Research On Role And Mechanism Of TRPC1 In Myocardial Ischemia/Reperfusion Injury And Screening And Evaluation Of The Active Ingredients Of Salvia Miltiorrhiza Bunge Targeting TRPC1

Background:Acute myocardial infarction（AMI）is a myocardial necrosis caused by acute,persistent ischemic and hypoxia in the coronary arteries.Reperfusion treatment is the most primary therapeutic measure for AMI.However,drop of blood pressure,cardiac dysfunction and other phenomena that occurred during a period of time after the recovery of blood flow not only cannot restore cardiac function,but also aggravate cardiac dysfunction and structural impairment,i.e.,myocardial ischemia/reperfusion（I/R）injury.Studies have indicated that myocardial injury caused by reperfusion injury accounts for more than 50%of the total myocardial injury after the recovery of blood flow recovery.Exploration and discovery of new targets for drugs and new strategies for effective intervention will be the hot and difficult issues to be urgently solved in this field.Transient receptor potential channel（TPRC）is an important non-selective cationic receptor channel located on the cell membrane.It is currently believed that during the reperfusion period,TRPC family mainly participates in Ca²⁺overloading process and regulates I/R injury by mediating Ca²⁺influx in cells.TRPC1 as the Ca²⁺channel mostly researched at the earliest time has not been recognized deep enough.Particularly,its role in the occurrence and development of heart disease remain unclear.Studying the molecular mechanisms and regulatory pathways of myocardial I/R injury induced by TRPC1 under pathological conditions will undoubtedly greatly help to design targeted drugs for TRPC1 channels.Prevention and treatment of myocardial I/R injury through traditional Chinese medicine（TCM）has been proved in basic and clinical experiments.Salvia miltiorrhiza Burge as one of the most commonly used drugs for promoting blood circulation and removing blood stasis in China can be used for AMI treatment.Researches show that Salvia miltiorrhiza Burge can reduce intracellular Ca²⁺overloading and myocardial I/R injury.Other reports show that various cardioprotective effects of Salvia miltiorrhiza Burge are closely correlated with TRPC1,suggesting the possibility for TRPC1 to participate in myocardial protection of Salvia miltiorrhiza Burge active ingredients as an important target.Therefore,based on the three-dimensional structure of TRPC1 protein,docking and virtual screening methods are used to select the active ingredients of Salvia miltiorrhiza Burge,and further verify the pharmacological effect and mechanism of active ingredients.This research aims to clarify the biological basis of TCM for treating diseases and new targets for drug intervention by modern medicine,biology,TCM and other multidisciplinary innovative technologies,and hopes to explore the active ingredients of TCM by a new model of new Chinese medicine discovery and evaluation“homologous modeling-virtual screening-molecular docking-affinity testing-activity evaluation”.It is expected that this research will provide an entry point for the future research on pharmacodynamic substance basis and coordinated action mechanism research according to TCM multi-component,multi-target.Objectives:To study the mechanism and regulatory pathways of TRPC1 mediating myocardial I/R injury under pathological conditions,and further provide a new drug target for the treatment of myocardial I/R injury.Finding of the exact active ingredients of Salvia miltiorrhiza Burge based on TRPC1 target,provide the research foundation and ideas for pharmacodynamic substance basis and coordinated action mechanism.Methods:1.In vivo experimental methodsThe left coronary artery ligation of wild type（WT）,Trpc1 and Trpc6 knockout mice(Trpc1^-/-,Trpc6^-/-)in the I/R group was performed for myocardial I/R injury（30 min/24 h）,while Sham group underwent Sham surgery.Rosmarinic acid（RosA）was administered at a dose of 100 mg/kg p.o.continuously for 1 week.（1）q PCR method was used to detect Trpc m RNA in tissue（Sham,I/R）of WT mice;Ogdh m RNA in tissue（I/R）of WT and Trpc1^-/-mice;Trpc1 and Ogdh m RNA in tissues（Sham,RosA,I/R,I/R+RosA）of WT mice.（2）Western blot method was used to detect TRPC proteins in tissue of WT mice（Sham,I/R）;TRPC1 protein in tissue（I/R）of WT,Trpc1^-/-mice;TRPC1 and OGDH（oxoglutarate dehydrogenase,OGDH）proteins in tissues of WT mice（Sham,RosA,I/R,I/R+RosA）.（3）RNA-Seq（transcriptome sequencing）technology was used to screen the differentially expressed genes of tissue（I/R）of WT,Trpc1^-/-mice for enrichment analysis.（4）M-type ultrasound imaging system was used to detect left ventricular ejection fraction（EF）and fractional shortening（FS）（Sham,I/R）of WT,Trpc1^-/-,Trpc6^-/-mice.（5）TTC+Evans blue double staining method was to determine the percentage of myocardial infarction（Sham,I/R）in WT,Trpc1^-/-,Trpc6^-/-mice;myocardial infarction（Sham,RosA,I/R,I/R+RosA）of WT mice.（6）H&E staining was used to observe the pathological changes in the AAR region of myocardium（Sham,I/R）in WT,Trpc1^-/-,Trpc6^-/-mice;pathological changes in the AAR region of myocardium（Sham,RosA,I/R,I/R+RosA）of WT mice.（7）TUNEL staining method was used to detect apoptosis in the AAR region of myocardium（Sham,I/R）of WT,Trpc1^-/-,Trpc6^-/-mice.（8）DHE-ROS active oxygen method was used to detect the changes of ROS in the AAR region of myocardium（Sham,I/R）of WT,Trpc1^-/-,Trpc6^-/-mice;ROS changes in the AAR region of myocardium（Sham,RosA,I/R,I/R+RosA）in WT mice（9）Cis aconitase（ACO）activity method was used to detect the changes in the aconitase activity in the AAR region of myocardium（Sham,I/R）of WT,Trpc1^-/-,Trpc6^-/-mice.（10）ELISA method was used to detect the activities of OGDH and PDH（pyruvate dehydrogenase,PDH）（Sham,I/R）in WT,Trpc1^-/-mice;the serum CK-MB（creatine kinase-MB,CK-MB）and c Tn I（cardiac troponin I,c Tn I）levels（Sham,RosA,I/R,I/R+RosA）in WT mice.2.In vitro experimental methodsHL-1 mouse cardiomyocytes were transfected with si RNA（si Con,si Trpc1,si Trpc3,si Trpc6）and plasmids（pc DNA3.1-vector,pc DNA3.1-Trpc1,pc DNA3.1-Ogdh）.OGD/R（9 h/6 h）was performed.The Normoxia group was cultured in the incubator for 15 h as a control.HL-1 cells were pretreated with RosA 50 m M for 6 h.（1）Fura-2/AM（calcium ion fluorescence probe）was used to detect the calcium ion concentration（Normoxia,OGD/R）in si Con,si Trpc1,si Trpc3,si Trpc6,pc DNA3.1-vector,pc DNA3.1-Trpc1 cells.（2）JC-1 staining was used to detect changes in mitochondrial membrane potential of apoptotic cells（Normoxia,OGD/R）in si Con,si Trpc1,si Trpc3,si Trpc6,pc DNA3.1-vector,pc DNA3.1-Trpc1 cells.（3）Annexin V-FITC/PI double staining flow cytometry was used to detect the apoptosis（Normoxia,OGD/R）in si Con,si Trpc1,si Trpc3,si Trpc6,pc DNA3.1-vector,pc DNA3.1-Trpc1 cells.（4）DHE-ROS（dihydroethidium-ROS,DHE-ROS）was used to detect ROS changes（Normoxia,OGD/R）in si Con,si Trpc1,si Ogdh,si Lgals3,si Trim30d,pc DNA3.1-vector,pc DNA3.1-Trpc1,si Trpc1+pc DNA3.1-Ogdh cells;ROS changes in ROS（control,RosA,OGD/R,OGD/R+RosA）in HL-1 cells.（5）ACO（cis aconitase）activity method was used to detect the aconitase activity of aconitase（Normoxia,OGD/R）in si Con,si Trpc1,si Trpc3,si Trpc6,pc DNA3.1-vector,pc DNA3.1-Trpc1 cells.（6）q PCR method was used to detect Ogdh m RNA（Normoxia,OGD/R）in si Con,si Trpc1,si Trpc3,si Trpc6,pc DNA3.1-vector,pc DNA3.1-Trpc1 cells;Trpc1 m RNA（control,RosA,OGD/R,OGD/R+RosA）in HL-1cells.（7）Western blot assay was used to detect Ca Nα,p-NFκB p65,p-IκB-α,OGDH protein（Normoxia,OGD/R）in si Con,si Trpc1,si Trpc3,si Trpc6,pc DNA3.1-vector,pc DNA3.1-Trpc1 cells;TRPC1,Ca Nα,p-NFκB p65,p-IκB-α,OGDH protein（control,RosA,OGD/R,OGD/R+RosA）in HL-1 Cells.（8）HEK293 cells overexpressing Nfκb p65were established,and the interaction between NFκB and Ogdh promoter was detected by Ch IP experiment.3.Data acquisition and virtual screening methodTraditional Chinese Medicine Systems Pharmacology Database and Analysis Platform（TCMSP）was used for the retrieval of Salvia miltiorrhiza Burge compounds for preliminary screening by referring to the Lipinski’s Rule of Five and other relevant screening conditions.A protein homology 3D model of TRPC1 was constructed,and the quality of the 3D model of TRPC1 structure was evaluated by Ramachandran plot.The compound group was comprehensively scored by Auto Dock Vina software based on shape matching and energy matching between receptor and ligand,and molecular docking binding mode analysis was further performed on the ideal compounds（highest score）screened.Furthermore,surface plasmon resonance（SPR）was used to determine the affinity of the compound to TRPC1 protein.Results:1.TRPC1 plays a critical role in myocardial I/R injury.（1）TRPC1 was closely correlated with myocardial I/R injury.q PCR results showed that Trpc1,Trpc3,and Trpc6 m RNA were increased in the AAR region of myocardium;Western blot results showed that the levels of TRPC1,TRPC3,and TRPC6 protein in the AAR region of myocardium were increased,among which,the changes in Trpc1 m RNA and protein were the most obvious.（2）TRPC1 mediated the outburst of Ca²⁺influx in the cardiomyocytes after OGD/R injury.Fura-2/AM results showed that inhibiting Trpc1could significantly reduce Ca²⁺influx after OGD/R injury,and Trpc1 overexpression could aggravate Ca²⁺influx.（3）TRPC1 played a key role in cell OGD/R injury.JC-1staining results showed that inhibiting Trpc1 could reduce the decrease ofΔΨm after OGD/R injury,and Trpc1 overexpression could induce the decrease ofΔΨm;Annexin V-FITC/PI results showed that inhibiting Trpc1 could alleviate apoptosis rate after OGD/R injury,while Trpc1 overexpression could increase apoptosis rate.（4）TRPC1 knockout improved the myocardial I/R injury in vivo.M-type ultrasound results showed that myocardial EF and FS function of Trpc1^-/-mice were improved;TTC+Evans blue results showed that the percentage of myocardial infarction size in Trpc1^-/-mice was significantly reduced;H&E results showed the myocardial pathological injury was markedly alleviated in the AAR region of myocardium;TUNEL results showed that the number of myocardial apoptotic positive cells was decreased significantly in the AAR region of myocardium.2.TRPC1 affects ROS production by regulating key molecules in the NFκB signaling pathway,and aggravates myocardial I/R damage.（1）RNA-Seq was used to screen gene differences in myocardial infarction areas between Trpc1^-/-mice and WT mice.Results showed that the expression levels of 1008genes in Trpc1^-/-mice group were changed significantly compared with the WT mice group,of which there were 731 upregulated genes（72.52%）and 277 down-regulated genes（27.48%）.GO-BP analysis results showed that oxidation reduction process,metabolic process and ROS generation process were the main biological process involved in the differential genes.Most of the genes involved in these processes are related to oxidative stress.（2）TRPC1 could mediate the generation of ROS in the AAR region of myocardium and HL-1 cells after OGD/R injury.DHE-ROS results showed that ROS generation in the AAR region of Trpc1 gene knockout mice was reduced,and silencing Trpc1 could reduce ROS generation after OGD/R injury in HL-1 cells;ACO results showed that the ACO activity in the AAR region of myocardium in Trpc1 gene knockout mice was enhanced,and silencing Trpc1 could also enhance the activity of ACO after OGD/R injury in HL-1 cells.（3）TRPC1 aggravated myocardial I/R injury by upregulating Ogdh and increasing ROS production.After analyzing the differential genes related oxidative stress in the GO-BP processes,the results showed that Ogdh gene difference was the most obvious.Ogdh was down-regulated by 29.53 times in the AAR region in Trpc1^-/-mice.The results of q PCR and western blot showed that the Ogdh m RNA and protein levels decreased in the AAR region in Trpc1^-/-mice,and the results of ELISA showed that the activity of OGDH also decreased in the AAR region in Trpc1^-/-mice.DHE-ROS staining found that silencing Ogdh gene could reduce ROS generation after OGD/R injury,and overexpression Ogdh gene reversed the reduction of ROS generation caused by silencing Trpc1 gene.（4）After myocardial I/R injury,TRPC1 regulated Ogdh through the NFκB signaling pathway.KEGG pathway results showed that TRPC1 significantly regulated the nuclear transcription factor NFκB signaling pathway after myocardial I/R injury;q PCR results showed that Nfκb p65 gene silencing could reduce Ogdh m RNA after OGD/R in HL-1 cells;Western blot results showed that Nfκb p65 gene silencing could reduce OGDH protein level after OGD/R in HL-1 cells,Trpc1 gene silencing reduced the protein levels of p-NFκB p65,p-IκB after OGD/R,and Trpc1 overexpression increased the protein levels of p-NFκB p65,p-IκB and Ca Nαafter OGD/R in HL-1 cells.Ch IP results showed that NFκB p65 transcription factor was combined with the promoter of Ogdh intranuclear target gene.3.Danshen’s active ingredient RosA can inhibit TRPC1 activity.（1）Establish a compound library of Danshen and initially screen 20 compounds with drug-like characteristics.Twenty compounds derived from Salvia miltiorrhiza Burge demonstrated good drug-like characteristics.TCMSP database was used for the retrieval of Salvia miltiorrhiza Burge compounds and 202 small molecule compounds were retrieved.By referring to the five drug-like principles and other relevant screening conditions,20 of compounds were found with good characteristic parameters.（2）RosA and TRPC1 had a stable combination conformation.The results of Auto Dock Vina software showed that the free energy of RosA and TRPC1 protein binding was-8.4 kcal/mol,apt to form a stable binding conformation.The docking pattern analysis revealed that the motif between the407-481 amino acids of the TRPC1 protein（hydrogen bonds,salt bridges andπ-πconjugated systems between amino acids and molecule）is essential for the binding of RosA to TRPC1,and the effect of this sequence may weaken the function of TRPC1.（3）RosA had high binding affinity to TRPC1 protein.The results of SPR showed that RosA and TRPC1 protein bind to each other.（4）RosA inhibited TRPC1 activity in vitro.Activity test results showed that RosA could inhibit the m RNA and protein levels of Trpc1after OGD/R in HL-1 cells and it could inhibit TRPC1-mediated Ca²⁺influx injury in HEK293 cells.4.RosA inhibits TRPC1-related signaling pathway protein levels and reduces myocardial I/R injury.（1）RosA could significantly reduce myocardial I/R injury.TTC+Evans blue results showed that RosA could reduce the size of myocardial infarction in mice;H&E results showed that RosA could reduce myocardial pathological injury;myocardial enzyme results showed that RosA could reduce the CK-MB and c Tn I levels in serum.（2）RosA reduced ROS generation both in vivo and in vitro.DHE-ROS results showed that RosA could reduce ROS generation in the AAR region of myocardium and HL-1 cells after OGD/R injury.（3）RosA could reduce Trpc1,Ogdh m RNA and protein levels in the AAR region of myocardium.q PCR results showed that RosA reduced Trpc1,Ogdh m RNA in the AAR region in mice.Western blot results showed that RosA reduced the protein levels of TRPC1 and OGDH in the AAR region of myocardium in mice.（4）RosA inhibited OGD/R-induced changes in TRPC1-related signaling pathway protein levels.Western blot results showed that RosA reduced TRPC1,Ca Nα,p-NFκB p65,p-IκB-αand OGDH protein levels after OGD/R injury in HL-1 cells.Conclusion:This study demonstrated for the first time that the Trpc1 gene and its encoded protein TRPC1 were significantly up-regulated after myocardial I/R injury;TRPC1 could significantly affect extracellular Ca²⁺influx,and then positively activate the Ca N/NFκB signaling pathway,prompt the transcription factor NFκB p65 into the nucleus,and bind to Ogdh gene promoter,affect Ogdh m RNA and protein levels,cause lots of intracellular ROS to be generated,and then aggravate myocardial injury.The active ingredient RosA in Salvia miltiorrhiza Burge had a strong affinity with TRPC1,which could inhibit the expression and activity of TRPC1 on OGD/R-induced injury in HL-1 cells.RosA could inhibit Ca²⁺-Ca N/NFκB signaling pathway,decrease OGDH protein levels,reduce ROS generation,and significantly alleviate myocardial I/R injury.