Based On The JAK2/STAT3 Signal Pathway To Explore The Pharmacological Mechanism Of Siegesbeckiae And Its Active Ingredients

Background:Myocardial hypoxia injury is the main pathological basis of cardiovascular disease.Up to now,its pathogenesis has not been fully elucidated,and there is no effective and specific drug therapy clinically.Studies have shown that TCM has unique advantages in the treatment of myocardial hypoxia injury,and a large number of relevant clinical and basic studies have been carried out at home and abroad.Studies have proved that water extracts and alcohol extracts of Herba Siegesbeckiae have pharmacological effects such as vasodilating,hypotensive,analgesia,antibacterial and anti-inflammatory,antioxidant and immunosuppression,and are often used in clinical treatment of cardiovascular and cerebrovascular diseases.Clinically,among the ingredients and proprietary Chinese drugs containing Herba Siegesbeckiae,the processing methods of Herba Siegesbeckiae vary.Currently,there is a lack of pharmacodynamics comparison of herba siegesbeckiae raw and alcoholic products and studies on myocardial hypoxia protective mechanism.By comparing the wine making and raw glandularstalk st.paulswortherb/common st.paulswort grass in H9c2 cell hypoxia injury to cells in lactate dehydrogenase(LDH)leakage rate,superoxide dismutase(SOD)content,reactive oxygen species(ROS)activity and cell apoptosis protein(Bcl-2,Bax)evaluation glandularstalk st.paulswortherb/common st.paulswort pharmacodynamic differences of different processed products of grass,and through the network pharmacology active ingredients and experimental verification of targets and carry on a cellular levelMETHOD:Experiment 1:Culture of H9c2 cardiomyocyte cell line and establishment of hypoxia modelTaking rat H9c2 cardiomyocytes as the research object,and the three-gas incubator(1%O2,5%CO2,94%N2)with different hypoxia time(12h,16h,20h,24h,48h)on cardiomyocytes,Cell survival rate was detected by Cell Counting Kit-8(CCK-8)method,appropriate hypoxia time was selected,and H9c2 cardiomyocyte hypoxia model was established.Experiment 2:Comparative study of effects of raw and alcoholic Herba Siegesbeckiae on Hypoxia damage of H9c2 myocardial cellsCells detected by drug toxicity by CCK-8,lyophilized powder screened raw pig Siegesbeckiae drug concentration and with wine Siegesbeckiae experiment.They were randomly divided into normal group,model group,Siegesbeckia sibiricum and Siegesbeckia distillosa group.After the model was administered,the cell viability was measured by the CCK-8 method,and the LDH leakage rate,SOD activity and ROS activity were measured by colorimetry.Western Blot detects the expression level of Bcl-2 and Bax protein in cardiomyocytes.Experiment 3:The pharmacological study on the treatment of myocardial hypoxic injuryAdopt TCMSP and previous literature reports to screen the active ingredients of Anoplophora by oral availability and drug similarity,and integrate targets by Swiss Target Prediction to construct potential active target information Data set,through Gene card and OMIM database to screen the target of treatment for myocardial hypoxic injury diseases,through the String database and Cytoscape software to draw protein interaction network,through R to carry on the signal pathway and gene ontology analysis of the tentacle grass target.Experiment 4:The pharmacodynamic effects of Siegesbeckiae Siegesbeckia and its active constituents qirenol and siegechinol on H9c2 cell hypoxia injury and its influence on JAK2/STAT3 signal pathwayCombined with the network pharmacology and literature reports,the raw stilts and their active ingredients Chirenol and stilbenol were taken as the research objects,the cell drug toxicity was detected by the CCK-8 method,and the raw stilts and Qiren used in the experiment were screened to determine Alcohol and fenspermyl alcohol concentration;based on H9c2 hypoxic injury model,randomly divided into blank control group,model group,raw scallion group,chirenol group,sperm alcohol group,using CCK-8 method to determine cells Vitality,colorimetric determination of LDH leakage rate,evaluation of its efficacy,Wesrern Blot detection of myocardial cell JAK2,STAT3 protein expression levels,to explain the mechanism of hypoxia protection.RESULT:Experiment 1:Culture of H9c2 cardiomyocyte cell line and establishment of hypoxia modelH9c2 cells hypoxia for 16h-48h can significantly reduce cell survival rate.H9c2 cells were hypoxic for 16h,the cell survival rate was 48.39%,the cell survival rate was moderately reduced,and the experimental results were reproducible(n=6).Therefore,16h was selected to establish a hypoxia injury model.Experiment 2:Comparative study on the efficacy of alcoholic and raw fennel grass against hypoxia injury of H9c2 cardiomyocytes(1)The CCK-8 drug toxicity results showed that:Compared with the normal group,there was no cytotoxicity in the alcohol and Siegesbeckia serrata administered at a concentration below 500?g·ml-1,so in subsequent experiments,both SHS and JHS used 500,300,100?g·ml-1 are the drug intervention concentrations.(2)The results of LDH leakage rate,SOD activity,ROS content and cell viability showed that compared with the normal group,the LDH leakage rate of the model group increased(P<0.05),ROS content increased(P<0.05),SOD activity decreased(P<0.05),Cell viability decreased(P<0.05).Compared with the model group,both the SHS group and the JHS group can reduce intracellular ROS content and LDH leakage(P<0.05),and increase SOD activity and cell viability(P<0.05).Comparison of SHS group and JHS group:The SOD activity and LDH leakage rate of SHS group were higher than those of JHS group(P<0.05);there was no significant difference in ROS level between the two groups.(3)Western Blot results showed that compared with the normal group,the expression of Bcl-2 in the model group was down-regulated(P<0.05),the expression of Bax was up-regulated(P<0.05),and the ratio of Bcl-2/Bax was down-regulated(P<0.05);compared with the model group,Both the SHS group and the JHS group can up-regulate the expression of Bcl-2(P<0.05)and the ratio of Bcl-2/Bax(P<0.05),but there is no significant difference in the Bax value;the Bax,Bcl-2 and Bcl-2/Bax values were compared,and there was no significant difference between the two groups.Experiment 3:Pharmacological study on network of Herba Siegesbeckiae in the treatment of Myocardial hypoxia injuryThere are 10 main active ingredients in idiocrema(Ivy saponin,stigmasterol,?-sitosterol,phenoxylinol,vernolic acid,crown beryllium,crotonic acid II,fennel ether acid,odd Ren alcohol and et.al),360 related target genes,205 target genes for the treatment of myocardial hypoxia were screened.The key compounds for the treatment of myocardial hypoxia in Jiujiao include crown berynine,stigmasterol,?-sitosterol,and Changchun(Vine glycosides)ligands,etc.,the key targets are mainly IL-6,MAPK3,VEGFA,SRC,CASP3,MAPK1,EGFR,TP53,PTGS2,STAT3,MAPK1,JUN,MTOR,PIK3CA,ESR1,MMP9,MAPK114,F2,FGF2,ACE,IL-2,PPARQ KDR,MMP2,AK,MDM2,NR3C1,Bcl2C1,JAK2,AGTR1,etc.Gene functions Herba treatment of myocardial hypoxia relationship between the major regulator of G protein coupled receptor,calcium transport,and synaptic receptors,myocardial epinephrine,acetylcholine,serotonin and other biologically received process.The main pathways include cancer pathway,apoptosis,calcium signaling pathway,p53 signaling pathway,cGMP-PKG signaling pathway and so on.Experiment 4:The pharmacodynamic effects of Siegesbeckiae Siegesbeckia and its active constituents qirenol and siegechinol on H9c2 cell hypoxia injury and its influence on JAK2/STAT3 signal pathway(1)The CCK-8 drug toxicity results showed that:compared with the normal group,the cell survival rate was the highest at the concentration of 10?g·ml-1,and the direct cell survival rate from to 50?g·ml-1 was normal.In the follow-up experiments,both qirenol and siegeline alcohol used 10?g·ml-1 as the drug intervention concentration(2)The results of LDH leakage rate showed that after 16 hours of hypoxia in H9c2 cells,compared with the normal group,the LDH leakage of the model group was significantly increased(p<0.01).Compared with the model group,Siegesbeckiae,Qirenol,Siegesbeckiae Essence alcohol can significantly reduce LDH leakage(P<0.01),but there is no significant difference in LDH leakage rate between the three groups.(3)Western Blot results showed:Compared with the normal group,the expression of STAT3 and JAK2 in the model group was down-regulated(P<0.05);compared with the model group,Siegesbeckiae,Chirenol and Siegesbeckiae could up-regulate STAT3 and JAK2(P<0.05),but there was no significant difference in the expression of JAK2 and STAT3 between the three groups.CONCLUSION:In this study,by comparing the effect of different hypoxia time on the hypoxia state of H9c2 cells,16h was determined as the hypoxia modeling time.By comparing the LDH leakage rate,SOD activity,ROS content,and Bax and Bcl-2 protein expression content,the initial evaluation of the difference in the efficacy of two kinds of raw wild velvet grass and wine-made wild velvet grass in the treatment of myocardial hypoxic injury,raw wild velvet grass It is better than wine-made Jiujiu.Using network pharmacology combined with the methods reported in the literature to screen out the active ingredients and protein targets,and experiments at the cellular level were verified.Chirenol and gerbilinol in A.sibiricum have better hypoxic protection and their effects The mechanism may play the role of myocardial hypoxia protection through the JAK2/STAT3 pathway.