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.

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.

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.

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.

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.

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.

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.

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.

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.

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.