The molecular targets of Kangai injection in gastric cancer by in silico network pharmacology approach and experiment confirmation.

INTRODUCTION: This study aimed to identify the phytochemical constituents that could target gastric cancer in Kangai injection using a network pharmacology-based approach. METHODS: Protein-protein interactions (PPI), Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were conducted utilizing String and OmicShare tools. In the in vitro experiments, the related mRNA and protein levels were assessed via real-time quantitative polymerase chain reaction and Western blotting, respectively. Cell proliferation was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay. RESULTS: Kangai injection comprises several compounds, which target multiple substrates and pathways related to gastric cancer. The PPI and Gene Ontology analyses revealed that tumor necrosis factor (TNF) was a hub gene. KEGG pathway enrichment analysis indicated that the the TNF pathway was significantly enriched. Kangai injection decreased the mRNA levels of TNFR2, TRAF2, PI3K, AKT, and IκBα and inhibited the phosphorylation of PI3K, AKT, and IκBα phosphorylations. Kangai injection inhibited cell proliferation, while TNFR2 overexpression or treatment with the PI3K activator 740 Y-P partially restored it. CONCLUSION: Kangai injection operates through multiple targets and pathways in gastric cancer, with the TNFR2/PI3K/AKT/NF-κB pathway playing a crucial role in its mechanism against gastric cancer.

Mycobacterium tuberculosis H37Rv Strain Increases the Frequency of CD3+TCR+ Macrophages and Affects Their Phenotype, but Not Their Migration Ability.

In mycobacterial infections, the number of cells from two newly discovered subpopulations of CD3+ myeloid cells are increased at the infection site; one type expresses the T cell receptor (CD3+TCRαß+) and the other does not (CD3+TCRαß-). The role of Mycobacterium tuberculosis (Mtb) virulence in generating these subpopulations and the ability of these cells to migrate remains unclear. In this study, monocyte-derived macrophages (MDMs) infected in vitro with either a virulent (H37Rv) or an avirulent (H37Ra) Mtb strain were phenotypically characterized based on three MDM phenotypes (CD3-, CD3+TCRαß+, and CD3+TCRαß-); then, their migration ability upon Mtb infection was evaluated. We found no differences in the frequency of CD3+ MDMs at 24 h of infection with either Mtb strain. However, H37Rv infection increased the frequency of CD3+TCRαß+ MDMs at a multiplicity of infection of 1 and altered the expression of CD1b, CD1c, and TNF on the surface of cells from both the CD3+ MDM subpopulations; it also modified the expression of CCR2, CXCR1, and CCR7, thus affecting CCL2 and IL-8 levels. Moreover, H37Rv infection decreased the migration ability of the CD3- MDMs, but not CD3+ MDMs. These results confirm that the CD3+ macrophage subpopulations express chemokine receptors that respond to chemoattractants, facilitating cell migration. Together, these data suggest that CD3+ MDMs are a functional subpopulation involved in the immune response against Mtb.

Temporal Splicing Switches in Elements of the TNF-Pathway Identified by Computational Analysis of Transcriptome Data for Human Cell Lines.

Alternative splicing plays an important role in numerous cellular processes and aberrant splice decisions are associated with cancer. Although some studies point to a regulation of alternative splicing and its effector mechanisms in a time-dependent manner, the extent and consequences of such a regulation remains poorly understood. In the present work, we investigated the time-dependent production of isoforms in two Hodgkin lymphoma cell lines of different progression stages (HD-MY-Z, stage IIIb and L-1236, stage IV) compared to a B lymphoblastoid cell line (LCL-HO) with a focus on tumour necrosis factor (TNF) pathway-related elements. For this, we used newly generated time-course RNA-sequencing data from the mentioned cell lines and applied a computational pipeline to identify genes with isoform-switching behaviour in time. We analysed the temporal profiles of the identified events and evaluated in detail the potential functional implications of alterations in isoform expression for the selected top-switching genes. Our data indicate that elements within the TNF pathway undergo a time-dependent variation in isoform production with a putative impact on cell migration, proliferation and apoptosis. These include the genes TRAF1, TNFRSF12A and NFKB2. Our results point to a role of temporal alternative splicing in isoform production, which may alter the outcome of the TNF pathway and impact on tumorigenesis.

CgA1AR-1 acts as an alpha-1 adrenergic receptor in oyster Crassostrea gigas mediating both cellular and humoral immune response.

We have now cloned an alpha-1 adrenergic receptor (A1AR) from the cDNA library of oyster Crassostrea gigas, designating as CgA1AR-1. The full length of CgA1AR-1 was 1149 bp and it encodes a protein of 382 amino acids containing a 7 transmembrane domain, whose putative topology was similar to the A1ARs in higher organisms and shared similarity of 19% with mammalian A1ARs according to the phylogenic analysis. After cell transfection of CgA1AR-1 into HEK293T cells and the incubation with its specific agonist norepinephrine (NE), the concentration of second messenger Ca2+ increased significantly (p < 0.05). But, this increasing of Ca2+ could be inhibited by adding A1AR antagonist DOX. Tissue distribution assays using qRT-PCR suggested that CgA1AR-1 mRNA was ubiquitously expressed in all the major tissues of oyster. LPS stimulation could induce the up-regulation of CgA1AR-1 mRNA in haemocytes from 12 h to 24 h post stimulation. Moreover, the blocking of CgA1AR-1 by DOX before LPS stimulation affected the mRNA expression of oyster TNF (CGI_10005109 and CGI_10006440) in haemocytes, resulting in the rise of haemocyte phagocytic rate and apoptosis index. In addition to cellular immunity, CgA1AR-1 was also involved in humoral immunity of oyster. Inhibition of CgA1AR-1 with DOX could repress the up-regulation of LZY and SOD activities caused by LPS stimulation. These results suggested that CgA1AR-1 acted as an α-1 adrenergic receptor in cetacholaminergic neuroendocrine-immune network mediating both cellular and humoral immune response.

Crocetin inhibits mast cell-dependent immediate-type allergic reactions through Ca2+/PLC/IP3 and TNF pathway.

Crocetin is a kind of glycocone naturally occurring in Crocus sativus L.. It is an active metabolite produced by biohydrolysis of Crocus sativus L.. Crocetin has anti-cardiovascular diseases and antioxidant effects, but its anti-allergic effect has not been reported. In this study, the inhibitory effect of crocetin on immunoglobulin E (IgE) - mediated allergic reaction and the mechanism of action were investigated. The passive cutaneous anaphylaxis (PCA) was used to elucidate the anti-allergic effects of crocetin in vivo. Degranulation assay, calcium imaging, and cytokine release assay were to evaluate the anti-allergic effect of crocetin in vitro. We found that crocetin IgE-mediated RBL-2H3 cell degranulation and allergy both in vitro and in vivo. The TNF pathway was inhibited by crocetin in our RNA-seq sequences, Furthermore, crocetin inhibits IgE-mediated calcium influx, and PLC / IP3 phosphorylation in RBL-2H3 cells. Our findings suggested that crocetin revealed prominent anti-allergy activity through TNF and Ca2+/PLC/IP3 pathway.

Experimental investigation for nonalcoholic fatty pancreas management using probiotics.

BACKGROUND: Nonalcoholic fatty pancreatitis (NAFP) presents a pressing challenge within the domain of metabolic disorders, necessitating further exploration to unveil its molecular intricacies and discover effective treatments. Our focus was to delve into the potential therapeutic impact of ZBiotic, a specially engineered strain of probiotic B. subtilis, in managing NAFP by targeting specific genes linked with necroptosis and the TNF signaling pathway, including TNF, ZBP1, HSPA1B, and MAPK3, along with their upstream epigenetic regulator, miR-5192, identified through bioinformatics. METHODS: Rats were subjected to either a standard or high-fat, high-sucrose diet (HFHS) for eight weeks. Subsequently, they were divided into groups: NAFP model, and two additional groups receiving daily doses of ZBiotic (0.5 ml and 1 ml/kg), and the original B. subtilis strain group (1 ml/kg) for four weeks, alongside the HFHS diet. RESULTS: ZBiotic exhibited remarkable efficacy in modulating gene expression, leading to the downregulation of miR-5192 and its target mRNAs (p < 0.001). Treatment resulted in the reversal of fibrosis, inflammation, and insulin resistance, evidenced by reductions in body weight, serum amylase, and lipase levels (p < 0.001), and decreased percentages of Caspase and Nuclear Factor Kappa-positive cells in pancreatic sections (p < 0.01). Notably, high-dose ZBiotic displayed superior efficacy compared to the original B. subtilis strain, highlighting its potential in mitigating NAFP progression by regulating pivotal pancreatic genes. CONCLUSION: ZBiotic holds promise in curbing NAFP advancement, curbing fibrosis and inflammation while alleviating metabolic and pathological irregularities observed in the NAFP animal model. This impact was intricately linked to the modulation of necroptosis/TNF-mediated pathway-related signatures.

Acquired semi-squamatization during chemotherapy suggests differentiation as a therapeutic strategy for bladder cancer.

Cisplatin-based chemotherapy remains the primary treatment for unresectable and metastatic muscle-invasive bladder cancers (MIBCs). However, tumors frequently develop chemoresistance. Here, we established a primary and orthotopic MIBC mouse model with gene-edited organoids to recapitulate the full course of chemotherapy in patients. We found that partial squamous differentiation, called semi-squamatization, is associated with acquired chemoresistance in both mice and human MIBCs. Multi-omics analyses showed that cathepsin H (CTSH) is correlated with chemoresistance and semi-squamatization. Cathepsin inhibition by E64 treatment induces full squamous differentiation and pyroptosis, and thus specifically restrains chemoresistant MIBCs. Mechanistically, E64 treatment activates the tumor necrosis factor pathway, which is required for the terminal differentiation and pyroptosis of chemoresistant MIBC cells. Our study revealed that semi-squamatization is a type of lineage plasticity associated with chemoresistance, suggesting that differentiation via targeting of CTSH is a potential therapeutic strategy for the treatment of chemoresistant MIBCs.

Single-Cell Profiles of Age-Related Osteoarthritis Uncover Underlying Heterogeneity Associated With Disease Progression.

Objective: Osteoarthritis (OA) is the most common chronic degenerative joint disease, which represents the leading cause of age-related disability. Here, this study aimed to depict the intercellular heterogeneity of OA synovial tissues. Methods: Single-cell RNA sequencing (scRNA-seq) data were preprocessed and quality controlled by the Seurat package. Cell cluster was presented and cell types were annotated based on the mRNA expression of corresponding marker genes by the SingleR package. Cell-cell communication was assessed among different cell types. After integrating the GSE55235 and GSE55457 datasets, differentially expressed genes were identified between OA and normal synovial tissues. Then, differentially expressed marker genes were overlapped and their biological functions were analyzed. Results: Totally, five immune cell subpopulations were annotated in OA synovial tissues including macrophages, dendritic cells, T cells, monocytes and B cells. Pseudo-time analysis revealed the underlying evolution process in the inflammatory microenvironment of OA synovial tissue. There was close crosstalk between five cell types according to the ligand-receptor network. The genetic heterogeneity was investigated between OA and normal synovial tissues. Furthermore, functional annotation analysis showed the intercellular heterogeneity across immune cells in OA synovial tissues. Conclusion: This study offered insights into the heterogeneity of OA, which provided in-depth understanding of the transcriptomic diversities within synovial tissue. This transcriptional heterogeneity may improve our understanding on OA pathogenesis and provide potential molecular therapeutic targets for OA.

Selected Clinical Features Fail to Predict Inflammatory Gene Expressions for TNF-α, TNFR1, NSMAF, Casp3 and IL-8 in Tendons of Patients with Rotator Cuff Tendinopathy.

The pathophysiology of rotator cuff tendinopathy is not fully understood, particularly in terms of the local inflammatory process. This study aimed to investigate the expression of selected molecules in the tumour necrosis factor (TNF)-α transduction pathway, including TNF-α, TNF receptor 1 (TNFR1), neutral sphingomyelinase activation associated factor (NSMAF), caspase 3 (Casp3), and interleukin (IL)-8, in patients with rotator cuff tendinopathy that had undergone surgical treatment. We included 44 participants that underwent arthroscopy, due to rotator cuff tendinopathy. Samples from the injured tendon were collected during arthroscopy, and RT-PCR was performed to determine gene expression. Pearson correlation analyses or U-Mann-Whitney test were performed to identify associations with the following parameters: sex, age at admission, body mass index, the presence of night pain, previous treatment (nonsteroidal anti-inflammatory drugs and/or steroids), medical history of the shoulder injury, upper subluxation of the humeral head, and the number of tendons injured. RT-PCR showed that the selected pro-inflammatory factors involved in the TNF-α signalling pathway expression levels were expressed in the tendon tissues. However, the levels of expression varied from patient to patient. Variations were over 250-fold for TNF-α, about 130-fold for TNFR1, NSMAF, and Casp3, and 1000-fold for IL-8. We could not confirm that any of the clinical parameters investigated were associated with the level of gene expression in the TNF-α pathway and IL-8.

CD3+ Macrophages Deliver Proinflammatory Cytokines by a CD3- and Transmembrane TNF-Dependent Pathway and Are Increased at the BCG-Infection Site.

Macrophages are essential cells of the innate immune response against microbial infections, and they have the ability to adapt under both pro- and anti-inflammatory conditions and develop different functions. A growing body of evidence regarding a novel macrophage subpopulation that expresses CD3 has recently emerged. Here, we explain that human circulating monocytes can be differentiated into CD3+TCRαß+ and CD3+TCRαß- macrophages. Both cell subpopulations express on their cell surface HLA family molecules, but only the CD3+TCRαß+ macrophage subpopulation co-express CD1 family molecules and transmembrane TNF (tmTNF). CD3+TCRαß+ macrophages secrete IL-1ß, IL-6 IP-10, and MCP-1 by both tmTNF- and CD3-dependent pathways, while CD3+TCRαß- macrophages specifically produce IFN-γ, TNF, and MIP-1ß by a CD3-dependent pathway. In this study, we also used a mouse model of BCG-induced pleurisy and demonstrated that CD3+ myeloid cells (TCRαß+ and TCRαß- cells) are increased at the infection sites during the acute phase (2 weeks post-infection). Interestingly, cell increment was mediated by tmTNF, and the soluble form of TNF was dispensable. BCG-infection also induced the expression of TNF receptor 2 on CD3+ myeloid cells, which increased after BCG-infection, suggesting that the tmTNF/TNFRs axis plays an important role in the presence or function of these cells in tuberculosis.

The cholinergic immune regulation mediated by a novel muscarinic acetylcholine receptor through TNF pathway in oyster Crassostrea gigas.

Muscarinic receptors, which selectively take muscarine as their ligand, are critical for the immunological and physiological processes in animals. In the present study, the open region frame (ORF) of a homologue of muscarinic acetylcholine (ACh) receptor (mAChR) was amplified from oyster Crassostrea gigas (named as CgmAChR-1), whose full length was 1983 bp and the protein it encoded contained 660 amino acids with a seven transmembrane region. Phylogeny analysis suggested that CgmAChR-1 shared homology with M5 muscarinic receptor found in invertebrates including Habropoda laboriosa, Acromyrmex echinatior and Echinococcus granulosus. After cell transfection of CgmAChR-1 into HEK293T cells and ACh incubation, the level of intracellular Ca(2+) and cAMP increased significantly (p < 0.05). Such trend could be reverted with the addition of M3 and M5 muscarinic receptor antagonists DAMP and DAR. The CgmAChR-1 transcripts were ubiquitously detectable in seven different tissues with the maximal expression level in adductor muscle. When the oysters received LPS stimulation, CgmAChR-1 mRNA expression in haemocyte was increased to the highest level (6.05-fold, p < 0.05) at 24 h, while blocking CgmAChR-1 using receptor antagonists before LPS stimulation promoted the expression of oyster TNF, resulting in the increase of haemocyte apoptosis index. These results suggested that CgmAChR-1 was the key molecule in cholinergic neuroendocrine-immune system contributing to the regulation of TNF expression and apoptosis process.

PKCδ-iPLA2-PGE2-PPARγ signaling cascade mediates TNF-α induced Claudin 1 expression in human lung carcinoma cells.

Claudin 1 (CLDN1) is a critical component of tight junction adhesion complexes that maintains the structural integrity of epithelial cell layers. Dysregulation of CLDN1 is associated with the growth and metastasis of human lung adenocarcinoma. TNF-α treatment was previously shown to increase expression of CLDN1 that mediated lung cancer cell morphology changes and migration. This study aimed to elucidate the molecular mechanisms involved in TNF-α induced CLDN1 expression in human lung carcinoma A549 cells. Chemical inhibition or siRNA downregulation of Src, PI3K, Akt, MAPKs, NFκB, caspase and PKC demonstrated that PKC, specifically PKCδ, is required for TNF-α induced CLDN1 expression. Further investigation of the PKC pathway revealed that CLDN1 expression is enhanced by the downstream molecules iPLA2, PGE2, 15-keto PGE2 and PPARγ. Conversely, inhibition of these molecules decreased CLDN1 expression. Additionally, a wound-healing assay demonstrated that TNF-α stimulation, PKC activation, prostaglandin treatment or PPARγ activation enhanced cell migration. In conclusion, TNF-α induced CLDN1 expression is regulated by the PKCδ-iPLA2-PGE2-PPARγ signaling cascade in human lung carcinoma A549 cells.

T11 target structure induced modulations of the pro-inflammatory and anti-infammatorycytokine expressions in experimental animals for glioma abrogation.

Glioma angiogenesis is the result of the interaction between cancer cells with endothelial cells, and the surrounding inflammatory cells. This interaction plays a crucial role in directing the neo-formation of blood vessels. In the carcinogenic milieu, inflammatory cytokines secreted from inflammatory cells affect endothelial cell functions that are indispensable for tumor growth and metastatic propagation. TNF-α, referred to as the 'inflammatory switch', has shown its potential as an inflammatory agent by activation of IL-8 and IL-6 through NF-κB mediated pathway. Therefore, inhibitors of angiogenesis appear to be promising therapeutic agents for advanced gliomas. Previous studies from our lab showed that T11TS, a membrane glycoprotein, has antiangiogenic and antineoplastic activities in experimental animals and human samples. The present experimental study was designed to evaluate the effect of T11TS therapy on inflammatory cytokine expression of TNF-α, IL-8, IL-6 and their downstream associated molecule NF-κB in vivo. Our results revealed that T11TS therapy induced downregulation of TNF-α, IL-8, IL-6, and NF-κB confirmed by FACS assay and ELISA. In situ-immunofluorescence results hint that T11TS has the efficacy to stop the inflammation related to angiogenesis. Moreover, upregulation of IL-4 and IL-10 in microglia after T11TS therapy helps in complete abrogation of glioma inflammation and angiogenesis. These effects might contribute to the antineoplastic activity of T11TS.