| Ischemia heart disease(IHD)is currently a major cause of death and disability worldwide,furthermore cardiovascular death in China is one of the leading causes of death in both urban and rural areas.Restoring perfusion is the most effective and important treatment for IHD,but myocardial ischemia reperfusion injury(MIRI)caused by restoring perfusion is an important factor that affect instant survival rate and long-term cardiac recovery after revascularisation in the treatment of ischaemic heart disease.There is no specific drug for the treatment of MIRI.Previous studies have found that the pathogenesis of MIRI includes oxidative stress imbalance,calcium overload,mitochondrial damage,energy metabolism disorders,autophagy,apoptosis,and inflammatory reactions,with these pathological mechanisms intertwined and causal to each other.In particular,the inflammatory response runs through the entire process of myocardial injury and repair after ischemia-reperfusion,is one of the key factors determining the final size of myocardial infarction and adverse cardiac remodeling,and is a key therapeutic target for improving the clinical outcomes of MIRI.Pyroptosis is a direct and close link between cell death and the inflammatory response and is also one of the main death modes that cause MIRI inflammatory reactions.The formation and activation of the NLRP3 inflammasome are key steps in mediating pyroptosis and inflammatory reactions in MIRI,and thus,regulating NLRP3 activation is a key strategy for regulating myocardial ischemia-reperfusion injury and inflammatory reactions.Shuangshen Ningxin Capsule(SSNX)is a traditional Chinese medicine composed of ginseng total saponins,total salvianolic acid,and total alkaloids of rhizoma corydalis,which have the functions of promoting qi and blood circulation,dispelling blood stasis,and relieving pain.It is mainly used to treat IHD.Previous studies have found that SSNX has a good protective effect on acute myocardial infarction,myocardial ischemia-reperfusion injury(MIRI),and coronary microcirculation disorders,and its mechanism mainly includes inhibition of cell autophagy,improvement of mitochondrial function,and reduction of cell apoptosis.However,there is little research on its anti-inflammatory effects and the mechanism of its anti-inflammatory action is unclear.Therefore,this study aims to establish an acute MIRI model in rats,explore the anti-inflammatory effects of SSNX and its effects on NLRP3 inflammasomes and pyroptosis,and screen and find the pathways and targets through which SSNX regulates NLRP3 using transcriptome and bioinformatics analysis techniques.Finally,the effects of SSNX and its effective components on regulating NLRP3-related pathways and key targets will be verified to elucidate the anti-inflammatory mechanism of SSNX in protecting against MIRI and understand the pharmacological mechanism of SSNX.Research aim①Clarifying the regulation of Shuangshen Ningxin Capsule on NLRP3 inflammasome as well as inflammation and pyroptosis meditated by NLRP3 inflammasome.②To explore and verify the pathway and target of Shuangshen Ningxin Capsule through regulating NLRP3 inflammasome,and clarify its mechanism against MIRI.Research Methods:1.Study on the protective and anti-inflammatory effects of SSNX on acute MIRI ratsEstablish an acute MIRI rat model and divide the rats into the Sham group,Model group,SSNX 90mg/kg group,SSNX 45mg/kg group,Nicorandil group(NIC),and Tongxinluo group(TXL).Analyze the tongue images of rats,use a pulse analyzer to detect pulse amplitude,use echocardiography to detect rat heart function,use acoustic imaging to detect the peak time of myocardial blood flow perfusion in rats,use HE staining to observe pathological changes in myocardial tissue,use Evan’s blue staining TTC staining sulfuric acid staining to observe myocardial infarct size and no-reflow area,and simultaneously detect changes in rat plasma coagulation parameters such as APTT,PT,TT,and FIB.Biochemical methods are used to detect the levels of CK,CKMB,LDH,AST,and ALT in rat serum,multiple factors are tested for changes in the levels of inflammatory factors such as IL-6,MCP-1,and TNF-α in rat serum,and Western blotting is used to detect the expression of NF-κB,TLR4,IL-1 β,and NLRP3 in myocardial tissue.2.Study on the effects of SSNX on NLRP3-mediated pyroptosis and inflammatory response in acute MIRI ratsEstablish an acute MIRI rat model and divide the rats into the Sham group,Model group,SSNX 90mg/kg group,and MCC950 group.ELISA is used to detect the levels of inflammatory factors such as IL-18 and IL-1β in rat serum,immunohistochemistry is used to detect changes in the expression of GSDMD,NLRP3,and Caspase-1 in rat myocardium.Immunofluorescence is used to detect the double-stained of ASC,NLRP3 and GSDMD in rat myocardium.Western blotting is used to detect the expression of ASC,NLRP3,Pro-Caspase-1,Caspase-1,GSDMD,GSDMD-N,IL-1β,and IL-18 proteins in rat myocardial tissue.3.Study on the regulatory effects of SSNX on the NLRP3/caspase-1/GSDMD pyroptosis pathway.NLRP3 overexpressing H9C2 cardiomyocyte cell line(NLRP3-H9C2)was established,and an oxygen-glucose deprivation/reoxygenation(OGD/R)injury model was established for H9C2 and NLRP3-H9C2 cells.The groups were divided into normal control group(NC),H9C2 cardiomyocyte cell group with OGD/R injury(OGD/R),SSNX intervention group with OGD/R injury(SSNX),NLRP3-H9C2 cardiomyocyte cell group with OGD/R injury(NLRP3+),SSNX intervention group with NLRP3+(SSNX+NLRP3+),and MCC950 intervention group with NLRP3+(MCC950+NLRP3+).Cell viability was detected using CCK-8,cell morphology was observed using transmission electron microscopy.The levels of inflammatory cytokines IL-18 and IL-1β in the cell supernatant were measured using multiplex assay.The double-stained of ASC,NLRP3 and GSDMD were detected using immunofluorescence.The expressions of ASC,NLRP3,Pro-Caspase-1,Caspase-1,GSDMD,and GSDMD-N in rat myocardial tissue were detected using Western blot.4.Exploring the upstream pathways and targets of SSNX regulation of NLRP3 inflammatory vesiclesNetwork pharmacology techniques were used to predict the pathways through which SSNX against MIRI and to screen for pathways that may be related to NLRP3 regulation.Transcriptional sequencing technology was then used to explore the genes through which SSNX protects against acute MIRI in rat myocardium,and to verify the regulatory effect of SSNX on the predicted pathways and the pathway in which NLRP3 is located.Key target gene expression levels were predicted and screened,and molecular docking technology was used to predict the possible targets.5.Verifying the regulatory effect of SSNX on the upstream pathways and targets of NLRP3The predicted upstream pathway which SSNX regulates NLRP3 is the cAMP/PKA signaling pathway,with PKA as a key target.Therefore,an OGD/Rinjured H9C2 cardiomyocyte model was mainly established,and PKA agonists and inhibitors were added to explore the relationship between PKA and the NLRP3 inflammasome.The experiment was divided into a normal control group(NC),an OGD/R-injured H9C2 cardiomyocyte group(OGD/R),an SSNX intervention group(SSNX),a Bucladesine(PKA agonist)intervention group(BUC),a H-89 dihydrochloride(PKA inhibitor)intervention group(H-89),and a PKA inhibitor intervention group with SSNX.Cell viability was detected by CCK-8,and the contents of ATP,cAMP,and cGMP in the cells were measured by ELISA.The expressions of PKA and NLRP3 were detected by immunofluorescence staining,and the expression of cAMP/PKA/NLRP3 pathway-related proteins PKA,P-PKA(Ser157),PKG,P-PKG(Ser239),NLRP3,P-NLRP3(Ser295),Pro-caspase-1,Caspase-1,and ASC were detected by Western blot.6.Validation of the regulatory effects of SSNX and its major component,total ginsenosides(TG),on targets of the cAMP/PKA/NLRP3 pathwayMolecular docking technology was used to predict that the active components in SSNX that primarily regulate the cAMP/PKA/NLRP3 pathway are derived from TG,which is closely related to abnormal myocardial energy metabolism.Therefore,a rat model of acute myocardial ischemia-reperfusion injury(MIRI)was established to investigate the protective effects of SSNX and TG on MIRI and their effects on targets of the cAMP/PKA/NLRP3 pathway.The experimental groups included the sham group,model group,SSNX 90mg/kg group(SSNX),and TG group.Echocardiography was used to detect cardiac function,HE staining was used to detect myocardial pathological changes in rats,Evan’s blue-TTC staining was used to detect the area of myocardial infarction in rats,biochemical methods were used to detect myocardial enzymes in rats,transmission electron microscopy was used to observe the ultrastructure of rat myocardial tissue,ELISA was used to detect the levels of cAMP,cGMP,ATP,ADP,and AMP in rat serum,matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF/MS)was used to detect the distribution and content changes of energy metabolism-related molecules such as ATP,ADP,AMP,GTP,GDP,GMP,and lipid metabolism-related molecules in rat myocardial tissue,and the distribution of active monomeric components in SSNX that target the myocardium was detected.Immunofluorescence was used to detect the fluorescence expression of PKA and NLRP3 double staining and PKA and PKG double staining in rat myocardium.Western blot was used to detect the expression of cAMP/PKA/NLRP3 pathway-related proteins,including PKA,P-PKA(Ser157),PKG,P-PKG(Ser239),NLRP3,P-NLRP3(Ser295),pro-caspase-1,caspase-1,and ASC.Research result1.SSNX has a significant protective effect on acute myocardial ischemiareperfusion injury in rats.SSNX can improve the degree of tongue cyanosis in MIRI rats,increase pulse amplitude,increase rat cardiac ejection fraction,shorten peak time of myocardial blood flow perfusion and reduce myocardial tissue edema,prolong activated partial thromboplastin time.SSNX can reduce serum levels of myocardial enzymes CK,CKMB,LDH,AST,and ALT,improve the ultrastructure of rat myocardial tissue and reduce myocardial infarct size and no-reflow area.2.SSNX can exert anti-inflammatory effects on acute MIRI by regulating the TLR4/NF-κB/NLRP3 signaling pathway.SSNX can reduce the levels of pro-inflammatory cytokines IL-6,TNF-α,and MCP-1 in the myocardium of MIRI rats and decrease the expression of proinflammatory proteins TLR4,NF-κB,p-NF-κB,NLRP3,and IL-1β,alleviate inflammation,which can protect the myocardium.3.SSNX exerts a protective effect on MIRI by regulating the NLRP3/Caspase1/GSDMD signaling pathway-mediated pyroptosis and inflammation.SSNX can reduce myocardial tissue edema in MIRI-injured rats,lower the expression levels of pyroptosis-related proteins NLRP3,ASC,Pro-Caspase-1,Caspase1,GSDMD-N and reduce the release of inflammatory factors IL-1β and IL-18,thereby alleviating inflammation and protecting myocardial tissue.Meanwhile,when NLRP3 is overexpressed in H9C2 myocardial cells,the expression levels of NLRP3,ASC,Pro-Caspase-1,Caspase-1 proteins,and pyroptosisrelated protein GSDMD,GSDMD-N are increased.The ultrastructure of cells under transmission electron microscopy shows a pyroptotic morphology,and the number of damaged cells increases,while IL-18 and IL-1 β release increases and cell inflammatory response worsens.SSNX can reduce the expression levels of NLRP3,ASC,ProCaspase-1,Caspase-1,GSDMD-N proteins and the release of inflammatory factors IL1β and IL-18 in OGD/R-injured H9C2 cells and NLRP3-H9C2 cells,reduce the degree of damage to cell morphology and structure,protect myocardial cells,and the protective effect is similar to that of NLRP3 inhibitor MCC950.4.SSNX regulates upstream pathways of NLRP3 inflammasome and is related to cAMP/PKA,with PKA being a key target.Through network pharmacology predictions,it was found that SSNX regulates NLRP3 inflammasome pathways was related to cAMP-related pathways.Transcriptome sequencing results validated that SSNX can regulate numerous genes related to NLRP3 and cAMP pathways.Analysis of the PPI interaction between genes in the NLRP3 pathway and cAMP pathway revealed that the genes Pkib,Pkia,and Pkig in the cAMP-related pathway are key genes in the PPI interaction with the pathway associated with NLRP3.Pkib,Pkia,and Pkig encoded proteins that are related to PKA inhibitors.PCR detection results showed that SSNX upregulates the expression of PKA genes in the myocardium of MIRI rats,while reducing the expression of NLRP3 genes.Therefore,SSNX regulates the cAMP/PKA/NLRP3 pathway,and PKA is a key molecule in this pathway.Using molecular docking technology to dock SSNX’s blood components and key targets in the cAMP/PKA/NLRP3 pathway,it was found that ginsenosides Rd,Re,Rf,Rg2,Rh2,and CK from ginseng saponins,Salvianolic acid B from total salvianolic acid,and protopine from total alkaloids of rhizoma corydalis all have good binding ability with key targets in the cAMP/PKA/NLRP3 pathway.Among the monomers with good binding ability,those from total ginsenosides(TG)of ginseng are the most.5.SSNX and TG can regulate the activation of NLRP3 inflammasome through the cAMP/PKA pathway,with PKA being a critical target.In H9C2 cardiomyocytes subjected to OGD/R injury and treated with BUC and SSNX,the levels of ATP and cAMP were increased and the level of cGMP was decreased.The fluorescence expression of PKA was increased and NLRP3 was decreased.An increase in PKA-related proteins PKA and P-PKA(Ser157)expression levels,and an increase in P-NLRP3(Ser295)protein expression levels(Ser295 can inhibit the assembly and activation of NLRP3 inflammasome).On the other hand,expression levels of PKG and P-PKG(Ser239),as well as the expression levels of NLRP3 inflammasome-related proteins NLRP3,Pro-caspase-1,Caspase-1,and ASC proteins,are decreased,and cell activity is enhanced.In contrast,in the OGD/R group treated with PKA inhibitor H-89,the effects of BUC and SSNX are reversed.After H89 intervention,SSNX activity decreases,ATP and cAMP levels decrease,cGMP levels increase,PKA fluorescence expression decreases,NLRP3 fluorescence expression increases,PKA-related proteins PKA and P-PKA(Ser157)expression levels decrease,and P-NLRP3(Ser295)protein expression levels decrease.PKG and P-PKG(Ser239)expression levels increase,and the expression levels of NLRP3 inflammasome-related proteins NLRP3,Pro-caspase-1,Caspase-1,and ASC proteins increase,along with an increase in inflammatory cytokine levels.At the same time,SSNX and TG can improve the energy metabolism state of rat myocardium with MIRI injury by increasing the ATP and cAMP levels in the heart tissue and serum of rats,reducing cGMP levels,and increasing the expression levels of PKA-related proteins PKA and P-PKA(Ser157),as well as P-NLRP3(Ser295)protein expression.They also reduce the expression levels of PKA antagonistic proteins PKG and P-PKG(Ser239),decrease the expression levels of NLRP3 inflammasome-related proteins NLRP3,Pro-caspase-1,Caspase-1,and ASC proteins,and reduce the levels of inflammatory cytokines.6.SSNX can regulate the distribution of energy metabolism substances and phospholipid molecules in the myocardium,and activate the cAMP/PKA pathway.SSNX and its component TG can improve the distribution of ATP,ADP,AMP,GTP,GDP,GMP in acute MIRI rat myocardial tissue,as well as the levels of cAMP,cGMP,ATP,ADP,and AMP in serum.They can also alter the cellular energy state,increase ATP generation,and change the distribution of phospholipid molecules such as PC,PA,PE,and PG in the myocardium.7.Various components in SSNX can target myocardial tissue.Mass spectrometry imaging technology shows that 18 monomeric components in SSNX target myocardial tissue,including 9 from total ginsenosides,3 from total salvianolic acids,and 6 from total alkaloids of rhizoma corydalis.These components are ginsenoside Rd,ginsenoside Re,ginsenoside Rf,ginsenoside Rf1,ginsenoside Rg2,ginsenoside F2,ginsenoside Rh1,ginsenoside Rh2,ginsenoside CK,salvianolic acid B,lithospermic acid,rosmarinic acid,morphine,tetrahydropalmatine,dehydrocorydaline,berberine,palmatine,and corydaline.Conclusion:1.SSNX can improve the cardiac function of acute MIRI rats,increase coronary blood flow,alleviate myocardial pathological injury,improve coagulation function,reduce myocardial enzyme levels,improve the ultrastructure of rat myocardium and reduce myocardial infarction area and non-reflow area,which protecting the myocardium.2.SSNX can regulate the TLR4/NF-κB/NLRP3 signaling pathway,reduce the content of inflammatory factors and the expression of inflammation-related proteins,and play an anti-inflammatory role in acute MIRI.3.SSNX can regulate the NLRP3/Caspase-1/GSDMD signaling pathway,reduce the content of inflammatory factors,lower the expression of inflammation-related proteins and pyroptosis-related proteins,alleviate inflammation response,reduce pyroptosis,and thus play a protective role in acute MIRI and OGD/R injury of H9C2 myocardial cells.4.SSNX can regulate the cAMP/PKA/NLRP3 signaling pathway,increase the content of ATP and activate cAMP signaling transduction.After that,SSNX can activate PKA,promote the phosphorylation of NLRP3 Ser295,thereby inhibiting the activation of NLRP3 inflammasome,reducing the expression of inflammation-related proteins,and reducing the content of inflammatory factors,thus exerting an antiinflammatory effect on acute MIRI in rats and OGD/R injury of H9C2 myocardial cells.5.SSNX can regulate the distribution of energy metabolic substances and phospholipid molecules in myocardium,improve the energy metabolism status of myocardium and the structure and permeability of cell membrane,and then regulate the cAMP/PKA signaling pathway.6.There are 18 components in SSNX that can target myocardial tissue.These components may have an important protective effect on acute MIRI myocardium.In summary,SSNX can regulate the TLR4/NF-κB/NLRP3 and cAMP/PKA/NLRP3 pathways to regulate NLRP3 inflammasome-mediated pyroptosis and inflammation response,thus exerting a protective effect on acute myocardial ischemia-reperfusion injury. |
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