of each group was detected by the GC-MS method.Chapter 3 Study on the protective effect of GXSTC components on cerebral ischemia injuryUsing OGD/R as a model in vitro,CCK-8 was used to screen the toxic effects of ellagic acid(E),salvianolic acid B(S),eugenol(D),and borneol(B)at a series concentration of 0~128 μg/m L on BMECs.Then,the safe dose ranges of four pharmacodynamic components on BMECs were determined.Based on this,the pharmacodynamic concentration and optimal compatibility ratio of four components were further screened.Finally,CCK8 was used to detect the effects of different doses of ESDB on the cell viability and cell morphology of OGD/R-injured BMECs.In the MCAO model,the SD rats were randomly divided into the sham,MCAO,Edeficiency(SDB),S-deficiency(EDB),D-deficiency(ESB),B-deficiency(ESD),ESDB,and nimodipine groups.After adaptive feeding for 7 days,except for the sham group,the other groups were all performed with the standard MCAO model.At the same time,each group was given the therapeutic drug or normal saline by intragastric administration,once a day,for 8 consecutive days.Subsequently,the blood was collected from the abdominal aorta and centrifuged,and then the brain was removed quickly.Then,the brain water content and neurological function was evaluated,TTC staining was carried out to detect cerebral infarction volume,and HE staining was performed to detect pathological damage.Finally,immunohistochemical staining was performed to detect the expression levels of tight junction proteins ZO-1 and Occludin in the brain tissue,evaluating the degree of damage to the blood-brain barrier.Chapter 4 Study on the effect and molecular mechanism of GXSTC components in promoting angiogenesis after cerebral ischemiaIn the MCAO model and OGD/R model,the SD rats were all randomly divided into the sham,MCAO,E deficiency(SDB),S deficiency(EDB),D deficiency(ESB),B deficiency(ESD),ESDB,and nimodipine groups.In vitro experiments,Ed U kit was used to detect cell proliferation,and cell scratching and Matrigel tube formation were performed to investigate their effects on the cell function of BMECs.To evaluate ESDB and its disassembled groups on the activation of the p-ERK/HIF-1α/VEGFA signal axis,westernblotting was performed to detect the expression levels of p-ERK,p-P70S6 K,HIF-1α,and its downstream related proteins(VEGFA and its receptors VEGFR2,e NOS,MMP2,MMP9).In vivo experiments,double-labeled immunofluorescence was used to detect the microvessel density of the cerebral cortex.Immunofluorescence and western blotting were used to detect the expression levels of p-ERK,HIF-1α,VEGFA and its receptor VEGFR2,to further evaluate the activation effect of ESDB and its disassembled groups on pERK/HIF-1α/VEGFA signal axis.Finally,HIF-1α silenced by si RNA technology,and pERK inhibited by a specific inhibitor(U01216)were performed to verify the regulatory mechanism of related proteins.ResultsChapter 1 Study on the effect and mechanism of GXSTC on cerebral ischemia injuryFirst,15 principal components of GXSTC were identified by HPLC and literature research.Then,the core targets of these components against cerebral ischemia injury were predicted through systems pharmacology and molecular docking,which were involved in PTGS2,RELA,TNF,JUN,MMP9,CASP3,MAPK1,NOS3,VEGFA,etc.The core pathways were involved in the regulation of AGE-RAGE,fluid shear stress and atherosclerosis,HIF-1,IL-17,and TNF signaling pathways.These results suggested that GXSTC can exert a protective effect on cerebral ischemia injury by a multi-component,multi-target,and multi-pathway synergy.In MCAO-injured rats,GXSTC significantly improved the neurological function,reduced the infarct volume,and decreased the rate of impaired neurons in a dose-dependent manner.In addition,we selected vascular function-related proteins(VEGFA and e NOS),inflammation-related proteins(COX-2),and neuronal apoptosis-related protein(c-Jun)from 12 core targets to further verify the molecular mechanisms.Finally,western blot analyses indicated that GXSTC markedly upregulated the expression of VEGFA and e-NOS,and downregulated the expression of COX-2 and c-Jun in MCAO-induced rats.These findings provide evidence that GXSTC can exert a multi-target synergetic effect on CBVDsby maintaining the vascular endothelial function,inhibiting neuronal apoptosis and inflammatory processes.Chapter 2 Screening the pharmacodynamic substances of GXSTC on cerebral ischemia injuryIn this chapter,we established a UPLC-MS/MS method for the determination of 14 components,and a GC-MS method for 2 volatile components in GXSTC,to investigate the distribution changes of these components in plasma and brain tissue between healthy rats and MCAO rats.The test results suggested that there were 10 components of GXSTC that can be prototyped into the blood.Among them,three components derived from Choerospondiatis Fructus,five components derived from Salviae miltiorrhizae Radix et Rhizoma,one component derived from Caryophylli Flos,and one component derived from Borneolum Syntheticum.Moreover,compared with the sham+GXSTC group,the contents of gallic acid,ellagic acid,protocatechuic acid,dihydrotanshinone I,tanshinone I,and tanshinone IIA decreased significantly,while the plasma levels of eugenol decreased significantly in rat plasma of MCAO+GXSTC group.In addition,ellagic acid,salvianolic acid B,and borneol can also be absorbed into the brain as prototypes,and their contents in the MCAO+GXSTC group were higher than those in the sham+GXSTC group,suggesting that the three components may be the key pharmacodynamic substances of GXSTC against cerebral ischemia injury.It was worth mentioning that eugenol was the only main component of Caryophylli Flos in rat plasma,so eugenol was also included in subsequent studies.Therefore,we finally determined ellagic acid,salvianolic acid B,eugenol,and borneol as the representative components of the parent drug,as the preferred compatibility group for the subsequent study on the mechanism and compatibility rules of GXSTC on cerebral ischemia injury.Chapter 3 Study on the protective effect of GXSTC components on cerebral ischemia injuryIn vitro study,we screened the toxic effects and pharmacodynamic concentration range of four components on BMECs,and determined that the optimal concentration points of ellagic acid,salvianolic acid B,eugenol,and borneol were 5,5,6.25,and 3.125 μM,in respectively.To maximize the efficacy of the components after compatibility,we use the optimal concentration point of each component as the compatibility ratio to obtain the molar concentration ratio of them as E:S:D:B = 8:8:10:5(equivalent to the mass concentration ratio E:S:D:B =3:7:2:1).To clarify the brain protective effect and synergistic effect of the four components after compatibility,the demolition study of equal radio on ESDB was also implemented.The results showed that ESDB and its disassembled groups could significantly improve the neurological function score and cerebral edema of MCAO rats,reduce the volume of cerebral infarction and pathological damage,and up-regulate the expression levels of tight junction protein(ZO-1 and Occludin)in the cerebral cortex of MCAO rats,further reduce BBB damage.In particular,the efficacy of the ESDB group was much better than that of its disassembled groups and the nimodipine group.The efficacy of the ESD group was comparable to that of the positive drug(nimodipine)group,while it was slightly inferior to that of the ESDB group.The brain protective effect of the E and S deficiency group was weaker,suggesting that ellagic acid and salvianolic acid B may be the indispensable components in the formula,and the addition of eugenol or borneol can further enhance the efficacy of these two components.Chapter 4 Study on the effect and molecular mechanism of GXSTC components in pro-angiogenesis after cerebral ischemiaThe effect and molecular mechanism of ESDB in promoting angiogenesis after cerebral ischemia were systematically investigated in vivo and in vitro.The results suggest that ESDB and its disassembled groups can significantly promote the proliferation,migration,increase lumen formation of BMECs,and increase the density of cerebral microvessels in MCAO rats,which suggested ESDB had a strong pro-angiogenesis effect.The results of immunofluorescence and western blot indicated that ESDB and itsdisassembled groups could regulate the expression levels of p-Erk1/2,p-70S6 K,HIF-1α,and VEGFA.At the same time,the further demolition study suggested that ellagic acid and salvianolic acid B may play an important role in the prescription,and were indispensable components for the prescription to exert “dispelling stasis to promote regeneration”.In the presence of ellagic acid or salvianolic acid B,the addition of eugenol or borneol can further increase the efficacy of these two components,and the synergistic effect of eugenol was stronger than that of borneol.The results of mechanism verification suggested that inhibiting the expression of HIF-1α could significantly reduce the effect of ESDB on promoting the proliferation,migration,and tube formation of BMECs.At the same time,inhibition of HIF-1α can reduce the effect of ESDB on up-regulating the expression of VEGFA and other related proteins,suggesting that the pro-angiogenesis effect of ESDB depended on the activation of HIF-1α.However,the inhibition of p-Erk1/2 can reduce the effect of ESDB on up-regulating the expression of HIF-1α and VEGFA.The above results suggested that ESDB can activate HIF-1α/VEGFA pathways through p-Erk1/2,then initiate the expression of downstream targets,and finally play a role in pro-angiogenesis after cerebral ischemiaConclusion1.GXSTC can exert a protective effect on cerebral ischemia injury by a multicomponent,multi-target,and multi-pathway,and the mechanism may be related to maintaining the vascular endothelial function,inhibiting neuronal apoptosis,and inflammation,and other processes.2.There were 10 components of GXSTC that can be prototyped into the blood,and 3 of them can also be distributed as prototypes into the brain tissue.Among them,eugenol,ellagic acid,salvianolic acid B,and borneol were the key pharmacodynamic substances for their TCMs,and can be used as the preferred compatibility group for the subsequent study.3.The combination of E,S,D and B exerted a protective effect and strong synergism on cerebral ischemia injury,and the efficacy of the whole formula group was much better than that of the disassembled groups,and nimodipine group.4.ESDB can promote angiogenesis after cerebral ischemia in rats,and its mechanism may be related to the up-regulation of the p-Erk1/2/HIF-1α/VEGFA signaling axis.At the same time,the further demolition study suggested that ellagic acid and salvianolic acid B were indispensable components for the prescription to exert “dispelling stasis to promote regeneration”.And the addition of eugenol or borneol can further increase the efficacy of these two components.