Inflammation-oriented montmorillonite adjuvant enhanced oral delivery of anti-TNF-α nanobody against inflammatory bowel disease.

Oral delivery of proteins faces challenges due to the harsh conditions of the gastrointestinal (GI) tract, including gastric acid and intestinal enzyme degradation. Permeation enhancers are limited in their ability to deliver proteins with high molecular weight and can potentially cause toxicity by opening tight junctions. To overcome these challenges, we propose the use of montmorillonite (MMT) as an adjuvant that possesses both inflammation-oriented abilities and the ability to regulate gut microbiota. This adjuvant can be used as a universal protein oral delivery technology by fusing with advantageous binding amino acid sequences. We demonstrated that anti-TNF-α nanobody (VII) can be intercalated into the MMT interlayer space. The carboxylate groups (-COOH) of aspartic acid (D) and glutamic acid (E) interact with the MMT surface through electrostatic interactions with sodium ions (Na+). The amino groups (NH2) of asparagine (N) and glutamine (Q) are primarily attracted to the MMT layers through hydrogen bonding with oxygen atoms on the surface. This binding mechanism protects VII from degradation and ensures its release in the intestinal tract, as well as retaining biological activity, leading to significantly enhanced therapeutic effects on colitis. Furthermore, VII@MMT increases the abundance of short-chain fatty acids (SCFAs)-producing strains, including Clostridia, Prevotellaceae, Alloprevotella, Oscillospiraceae, Clostridia_vadinBB60_group, and Ruminococcaceae, therefore enhance the production of SCFAs and butyrate, inducing regulatory T cells (Tregs) production to modulate local and systemic immune homeostasis. Overall, the MMT adjuvant provides a promising universal strategy for protein oral delivery by rational designed protein.

Critical role of msgA in invasive capacity and intracellular survivability of Salmonella.

Salmonella enterica serovar Typhimurium, which is a common foodborne pathogen, causes both intestinal and systemic infections in hosts. Salmonella has a complex pathogenic mechanism that involves invasive capacity and intracellular survivability, which hampers research on virulence of Salmonella. The virulence of Salmonella is primarily studied through Salmonella pathogenicity islands (SPIs). However, there are also genes outside these SPIs that significantly impact virulence. Macrophage survival gene msgA is positioned at a region independent of the SPIs and conserved in Salmonella. However, there has been limited research on msgA to date. This study aims to investigate the virulent function of msgA to deepen our understanding of Salmonella virulence. Proteomic and RT-qPCR analyses reveal that MsgA influences multiple metabolic pathways and the expression of SPIs. The depletion of msgA led to the significantly reduced invasive capacity and intracellular survivability, and thus the decreased virulence of Salmonella. In conclusion, our study suggests that MsgA is an important regulator that mainly regulates virulence. Further research into the function of MsgA will enhance the understanding of Salmonella pathogenesis and promote the application of Salmonella for medical treatment. IMPORTANCE: Salmonella enterica serovar Typhimurium is a common foodborne pathogen, it has a complex pathogenic mechanism that involves invasive capacity and intracellular survivability. The virulence of Salmonella is primarily studied through its pathogenicity islands. In contrast, virulence genes located outside the Salmonella pathogenicity islands (SPIs) have received less attention. Macrophage survival gene (MsgA) is positioned at a region independent of the SPIs and conserved in Salmonella. Our research indicates that MsgA is a novel global regulator influencing the metabolic pathways and SPIs. Further research into the function of MsgA will enhance the understanding of Salmonella pathogenesis and promote the application of Salmonella for medical treatment.

Dual-Engineered Macrophage-Microbe Encapsulation for Metastasis Immunotherapy.

Lung metastases are the leading cause of death among cancer patients. The challenges of inefficient drug delivery, compounded by a robust immunosuppressive microenvironment, make effective treatment difficult. Here, an innovative dual-engineered macrophage-microbe encapsulation (Du-EMME) therapy is developed that integrates modified macrophages and engineered antitumor bacteria. These engineered macrophages, termed R-GEM cells, are designed to express RGD peptides on extracellular membranes, enhancing their tumor cell binding and intratumor enrichment. R-GEM cells are cocultured with attenuated Salmonella typhimurium VNP20009, producing macrophage-microbe encapsulation (R-GEM/VNP cells). The intracellular bacteria maintain bioactivity for more than 24 h, and the bacteria released from R-GEM/VNP cells within the tumor continue to exert bacteria-mediated antitumor effects. This is further supported by macrophage-based chemotaxis and camouflage, which enhance the intratumoral enrichment and biocompatibility of the bacteria. Additionally, R-GEM cells loaded with IFNγ-secreting strains (VNP-IFNγ) form R-GEM/VNP-IFNγ cells. Treatment with these cells effectively halts lung metastatic tumor progression in three mouse models (breast cancer, melanoma, and colorectal cancer). R-GEM/VNP-IFNγ cells vigorously activate the tumor microenvironment, suppressing tumor-promoting M2-type macrophages, MDSCs, and Tregs, and enhancing tumor-antagonizing M1-type macrophages, mature DCs, and Teffs. Du-EMME therapy offers a promising strategy for targeted and enhanced antitumor immunity in treating cancer metastases.

Programmable Bacteria with Dynamic Virulence Modulation System for Precision Antitumor Immunity.

Engineered bacteria-mediated antitumor approaches have been proposed as promising immunotherapies for cancer. However, the off-target bacterial toxicity narrows the therapeutic window. Living microbes will benefit from their controllable immunogenicity within tumors for safer antitumor applications. In this study, a genetically encoded microbial activation strategy is reported that uses tunable and dynamic expression of surface extracellular polysaccharides to improve bacterial biocompatibility while retaining therapeutic efficacy. Based on screening of genes associated with Salmonella survival in macrophages, a novel attenuated Salmonella chassis strain AIS (htrA gene-deficient) highly enriched in tumors after administration and rapidly cleared from normal organs are reported. Subsequently, an engineered bacterial strain, AISI-H, is constructed based on the AIS strain and an optimized quorum-sensing regulatory system. The AISI-H strain can achieve recovery of dynamic tumor-specific bacterial virulence through a novel HTRA-RCSA axis-based and quorum-sensing synthetic gene circuit-mediated increase in extracellular polysaccharide content. These strains act "off" in normal organs to avoid unwanted immune activation and "on" in tumors for precise tumor suppression in mice. The AISI-H strain shows significant tumor inhibition and potent activation of anticancer immunity in a melanoma mouse model. The AISI-H strain exhibits excellent biocompatibility. This bacterial regulation strategy expands the applications of microbe-based antitumor therapeutics.

Different depths of food restriction and high-fat diet refeeding in mice impact host obesity and metabolic phenotypes with correlative changes in the gut microbiota.

Overweight and obesity affect almost 2 billion adults worldwide, and food restriction (FR) is commonly used to reduce body fat. Whether refeeding (Re) after FR at different ages and to different degrees leads to overweight and its possible mechanisms are uncertain. In this study, adult and young mice were both restricted to 15% and 40% of their casual food intake, and then were fed 60% high-fat chow (FR15%-Re, FR40%-Re), whereas the control groups(CON) consumed high-fat or normal food throughout, respectively. The results of the study suggest that mild FR-heavy feeding may lead to more significant abnormal fat accumulation, liver damage, and increased recruitment of intestinal inflammatory factors and immune cells in mice of different ages and involves multiple types of alterations in the gut microbiota. Further fecal transplantation experiments as well as serum and liver enzyme-linked immunosorbent assay experiments preliminarily suggest that the link between lipid metabolism and inflammatory responses and the gut microbiota may be related to the regulation of the gut and live by Lipopolysaccharides(LPS) and Peroxisome Proliferator-Activated Receptor-Alpha(PPAR-α). In addition, our study may also serve as a reference for studying obesity prevention and treatment programs at different ages.

ADT-OH exhibits anti-metastatic activity on triple-negative breast cancer by combinatorial targeting of autophagy and mitochondrial fission.

High basal autophagy and enhanced mitochondrial fission in triple-negative breast cancer (TNBC) cells support cell migration and promote plasticity of cancer cell metabolism. Here, we suggest a novel combination therapy approach for the treatment of TNBC that targets Drp1-mediated mitochondrial fission and autophagy pathways. Hydrogen sulfide (H2S) mediates a myriad of biological processes, including autophagy and mitochondrial function. In this study, we demonstrated that 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADT-OH), one of the most widely utilized sustained-release H2S donors, effectively suppresses metastasis of TNBC cells in the absence of proliferation inhibition in vitro and in vivo. ADT-OH treatment ameliorated autophagy flux by suppressing autophagosome formation and induced mitochondrial elongation through decreasing expression of dynamin-related protein 1 (Drp1) and increasing expression of mitochondrial fusion protein (Mfn2). At the same time, ADT-OH downregulated mitophagy flux and inhibited mitochondrial function, eventually leading to the inhibition of migration and invasion in TNBC cells. In vivo, intraperitoneal administration of ADT-OH revealed a potent anti-metastatic activity in three different animal models, the MDA-MB-231 orthotopic xenograft model, the 4T1-Luci orthotopic model and the 4T1-Luci tail vein metastasis model. However, ADT-OH has an extremely low water solubility, which is a significant barrier to its effectiveness. Thus, we demonstrated that the solubility of ADT-OH in water can be improved significantly by absorption with hydroxypropyl-ß-cyclodextrin (CD). Remarkably, the obtained CD-ADT-OH demonstrated superior anti-cancer effect to ADT-OH in vivo. Altogether, this study describes a novel regulator of mammalian mitochondrial fission and autophagy, with potential utility as an experimental therapeutic agent for metastatic TNBC.

Oncolytic bacteria VNP20009 expressing IFNß inhibits melanoma progression by remodeling the tumor microenvironment.

In the tumor microenvironment (TME), tumor-associated NEs (TANs) have the potential to be protumorigenic or antitumorigenic within the TME in response to environmental cues. The diversity and plasticity of NEs (NEs) underlie the dual potential of TANs in the TME. Here, we utilized the tumor-targeting bacterium VNP20009 (VNP) to carry a plasmid expressed IFNß (VNP-IFNß), which can deliver IFNß and remodel TANs to an antitumorigenic phenotype, and performed preclinical evaluations in the B16F10 lung metastasis model and the B16F10 subcutaneous xenograft model. Compared with VNP, VNP-IFNß recruited more NEs and macrophages (Mφs) with antitumor phenotypes in lung metastases and activated dendritic cells (DCs) differentiation, which activated antitumor immune responses of CD4+ T cells, and ultimately inhibited melanoma progression. This study enriches the bacterial-mediated tumor therapy by using tumor-targeting bacteria to deliver IFNß to the tumor site and inhibit melanoma growth and metastasis by remodeling the tumor immune microenvironment.

Mechanism of Mutation-Induced Effects on the Catalytic Function of TEV Protease: A Molecular Dynamics Study.

Tobacco etch virus protease (TEVp) is wildly exploited for various biotechnological applications. These applications take advantage of TEVp's ability to cleave specific substrate sequences to study protein function and interactions. A major limitation of this enzyme is its relatively slow catalytic rate. In this study, MD simulations were conducted on TEV enzymes and known highly active mutants (eTEV and uTEV3) to explore the relationship between mutation, conformation, and catalytic function. The results suggest that mutations distant from the active site can influence the substrate-binding pocket through interaction networks. MD analysis of eTEV demonstrates that, by stabilizing the orientation of the substrate at the catalytic site, mutations that appropriately enlarge the substrate-binding pocket will be beneficial for Kcat, enhancing the catalytic efficiency of the enzyme. On the contrary, mutations in uTEV3 reduced the flexibility of the active pocket and increased the hydrogen bonding between the substrate and enzyme, resulting in higher affinity. At the same time, the MD simulation demonstrates that mutations outside of the active site residues could affect the dynamic movement of the binding pocket by altering residue networks and communication pathways, thereby having a profound impact on reactivity. These findings not only provide a molecular mechanistic explanation for the excellent mutants, but also serve as a guiding framework for rational computational design.

Zinc as a potential regulator of the BCR-ABL oncogene in chronic myelocytic leukemia cells.

BACKGROUND: Generally, decreased zinc in the serum of tumor patients but increased zinc in tumor cells can be observed. However, the role of zinc homeostasis in myeloid leukemia remains elusive. BCR-ABL is essential for the initiation, maintenance, and progression of chronic myelocytic leukemia (CML). We are currently investigating the association between zinc homeostasis and CML. METHODS: Genes involved in zinc homeostasis were examined using three GEO datasets. Western blotting and qPCR were used to investigate the effects of zinc depletion on BCR-ABL expression. Furthermore, the effect of TPEN on BCR-ABL promoter activity was determined using the dual-luciferase reporter assay. MRNA stability and protein stability of BCR-ABL were assessed using actinomycin D and cycloheximide. RESULTS: Transcriptome data mining revealed that zinc homeostasis-related genes were associated with CML progression and drug resistance. Several zinc homeostasis genes were affected by TPEN. Additionally, we found that zinc depletion by TPEN decreased BCR-ABL mRNA stability and transcriptional activity in K562 CML cells. Zinc supplementation and sodium nitroprusside treatment reversed BCR-ABL downregulation by TPEN, suggesting zinc- and nitric oxide-dependent mechanisms. CONCLUSION: Our in vitro findings may help to understand the role of zinc homeostasis in BCR-ABL regulation and thus highlight the importance of zinc homeostasis in CML.

The oncolytic bacteria-mediated delivery system of CCDC25 nucleic acid drug inhibits neutrophil extracellular traps induced tumor metastasis.

BACKGROUND: Neutrophil extracellular traps (NETs), antibacterial weapons of neutrophils (NEs), have been found to play a crucial role in cancer metastasis in recent years. More and more cancer research is focusing on anti-NETs. However, almost all anti-NETs treatments have limitations such as large side effects and limited efficacy. Therefore, exploring new anti-NETs therapeutic strategies is a long-term goal. RESULTS: The transmembrane protein coiled-coil domain containing 25 (CCDC25) on tumor cell membranes can bind NETs-DNA with high specificity and affinity, enabling tumor cells to sense NETs and thus promote distant metastasis. We transformed shCCDC25 into VNP20009 (VNP), an oncolytic bacterium, to generate VNP-shCCDC25 and performed preclinical evaluation of the inhibitory effect of shCCDC25 on cancer metastasis in B16F10 lung metastasis and 4T1 orthotopic lung metastasis models. VNP-shCCDC25 effectively blocked the downstream prometastatic signaling pathway of CCDC25 at tumor sites and reduced the formation of NETs while recruiting more neutrophils and macrophages to the tumor core, ultimately leading to excellent metastasis inhibition in the two lung metastasis models. CONCLUSION: This study is a pioneer in focusing on the effect of anti-NET treatment on CCDC25. shCCDC25 is effectively delivered to tumor sites via the help of oncolytic bacteria and has broad application in the inhibition of cancer metastasis via anti-NETs.

Camouflaging attenuated Salmonella by cryo-shocked macrophages for tumor-targeted therapy.

Live bacteria-mediated antitumor therapies mark a pivotal point in cancer immunotherapy. However, the difficulty in reconciling the safety and efficacy of bacterial therapies has limited their application. Improving bacterial tumor-targeted delivery while maintaining biosafety is a critical hurdle for the clinical translation of live microbial therapy for cancer. Here, we developed "dead" yet "functional" Salmonella-loaded macrophages using liquid nitrogen cold shock of an attenuated Salmonella typhimurium VNP20009-contained macrophage cell line. The obtained "dead" macrophages achieve an average loading of approximately 257 live bacteria per 100 cells. The engineered cells maintain an intact cellular structure but lose their original pathogenicity, while intracellular bacteria retain their original biological activity and are delay freed, followed by proliferation. This "Trojan horse"-like bacterial camouflage strategy avoids bacterial immunogenicity-induced neutrophil recruitment and activation in peripheral blood, reduces the clearance of bacteria by neutrophils and enhances bacterial tumor enrichment efficiently after systemic administration. Furthermore, this strategy also strongly activated the tumor microenvironment, including increasing antitumor effector cells (including M1-like macrophages and CD8+ Teffs) and decreasing protumor effector cells (including M2-like macrophages and CD4+ Tregs), and ultimately improved antitumor efficacy in a subcutaneous H22 tumor-bearing mouse model. The cryo-shocked macrophage-mediated bacterial delivery strategy holds promise for expanding the therapeutic applications of living bacteria for cancer.

Sulfide:quinone oxidoreductase alleviates ferroptosis in acute kidney injury via ameliorating mitochondrial dysfunction of renal tubular epithelial cells.

Ferroptosis is iron-dependent and regulates necrosis caused by lipid peroxidation and mitochondrial damage. Recent evidence has revealed an emerging role for ferroptosis in the pathophysiology of acute kidney injury (AKI). Sulfide:quinone oxidoreductase (SQOR) is a mitochondrial inner membrane protein highly expressed in the renal cortex. However, the effects of SQOR on ferroptosis and AKI have not been elucidated. In this study, we evaluated the effects of SQOR in several AKI models. We observed a rapid decrease in SQOR expression after cisplatin stimulation in both in vivo and in vitro models. SQOR-deletion mice exhibit exacerbated kidney impairment and ferroptosis in renal tubular epithelial cells following cisplatin injury. Additionally, our results showed that the overexpression of SQOR or ADT-OH (the slow-releasing H2S donor) preserved renal function in the three AKI mouse models. These effects were evidenced by lower levels of serum creatinine (SCr), blood urea nitrogen (BUN), renal neutrophil gelatinase-associated lipocalin (NGAL), and kidney injury molecule 1 (KIM-1). Importantly, SQOR knockout significantly aggravates cisplatin-induced ferroptosis by promoting mitochondrial dysfunction in renal tubular epithelial cells (RTECs). Moreover, online database analysis combined with our study revealed that SYVN1, an upregulated E3 ubiquitin ligase, may mediate the ubiquitin-mediated degradation of SQOR in AKI. Consequently, our results suggest that SYVN1-mediated ubiquitination degradation of SQOR may induce mitochondrial dysfunction in RTECs, exacerbating ferroptosis and thereby promoting the occurrence and development of AKI. Hence, targeting the SYVN1-SQOR axis could be a potential therapeutic strategy for AKI treatment.

Resveratrol, a novel inhibitor of fatty acid binding protein 5, inhibits cervical cancer metastasis by suppressing fatty acid transport into nucleus and downstream pathways.

BACKGROUND AND PURPOSE: Because of cervical cancer (CC) metastasis, the prognosis of diagnosed patients is poor. However, the molecular mechanisms and therapeutic approach for metastatic CC remain elusive. EXPERIMENTAL APPROACH: In this study, we first evaluated the effect of resveratrol (RSV) on CC cell migration and metastasis. Via an activity-based protein profiling (ABPP) approach, a photoaffinity probe of RSV (RSV-P) was synthesized, and the protein targets of RSV in HeLa cells were identified. Based on target information and subsequent in vivo and in vitro validation experiments, we finally elucidated the mechanism of RSV corresponding to its antimetastatic activity. KEY RESULTS: The results showed that RSV concentration-dependently suppressed CC cell migration and metastasis. A list of proteins was identified as the targets of RSV, through the ABPP approach with RSV-P, among which fatty acid binding protein 5 (FABP5) attracted our attention based on The Cancer Genome Atlas (TCGA) database analysis. Subsequent knockout and overexpression experiments confirmed that RSV directly interacted with FABP5 to inhibit fatty acid transport into the nucleus, thereby suppressing downstream matrix metalloproteinase-2 (MMP2) and matrix metalloproteinase-9 (MMP9) expression, thus inhibiting CC metastasis. CONCLUSIONS AND IMPLICATIONS: Our study confirmed the key role of FABP5 in CC metastasis and provided important target information for the design of therapeutic lead compounds for metastatic CC.

Oncogenic KRAS effector USP13 promotes metastasis in non-small cell lung cancer through deubiquitinating ß-catenin.

KRAS mutations are frequently detected in non-small cell lung cancers (NSCLCs). Although covalent KRASG12C inhibitors have been developed to treat KRASG12C-mutant cancers, effective treatments are still lacking for other KRAS-mutant NSCLCs. Thus, identifying a KRAS effector that confers poor prognosis would provide an alternative strategy for the treatment of KRAS-driven cancers. Here, we show that KRAS drives expression of deubiquitinase USP13 through Ras-responsive element-binding protein 1 (RREB1). Elevated USP13 promotes KRAS-mutant NSCLC metastasis, which is associated with poor prognosis in NSCLC patients. Mechanistically, USP13 interacts with and removes the K63-linked polyubiquitination of ß-catenin at lysine 508, which enhances the binding between ß-catenin and transcription factor TCF4. Importantly, we identify 2-methoxyestradiol as an effective inhibitor for USP13 from a natural compound library, and it could potently suppress the metastasis of KRAS-mutant NSCLC cells in vitro and in vivo. These findings identify USP13 as a therapeutic target for metastatic NSCLC with KRAS mutations.

Stilbenes: a promising small molecule modulator for epigenetic regulation in human diseases.

Stilbenes are characterized by a vinyl group connecting two benzene rings to form the basic parent nucleus. Hydrogen atoms on different positions of the benzene rings can be substituted with hydroQxyl groups. These unique structural features confer anti-inflammatory, antibacterial, antiviral, antioxidant, anticancer, cardiovascular protective, and neuroprotective pharmacological effects upon these compounds. Numerous small molecule compounds have demonstrated these pharmacological activities in recent years, including Resveratrol, and Pterostilbene, etc. Tamoxifen and Raloxifene are FDA-approved commonly prescribed synthetic stilbene derivatives. The emphasis is on the potential of these small molecules and their structural derivatives as epigenetic regulators in various diseases. Stilbenes have been shown to modulate epigenetic marks, such as DNA methylation and histone modification, which can alter gene expression patterns and contribute to disease development. This review will discuss the mechanisms by which stilbenes regulate epigenetic marks in various diseases, as well as clinical trials, with a focus on the potential of small molecule and their derivatives such as Resveratrol, Pterostilbene, and Tamoxifen.

Antibacterial effect of protease-responsive cationic eugenol liposomes modified by gamma-polyglutamic acid against Staphylococcus aureus.

Eugenol, as a natural antibacterial agent, has been widely studied for its inhibitory effect on the common food-borne pathogen Staphylococcus aureus (S. aureus). However, the widespread application of eugenol is still limited by its instability and volatility. Herein, γ-polyglutamic acid coated eugenol cationic liposomes (pGA-ECLPs) were successfully constructed by self-assembly with an average particle size of 170.7 nm and an encapsulation efficiency of 36.2%. The formation of pGA shell significantly improved the stability of liposomes, and the encapsulation efficiency of eugenol only decreased by 20.7% after 30 days of storage at 4 °C. On the other hand, the pGA layer can be hydrolyzed by S. aureus, achieving effective control of release through response to bacterial stimuli. The application experiments further confirmed that pGA-ECLPs effectively prolonged the antibacterial effect of eugenol in fresh chicken without causing obvious sensory effects on the food. The above results of this study provide an important reference for extending the action time of natural antibacterial substances and developing new stimuli-responsive antibacterial systems.

Neutrophil-Mediated Tumor-Targeting Delivery System of Oncolytic Bacteria Combined with ICB for Melanoma Lung Metastasis Therapy.

Oncolytic bacteria are the most promising tumor target vector. Questions also remain regarding finding a balance between the therapeutic efficacy and safety of oncolytic bacteria. The critical measure of how this balance is maintained is the improvement in tumor colonization. Attenuated Salmonella typhimurium (VNP20009) as the only Salmonella strain to be evaluated in a clinical trial is a potential tumor therapeutic bacterium. A delivery system with controlled release of VNP after being loaded into neutrophils, which significantly increases the tumor-targeting of VNP and enhances its therapeutic efficacy in a melanoma lung metastasis model is constructed. To improve the synergistic therapeutic effect, a PD1 nanobody is applied to this system (NE(PD1nb)). NE(PD1nb) activate dendritic cells (DCs) differentiation and stimulate the M1-like differentiation of macrophages, and induce CD4+ T-cells maturity and cytotoxic CD8+ T-cells activation through DCs tumor antigen presentation.

ADT-OH improves intestinal barrier function and remodels the gut microbiota in DSS-induced colitis.

Owing to the increasing incidence and prevalence of inflammatory bowel disease (IBD) worldwide, effective and safe treatments for IBD are urgently needed. Hydrogen sulfide (H2S) is an endogenous gasotransmitter and plays an important role in inflammation. To date, H2S-releasing agents are viewed as potential anti-inflammatory drugs. The slow-releasing H2S donor 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADT-OH), known as a potent therapeutic with chemopreventive and cytoprotective properties, has received attention recently. Here, we reported its anti-inflammatory effects on dextran sodium sulfate (DSS)-induced acute (7 days) and chronic (30 days) colitis. We found that ADT-OH effectively reduced the DSS-colitis clinical score and reversed the inflammation-induced shortening of colon length. Moreover, ADT-OH reduced intestinal inflammation by suppressing the nuclear factor kappa-B pathway. In vivo and in vitro results showed that ADT-OH decreased intestinal permeability by increasing the expression of zonula occludens-1 and occludin and blocking increases in myosin II regulatory light chain phosphorylation and epithelial myosin light chain kinase protein expression levels. In addition, ADT-OH restored intestinal microbiota dysbiosis characterized by the significantly increased abundance of Muribaculaceae and Alistipes and markedly decreased abundance of Helicobacter, Mucispirillum, Parasutterella, and Desulfovibrio. Transplanting ADT-OH-modulated microbiota can alleviate DSS-induced colitis and negatively regulate the expression of local and systemic proinflammatory cytokines. Collectively, ADT-OH is safe without any short-term (5 days) or long-term (30 days) toxicological adverse effects and can be used as an alternative therapeutic agent for IBD treatment.

Zinc depletion induces JNK/p38 phosphorylation and suppresses Akt/mTOR expression in acute promyelocytic NB4 cells.

BACKGROUND: Myeloid leukemia is associated with reduced serum zinc and increased intracellular zinc. Our previous studies found that zinc depletion by TPEN induced apoptosis with PML-RARα oncoprotein degradation in acute promyelocytic NB4 cells. The effect of zinc homeostasis on intracellular signaling pathways in myeloid leukemia cells remains unclear. OBJECTIVE: This study examined how zinc homeostasis affected MAPK and Akt/mTOR pathways in NB4 cells. METHODS: We used western blotting to detect the activation of p38 MAPK, JNK, ERK1/2, and Akt/mTOR pathways in NB4 cells stimulated with the zinc chelator TPEN. Whether the effects of TPEN on these pathways could be reversed by zinc or the nitric oxide donor sodium nitroprusside (SNP) was further explored by western blotting. We used Zinpyr-1 staining to assess the role of SNP on labile zinc levels in NB4 cells treated with TPEN. In additional, we evaluated expressional correlations between the zinc-binding protein Metallothionein-2A (MT2A) and genes related to MAPKs and Akt/mTOR pathways in acute myeloid leukemia (AML) based on the TCGA database. RESULTS: Zinc depletion by TPEN activated p38 and JNK phosphorylation in NB4 cells, whereas ERK1/2 phosphorylation was increased first and then decreased. The protein expression levels of Akt and mTOR were downregulated by TPEN. The nitric oxide donor SNP promotes zinc release in NB4 cells under zinc depletion conditions. We further found that the effects of zinc depletion on MAPK and Akt/mTOR pathways in NB4 cells can be reversed by exogenous zinc supplementation or treatment with the nitric oxide donor SNP. By bioinformatics analyses based on the TCGA database, we demonstrated that MT2A expression was negatively correlated with the expression of JNK, and was positively correlated with the expression of ERK1 and Akt in AML. CONCLUSION: Our findings indicate that zinc plays a critical role in leukemia cells and help understanding how zinc depletion induces apoptosis.