| Purpose:Xin-Ji-Er-Kang（XJEK）is proved as an efficient prescription in the therapy of various cardiovascular diseases.This herb consist of Ginseng,Astragalus astragali,Sophora flavescens and etc.While the material basis and potential mechanism of XJEK was still unclear,which hampered the development of XJEK.Clinically,MIR injury is an unavoidable phenomenon in the therapy of ischemic heart disease which is a serious threat to human health.Therefore,detail clarified the therapeutic potential and mechanism of XJEK for MIR injury are of great importance.Methods:（1）A rapid analysis method for identification of chemical composition in XJEK was established based on Q-Exactive plus hybrid quadrupole-orbitrap mass spectrometer and Xcalibur 4.1 software platform.Combining with the information of Compound Database,the migration components in the plasma and heart after gavaging administration of XJEK in MIR mice were identified.Next,these key components in the plasma and heart were quantitatively analyzed by liquid chromatography-triple quadrupole mass spectrometry（LC/MS/MS）.The common targets of XJEK and MIR disease are studied based on Bioinformatics.（2）In vivo experiments,80 male Kunming mice were subjected to MIR surgery.Left anterior descending coronary artery was ligated 30 minutes for ischemic,and then the ligation line was released to obtain a MIR model.72 hours after surgery,surviving mice were randomly divided into sham group,sham-XJEK group,MIR group,XJEK group,fosinopril group and matrine group.Sham group and MIR group were intragastric administration with normal saline,and other groups were given corresponding doses of drug by gavaging.After 6 weeks of drug intervention,echocardiography,cardiac morphology,heart weight index（heart weight/body weight,HW/BW）were analyzed in each group.TUNEL staining evaluted the cardiomyocytes apoptosis.Hematoxylin and eosin（HE）and Masson staining analyzed pathological changes in mice myocardial tissue.Enzyme-linked immunosorbent assay（El ISA）determined changes in N terminal pro B type natriuretic peptide（NT-pro BNP）and cardiac troponin I（c Tn I）in the serum of mice.The expression of JAK2,p-JAK2,STAT3,p-STAT3,STAT1,p-STAT1,Bax,Bcl-2 in myocardial tissue were analzed by Western Blot（WB）.（3）In vitro experiments,human-derived cardiomyocytes（AC16）received a cellular hypoxia-reoxygenation injury model.The experiment is grouped as follows:control group（Control group）,hypoxia/reoxygenation group（Hypoxia/reoxygenation,H/R group）,XJEK group,matrine group.After H/R and drug treatment,CCK8 detects cell viability;Western blotting detects the related proteins expression of JAK-STAT signaling pathway;Flow cytometry detects the level of cardiomyocytes apoptosis;Live cell imaging detects cell proliferation and morphological changes.JAK2 inhibitor AG490 or knockdown of JAK2 were used to explore the potential mechanism of XJEK.Results:（1）Combined QE-plus with Xcalibur 4.1 analysis strategy,the chemical components of XJEK was identified.A total of 52 monomer components were identified.And the source of its single medicine was classified.Among them,five species were from ginseng,four species were from Polygonatum,six species were from Salvia,three species were from Angelica,three species were from Licorice,three species were from Trichosanthestric,eight species were from Astragalus,four species were from Sophora,one species were from Ophiopogon,three species were from Notoginseng,four species were from Schisandra fruit,one species were from Xeibai and seven species were from Epimedium.After gavage administration of XJEK for 6 weeks,fifteen and four migration components were detected in the plasma and heart of mice,respectively.The results showed that this method can quickly and comprehensively identify the chemical components of XJEK in vivo and in vitro.（2）Mice subjected to MIR surgery,then gavage administration of XJEK for 1 week and 6 weeks.Finally,matrine,oxymatrine and sophocarpine in the plasma and heart of mice was quantitatively analyzed based on LC/MS/MS.After 1 week of gavaging administration,the contents of matrine,oxymatrine and sophorine in plasma were 128.76±44.41 ng/m L,31.73±7.07 ng/m L,14.17±7.05 ng/m L;the contents of matrine,oxymatrine and sophorine in heart were 13.15±5.95 ng/100mg,3.03±0.73 ng/100mg,1.79±0.92 ng/100mg;After 6 weeks of gavaging administration,the contents of matrine,oxymatrine and sophorine in plasma were 201.01±42.79 ng/m L,50.78±6.67 ng/m L,24.33±7.54 ng/m L;the contents of matrine,oxymatrine and sophorine in heart were13.88±4.21 ng/100mg,4.53±0.81 ng/100mg,2.06±0.67 ng/100mg.（3）Based on bioinformatics technology,we discovered that matrine,oxymatrine and sophocarpine have 18 common targets with MIR disease.According to the RPKM value of each protein in the heart tissue,JAK2 was finally selected for our study.Furthermore,molecular dynamics simulation results show that matrine can be stably combined with the JH2 domain of JAK2 through hydrogen bonds.（4）The results of in vivo experiments showed that compared with the MIR group,the cardiac function parameters and morphology in the MIR mice which treated with XJEK are significantly improved,and the heart weight index are reduced.For the serological indicators,it was found that compared with MIR group,XJEK and matrine can down-regulate the levels of NT-pro BNP and c Tn I in serum.The detection of the JAK-STAT signaling pathway shows that compared with the sham group,the phosphorylation levels of JAK2 and STAT1 in the MIR group were significantly increased;the phosphorylation level of JAK2 further increased after the administration of XJEK and matrine,the level of p-STAT3 increasing accompany with the decreasing level of p-STAT1.As for apotosis,the increase of the ratio of Bax/Bcl-2 and TUNEL+staining cell in MIR group demonstrated that the cardiomyocyte apotosis level is enchanced.After the treatment of XJEK and matrine the level of cardiomyocyte apoptosis was significantly reduced（5）Compared the H/R group with the control group,the cell viability was obviously decreased;the results of flow cytometry and WB showed that the level of cardiomyocytes apoptosis was notably rised.After XJEK and matrine treatment,it can significantly improve cell viability and reduce the rate of cardiomyocyte apoptosis.The detection of JAK-STAT signal pathway showed that compared with the control group,the phosphorylation level of JAK2/STAT1/STAT3 in the H/R group was significantly decreased,and the level of p-JAK2/p-STAT3 was distinguished increased after treatment with XJEK and matrine.When AG490 is used to inhibit the phosphorylation of JAK2 or down-regulate the expression of JAK2,the cardioprotective effects of XJEK and matrine are partially cancelled.Conclusions:The obtained results confirm our hypothesis that XJEK exerts cardioprotective effect on MIR injury,which may be associated with the activation of JAK2/STAT3 signaling pathway.Moreover,matrine is one of the potential active ingredients of XJEK. |
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