Long-term swimming exercise attenuates right ventricular hypertrophy via modulating meta-inflammation in monocrotaline-induced pulmonary hypertensive rats.

Pulmonary arterial hypertension (PAH) is a complex disease characterized by elevated pulmonary vascular resistance, resulting in right ventricular (RV) hypertrophy and, eventually, failure, which remains the primary cause of mortality in PAH patients. While current PAH therapies primarily target vascular abnormalities, most fail to address RV dysfunction. Therefore, improving RV function is a critical treatment goal. Exercise has emerged as an effective intervention for PAH, but the specific impact of swimming exercise on this disease and its associated pathological changes has been less extensively studied. In this study, we investigated the effects of swimming training (60 min/day, 5 days/week for 4 weeks) on monocrotaline (MCT; 60 mg/kg, i. p.)-induced PAH in rats. Our findings demonstrate that swimming significantly attenuates RV hypertrophy and reduces mean pulmonary arterial pressure (MPAP), mitigating the detrimental effects of PAH. Furthermore, we observed structural remodeling in the right ventricle, including increased myocardial necrosis, collagen deposition, and fibrosis-related protein expression. Swimming exercise training was found to reduce these pathological changes, suggesting a protective effect on the right ventricle. Mechanistically, our study revealed the crucial role of meta-inflammation in PAH and the anti-PAH effects of exercise. Swimming training attenuated macrophage accumulation, reduced serum inflammatory cytokines, and improved systemic and RV insulin sensitivity, highlighting its potential to modulate meta-inflammatory processes. In summary, our study suggests that swimming training exerts a beneficial effect on RV function and hypertrophy in MCT-induced PAH rats by targeting meta-inflammation. These results underscore the potential value of exercise-based rehabilitation as a complementary therapy for PAH patients.

Petroleum hydrocarbons and colored dissolved organic matter shape marine oil-degrading microbiota in different patterns.

Both petroleum hydrocarbons (PHCs) from oil pollution and colored dissolved organic matter (CDOM) have great influences on the marine microbial community as carbon source factors. However, their combined effects and the specific influence patterns have been kept unclear. This study selected the northeastern South China Sea (NSCS), a typical oil contaminated area, and investigated the characteristics of oil-degrading microbiota in the seawaters by high-throughput sequencing and the relationships with PHCs and CDOM as well as other environmental factors. The results showed the oil pollution had induced the enrichment of oil-degrading bacteria and oil-degrading functional genes, resulting in the core function of oil-degrading microbiota for shaping the microbial community. The Mantel test indicated carbon source factors played the dominant role in shaping the oil-degrading microbiota, compared with geographical distance and other non­carbon source factors. The influence patterns and strength of PHCs and CDOM on oil-degrading microbiota were further comprehensively analyzed. PHCs played a driving role in the differentiation of oil-degrading microbiota, while CDOM played a stabilizing role for the community similarity. The constructed structural equation model confirmed their distinct influence patterns and also explored the mediating effects of bulk organic carbon. This work not only revealed the important impact of oil pollution on marine microbial communities, but also made people realize the self-regulation ability of the marine environment through the endogenous organic matter.

Bioproduction of 3-Hydroxypropionic Acid by Enhancing the Precursor Supply with a Hybrid Pathway and Cofactor Regeneration.

3-Hydroxypropionic acid (3-HP) is one of the 12 valuable platform chemicals with versatile applications in the chemical, food, and cosmetic industries. However, the biosynthesis of 3-HP faces challenges due to the lack of robust chassis and the high costs associated with the fermentation process. To address these challenges, we made efforts to augment the robustness of 3-HP-producing chassis by exploiting metabolic regulation, controlling carbon flux, balancing cofactor generation, and optimizing fermentation conditions. First, the malonyl-CoA (MCA) pathway was recruited and rebalanced in Escherichia coli. Subsequently, a hybrid pathway integrating the Embden-Meyerhof-Parnas pathway with the nonoxidative glycolysis pathway was systematically modulated to enhance carbon flux to the MCA pathway, followed by fine-tuning NADPH regeneration. Then, by optimizing the fermentation conditions, 3-HP production was significantly improved, reaching 6.8 g/L. Finally, in a fed-batch experiment, the final chassis produced 42.8 g/L 3-HP, corresponding to a 0.4 mol/mol yield and 0.6 g/(L·h) productivity.

Sodium octanoate mediates GPR84-dependent and independent protection against sepsis-induced myocardial dysfunction.

INTRODUCTION: This study aims to evaluate the therapeutic effects of sodium octanoate (SO), a medium-chain fatty acid salt, on SIMD in a murine model and to explore its underlying mechanisms. METHODS: Male mice were subjected to sepsis models through two methods: intraperitoneal injection of lipopolysaccharide (LPS) and cecal ligation and punction (CLP). Mice received interval doses of SO every 2 hours or 4 hours for a total of six times or three times after LPS treatment. The relationship between SO and G protein-coupled receptor 84 (GPR84) was evaluated through GEO data analysis and molecular docking studies. DBA/2 mice were used to study the role of the GPR84 protein in the SO-mediated protection. Energy metabolomics was utilized to comprehensively assess the impact of SO on the levels of cardiac energy metabolic products in septic mice. histone modification identification techniques were used to further identify the specific sites of histone modification in the hearts of SO-treated septic mice. RESULTS: SO treatment significantly improved myocardial contractile function, restored the oxidative stress imbalance and enhanced the myocardium's resistance to oxidative injury. SO significantly promotes the expression of GPR84. The loss of GPR84 function markedly attenuates the protective effects of SO. SO enhanced myocardial energy metabolism by promoting the synthesis of acetyl-CoA and upregulating genes involved in fatty acid ß-oxidation which were abolished by medium-chain acyl-CoA dehydrogenase (MCAD) knockdown. SO induced histone acetylation, particularly at H3K123 and H3K80. CONCLUSION: Our study demonstrates that SO exerts protective effects against SIMD through both GPR84-mediated anti-inflammatory and antioxidant actions and GPR84-independent enhancement of myocardial energy metabolism, possibly mediated by MCAD.

TRIM24-DTNBP1-ATP7A mediated astrocyte cuproptosis in cognition and memory dysfunction caused by Y2O3 NPs.

Yttrium oxide nanoparticles (Y2O3 NPs), extensively utilized rare earth nanoparticles, exhibited a diverse range of applications across various fields, which leading to increased human exposure. Moreover, potential neurotoxic risks have been associated with their use, yet the underlying mechanism remains unclear. The present study aimed to investigate the effects of Y2O3 NPs on cognitive function in rats with a particular focus on elucidating the pivotal role played by astrocytes in this process. The results demonstrated that Y2O3 NPs induced cognitive and memory impairment in rats, copper (Cu) accumulation and cuproptosis of astrocytes as contributing factors. Furthermore, we elucidated that Y2O3 NPs induced astrocytes cuproptosis by inhibiting TRIM24/DTNBP1/ATP7A signaling pathway-mediated cellular Cu efflux. We provide, for the first time, the important involvement of astrocytes in Y2O3 NPs-induced neurotoxicity, elucidating that cuproptosis as the primary mode of cell death. These results offer valuable insights for the future safe application of rare earth nanoparticles in field of neurology.

Characteristics of organic matter driven by Eichhornia crassipes during co-contamination with per(poly)fluoroalkyl substances (PFASs) and microplastics (MPs).

Co-contamination with MPs and PFASs has been recorded, particularly in surface-water environments. Floating macrophyte microcosms are an important part of the surface water ecosystem, and dissolved organic matter (DOM) driven by floating macrophytes (FMDDOM) is critical for maintaining material circulation. However, knowledge gaps remain regarding the impact of MPs and PFASs co-pollution on FMDDOM. An greenhouse simulation experiment was conducted in this study to investigate the effects of four PFASs, perfluorooctanoic acid (PFOA), perfluoro-octane-sulfonic acid (PFOS), perfluoro-2-methyl-3-oxahexanoic acid (Gen X), and potassium 9-chlorohexadecafluoro-3-oxanonane-1-sulfonate (F-53B), on FMDDOM sourced from Eichhornia crassipes (E. crassipes), a typical floating macrophyte, in the presence and absence of polystyrene (PS) MPs. Four PFASs increased FMDDOM release from E. crassipes, leading to a 32.52-77.49 % increase in dissolved organic carbon (DOC) levels. PS MPs further increased this, with results ranging from -21.28 % to 26.49 %. Based on the parallel factor analysis (PARAFAC), FMDDOM was classified into three types of fluorescent components: tryptophan-like, humic-like, and tyrosine-like compounds. Contaminants of MPs and PFASs modified the relative abundance of these three components. Protein secondary structure analysis showed that fluorocarbon bonds tended to accumulate on the α-helix of proteins in FMDDOM. The relative abundance of fluorescent and chromophorous FMDDOMs varied from 0.648 ± 0.044 to 0.964 ± 0.173, indicating that the photochemical structures of the FMDDOM were modified. FMDDOM exhibits decreased humification and increased aromaticity when contaminated with MPs and PFASs, which may be detrimental to the geochemical cycling of carbon. This study offers a theoretical basis for assessing the combined ecological risks of MPs and PFASs in floating macrophyte ecosystems.

Microbe-disease associations prediction by graph regularized non-negative matrix factorization with L 2 , 1 $$ {L}_{2,1} $$ norm regularization terms.

Microbes are involved in a wide range of biological processes and are closely associated with disease. Inferring potential disease-associated microbes as the biomarkers or drug targets may help prevent, diagnose and treat complex human diseases. However, biological experiments are time-consuming and expensive. In this study, we introduced a new method called iPALM-GLMF, which modelled microbe-disease association prediction as a problem of non-negative matrix factorization with graph dual regularization terms and L 2 , 1 $$ {L}_{2,1} $$ norm regularization terms. The graph dual regularization terms were used to capture potential features in the microbe and disease space, and the L 2 , 1 $$ {L}_{2,1} $$ norm regularization terms were used to ensure the sparsity of the feature matrices obtained from the non-negative matrix factorization and to improve the interpretability. To solve the model, iPALM-GLMF used a non-negative double singular value decomposition to initialize the matrix factorization and adopted an inertial Proximal Alternating Linear Minimization iterative process to obtain the final matrix factorization results. As a result, iPALM-GLMF performed better than other existing methods in leave-one-out cross-validation and fivefold cross-validation. In addition, case studies of different diseases demonstrated that iPALM-GLMF could effectively predict potential microbial-disease associations. iPALM-GLMF is publicly available at https://github.com/LiangzheZhang/iPALM-GLMF.

Resveratrol reinforces the therapeutic effect of mesenchymal stem cell (MSC)-derived exosomes against renal ischemia‒reperfusion injury (RIRI)-associated fibrosis by suppressing TGF-ß-induced epithelial-mesenchymal transition.

Resveratrol (RSV) has been shown to prevent epithelial-mesenchymal transition (EMT) in different diseases by modulating several signaling pathways, and RSV can prevent EMT by modulating the signaling of the TGF-ß/Smad axis. In the development of renal ischemia‒reperfusion injury (RIRI), RSV and MSC-derived exosomes could ameliorate RIRI via different signaling pathways. In this study, we aimed to investigate the effect of RSV plus MSC-derived exosomes on the prognosis of RIRI. Quantitative real-time polymerase chain reaction (PCR) was performed to measure the expression of E-CAD, SMA, COL10A1, VMT and MMP-7 mRNA in TCMK-1 cells and mice under various conditions. HE and Masson staining were used to evaluate kidney injury and fibrosis in mice under various conditions. RSV effectively maintained the TGF-ß- and AA-induced upregulation of E-CAD, SMA, COL10A1, VMT and MMP-7 mRNA expression in TCMK-1 cells. Moreover, MSC-derived exosomes effectively reinforced the effect of RSV on reducing the TGF-ß- and AA-induced upregulation of E-CAD, SMA, COL10A1, VMT and MMP-7 mRNA expression in TCMK-1 cells. Furthermore, MSC-derived exosomes enhanced the capability of RSV to maintain the RIRI-induced increases in Cr and BUN, as well as the upregulation of E-CAD, SMA, COL10A1, VMT and MMP-7 mRNA expression in mice. In addition, MSC-derived exosomes enhanced the capability of RSV to decrease RIRI-induced kidney injury and fibrosis in mice. Our findings showed that the administration of MSC-derived exosomes and RSV could suppress the TGF-ß-induced epithelial-mesenchymal transition. This suppressive effect was promoted by the coadministration of MSC-derived exosomes and RSV.

Engineered Macrophage Membrane-Coated S100A9-siRNA for Ameliorating Myocardial Ischemia-Reperfusion Injury.

Despite the widespread adoption of emergency coronary reperfusion therapy, reperfusion-induced myocardial injury remains a challenging issue in clinical practice. Following myocardial reperfusion, S100A8/A9 molecules are considered pivotal in initiating and regulating tissue inflammatory damage. Effectively reducing the S100A8/A9 level in ischemic myocardial tissue holds significant therapeutic value in salvaging damaged myocardium. In this study, HA (hemagglutinin)- and RAGE (receptor for advanced glycation end products)- comodified macrophage membrane-coated siRNA nanoparticles (MMM/RNA NPs) with siRNA targeting S100A9 (S100A9-siRNA) are successfully prepared. This nanocarrier system is able to target effectively the injured myocardium in an inflammatory environment while evading digestive damage by lysosomes. In vivo, migration of MMM/RNA NPs to myocardial injury lesions is confirmed in a myocardial ischemia-reperfusion injury (MIRI) mouse model. Intravenous injection of MMM/RNA NPs significantly reduced S100A9 levels in serum and myocardial tissues, further decreasing myocardial infarction area and improving cardiac function. Targeted reduction of S100A8/A9 by genetically modified macrophage membrane-coated nanoparticles may represent a new therapeutic intervention for MIRI.

Achieving endogenous partial denitrification by cultivating denitrifying glycogen-accumulating organisms.

Although anaerobic ammonia oxidation (anammox) is considered a promising process due to its high efficiency and low energy in nitrogen removal, nitrite inadequacy was one of the bottlenecks for the application of anammox. However, endogenous partial denitrification (EPD) has been emerging as a stable pathway to provide nitrite for anammox. Furthermore, denitrifying glycogen-accumulating organisms (DGAOs) are believed to be associated with EPD. In this study, firstly, GAOs were gradually enriched in a sequencing batch reactor (SBR) with the dual strategy of influent phosphorus limitation and withdrawal after the anaerobic stage. DGAOs were successfully induced by adding sodium nitrate solution at the end of the anaerobic stage, resulting in NO3--N concentration increasing from 15 to 30 mg/L. During a typical SBR cycle, DGAOs contributed up to 96% of the conversion of intracellular carbon sources and up to around 95% of nitrate reduction during the anoxic stage. The maximum nitrate-to-nitrite transformation ratio (NTR) of the system reached 80%. Microbial community analysis demonstrated that the Ca. Compatibactors were the dominant functional bacteria for EPD, with a relative abundance of 31.12%. However, the relative abundance of phosphorous-accumulating organisms (PAOs) was only 1.02%. This study reveals the important role of DGAOs in the EPD process, which can provide a stable nitrite for anammox.

Three classes of propofol binding sites on GABAA receptors.

Propofol is a widely used anesthetic and sedative that acts as a positive allosteric modulator (PAM) of gamma-aminobutyric acid type A (GABAA) receptors. Several potential propofol binding sites that may mediate this effect have been identified using propofol-analogue photoaffinity labeling. o-PD labels ß-H267, a pore-lining residue, whereas AziPm labels residues ß-M286, ß-M227 and α-I239 in the two membrane-facing interfaces (ß(+)/α(-) and α(+)/ß(-)) between α and ß subunits. This study used photoaffinity labeling of α1ß3 GABAA receptors to reconcile the apparently conflicting results obtained with AziPm and o-PD labeling, focusing on whether ß3-H267 identifies specific propofol binding site(s). The results show that propofol, but not AziPm protects ß3-H267 from labeling by o-PD, whereas both propofol and o-PD protect against AziPm labeling of ß3-M286, ß3-M227 and α1I239. These data indicate that there are three distinct classes of propofol binding sites, with AziPm binding to two of the classes and o-PD to all three. Analysis of binding stoichiometry using native mass spectrometry in ß3 homomeric receptors, demonstrated a minimum of five AziPm labeled residues and three o-PD labeled residues per pentamer, suggesting that there are two distinct propofol binding sites per ß-subunit. The native MS data, coupled with photolabeling performed in the presence of zinc, indicate that the binding site(s) identified by o-PD are adjacent to, but not within the channel pore, since the pore at the 17' H267 residue can accommodate only one propofol molecule. These data validate the existence of three classes of specific propofol binding sites on α1ß3 GABAA receptors.

Effects of fermentation conditions on molecular weight, production, and physicochemical properties of gellan gum.

Gellan gum has been widely used in many industries due to its excellent physical properties. In this study, the effects of different fermentation conditions on molecular weight and production of gellan gum were analyzed, and the optimized fermentation conditions for a high molecular weight gellan gum (H-GG: 6.42 × 105 Da) were obtained, which increased the molecular weight and yield of gellan gum by 201.4 % and 44.9 % respectively. Fourier transform infrared spectroscopy (FT-IR) and x-ray diffraction (XRD) analysis indicated that H-GG has similar characteristic absorption and semi-crystalline structures with the initial gellan gum (I-GG), and it was composed of glucose, rhamnose, and glucuronic acid showing no obvious changes in the molecular structure. Scanning electron microscope (SEM) observation revealed that the filaments of H-GG were slender, longer, and looser with larger pores. Importantly, gel properties analysis showed that the gel strength, viscoelasticity, and water-holding capacity of H-GG were better than those of I-GG, and the rheological results revealed that the H-GG is a pseudoplastic fluid with higher apparent viscosity and stable viscoelasticity at 20-70 °C. Therefore, the molecular weight and yield of gellan gum are significantly affected by fermentation conditions, and the obtained H-GG demonstrates improved gel and rheological properties.

Neoadjuvant envafolimab in a patient with MSI-H/dMMR colon cancer: a case report and literature review.

Clinical evidences of neoadjuvant immunotherapy in patients with mismatch repair deficient/microsatellite instability-high status (dMMR/MSI-H) colorectal cancer have not been well received. A 36-year-old man complained of recurrent right upper quadrant pain for more than 1 year, and the symptoms were not significantly relieved after 10 days of oral Changyanning tablet. The patient was finally diagnosed as dMMR/MSI-H colon cancer. Tumor regression was achieved after seven cycles of envafolimab treatment, and the patient obtained postoperative pathological complete response (pCR). Here, we report a case of MSI-H/dMMR transverse colon cancer, who obtained pCR after neoadjuvant envafolimab (a novel subcutaneous single-domain anti-PD-L1 antibody) with a favorable safety profile, aiming to enhance the experiences of comprehensive diagnosis and treatment of colon cancer.

Immune checkpoint inhibitors (ICIs) are a type of immunotherapy which can be used in the treatment of colorectal cancer. The authors here report the functions of envafolimab (a type of ICI) used before surgery to shrink tumor volume in colorectal cancer. A 36-year-old man suffered from repeated illness of right upper quadrant for over 1 year, and the illness were not recovered after 10 days of oral Changyanning tablet. The patient was finally diagnosed with colorectal cancer. After seven cycles of envafolimab treatment, tumor volume was significantly decreased, and the patient obtained favorable surgical outcomes with tolerable safety after surgery.

Exploring Synergistic GHG Emissions Mitigation Potential and Costs of Room Air Conditioners.

This study explores the potential of synergistically reducing direct (refrigerant) and indirect (electricity) greenhouse gas (GHG) emissions in the global room air conditioning (RAC) sector, based on 80% of global RAC manufactured in China. Three scenarios are evaluated: Business-as-usual (BAU) based on maintaining refrigerant and energy efficiency levels from 2021 China RAC sales shares, Kigali Amendment compliant with 10% energy efficiency improvement by 2025 (KAE), and accelerated refrigerant transition and energy efficiency improvement (ATE). Each scenario considers the costs of refrigerant and efficiency measures for export market groups based on Kigali Amendment classifications. BAU predicts around 1 Gt CO2-eq average annual global RAC emissions (2022-2060). Cumulative emission reductions in China's RAC manufacturing under KAE and ATE are 12.2 and 17.2 Gt CO2-eq A5II and A5I (except China), presenting cost-effective abatement measures, with average costs of -$51.4 and -$68.8/t CO2-eq in KAE and ATE. Cumulative average abatement costs are around $18 and $4/t CO2-eq globally. KAE and ATE scenarios would avoid surface temperature rises of 0.023 (±0.002) °C and 0.027 (±0.003) °C, respectively, versus BAU. Collaboration between China and importing countries is urged to enhance energy efficiency in RACs traded, ensuring sustainable mitigation aligned with the Kigali Amendment.

Supplemental L-arginine promotes hepatocyte proliferation and alters liver fatty acid metabolism in the late embryonic phase: an RNA-seq analysis.

The in ovo feeding (IOF) of L-arginine (L-Arg) to chick embryos is a viable method for improving early intestinal development, subsequently leading to an acceleration in growth rate during the posthatch stage. However, the liver, being the pivotal organ for energy metabolism in poultry, the precise effects and mechanisms of L-Arg on the liver development and metabolism remain unclear. To elucidate these, the present study injected 2 doses of L-Arg (10 mg/egg and 15 mg/egg) into the embryos of Hongyao chickens at 17.5 d of incubation, subsequently incubating them until d 19 for further analysis. IOF of 15 mg L-Arg/egg significantly increased the organ indices of liver and small intestine, as well as the duodenal villus height/crypt depth. RNA-Seq analysis of liver tissues showed that the metabolism of xenobiotics, amino acid metabolism, and the fatty acid metabolism were significantly enriched in L-Arg injection group. The core differentially expressed genes (DEGs) were primarily involved in cell proliferation and fatty acid metabolism. The CCK8 assays revealed that supplemental L-Arg significantly enhanced the proliferation of primary embryo hepatocytes and leghorn male hepatoma (LMH) cells. Upregulation of core DEGs, including HBEGF, HES4, NEK3, EGR1, and USP2, significantly promoted the proliferation of liver cells. Additionally, analysis of triglyceride and total cholesterol content, as well as oil red O staining, indicated that supplemental L-Arg effectively reduced lipid accumulation. Overall, L-Arg supplementation in late chick embryos may promote early liver and small intestine development by reducing liver lipid deposition and enhancing energy efficiency, necessitating further experimental validation. This study provides profound insights into the molecular regulatory network of L-Arg in promoting the development of chicken embryos. The identified DEGs that promote cell proliferation and lipid metabolism can serve as novel targets for further developing methods to enhance early development of chicken embryos.

Unraveling the atomic-scale mechanism of interfacial alkali ion close packing in nano glassy fibers driven by CO2-mediated attraction.

The poor alkali resistance of nano-glassy fiber (NGF) hinders its application in aggressive alkaline environments like cement and alkaline wastewater. This study introduced a potential strategy to enhance the alkali resistance of NGF based on the distinct atomic-scale interactions between CO2 molecules and NGF ions at high temperatures. The short-range structure in the NGF-CO2 system was elucidated through the pair distribution functions and coordination numbers of ion-oxygen pairs and second-nearest-neighbor ion-ion pairs. High-mobility Na ions were observed to exhibit a strong interaction with CO2, penetrating through the melt skeleton and accumulating at the interface. The enrichment of Na ions as network modifiers enhanced the alkali resistance of NGF, with the maximum thickness of the enrichment layer reaching ∼5 Šat 1173 K. These findings present a straightforward production approach for alkali-resistant NGF, not requiring additional costly materials or processes.

Microplastics and benthic animals reshape the geochemical characteristics of dissolved organic matter by inducing changes in keystone microbes in riparian sediments.

Dissolved organic matter (DOM) in riparian sediments plays a vital role in regulating element cycling and pollutant behavior of river ecosystems. Microplastics (MPs) and benthic animals (BAs) have been frequently detected in riparian sediments, influencing the substance transformation in river ecosystems. However, there is still a lack of systematic investigation on the effects of MPs and BAs on sediment DOM. This study investigated the impact of MPs and BAs on the geochemical characteristics of DOM in riparian sediments and their microbial mechanisms. The results showed that MPs and BAs increased sediment DOC concentration by 34.24%∼232.97% and promoted the conversion of macromolecular components to small molecular components, thereby reducing the humification degree of DOM. Mathematical model verified that the changes of keystone microbes composition in sediments were direct factors affecting the characteristics of DOM in riparian sediment. Especially, MPs tolerant microbes, including Planctomicrobium, Rhodobacter, Hirschia and Lautropia, significantly increased DOC concentration and decreased humification degree (P < 0.05). In addition, MPs and BAs could also influence keystone microbes in sediments by altering the structure of microbial network, thereby indirectly affecting DOM characteristics. The study demonstrates the pollution behavior of MPs in river ecosystems and provides a basis for protecting the ecological function of riparian sediments from MPs pollution.

Establishment of a Steatosis Model in LMH Cells, Chicken Embryo Hepatocytes, and Liver Tissues Based on a Mixture of Sodium Oleate and Palmitic Acid.

Research on hepatic steatosis in animal husbandry has been a prominent area of study. Developing an appropriate in vitro cellular steatosis model is crucial for comprehensively investigating the mechanisms involved in liver lipid deposition in poultry and for identifying potential interventions to address abnormalities in lipid metabolism. The research on the methods of in vitro liver steatosis in chickens, particularly the effects of different fat mixtures, is still lacking. In this study, LMH cells were utilized to investigate the effects of OA, SO, PA, SP, and their pairwise combinations on steatosis development, with the aim of identifying the optimal conditions for inducing steatosis. Analysis of triglyceride (TG) content in LMH cells revealed that OA and SP had limited efficacy in increasing TG content, while a combination of SO and PA in a 1:2 ratio exhibited the highest TG content. Moreover, Oil Red O staining results in LMH cells demonstrated that the combination treatment had a more pronounced induction effect compared to 0.375 mM SO. Additionally, RNA-seq analysis showed that 0.375 mM SO significantly influenced the expression of genes associated with fatty acid metabolism compared to the control group, whereas the combination of SO and PA led to an enrichment of key GO terms associated with programmed cell death. These findings suggest that varying conditions of cellular steatosis could lead to distinct disruptions in gene expression. The optimal conditions for inducing steatosis in LMH cells were also tested on chicken embryonic liver cells and embryos. TG detection and Oil Red O staining assays showed that the combination of SO and PA successfully induced steatosis. However, the gene expression pattern differed from that of LMH cells. This study lays the foundations for further investigations into avian hepatic steatosis.

Screening of reliable reference genes for the normalization of RT-qPCR in chicken liver tissues and LMH cells.

The liver plays a vital role in lipid synthesis and metabolism in poultry. To study the functional genes more effectively, it is essential to screen of reliable reference genes in the chicken liver, including females, males, embryos, as well as the Leghorn Male Hepatoma (LMH) cell line. Traditional reference gene screening involves selecting commonly used housekeeping genes (HKGs) for RT-qPCR experiments and using different algorithms to identify the most stable ones. However, this approach is limited in selecting the best reference gene from a small pool of HKGs. High-throughput sequencing technology may offer a solution to this limitation. This study aimed to identify the most consistently expressed genes by utilizing multiple published RNA-seq data of chicken liver and LMH cells. Subsequently, the stability of the newly identified reference genes was assessed in comparison to previously validated stable poultry liver expressed reference genes and the commonly employed HKGs using RT-qPCR. The findings indicated that there is a higher degree of similarity in stable expression genes between female and male liver (such as LSM14A and CDC40). In embryonic liver, the optimal new reference genes were SUDS3, TRIM33, and ERAL1. For LMH cells, the optimal new reference genes were ALDH9A1, UGGT1, and C21H1orf174. However, it is noteworthy that most HKGs did not exhibit stable expression across multiple samples, indicating potential instability under diverse conditions. Furthermore, RT-qPCR experiments proved that the stable expression genes identified from RNA-seq data outperformed commonly used HKGs and certain validated reference genes specific to poultry liver. Over all, this study successfully identified new stable reference genes in chicken liver and LMH cells using RNA-seq data, offering researchers a wider range of reference gene options for RT-qPCR in diverse situations.