Aptamer Protein Research Articles

DNA logic gate for cancer cells identification, detection, and modulation of cell–cell interactions

The precise detection of cancer cells and modulation of cell–cell interactions would be highly desired for noninvasive and nontoxic cancer treatments but it remains challenging due to the existence of biomarkers on both normal and tumor cells. In this work, we engineered a logic-gated DNA molecular machine incorporating a membrane protein aptamer and an extracellular acid responsive split i-motif for the precise identification and detection of cancer cells, as well as the mediation of cell–cell interactions. In this system, only in the presence of both the membrane protein and protons, the logic-gated DNA molecular machine is activated to perform the “AND” logic operation to induce catalytic hairpin assembly (CHA) on the electrode, leading to the formation of G-quadruplex/hemin and the generation of an amplified electrochemical signal. On the other hand, the formed “AND” logic could hybridize with the DNA fragment on T-cell, effectively shorting the distance between T-cell and cancer cells. Benefiting from the DNA logic gate and CHA, this strategy could not only precisely identify cancer cells, but also show a sensitive analysis of cancer cells with a detection limit of 15 cells/mL. The logic-gated DNA molecular machine achieved the identification of cancer cells and modulation of intercellular interactions, providing a new idea for precise disease diagnosis and therapy.

Proteomic Risk Score of Increased Respiratory Susceptibility: A Multi-Cohort Study.

Accelerated decline in lung function is associated with incident COPD, hospitalizations and death. However, identifying this trajectory with longitudinal spirometry measurements is challenging in clinical practice. To determine whether a proteomic risk score trained on accelerated decline in lung function can assess risk of future respiratory disease and mortality. In CARDIA, a population-based cohort starting in young adulthood, longitudinal measurements of FEV1 percent predicted (up to six timepoints over 30 years) were used to identify accelerated and normal decline trajectories. Protein aptamers associated with an accelerated decline trajectory were identified with multivariable logistic regression followed by LASSO regression. The proteomic respiratory susceptibility score was derived based on these circulating proteins and applied to the UK Biobank and COPDGene studies to examine associations with future respiratory morbidity and mortality. Higher susceptibility score was independently associated with all-cause mortality (UKBB: HR 1.56, 95%CI 1.50-1.61; COPDGene: HR 1.75, 95%CI 1.63-1.88), respiratory mortality (UKBB: HR 2.39, 95% CI 2.16-2.64; COPDGene: HR 1.83, 95%CI 1.33-2.51), incident COPD (UKBB: HR 1.84, 95%CI 1.71-1.98), incident respiratory exacerbation (COPDGene: OR 1.11, 95%CI 1.03-1.20), and incident exacerbation requiring hospitalization (COPDGene: OR 1.18, 95%CI 1.08-1.28). A proteomic signature of increased respiratory susceptibility identifies people at risk of respiratory death, incident COPD, and respiratory exacerbations. This susceptibility score is comprised of proteins with well-known and novel associations with lung health and holds promise for the early detection of lung disease without requiring years of spirometry measurements.

A microfluidic-based chemiluminescence biosensor for sensitive multiplex detection of exosomal microRNAs based on hybridization chain reaction

The analysis of microRNAs (miRNAs) in exosomes is of great importance for noninvasive early disease diagnosis. However, current techniques to detect exosomal miRNAs is hampered either by laborious exosome isolation or low abundance of miRNAs in exosomes. Here, we developed a microfluidic chemiluminescence (CL) analysis method for the multiplexed detection of exosomal miR-21 and miR-155. The microfluidic device contained three parts: a snake-shaped channel for fully mixing chemiluminescent reagents, a ship-shaped channel modified with CD63 protein aptamer for capturing exosomes, and another two parallel ship-shaped channels for hybridization chain reaction (HCR) amplification and CL detection. The multiple signal amplification was realized by Y-shaped arrays, HCR amplification, and poly-HRP catalyzed CL reaction. Using this multiple signal amplification method, our microfluidic CL biosensor achieves a limit of detection of miRNAs of 0.49 fM, with a linear range of 1 fM–10 pM, which is better or comparable to previously reported biosensors. What's more, the proposed microfluidic biosensor exhibits great specificity and selectivity to the target miRNA. Moreover, the microfluidic CL strategy exhibited excellent accuracy and could significantly distinguish different cancer subtypes as well as cancer patients and healthy people. These results suggest that this simple, high sensitive, and more accurate analytical strategy by analyzing different types of exosomal miRNAs has the potential applications in cancer diagnosis and stage monitoring.

Correction: Intuitionistic fuzzy rough set model based on k-means and its application to enhance prediction of aptamer–protein interacting pairs

Correction: Intuitionistic fuzzy rough set model based on k-means and its application to enhance prediction of aptamer–protein interacting pairs

Intuitionistic fuzzy rough set model based on k-means and its application to enhance prediction of aptamer–protein interacting pairs

Intuitionistic fuzzy rough set model based on k-means and its application to enhance prediction of aptamer–protein interacting pairs

Nuclease-assisted selection of slow-off rate aptamers.

Conventional directed evolution methods offer the ability to select bioreceptors with high binding affinity for a specific target in terms of thermodynamic properties. However, there is a lack of analogous approaches for kinetic selection, which could yield affinity reagents that exhibit slow off-rates and thus remain tightly bound to targets for extended periods. Here, we describe an in vitro directed evolution methodology that uses the nuclease flap endonuclease 1 to achieve the efficient discovery of aptamers that have slow dissociation rates. Our nuclease-assisted selection strategy can yield specific aptamers for both small molecules and proteins with off-rates that are an order of magnitude slower relative to those obtained with conventional selection methods while still retaining excellent overall target affinity in terms of thermodynamics. This new methodology provides a generalizable approach for generating slow off-rate aptamers for diverse targets, which could, in turn, prove valuable for applications including molecular devices, bioimaging, and therapy.

Plasma proteomics in the UK Biobank reveals youthful brains and immune systems promote healthspan and longevity.

Organ-derived plasma protein signatures derived from aptamer protein arrays track organ-specific aging, disease, and mortality in humans, but the robustness and clinical utility of these models and their biological underpinnings remain unknown. Here, we estimate biological age of 11 organs from 44,526 individuals in the UK Biobank using an antibody-based proteomics platform to model disease and mortality risk. Organ age estimates are associated with future onset of heart failure (heart age HR=1.83), chronic obstructive pulmonary disease (lung age HR=1.39), type II diabetes (kidney age HR=1.58), and Alzheimer's disease (brain age HR=1.81) and sensitive to lifestyle factors such as smoking and exercise, hormone replacement therapy, or supplements. Remarkably, the accrual of aged organs progressively increases mortality risk while a youthful brain and immune system are uniquely associated with disease-free longevity. These findings support the use of plasma proteins for monitoring organ health and the efficacy of drugs targeting organ aging disease.

High Throughput Plasma Proteomics and Risk of Heart Failure and Frailty in Late Life

ImportanceHeart failure (HF) and frailty frequently coexist and may share a common pathobiology, although the underlying mechanisms remain unclear. Understanding these mechanisms may provide guidance for preventing and treating both conditions.ObjectiveTo identify shared pathways between incident HF and frailty in late life using large-scale proteomics.Design, Setting, and ParticipantsIn this cohort study, 4877 aptamers (Somascan v4) were measured among participants in the community-based longitudinal Atherosclerosis Risk In Communities (ARIC) cohort study at visit 3 (V3; 1993-1995; n = 10 638) and at visit 5 (V5; 2011-2013; n = 3908). Analyses were externally replicated among 3189 participants in the Cardiovascular Health Study (CHS). Data analysis was conducted from February 2022 to June 2023.ExposuresProtein aptamers, measured at study V3 and V5.Main Outcomes and MeasuresOutcomes assessed included incident HF hospitalization after V3 and after V5, prevalent frailty at V5, and incident frailty between V5 and visit 6 (V6; 2016-2017; n = 4131). Frailty was assessed using the Fried criteria. Analyses were adjusted for age, gender, race, field center, hypertension, diabetes, smoking status, body mass index, estimated glomerular filtration rate, prevalent coronary heart disease, prevalent atrial fibrillation, and history of myocardial infarction. Mendelian randomization (MR) analysis was performed to assess potential causal effects of candidate proteins on HF and frailty.ResultsA total of 4877 protein aptamers were measured among 10 638 participants at V3 (mean [SD] age, 60 [6] years; 4886 [46%] men). Overall, 286 proteins were associated with incident HF after V3 (822 events; P < 1.0 × 10−5), 83 of which were also associated with incident after V5 (336 events; P < 1.7 × 10−4). Among HF-free participants at V5 (n = 3908; mean [SD] age, 75 [5] years; 1861 [42%] men), 48 of 83 HF-associated proteins were associated with prevalent frailty (223 cases; P < 6.0 × 10−4), 18 of which were also associated with incident frailty at V6 (152 cases; P < 1.0 × 10−3). These proteins enriched fibrosis and inflammation pathways and demonstrated stronger associations with incident HF with preserved ejection fraction (HFpEF) than HF with reduced ejection fraction. All 18 proteins were associated with both prevalent frailty and incident HF in CHS. MR identified potential causal effects of several proteins on frailty and HF.Conclusions and RelevanceIn this study, the proteins associated with risk of HF and frailty enrich for pathways related to inflammation and fibrosis as well as risk of HFpEF. Several of these proteins could potentially contribute to the shared pathophysiology of frailty and HF.

Membrane protein guided and ATP-gated dual module DNA nanodevice for amplified fluorescence labeling of cancer cells

The occurrence and progression of tumors are accompanied by changes in both the microenvironment and cancer cells. Therefore, the detection of extracellular alert molecule accumulation, as well as the identification of abnormal cancer biomarkers both contribute to the accurate localization of individual cancer cells. Here, we have designed a cancer cell fluorescence labeling nanodevice with a membrane protein targeting domain and extracellular ATP-fueled amplification switch. The AND logic-based nanodevice light-up the cancer cell membrane only in the coexistence of target membrane proteins and abnormal ATP concentration. We demonstrated the ability of the nanodevice to selectively label target cells in a cell mixture supplemented with ATP at concentrations within the range in the tumor microenvironment (1–5×10−4 M). Additionally, the modular design of DNA nanodevices enables broad application to different cancer cells by replacing them with corresponding membrane protein aptamers. Our fluorescence labeling nanodevice with signal amplification shows promise in serving as an imaging tool for visualizing the impact of ATP regulation on cancer evolution.

Surface plasmon resonance aptasensing and computational analysis of Staphylococcus aureus IsdA surface protein.

Staphylococcus aureus (S. aureus), a common foodborne pathogen, poses significant public health challenges due to its association with various infectious diseases. A key player in its pathogenicity, which is the IsdA protein, is an essential virulence factor in S. aureus infections. In this work, we present an integrated in-silico and experimental approach using MD simulations and surface plasmon resonance (SPR)-based aptasensing measurements to investigate S. aureus biorecognition via IsdA surface protein binding. SPR, a powerful real-time and label-free technique, was utilized to characterize interaction dynamics between the aptamer and IsdA protein, and MD simulations was used to characterize the stable and dynamic binding regions. By characterizing and optimizing pivotal parameters such as aptamer concentration and buffer conditions, we determined the aptamer's binding performance. Under optimal conditions of pH 7.4 and 150 mM NaCl concentration, the kinetic parameters were determined; ka = 3.789 × 104/Ms, kd = 1.798 × 103/s, and KD = 4.745 × 10-8 M. The simulations revealed regions of interest in the IsdA-aptamer complex. Region I, which includes interactions between amino acid residues H106 and R107 and nucleotide residues 9G, 10U, 11G and 12U of the aptamer, had the strongest interaction, based on ΔG and B-factor values, and hence contributed the most to the stability of the interaction. Region II, which covers residue 37A reflects the dynamic nature of the interaction due to frequent contacts. The approach presents a rigorous characterization of aptamer-IsdA binding behavior, supporting the potential application of the IsdA-binding aptamer system for S. aureus biosensing.

Cost-effective evaluation of Aptamer candidates in SELEX-based Aptamer isolation

Aptamers are short, single-stranded nucleic acids with high affinity and specificity for various targets, making them valuable in diagnostics and therapeutics. Their isolation traditionally involves a time-consuming and costly process called SELEX. While SELEX methods have evolved to improve binding and amplification, the crucial step of aptamer identification from sequencing data remains expensive and often overlooked. Common identification methods require modification of aptamer candidates with labels like biotin or fluorescent dyes, which becomes costly and cumbersome for high-throughput sequencing data. This paper presents an efficient and cost-effective approach to streamline aptamer identification. It employs asymmetric polymerase chain reaction (PCR) to generate modified single-stranded DNA copies of aptamer candidates, simplifying the modification process. By using excess modified forward primers and limited reverse primers, this method reduces costs since only unmodified candidates need to be synthesized initially. The approach was demonstrated with an IgE protein aptamer and successfully applied to identify aptamers from a pool of 12 candidates against a monoclonal antibody. The validity of the results was further confirmed through the direct synthesis of fluorophore-conjugated aptamer candidates, yielding consistent outcomes while reducing the cost by threefold. This approach addresses a critical bottleneck in aptamer discovery by significantly reducing the time and cost associated with aptamer identification, facilitating aptamer-based research and making aptamers more accessible for various applications in diagnostics and therapeutics.

Single-Round Circular Aptamer Discovery Using Bioinspired Magnetosome-Like Magnetic Chain Cross-Linked Graphene Oxide.

Circular aptamers are promising candidates for analytical and therapeutic applications due to their enhanced biological and structural stability. However, the process of circular aptamer selection remains a great challenge, as it requires multiple rounds of binding-separation-amplification that involves issues with nonspecific binding and amplification bias. Here, we develop a highly practical solution for reliable selection of circular aptamers in a single round based on magnetosome-like magnetic chain cross-linked graphene oxide (separation efficiency ≈ 105). High-affinity aptamer candidates can be rapidly selected from a preenriched circular DNA library, while low-affinity candidates are effectively adsorbed and separated by magnetosome-like magnetic chain cross-linked graphene oxide. With lipopolysaccharide as a representative model, the single-round selected lipopolysaccharide circular aptamer has been identified to have a high binding affinity with a Kd value of low to nanomolar range. Using this method, circular aptamers for protein and small-molecule targets were also successfully generated. We envision that this approach will accelerate the discovery of various new circular aptamers and open up a new avenue for analytical and therapeutic studies.

Screening and Application of DNA Aptamers for Heparin-Binding Protein.

Rapid detection of heparin-binding protein (HBP) is essential for timely intervention in sepsis cases. Current detection techniques are usually antibody-based immunological methods, which have certain problems, such as complexity and slow detection, and fall short in meeting the urgency of clinical needs. The application of an aptamer can address these concerns well. In this study, HBP-specific DNA aptamers were screened first. Among which, Apt-01, Apt-02, and Apt-13 had a high affinity for HBP, exhibiting impressive KD values of 3.42, 1.44, and 1.04 nmol/L, respectively. Then, the aptamer of HBP and its partially complementary primer probe were combined to form double-stranded DNA (dsDNA) and synthesize a circular DNA template. The template is complementary to the primer probe, but due to the presence of dsDNA, ExoIII cleaves C2-13 as an RCA primer probe, rendering the template unable to recognize the primer probe and preventing the RCA reaction from proceeding. When the target is present, it competes with the adapter for recognition and releases C2-13, exposing its 3' end. After initiating the RCA at room temperature and reacting with SYBR GreenII at 37 °C for 20 min, fluorescence changes can be observed and quantitatively analyzed at a 530 nm wavelength, achieving quantitative biological analysis. Apt-01 was used to develop a fluorescent biosensor for HBP detection, which exhibited a good linear range (0.01 nmol/L to 10 nmol/L) and detection limit (0.0056 nmol/L). This advancement holds the potential to lay a solid groundwork for pioneering sensitive and specific methods for HBP detection and to significantly enhance the diagnostic processes for sepsis.

Intrinsic tenase blood biomarker imprinted polymer-aptasensor with carbon nanohorn and gold nano-urchin construct for primitive-phase diagnosis of haemophilia B

The selective biomimetic aptasensor for blood coagulation factor IX protein (FIX) detection was developed using an interdigitated electrode with an Archimedean spiral pattern. In contrast to conventional molecularly imprinted polymer (MIP) techniques, aptamer was employed as a macromonomer to accelerate double binding affinity. To preserve the aptamer profile in its protein-binding orientation, FIX protein and thiol-modified RNA aptamers were complexed prior to MIP fabrication. The immobilized aptamer-FIX complex was surrounded by a polymer generated by the electropolymerization of 3-thiophene acetic acid (3TAA). Subsequent to FIX protein removal, leaves imprinted cavities facilitate selective FIX protein detection in conjunction with the affinity of embedded aptamer affinity. The Archimedean IDE surface was functionalized with carbon nanohorn (CNH) and gold nanourchin (GNU) to increase the imprinting ratios and sensor sensitivity. The developed FIX-aptasensor shows a detection limit of 0.06 fM, which is 660-fold higher than aptamer-embedded MIP nanoparticles. Moreover, the sensor exhibited greater selectivity for FIX, discriminating IgG and thrombin. As a preliminary study for clinical use, the sensor was used to analyze human serum without target spiking and detected FIX-protein with a relative standard deviation of 9.18%. It was ascertained that the sensor maintained 85% sustained performance for a duration of five weeks.

Analysis of human epidermal growth factor receptor 2 interaction on aptamer-probed interdigitated electrode for breast cancer diagnosis.

Breast cancer has been reported to be high in its incidence with women, and early identification of breast cancer helps to improve and provide an effective treatment. Tumor markers are active substances; in particular, human epidermal growth factor receptor 2 (HER2) is over-expressed at the level of 20%-30%. This research work developed a highly sensitive HER2 biosensor on the interdigitated electrode (IDE) by using aptamer as a detection probe. To enhance the analytical performances, aptamer was attached to the gold nanoparticle and immobilized on the IDE through a chemical linker [(3-aminopropyl)triethoxysilane]. On the aptamer conjugation, HER2 was quantified through current-volt measurements, and the limit of detection of HER2 was calculated as 1pg/mL on a linear range from 0.1 to 3000pg/mL at an R2 (regression coefficient) of 0.9657. Further, a selective performance with human serum increased the current responses by increasing HER2 concentrations. Specific experiments with control protein and complementary aptamer sequence failed to enhance the current responses. This HER2 biosensor reflects the occurrence of breast cancer at its lower abundance and helps to identify the associated complications.

Aptamer‐Assisted Blockade of the Immune Suppressor Sialic Acid‐Binding Immunoglobulin‐Like Lectin‐15 for Cancer Immunotherapy

AbstractThe percentage of low response and adaptive resistance to current antibody‐based immune checkpoint blockade (ICB) therapy requires the development of novel immunotherapy strategies. Here, we developed an aptamer‐assisted immune checkpoint blockade (Ap‐ICB) against sialic acid‐binding immunoglobulin‐like lectin‐15 (Siglec‐15), a novel immune suppressor broadly upregulated on cancer cells and tumor infiltrating myeloid cells, which is mutually exclusive of programmed cell death ligand 1 (PD‐L1). Using protein aptamer selection, we identified WXY3 aptamer with high affinity against Siglec‐15 protein/Siglec‐15 positive cells. We demonstrated that WXY3 aptamer rescued antigen‐specific T cell responses in vitro and in vivo. Importantly, the WXY3 Ap‐ICB against Siglec‐15 amplified anti‐tumor immunity in the tumor microenvironment and inhibited tumor growth/metastasis in syngeneic mouse model, which may result from enhanced macrophage and T cell functionality. In addition, by using aptamer‐based spherical nucleic acids, we developed a synergetic ICB strategy of multivalent binding and steric hindrance, which further improves the in vivo anti‐tumor effect. Taken together, our results support Ap‐ICB targeted Siglec‐15 as a potential strategy for normalization cancer immunotherapy.

Aptamer-Assisted Blockade of the Immune Suppressor Sialic Acid-Binding Immunoglobulin-Like Lectin-15 for Cancer Immunotherapy.

The percentage of low response and adaptive resistance to current antibody-based immune checkpoint blockade (ICB) therapy requires the development of novel immunotherapy strategies. Here, we developed an aptamer-assisted immune checkpoint blockade (Ap-ICB) against sialic acid-binding immunoglobulin-like lectin-15 (Siglec-15), a novel immune suppressor broadly upregulated on cancer cells and tumor infiltrating myeloid cells, which is mutually exclusive of programmed cell death ligand 1 (PD-L1). Using protein aptamer selection, we identified WXY3 aptamer with high affinity against Siglec-15 protein/Siglec-15 positive cells. We demonstrated that WXY3 aptamer rescued antigen-specific T cell responses in vitro and in vivo. Importantly, the WXY3 Ap-ICB against Siglec-15 amplified anti-tumor immunity in the tumor microenvironment and inhibited tumor growth/metastasis in syngeneic mouse model, which may result from enhanced macrophage and T cell functionality. In addition, by using aptamer-based spherical nucleic acids, we developed a synergetic ICB strategy of multivalent binding and steric hindrance, which further improves the in vivo anti-tumor effect. Taken together, our results support Ap-ICB targeted Siglec-15 as a potential strategy for normalization cancer immunotherapy.

Structural Insights into Protein-Aptamer Recognitions Emerged from Experimental and Computational Studies.

Aptamers are synthetic nucleic acids that are developed to target with high affinity and specificity chemical entities ranging from single ions to macromolecules and present a wide range of chemical and physical properties. Their ability to selectively bind proteins has made these compounds very attractive and versatile tools, in both basic and applied sciences, to such an extent that they are considered an appealing alternative to antibodies. Here, by exhaustively surveying the content of the Protein Data Bank (PDB), we review the structural aspects of the protein-aptamer recognition process. As a result of three decades of structural studies, we identified 144 PDB entries containing atomic-level information on protein-aptamer complexes. Interestingly, we found a remarkable increase in the number of determined structures in the last two years as a consequence of the effective application of the cryo-electron microscopy technique to these systems. In the present paper, particular attention is devoted to the articulated architectures that protein-aptamer complexes may exhibit. Moreover, the molecular mechanism of the binding process was analyzed by collecting all available information on the structural transitions that aptamers undergo, from their protein-unbound to the protein-bound state. The contribution of computational approaches in this area is also highlighted.

Aptamer-Protein Interactions: From Regulation to Biomolecular Detection.

Serving as the basis of cell life, interactions between nucleic acids and proteins play essential roles in fundamental cellular processes. Aptamers are unique single-stranded oligonucleotides generated by in vitro evolution methods, possessing the ability to interact with proteins specifically. Altering the structure of aptamers will largely modulate their interactions with proteins and further affect related cellular behaviors. Recently, with the in-depth research of aptamer-protein interactions, the analytical assays based on their interactions have been widely developed and become a powerful tool for biomolecular detection. There are some insightful reviews on aptamers applied in protein detection, while few systematic discussions are from the perspective of regulating aptamer-protein interactions. Herein, we comprehensively introduce the methods for regulating aptamer-protein interactions and elaborate on the detection techniques for analyzing aptamer-protein interactions. Additionally, this review provides a broad summary of analytical assays based on the regulation of aptamer-protein interactions for detecting biomolecules. Finally, we present our perspectives regarding the opportunities and challenges of analytical assays for biological analysis, aiming to provide guidance for disease mechanism research and drug discovery.

Resveratrol improves emamectin benzoate-induced pyroptosis and inflammation of Ctenopharyngodon idellus hepatic cells by alleviating oxidative stress/endoplasmic reticulum stress

Emamectin benzoate (EMB) is the most widely used pesticide in the world and contributes to water pollution. Owing to the lack of a specific antidote, EMB has a severe negative impact on the health of aquatic organisms. Resveratrol (RES), a substance with antioxidant capacity, is secreted by the fruits of many plants. This study was to explore the protection of RES against EMB-induced pyroptosis and inflammatory response in grass carp (Ctenopharyngodon idellus) hepatic liver (L8824) cells by oxidative stress/endoplasmic reticulum (ER) stress. The results showed that compared to the CON group, EMB induced oxidative stress in L8824 cells with the increase of reactive oxygen species (ROS), methane dicarboxylic aldehyde (MDA), and hydrogen peroxide (H2O2) contents and the decrease of total superoxide dismutase (t-sod) and glutathione peroxidase (gsh-px) activities (P < 0.05). In addition, EMB triggered ERS, increasing the relative mRNA expression of protein kinase R-like endoplasmic reticulum kinase (perk), inositol requiring enzyme 1 alpha (ire1α), glucose-regulated protein 78 (grp78), activating transcription factor 4 (atf4), activating transcription factor 6 (atf6), and CCAAT-enhancer-binding protein homologous protein (chop) and the protein expression of eukaryotic initiation factor 2α (eif2α), chop, atf6, and atf4. Meanwhile, EMB further induced pyroptosis by upregulating the mRNA and protein expression of nlrp3, aptamer protein (asc), caspase-1, gsdmd, interleukin-1β (il-1β), and interleukin-18 (il-18). EMB also induced inflammation in L8824 cells by increasing the mRNA expression of interleukin-2 (il-2), interleukin-6 (il-6), tumor necrosis factor-α (tnf-α), and ifn-γ and decreasing the content of interleukin-10 (il-10). However, compared to the EMB group, the oxidant indices and expression of genes related to ER stress, pyroptosis, and pro-inflammatory factors were significantly down-regulated (P < 0.05), whereas the antioxidant indicators and anti-inflammatory factor were significantly up-regulated in the EMB + RES group (P < 0.05). In conclusion, EMB caused hepatocytes pyroptosis and inflammation in grass carp, and RES could alleviate EMB-induced pyroptosis and inflammation in L8824 cells by ameliorating oxidative stress/ER stress.