Live biotherapeutic products (LBPs) represent a distinct category of biological products containing viable organisms, such as bacteria, utilized for the prevention and treatment of human diseases (excluding vaccines). Presently, research and development efforts in LBPs are predominantly centered on live bacteria. Compared to traditional drugs, the LBPs demonstrate unique characteristics, including replicability, target specificity, and responsiveness. Owing to these properties, LBPs have emerged as hotspots in the development of specialized treatments for various major diseases, with applications spanning malignant tumors, metabolic disorders, inflammatory bowel diseases, genetic defects, and more. Nevertheless, natural bacteria face inherent limitations—such as low activity, instability, and safety concerns—that hinder their pharmacological potential. As a result, engineering strategies have become essential for enhancing the properties of bacteria and facilitating their clinical applications. This article delves into recent advancements in LBPs derived from engineered bacteria, offering a systematic review of reported engineering strategies, which are broadly categorized into chemical, physical, and genetic modifications. The findings indicate that no single engineering approach can comprehensively address all the challenges associated with converting viable bacteria into effective LBPs. To overcome this limitation, a concept of "multi-engineered bacteria" is introduced. This framework advocates for the integration of physical, chemical, and biological engineering strategies to develop next-generation LBPs with enhanced functionality and clinical potential. This article provides a concise review of current research on LBPs based on engineered bacteria and outlines forward-looking perspectives for advancing their development through innovative engineering approaches.

Bacterial infectious diseases persistently pose severe threats to human health, development of livestock and aquaculture industries, and ecological stability. The extensive use of conventional antibiotics has led to increasingly critical issues of bacterial resistance, making the development of novel and effective strategies for preventing and treating bacterial infections an urgent priority. Bdellovibrio bacteriovorus, as a genus of parasitic bacteria that prey on other bacteria, exhibits lytic activity against various pathogenic species and demonstrates potential for combating bacterial infections. However, the direct application of B. bacteriovorus suspensions or powders faces challenges including rapid clearance, susceptibility to immune system elimination, difficulty in maintaining their vitality, and poor user compliance. Recent advancements in engineered B. bacteriovorus technology have created new opportunities for more precise and efficient utilization of these predators in infection control. This paper reviews recent advances in engineered B. bacteriovorus for bacterial infection control, with particular emphasis on engineering strategies based on formulation design, surface modification, and genetic editing, along with their therapeutic applications. The review aims to provide valuable insights for advancing research on engineered B. bacteriovorus technologies.

Attenuated Salmonella typhimurium VNP20009 is a novel oncolytic bacterium with high tumor-targeting properties. One of its anti-tumor mechanisms is the induction of tumor cell apoptosis, although the specific molecular mechanisms remain unclear. Melanoma, the deadliest form of skin cancer, is associated with significant challenges, such as severe side effects and high recurrence rates in current treatments. This study used the B16F10 mouse melanoma cell line as a model to explore the regulatory mechanism of VNP20009-induced apoptosis in melanoma cells. The results showed that VNP20009 significantly induced apoptosis in B16F10 cells in a time- and concentration-dependent manner. Transcriptomic analysis revealed that the p53 signaling pathway was significantly enriched in the VNP20009-treated group, suggesting that this pathway might mediate the pro-apoptotic effects of VNP20009. Further investigations demonstrated that VNP20009 induces apoptosis by activating key genes in the p53 pathway, including PUMA, and its upstream and downstream molecules, such as p53, CytC, CASP9, and CASP3, forming a cascade reaction. In conclusion, this study elucidates the molecular mechanism by which VNP20009 induces apoptosis in B16F10 melanoma cells through the p53-PUMA axis, providing new theoretical insights for melanoma treatment based on attenuated Salmonella bacteria.

Oral probiotics are susceptible to the gastrointestinal environment, so the number of probiotics reaching the intestine is small and difficult to colonize, limiting the application of probiotic therapy. In this study, Lactobacillus rhamnosus (LGG), a common probiotic, was chosen as a model, and layer-by-layer encapsulated LGG-loaded porous microspheres with glycol chitosan (GCS) and sodium alginate (SA) were prepared to investigate it's in vitro properties. Poly-L-lactic acid porous microspheres (PLPM) were prepared by the complex milk-solvent evaporation method, with rounded morphology, uniform size, open and connected porous structure, and the average particle size of 138.5 μm. The PLPM were co-incubated with LGG for 8 h at 37 ℃ to obtain the LGG-loaded porous microspheres (LPM) with high bacterial loadings. The surface of the LPM were wrapped with GCS and SA layer by layer by electrostatic action to obtain the layer-by-layer encapsulated LGG-loaded porous microspheres with GCS and SA (AGLPM). In vitro experiments demonstrated that AGLPM could tolerate simulated gastric fluid at pH 1.2 and simulated intestinal fluid at pH 7.4 for 2 h, and its stability was significantly better than that of bare LGG. AGLPM was a better probiotic dosage form.

Radiation-induced intestinal injury (RIII) is a common syndrome in the radiotherapy of abdominal and pelvic malignant tumors, heavily influencing the living quality, but no specific clinical regimens are available. Inulin is a naturally soluble dietary fiber. Clostridium butyricum (Cb) is a strict Gram-positive anaerobic bacillus, which can secrete and produce a large number of butyric acid, improving the intestinal barrier function and reducing the growth and colonization of opportunistic bacteria. A synbiotic of inulin gels (IG) and Cb exerts the synergistic effect of prebiotic intestinal retention and probiotics. In this study, an oral synbiotic of IG and Cb was applied for the prevention and treatment of RIII and the mechanisms were explored. Animal experiments were approved by the Ethics Committee of the Academy of Military Medical Sciences and the experiments were conducted in accordance with relevant guidelines and regulations (authorizing number: IACUC-DWZX-2022-525). An RIII mouse model was established after whole abdominal γ-ray radiation of 13 Gy. The synbiotic was intragastrically administered to the mice 1 h pre-radiation. Compared to the models, the mice of the synbiotic group had more regenerated intestinal crypts, longer villus and colon, and more tight junction protein on day 3.5. Moreover, the mice of the synbiotic group had an obvious increase in the relative abundance of gut microbiota on day 7, especially the amounts of multiple probiotics and short-chain fatty acid metabolites. On day 14, the mice of the synbiotic group had highly low permeability of intestinal mucosa according to the fluorescence labeling experiment, which was close to the normal level. Moreover, the mice of the synbiotic group showed a high decrease of proinflammatory factors including tumor necrosis factor-α and interleukin-6, close to the normal levels. Therefore, the oral synbiotic can alleviate the syndromes of RIII and improve the repair of damaged intestinal and colon tissues, and its protective effect is better than IG and Cb. The oral synbiotic is a safe and effective biological drug against RIII.

Radiation enteritis (RE) is the most common complication during radiotherapy, although safe and effective drugs for the treatment of RE are deficient. Probiotics have been demonstrated to own antiradiation function. Synbiotics are composed of probiotics and prebiotics, which enhance the ability of probiotics. Here, inulin gel (IG) that own the ability to resist gastric acid and retain in the colon were used to load three types of probiotics, including Bacillus cereus (BC), Bacillus licheniformis (BL), and Lactobacillus reuteri (LR), respectively. The RE pharmacodynamic studies of them were conducted. The probiotics were embedded in the IG by scanning electron microscopy and confocal laser scanning microscopy. IG promoted the growth of probiotics compared to probiotics alone. Animal experiments were approved by the Ethics Committee of the Academy of Military Medical Sciences and the experiments were conducted in accordance with relevant guidelines and regulations (No. IACUC-DWZX-2024-P510). Mice experienced the whole abdominal irradiation of 13 Gy γ rays to form RE models. Compared to correspondingly probiotics alone, all probiotic-loaded IG synbiotics had the better prevention and treatment efficiencies in repairing the intestinal barriers, improving the structure of crypts, oxidative stress, inflammation, and imbalanced gut microbiota and promoting the recovery of intestinal villus, where the Bacillus cereus-loaded IG was the best. This study provides a novel therapeutic for the prevention and treatment of RE.

Whole body irradiation (WBI) injury is defined multi-organ damages caused by whole-body exposure to ionizing radiation. The traditional radioprotective drug, amifostine, has significant adverse effects. Probiotics are reported to have radioprotective function, although their therapeutic efficacy is low due to poor gastrointestinal tolerance and the insufficient retention and colonization in the colon. In this study, chitosan/tannic acid double-layer-coated Lactobacillus reuteri was prepared, which was encapsulated in calcium alginate hydrogel microspheres to get an engineered probiotic-loaded microsphere formulation. The bilayer coating was confirmed by twice inversions of zeta potentials. Moreover, the coating improved bacterial adhesion and aggregation. Optical microscopy revealed the smooth morphology of microspheres, laser confocal imaging showed the uniform distribution of coated bacteria in microspheres, and scanning electron microscopy exhibited pores in the surface. The microspheres exhibited in vitro gastrointestinal resistance with rapidly swelling in the colonic environment to release bacteria. All the animal experiments were approved by Academy of Military Medicine Sciences (Approval No: IACUC-DWZX-2024-P510) and conducted in compliance with relevant guidelines. The 6.5 Gy whole-body irradiated mouse model was established. Starting from 2 days prior to irradiation, probiotic-loaded microspheres were administered via oral gavage consecutively for 6 days. Compared with the model group, the bacteria-loaded microspheres demonstrated protective effects on the hematopoietic system by promoting the recovery of red blood cells and platelets, maintaining the morphology of splenic red pulp and white pulp, and preserving bone marrow nucleated cells along with their proliferative capacity. Engineered probiotics have expanded the spectrum of radioprotective drugs, offering novel insights for the development of live biotherapeutic products aimed at preventing and treating radiation-induced injury.

High-altitude sleep disturbance is a common acute high-altitude disease that can trigger physiological discomfort such as acute high-altitude reactions, with a lack of safe and effective preventive medications in clinical practice. Based on the gut-brain axis theory, this study designed and prepared a synbiotics combining Lactobacillus rhamnosus (LGG)-Lycium barbarum polysaccharide (LBP). First, the LGG-LBP synbiotics was prepared and evaluated. The mice were randomly divided into healthy, model, positive control (acetazolamide), LBP, LGG and LGG-LBP synbiotics group. After 7 days of administration, the mouse model of high-altitude sleep disturbance was established, the treating effects were evaluated through sleep duration, behavioral, hemogram test, and the content of tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) in blood. 16S rRNA sequencing was used to analyze the changes of gut microbiota, and the pathological changes of small intestine were observed. The LGG-LBP synbiotics prolonged sleep duration, improved exploratory ability and short-term memory, promoted blood cell recovery. Moreover, LGG-LBP synbiotics enhanced the abundance of probiotics in the gut, and reduced intestinal inflammation. LGG-LBP synbiotics may be a potential prophylactic drug for high-altitude sleep disturbance. The animal operation was approved by the Ethics Committee of the Academy of Military Medical Sciences, Academy of Military Science (Approval number: IACUC-DWZX-2022-511). All experiments were conducted in accordance with relevant guidelines and regulations.

Combined radiation and burn injury (CRBI) is induced by simultaneous or sequential ionizing radiation damage and skin burns. CRBI weakens the immune ability, leading to drug-resistant bacterial infections and delayed wound healing. Bdellovibrio-and-like organisms (BALO) are naturally predatory bacterium that can prey on most Gram-negative bacteria by entering the periplasmic space of their prey and degrading the biomolecules of host cells. In this study, we combined gelatin, calcium alginate, and activated BALO water samples to form bio-inks to three-dimensional (3D)-print BALO-loaded hydrogels (TDBG) for the treatment of CRBI combined with multidrug-resistant Acinetobacter baumannii (MRAB) infection. The freeze-dried 3D-printed hydrogel exhibited a 3D network structure attached with gelatin films, and owned good printability and biocompatibility. The printability improved adaptation to wound shapes for the personalized treatment of infected wounds. The 3D network structure allowed the surviving and motion of BALO, favoring its high predatory activity. All animal experiments were approved by the Ethics Committee of Academy of Military Medical Sciences, and the experiments were conducted in accordance with relevant guidelines and regulations (approval number: IACUC-DWZX-2022-834). TDBG treatment improved wound healing by accelerating the mouse wound closure rate of CRBI combined with MRAB infection, reducing the expression of pro-inflammatory cytokines in the wound tissues, and increasing collagen deposition. This study expands the application scope of live biological products and provides a basis for their development and clinical applications.

Current physical and chemical dosimeters are limited in that they cannot directly measure the biological effects of radiation or detect it within the body. Biosensors based on engineered probiotics demonstrate high stability and safety, can be used to detect ionizing radiation in vivo. In this study, an oral engineered microbial sensor for ionizing radiation detection has been developed. The Escherichia coli Nissle 1917 (EcN) was selected as the chassis strain. Using CRISPR/Cas9 gene-editing technology, the cryptic plasmids of EcN were successfully removed to yield the chassis strain ΔEcN. To design a radiation-responsive gene circuit, the recA promoter from the SOS response pathway was utilized as the radiation response element, while a fluorescent protein served as the reporter element. This system was designed to be induced by mitomycin C and γ-rays. The performance of engineered bacteria with various gene circuits was characterized and optimized, resulting in the selection of improved candidate strains. Animal experiments were approved by the Ethics Committee of Academy of Military Medical Sciences and the experiments were conducted in accordance with relevant guidelines and regulations (approval number: IACUC-DWZX-2022-521). After comparing the in vivo radiation detection capabilities of these strains, the EC-8, which exhibited higher sensitivity, was identified as the final oral microbial sensor. This research applies synthetic biology principles to design and engineer a probiotic capable of detecting ionizing radiation within the body. The findings offer a novel method for in vivo ionizing radiation detection and lay the foundation for the development of live biotherapeutics for the precise diagnosis of radiation damage.

Human and animal health will be seriously harmed by myocardial infarction, the diagnostic speed and the therapeutic effect of this disease need to be improved urgently. As the natural carrier for delivering cell information, some microRNAs (miRNAs) found in exosomes can reflect and act on the pathological changes caused by myocardial infarction for effective diagnosis and treatment. The feasibility of exosomal miRNAs (e.g. miR-4516, miR-203, and miR-1915-3p) from different sources as diagnostic agents for myocardial infarction, as well as the research progresses in relief of cell death via apoptosis (e.g. miR-21a-5p, miR-30e, and miR-210), autophagy (e.g. miR-125b-5p, miR-301, and miR-143-3p), pyroptosis (e.g. miR-182-5p, miR-133a, and miR-100-5p), and ferroptosis (e.g. miR-26b-5p and miR-23a-3p), promotion of forming new blood vessels (e.g. miR-29b-3p, miR-210-3p, and miR-494-3p), and inhibition of inflammatory response (e.g. miR-25-3p, miR-182-5p, and miR-671) for intervention therapy of myocardial infarction were reviewed here to provide new strategies for the diagnosis and treatment of myocardial infarction.

Depression disorder is a prevalent psychiatric disorder characterized by high incidence, recurrence and disability rates, which imposes significant social and economic burdens globally. The etiology of depression is still unclear, and treatment options remain limited. Various animal models have been developed for screening and evaluation of antidepressants. This paper reviewed the current trends in depression research and typical non-clinical animal models, summarized the important issues in standardized non-clinical research of depression disorders and proposed criteria for the selection of appropriate R&D models.

Lymphocyte activation gene 3 (LAG-3) is an important inhibitory receptor on T cells, which plays a crucial role in tumor immune evasion. LAG-3 is primarily expressed on activated T cells, natural killer (NK) cells and B cells, et al. By binding to its ligands, LAG-3 inhibits T cell proliferation, activation, and effector functions. LAG-3 has emerged as the third immune checkpoint protein (ICP) used in clinical practice, following programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). Currently, there has been at least 20 LAG-3-targeted drugs undergoing clinical trials. This article mainly reviews the structure, expression regulation, ligands, co-expressed ICP of LAG-3, as well as its application in tumor immunotherapy, and discusses the current challenges of targeting LAG-3 research.

Neurodegenerative diseases are one of the leading causes of death and disability worldwide, with complex pathogenesis and lacking effective therapeutic drugs. Increasing researches have shown that most neurodegenerative diseases involve abnormalities in iron homeostasis and activation of immune cells in the brain. Iron accumulation in microglia promotes ferroptosis, leading to cellular dysfunction and death. In contrast, inhibiting ferroptosis can alleviate neuroinflammation, protect neurons, and slow disease progression, highlighting the pivotal roles of ferroptosis and neuroinflammation in neurodegenerative diseases. This review summarizes the roles of ferroptosis and neuroinflammation in neurodegenerative diseases, further discusses the related targets regulating these processes, and reviews the therapeutic potential of drugs targeting ferroptosis and neuroinflammation in neurodegenerative diseases. This review aims to provide novel targets and therapeutic drugs for the treatment of neurodegenerative disease, offering new strategies for clinical management and improving the symptoms and prognosis of neurodegenerative disease.

Pulmonary fibrosis (PF) is a lung disease with a very poor prognosis that seriously affects the quality of life of patients and is characterized by scarring and thickening of the tissue surrounding the alveolar walls, ultimately leading to respiratory failure. Currently, the Food and Drug Administration (FDA) approved drugs for the treatment of PF include pirfenidone and nidazanib, however, these two drugs can only delay the progression of the disease but cannot achieve the reversal of PF, and their clinical application is limited due to high price and multiple adverse effects. The pathogenesis of PF has not been fully elucidated, and studies have demonstrated that aberrant immune cell activation and regulation play an important role in PF. This review aims to discuss the role of immune cell activation and regulation in PF in recent years. The aim of this review is to discuss recent advances in the study of the role of immune cells in the process of PF, with the aim of providing theoretical guidance for the development of novel immunotherapies.

Tigliane-type diterpenoids are the main active components of croton and a diverse group of macrocyclic diterpenes with diverse biological activities. Their anti-HIV activity have unique advantages and great potential in the treatment of acquired immunodeficiency syndrome (AIDS), and were expected to be developed as a candidate drug for clinical trials of anti-AIDS. This article reviews the research progress on the components of tigliane-type diterpenoids in croton and their anti-HIV activity in recent years, conducts systematic comparisons, summarizes the currently discovered tigliane-type diterpenoids in croton. Their chemical structures and biological activity relationship were summarized. The research progress on the mechanism of tigliane-type diterpenoids' anti-HIV activity was briefly discussed. This research could provide guidance for further development and utilization of coumarin type diterpenes, as well as new ideas for the application of croton.

With the increasing abuse of antibiotics and the growing resistance of bacteria, it is urgent to find new antibacterial agents. Numerous constituents of traditional Chinese medicine exhibit pronounced antibacterial, anti-inflammatory, and antioxidant pharmacological properties, often operating through multiple mechanisms, thereby positioning them as a vital source for the development of novel antibacterial agents in the future. Nevertheless, the antibacterial constituents of traditional Chinese medicine exhibit challenges such as inadequate stability, low solubility, and suboptimal intelligent release capabilities, which hinder their extensive application in antibacterial formulations. Metal-organic framework materials serve as highly effective drug carriers for antibacterial constituents of traditional Chinese medicine, attributed to their high specific surface area, elevated porosity, controllable pore dimensions, and responsive release properties. Furthermore, they not only enhance the stability and solubility of these antibacterial constituents while also exhibiting inherent antibacterial activity and responsive release capabilities. This paper presents a comprehensive overview of bacterial resistance mechanisms and the action pathways of antibacterial constituents of traditional Chinese medicine against resistant bacteria. Additionally, it highlights the current advancements in metal-organic framework materials and their application in the delivery systems for these antibacterial constituents, aiming to provide valuable insights for the research and innovation of formulations based on traditional Chinese medicine.

Tissue factor (TF), a transmembrane glycoprotein expressed in normal tissues, has a variety of physiological functions in embryonic development, hemostasis and non hemostasis pathways. Studies have found that TF is overexpressed in a variety of tumor tissues and promotes tumor progression. Kaplan Meier (K-M) survival analysis showed that high expression of TF gene was associated with poor prognosis in renal and pancreatic cancer. Therefore, TF has received extensive attention as a target of tumor immunotherapy, and a number of antibody-drug conjugates (ADC) drugs have entered the clinical research stage. In this paper, the gene structure, expression, biological function and the correlation with tumor of TF were systematically elaborated, and the direction of drug design for the new generation of TF-ADC was proposed, in order to provide theoretical support and development direction for the drug research and development of this target.

In recent years, a large number of peptide compounds have been obtained from natural sources or synthesized chemically, which have attracted significant interest due to their high biological activity and low side effects. However, linear peptides encounter many challenges in the field of drug development because they are easily broken down by enzymes and do not pass through cell membranes well. Cyclic peptides, on the other hand, have a stable structure, strong binding to targets, and lower toxicity. They combine the advantages of natural peptides and small molecule drugs in terms of biological activity and drug metabolism, addressing the shortcomings of linear peptides and becoming increasingly important in drug research. This article focuses on the development history of cyclic peptides, discusses the sources, acquisition methods, and specific applications in the field of pharmacology in recent years, and prospects for their future development potential, aiming to provide a theoretical and practical basis for the clinical application of cyclic peptides.

Nutlin-3 is a representative small molecule MDM2-p53 antagonist, which can stabilize the p53 state by disrupting the interaction between p53 and MDM2, thereby inducing the p53 signaling pathway to exert antitumor effects. In this study, six wild-type p53 tumor cell lines, HCT-116, H460, HepG2, MCF-7, A549 and SJSA-1, were used as research objects, and the effects of nutlin-3 on the proliferation of six wild-type p53 cancer cells were detected by methyl thiazolyl tetrazolium (MTT) method and plate cloning assay. The effects of nutlin-3 on H460 cell cycle and apoptosis were detected by flow cytometry. Western blot assay was used to detect ubiquitin-specific protease 7 (USP7), death domain-associated protein (DAXX), murine double minute 2 (MDM2), murine double minute 4 (MDMX/ MDM4), p53, to explore the anti-tumor mechanism of nutlin-3; co-immunoprecipitation (Co-IP) assay was used to detect the effect of nutlin-3 on the interaction between MDM2, MDMX and p53. The results showed that nutlin-3 inhibited the proliferation of H460 in a time- and concentration-dependent manner. The results of cell cycle and apoptosis showed that nutlin-3 could block the H460 cell cycle in the G0/G1 phase, and induce apoptosis by activating cleaved-PARP. Western blot results showed that nutlin-3 could up-regulate the expression of USP7, DAXX, MDM2, MDMX and p53 in H460 cells. Co-IP results showed that nutlin-3 inhibited the protein interactions between MDM2 and p53 and MDM2 and MDMX. In conclusion, nutlin-3 can significantly inhibit the proliferation of wild-type p53 cancer cells and induce cell cycle arrest and apoptosis, which may be related to the disruption of MDM2/MDMX's interaction with p53 to activate the MDM2-p53 signaling pathway.

In the early stage of the project, it was found that natural pentacyclic triterpenes liquidambaric acid regulates the NEDD8 modification of Cullin2. This study aims to find more triterpenoid natural active molecules targeting Cullin family members and reveal its mechanism of action. Western blot was used to detect natural products that can significantly change the total protein NEDD8 modification and specific Cullin protein NEDD8 modification in cells; microscale thermophoresis (MST) was used to detect the direct binding of candidate small molecule oleanonic acid to TRAF family proteins, and the binding at the level of living cells was verified by cellular thermal shift assay (CETSA). Proximity ligation assay (PLA) was used to investigate the regulatory effect of oleanonic acid on the protein interaction between TNF receptor-associated factor 1 (TRAF1) and Cullin1 NEDD8 modified complex. Three pentacyclic triterpenoids were found to significantly inhibit NEDD8 modification in cells, among which oleanonic acid had the strongest effect on blocking NEDD8 modification. Different from the previous identification that liquidambaric acid regulates Cullin2/5, oleanonic acid can also specifically induce NEDD8-modified Cullin1 to transform into its unmodified form. And binding experiments showed that oleanonic acid could directly bind to TRAF1 at the level of cell lysate and living cells. Further mechanism studies found that oleanonic acid significantly changed the protein interaction between TRAF1 and Cullin1 NEDD8 modified complex. The above results indicate that oleanonic acid targets TRAF1 and regulates its interaction with NEDD8 modification complex to inhibit NEDD8 modification of Cullin.

2',4'-Dimethoxychalcone (DMC) is a structural modifier of carvacrol B. In this study, gastric cancer cells MGC-803 and HGC-27 were used as the subjects to investigate the anti-tumor effect and mechanism of DMC on gastric cancer (GC) cells both in vitro and in vivo. DMC inhibited cell viability and cell proliferation and promoted cell apoptosis in GC cells, detected by CCK-8 assay, EdU staining and Annexin V-FITC/PI double-staining flow cytometry. A nude mouse model of GC cell xenograft was constructed by subcutaneous injection with MGC-803 cells, for measuring the effect of DMC on the growth of GC in vivo, and DMC inhibited the growth of subcutaneous transplantation tumor in nude mice. The animal experiments were approved by the Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine under the ethical number PZSHUTCM2310110002. The effect of DMC on the RNA expression of MGC-803 cells was detected by RNA-seq assay, and it was found that the biological function of DMC was enriched in glycolysis. DMC inhibited the glucose uptake capacity and lactate production and efflux of gastric cancer cells, detected by using 2-NBDG probe with flow cytometry and lactate (LD) test kit. Western blot assay was performed to detect the protein expression of proliferation, apoptosis, and glycolysis-related proteins in gastric cancer cells, and the results demonstrated that DMC up-regulated the protein expression of cleaved caspase-9, cleaved caspase-3, cleaved PARP, down-regulated Ki-67 protein expression, and inhibited the protein expression of c-Myc, LDHA, GLUT3, PDHK1 and MCT1 in gastric cancer cells. The Seahorse energy metabolism analyser was used to measure the rate of glycolysis, and it was found that DMC could down-regulate the basal glycolysis rate and compensatory glycolysis in gastric cancer cells. The c-Myc overexpressing cell line MGC-803 was used in the reversal experiment to further confirm that DMC suppressed gastric cancer growth through inhibiting c-Myc mediated glucose uptake and glycolysis. In conclusion, DMC may inhibit the protein expression of c-Myc and its target glycolysis-related genes, suppressed c-Myc-mediated glucose uptake and glycolysis in gastric cancer cells, thereby inhibited the cell proliferation and promoted cell apoptosis of gastric cancer cells, and thus finally inhibited the growth of gastric cancer in vivo and in vitro.

Oxaliplatin (Oxa) is a chemotherapy drug commonly used for advanced colorectal cancer, however most patients develop resistance after treatment while the mechanisms of which have not been fully elucidated. In this study, oxaliplatin resistant cell lines were constructed from human colorectal cancer HCT116 cells through concentration gradient induction. On this basis, we investigated the expression profiling of HCT116/Oxa cells based on quantitative proteomics. Gene ontology (GO) analysis was conducted via The Database for Annotation, Visualization, and Integrated Discovery Database (DAVID), and pathway enrichment analysis was done using GeneAnalytics database. The potential targets and molecular mechanisms of oxaliplatin resistance in colorectal cancer were further studied by inhibitors, Western blot and siRNA. The results showed that the oxaliplatin resistance index of HCT116/Oxa cells was 10.2. HCT116/Oxa cells demonstrated stronger proliferation potential and anti-apoptotic capacity to oxaliplatin compared with HCT116 cells. Proteomic data demonstrated significant expression change of 717 genes in HCT116/Oxa cells, among which 399 genes were up-regulated while 318 ones down-regulated comparing with HCT116 cells. GO enrichment analysis showed that differentially expressed genes were mainly related to biological processes such as oxidative stress response, iron metabolism, lipid metabolism, apoptosis and cell cycle progression. Pathway analysis displayed notable changes of cell metabolism, ferroptosis, Nrf2-ARE signaling, fatty acid and glutathione metabolism in HCT116/Oxa cells. Quantitative results indicated that the expression of proteins directly related to ferroptosis, including glutathione peroxidase 4 (GPX4), glutamate-cysteine ligase regulatory subunit (GCLM), ferritin light chain (FTL), ferritin heavy chain (FTH1), heme oxygenase 1 (HMOX1), glutathione reductase (GSR) and NADH dehydrogenase 1 (NQO1) increased, while long chain fatty acid-CoA ligase (ACSL) 4 and ACSL1 decreased significantly in HCT116/Oxa cells. Functional studies showed that RSL3, a specific inhibitor of GPX4, decreased the viability of drug-resistant cells, improved lipid peroxidation, increased the concentration of ferrous ions, malondialdehyde, and decreased the concentration of glutathione (GSH). Western blot showed that the expressions of GPX4, FTH1, FTL and GSR increased in HCT116/Oxa, while ACSL4 decreased. RSL3 reversed the levels of GPX4, FTH1, FTL, GSR and ACSL4. It was further found that knockdown of GPX4 decreased the viability of drug-resistant cells, increased lipid peroxidation levels and decreased GSH concentration. These results suggest that ferroptosis resistance mediated by GSH/GPX4 pathway may be a potential mechanism of oxaliplatin resistance in HCT116/Oxa, and inhibition of GSH/GPX4 signaling could be an effective approach to reverse oxaliplatin resistance in colorectal cancer.

Polydatin (PD) is a natural active crystalline compound extracted from the roots and stems of Polygonum cuspidatum, and is a natural precursor of resveratrol. This study aims to investigate the therapeutic effects of PD on monosodium urate (MSU)-induced gouty arthritis in mice and its potential mechanisms. The animal experiment has been approved by the Ethics Committee of Nanjing University (approval number: 2407002). A gouty arthritis model was established by injecting 20 μL of MSU (25 mg·mL^-1) suspension into the mouse plantar. The effect of PD on pathological changes in the mouse plantar was evaluated. The treatment group received daily intraperitoneal injections of different doses of PD (low dose: 5 mg·kg^-1, medium dose: 10 mg·kg^-1, high dose: 20 mg·kg^-1) for 3 days before model induction. The thickness of the mouse plantar was measured and photographed at 3, 6, 9, 12, and 24 h after MSU suspension injection. Histopathological damage to the plantar tissue was observed using hematoxylin-eosin (H&E) staining. Immunohistochemistry and immunofluorescence were used to detect the expression of NLRP3 and CASP1 p20 to assess NLRP3 inflammasome activation in the plantar tissue. At the cellular level, lipopolysaccharide (LPS) combined with adenosine triphosphate (ATP)/MSU/nigericin was used to construct a cellular activation model of the NLRP3 inflammasome. ELISA was used to detect the effect of PD on interleukin-1β (IL-1β) secretion after NLRP3 inflammasome activation in macrophages. Flow cytometry was employed to measure CASP1 p20 activation in macrophages. Immunofluorescence was used to examine NLRP3 inflammasome assembly in macrophages. The results of the study indicate that, compared to the model group, the PD-treated group exhibited a significant reduction in the swelling of the mouse plantar. H&E staining showed a notable reduction in tissue damage in the mouse plantar, suggesting that PD has a therapeutic effect on plantar damage in mice. Immunohistochemistry and immunofluorescence results revealed a significant decrease in the expression of CASP1 p20 and NLRP3, indicating that PD significantly inhibits the activation of the NLRP3 inflammasome, thereby attenuating the local inflammatory response in the mouse plantar. At the cellular level, PD treatment significantly reduced the secretion of IL-1β and activation of CASP1 p20, both of which are mediated by NLRP3 inflammasome activation. Furthermore, NLRP3 inflammasome assembly was inhibited. In summary, PD exerts its anti-inflammatory effect by suppressing the assembly and activation of the NLRP3 inflammasome, reducing the production and release of the pro-inflammatory cytokine IL-1β, thereby alleviating joint damage in mouse gouty arthritis. This provides a novel strategy for the treatment of gout.

Liver fibrosis is a chronic liver injury caused by various pathogenic factors, leading to excessive accumulation of extracellular matrix such as collagen. It represents a common pathological hallmark during the progression of most chronic liver diseases. However, there is currently no universally recognized specific and effective drug for the clinical treatment of liver fibrosis. Therefore, this study investigates the effects of Alisma Rhizoma on bile duct ligation (BDL)-induced liver fibrosis and explores the potential pharmacological mechanisms. The animal experimental protocol was reviewed and approved by the Animal Welfare and Ethics Committee of Shanghai University of Traditional Chinese Medicine (registration No. PZSHUTCM2303280007), in compliance with relevant animal welfare and ethical standards. Mice were subjected to BDL to induce liver fibrosis. Mice were divided into five groups: sham operation group (Sham), model group (BDL), ethanol extract protection group (BDL+EE, 1.6 g·kg^-1), water extract protection group (BDL+WE, 4.0 g·kg^-1), obeticholic acid protection group (BDL+OCA, 10 mg·kg^-1). The results showed that both of EE and WE could attenuate BDL-induced liver fibrosis as evident by reduced serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyltransferase (GGT) activities, total bile acids (TBA) levels, and improved pathological conditions such as cholestasis, collagen deposition, inflammatory cell infiltration, and liver tissue necrosis. Notably, EE showed better efficacy than WE. Further studies showed that EE improved liver fibrosis dose dependently. EE treatment impaired the bile acids homeostasis in serum and liver, and recovered the hepatic mRNA expression of farnesoid X receptor (FXR) as well as the downstream genes including small heterodimer partner (SHP), cholesterol 7-alpha hydroxylase (CYP7A1) and bile salt export pump (BSEP). Further study also proved that the four major triterpenes in EE increased the transcriptional activities of FXR in vitro. This study provides a theoretical basis for the clinical application of Alisma Rhizoma in the prevention and treatment of liver fibrosis.

Fusobacterium nucleatum (Fn) is closely associated with the occurrence and progression of colorectal cancer (CRC). The development of specific antibacterial agents targeting Fn is crucial for the prevention and treatment of CRC. Based on the preliminary phenotypic screening results from our research group, dimetridazole was successfully identified as a hit compound with antibacterial activity against Fn. In this preliminary structural optimization study, we designed and synthesized seven novel nitroimidazole derivatives comprising three structural types, followed by antimicrobial evaluation of all target compounds. Among them, compound CL6 exhibited excellent antibacterial activity against Fn (MIC = 0.5 μg·mL^-1) and demonstrated good selectivity towards intestinal bacteria and normal cells. Compound CL6 significantly inhibited the migration of CRC cells (HCT116) induced by Fn preliminary mechanistic studies suggest that compound CL6 disrupts the integrity of the Fn bacterial biofilm and cell wall, providing a promising lead compound for the development of novel anti-Fn drugs.

The 95% ethanol extract of Curcuma longa was isolated and purified by silica gel column chromatography, polyamide column chromatography, preparative thin layer chromatography and semi-preparative HPLC. Then, the structures of the obtained compounds were identified by HR-MS, IR, and NMR. Absolute configuration of the new compound was determined by calculating ECD. Finally, two bisabolane-type sesquiterpenoids were obtained from C. longa and identified as (7S)-1,3,5,10-bisabolatetraen-3-nitro-9-one (1) and turmeronol A (2). Compound 1 was a novel nitro-substituted bisabolane-type sesquiterpenoid.

Four previously undescribed lanostane tetracyclic triterpenoids baoslingzhines T-W (1-4) were isolated from Ganoderma lucidum. Their structures including relative and absolute configurations were assigned by spectroscopic methods and ECD calculations.

The methanol extract of Pteris wallichiana was separated and purified by MCI gel, sephadex LH 20, flash C18 and silica gel column chromatography combined with semi-pre HPLC. The chemical structures of the isolated compounds were identified by MS, IR, NMR, etc. Five sesquiterpene compounds were isolated from Pteris wallichiana and identified as 6,7-tetrahydrofuran-(2S, 3S)-pterosin C-3-O-β-D-(6′-acetyl)-Glu (1), (2S, 3S)-pterosin C-3-O-β-D-Glu (2), (2S)-pterosin A (3) and (2S)-13-hydroxyl-pterosin A (4), (2R, 3S)-2-hydroxyl-pterosin C (5). Compound 1 is a new sesquiterpene, compounds 3-5 were isolated for the first time. In vitro bioactivity assay showed that compound 1 was able to inhibit the proliferation of 4T1 and EMT6 cells, and possessed significant anti-triple-negative breast cancer bioactivity.

Four terpenoids were isolated from the neutral portion of petroleum ether extract of Artemisia annua by several chromatographic methods, such as silica gel, MCI Gel CHP-20, ODS, Sephadex LH-20 and semi-preparative HPLC. Their structures were identified by HR-MS and nuclear magnetic resonance spectroscopy. These compounds were defined as (1S,4S,5R,6S,9R,10R)-4-ethoxy-9,10-dimethyloctahydrofuro-(3,2-i)-isochromen-11(4H)-one (1), 3a,4,5,6,6a,7-hexahydro-3,6-dimethyl-9-methyl-2H-naphtho[8a,1-b]furan-2,8(3H)-dione (2), kobusone (3) and 1,2-campholide (4). Compound 1 is a new compound, of which the absolute configuration was established by single crystal X-ray crystallographic analysis. Compound 2 is a new natural product. Compounds 3 and 4 are first isolated from the Artemisia genus.

Six compounds including three new compounds were obtained from the fruiting bodies of Ganoderma lucidum in Baoshan area. These structures were identified as baosacid A (1), baosside A (2), ethyl 2,5-dihydroxy-γ-oxobenzenebutanoate (3), australins A (4), 2,5-dihydroxy-γ-oxobenzenebutanoic acid (5), and methyl 2,5-dihydroxy-γ-oxobenzenebut anoate (6) on the basis of spectroscopic methods. In addition, compound 5 is a newly occurring natural product.

The genus Gynostemma, with abundant plant resources, is widely distributed in China. Gynostemma plants have gathered widespread attention both domestically and internationally due to their abundant contents of diverse dammarane triterpenoid saponins and various promising pharmacological activities. At present, the studies on the chemical constituents and pharmacological activities of Gynostemma plants mainly focus on G. pentaphyllum (Thunb.) Makino and G. longipes C. Y. Wu ex C. Y. Wu & S. K. Chen, with less attention given to other species within genus. In this study, the chemical constituents of G. burmanicum King ex Chakrav were systematically identified by ultra-high performance liquid chromatography-quadrupole-time of flight-mass spectrometry (UHPLC-Q-TOF-MS). Firstly, the LC-MS analysis of G. burmanicum from different sources was carried out to evaluate the consistency. According to the mass spectrometry fragmentation pattern of dammarane triterpenoid saponins from Gynostemma, the fragmentation characteristics of malonylated and acetylated saponins, combined with the self-built database and online database such as ChemSipder, SciFinder, PubChem, the chemical components of G. burmanicum were identified. The similarities and differences in the components between G. burmanicum and G. longipes were further assessed by comparing their base peak chromatogram. The experimental results indicated a good consistency in the composition of G. burmanicum samples from different sources. A total of 47 chemical components were identified from G. burmanicum, including 12 flavonoids and 35 triterpenoid saponins, among which 6 were new compounds. Saponins in G. burmanicum generally exhibited malonylation and acetylation, appearing after the corresponding prototypical saponins on a reversed phase chromatography. The base peak ion (BPI) chromatograms showed that the saponins of G. burmanicum were highly consistent with those of G. longipes, with comparable contents of the main components gypenoside XLIX and gypenoside A and their malonylated derivatives. In summary, this study comprehensively clarified the chemical composition and characteristics of G. burmanicum, which provided an experimental basis for the development and utilization of G. burmanicum.

The study aims to provide an important basis for IMH020 production process and quality control and develop an HPLC method for the determination of related substances in IMH020. The analysis was performed on a Waters Symmetry C₁₈ column (250 mm × 4.6 mm, 5 µm). The mobile phase A was acetonitrile and the mobile phase B was water-0.2% glacial acetic acid solution with isocratic elution. The flow rate was 1.0 mL·min^-1 and the column temperature was maintained at 30 ℃. The wavelength was 270 and 300 nm. The injection volume was 10 μL. The results show that all the related substances gained a completely chromatographic separation. Good linear relationships of all the related substances (I-1, I-2, I-3a, I-3b, I-4 and I-5) were obtained (r ≥ 0.998 9) and recoveries were in the range of 93.1%-97.8% (RSD ≤ 2%, n = 9). Only I-3a and I-3b were detected below the limit for specified impurities in the three batches of samples, and the largest unspecified single impurity and the total impurities were within the limit. The HPLC method established in this paper is simple, sensitive and accurate, and can be used for the determination of related substances of IMH020.

Based on mass spectrometry imaging method, we investigated the effects of Panax notoginseng in improving diabetic retinopathy (DR) and interfering with corneal, vitreous and retinal metabolites, to reveal the mechanism of Panax notoginseng's action in improving DR. All animal experiments were approved by the Experimental Animal Ethics Committee of Beijing University of Chinese Medicine (Approval No.: BUCM-2023052204-2117). Streptozotocin (STZ)-induced diabetes mellitus (DM) rat model was used, and fasting blood glucose (FBG) and glucosylated serum protein (GSP) levels were measured in each group of rats. Occludin and zonula occludens-1 (ZO-1) were detected by immunofluorescence staining; air flow-assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI) was used to detect endogenous metabolites in the cornea, vitreous, and retinal microregions of the eyes of rats in the DM group and Panax notoginseng group. Endogenous metabolites were detected in the cornea, vitreous, and retinal microregions of the DM and Panax notoginseng groups, and the DM and Panax notoginseng groups were screened for different metabolites by principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). Differential metabolites were screened in the DM and Panax notoginseng groups, the in situ spatial information of differential metabolites in each microregion was analyzed, and the related metabolic pathways were analyzed by the Kyoto encyclopedia of genes and genomes (KEGG) database. The results showed that compared with the DM group, diabetic rats in the Panax notoginseng group showed a decreasing trend in both FBG and GSP, and an increase in the expression of ZO-1 and occludin in the retina (P < 0.001); AFADESI-MSI analysis showed that there were a total of 34 differential metabolites in the cornea, vitreous body, and retinal microregion in the Panax notoginseng group, of which Panax notoginseng called back 13 differential metabolites. In the retinal microregion, Panax notoginseng significantly regulated lysophosphatidylserine (18∶0), phosphatidylethanolamine (34∶2) and phosphatidylserine (40∶7/42∶7). The metabolic pathway enrichment results indicated that Panax notoginseng mainly regulated glycerophospholipid metabolism, glycosylphosphatidylinositol synthesis, niacin and nicotinamide metabolism as well as glycerol ester metabolic pathways. In conclusion, Panax notoginseng improves the blood-retinal barrier (BRB) in diabetic rats, and its mechanism of action may be closely related to glycerophospholipid metabolism. This study provides scientific evidence for the mechanism of action of Panax notoginseng in improving DR, and demonstrates the potential of mass spectrometry imaging technology applied to the study of pharmacological mechanisms.

Dendritic cells (DCs) play a critical role in both innate and adaptive immunity, particularly in regulating antitumor immune responses. However, immunosuppressive cytokines in the tumor microenvironment and lipid peroxidation imbalance within DCs limit their ability to activate tumor-specific T cells effectively. To address this, we developed a novel biomimetic nanodrug delivery platform using artificial intelligence (AI). This platform encapsulates curcumin nanoparticles in bacterial outer membrane vesicles (OMVs) to enhance DCs function through a dual approach: targeted drug delivery and immune activation. In vitro experiments demonstrated that curcumin reduced lipid peroxidation stress in DCs by modulating the IRE1α-XBP1 signaling pathway, thereby restoring their antigen-presenting function. Additionally, OMVs not only acted as efficient drug carriers but also as immune activators, promoting DCs maturation and enhancing tumor-specific immune responses. This study presents a promising strategy for improving antitumor immunotherapy and offers new insights into the application of AI in drug delivery systems.

Plasma exosomes (Pla-Exos) were extracted from rats by ultracentrifugation. The ultracentrifugation method extracted rat Pla-Exos at a speed of 150 000 ×g for 2.5 h. Various purification methods including ultracentrifugation, magnetic bead capture method, and ultrafiltration would be employed to purify Pla-Exos. The Pue-Exos were prepared via sonication-assisted and co-incubation methods. The influence factors and levels of the preparation of plasma exosomes carrying puerarin (Pue-Exos) were investigated by RSM plus CCD method with encapsulation rate as an index. Then the characterization, the stability, and in vitro release of Pue-Exos were determined. The particle sizes of exosomes purified by ultracentrifugation, ultrafiltration, or magnetic bead capture method were all within the range of 30-150 nm. The Western blot results showed that purified Pla-Exos and Pue-Exos contained marker proteins TSG101, CD63, and CD81. The TEM showed that purified Pla-Exos and Pue-Exos exhibited well-defined double-layered membrane vesicle structures. The optimal prescription conditions for preparing Pue-Exos were finally determined as follows: 1 h ultrasound time, 39 W ultrasound power, mass ratio of 10∶1. The prepared Pue-Exos exhibit good stability and sustained release, significantly enhancing the in vitro transpermeability of puerarin across the blood-brain barrier (P < 0.01). This study was approved by the Experimental Animal Ethics Review Committee of Hebei North University, and ethics approval number was HBNV202307012103.

Polygonatum Mill. (Asparagaceae) is a pharmaceutically important genus with many species are of significant medicinal value. Taxonomy and interspecific identification of Polygonatum species have long been controversial due to their considerable morphological variation, wide geographic distribution, complex speciation processes, and lacking of high-resolution molecular markers. To evaluate species discrimination power of 14 plastid divergence hotspot regions (candidate sequences) and their combinations in Polygonatum, a total of 166 individuals from 32 populations representing 15 medicinal Polygonatum species distributed in China were sampled for study. The interspecific and intraspecific genetic variation of each sequence and sequence combination were estimated, and tree-based and pairwise genetic distance (PWG-distance) methods were applied. The results indicated that except for trnT-trnL, the designed primers for all the other 13 candidate sequences showed good universality. Varying degrees of overlaps were detected between intraspecific and interspecific genetic distances in each of the 14 single candidate sequences and their combinations. Nonetheless, overlaps in the combined sequences were significantly lower than those in single sequences. Species resolution of the 14 single sequences were 6.67%-40% and 20%-60% based on tree-based and PWG-distance methods, separately. The combined sequences possessed higher species-resolving power with 40%-73.33% by tree-based method and 46.67%-73.33% by PWG-distance method, accordingly. Among them, the combined sequences C0 and C1 (in both tree-based and PWG-distance methods), C2 and C3 (in tree-based method), and C25 (in PWG-distance method) all showed the best resolution degree of 73.33%, indicating that combination of sequences could effectively improve species discrimination power. In addition, sequences psaJ-rpl33, rps16-trnQ, trnF-ndhJ, trnT-trnL, trnK-matK and atpF all exhibited relatively higher species-resolving degree, which could be used as specific molecular markers for the identification of medicinal Polygonatum species, and we propose the combination of psaJ-rpl33+rps16-trnQ+trnF-ndhJ+trnK-matK+atpF as the most ideal high-resolution molecular marker for discriminating the medicinal Polygonatum. This study will provide a basis for conservation and utilization of germplasm resources and accurate identification of medicinal Polygonatum, as well as standardizing the market for Polygonati Rhizoma.

L-Asparaginase (ASNase), an aminohydrolase, is widely utilized in the pharmaceutical and food industries. Among its various sources, Escherichia coli K12-derived EcASNase has been employed as a clinical drug for the treatment of acute lymphoblastic leukemia (ALL). However, the limited catalytic activity and stability of EcASNase have restricted its broader application in medicine and food processing. In this study, a random mutagenesis library was constructed via error-prone PCR, followed by high-throughput screening using a coupled bacterial growth strategy. Three positive mutants with enhanced activity were identified: G38S, Q212Y, and S274P, exhibiting activities 1.4-, 1.1-, and 1.2-fold higher than the wild type (WT), respectively. Saturation mutagenesis libraries were subsequently generated for positions 38, 212, and 274, leading to the identification of mutants G38A, G38S, G38Q and G38V, with k_cat/K_m values 1.7-, 1.5-, 2.1-, and 2.2-fold higher than WT, respectively. Among these, G38V emerged as the most active mutant, with a T_m value increased by 8.4 ℃ compared to WT. Combination mutations, such as G38V/Q212F and G38V/S274P, failed to yield further activity improvements. This research elucidates the contributions of critical residues to the enzyme's activity and stability, providing novel insights into the rational design and development of therapeutic enzymes.