Exploring The Neuroprotective Mechanism Of Mongolian Medicine Zhachong Thirteen-flavor Pill Against AIS From The Perspective Of TRPV1/NMDAR-mediated Excitotoxicit

Zachong Shisanwei Pill(ZSP),a classical formula of Mongolian medicine commonly used in the treatment of ischemic stroke(IS),has good clinical efficacy in both the acute and sequelae stages of IS.Research related to ZSP mostly focuses on the recovery of sequelae of IS,at present.There is a lack of research on the neuroprotection of ZSP to neurological damage in the acute stage,and its mechanism has not yet been elucidated.Our group previously explored the regulatory effect of ZSP on oxidative stress damage in neuronal cells and found that the formula can inhibit endogenous apoptosis,thereby protecting neuronal cells from oxidative stress damage.Therefore,with the aim of investigating the neuroprotective mechanism of action of ZSP against acute ischemic stroke(AIS),we analyze the formula of ZSP from the perspective of the theoretical system of Mongolian and Chinese medicine to figure out its theoretical mechanism of ZSP,and with the basis of clarifying the characteristics and efficacy of its formula,investigate its mechanism of action against AIS.In this study,we first analyze the composition and compatibility of ZSP based on the theoretical system of Mongolian and Chinese medicine,to explore the characteristics and therapeutic effects of the formula.Then the therapeutic effect of ZSP on AIS was verified on middle cerebral artery occlusion/reperfusion(MCAO/R)model rats,using bibliometric methods,to explore the relevant research on transient receptor potential(TRP)channels in the field of traditional Chinese medicine,and based on this to fit the relationship between them and the key receptor N-methyl-D-aspartate receptor(NMDAR)in the excitatory toxicity process of IS.Subsequently,whole cell patch clamp technique was used to verify the regulatory effect of ZSP on TRPV1-HEK293 cells.Untargeted metabolomics technology was used to predict and search for the intervention molecular pathways of this formula on AIS,and explore whether it contains components that can regulate TRPV1.Thereby,at the molecular level,explore the effect of this formula on the apoptosis of hippocampal neurons on the infarcted side of MCAO/R model rats as well as the correlation regulation of TRPV1/NMDAR by this formula,providing possible evidence for clarifying the neuroprotective mechanism of ZSP.Methods1.Theoretical analysis,analyzing the formula of ZSP from the perspective of the theoretical system of Mongolian and Chinese medicine respectively,digging into the characteristics of its prescription,and investigating its mechanism for AIS.2.Verify the protective effect of Zhachong Thirteen Flavor Pills on brain tissue in a rat MCAO/R model.The MCAO/R model rats were established using the suture-occluded method and divided into the sham group,the model group,the Edaravone(EDA)group,and the equivalent dose and high dose of ZPS groups(ZSP-M、ZSP-H).The EDA group was given an intraperitoneal injection of the Edaravone(2.7 mL-kg-1-d-1),ZSP-M and ZSP-H received a ZSP gavage with(0.18 g ·kg-1 ·d-1,0.036·kg-1·d-1)respectively.The Sham and Model groups received a corresponding volume of pure water gavage and a corresponding volume of physiological saline intraperitoneally,the EDA group received an equal volume of purified water gavage,while the ZSP-M and ZSP-H groups were given an equal volume of physiological saline by intraperitoneal injection.And evaluate the effect of ZSP on neurological deficits and upper limb muscle strength in MCAO/R model rats through mNSS neurological deficit score and grip traction test;Evaluate the effect of ZSP on cerebral infarction volume in MCAO/R model rats through TTC staining.Simultaneously,Western Blot was used to investigate the effect of ZSP on the expression changes of NF-200 and NeuN in the infarcted hippocampal tissue of MCAO/R model rats.3.Using bibliometric methods to fit the relationship between TRP channels and NMDAR.Based on Citespace and VOSviewer software,bibliometric analysis was conducted on the research progress of TRP channels in the field of traditional Chinese medicine and NMDAR in IS,and the relationship between TRP channels and NMDAR was fitted.4.Using whole cell patch clamp at the cellular level to demonstrate the effect of ZSP on the electrophysiology of TRPV1.The whole cell patch clamp technique was used to detect the effect of ZSP on the transmembrane current of TRPV1-HEK293 cells,in order to investigate whether this formula has a regulatory effect on TRPV1.5.Confirm the presence of TRP channel modulators in the brain tissue of MCAO/R model rats after administration of ZSP.MCAO/R model rats were established using the suture-occluded method and divided into the ZSP group and the blank group.The ZSP group received a ZSP gavage,while the blank group received an equal volume of pure water.Untargeted metabolomics technology was used to detect and compare the components in the brain tissues of two groups of rats,in order to determine the distribution of components in the brain tissues of MCAO/R model rats.Then molecular docking was used to explore whether there are components that can target TRPV1 and predict the intervention mechanism of this formula on AIS through analysis.6.Investigating the regulatory effect of TRPV1 on NMDAR in the brain tissue of MCAO/R model rats after the intervention of ZSP at the molecular level.MCAO/R model rats were established using the suture-occluded method and divided into the sham group,the model group,and the ZSP group.ZSP and purified water were administered orally,respectively.The effects of ZSP on the expression of Bax,Bcl-2,cytochrome C,Cleared Caspase-3,TRPV1,NMDAR1,NMDAR2B,pNMDAR2B,PSD-95,nNOS,CaMKII,pCaMKII in the infarcted hippocampal tissue of MCAO/R model rats were detected using RT-qPCR and Western Blot methods.Results1.From the theory of Mongolian medicine,the characteristics of ZSP are mainly based on the principles of removing stickiness,calming Heyi,regulating stomach fire,and repairing white vein damage,and it is consistent with the pathogenesis of Badagan(mucus)blocking the white pulse and causing disorder of Heyi within the pulse in Wusun sa disease.From the perspective of traditional Chinese medicine,the formulation of ZSP focuses on opening the orifices and inducing resuscitation.A large number of aromatic herbs that are mobile and penetrating are used together to strengthen the function of promoting qi and opening the body,dispelling impurities and turbidity.It is also supplemented with astringent and heavy-settling herbs to prevent the disadvantage of excessive dissipation of acrid warm herbs.The primary goal of the entire formula is to open the orifices and induce resuscitation,which is consistent with the primary goal of traditional Chinese medicine for stroke.2.(1)The weight of the Sham group rats showed a positive increase compared to before modeling,with no neurological deficits and normal upper limb muscle strength.However,the weight of the Model group,Model group,EDA group,ZSP-M group,and ZSP-H group rats showed a negative increase compared to before modeling,and showed significant neurological deficits and decreased upper limb muscle strength.Compared to the Model group,the EDA group,ZSP-M group,and ZSP-H group showed less weight loss,lighter neurological deficits,and improved upper limb muscle strength.This suggests that ZSP can improve neurological deficits and alleviate limb dysfunction in MCAO/R model rats,and there is no significant difference in the improvement of neurological deficits and limb dysfunction between high-dose and equivalent doses of ZSP.(2)Compared with the Model group rats,the infarct volume of brain tissue in the EDA group,ZSP-M group,and ZSP-H group rats was significantly reduced(p<0.001,p<0.001,p<0.001).However,there was no significant difference in infarct volume among the three intervention groups(p>0.05),indicating that ZSP can reduce the infarct volume of the brain,and there was no significant difference between the high dose and equivalent dose of ZSP.(3)Compared with the Sham group,the protein expression of NF-200(p<0.001,p<0.001,P<0.001,P<0.001)and NeuN(p<0.001,P<0.01,P<0.01,p<0.05)in the infarcted hippocampal tissue of rats in the Model group,Model group,EDA group,ZSP-M group,and ZSP-H group significantly decreased,indicating that the MCAO/R model can cause neuronal damage in rats.Compared with the Model group rats,the expression of NF-200(p<0.001,p<0.001,p<0.01)and NeuN(p<0.01,p<0.01,p<0.01)in the right hippocampal tissue of rats in the EDA group,ZSP-M group,and ZSP-H group was upregulated,indicating that ZSP can alleviate neuronal damage in the infarcted hippocampal tissue of MCAO/R model rats and play a neuroprotective role,and there was no significant difference between the high magnification dose and the equivalent dose ZSP(p>0.05,p>0.05).3.(1)In the field of traditional Chinese medicine in China,research has been conducted based on the TRP channels and it has been found that traditional Chinese medicine which was warm acrid can target and regulate TRPV1 to exert its therapeutic effect.and the activation temperature required for different Thermo-sensing TRP Channels is different,corresponding to the "heat and cold" theory of traditional Chinese medicine,which is used to explain some disease symptoms and treatment principles in traditional Chinese medicine theory.In terms of diseases,kidney disease,respiratory and digestive diseases are hot topics in the application of TRP channels.Relatively speaking,there is a lack of research on neurological diseases.(2)In the research of NMDAR in IS from 2012 to 2022,the role of NMDAR in excitotoxicity and the exploration of drug neuroprotective effects based on this have become hot topics in recent years.The NMDAR2B/PSD-95/nNOS signal transduction trimer is a hot topic in the study of NMDAR-mediated excitotoxicity.4.(1)ZSP can induce the production of transmembrane currents in TRPV1-HEK293 cells,in two independent experiments,the peak values of the ZSP-induced transmembrane current were 23.48%and 23.54%of the capsaicin-induced peak values,respectively.(2)ZSP can inhibit the transmembrane currents of TRPV1-HEK293 cells induced by capsaicin,in two independent experiments,The inhibition rates of ZSP on the transmembrane current induced by Capsaicin are 42.54%and 54.78%,respectively.5.(1)According to Untargeted metabolomics analysis,there are 20 positive ion components in the brain tissue of MCAO/R model rats,including moracin O,glyceryl linolenate,4-aminophenol,acetylpterosin C,3,4,5-trimethoxycinnamic acid,[12]-gingerdione、monoolein,thymol,2-nonanone,3-hydroxy-28,13-lupanolide,(1alpha,6alpha,7alphaH)-2,4(15)-copadiene,2-phenylethyl beta-D-glucopyranoside,3,5-dimethoxy-4-hydroxybenzaldehyde,camelledionol,6-O-methylcodeine,(6beta,24R)-6-hydroxystigmast-4--en-3-one,vanillic acid,licoisoflavone A,egonol,quinoside D,two anionic components,namely capsaicin and 12,13-EODE.(2)There was a total of 398 common targets,between the targets related to AIS,and the targets which correspond to component distribution of ZSP in the brain tissue of MCAO/R model rats.GO enrichment analysis was conducted on common targets,with a total of 2410 entries under the biological process(BP)category,169 entries under the cellular component(CC)category,and 280 entries under the molecular function(MF)category.(3)KEGG enrichment analysis was carried out for common targets with p<0.05 as the boundary,a total of 216 pathways could be enriched.The pathways named after tumors/cancer and infectious diseases,which were unrelated to AIS,were screened out,and the remaining 155 pathways were ranked according to p-value from small to large.The top 20 entries were respectively:Neuroactive ligand-receptor interaction,PI3K-Akt signaling pathway,cAMP signaling pathway,Rapl signaling pathway,Calcium signaling pathway,Focal adhesion,Inflammatory mediator regulation of TRP channels,Sphingolipid signaling pathway,Ras signaling pathway,Endocrine resistance,Insulin resistance,Th17 cell differentiation,MAPK signaling pathway,HIF-1 signaling pathway,Serotonergic synapse,Regulation of actin cytoskeleton,FoxO signaling pathway,ErbB signaling pathway,Phospholipase D signaling pathway,Neurotrophin signaling pathway.(4)Through molecular docking,it was found that Moracin O,Licoisoflavone A,Egonol,capsaicin,vanillic acid,and thymol may regulate their expression by binding to TRPV1,thereby exerting corresponding effects.5.(1)In two independent patch clamp tests,the peak transmembrane current of TRPV1 induced by ZSP was 23.48%and 23.54%of the peak induced by Capsaicin,respectively.(2)In two independent patch clamp tests,Capsazepine reduced the transmembrane current induced by Capsaicin by 88.21%and 91.89%,while ZZP reduced the transmembrane current induced by Capsaicin by 42.54%and 54.78%,respectively.6.(1)Compared with the Sham group,the Model group showed a significant increase in the protein expression level of increase in Bax/Bcl-2,cytochrome C,and Cleared-Caspase-3.Compared to the Model group(p<0.001,p<0.001,p<0.01),the protein expression of Bax/Bcl-2,cytochrome C,and Cleared-Caspase-3 in the ZSP group was significantly downregulated(p<0.001,p<0.001,p<0.01).(2)At the expression level of mRNA,compared to the Sham group,the expression of TRPV1,NMDAR1 and NMDAR2B in the Model group rats were significantly increased(p<0.05,p<0.001,p<0.01)while the expression of PSD-95 was decreased(p<0.001).However,compared to the Model group,the expression of TRPV1,NMDAR1,and NMDAR2B in the ZSP group was significantly downregulated(p<0.05,p<0.001,p<0.05)while the expression of PSD-95 was upregulated(p<0.05).At the expression level of protein,compared to the Sham group,the expression of TRPV1,NMDAR1,NMDAR2B,pNMDAR2B,nNOS,CaMKII,pCaMKII increased(p<0.01,p<0.001,p<0.01,p<0.01,p<0.05,p<0.01,p<0.001),while the expression of PSD-95 decreased(p<0.001)in the Model group.When the ZSP group compared to the Model group,the expression of TRPV1,NMDAR1,NMDAR2B,pNMDAR2B,nNOS,CaMKⅡ,and pCaMKⅡ was downregulated p<0.001,p<0.01,p<0.05,p<0.05,p<0.01,p<0.05,p<0.01),and the expression of PSD-95 was increased(p<0.05)in increased.ConclusionFrom the different perspectives of the theoretical systems of Mongolian medicine and traditional Chinese medicine,the composition and compatibility of the Zhachong Shisanwei Pill are consistent with the treatment principles for the acute phase of AIS in the corresponding theories,indicating the rationality of the application of ZSP in the treatment of AIS has been theoretically confirmed.ZSP has been validated on MCAO/R model rats that it can reduce neuronal damage,reduce cerebral infarction volume,improve neurological deficits and limb dysfunction,to exert neuroprotective effects.Traditional Chinese medicine which was warm acrid can target and regulate TRPV1,and NMDAR2B and excitotoxicity biomarkers NMDAR2B/PSD-95/nNOS constituted of which,are cutting-edge hotspots in the research of NMDAR in IS.ZSP can inhibit the activation of TRPV1 on TRPV1-HEK293 cells and reduce its mediated transmembrane current.This effect is achieved through the synergistic action of ZSP through multiple components,targets,and pathways.ZSP can exert its neuroprotective effect by inhibitting TRPV1,regulating NMDAR/PSD-95/nNOS,downregulating the expression of nNOS,and alleviating neuronal apoptosis caused by excitotoxicity.