| Background:Depression after acute myocardial infarction(AMI)predicted adverse cardiac events and death,the reported prevalence of which for the last few years generally ranged from 20%to 40%.However,the effect of antidepressants on cardiac prognosis is still controversial,and current therapeutic strategies cannot fully satisfy clinical needs.Shuangxinfang(Psycho-cardiology Formula,PCF)could activate blood circulation and regulate spirit,increase energy and restore deficiency,as well as improve angina pectoris and relieve depression,the clinical effects of which has been confirmed by our team.But there is a lack of research on the pharmacological components and protein targets of PCF,and the accurate mechanism by which it plays the pivotal roles remains to be further elucidated.UPLC-Q-Exactive Orbitrap MS/MS(LC-MS/MS)has been widely used in the qualitative and quantitative analysis of the components of traditional Chinese medicine(TCM),and also applied to identify protein expression.In this study,the pharmacodynamic components and protein targets of PCF were analyzed by LC-MS/MS and bioinformatics methods,and the anti-depressive mechanism was explored in vivo and in vitro experiments.Besides,we applied literature mining in pattern of syndrome,syndrome element and medication rule,as well as meta-analysis to evaluate psycho-cardiology effects.Study 1 The syndrome type and medication regularity of coronary heart disease(CHD)with depression based on the literature reviewObjective:To analyze the syndrome type and medication regularity of CHD with depression based on the literature review.Methods:We searched the published randomized controlled trials(RCTs)of Chinese herbs in the treatment of CHD comorbidized depression by PubMed,Embase,Cochrane Library,CNKI,Wanfang and VIP databases.Frequency analysis was used in TCM syndrome types and syndrome elements of coronary heart disease with depression.Also,we analyzed the frequency of herbs and association rules among herbs on the Traditional Chinese Medicine Inheritance Assist Platform(TCMISS),further developed potential new medicine pairs and medicine teams,in order to dig out prescription regularity.Results:A total of 88 prescriptions were screened out.The syndromes mainly included qi stagnation and blood stasis syndrome and qi deficiency and blood stasis syndrome.The key syndrome elements included blood stasis,qi stagnation and qi deficiency.Most of the herbs entered the liver meridian and heart meridian.The medicinal properties were mainly warm,bitter and acrid.Bupleurum was the most frequently used herbs.The core prescriptions included bupleurum,radix paeoniae alba,radix curcumae and szechuan lovage rhizome.Among the candidate medicine teams,there were more combinations of herbs to tonify qi and yin,promote blood circulation and regulate the heart.Conclusions:The basic pathogenesis of CHD combined with depression might be "stasis of heart arteries and qi stagnation".Most of doctors treated CHD comorbid with depression from the angle of "the liver" or "psycho-cardiology".For the former principles,the basic therapeutic methods were regulating the liver and activating blood circulation.On condition of psycho-cardiology forum,the significant contents were managing qi and activating blood,reinforcing qi and nourishing yin,as well as regulating mental states.Study 2 Systematic evaluation and meta-analysis of the traditional Chinese medicine on coronary heart disease with depression for psycho-cardiology effects and inflammatory factorsObjective:To systematically evaluate the effects of traditional Chinese medicine combined with conventional western medicine on the efficacy and inflammatory factors of coronary heart disease with depression,and to provide evidence-based reference for clinical practice.Methods:We searched randomized controlled trials(RCTs)in PubMed,Cochrane Library,CNKI,Wanfang database and Chinese biomedical literature service system(CBM)until March 1,2021.According to the Jadad criteria,we assessed the quality of the selected studies,and carried out meta analysis with the help of Stata16.0 software.Results:A total of 1574 patients were enrolled in 16 studies.Meta analysis showed that compared with conventional western medicine,TCM combined with western medicine treatment could reduce C-reactive protein(CRP)level(SMD=-1.42,95%CI[-1.79,-1.05],P=0.00),reduce IL-17 content(SMD=-0.89,95%CI[-1.48,-0.29],P=0.003),decrease TNFα expression(SMD=-2.41,95%CI[-3.48,-1.34],P=0.00),lower Hamilton depression scale(HAMD)score(SMD=-1.72,95%CI[-2.24,-1.20],P=0.00),reduce self-rating depression scale(SDS)score(SMD=-1.53,95%CI[-2.34,-0.73],P=0.00),and improve the effective rate of angina pectoris(SMD=3.22,95%CI[1.94,5.34],P=0.00).Conclusions:Traditional Chinese medicine can reduce the levels of CRP,IL-17 and TNF-α,decrease the score of HAMD and SDS,and increase the effective rate of angina pectoris in patients of coronary heart disease complicated with depression.However,due to the limited number of high-quality literatures included,and the high heterogeneity and publication bias of secondary research evidence,more high-quality literatures are needed to verify the reliability of the results in the future.Study 3 HPLC-Orbitrap MS/MS analysis on the proteins in the peri-infarct border zone and hippocampus of rats with depression-like behavior after myocardial infarctionObjective:Proteomics and bioinformatics techniques were adopted to explore the targets of PCF and infer the mechanism.Methods:AMI models were established by coronary ligation,and the rats were randomly divided into the sham group,AMI group,and PCF group.The depression-like behavior was then evaluated by open field test(OFT)and forced swimming test(FST)at 5-7 days after surgery.Myocardium and hippocampus were collected for tissue lysis,protein quantification and enzymolysis.Easy Nano-flow HPLC system combined with Orbitrap Fusion Lumos mass spectrometry with electrospray ion source was used to collect LC-MS/MS data.The original data were searched by Proteome Discoverer 2.4 software.The difference between groups was evaluated by Fold change(FC)in protein abundance.Differentially expressed proteins(DEPs)were screened out according to the condition of |log2(FC)|≥1.0 or P<0.05.The pheatmap package of R software was used to draw the cluster analysis heatmap.The interaction network of differential proteins was constructed by STRING database and Cytoscape3.8.2.KOBAS database was used for GO annotation classification and pathway enrichment analysis of DEPs.Western blotting was used to test the protein expression.Results:Compared with the sham group,the total movement distance and average speed of the rats from the AMI group in the OFT were significantly declined(P<0.0001),and the immobility time in the FST was significantly prolonged(P<0.01).Compared with the AMI group,the total movement distance(P<0.01)and the average speed(P<0.0001)of rats in the OFT were significantly increased,and the immobility time in the FST was shortened in PCF group(P<0.05).There were 328 differentially expressed proteins in the AMI group compared with the sham group,and a total of 208 DEPs were found between PCF group and AMI group.131 DEPs in peri-infarct border zone(BZ)were obtained by intersection from three groups.KOBAS database was performed to enrich 131 differential proteins,and 117 signal pathways were screened out(P<0.05).Altered molecules were discovered to be enriched in innate immune system,immune system,neutrophil degranulation,arginine biosynthesis and others.338 GO items were enriched(P<0.05),and the top 20 enriched GO terms were relevant to the cytoplasm,exosomal secretion,protein transport,apoptotic process,arachidonic acid binding,innate immune response,and nuclear factor kappa-B(NF-κB)transcription factor activity.Compared with the sham group,157 DEPs were found in the AMI group.There were 137 DEPs found between PCF group and AMI group.A total of 64 DEPs were obtained by intersection from 3 groups in hippocampus.KOBAS database was used to screen out 106 signaling pathways(P<0.05).The DEPs were largely related to ABCfamily proteins mediated transport,integrin signaling,integrin alpha Ⅱb beta3 signaling,and downstream signaling events of B cell receptor.207 GO items were obtained(P<0.05),and regulation of neuron death,chemical synaptic transmission,axon,collagen binding,cell adhesion were found to be enriched.Protein S100A9 was the only DEP shared by myocardium and hippocampus as shown in venn diagram.The results of western blotting showed that compared with sham operation,the expression of S100A9 protein in myocardium,hippocampus and cortex was significantly increased induced by coronary ligation(P<0.05,P<0.01,P<0.0001).On the contrast,the expression of S100A9 in hippocampus and cortex was significantly down-regulated after the administration of PCF.(P<0.05,P<0.01).Conclusions:Proteomics demonstrated that protein S100A9 might be a pivotal target of depression-like behavior in MI rats.Combined with biological function and pathway enrichment analysis,the pathological mechanism might involve macrophage/microglia activation.Study 4 Identification of components in shuangxinfang and molecular docking to protein S100A9Objective:LC-MS/MS was used to determine the components and the blood migration components of PCF,which laid a foundation for the research of pharmacodynamic substances.Methods:AMI rat models were established,and the rats were randomly divided into the control group,control/PCF administration group,and AMI/PCF group.Then we employed UPLC-Q-Exactive Orbitrap-MS to determine the effective components and blood transfer components of PCF.HPLC separation was performed on AQUITY UPLC C18 column with gradient elution of 0.1%formic acid aqueous solution(A)and acetonitrile solution(B)(0-3 min,0%B;45 min,3-0%-75%B;45-45.1 min,75%0%B;45.1-50 min,0%B),and the flow rate was 0.3 ml/min.Q-Exactive Orbitrap MS with electrospray ionization(ESI)ion source was applied for qualitative analysis under positive and negative ion mode.Combining with MS data,literatures and database matching,ion peak number and peak area were confirmed.AutoDock Vina software was used to conduct molecular docking between the blood migration components of PCF and the target protein,and visual processing was performed on Pymol and Discovery Studio V20 software.Results:Under the anion mode,91 chemical components were identified by PCF,while a total of 92 compounds were identified in positive ion mode.After removing the repeated components,142 chemical components were identified in total ion mode,including 74 from salvia miltiorrhiza,40 from Ligusticum chuanxiong,14 from jujube seed and 18 from lily.A total of 7 components were identified in vivo,2 of which were identified as Miltionone I and neocryptotanshinone in the control/PCF plasma.A total of 7 compounds were introduced into the plasma of AMI/PCF group,including Miltionone I,neocryptotanshinone,Danshenxinkun A,ferulic acid,Valerophenone,vanillic acid and senkyunolide-D.The binding force between the PCF constituents absorbed into blood and target protein S100A9 was less than-5.0 KJ/mol.Conclusions:UPLC-Q-Exactive Orbitrap MS analysis can quickly identify and analyze the components of PCF.Moreover,the components had strong binding force with S100A9 protein.There are relatively abundant results on the improvement of myocardial infarction and depression supported by ferulic acid,which provides a reference for the research of pharmacodynamic substance and subsequent experiments.Study 5 To explore the mechanism of Shuangxinfang on depression-like behavior in myocardial infarction rats from the perspective of S100A9-mediated microglial inflammationObjective:To explore the mechanism of PCF on depression-like behavior in myocardial infarction rats from the perspective of macrophage/microglial inflammation mediated by protein S100A9.Methods:The rats were divided into the sham group,AMI group,ABR-215757(S100A9 inhibitor)group and PCF group.After the last dose,the rats underwent behavioral tests and echocardiography.A half of the rats were sacrificed.Pathological damage of the heart was observed by HE staining and TUNEL staining,and hippocampal neurotransmitters were detected by liquid chromatography-mass spectrometry.Western blotting was used to detect the expression of TLR4 and NF-κB protein in myocardium and hippocampus,and RT-qPCR was performed to test the expression of S100A9 and NF-κB gene in myocardium and hippocampus.Also,we detected the levels of S100A9,TNF-α and IL-1β in peripheral blood,myocardium and hippocampus by ELISA.The location and quantification of S100A9 in hippocampus were processed by immunofluorescence.Moreover,the number of CD68 positive cells in myocardium and Iba1 positive cells in hippocampus were detected by immunofluorescence technique.The remaining rats were sacrificed on the 21st day after surgery.Masson staining was operated to observe cardiac fibrosis,while western blotting was applied to test BDNF expression and Nissl staining was used to observe hippocampal neurogenesis.Results:Compared with the sham group,left ventricular ejection fraction(LVEF)and left ventricular fractional shortening(LVFS)in the AMI group were significantly decreased(P<0.00001),and an increasement in left ventricular end-systolic inner diameter(LVIDs),left ventricular end-diastolic inner diameter(LVIDd),left ventricular end-systolic volume(LVESV)and left ventricular end-diastolic volume(LVEDV)were observed(P<0.00001,P<0.05,P<0.05,P<0.001).Compared with the AMI group,LVEF and LVFS group were significantly increased(P<0.01),and a decline in LVIDs,LVIDd and LVEDV in ABR215757 group were found(P<0.05,P<0.01,P<0.05).Compared with the AMI group,LVEF and LVFS in PCF group were significantly elevated(P<0.05),while LVIDd and LVEDV were decreased(P<0.05).Hematoxylin-eosin(HE)staining showed orderly arrangement of myocardial fibers,uniform cytoplasm,and clear nucleus in the sham group.Myocardial cells in the AMI group were characterized by massive degeneration and necrosis,disorder of arrangement,nuclear pyrosis,loose edema,inflammatory cell infiltration and capillary hyperplasia in the infarct area.Compared with the AMI group,the myocardial fiber disorder,loose and edema in PCF group and ABR-215757 group were alleviated.TUNEL staining results showed that a small number of positive cells were scattered in the sham group,while a large number of TUNEL positive cells were expressed in the AMI group,and the apoptosis index was significantly increased(P<0.001).Compared with AMI group,the apoptosis index in PCF group and ABR-215757 group was significantly decreased(P<0.01,P<0.05).In Masson staining,myocardial cells in the sham group were stained red and arranged neatly,without obvious fibrosis.At 21 days after coronary ligation,the myocardial structure of the model group was disorganized,and the fibrosis area was significantly enlarged(P<0.0001).Compared with the AMI group,the fibrosis area decreased in PCF group and ABR-215757 group significantly(P<0.001).Compared with the sham group,the number of verticality and total distance of rats in the AMI group were significantly decreased(P<0.01).These two indicators in PCF group were significantly increased(P<0.05).The results of FST showed that compared with sham group,the immobility time of rats in AMI group was significantly longer(P<0.01).After the administration of PCF or ABR-215757,rats had a much shorter immobility time in the FST(P<0.0001).Under the microscope,the nerve cells in the sham group were arranged neatly and densely,and the number of nissl bodies was abundant.However,the neurons in the hippocampal dentate gyrus(DG)region of the AMI group were obviously lost and arranged loosely.The number of nissl bodies was reduced,and the IOD value of the AMI group was significantly lower than that of the sham group(P<0.05).The level of 5-HT in the AMI group was significantly decreased(P<0.01),and significantly increased in ABR-215757 group and PCF group(P<0.05).The hippocampal Kyn/Try ratio was increased in the AMI group(P<0.05),and declined significantly after the administration of ABR-215757(P<0.05).Compared with the sham group,the hippocampal BDNF of AMI group was decreased,and PCF or ABR-215757 could reverse the expression of it,but there was no statistical difference between groups.The expression level of TLR4 and NF-κB protein was elevated in the AMI group compared to the sham group and was returned to the basal level by the treatment of PCF,but there was no significant statistical difference between groups.Compared with the AMI group,the expression of NF-κB protein in ABR-215757 group was down-regulated(P<0.05).The gene expression of S100A9 and NF-κB in the AMI group was elevated than that in the sham group,and showed a down-trend after the treatment of PCF,but there was no significant statistical difference between groups.Compared with the AMI group,the expression of S100A9 gene of hippocampus in ABR-215757 group was down-regulated(P<0.05).The results of immunofluorescence showed that the expression of S100A9 in the AMI hippocampus was significantly increased compared with that of the sham group(P<0.001),and decreased significantly in PCF group or ABR-215757 group(P<0.0001,P<0.05).ELISA results showed that compared with the sham group,coronary ligation significantly increased the expressions of S100A9,TNF-α and IL-1β in serum(P<0.0001,P<0.01,P<0.0001),myocardium(P<0.01,P<0.0001,P<0.01)and hippocampus(P<0.0001,P<0.0001,P<0.01).PCF inhibited the expression of S100A9(P<0.0001,P<0.05),TNF-α(P<0.01,P<0.0001)and IL-1β(P<0.01,P<0.01)in serum and hippocampus,and decreased the level of IL-1β in myocardium(P<0.05).ABR-215757 inhibited the expression of S100A9 and TNFa in serum(P<0.0001,P<0.01),myocardium(P<0.05,P<0.001)and hippocampus(P<0.01,P<0.001).The levels of IL-1β in serum(P<0.001)and hippocampus(P<0.05)were decreased.Acute myocardial ischemia significantly increased the number of CD68 positive cells(P<0.01).Instead,the administration of PCF and ABR-215757 reduced the number of CD68 positive cells(P<0.01).Coronary ligation increased the number of activated microglia in hippocampus(P<0.01),while PCF and ABR-215757 decreased the number of Ibal positve cells in hippocampus(P<0.05,P<0.01).Conclusions:In vivo studies showed that PCF might improve cardiac function and depression-like behavior after AMI by inhibiting S100A9-induced macrophage/microglial inflammation.Study 6 Effect of Shuangxinfang on S100A9-mediated microglial inflammationObjective:In vitro experiment was performed to explore the effect of PCF serum and ferulic acid on S100A9-mediated microglial inflammation.Methods:BV2 microglia were divided into control group,recombinant S100A9 group,C34(TLR4 inhibitor)group,ferulic acid(FA)group,PCF serum group,and control serum group.After intervention with 0.1 μM recombinant S100A9 protein for 6h,BV2 microglia cell lines were incubated with C34,ferulic acid,PCF serum,or control serum.The morphological changes of microglia were observed by fluorescence microscope,and cell viability was tested by CCK8 assay.The levels of S100A9,TNF-α and IL-1β in cell supernatant were detected by ELISA.Results:Low and medium doses of PCF serum had no significant effect on microglia cell viability,while the high dose group could reduce the cell viability to a certain extent,but there was no statistical significance.The protein S100A9 at a concentration of 0.01 μM had no significant effect on microglia cell viability,while 0.1 μM S100A9 protein significantly increased the number of microglia cell(P<0.05).Under reversed microscope,resting microglia cells have oblate bodies with extending long synapses.Under the activation of recombinant S100A9 protein,microglia cells become enlarged,retract their processes,form new motile protrusions,and transform into spherical or ameboid form.Compared with the control group,the recombinant S100A9 protein significantly reduced the cell radius ratio(P<0.05).The levels of S100A9,TNF-α and IL-1β in recombinant S100A9 group were significantly elevated compared with control group(P<0.05).The level of S100A9 in PCF serum group was decreased(P<0.05),while the level of inflammatory factors in control serum group was not significantly changed.Ferulic acid could not only decrease S100A9 content,but also inhibit the expression of TNF-α and IL-1β(P<0.05).C34(TLR4 inhibitor)could reverse the proinflammatory effect of S100A9(P<0.05).Conclusions:In vitro studies suggested that S100A9,as an upstream target of inflammatory cascade,might be a potential biomarker in neuroinflammation-related diseases.TLR4 is one of the receptors of S100A9 to activate microglia.PCF serum,as well as ferulic acid,could be promising therapeutic candidates for psycho-cardiology diseases and strategies to block S100A9-induced inflammatory outburst. |
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