RESUMO
BACKGROUND: The purpose of this study was to examine the in vivo activity of rosmarinic acid (RA) - a phytochemical with antioxidant, anti-inflammatory, and antiviral properties - against influenza virus (IAV). An antibody-based kinase array and different in vitro functional assays were also applied to identify the mechanistic underpinnings by which RA may exert its anti-IAV activity. METHODS: We initially examined the potential efficacy of RA using an in vivo mouse model. A time-of-addition assay and an antibody-based kinase array were subsequently applied to investigate mechanism-of-action targets for RA. The hemagglutination inhibition assay, neuraminidase inhibition assay, and cellular entry assay were also performed. RESULTS: RA increased survival and prevented body weight loss in IAV-infected mice. In vitro experiments revealed that RA inhibited different IAV viruses - including oseltamivir-resistant strains. From a mechanistic point of view, RA downregulated the GSK3ß and Akt signaling pathways - which are known to facilitate IAV entry and replication into host cells. CONCLUSIONS: RA has promising preclinical efficacy against IAV, primarily by interfering with the GSK3ß and Akt signaling pathways.
Assuntos
Vírus da Influenza A , Influenza Humana , Animais , Antivirais , Cinamatos , Depsídeos , Glicogênio Sintase Quinase 3 beta , Humanos , Camundongos , Oseltamivir , Proteínas Proto-Oncogênicas c-akt , Replicação Viral , Ácido RosmarínicoRESUMO
Glioblastoma is the most common primary brain malignancy with limited treatment options. EphA2 is a tumor-associated-antigen overexpressed in glioblastoma. Pre-clinical studies have demonstrated the promise of EphA2-redirected CAR T-cells against glioblastoma. We conduct the first-in-human trial of EphA2-redirected CAR T-cells in patients with EphA2-positive recurrent glioblastoma and report the results of three patients enrolled as the first cohort receiving the starting dosage (1×106 cells/kg). A single infusion of EphA2-redirected CAR T-cells was administrated intravenously, with the lymphodepletion regimen consisting of fludarabine and Cyclophosphamide. In two patients, there was grade 2 cytokine release syndrome accompanied by pulmonary edema, which resolved completely with dexamethasone medication. Except that, there was no other organ toxicity including neurotoxicity. In both the peripheral blood and cerebral-spinal-fluid, we observed the expansion of CAR T-cells which persisted for more than four weeks. In one patient, there was a transit diminishment of the tumor. Among these three patients, one patient reported SD and two patients reported PD, with overall survival ranging from 86 to 181 days. At the tested dose level (1×106 cells/kg), intravenously infusion of EphA2-rediretected CAR T-cells were preliminary tolerable with transient clinical efficacy. Future study with adjusted dose and infusion frequency of CAR T-cells is warranted. TRIAL REGISTRATION NUMBERS: NCT03423992.
RESUMO
Although chimeric antigen receptor T (CAR-T) cell therapy achieves high remission rates, challenges (e.g., toxicity management and relapse prevention) remain. The major risks are cytokine release syndrome and related neurological toxicity. The influence of the CAR surface density on the efficacy/safety of CAR-T cell therapy and the factors determining CAR density were not elucidated comprehensively. Here, we discovered that the use of the MND promoter increased the transduction rate and reduced the CAR surface density. Additionally, MND-driven CAR-T cells had prolonged antileukemia activity in a mouse model. In an initial dual-armed anti-CD19 CAR-T cell pilot study (ClinicalTrials.gov: NCT03840317), eight and six subjects were infused with MND and EF1α promoter-driven autologous CAR-T cells (3 × 105 CAR-T cells/kg), respectively. MND subjects developed mild fever and lower interferon gamma (IFN-γ) concentrations than in the EF1A19 group. All but one subject in each cohort reached minimal residual disease (MRD)-negative complete remission after the first month of evaluation. These results represent the first comprehensive study on the promoter-driven modulation of CAR-T cell functionality. These findings encourage further evaluation of the potential of the MND promoter to drive CAR-T cells as a broadly applicable cellular product for anticancer immunotherapy.
RESUMO
Clinical trials of chimeric antigen receptors (CARs) targeting CD19 have produced impressive results in hematological malignancies, including diffuse large B-cell lymphoma (DLBCL). However, a notable number of patients with DLBCL fail to achieve remission after CD19 CAR T-cell therapy and may therefore require a dual targeted CAR T-cell therapy. A 31-year-old man with refractory DLBCL was assessed in the present case report. The patient was treated with sequential infusion of single CD19 CAR T cells followed by dual CD19/CD22-targeted CAR T cells. The outcome was that the patient achieved partial remission after the first single CD19 CAR T-cell infusion and complete remission after the dual CD19/CD22-targeted CAR T-cell infusion. Grade 1 cytokine release syndrome (CRS) was observed after the single CD19 CAR T-cell infusion, while grade 3 CRS and hemophagocytic syndrome were observed after the dual targeted CAR T-cell infusion, but these adverse effects alleviated after the treatments. To the best of our knowledge, the present case report is the first to describe the successful application of dual CD19/CD22-targeted CAR T-cell therapy for the treatment of refractory DLBCL. The report suggests that dual CD19/CD22-targeted CAR T-cell therapy may represent a promising option for the treatment of refractory DLBCL; however, caution should be taken due to potential CRS development.
RESUMO
Enterovirus A71 (EV-A71), a positive-stranded RNA virus of the Picornaviridae family, may cause neurological complications or fatality in children. We examined specific factors responsible for this virulence using a chemical genetics approach. Known compounds from an anti-EV-A71 herbal medicine, Salvia miltiorrhiza (Danshen), were screened for anti-EV-A71. We identified a natural product, rosmarinic acid (RA), as a potential inhibitor of EV-A71 by cell-based antiviral assay and in vivo mouse model. Results also show that RA may affect the early stage of viral infection and may target viral particles directly, thereby interfering with virus-P-selectin glycoprotein ligand-1 (PSGL1) and virus-heparan sulfate interactions without abolishing the interaction between the virus and scavenger receptor B2 (SCARB2). Sequencing of the plaque-purified RA-resistant viruses revealed a N104K mutation in the five-fold axis of the structural protein VP1, which contains positively charged amino acids reportedly associated with virus-PSGL1 and virus-heparan sulfate interactions via electrostatic attraction. The plasmid-derived recombinant virus harbouring this mutation was confirmed to be refractory to RA inhibition. Receptor pull-down showed that this non-positively charged VP1-N104 is critical for virus binding to heparan sulfate. As the VP1-N104 residue is conserved among different EV-A71 strains, RA may be useful for inhibiting EV-A71 infection, even for emergent virus variants. Our study provides insight into the molecular mechanism of virus-host interactions and identifies a promising new class of inhibitors based on its antiviral activity and broad spectrum effects against a range of EV-A71.
Assuntos
Antivirais/administração & dosagem , Proteínas do Capsídeo/genética , Cinamatos/administração & dosagem , Depsídeos/administração & dosagem , Enterovirus Humano A/patogenicidade , Infecções por Enterovirus/tratamento farmacológico , Salvia miltiorrhiza/química , Animais , Antivirais/farmacologia , Proteínas do Capsídeo/antagonistas & inibidores , Proteínas do Capsídeo/química , Linhagem Celular , Cinamatos/farmacologia , Depsídeos/farmacologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Enterovirus Humano A/efeitos dos fármacos , Enterovirus Humano A/metabolismo , Infecções por Enterovirus/virologia , Heparitina Sulfato/metabolismo , Humanos , Células Jurkat , Glicoproteínas de Membrana/metabolismo , Camundongos , Mutação , Extratos Vegetais/administração & dosagem , Extratos Vegetais/farmacologia , Ligação Proteica/efeitos dos fármacos , Eletricidade Estática , Fatores de Virulência/antagonistas & inibidores , Fatores de Virulência/química , Fatores de Virulência/genética , Ácido RosmarínicoRESUMO
Ninety percent of relapse/refractory B-cell acute lymphatic leukemia (R/R B-ALL) patients can achieve complete remission (CR) after CD19-targeting chimeric antigen receptor T (CAR-T) cell therapy. However, around 50% of them relapse in 1 year. Persistent CAR-T cell engraftment is considered as the key to remain durable remission. Here, we initiated a phase I study to treat 10 pediatric B-ALL patients using a CD19-targeted second generation CAR with a 4-1BB intracellular costimulatory domain. All patients received a standard fludarabine and cyclophosphamide (FC) preconditioning regiment, followed by a CAR-T infusion with a median number of 0.5 (0.3-1.58) × 106 CAR+ T cells/kg. The pretreatment tumor burdens were high with a median bone marrow (BM) blasts percentage of 59.2% (7.31%-86.2%), excluding one patient only with brain infiltration of leukemia cells (0% BM blasts). The initial CR rate was 80% (n = 8/10). Four patients (40%) experienced serious (grade > 2) cytokine release syndrome (CRS) and three patients (30%) with obvious neurotoxicity. Monthly assessments of CD19+ minimal residual disease (MRD) and CAR-T engraftment demonstrated the anti-CD19 activity of long-term engrafted CAR-T cell clones in one patient for more than 2 years.
Assuntos
Antígenos CD19/imunologia , Imunoterapia Adotiva , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Criança , Pré-Escolar , Feminino , Humanos , Imunoterapia Adotiva/efeitos adversos , Imunoterapia Adotiva/métodos , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidade , Prognóstico , Receptores de Antígenos de Linfócitos T/genética , Recidiva , Resultado do TratamentoRESUMO
Influenza virus remains an emerging virus and causes pandemics with high levels of fatality. After screening different plant extracts with potential anti-influenza activity, a water extract of Taxodium distichum stems (TDSWex) showed excellent activity against influenza viruses. The EC50 of TDSWex was 0.051 ± 0.024 mg/mL against influenza virus A/WSN/33. TDSWex had excellent antiviral efficacy against various strains of human influenza A and B viruses, particularly oseltamivir-resistant clinical isolates and a swine-origin influenza strain. We observed that the synthesis of viral RNA and protein were inhibited in the presence of TDSWex. The results of the time-of-addition assay suggested that TDSWex inhibited viral entry and budding. In the hemagglutination inhibition assay, TDSWex inhibited the hemagglutination of red blood cells, implying that the extract targeted hemagglutin-related functions such as viral entry. In the attachment and penetration assay, TDSWex showed antiviral activity with EC50s of 0.045 ± 0.026 and 0.012 ± 0.003 mg/mL, respectively. In addition, TDSWex blocked neuraminidase activity. We conclude that TDSWex has bimodal activities against both hemagglutinin and neuraminidase during viral replication.
Assuntos
Hemaglutininas/metabolismo , Neuraminidase/metabolismo , Orthomyxoviridae/metabolismo , Extratos Vegetais/metabolismo , Taxodium/química , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cães , Hemaglutininas/química , Humanos , Células Madin Darby de Rim Canino , Microscopia de Fluorescência , Neuraminidase/antagonistas & inibidores , Orthomyxoviridae/enzimologia , Extratos Vegetais/química , Extratos Vegetais/toxicidade , RNA Viral/química , RNA Viral/metabolismo , Taxodium/metabolismo , Proteínas Virais/metabolismo , Internalização do Vírus/efeitos dos fármacos , Liberação de Vírus/efeitos dos fármacosRESUMO
OBJECTIVES: Enterovirus 71 (EV-A71) is an important pathogen that can cause severe neurological symptoms and even death. Our aim was to identify potent anti-EV-A71 compounds and study their underlying mechanisms and in vivo activity. METHODS: We identified a potent imidazolidinone derivative (abbreviated to PR66) as an inhibitor of EV-A71 infection from the screening of compounds and subsequent structure-based modification. Time-course treatments and resistant virus selection of PR66 were employed to study the mode of mechanism of PR66. In vivo activity of PR66 was tested in the ICR strain of new-born mice challenged with EV-A71/4643/MP4. RESULTS: PR66 could impede the uncoating process during viral infection via interaction with capsid protein VP1, as shown by a resistant virus selection assay. Using site-directed mutagenesis, we confirmed that a change from valine to phenylalanine in the 179th amino acid residue of the cDNA-derived resistant virus resulted in resistance to PR66. PR66 increased the virion stability of WT viruses, but not the PR66-resistant mutant, in a particle stability thermal release assay. We further showed that PR66 had excellent anti-EV-A71 activity in an in vivo mouse model of disease, with a dose-dependent increase in survival rate and in protection against virus-induced hind-limb paralysis following oral or intraperitoneal administration. This was associated with reductions of viral titres in brain and muscle tissues. CONCLUSIONS: We demonstrated here for the first time that an imidazolidinone derivative (PR66) could protect against EV-A71-induced neurological symptoms in vivo by suppressing EV-A71 replication. This involved binding to and restricting viral uncoating.
Assuntos
Antivirais/metabolismo , Antivirais/farmacologia , Capsídeo/efeitos dos fármacos , Enterovirus Humano A/efeitos dos fármacos , Animais , Antivirais/isolamento & purificação , Linhagem Celular , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Infecções por Enterovirus/tratamento farmacológico , Infecções por Enterovirus/virologia , Humanos , Concentração Inibidora 50 , Camundongos Endogâmicos ICR , Análise de SobrevidaRESUMO
Endoplasmic reticulum (ER) stress is a general term for representing the pathway by which various stimuli affect ER functions. ER stress induces the evolutionarily conserved signaling pathways, called the unfolded protein response (UPR), which compromises the stimulus and then determines whether the cell survives or dies. In recent years, ongoing research has suggested that these pathways may be linked to the autophagic response, which plays a key role in the cell's response to various stressors. Autophagy performs a self-digestion function, and its activation protects cells against certain pathogens. However, the link between the UPR and autophagy may be more complicated. These two systems may act dependently, or the induction of one system may interfere with the other. Experimental studies have found that different viruses modulate these mechanisms to allow them to escape the host immune response or, worse, to exploit the host's defense to their advantage; thus, this topic is a critical area in antiviral research. In this review, we summarize the current knowledge about how RNA viruses, including influenza virus, poliovirus, coxsackievirus, enterovirus 71, Japanese encephalitis virus, hepatitis C virus, and dengue virus, regulate these processes. We also discuss recent discoveries and how these will produce novel strategies for antiviral treatment.
RESUMO
Bai Shao (BS, the root of Paeonia lactiflora Pall.), a common Chinese herb in many recipes used to treat viral infection and liver diseases, is recognized for its ability to nourish menstruation, its Yin convergence, and as an antiperspirant. However, the mechanism and components for its antiviral function remain to be elucidated. In this study, an ethanolic extract of BS was further partitioned into aqueous and organic parts (EAex) for in vitro functional study and in vivo efficacy testing. EAex exhibited an IC50 of 0.016 ± 0.005 mg/mL against influenza virus A/WSN/33 (H1N1), with broad-spectrum inhibitory activity against different strains of human influenza A viruses, including clinical oseltamivir-resistant isolates and an H1N1pdm strain. The synthesis of both viral RNA and protein was profoundly inhibited when the cells were treated with EAex. A time-of-addition assay demonstrated that EAex exerted its antiviral activity at various stages of the virus replication cycle. We addressed its antiviral activity at virus entry and demonstrated that EAex inhibits viral hemagglutination and viral binding to and penetration into host cells. In vivo animal testing showed that 200 mg/kg/d of EAex offered significant protection against viral infection. We conclude that BS possesses antiviral activity and has the potential for development as an anti-influenza agent.
Assuntos
Antivirais/farmacologia , Medicina Herbária , Vírus da Influenza A/efeitos dos fármacos , Paeonia/química , Extratos Vegetais/farmacologia , Animais , Antivirais/administração & dosagem , Antivirais/isolamento & purificação , Modelos Animais de Doenças , Vírus da Influenza A/fisiologia , Concentração Inibidora 50 , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/prevenção & controle , Extratos Vegetais/administração & dosagem , Extratos Vegetais/isolamento & purificação , Resultado do Tratamento , Internalização do Vírus/efeitos dos fármacosRESUMO
Streptothricin-F (STT-F), one of the early-discovered antibiotics, consists of three components, a ß-lysine homopolymer, an aminosugar D-gulosamine, and an unusual bicyclic streptolidine. The biosynthesis of streptolidine is a long-lasting but unresolved puzzle. Herein, a combination of genetic/biochemical/structural approaches was used to unravel this problem. The STT gene cluster was first sequenced from a Streptomyces variant BCRC 12163, wherein two gene products OrfP and OrfR were characterized inâ vitro to be a dihydroxylase and a cyclase, respectively. Thirteen high-resolution crystal structures for both enzymes in different reaction intermediate states were snapshotted to help elucidate their catalytic mechanisms. OrfP catalyzes an Fe(II) -dependent double hydroxylation reaction converting L-Arg into (3R,4R)-(OH)2 -L-Arg via (3S)-OH-L-Arg, while OrfR catalyzes an unusual PLP-dependent elimination/addition reaction cyclizing (3R,4R)-(OH)2 -L-Arg to the six-membered (4R)-OH-capreomycidine. The biosynthetic mystery finally comes to light as the latter product was incorporation into STT-F by a feeding experiment.
Assuntos
Aminoácidos/síntese química , Estreptotricinas/síntese química , Aminoácidos/química , Hidroxilação , Oxigenases de Função Mista/química , Estreptotricinas/químicaRESUMO
The growing number of Klebsiella pneumoniae infections, commonly acquired in hospitals, has drawn great concern. It has been shown that the K1 and K2 capsular serotypes are the most detrimental strains, particularly to those with diabetes. The K1 cps (capsular polysaccharide) locus in the NTUH-2044 strain of the pyogenic liver abscess (PLA) K. pneumoniae has been identified recently, but little is known about the functions of the genes therein. Here we report characterization of a group of cps genes and their roles in the pathogenesis of K1 K. pneumoniae. By sequential gene deletion, the cps gene cluster was first re-delimited between genes galF and ugd, which serve as up- and down-stream ends, respectively. Eight gene products were characterized in vitro and in vivo to be involved in the syntheses of UDP-glucose, UDP-glucuronic acid and GDP-fucose building units. Twelve genes were identified as virulence factors based on the observation that their deletion mutants became avirulent or lost K1 antigenicity. Furthermore, deletion of kp3706, kp3709 or kp3712 (ΔwcaI, ΔwcaG or Δatf, respectively), which are all involved in fucose biosynthesis, led to a broad range of transcriptional suppression for 52 upstream genes. The genes suppressed include those coding for unknown regulatory membrane proteins and six multidrug efflux system proteins, as well as proteins required for the K1 CPS biosynthesis. In support of the suppression of multidrug efflux genes, we showed that these three mutants became more sensitive to antibiotics. Taken together, the results suggest that kp3706, kp3709 or kp3712 genes are strongly related to the pathogenesis of K. pneumoniae K1.
Assuntos
Cápsulas Bacterianas/genética , Vias Biossintéticas/genética , Genes Bacterianos/genética , Klebsiella pneumoniae/genética , Antibacterianos/farmacologia , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Cápsulas Bacterianas/química , Cápsulas Bacterianas/efeitos dos fármacos , Cápsulas Bacterianas/imunologia , Vias Biossintéticas/efeitos dos fármacos , Configuração de Carboidratos , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Frutose/biossíntese , Deleção de Genes , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Teste de Complementação Genética , Klebsiella pneumoniae/crescimento & desenvolvimento , Klebsiella pneumoniae/imunologia , Klebsiella pneumoniae/patogenicidade , Testes de Sensibilidade Microbiana , Mutagênese Insercional/genética , Fases de Leitura/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismoAssuntos
Oxirredutases do Álcool/química , Mononucleotídeo de Flavina/química , Glicopeptídeos/biossíntese , Glicopeptídeos/química , Teicoplanina/análogos & derivados , Oxirredutases do Álcool/metabolismo , Antibacterianos/biossíntese , Antibacterianos/química , Conformação Molecular , Especificidade por Substrato , Teicoplanina/biossíntese , Teicoplanina/químicaRESUMO
The unique pharmacokinetic/pharmacodynamic activities of glycopeptide antibiotics are conferred from the tailoring steps occurring on the aglycone. It was hypothesized that the sugar moiety attached to the aglycone is derived from an unusual UDP-glucosamine and is followed by an acylation reaction in the biosynthesis of teicoplanin/A40926. Here we report that three homologous (>65% identical) proteins Dbv21, Orf2*, and Orf15, previously assigned as hypothetical proteins in the biosynthesis of A40926, teicoplanin, and chloroeremomycin, respectively, are novel deacetylases (Dbv21 and Orf2*) and thymidyltransferase (Orf15). Dbv21 and Orf2* catalyze the deacetylation reaction of N-acetylglucosaminyl-teicoplanin pseudoaglycone, while Orf15 catalyzes the formation of dTDP-glucose that is required for the epi-vancosamine/vancosamine decoration of chloroeremomycin/vancomycin.
Assuntos
Amidoidrolases/metabolismo , Antibacterianos/biossíntese , Glicopeptídeos/biossíntese , Família Multigênica/genética , Nucleotidiltransferases/metabolismo , Timidina/metabolismo , Acilação , Amidoidrolases/genética , Antibacterianos/química , Bioensaio , Catálise , Cromatografia Líquida , Glicopeptídeos/química , Cinética , Espectrometria de Massas , Modelos Químicos , Família Multigênica/fisiologia , Nucleotidiltransferases/genética , Proteínas Virais/genética , Proteínas Virais/fisiologiaRESUMO
Tir of enteropathogenic Escherichia coli (EPEC) or enterohemorrahgic E. coil (EHEC) is translocated by a type III secretion system to the host cell membranes where it serves as a receptor for the binding of a second bacterial membrane protein. In response to the binding, EPEC Tir is phosphorylated at Tyr474, and this phosphorylation is necessary for the signaling of pedestal formation. Tir of EHEC has no equivalent phosphorylation site but it is similarly needed for cytoskeleton rearrangement. How these two Tir molecules achieve their function by apparently different mechanisms is not completely clear. To examine their intrinsic differences, the two Tirs were expressed in HeLa cells and compared. Actin in complexes could be pelleted down from the lysate of cells expressing EHEC Tir but not EPEC Tir. By immunostaining, neither Tir molecule was found in phosphorylated state. In the cytoplasm, EHEC Tir was frequently found in fibrous structures whereas EPEC Tir was observed completely in a diffusive form. The determinant critical for the EHEC Tir fibrous formation was mapped to the C-terminal region of the molecule that deviates from the EPEC counterpart. This region may play a role in taking an alternative route different from Tyr474 phosphorylation to transduce signals.
Assuntos
Escherichia coli O157 , Proteínas de Escherichia coli/metabolismo , Escherichia coli , Receptores de Superfície Celular/metabolismo , Sequência de Aminoácidos , Escherichia coli/química , Escherichia coli/metabolismo , Escherichia coli/patogenicidade , Escherichia coli O157/química , Escherichia coli O157/metabolismo , Escherichia coli O157/patogenicidade , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Células HeLa , Humanos , Dados de Sequência Molecular , Fosforilação , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Ischemia-reperfusion (I/R) injury of the heart can be attenuated by application of median nerve stimulation (MNS) through either electroacupuncture (EA) or local somatothermal stimulation (LSTS). This study is aimed to investigate the differences of myocardial protein expression between MNS by EA and by LSTS. Twenty-four male Sprague-Dawley rats were randomly allocated to 8 groups. In MNS by EA groups, animals were EA-stimulated for 30 min and the hearts were taken for proteomics analysis at an interval of 0 min (group 1), 30 min (group 2), 60 min (group 3) and control group (group 4); whereas in MNS by LSTS groups, following 1 dose (group 5), 2 doses (group 6), 3 doses (group 7) and control group (group 8) of LSTS, the hearts were taken for proteomics analysis. Myocardial protein expression profiles were analyzed and validated by Western blot analysis. Finally, the biological responses were also evaluated. The results showed that either MNS by EA followed by a 30-min rest period (group 2) and by 3 doses of LSTS (group 7) had cardioprotective effect against I/R injury. However, the myocardial protein expression profiles are quite different between the above-mentioned groups. We conclude that MNS by either EA or LSTS attenuates I/R injury in rat hearts through different protective mechanisms and that EA and LSTS might provide an alternative treatment strategy for ischemic heart disease.