Injury of Macrophages Induced by Clostridium perfringens Type C Exotoxins.

Clostridium perfringens is a kind of anaerobic Gram-positive bacterium that widely exists in the intestinal tissue of humans and animals. And the main virulence factor in Clostridium perfringens is its exotoxins. Clostridium perfringens type C is the main strain of livestock disease, its exotoxins can induce necrotizing enteritis and enterotoxemia, which lead to the reduction in feed conversion, and a serious impact on breeding production performance. Our study found that treatment with exotoxins reduced cell viability and triggered intracellular reactive oxygen species (ROS) in human mononuclear leukemia cells (THP-1) cells. Through transcriptome sequencing analysis, we found that the levels of related proteins such as heme oxygenase 1 (HO-1) and ferroptosis signaling pathway increased significantly after treatment with exotoxins. To investigate whether ferroptosis occurred after exotoxin treatment in macrophages, we confirmed that the protein expression levels of antioxidant factors glutathione peroxidase 4/ferroptosis-suppressor-protein 1/the cystine/glutamate antiporter solute carrier family 7 member 11 (GPX4/FSP1/xCT), ferroptosis-related protein nuclear receptor coactivator 4/transferrin/transferrin receptor (NCOA4/TF/TFR)/ferritin and the level of lipid peroxidation were significantly changed. Based on the above results, our study suggested that Clostridium perfringens type C exotoxins can induce macrophage injury through oxidative stress and ferroptosis.

Thrombomodulin reduces α-synuclein generation and ameliorates neuropathology in a mouse model of Parkinson's disease.

The neurotoxic α-synuclein (α-syn) oligomers play an important role in the occurrence and development of Parkinson's disease (PD), but the factors affecting α-syn generation and neurotoxicity remain unclear. We here first found that thrombomodulin (TM) significantly decreased in the plasma of PD patients and brains of A53T α-syn mice, and the increased TM in primary neurons reduced α-syn generation by inhibiting transcription factor p-c-jun production through Erk1/2 signaling pathway. Moreover, TM decreased α-syn neurotoxicity by reducing the levels of oxidative stress and inhibiting PAR1-p53-Bax signaling pathway. In contrast, TM downregulation increased the expression and neurotoxicity of α-syn in primary neurons. When TM plasmids were specifically delivered to neurons in the brains of A53T α-syn mice by adeno-associated virus (AAV), TM significantly reduced α-syn expression and deposition, and ameliorated the neuronal apoptosis, oxidative stress, gliosis and motor deficits in the mouse models, whereas TM knockdown exacerbated these neuropathology and motor dysfunction. Our present findings demonstrate that TM plays a neuroprotective role in PD pathology and symptoms, and it could be a novel therapeutic target in efforts to combat PD. Schematic representation of signaling pathways of TM involved in the expression and neurotoxicity of α-syn. A TM decreased RAGE, and resulting in the lowered production of p-Erk1/2 and p-c-Jun, and finally reduce α-syn generation. α-syn oligomers which formed from monomers increase the expression of p-p38, p53, C-caspase9, C-caspase3 and Bax, decrease the level of Bcl-2, cause mitochondrial damage and lead to oxidative stress, thus inducing neuronal apoptosis. TM can reduce intracellular oxidative stress and inhibit p53-Bax signaling by activating APC and PAR-1. B The binding of α-syn oligomers to TLR4 may induce the expression of IL-1ß, which is subsequently secreted into the extracellular space. This secreted IL-1ß then binds to its receptor, prompting p65 to translocate from the cytoplasm into the nucleus. This translocation downregulates the expression of KLF2, ultimately leading to the suppression of TM expression. By Figdraw.

Inhibition of SFTSV replication in humanized mice by a subcutaneously administered anti-PD1 nanobody.

Severe fever with thrombocytopenia syndrome (SFTS) is a life-threatening disease caused by a novel bunyavirus (SFTSV), mainly transmitted by ticks. With no effective therapies or vaccines available, understanding the disease's mechanisms is crucial. Recent studies found increased expression of programmed cell death-1 (PD-1) on dysfunctional T cells in SFTS patients. However, the role of the PD-1/programmed cell death-ligand 1 (PD-L1) pathway in SFTS progression remains unclear. We investigated PD-1 blockade as a potential therapeutic strategy against SFTSV replication. Our study analyzed clinical samples and performed in vitro experiments, revealing elevated PD-1/PD-L1 expression in various immune cells following SFTSV infection. An anti-PD-1 nanobody, NbP45, effectively inhibited SFTSV infection in peripheral blood mononuclear cells (PBMCs), potentially achieved through the mitigation of apoptosis and the augmentation of T lymphocyte proliferation. Intriguingly, subcutaneous administration of NbP45 showed superior efficacy compared to a licensed anti-PD-1 antibody in an SFTSV-infected humanized mouse model. These findings highlight the involvement of the PD-1/PD-L1 pathway during acute SFTSV infection and suggest its potential as a host target for immunotherapy interventions against SFTSV infection.

Establishment of an Indirect ELISA Method for the Detection of the Bovine Rotavirus VP6 Protein.

The objective of this study was to develop an indirect ELISA utilizing a polyclonal antibody against bovine rotavirus (BRV) VP6 protein. To achieve this, pcDNA3.1-VP6, a recombinant eukaryotic expression plasmid, was constructed based on the sequence of the conserved BRV gene VP6 and was transfected into CHO-K1 cells using the transient transfection method. The VP6 protein was purified as the coating antigen using nickel ion affinity chromatography, and an indirect ELISA was subsequently established. The study found that the optimal concentration of coating for the VP6 protein was 1 µg/mL. The optimal blocking solution was 3% skim milk, and the blocking time was 120 min. The secondary antibody was diluted to 1:4000, and the incubation time for the secondary antibody was 30 min. A positive result was indicated when the serum OD450 was greater than or equal to 0.357. The coefficients of variation were less than 10% both within and between batches, indicating the good reproducibility of the method. The study found that the test result was positive when the serum dilution was 217, indicating the high sensitivity of the method. A total of 24 positive sera and 40 negative sera were tested using the well-established ELISA. The study also established an indirect ELISA assay with good specificity and sensitivity for the detection of antibodies to bovine rotavirus. Overall, the results suggest that the indirect ELISA method developed in this study is an effective test for detecting such antibodies.

Comparative proteome analysis revealed the differences in response to both Mycobacterium tuberculosis and Mycobacterium bovis infection of bovine alveolar macrophages.

Tuberculosis (TB), attributed to the Mycobacterium tuberculosis complex, is one of the most serious zoonotic diseases worldwide. Nevertheless, the host mechanisms preferentially leveraged by Mycobacterium remain unclear. After infection, both Mycobacterium tuberculosis (MTB) and Mycobacterium bovis (MB) bacteria exhibit intimate interactions with host alveolar macrophages; however, the specific mechanisms underlying these macrophage responses remain ambiguous. In our study, we performed a comparative proteomic analysis of bovine alveolar macrophages (BAMs) infected with MTB or MB to elucidate the differential responses of BAMs to each pathogen at the protein level. Our findings revealed heightened TB infection susceptibility of BAMs that had been previously infected with MTB or MB. Moreover, we observed that both types of mycobacteria triggered significant changes in BAM energy metabolism. A variety of proteins and signalling pathways associated with autophagy and inflammation-related progression were highly activated in BAMs following MB infection. Additionally, proteins linked to energy metabolism were highly expressed in BAMs following MTB infection. In summary, we propose that BAMs may resist MTB and MB infections via different mechanisms. Our findings provide critical insights into TB pathogenesis, unveiling potential biomarkers to facilitate more effective TB treatment strategies. Additionally, our data lend support to the hypothesis that MTB may be transmitted via cross-species infection.

Correction: Nie et al. Endoplasmic Reticulum Stress Mediated NLRP3 Inflammasome Activation and Pyroptosis in THP-1 Macrophages Infected with Bacillus Calmette-Guérin. Int. J. Mol. Sci. 2023, 24, 11692.

Lei Liu was not included as an author in the original publication [...].

[A Novel Chinese Medicine Formula Inhibits Non-small Cell Lung Cancer by Triggering Oxidative Stress Dependent on Pentose Phosphate Pathway].

BACKGROUND: Non-small cell lung cancer (NSCLC) is one of the most lethal malignancies worldwide. A novel Chinese medicine formula-01 (NCHF-01) has shown significant clinical efficacy in the treatment of NSCLC, but the mechanism of this formula in the treatment of NSCLC is not fully understood. The aim of this study is to investigate the molecular mechanism of NCHF-01 in inhibiting NSCLC. METHODS: Lewis lung cells (LLC) tumor bearing mice were established to detect the tumor inhibitory effect of NCHF-01. The morphological changes of tissues and organs in LLC tumor-bearing mice were detected by hematoxylin-eosin (HE) staining. NSCLC cells were treated by NCHF-01. The effects of cell viability and proliferation were detected by MTT and crystal violet staining experiment. Flow cytometry was used to detect cell cycle, apoptosis and reactive oxygen species (ROS). Network pharmacology was used to predict the mechanism of its inhibitory effect of NSCLC. Western blot and immunohistochemistry (IHC) were used to detect the expression of related proteins. RESULTS: NCHF-01 can inhibit tumor growth in LLC tumor-bearing mice, and has no obvious side effects on other tissues and organs. NCHF-01 could inhibit cell viability and proliferation, induce G2/M phase arrest and apoptosis, and promote the increase of ROS level. Network pharmacological analysis showed that NCHF-01 exerts anti-NSCLC effects through various biological processes such as oxidative stress and central carbon metabolism. NCHF-01 can reduce the protein expression and enzyme activity of the key enzymes 6-phosphate glucose dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) in the pentose phosphate pathway (PPP). CONCLUSIONS: NCHF-01 can inhibit NSCLC through oxidative stress dependent on the PPP.

S100A4 Promotes BCG-Induced Pyroptosis of Macrophages by Activating the NF-κB/NLRP3 Inflammasome Signaling Pathway.

Pyroptosis is a host immune strategy to defend against Mycobacterium tuberculosis (Mtb) infection. S100A4, a calcium-binding protein that plays an important role in promoting cancer progression as well as the pathophysiological development of various non-tumor diseases, has not been explored in Mtb-infected hosts. In this study, transcriptome analysis of the peripheral blood of patients with pulmonary tuberculosis (PTB) revealed that S100A4 and GSDMD were significantly up-regulated in PTB patients' peripheral blood. Furthermore, there was a positive correlation between the expression of GSDMD and S100A4. KEGG pathway enrichment analysis showed that differentially expressed genes between PTB patients and healthy controls were significantly related to inflammation, such as the NOD-like receptor signaling pathway and NF-κB signaling pathway. To investigate the regulatory effects of S100A4 on macrophage pyroptosis, THP-1 macrophages infected with Bacillus Calmette-Guérin (BCG) were pre-treated with exogenous S100A4, S100A4 inhibitor or si-S100A4. This research study has shown that S100A4 promotes the pyroptosis of THP-1 macrophages caused by BCG infection and activates NLRP3 inflammasome and NF-κB signaling pathways, which can be inhibited by knockdown or inhibition of S100A4. In addition, inhibition of NF-κB or NLRP3 blocks the promotion effect of S100A4 on BCG-induced pyroptosis of THP-1 macrophages. In conclusion, S100A4 activates the NF-κB/NLRP3 inflammasome signaling pathway to promote macrophage pyroptosis induced by Mtb infection. These data provide new insights into how S100A4 affects Mtb-induced macrophage pyroptosis.

Endoplasmic Reticulum Stress Mediated NLRP3 Inflammasome Activation and Pyroptosis in THP-1 Macrophages Infected with Bacillus Calmette-Guérin.

Tuberculosis (TB) is a zoonotic infectious disease caused by Mycobacterium tuberculosis (Mtb). Mtb is a typical intracellular parasite, and macrophages are its main host cells. NLRP3 inflammasome-mediated pyroptosis is a form of programmed cell death implicated in the clearance of pathogenic infections. The bidirectional regulatory effect of endoplasmic reticulum stress (ERS) plays a crucial role in determining cell survival and death. Whether ERS is involved in macrophage pyroptosis with Mtb infection remains unclear. This article aims to explore the regulation of the NLRP3 inflammasome and pyroptosis by ERS in THP-1 macrophages infected with Mycobacterium bovis Bacillus Calmette-Guérin (BCG). The results showed that BCG infection induced THP-1 macrophage ERS, NLRP3 inflammasome activation and pyroptosis, which was inhibited by ERS inhibitor TUDCA. NLRP3 inhibitor MCC950 inhibited THP-1 macrophage NLRP3 inflammasome activation and pyroptosis caused by BCG infection. Compared with specific Caspase-1 inhibitor VX-765, pan-Caspase inhibitor Z-VAD-FMK showed a more significant inhibitory effect on BCG infection-induced pyroptosis of THP-1 macrophages. Taken together, this study demonstrates that ERS mediated NLRP3 inflammasome activation and pyroptosis after BCG infection of THP-1 macrophages, and that BCG infection of THP-1 macrophages induces pyroptosis through canonical and noncanonical pathways.

Gene editing tools for mycoplasmas: references and future directions for efficient genome manipulation.

Mycoplasmas are successful pathogens that cause debilitating diseases in humans and various animal hosts. Despite the exceptionally streamlined genomes, mycoplasmas have evolved specific mechanisms to access essential nutrients from host cells. The paucity of genetic tools to manipulate mycoplasma genomes has impeded studies of the virulence factors of pathogenic species and mechanisms to access nutrients. This review summarizes several strategies for editing of mycoplasma genomes, including homologous recombination, transposons, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system, and synthetic biology. In addition, the mechanisms and features of different tools are discussed to provide references and future directions for efficient manipulation of mycoplasma genomes.

Pyroptosis of Macrophages Induced by Clostridium perfringens Beta-1 Toxin.

Clostridium perfringens beta-1 toxin (CPB1) is responsible for necrotizing enteritis and enterotoxemia. However, whether the release of host inflammatory factors caused by CPB1 is related to pyroptosis, an inflammatory form of programmed cell death, has not been reported. A construct expressing recombinant Clostridium perfringens beta-1 toxin (rCPB1) was created, and the cytotoxic activity of the purified rCPB1 toxin was assessed via CCK-8 assay. The rCPB1-induced macrophage pyroptosis by assessing changes to the expression of pyroptosis-related signal molecules and the pyroptosis pathway of macrophages using quantitative real-time PCR, immunoblotting, ELISA, immunofluorescence, and electron microscopic assays. The results showed that the intact rCPB1 protein was purified from an E. coli expression system, which exhibited moderate cytotoxicity on mouse mononuclear macrophage leukemia cells (RAW264.7), normal colon mucosal epithelial cells (NCM460), and human umbilical vein endothelial cells (HUVEC). rCPB1 could induce pyroptosis in macrophages and HUVEC cells, in part through the Caspase-1-dependent pathway. The rCPB1-induced pyroptosis of RAW264.7 cells could be blocked by inflammasome inhibitor MCC950. These results demonstrated that rCPB1 treatment of macrophages promoted the assembly of NLRP3 inflammasomes and activated Caspase 1; the activated Caspase 1 caused gasdermin D to form plasma membrane pores, leading to the release of inflammatory factors IL-18 and IL-1ß, resulting in macrophage pyroptosis. NLRP3 may be a potential therapeutic target for Clostridium perfringes disease. This study provided a novel insight into the pathogenesis of CPB1.

Human embryonic stem cell-derived mesenchymal stem cell secretome reverts silica-induced airway epithelial cell injury by regulating Bmi1 signaling.

Silicosis is an irreversible chronic pulmonary disease caused by long-term inhalation and deposition of silica particles, which is currently incurable. The exhaustion of airway epithelial stem cells plays a pathogenetic role in silicosis. In present study, we investigated therapeutic effects and potential mechanism of human embryonic stem cell (hESC)-derived MSC-likes immune and matrix regulatory cells (IMRCs) (hESC-MSC-IMRCs), a type of manufacturable MSCs for clinical application in silicosis mice. Our results showed that the transplantation of hESC-MSC-IMRCs led the alleviation of silica-induced silicosis in mice, accompanied by inhibiting epithelia-mesenchymal transition (EMT), activating B-cell-specific Moloney murine leukemia virus integration site 1 (Bmi1) signaling and airway epithelial cell regeneration. In consistence, the secretome of hESC-MSC-IMRC exhibited abilities to restore the potency and plasticity of primary human bronchial epithelial cells (HBECs) proliferation and differentiation following the SiO2 -induced HBECs injury. Mechanistically, the secretome resolved the SiO2 -induced HBECs injury through the activation of BMI1 signaling and restoration of airway basal cell proliferation and differentiation. Moreover, the activation of BMI1 significantly enhanced the capacity of HBEC proliferation and differentiation to multiple airway epithelial cell types in organoids. Cytokine array revealed that DKK1, VEGF, uPAR, IL-8, Serpin E1, MCP-1 and Tsp-1 were the main factors in the hESC-MSC-IMRC secretome. These results demonstrated a potential therapeutic effect of hESC-MSC-IMRCs and their secretome for silicosis, in part through a mechanism by activating Bmi1 signaling to revert the exhaustion of airway epithelial stem cells, subsequentially enhance the potency and plasticity of lung epithelial stem cells.

Critical Role of 6-Phosphogluconate Dehydrogenase in TAp73-Mediated Cancer Cell Proliferation.

Cancer cells frequently alter their metabolism to support biogenesis and proliferation and survive specific metabolic stressors. The glucose-associated pentose phosphate pathway (PPP) is crucial for cancer cell proliferation. In particular, 6-phosphogluconate dehydrogenase (6PGD), the second dehydrogenase in the PPP, catalyzes the decarboxylation of 6-phosphogluconate into ribulose 5-phosphate (Ru5P). However, the mechanisms controlling 6PGD expression in cancer cells remain unclear. Herein, we show that TAp73 increases Ru5P and NADPH production through 6PGD activation to counteract reactive oxygen species and protects cells from apoptosis. Moreover, 6PGD overexpression rescues the proliferation and tumorigenic ability of TAp73-deficient cells. These findings further establish the critical role of TAp73 on glucose metabolism regulation, demonstrating that TAp73 can activate 6PGD expression to support oncogenic cell growth. IMPLICATIONS: By transcriptional upregulation of 6PGD, TAp73 promotes the generation of Ru5P and NADPH, and enhances tumor cell proliferation.

Sensitive fluorescence assay for the detection of glyphosate with NACCu2+ complex.

Glyphosate is widely used as an herbicide in weed control. However, the excessive use and residue of glyphosate adversely affect the environment. Thus, a rapid and highly sensitive system must be developed for glyphosate detection. Herein, a novel turn-on fluorescent probe was designed and synthesized for glyphosate, that is N-butyl-1,8-naphthalimide-4-hydrazino-6-isopropyl-chromone (NAC). The fluorescence of NAC was quenched by the addition of Cu2+ to form NACCu2+ complex in dimethyl sulfoxide/2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (DMSO/HEPES, 9/1, v/v, pH = 7.0). Upon the addition of glyphosate, the fluorescence of NACCu2+ was recovered through chelation between Cu2+ and glyphosate. The NACCu2+ complex exhibited the desired linearity of glyphosate concentration under optimum conditions in the range of 0-40 µM with a low detection limit of 36 nM. Based on competitive coordination, NACCu2+ exhibited good sensitivity and selectivity for glyphosate. Moreover, NAC was successfully utilized to detect glyphosate in tap water, local water from Songhua River, soil, maize, and soybean with convenient operations, indicating a promising application in pesticide residue detection.

Secretome of hESC-Derived MSC-like Immune and Matrix Regulatory Cells Mitigate Pulmonary Fibrosis through Antioxidant and Anti-Inflammatory Effects.

Oxidative stress and inflammation are major drivers in the pathogenesis and progression of pulmonary fibrosis (PF). The mesenchymal stem cell (MSC) secretome has regenerative potential and immunomodulatory functions. Human embryonic stem cell (hESC)-derived MSC-like immune and matrix regulatory cells (IMRCs) are manufacturable with large-scale good manufacturing practice (GMP) preparation. In the present study, the antioxidative and anti-inflammatory properties and the therapeutic effect of the secretome of hESC-MSC-IMRC-derived conditioned culture medium (CM) (hESC-MSC-IMRC-CM) were investigated. Results revealed the capacities of hESC-MSC-IMRC-CM to reduce bleomycin (BLM)-induced reactive oxygen species (ROS), extracellular matrix (ECM) deposition, and epithelial-mesenchymal transition (EMT) in A549 cells. The administration of concentrated hESC-MSC-IMRC-CM significantly alleviated the pathogenesis of PF in lungs of BLM-injured mice, as accessed by pathohistological changes and the expression of ECM and EMT. A mechanistic study further demonstrated that the hESC-MSC-IMRC-CM was able to inhibit BLM-induced ROS and pro-inflammatory cytokines, accompanied by a reduced expression of Nox4, Nrf2, Ho-1, and components of the Tlr4/MyD88 signaling cascade. These results provide a proof of concept for the hESC-MSC-IMRC-derived secretome treatment of PF, in part mediated by their antioxidative and anti-inflammatory effects. This study thus reinforces the development of ready-to-use, cell-free hESC-MSC-IMRC secretome biomedicine for the treatment of PF in clinical settings.

[Knockdown of receptor tyrosine kinase-like orphan receptor 2 (ROR2) inhibits the autophagy of RAW264.7 mouse macrophages induced by BCG vaccine].

Objective To investigate the regulation of Wnt5a/receptor tyrosine kinase-like orphan receptor 2 (ROR2) signaling pathway on macrophage autophagy induced by Bacille Calmette Guerin (BCG) infection. Methods RAW264.7 cells were infected with BCG at 0, 2, 6, 12 and 24 hours, and the expressions of Wnt5a, ROR2 and autophagy-related protein microtubule-associated protein 1 light chain 3II (LC3II ) were detected by Western blot analysis. After RAW264.7 cells were treated with ROR2 small interfering RNA and BCG infection respectively or together, the protein expressions of autophagy-related genes 5 (ATG5), P62, beclin-1, ATG7 and LC3II in RAW264.7 cells were tested by Western blot analysis. Autophagy flux was detected by mRFP-GFP-LC3 double-label adenovirus assay. Results Compared with the control group, Wnt5a, ROR2 and LC3II had the highest expression in RAW264.7 cells 6 hours after BCG infection. Compared with the non-infected control group, the expressions of autophagy-related proteins ATG5, P62, beclin-1, ATG7 and LC3II showed an increase, along with increased number of autophagosomes and autophagolysosomes in RAW264.7 cells infected with BCG. Compared with BCG infected group, the expressions of the above proteins observed a decrease, and the number of autophagosomes and autophagolysosomes both descended in the co-treatment group with knockdown ROR2 and BCG infection. Conclusion Knockdown of ROR2 can inhibit autophagy in macrophages induced by BCG infection.

Vitamin C inhibits apoptosis in THP­1 cells in response to incubation with Mycobacterium tuberculosis.

Tuberculosis (TB) is a chronic and fatal zoonotic infectious disease caused by Mycobacterium tuberculosis (M. tb) infection. The THP-1 cell line is a cell model for studying the function, mechanism and signaling pathways of macrophages; macrophages are the primary host cells of M. tb. Macrophages are important for the progression of tuberculosis, as they affect the release of various inflammatory cytokines, including IL-1ß, IL-6 and TNF-α. Vitamin C is a trace element for the human body. Its biological efficacy depends on its redox abilities and its role as a cofactor in several enzymatic reactions. However, whether vitamin C can protect THP-1 cells from M. tb infection has not yet been reported. The present study aimed to further investigate the effects of vitamin C on M. tb infection-induced THP-1 cell injury and its mechanism. In the present study, MTT assay, reverse transcription-quantitative PCR, EdU cell proliferation assay, western blotting, immunohistochemistry, flow cytometry and TUNEL staining assays were used to assess the cell viability, inflammation and apoptotic levels of THP-1 cells induced by M. tb following vitamin C treatment. The effect of vitamin C on M. tb infection was also assessed using Balb/c mice; pulmonary injury was assessed by H&E staining of the lung tissue. The results demonstrated that vitamin C markedly attenuated cellular damage caused by M. tb infection. The results demonstrated that vitamin C reduced the expression of M. tb-induced apoptosis-related proteins (Cleaved-caspase-9, Cleaved-caspase-3, Bcl-2, Cyt-c) and inflammatory factors (IL-1ß, IL-6, NLRP3, TNF-α, IL-8, NF-κB) in THP-1 cells and reduced apoptosis. Overall, these results suggested that vitamin C may reduce lung damage caused by M. tb infection.

Ror2-mediated cholesterol accumulation regulates autophagic activity within BCG-infected macrophages.

Ror2 is a primary binding partner for the non-classical Wnt signaling pathway regulator Wnt5a that plays a central role in regulating the metabolic processing of lipids within the cell. Mycobacterium tuberculosis is an intracellular pathogen that utilizes the lipid substrate cholesterol as its primary source of carbon. Cholesterol accumulation can regulate autophagy, which is in turn associated with a variety of pathological conditions. This study was designed to explore the pathways that modulate Ror2-regulated cholesterol accumulation within macrophages infected by the mycobacterium Bacillus Calmette-Guerin (BCG). BCG infection of RAW264.7 cells resulted in increased Ror2 expression, cholesterol accumulation, and autophagic activity in addition to promoting the upregulation of cholesterol synthesis-related proteins and the downregulation of cholesterol transporter proteins. Ror2 knockdown, in contrast, reversed these phenotypic changes. Treatment with T0901317 decreased the aggregation of cholesterol within cells and suppressed BCG-induced autophagy, while OX-LDL had the opposite effect. Knocking down Ror2 further reduced cholesterol levels in the context of T0901317 or OX-LDL pretreatment, alleviating BCG-induced autophagy irrespective of either of these pretreatments. Together, these data indicate that Ror2 can shape the autophagic activity induced within macrophages upon BCG infection by modulating intracellular cholesterol levels.

Lycium barbarum polysaccharide attenuates Pseudomonas-aeruginosa pyocyanin-induced cellular injury in mice airway epithelial cells.

Background: Lycium barbarum berries have been utilized in Asia for many years. However, the mechanisms of its lung-defensive properties are indeterminate. Objective: We investigate whether L. barbarum polysaccharide (LBP) could weaken Pseudomonas aeruginosa infection-induced lung injury. Design: Mice primary air-liquid interface epithelial cultures were pretreated with LBP and subsequently treated with pyocyanin (PCN). Lung injury, including apoptosis, inflammation, and oxidative stress, was estimated by western blot, enzyme-linked immunosorbent assay, and real-time quantitative polymerase chain reaction, Real-time qPCR (Q-PCR). Flow cytometry was used to test cell apoptosis. Moreover, Balb/c mice were used to evaluate the tissue injury. We used hematoxylin-eosin staining and immunofluorescence to detect the expression of related proteins and tissue damage in mouse lungs and spleen. Results: The flow cytometric analysis shows the potential of LBP to reduce time-dependent cell death by PCN. Mechanistically, LBP reduces PCN-induced expression of proapoptotic proteins and caspase3 and induces the activation of Bcl-2 in mice bronchial epithelial cells. Similarly, LBP reduces PCN-induced intracellular reactive oxygen species (ROS) production. Moreover, LBP inhibits the production of inflammatory cytokines, Interleukin (IL-1ß), Tumor Necrosis Factor (TNF), IL-6, and IL-8. Our study confirms the ability of LBP to retard PCN-induced injury in mice lung and spleen. Conclusions: The inhibition of PCN-induced lung injury by LBP is capable of protecting mice cells from injury.

Vitamin D3 promotes autophagy in THP-1 cells infected with Mycobacterium tuberculosis.

Tuberculosis (TB) is a major disease that causes mortality worldwide. The lethality of this disease is a result of the contagious bacteria Mycobacterium tuberculosis (M.tb). Infection can inhibit phagosomal maturation, with M.tb mainly attacking macrophages and inhibiting autophagy and apoptosis. Vitamin D has been used to treat tuberculosis, whereby the active metabolite, 1,25-dihydroxyvitamin D, may enhance the immune response to M.tb. Moreover, macrophages infected with M.tb have a high demand for Ca2+. However, the mechanisms by which vitamin D3 protects against and treats TB remain unclear. In the present study, MTT assay showed that vitamin D3 decreased the viability of THP-1 cells in a dose- and time-dependent manner. Autophagy-related factors in THP-1 cells infected with M.tb were analyzed by western blotting and RT-qPCR and the results demonstrated that vitamin D3 significantly increased the expression level of p62, LC3Ⅱ/LC3Ⅰ, Beclin-1, ATG-5 and AMPK in THP-1 cells following M.tb infection. The Ca2+ concentration assay demonstrated that vitamin D3 may promoted cellular autophagy by inhibiting the concentration of Ca2+. Furthermore, the effect of vitamin D3 on M.tb infection was also assessed using Balb/c mice; pulmonary injury was assessed by H&E staining of the lungs tissue. The results demonstrated that vitamin D3 markedly attenuated cellular damage caused by M.tb infection. In conclusion, the present study indicated that vitamin D3 may activate cell autophagy signals by inhibiting the concentration of Ca2+. These data may improve understanding of the effect of vitamin D3 on M.tb infection and help determine the underlying mechanism of vitamin D3 to alleviate and treat the inflammatory response caused by TB.