ETHNOPHARMACOLOGICAL RELEVANCE:Paeoniae Decoction (PD) is a classic Chinese medicine formula for treating intestinal diseases. However, its direct intervention effect on intestinal fibrosis and the underlying molecular mechanisms have not been fully elucidated. AIM OF THE STUDY:To evaluate the effect of PD on inflammatory bowel disease (IBD) associated intestinal fibrosis and to systematically investigate its potential molecular mechanisms utilizing integrated multi-omics. MATERIALS AND METHODS:The chemical constituents of PD were characterized and quantified utilizing ultra-high performance liquid chromatography-Quadrupole-Orbitrap mass spectrometry (LC-Q-Orbitrap-MS) and LC-MS/MS. A chronic experimental colitis-associated intestinal fibrosis mouse model lasting 56 days and involving four repeated cycles of dextran sulfate sodium (DSS) injury was established. The therapeutic efficacy of PD was evaluated through endoscopy, histopathological analysis, and collagen staining. Label-free quantitative proteomics and untargeted metabolomics and network pharmacology were employed to elucidate the target networks. In vitro mechanisms were further validated in TGF-β1-stimulated NCM460 cells using wound healing assays, immunofluorescence, and Western blotting to evaluate the epithelial-mesenchymal transition (EMT) process and the TGF-β/Smad signaling pathway. Finally, molecular docking was used to verify the direct binding of the main bioactive components of PD to the key pathway targets. RESULTS:Chemical profiling identified 16 common constituents across independent batches of PD. In vivo, PD administration significantly ameliorated intestinal fibrosis and reduced massive extracellular matrix (ECM) deposition in the mucosal and submucosal layers. Integrated omics analyses revealed that PD profoundly modulated the TGF-β signaling pathway, ECM-receptor interaction, and the Sphingolipid signaling pathway. Mechanistically, PD dose-dependently inhibited myofibroblast activation, downregulating the expression of α-SMA, Vimentin, Collagen I, and Collagen III. In vitro, PD serum suppressed TGF-β1-induced cell migration, preserved the epithelial marker E-cadherin, and inhibited the nuclear translocation of Snail by significantly decreasing the phosphorylation of Smad2 and Smad3. Furthermore, PD reversed pro-fibrotic sphingolipid metabolism and upregulated taurine and glutathione metabolism associated with antioxidant defense. CONCLUSION:PD effectively alleviates chronic colitis-associated intestinal fibrosis by inhibiting the TGF-β/Smad signaling pathway-mediated EMT process, reducing ECM deposition, and reprogramming the fibrotic metabolic microenvironment.

Polysaccharides from Lycium barbarum L., particularly LBP3, exhibit immunomodulatory and anti-tumor properties, yet their active constituents remain unclear. Here, LBP3 was fractionated into PolyA, PolyB, and PolyC. PolyC emerged as the key bioactive component, enhancing phagocytosis in vitro and showing superior anti-tumor efficacy to PolyA in vivo (125 mg/kg). Mechanistically, PolyC upregulates gp91 and p47 expression involving Dectin-1, driving M2 macrophages toward an M1 phenotype and enhancing antigen presentation and adaptive immunity. Purification of PolyC yielded its major subfraction, PolyD, which was structurally characterized as a predominant α-(1 → 4)-linked glucan with α-(1 → 6)-branching and a terminal β-linkage. Despite its α-configuration, PolyD retains Dectin-1 agonistic activity, suggesting that Dectin-1 may recognize specific α-glucan conformations. In summary, this study establishes PolyC as the key bioactive component of LBP3 and PolyD as its structurally defined α-glucan, demonstrating that PolyC's anti-tumor activity involves Dectin-1-mediated NOX2 upregulation in TAMs, expanding the chemical diversity of Dectin-1 agonists and informing immunotherapy design.

Accumulation of neutrophil extracellular traps (NETs) in ulcerative colitis (UC) is associated with impaired intestinal epithelial barrier integrity. However, little is known about how NETs affect intestinal epithelial repair. This study sheds light on the molecular mechanisms through which excess NETs cause intestinal epithelial damage in UC mice. We found that UC mice had elevated levels of circulating cell-free DNA (cfDNA), mainly from NET byproducts (e.g., NET-DNA). NET-DNA in the intestine worsened UC symptoms, while DNase I treatment to eliminate it alleviated these symptoms. RNA-seq analysis revealed significant changes in IL-22 mRNA between wild-type and peptidylarginine deiminase 4 knockout (PAD4-/-) mice. Flow cytometry results indicated that NET-DNA mainly affected IL-22 secretion by group 3 innate lymphoid cells (ILC3s), while other forms of DNA had little influence on IL-22 expression. The IL-22+ILC3s ratio was restored in both DNase I-treated and PAD4-/- mice; moreover, levels of mucin, tight junction proteins, and Ki67 were significantly increased. Co-incubating ILC3s or the mouse lymphocyte cell line MNK3 with NET-DNA decreased IL-22 levels. ILC3s expressed the NET-DNA receptor coiled-coil domain containing protein 25 (CCDC25); however, NET-DNA did not affect IL-22 secretion in shCCDC25-MNK3 cells. Additionally, inhibiting ILK-HIF-1α proteins, downstream of CCDC25, increased IL-22 production in MNK3 cells. Finally, we established an in vitro culture system using MNK3 and Caco-2 cells. The supernatant from NET-DNA-treated MNK3 cells increased FITC-dextran permeability and reduced ZO-1 expression in Caco-2 cells. Thus, CCDC25 in ILC3s responds to NET-DNA by reducing IL-22 levels in UC mice, negatively impacting mucosal healing.

Polysaccharides from Lycium barbarum L., particularly the LBP3 fraction, exhibit immunomodulatory and anti-tumor properties, yet their active constituents and mechanisms remain unclear. Here, LBP3 was fractionated into three subfractions: PolyA (200–350 kDa), PolyB (100–200 kDa), and PolyC (40–100 kDa). In vitro assays showed that all fractions induced macrophage activation, but PolyC uniquely exhibited potent phagocytosis-enhancing activity. In a murine hepatocellular carcinoma model, PolyC exhibited superior in vivo anti-tumor efficacy compared to PolyA, even at a lower dosage. Mechanistically, PolyC activates the NADPH oxidase pathway by acting on the Dectin-1 receptor on macrophages, thereby reprogramming macrophages from M2 phenotype to M1 phenotype. This phenotypic shift enhanced phagocytic and antigen-presenting capacities, thereby triggering adaptive immune activation. Subsequent purification of PolyC yielded its major active subfraction, designated PolyD. Structural analysis revealed that PolyC and PolyD both are primarily composed of glucose residues linked via β-glycosidic bonds, which may constitute a key structural determinant for Dectin-1 recognition. In summary, this study identifies PolyC as the key bioactive component of LBP3, characterizes its structural features, and elucidates the molecular mechanism by which it reprograms tumor-associated macrophages (TAMs) via the Dectin-1/NADPH oxidase pathway. These findings provide a theoretical foundation for the development of polysaccharide-based tumor immunotherapies.

Background Promoting damaged intestinal mucosal repair and restoring epithelial barrier function are critical strategies for treating ulcerative colitis (UC). Interleukin-22 (IL-22), a key cytokine for intestinal epithelial homeostasis, is mainly secreted by group 3 innate lymphoid cells (ILC3s). ILC3s exhibit substantial plasticity: NCR⁻ILC3 preferentially secrete IL-17A, whereas NCR⁺ILC3 primarily produce IL-22; under inflammatory conditions, NCR⁺ILC3 and ILC1 can undergo reciprocal conversion. Hypoxia-inducible factor-1α (HIF-1α) signaling is involved in regulating ILC3 subset balance and influences intestinal immune homeostasis. Epicatechin, a common dietary flavonoid, possesses antioxidant and anti-inflammatory activities; however, whether it modulates ILC3 biological functions through the HIF-1α pathway remains unknown. Objective To investigate whether epicatechin ameliorates UC through HIF-1α-mediated restoration of NCR⁺ILC3 cells and to identify its direct molecular target. Methods Primary lymphocytes isolated from mesenteric lymph nodes of C57BL/6 mice and the ILC3-derived MNK3 cell line were used to validate the effect of HIF-1α modulation on NCR⁺ILC3 numbers. An acute UC model was induced by 3% DSS in C57BL/6 mice. Mice were treated with epicatechin (37.5, 75, or 150 mg/kg/d) or mesalazine (500 mg/kg/d) by gavage, or with roxadustat (25.0 mg/kg) by intraperitoneal injection. Intestinal epithelial barrier function was assessed by Western blot, immunofluorescence, and histopathology. Flow cytometry was used to detect changes in HIF-1α levels and NCR⁺ILC3 abundance. The direct target of epicatechin was identified by integrating DARTS, CETSA, and mass spectrometry, and the mechanism was validated by shRNA-mediated knockdown combined with co-immunoprecipitation. Results HIF-1α levels correlated positively with NCR⁺ILC3 numbers. In DSS-induced acute colitis, epicatechin dose-dependently alleviated body weight loss, colon shortening, and systemic inflammation; improved histopathological injury; restored tight junction protein expression (Occludin, ZO-1) and mucin secretion; and reduced intestinal permeability. Epicatechin restored NCR⁺ILC3 numbers in the colonic lamina propria and mesenteric lymph nodes while concomitantly restoring HIF-1α levels. In vitro, epicatechin dose-dependently upregulated the mRNA expression of Il22, Rorc, HIF-1A, and Ncr1, as well as NICD and HIF-1α protein levels in MNK3 cells, whereas total NLE1 protein remained unaffected. Mechanistically, epicatechin bound NLE1 (binding energy −8.5 kcal/mol), enhanced the interaction between NLE1 and NICD, and simultaneously disrupted the binding of NICD to FBXW7, the substrate recognition subunit of the E3 ubiquitin ligase complex, thereby reducing NICD ubiquitination and stabilizing HIF-1α signaling. NLE1 knockdown completely abolished epicatechin-induced activation of the NICD/HIF-1α pathway, IL-22 secretion, and target gene upregulation, confirming NLE1 as its critical molecular target. Conclusion Epicatechin alleviates DSS-induced colitis by directly targeting NLE1, enhancing NLE1–NICD binding and reducing NICD–FBXW7 interaction to stabilize NICD protein, thereby activating the NLE1/NICD/HIF-1α signaling axis and restoring NCR⁺ILC3 numbers and IL-22 secretion. These findings establish a molecular link between dietary flavonoids and ILC3-mediated immunity, positioning epicatechin as a phytochemical lead for UC mucosal repair.

Glutamine metabolism is essential for infectious spleen and kidney necrosis virus (ISKNV) replication. Glutaminase 1 (GLS1), the key enzyme of the glutamine metabolism, and c-Myc positively regulate ISKNV infection, while c-Myc is closely correlated with GLS1. However, the regulatory mechanism among ISKNV, c-Myc and glutamine metabolism remains unclear. Here, we indicated that c-Myc increased glutamine uptake by increasing the GLS1, glutamate dehydrogenase (GDH) and isocitrate dehydrogenase (IDH2) expression of glutamine metabolism. ISKNV ORF102R, ORF093R and ORF118L co-located with c-Myc in CPB cells. Co-IP results showed that ISKNV ORF102R and ORF093R interacted with c-Myc, while ORF118L did not interact with c-Myc. The expression levels of c-Myc, GLS1 and IDH2 were increased in ISKNV ORF093R expression cells, and the mRNA and protein levels of GLS1 were upregulated in ISKNV 102R-expressing cells. These results indicated that ISKNV reconstructed glutamine metabolism to satisfy the energy and macromolecule requirements for virus proliferation by ORF093R and ORF102R interacting with c-Myc, which provides the foundation for innovative antiviral strategies.

ETHNOPHARMACOLOGICAL RELEVANCE:Dahuang Mudan Decoction is a classic Chinese medicine prescription for treating ulcerative colitis (UC). Previous studies have shown that Dahuang Mudan Decoction has preventive and therapeutic effects on mice with dextran sulfate sodium (DSS) induced colitis. AIM OF THE STUDY:The objective of this research endeavor was to ascertain the most efficacious synergistic blend of Emodin, Luteolin, and Paeonol, the main active ingredients in Dahuang Mudan Decoction, in alleviating UC. Additionally, it sought to elucidate the underlying therapeutic mechanisms and evaluate the safety of the combined components. MATERIALS AND METHODS:Employing Emodin, Luteolin, and Paeonol as starting materials, the optimal combination was selected by orthogonal design. Basic pharmacodynamics was observed in mouse model of UC induced by DSS. The pathological changes of the colon were observed using hematoxylin and eosin (H&E) staining. The changes of cytokines and proteins related to inflammation and intestinal barrier function were detected by WB, Alcian blue staining, immunofluorescence, immunohistochemistry and related kits. Subsequently, 16S rRNA sequencing was used to observe changes in the intestinal flora. To evaluate the therapeutic effect and potential mechanism of the optimal monomer composition on UC mouse model. Finally, we performed toxicity tests as part of the safety assessment of the combination of the three monomers. RESULTS:The different combinations of Emodin, Luteolin, and Paeonol alleviated DSS-induced colitis to varying degrees. The ELP5 group (Emodin 5 mg/kg + Luteolin 5 mg/kg + Paeonol 15 mg/kg) and ELP9 group (Emodin 15 mg/kg + Luteolin 15 mg/kg + Paeonol 75 mg/kg) had the most significant mitigation effect on UC mice. Mechanistically, the monomeric composition provides a comprehensive treatment for UC by addressing multiple aspects, including anti-inflammatory and antioxidant effects, repairing the damaged intestinal barrier, restoring the intestinal flora structure, and regulating short-chain fatty acid levels. In addition, the combination of Emodin, Luteolin and Paeonol exhibited a more significant effect on DSS-induced colitis compared to the individual components, indicating a synergistic effect among them. In the single-dose toxicity test, no obvious abnormalities were found in the general state or major organs of the mice. In repeated toxicity tests, it was found that the combined use of three monomers had less effect on organ index, hematology and serum biochemical indexes than that of a single compound. Pathological examination showed that the three monomers had certain toxicity to mouse liver, kidney and lung when used alone and in large doses for a long time, and the toxicity was significantly reduced after combined use. CONCLUSIONS:We have determined the optimal combination of three active ingredients in Dahuang Mudan Decoction to alleviate DSS induced colitis in mice by inhibiting intestinal inflammation and oxidative stress, repairing impaired intestinal barrier function, and regulating intestinal flora disturbance. The results of single administration toxicity test proved the safety of the three monomers combined, and repeated administration toxicity test clarified the safe dose range of the combined administration, and also revealed that the combined therapy exhibited superior safety compared to monotherapy.

The incidence of acute liver injury is increasing and poses a significant threat to human health. Ganoderma lucidum spore oil (GLSO), a lipid substance extracted from Ganoderma lucidum spore powder using supercritical CO2 technology, has been investigated for its potential to prevent acute liver injury. However, the specific mechanism underlying the protective effects of GLSO remains incompletely understood. In this study, we investigated the preventive effect of GLSO on acute liver injury in rats, focusing on the gut microbiome and serum metabolomics. GLSO effectively alleviated liver dysfunction and reduced inflammation, leading to the prevention of acute liver injury in rats. Serum metabolomics analysis revealed that GLSO primarily modulated lipid metabolic pathways related to glycerophospholipid metabolism and sphingolipid metabolism. Specifically, GLSO decreased the levels of metabolites such as lysophosphatidylcholine (LPC), glycerophosphatidylcholine (GPC), and sphinganine 1-phosphate (SA1P), while increasing the levels of phosphatidylglycerol (PG) and digalactosylceramide (DGC). Gut microbiomics data indicated that GLSO effectively regulated the composition of the gut microbiota in rats with acute liver injury. Specifically, it increased the abundance of Firmicutes and decreased the abundance of Proteobacteria. Mantel test correlation analysis revealed a close relationship between gut microbial Burkholderiales and lipid metabolites in GLSO-mediated prevention of acute liver injury. GLSO exerts its preventive effects on acute liver injury by remodeling the gut microbiota and regulating lipid metabolism. These findings provide novel insights and potential directions for the development of new drugs targeting acute liver injury.

Metabolic-associated fatty liver disease (MAFLD) has emerged as a prevalent chronic liver disease. Our review of the existing literature reveals that the interplay between gut microbiota, mitochondria, and the liver is a key mechanism in the development of MAFLD. This paper distills the pathogenic role of gut microbiota in MAFLD through its influence on mitochondria and outlines the therapeutic mitochondrial mechanisms of MAFLD that leverage gut microbiota. It also touches on the traditional Chinese medicine perspective on the liver-intestine connection and the concept of ''qi'' in relation to mitochondria, as well as its modern medical counterpart. We conclude that the gut microbiota and their metabolites can directly or indirectly affect the intestinal mitochondria, leading to structural and functional changes. These changes include shifts in mitochondrial membrane potential, changes in permeability, and dysregulation of signaling pathways. As a result, the permeability of intestinal epithelial cells may be increased, and the integrity of the intestinal barrier may be compromised. The gut microbiota and their metabolites can then influence hepatic mitochondria through the hepatic-intestinal axis, triggering liver pathology. When liver damage occurs, their metabolites can enter the intestine and affect intestinal mitochondria and microbiota, which in turn can lead to a disrupted intestinal barrier and microbiota and a dysregulated homeostatic balance. Our extensive literature review suggests that the gut microbiota may mediate the treatment of MAFLD through mitochondrial pathways. The therapeutic approach of modulating the gut microbiota to regulate mitochondrial function and restore liver health is promising. Traditional Chinese medicine diets are particularly well suited for this strategy. Further research is warranted to fully elucidate the underlying mechanisms. By protecting the body's own mitochondrial function through the gut microbiota, we can effectively combat liver injury, providing a novel therapeutic avenue for the treatment of liver disease.

In this study, we assessed the therapeutic effects of Bifidobacterium animalis subsp. lactis GOLDGUT-BB69 (BB69) on diarrhea in mice by monitoring diarrhea scores, intestinal histopathology, detecting cytokine and protein expression, and intestinal microbial composition and metabolism. The results showed that BB69 significantly reduced the disease symptoms of diarrhea in mice, attenuated the pathological features of the intestine, decrease the protein expression in the TLR4/NF-κB signaling pathway, reduced intestinal inflammation, lowered intestinal permeability, and significantly increased the expression of mucin and tight junction proteins (ZO-1, occludin), which repaired intestinal mucosal injuries and enhanced the intestinal barrier function. In addition, BB69 significantly reduced the gut dysbiosis of mice with diarrhea by increasing the abundance and diversity of intestinal microorganisms and the content of short-chain fatty acids (SCFAs). Our findings suggest that BB69 can act as a functional probiotic, alleviating diarrhea-related symptoms by inhibiting the inflammatory response and promoting the restoration of the intestinal mucosal barrier and intestinal flora.

Background Chemotherapy-induced intestinal injury caused by 5-Fluorouracil (5-FU) severely impairs intestinal barrier integrity through oxidative stress and mitochondrial dysfunction. Currently, effective preventive strategies are lacking. This study investigated the protective effects and mechanisms of LBP3, a bioactive fragment of Lycium barbarum polysaccharides, against chemotherapy-induced intestinal injury. Methods 5-FU-induced chemotherapy-induced intestinal injury mouse model and IEC-6 cell model were used to evaluate weight loss, colon pathology, intestinal barrier function, oxidative stress, and mitochondrial dysfunction. The role of the Nrf2 pathway was studied using the inhibitor ML385. Results LBP3 significantly alleviated body weight loss, colon tissue damage, and inflammatory cell infiltration, and restored the expression of tight junction proteins (Occludin, ZO-1) and barrier integrity. LBP3 reduced reactive oxygen species (ROS) levels, restored superoxide dismutase (SOD) and glutathione (GSH) activity, and decreased malondialdehyde (MDA) accumulation, mitigating oxidative stress. Furthermore, LBP3 improved mitochondrial function by restoring membrane potential and morphology. Conclusion LBP3 alleviates chemotherapy-induced intestinal injury by enhancing antioxidant defenses and mitochondrial protection, has been strongly associated with the activation of Nrf2 pathway. This study highlights LBP3's potential as a therapeutic agent for chemotherapy-induced intestinal injury.

ETHNOPHARMACOLOGICAL RELEVANCE:In traditional Chinese medicine, rhei radix et rhizoma, is derived from the dried roots and rhizomes of Rheum palmatum L., Rheum tanguticum Maxim. ex Balf. or medicinal Rheum officinale Baill, are used to treat intestinal carbuncle and abdominal pain, as well as damp-heat dysentery. Chrysophanein is a natural compound extracted from rhei radix et rhizoma with significant anti-inflammatory properties. AIM:Ulcerative colitis (UC) is characterized by chronic mucosal inflammation. Current therapies alleviate symptoms via anti-inflammatory effects but fail to prevent relapse. Restoring the intestinal mucosal barrier represents a novel therapeutic strategy beyond conventional approaches. This study aims to show that chrysophanein ameliorates UC symptoms by targeting the coiled-coil domain containing protein 25/integrin-linked kinase/hypoxia-inducible factor-1α(CCDC25/ILK/HIF-α) pathway to repair mucosal barrier integrity. METHODS:We established a CCDC25-overexpressing UC mouse model through the intraperitoneal injection of adeno-associated virus (AAV)-CCDC25, alongside providing the mice with free access to dextran sulfate sodium(DSS). Disease progression was evaluated through body weight, survival status, colon length, endoscopy, and histopathology. chrysophanein-treated UC mice were assessed via Alcian blue staining (mucin quantification), WB (Occludin/ZO-1/claudin-1), in vivo imaging (FITC-dextran 4000 distribution), limulus amebocyte lysate assay (serum LPS), bacterial translocation assays, WB (CCDC25/ILK/HIF-1α pathway), and flow cytometry (IL-22+ILC3s). Transmission electron microscopy (TEM) evaluated epithelial morphology, and co-culture experiments examined chrysophanein's protective effects on enterocytes. RESULTS:Compared to control AAV-UC mice, CCDC25-overexpressing mice exhibited exacerbated symptoms. chrysophanein significantly attenuated weight loss, hematochezia, and colon damage, enhanced mucosal barrier function (mucin, ZO-1/Occludin/Claudin-1), reduced bacterial translocation and serum LPS, suppressed CCDC25/ILK/HIF-1α, and elevated IL-22+ILC3 proportions in UC mice. However, CCDC25 overexpression diminished chrysophanein's efficacy, correlating with reduced IL-22+ILC3s. chrysophanein-MNK3-conditioned media increased TEER, lowered FD4 permeability, and upregulated ZO-1 (mimicking IL-22 recombinant protein), whereas OECCDC25-MNK3 media reversed these effects, confirming CCDC25's overexpression interferes with chrysophanein-mediated intestinal epithelial barrier repair via the IL-22 pathway. CONCLUSION:Chrysophanein rescues ILC3-derived IL-22 by blocking CCDC25/ILK/HIF-1α for mucosal healing in UC.

Ethnopharmacological relevance In traditional Chinese medicine, the radices of Glycyrrhiza uralensis Fisch, known as liquorice, have been used for relieving cough, alleviating pain and harmonizing the actions of all medicinals in a formula. Glycyrrhizic acid (GA), a natural compound derived from licorice, exhibits notable anti-inflammatory properties. Aim Neutrophil extracellular trap (NET) generated by peptidylarginine deiminase 4 (PAD4) has been implicated in the progression of colitis to colitis-associated colorectal cancer (CAC). This study aims to investigate whether GA can ameliorate CAC through the inhibition of PAD4 activity and reduction of NET formation. Methods We investigated the correlation between PAD4 expression levels and immune cell infiltration in colorectal cancer utilizing the TIMER database, while also assessing PAD4 levels and activity in human CAC biopsy samples. To evaluate the therapeutic potential of licorice acid on CAC in vivo, we employed the AOM/DSS model and confirmed its inhibitory effects on NET formation in vitro. Furthermore, we explored whether licorice acid can restore immune cell cytotoxicity by diminishing NET formation through fluorescence transfection of CT26 cell lines and subsequent sorting of CD8+ T cells. Additionally, we elucidated the detrimental role of PAD4 in CAC progression using PAD4-/- mice. Results We observed that GA ameliorated colonic inflammation, reduced tumorigenicity, and decreased NET formation, as evidenced by decreased levels of PAD4, citH3, MPO and MMP-9. In vitro experiments demonstrated that GA effectively bound to PAD4 and inhibited its enzyme activity. Furthermore, GA prevented epithelial cell destruction while enhancing CD8+ T-cell-mediated tumor killing through the suppression of NET formation in a coculture system. Conclusions We demonstrate that GA inhibits CAC occurrence by suppressing PAD4 activity and reducing NET formation.

ETHNOPHARMACOLOGICAL RELEVANCE:Ganoderma lucidum spore oil (GLSO) is a well-known health product that is beneficial for immuno-enhancement, which stems from a medicine fungus: Ganoderma lucidum (Curtis) P. Karst. Ganoderma lucidum has been used as tonic in China for more than 2000 years. Modern pharmacological studies have shown that it has effects with immunomodulatory, hypoglycemic, hypolipidemic, anti-oxidation, anti-aging and anti-tumor. AIM OF THE STUDY:The immuno-enhancement ability of GLSO in hepatoma H22-bearing mice was investigated in this study, and our work aimed to reveal the potential mechanisms of the antitumor efficacy of GLSO. MATERIALS AND METHODS:The GLSO components were identified via UHPLC-Q-Orbitrap HRMS. A H22 cell subcutaneously transplanted tumor mouse model was constructed, and GLSO was preadministered. The antitumor efficacy of GLSO in hepatoma H22-bearing mice was evaluated according to tumor size, tumor growth curves, tumor inhibition rates and Ki67 level. T and B lymphocyte proliferation, delayed hypersensitivity, NK cell killing activity, macrophage phagocytotic activity and macrophage polarization, cytokine levels and CD69 molecule expression were detected to estimate immune function. Network pharmacology analysis, flow cytometry and Pro-DIA quantitative proteomics analysis were performed to investigate the potential mechanism of GLSO in tumor inhibition, which was verified by WB and RT-PCR. RESULTS:Thirty-eight compounds including triterpenoids, fatty acids and esters, were identified from GLSO. Mice treated with GLSO showed the smaller initial and final tumor volumes and lower Ki67 expression, GLSO treatment could prevented tumor occurrence and inhibited tumor growth. Treatment with GLSO promoted a strong immune response including macroregulation in immune organs, enhancement of macrophage phagocytosis, NK cell cytotoxicity,T cells and B cells proliferation activity, delayed-type hypersensitivity reaction, reducing the production of M2 macrophages and regulation of cytokine secretion in hepatoma H22-bearing mice. Network pharmacology analysis and flow cytometry results showed that treatment with GLSO might have beneficial effects on improving the tumor immune microenvironment. Proteomics analysis showed that GLSO inhibited eicosanoid metabolism pathway, WB and RT-PCR re-check these results. CONCLUSION:These findings support that GLSO enhances immunity in hepatoma H22-bearing mice and we first report that GLSO restricts tumor growth by inhibiting eicosanoid metabolism pathway.

BACKGROUND:Ulcerative colitis (UC) is a chronic inflammatory disease driven by intestinal immune imbalance. In addition, its pathogenesis includes environment, genetics, microbiota, genes, and diet. Epidemiological and related studies have shown that a high-fat diet habit can promote the progression of UC. The trained immunity induced by a high-fat diet is a key factor in the occurrence and development of chronic inflammatory diseases. As a chronic inflammatory disease, the research on how the trained immunity induced by a high-fat diet promotes the progression of UC is still unclear. Previous studies have shown that Dahuang Mudan Decoction can effectively alleviate the progression of UC, but whether its mechanism of action can also inhibit trained immunity is not clear. METHODS:To explore the promoting effect of trained immunity induced by a high-fat diet on UC, in this study, mice were fed a high-fat diet for 4 weeks and then modeled with 2 % DSS. Subsequently, by transplanting the bone marrow of a high-fat diet, it was repeatedly verified that a high-fat diet can promote the progression of UC through trained immunity. Then, to explore the therapeutic effect of Dahuang Mudan Decoction (DMD) and its mechanism, the above-mentioned model was intervened with DMD (125, 250, 500mg/kg). By transplanting the bone marrow of a high-fat diet and intervening with DMD, it was repeatedly verified that DMD can alleviate UC promoted by a high-fat diet by inhibiting trained immunity. RESULTS:The results of this study showed that the intervention of a high-fat diet promoted the progression of UC, aggravated the disease-related indicators of UC, promoted the damage of the colonic mucosal structure, reduced the expression of colonic mucin tight junction proteins, increased the number of bone marrow hematopoietic stem and progenitor cells (HSPCs), granulocyte-monocyte progenitor cells (GMPs), splenic monocytes, and colonic macrophages; promoted the expression of colonic inflammatory factors (IL-6, TNF-α). After transplanting the bone marrow intervention with a high-fat diet can promote the progression of UC, which is consistent with the trend of the above indicators. After the intervention of DMD, the above indicators were alleviated, and the subsequent transplantation of the bone marrow from a high-fat diet intervened with DMD can effectively alleviate the UC-related symptoms. CONCLUSION:A high-fat diet promotes the progression of UC disease by promoting trained immunity and increasing the proportion of bone marrow HSPCs and GMPs, thereby increasing the proportion of splenic monocytes, the number of colonic tissue macrophages, and the level of inflammatory factors. DMD can alleviate the inflammatory response of UC mice intervened with a high-fat diet by inhibiting bone marrow trained immunity.

The incomplete degradation of tumour cells by macrophages (Mφ) is a contributing factor to tumour progression and metastasis, and the degradation function of Mφ is mediated through phagosomes and lysosomes. In our preliminary experiments, we found that overactivation of NADPH oxidase 2 (NOX2) reduced the ability of Mφ to degrade engulfed tumour cells. Above this, we screened out liquiritin from Glycyrrhiza uralensis Fisch, which can significantly inhibit NOX2 activity and inhibit tumours, to elucidate that suppressing NOX2 can enhance the ability of Mφ to degrade tumour cells. We found that the tumour environment could activate the NOX2 activity in Mφ phagosomes, causing Mφ to produce excessive reactive oxygen species (ROS), thus prohibiting the formation of phagolysosomes before degradation. Conversely, inhibiting NOX2 in Mφ by liquiritin can reduce ROS and promote phagosome-lysosome fusion, therefore improving the enzymatic degradation of tumour cells after phagocytosis, and subsequently promote T cell activity by presenting antigens. We further confirmed that liquiritin down-regulated the expression of the NOX2 specific membrane component protein gp91 phox, blocking its binding to the NOX2 cytoplasmic component proteins p67 phox and p47 phox, thereby inhibiting the activity of NOX2. This study elucidates the specific mechanism by which Mφ cannot degrade tumour cells after phagocytosis, and indicates that liquiritin can promote the ability of Mφ to degrade tumour cells by suppressing NOX2.

Tripartite Motif-Containing 44 (TRIM44) is responsible for cancers, neurodegenerative diseases, and viral infections. However, the role of Siniperca chuatsi TRIM44 (scTRIM44) during viral infection remains unclear. In the present study, we analyzed the molecular characteristics of scTRIM44 and its role in infectious spleen and kidney necrosis virus (ISKNV), largemouth bass virus (LMBV), and Siniperca chuatsi rhabdovirus (SCRV) infection. ScTRIM44 contained one B-box domain (B, 166–207 aa) and a coiled-coil domain (CC, 279–309 aa), but lacked the canonical RING domain of E3 ubiquitin ligases. The scTRIM44 mRNA was expressed relatively high in immune-related tissues. The mRNA expression of scTRIM44 significantly decreased in vivo and vitro post-ISKNV and -LMBV infection. However, the expression of scTRIM44 mRNA showed significant up-regulation post-SCRV infection. ScTRIM44 positively regulated SCRV infection in CPB cells, but copies of ISKNV and LMBV showed no significant alteration in over-expressed or knocked-down scTRIM44 cells. Moreover, scTRIM44 positively regulated RIG-I- and MDA5-mediated interferon molecule signaling. These data suggested that scTRIM44 promoted SCRV infection by positively regulating RIG-I- and MDA5-mediated interferon molecule signaling, but didn’t regulate ISKNV and LMBV infection. This research provided a comprehensive insight into the antiviral activity of scTRIM44.

Inhibiting the death receptor 3 (DR3) signaling pathway in group 3 innate lymphoid cells (ILC3s) presents a promising approach for promoting mucosal repair in individuals with ulcerative colitis (UC). Paeoniflorin, a prominent component of Paeonia lactiflora Pall., has demonstrated the ability to restore barrier function in UC mice, but the precise mechanism remains unclear. In this study, we aimed to delve into whether paeoniflorin may promote intestinal mucosal repair in chronic colitis by inhibiting DR3 signaling in ILC3s. C57BL/6 mice were subjected to random allocation into 7 distinct groups, namely the control group, the 2% dextran sodium sulfate (DSS) group, the paeoniflorin groups (25, 50, and 100 mg/kg), the anti-tumor necrosis factor-like ligand 1A (anti-TL1A) antibody group, and the IgG group. We detected the expression of DR3 signaling pathway proteins and the proportion of ILC3s in the mouse colon using western blot and flow cytometry, respectively. Meanwhile, DR3-overexpressing MNK-3 cells and 2% DSS-induced Rag1-/- mice were used for verification. The results showed that paeoniflorin alleviated DSS-induced chronic colitis and repaired the intestinal mucosal barrier. Simultaneously, paeoniflorin inhibited the DR3 signaling pathway in ILC3s and regulated the content of cytokines (Interleukin-17A, Granulocyte-macrophage colony stimulating factor, and Interleukin-22). Alternatively, paeoniflorin directly inhibited the DR3 signaling pathway in ILC3s to repair mucosal damage independently of the adaptive immune system. We additionally confirmed that paeoniflorin-conditioned medium (CM) restored the expression of tight junctions in Caco-2 cells via coculture. In conclusion, paeoniflorin ameliorates chronic colitis by enhancing the intestinal barrier in an ILC3-dependent manner, and its mechanism is associated with the inhibition of the DR3 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE:Ulcerative colitis (UC) is one of non-specific inflammatory bowel disease that mainly affects the colon. Recently, UC has become a significant social and economic problem worldwide. Baitouweng decoction (BD), a traditional Chinese medicine described in the "Treatise on Febrile Diseases", has been used for centuries to treat intestinal diseases. However, its underlying mechanism remains largely unexplored.AIM OF STUDY:In this study, we aimed to investigate the effect of BD on autophagy for repairing the colonic barrier in DSS-induced colitis mice and explored its role in regulating the autophagic signaling pathway AMPK/mTOR.MATERIALS AND METHODS:Mice with colitis were treated with 3% dextran sulfate sodium (DSS) for 7 days. The effectiveness of BD in treating DSS-induced colitis was evaluated through body weight, disease activity index (DAI), colon length, pathological changes, organ index, and proportion of blood cells. Moreover, intestinal epithelial permeability was analyzed by examining FITC-dextran leakage, the bacterial load of mesenteric lymph nodes (MLNs), and bacterial infiltration of colon tissues. Barrier function was evaluated by assessing the number and proportion of colonic goblet cells and the expression of tight junction proteins, including ZO-1, claudin-1, and occludin. Furthermore, the levels of autophagy were assessed by examining the number of autophagosomes and the expression of the autophagy-related proteins LC3, Beclin1, and P62. Additionally, network pharmacology research was conducted to analyze the potential mechanisms underlying the medicinal effects, as indicated by the role of AMPK/mTOR in regulating the autophagic signaling pathway.RESULTS:BD improved colitis symptoms in mice by restoring body weight and colon length and reducing inflammatory cell infiltration. Additionally, BD decreased the diffusion of FITC-dextran and bacterial translocation in MLNs, as well as bacterial infiltration of the colonic mucosa. The number and proportion of colonic goblet cells, the expression of ZO-1, Claudin-1, and Occludin, and the levels of autophagy were also increased by BD. Network pharmacology analysis suggested that BD might affect intestinal autophagy through the AMPK signaling pathway, which was confirmed by the activation of AMPK phosphorylation and the downregulation of mTOR expression following BD treatment.CONCLUSION:Our study demonstrated that BD repaired the intestinal epithelial barrier in DSS-induced colitis mice by activating AMPK phosphorylation and inhibiting mTOR expression to promote autophagy.