This experiment aimed to investigate the effects of the Xianshanhuang compound preparation on the growth performance and immune function of Trachinotus ovatus. A total of 630 healthy Trachinotus ovatus with an initial body weight of (4.57±0.31) g were selected and randomly divided into seven groups, with three replicates per group and 30 fish per replicate. The control group was fed with basic diet, while the experimental groups were added with 2.5, 5.0, 10.0, 15.0, 20.0, and 25.0 g/kg of Xianshanhuang compound preparation to the basic diet. The pre-test period was seven days, and the formal test period was 30 days. The results showed that on the 30th day of the experiment, compared with the control group, the weight gain rate (WGR) and specific growth rate (SCR) of the group 10.0 were significantly increased (P<0.05). There was no significant difference in the blood routine indicators among all groups (P>0.05). Histological sections of the liver and spleen displayed clear structures, with no significant pathological changes observed in each group. In the liver and spleen tissues of group 10.0, the expression level of tumor necrosis factor-α (TNF-α) was decreased, while the expression levels of interleukin-1β (IL-1β), nuclear factor kappa B (NF-κB), and interferon alpha-3 reverse transcription product (IFNa3-RT) were increased, but there was no significant difference (P>0.05). The study shows that feeding Trachinotus ovatus with the Xianshanhuang compound preparation at 10.0 g/kg for 30 days has no toxic or side effects, demonstrates good safety, and can improve the growth performance of Trachinotus ovatus.

This study aimed to investigate the regulatory effect and mechanism of polysaccharide component 1 from Arthrospira platensis (PAP-1) on the inflammatory response induced by porcine pseudorabies virus (PRV) in porcine alveolar macrophages cells (3D4/2). The cytotoxic effect of PAP-1 on 3D4/2 cells and its influence on cell viability after PRV infection were assessed using the CCK-8 assay. Treat 3D4/2 cells infected with PRV with PAP-1 at different concentrations (25, 50, 100 mg/L), and set up a cell control group and a PRV group, with three replicates per group. The content of inflammatory factors, expression of inflammatory genes, and phosphorylation levels of key proteins were measured. The results showed that compared with the PRV group, the concentrations of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1) in the 25~100 mg/L PAP-1 groups were extremely decreased (P<0.01). The gene expression levels of TNF-α, IL-1β, IL-8, inducible nitric oxide synthase (iNOS), and MCP-1 were extremely down-regulated (P<0.01). The phosphorylated p38 mitogen activated protein kinase/p38 mitogen activated protein kinase (p-p38/p38) and phosphorylated c-jun N-terminal kinase/c-jun N-terminal kinase (p-c-jun/c-jun) in the 25 mg/L PAP-1 group were significantly reduced (P<0.05), while p-p38/p38 and p-c-jun/c-jun in the 50 mg/L PAP-1 group were extremely reduced (P<0.01). The p-p38/p38 in the 100 mg/L PAP-1 group was significantly decreased (P<0.05), and p-c-jun/c-jun was extremely decreased (P<0.01). The study shows that PAP-1 alleviates the inflammatory response in 3D4/2 cells by inhibiting the activation of the mitogen-activated protein kinase (MAPK) pathway, reducing the levels of inflammatory factors, and down-regulating the expression of inflammation-related genes.

The experiment aimed to investigate the effects of Gynostemma pentaphyllum extract (GPE) on the intestinal health of weaned Bama miniature pigs. A total of 50 weaned Bama miniature piglets aged 30 to 35 days with an initial body weight of (5.34±0.25) kg were randomly divided into five groups, with five replicates per group and two piglets per replicate. The control group (CON group) was fed a basal diet, the experimental groups were fed the basal diet supplemented with 0.4%, 0.8%, and 1.6% GPE, respectively, and the Astragalus extract (APE) group was fed the basal diet supplemented with 0.2% APE. The pre-test period lasted for seven days, and the formal test period lasted for 28 days. The results showed that compared with the CON group, the 0.8% GPE and 1.6% GPE groups significantly improved the morphological structure of various segments of the small intestine. The mRNA expression levels of Toll-like receptor 4 (TLR4) and tumor necrosis factor receptor-associated factor 6 (TRAF6) in the jejunum and ileum of the experimental groups were significantly down-regulated (P0.05). In the 1.6% GPE group, the mRNA expression levels of interleukin-10 (IL-10) in the ileum, as well as Toll-like receptor 9 (TLR9), tight junction protein 1 (ZO-1), and occludin (OCLN) in the jejunum were significantly up-regulated (P0.05). Compared with the CON group, the relative abundance of Firmicutes in the cecum was significantly increased (P0.05), the relative abundance of Verrucomicrobiota in the cecum and Streptococcus in the colon were significantly decreased of the experimental groups (P0.05). The relative abundance of Bacteroidetes in the colon of the 0.4% GPE and 1.6% GPE groups was significantly increased (P0.05), and the relative abundance of Streptococcus in the cecum was significantly decreased (P0.05). The study shows that GPE can improve intestinal morphology, regulate inflammatory responses, and optimize microbiota structure in Bama miniature pigs, and the recommended supplementation dose is 1.6%.

The emergence and spread of multidrug-resistant Klebsiella pneumoniae (MDR-KP), which leads to acute lung injury (ALI) and severe pneumonia, pose a huge challenge to clinical treatment and public health. Non-antibiotic strategies have recently garnered significant attention as promising avenues for combating antibiotic resistance. In this study, whole-genome sequencing of an MDR-KP strain isolated from a deceased goat revealed key genetic features underlying its pathogenicity, including 31 resistance genes mediating broad-spectrum resistance to 14 antibiotic classes, 71 pathogen-host interaction genes supporting cross-organ infection, and 66 virulence factors enhancing colonization and immune evasion. Importantly, this clinically isolated strain was found to be pathogenic to both mice and chickens. Poria cocos polysaccharide (PCP) is a natural immunomodulator, but its effects on MDR-KP-induced ALI have not been explored. Herein, we investigated the protective effect of oral PCP and its dependence on gut microbiota remodeling using murine models of intranasal MDR-KP infection and antibiotic-induced pseudo-germ-free mice. The results showed that PCP pretreatment significantly alleviated MDR-KP-induced ALI by reducing pulmonary bacterial load (P < 0.01) and suppressing TNF-α and IL-1β levels in bronchoalveolar lavage fluid (P < 0.05). Mechanistically, PCP restored intestinal barrier function by upregulating tight junction proteins (occludin and claudin-1) and mucosal defense factors (SIgA and MUC-2), and reversed gut dysbiosis by restoring microbial diversity and enriching beneficial taxa. These protective effects were largely attenuated in pseudo-germ-free mice, suggesting that the protection offered by PCP against MDR-KP-induced ALI is dependent on the gut microbiota. In conclusion, oral PCP alleviates MDR-KP-induced ALI through a gut microbiota-mediated pathway by remodeling the gut microbiota, reinforcing intestinal barrier integrity, and regulating the gut-lung axis, providing a novel gut-targeted therapeutic strategy against drug-resistant KP infections.

This experiment aimed to investigate the effects of Spirulina polysaccharide extract on the immune, and antioxidant function, and nitrite stress of Litopenaeus vannamei. A total of 1 800 Litopenaeus vannamei were randomly divided into six groups, with three replicates per group and 100 shrimp per replicate. The blank control group and the nitrite stress control group were fed the basal diet. The high-dose, medium-dose, and low-dose groups received basal diets supplemented with 4.0, 2.0, and 1.0 g/kg of Spirulina polysaccharide extract, respectively. The positive control group (Astragalus polysaccharide group) supplemented with 2.0 g/kg of Astragalus polysaccharide in the basal diet. The pre-test period was seven days, and the formal test period was 30 days. After the feeding test concluded, a 72-hour nitrite stress test was conducted. The results showed that the activities of acid phosphatase (ACP), alkaline phosphatase (ALP), and superoxide dismutase (SOD) in the high-dose, medium-dose, and low-dose groups were higher than those in the blank control group. After 24 hours of nitrite stress, the activities of ACP, ALP, phenoloxidase (PO), aspartate aminotransferase (AST), and total antioxidant capacity (T-AOC) in the nitrite stress control group were significantly higher than those in the blank control group (P<0.05), while ACP, ALP activities, and T-AOC in the high-dose and medium-dose groups were significantly lower than those in the nitrite stress control group (P<0.05). After 48 hours of stress, the ACP, ALP activities, and T-AOC in the nitrite stress control group were significantly lower than those in the blank control group (P<0.05), and the ALP activity and T-AOC in the medium-dose group were significantly higher than those in the nitrite stress control group (P<0.05). The study show that Spirulina polysaccharide extract enhances nonspecific immune and antioxidant function, regulates physiological and biochemical responses under stress, and alleviates nitrite-induced damage in Litopenaeus vannamei.

Bacillus subtilis is widely used as a probiotic to mitigate alcohol-induced gut-liver injury, yet its therapeutic potential is limited by poor survival under gastrointestinal and ethanol stress, often necessitating encapsulation for viable delivery. Here, we isolated food-borne B. subtilis strains carrying DNA phosphorothioation (PT)—a DNA backbone modification in which sulfur replaces a non-bridging oxygen. The PT strain exhibited markedly enhanced tolerance to multiple stresses compared with the PT-deficient mutant, an effect we attribute to PT-mediated neutralization of reactive oxygen species (ROS) generated by ethanol and gastrointestinal conditions. In an acute mouse model of alcohol-induced injury, the PT strain showed a favorable safety profile and superior efficacy in reducing oxidative damage. In a chronic weaned piglet model, the PT strain achieved higher gastrointestinal survival and conferred stronger hepato-intestinal protection, reversing gut microbiota dysbiosis, preserving mucosal barrier integrity, and attenuating hepatic steatosis. Mechanistically, B. subtilis converts luminal taurochenodeoxycholic acid (TCDCA) into the cytoprotective bile acid tauroursodeoxycholic acid (TUDCA), which activates host TGR5 signaling to reinforce the intestinal barrier. This barrier fortification blocks lipopolysaccharide (LPS) translocation into the portal circulation, thereby suppressing the hepatic IFN-γ/JAK-STAT cytokine cascade and ameliorating liver damage. Our findings establish DNA phosphorothioation as a built-in stress shield that enables robust probiotic delivery in the alcohol-exposed gastrointestinal tract, providing a strategy for engineering resilient live biotherapeutics against gut-liver axis disorders.

Non-alcoholic fatty liver disease (NAFLD) is a primary metabolic disorder that threatens adolescent health globally, with no effective therapeutic agents currently available. Bellamya purificata is a traditional Chinese medicine categorized as "medicinal food", and polysaccharides are among its active components. However, its physicochemical structure remains poorly characterized, and no study has evaluated its effects on NAFLD. In this study, a homogeneous neutral polysaccharide, α-D-glucan (Mw = 6412.704 kDa), was isolated from B. purificata. The structure of the polysaccharide was characterized using monosaccharide composition analysis, methylation analysis, NMR spectroscopy, and scanning electron microscopy. The backbone structure of the polysaccharide comprises →4)-α-D-Glcp-(1→ and →4,6)-α-D-Glcp-(1→, with side chains of α-D-Glcp-(1→ attached to the O-6 position of the 1→4,6)-α-D-Glcp-(1→ sugar residues. Additionally, QSPS-1D effectively reduced weight gain, hepatic lipid accumulation (TC and TG), and inflammatory responses (tnf-α and il-1β) in NAFLD zebrafish. Moreover, QSPS-1D alleviated dysbiosis by inhibiting harmful bacteria (e.g., Stenotrophomonas, Agrobacterium, and Chryseobacterium) and promoting beneficial microbiota (e.g., Rothia), which restored the Firmicutes-to-Bacteroidetes ratio. In parallel, it enhanced the expression of tight junction proteins (zo-1 and claudin-1), leading to the repair of the intestinal mucosal barrier. These findings suggest that B. purificata polysaccharides may be a potential functional food for early NAFLD intervention, with effects potentially associated with the modulation of the gut microbiota.

Infection with Pseudorabies virus (PRV) triggers a state of oxidative distress in host cells, which consequently results in cellular damage. Although the normal butanol fraction of flavonoids derived from Polygonum hydropiper L. (FNB) is recognized for its strong antioxidant properties, it remains unclear whether FNB can mitigate PRV-induced oxidative damage and whether this protective effect involves long non-coding RNA (lncRNA)-mediated regulation during PRV infection. This study aimed to investigate the protective effect of FNB against oxidative stress in PRV-infected porcine alveolar macrophage cell line (3D4/2) and to elucidate the associated lncRNA regulatory network. The safe concentration of FNB was determined using the CCK-8 assay, and oxidative stress-related parameters were measured using commercial kits. The expression levels of key factors in oxidative stress signaling pathways were analyzed by qRT-PCR and Western blotting. Furthermore, transcriptome sequencing was performed to identify differentially expressed mRNAs and lncRNAs, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses to construct a lncRNA-mRNA interaction network. The results showed that treatment with 50 μg/mL FNB for 12 h significantly restored the PRV-suppressed activity of catalase (CAT) (p < 0.05), whereas treatment with 12.5 μg/mL FNB for 12 h markedly increased the activities of glutathione peroxidase (GSH-Px) and heme oxygenase-1 (HO-1) in PRV-infected cells (p < 0.01). At the molecular level, treatment with 50 μg/mL FNB for 12 h significantly increased the mRNA and protein expression levels of HO-1 and NAD(P)H:quinone oxidoreductase 1 (NQO1) (p < 0.01). Transcriptomic analysis identified 6948 differentially expressed messenger RNAs (mRNAs) after PRV infection and 4400 differentially expressed mRNAs after FNB intervention compared with the PRV group, with these genes being mainly enriched in oxidative stress-related processes. By screening for key antioxidant-related mRNAs and their potentially interacting lncRNAs, a lncRNA-mRNA network was constructed. In conclusion, FNB may alleviate PRV infection-induced oxidative damage by activating the Nrf2/HO-1 signaling pathway and modulating specific lncRNA-mRNA networks. This study provides novel insights into the antioxidant effect of FNB against PRV infection-induced oxidative damage and the regulatory role of lncRNAs.

Single-nucleus sequencing is a powerful tool for studying ovarian development. However, a standardized protocol for single-nucleus isolation in crustaceans is currently lacking. In this study, the key parameters for preparing nuclei suspension of Litopenaeus vannamei were optimized systematically for the first time, employing evaluation methods such as HE staining, trypan blue staining, scanning electron microscopy, 10 × Genomics quality control data, and t-SNE plots. The results showed that the optimal lysis efficiency of fresh ovarian tissue was achieved by manually grinding with 0.1% NP40 for 5 min, and the nuclei quality was ensured. Meanwhile, an iodixanol-assisted approach was used to reduce the mechanical damage to nuclei caused by centrifugation, significantly enhancing nuclear recovery. To capture as many nuclear types as possible, we employed a combination of high- and low-speed centrifugation, improving nuclear recovery and reducing data bias. Shrimp ovarian samples can be processed within 40 min or less. The single nuclei isolated from shrimp ovaries by the optimized method were uniformly round with clear boundaries, a significant reduction in clumping and debris, and minimal nuclear residue in the supernatant. The nuclear recovery rate increased from 4.78% to 40.56%. This study bridges the gap in single-cell research on the reproductive system of L. vannamei and provides new directions and insights for the preparation of nuclei suspension from complex tissue samples in the crustacean research.

Pseudorabies virus (PRV) can infect a wide range of animal species, including swine and rodents. Infection in pigs is associated with significant economic losses in the global pork industry and is characterized by acute, often fatal disease, as well as central nervous system (CNS) invasion, which leads to neurological manifestations. Although PRV replication has been extensively characterized in certain murine neuronal cell lines such as Neuro-2a, the mechanisms underlying PRV-induced neuroinflammatory injury and necroptosis remain largely unclear. In this study, Kunming mice and mouse astrocytes (C8-D1A) were infected with PRV-GXLB-2013 at different doses to evaluate neurological injury and inflammatory responses. Given that the NF-κB/MLKL signaling pathway was found to be activated during PRV infection, a selective MLKL inhibitor, necrosulfonamide (NSA), was applied to investigate the role of necroptosis in PRV-induced neuroinflammatory damage. Mice infected with higher viral doses showed increased mortality, severe neurological symptoms, elevated brain inflammation, and pathological changes. In C8-D1A cells, PRV infection significantly upregulated inflammatory cytokines and key components of the NF-κB/MLKL pathway. Importantly, NSA treatment markedly reduced these inflammatory responses, mitochondrial damage, and cellular necrosis. Collectively, these findings suggest that PRV infection triggers neuroinflammatory injury through the activation of necroptosis and the NF-κB/MLKL signaling pathway. This study provides novel mechanistic insights into PRV-induced neurological damage and highlights potential therapeutic targets for intervention.

IntroductionThe normal butanol fraction of Polygonum hydropiper L. flavonoids (FNB) exhibits significant anti-inflammatory effects. This study investigated FNB's impact on inflammatory responses induced by Porcine circovirus type 2 (PCV2) in cell and mouse models.MethodsAn inflammatory model was established in RAW264.7 cells infected with varying PCV2 concentrations. And assigning both RAW264.7 cells and 108 SPF-grade KM mice to Control, PCV2, Rutin, and various dosages of FNB groups. Inflammatory factors such as Monocyte Chemoattractant Protein-1 (MCP-1), interleukin-6 (IL-6), IL-8, IL-10, Tumor Necrosis Factor-alpha (TNF-α), Reactive Oxygen Species (ROS), and Nitric Oxide (NO) were quantified using ELISA, RT-qPCR and immunohistochemistry.ResultsResults showed that a PCV2 titer of 104.5 TCID50/0.1 mL when applied to RAW264.7 cells effectively established an in vitro inflammatory model at 12 and 24 h post-infection. Following PCV2 infection, all the inflammatory factors displayed a significant increased both in culture supernatant and intracellular mRNA expression levels (p < 0.05 or p < 0.01), but these levels were reduced by FNB treatment (p < 0.05 or p < 0.01). In mouse sera post-PCV2 infection also showed elevated levels of IL-6, IL-8 IL-10, TNF-α, and MCP-1 (p < 0.05 or p < 0.01). Additionally, mRNA and protein levels for TNF-α, IL-8, IL-10, IL-6, and iNOS rose significantly in lung tissues (p < 0.01) but decreased with FNB treatment (p < 0.05 or p < 0.01).DiscussionThese findings suggest that FNB reduces inflammatory factor production and modulates the inflammatory response triggered by PCV2 infection, potentially enhancing host resistance against it.

Sepsis, a life-threatening systemic inflammatory syndrome caused by dysregulated host responses to infection, frequently originates from Gram-negative pathogens such as Escherichia coli (E. coli). While antibiotics remain the mainstay treatment, rising antimicrobial resistance necessitates alternative therapeutic strategies. Formononetin (FMN), a bioactive isoflavone derived from traditional Chinese medicine, exhibit potent anti-inflammatory and antioxidant properties. This study employed an integrative approach combining in vivo pharmacodynamic evaluation, network pharmacology, and experimental validation to systematically investigate FMN's therapeutic potential against E. coli induced sepsis. In vivo studies in a murine sepsis model showed FMN significantly reduced E. coli sepsis induced body weight loss, clinical manifestations, histopathological changes, and suppressed proinflammatory cytokines (e.g. IL-6 and TNF-α). Network pharmacology identified 28 putative targets interacted with the E. coli sepsis-related site and molecular docking suggested possible interactions between FMN and key inflammatory mediators such as Toll-like receptor 4 (TLR4) and tumor necrosis factor (TNF). qRT-PCR indicated that FMN modulated the transcription of TLR4/MyD88-related genes, with downstream reductions in the mRNA levels of TLR4, MyD88, iNOS and IL-6. These findings suggest that FMN may be a promising candidate for further investigation as a multi-target modulator in sepsis.

Arthrospira platensis polysaccharide component 1 (PAP-1), a purified polysaccharide monomer isolated from Arthrospira platensis, exhibits pronounced antioxidant activity. To investigate the in vivo and in vitro regulatory effects of PAP-1 on antioxidant enzyme activities and inflammatory mediators in mice and RAW264.7 cells, the mice were administered PAP-1 by gavage, and the cells were cultured with PAP-1. Subsequently, serum, lung, spleen, and thymus tissues from mice, as well as the cultured RAW264.7 cells, were collected for analysis using RNA sequencing, commercial assay kits, immunohistochemistry, RT-qPCR, and Western blotting. The results demonstrated that PAP-1 significantly reduced the levels of oxidative stress-related indicators (NO, iNOS, MDA, MPO, and XOD), while markedly enhancing the activities of antioxidant enzymes (SOD, CAT, and GSH-Px) (p < 0.05), a trend consistently observed in both in vivo and in vitro experiments. Furthermore, PAP-1 upregulated the expression of key antioxidant genes and proteins, including HO-1, NQO1, GCLM, p62, Prdx1, and SLC7A11. Collectively, these findings indicate that PAP-1 exerts regulatory antioxidant effects in mice and RAW264.7 cells by enhancing antioxidant enzyme activity and suppressing oxidative stress responses, underscoring its potential as a natural antioxidant agent.

To solve the aquaculture farmers' difficulty in choosing suitable probiotics, we evaluated the five-probiotics sold in the market. Here, Bacillus subtilis-based product, compound Bacillus-based product (BC), nitrifying bacteriabased product (NB), photosynthetic bacteria-based product (PB) and aquatic lactic acid bacteria-based product (AL) were sprayed into the aquaculture water for 42 days, and shrimp performance and the water environment were measured. The results showed that NB, PB, and AL significantly increased the shrimp growth performance (p < 0.05). The five probiotics enhanced AMS activity significantly (p < 0.01). BC, PB, and AL reduced MDA, GOT, and GPT activity, while BS increased the Vibrio resistance and SOD, CAT, GSH-PX activity. The 16 s sequencing analysis revealed that compared to the control group, the AL and PB groups had significantly different microbial compositions, fewer unique ASVs, and lower intestinal microbiota abundance. Prevotella_7, Muribaculaceae_unclassified, and Veillonella were negative correlation with growth performance significantly, and Enterobacter, Hafnia-Obesumbacterium, Pseudomonas, and Klebsiella were positive correlation with immunity index (p < 0.05). In the control group, 72 % of the gut microbiota originated from the culture water, and this proportion increased after probiotics were added. All five probiotics improved water quality, especially AL. These data suggested that different probiotics have distinct preferences. AL showed the best overall performance, including high growth, favorable gut microbiota, and improved water quality. BS played a key role in enhancing shrimp resistance to Vibrio and nonspecific immunity. This study helped improve the efficacy assessment of aquatic probiotics and provides a theoretical basis for selecting suitable probiotics.

The Sanhuanglianqiao mixture (SHLQ) was prepared from a certain proportion of Scutellaria baicalensis Georgi, Forsythia suspensa, and Rheum palmatum L and obtained by water extraction and alcohol precipitation. This study explored the effects of SHLQ on Penaeus vannamei and its protective role against Vibrio parahaemolyticus (V. parahaemolyticus) infection. The results indicated that the main active components of SHLQ, namely baicalin, phillyrin, and emodin, had concentrations of 7.34 mg/mL, 4.01 mg/mL, and 32.95 μg/mL, respectively. SHLQ significantly enhanced shrimp growth performance and digestive enzyme activities. Mechanistically, SHLQ augmented innate immunity by upregulating immune-related genes and boosting antioxidant defenses in hepatopancreas and hemolymph. Concurrently, SHLQ reshaped gut microbiota diversity, suppressing pathogenic Vibrionaceae while enriching beneficial taxa, thereby reinforcing metabolic and immune functions. Critically, SHLQ alleviated V. parahaemolyticus-induced intestinal/hepatopancreatic damage, increased hemolymph cell viability, and reduced apoptosis caused by the virulence factor rPirB. These synergistic effects-combining immunomodulation, microbiota regulation, and tissue protection-collectively improved shrimp survival rates. Our findings highlight SHLQ as a promising multi-target agent against V. parahaemolyticus infection in aquaculture.

BACKGROUND:Lophatherum gracile Brongn., also known as Dan-Zhu-Ye, is traditionally used to clear heat and purge fire, relieve irritability and thirst, diuresis and catharsis. Our preliminary research revealed that total flavonoid extracts from Lophantherum gracile Brongn. (TFLG) can reverse the inflammatory response induced by LPS in mice, but the exact mechanism is still unclear. METHODS:The main chemical constituents of TFLG were first analyzed via liquid chromatography tandem high-resolution mass spectrometry (LC-HRMS). We then used network pharmacology to screen potential targets of TFLG to treat inflammation. Subsequently, inflammatory models were established in 3D4/2 cells and SPF Kunming mice via lipopolysaccharide. The predicted potential targets and associated signaling pathways were further validated through ELISA, western blot analysis, and qRT-PCR. RESULTS:The LC-HRMS results revealed that approximately 36 compounds with contents greater than 0.1 % were identified in TFLG. Network pharmacology analysis revealed that 103 common targets of TFLG are associated with inflammation. The results of molecular docking indicated that the main ingredients of L. gracile (quercetin, kaempferol, and luteolin) can exert anti-inflammatory effects through binding with the inflammatory targets TNF, IL-6, and IL-1β. Our experimental results demonstrated that TFLG reversed the upregulation of IL-6, IL-1β, IL-12, and TNF-α and the downregulation of IL-10 in both in vitro and in vivo inflammatory models induced by LPS. The qRT-PCR results were consistent with the ELISA results above. Western blot analysis revealed that TFLG reduced the expression levels of p65, p-p65, p-IκB-α, p38, and p-p38 and increased the expression of IκB-α. CONCLUSIONS:TFLG can exert an anti-inflammatory effect by inhibiting the NF-κB and MAPK signaling pathways, which aligns with the anticipated outcomes of network pharmacology. This study offers new evidence supporting the potential application of TFLG as a medication for reducing inflammation.

This study investigated the immunomodulatory effects and underlying mechanisms of total flavonoid of Spatholobus suberectus Dunn (TFSD) and its primary constituent formononetin (FMN) in immunosuppressed mouse models. Spatholobus suberectus Dunn (S. suberectus Dunn) was first analyzed qualitatively and quantitatively by broadly targeted metabolomics using Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Integrated network pharmacology and molecular docking approaches were used to investigate the potential mechanisms underlying S. suberectus Dunn’s immunomodulatory effects. Subsequently, mice were pretreated with TFSD and FMN for seven days before constructing an immunosuppression model through intraperitoneal injection of 200 mg/kg.bw cyclophosphamide (CTX). In vivo experiments validated the findings and investigated the mechanisms underlying the effects of TFSD and FMN on immunosuppression. Metabolomic analysis identified 501 distinct flavonoids in S. suberectus Dunn, with FMN exhibiting the highest relative abundance among all detected compounds. The primary active components of S. suberectus Dunn against immunosuppression are flavonoids, including FMN and vestitol. The core targets of these components were identified as NF-κB and IKKβ, with the NF-κB signaling pathway being suggested as the most probable mechanism of action. FMN exhibited strong binding affinity to the core targets, NF-κB p65 and IKKβ. In vivo experiments indicated that pretreatment with TFSD and FMN prevented CTX-induced pathological damage in the spleen and thymus and increased immune cell counts and immunoglobulin levels. Additionally, it significantly upregulated the secretion and expression of key cytokines and the mRNA expression of NF-κB p65, IKKα, and IKKβ (p < 0.05 or p < 0.01). In conclusion, TFSD and FMN can protect mice from CTX-induced immunosuppression by regulating the NF-κB signaling pathway, making them promising drug candidates for preventing and treating immunosuppression-related diseases.

Pseudorabies virus (PRV) poses a great danger to pig farming and human public health safety. Previous studies have shown that the first component of Arthrospira platensis polysaccharide (PAP-1) may alleviate the inflammatory response of RAW264.7 cells induced by PRV infection through the IL-17RA signaling pathway and its circRNA-miRNA-mRNA network, but the specific mechanism is not clear. In this study, we screened the LncRNA sequences related to IL-17RA signaling pathway by transcriptomics data. The mechanism was verified by gene interference or overexpression and dual luciferase experiments to investigate the regulatory mechanism of PAP-1 on IL-17RA inflammatory signaling pathway in PRV infection. In vitro studies showed that PAP-1 significantly reduced the PRV-induced elevated expression of IL-17RA signaling pathway inflammatory factors such as IL-17RA, IL-1 beta, CXCL10, and iNOS in RAW264.7 cells (P < 0.05). On this basis, the key regulatory factor LncRNA Dhx35 of IL-17 RA signaling pathway was screened by whole transcriptome sequencing analysis. It was found that PAP-1 could reduce the transcriptional activity of IL-17RA by down-regulating PRV-induced LncRNA Dhx35 as a competing endogenous RNA to bind to mmu-miR-185-3p, which decreased the inflammatory response. In vivo experiments revealed that PAP-1 down-regulated the expression of LncRNA Dhx35, up-regulated the expression of mmu-miR-185-3p, and significantly down-regulated the expression of IL-17RA, IL-1 beta, CXCL10, and iNOS in lung tissues of PRV-infected mice (P < 0.05). Taken together, our data suggest that PAP-1 reduces PRV infection-induced inflammatory responses in RAW264.7 cells and mouse lung tissues by regulating LncRNA Dhx35/mmu-miR-185-3p/IL-17RA.

Flavonoid n-butanol (FNB) possess diverse pharmacological properties. This study aimed to explore the mechanism of FNB in regulating oxidative response in PCV2-infected RAW264.7 cells. PCV2-infected macrophages were treated with FNB, and oxidative stress markers, antioxidant enzyme activities, as well as related gene and protein expression were assessed to evaluate FNB's regulatory effects. Specifically, the level of Nitric Oxide (NO), Total antioxidant capacity (T-AOC), anti-hydroxyl radical capacity, anti-superoxide anion capacity, L-Glutathione (GSH) level, Super Oxide Dismutase (SOD) and Catalase (CAT) were detected. The expression of key oxidative stress-related and signaling pathway genes and proteins was determined by qPCR and western blotting, respectively. The results indicated that FNB reduced intracellular ROS, increased SOD and CAT activities, improved antioxidant capacity, upregulated the mRNA expression levels of HO-1, NQO1, Nrf2, Pi3kca, SOD, and HDAC1, downregulated AKT, Keap1, and HAT1, enhanced HDAC1 activity, and inhibited HAT activity. In conclusion, FNB protects against PCV2-induced oxidative damage by activating the PI3K/AKT pathway and inhibiting Keap1, which collectively enhance the Nrf2/HO-1 antioxidant response.