Phosphorylation Levels Of STAT3 Research Articles

Farnesoid X Receptor Attenuates the Tumorigenicity of Liver Cancer Stem Cells by Inhibiting STAT3 Phosphorylation

The Farnesoid X receptor (FXR) has recently been identified as being closely associated with the progression of primary hepatocellular carcinoma. Cancer stem cells (CSCs) play a crucial role in tumor initiation, progression, invasion, metastasis, recurrence, and drug resistance. The elucidation of the role and regulatory mechanism of FXR in CSCs is therefore deemed significant. Here, bioinformatics analysis has revealed a downregulation of FXR in hepatocellular carcinoma (HCC), which showed a negative correlation with HCC malignancy. This result was further confirmed through clinical sample analysis. Subsequently, CSCs were isolated from HCC cell lines and exhibited a significant decrease in the expression of FXR. The activation of FXR resulted in a remarkable inhibition of the proliferation, invasion, and tumorigenicity of CSCs. Furthermore, activated FXR prominently upregulated the expression of SOCS3 while suppressing STAT3 phosphorylation in CSCs. To further investigate this discovery, we established a DEN-induced HCC model in mice and observed that FXR-deficient mice exhibited heightened susceptibility to HCC. This was accompanied by decreased expression levels of SOCS3 and elevated expression and phosphorylation levels of STAT3, as well as significantly enhanced HCC CSCs markers and stemness-related genes expression in DEN-induced HCC tissues of FXR-deficient mice. Additionally, we also found a significant upregulation of CSCs markers and stemness-related genes within HCC clinical samples. Based on these findings, we postulated that targeted regulation of SOCS3 by FXR inhibits STAT3 phosphorylation, thereby exerting an inhibitory effect on CSCs.

Huaier inhibits the proliferation and migration of gastrointestinal stromal tumors by regulating the JAK2 / STAT3 signaling pathway.

Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the digestive tract, often accompanied by a high risk of recurrence and drug resistance. Huaier (Trametes robiniophila Murr), a traditional Chinese medicinal fungus, has demonstrated potent anticancer properties and is widely used as an adjuvant treatment for liver, breast, gastric, colon, and non-small cell lung cancers. However, its effects and molecular mechanisms in GIST remain unclear. This study aims to explore the inhibitory effects and underlying mechanisms of Huaier on GIST through network pharmacology and experimental validation. Initially, we utilized a publicly accessible database to identify the core targets and principal pathways associated with Huaier's therapeutic effects on gastrointestinal stromal tumors. To further evaluate its biological impact, cell viability, proliferation, migration, and invasion were assessed through CCK-8 and EdU assays, wound healing tests, and Transwell experiments. Apoptotic cell death was quantified using flow cytometry analysis. Additionally, the influence of Huaier extract on the expression levels of JAK2 and STAT3 proteins was examined via Western blotting. Finally, a subcutaneous xenograft mouse model was employed to investigate the anti-tumor efficacy of Huaier in vivo. In this study, GAPDH, TNF, STAT3, ESR1, EGFR, IL6, CCND1, PTGS2, BCL2L1, and MAPK3 were identified as shared molecular targets, with the JAK/STAT signaling pathway recognized as the pivotal regulatory mechanism. Experimental findings demonstrated that Huaier exerted inhibitory effects on the proliferation, migration, and invasion of GIST-T1 and GIST-882 cells, exhibiting both dose- and time-dependent responses. Furthermore, Huaier was found to promote apoptosis in these cells. Western blot analysis revealed that treatment with Huaier extract significantly decreased the phosphorylation levels of JAK2 and STAT3, thereby suppressing the activation of the JAK2 / STAT3 signaling cascade. In vivo experiments further substantiated these findings, showing that Huaier treatment markedly reduced tumor size and inhibited tumor progression. Our results suggest that Huaier may inhibit the growth of GIST cells by inhibiting the JAK2/STAT3 signaling pathway, reduce cell proliferation, induce apoptosis, reduce cell migration and invasion, and show anti-tumor effects in vivo and in vitro.

The JAK1/3 Inhibitor Tofacitinib Regulates Th Cell Profiles and Humoral Immune Responses in Myasthenia Gravis.

Tofacitinib, a first-generation Janus kinase (JAK) 1/3 inhibitor, is commonly used for treating ulcerative colitis and rheumatoid arthritis. However, its role in myasthenia gravis (MG) remains unclear. This study aimed to evaluate the immunomodulatory effects of tofacitinib on experimental autoimmune myasthenia gravis (EAMG) and peripheral blood mononuclear cells (PBMCs) from patients with MG. Flow cytometry, enzyme-linked immunosorbent assay (ELISA), quantitative reverse transcription polymerase chain reaction (qRT-PCR), and Western blot were used to evaluate the effects of tofacitinib on T helper (Th) cell profiles, humoral immune responses, and the JAK-signal transducer and activator of transcription (STAT) pathway proteins. In vivo, tofacitinib significantly ameliorated EAMG severity in rats, reducing the proportions of Th1, Th17 and memory B cells, and anti-acetylcholine receptor (AChR) antibodies levels, while increasing the proportions of regulatory T (Treg) cells. Invitro, tofacitinib administration resulted in a significant decrease in the proportions of Th1 and IgG-secreting B cell, and a significant upregulation of Treg cells in mononuclear cells (MNCs) from EAMG rats, which was consistent with findings in PBMCs from MG patients. Further analysis revealed that tofacitinib inhibited CD4+ T cell differentiation into Th1 by decreasing phosphorylated STAT1 levels, while promoting Treg differentiation via increased phosphorylated STAT5 levels in MNCs from EAMG rats. Tofacitinib modulates Th cell profiles and humoral immune responses by targeting the JAK-STAT pathway, suggesting its potential as a therapeutic candidate for MG. Further clinical studies are warranted to evaluate the efficacy and safety of tofacitinib in MG patients.

Botulinum toxin type A inhibits the formation of hypertrophic scar through the JAK2/STAT3 pathway.

Hypertrophic scar (HS) is a fibrous proliferative disorder that occurs in the dermis after skin injury. Studies have confirmed that Botulinum toxin type A (BTA) is effective in scar prevention and treatment. However, the specific mechanism remains uncertain. Hypertrophic scar fibroblasts (HSFs) and normal skin fibroblasts (NSFs) from the skin tissues of HS patients were isolated and cultured. Western blot analysis was conducted to measure the expression of JAK2/STAT3 pathway-related proteins. HSFs were treated with the JAK2 inhibitor (AG490) or agonist (C-A1). The CCK-8 assay, EdU staining, scratch-wound assay and transwell assay were used to examine the biological properties of HSFs. Western blot, immunofluorescence, and Sirius red staining were used to assess the fibrosis of HSFs. Additionally, a mouse full-thickness wound model was constructed to investigate the role of BTA in wound healing. The results showed that the JAK2 and STAT3 phosphorylation levels were markedly increased in HS tissues and HSFs. AG490 treatment reduced cell viability, proliferation and migration capacity, and inhibited the fibrosis of HSFs, whereas C-A1 treatment had the opposite effect. BTA treatment inhibited the JAK2/STAT3 pathway. BTA reduced cell viability, proliferation and migration ability, and inhibited the fibrosis of HSFs, while C-A1 intervention weakened the impact of BTA. Meanwhile, BTA promoted wound healing and reduced collagen deposition in vivo. In conclusion, BTA inhibited the JAK2/STAT3 pathway, which in turn hindered the proliferation, migration and fibrosis of HSFs, and promoted wound healing in mice.

LRP1B Suppresses Immunotherapy Efficacy in Lung Adenocarcinoma by Preventing Ferroptosis.

Immune biomarkers for non-small-cell lung cancer (NSCLC) are programmed death ligand 1 (PD-L1) and tumor mutational burden (TMB). However, they cannot accurately predict the effectiveness of immunotherapy. Identifying appropriate biomarkers that can differentiate between beneficiary groups is imperative. We identified lipoprotein receptor-related protein 1B (LRP1B) mutation as a potential biomarker for immunotherapy by analyzing clinical data, combined with bioinformatics analysis. The effects of LRP1B on ferroptosis were assessed using qRT-PCR, Western blotting, CCK-8 assay, and flow cytometry. The potential mechanism underlying the regulation of ferroptosis by LRP1B was elucidated using qRT-PCR, Western blotting, ChIP, and dual-luciferase reporter gene assays. Through the collection and analysis of clinical data, we had established that LRP1B mutations are closely associated with immunotherapy. Bioinformatics analysis revealed significant differences in the expression levels of PD-L1 and TMB between patients with LRP1B mutation and wild-type patients in lung adenocarcinoma (LUAD). Furthermore, we observed that patients with LRP1B mutation in LUAD had significantly higher levels of tumor-infiltrating lymphocytes (TILs) than wild-type patients. In addition, we found that patients with LRP1B mutation in LUAD had significantly prolonged progression-free survival (PFS) compared to wild-type patients. However, the differences of PD-L1 expression, TILs, and PFS were not observed in patients with LRP1B mutation in lung squamous cell carcinoma (LUSC). These findings provided strong evidence that LRP1B mutation was a potential biomarker for immunotherapy in LUAD. Moreover, our invivo experiments indicated that knockdown of LRP1B enhanced the efficacy of mPD-1, and mechanistic studies revealed that LRP1B regulated the sensitivity of cells to ferroptosis by modulating the expression of SLC7A11 through altering the phosphorylation level of STAT3. Further analysis revealed that LRP1B knockdown promoted immunotherapy invivo. Our results confirmed that LRP1B affected the efficacy of immunotherapy by modulating the sensitivity of NSCLC cells to ferroptosis. LRP1B mutations represent a highly promising immunotherapeutic biomarker for NSCLC.

MiR-144/451 attenuates lipopolysaccharide-induced lung inflammation by downregulating Rac1 and STAT-3 in macrophages.

MicroRNAs have been shown to play a critical role in lung inflammatory diseases. Here, we report that knocking out miR-144/451 in mice exacerbates lipopolysaccharide (LPS)-induced lung inflammation. The lung inflammation in mice was induced by intratracheal instillation of LPS. Loss-of-function experiments demonstrated that miR-144/451 gene knockout (KO) increased LPS-induced lung inflammation and oxidant stress compared with wild-type (WT) mice, as manifested by increased total bronchoalveolar lavage fluid cells and neutrophil counts, elevated TNF-α and IL-6 levels in bronchoalveolar lavage fluid, enhanced myeloperoxidase activity, and reduced catalase and glutathione peroxidase activity in lung tissues. We also found that LPS significantly decreased miR-451 expression in lung tissues and macrophages; while miR-451 overexpression in LPS-induced RAW264.7 cells remarkably reduced TNF-α and IL-6 levels as well as reactive oxygen species (ROS) production, suggesting a feedback loop might exist in inflammatory cells. Rac1 mRNA and protein levels were downregulated in miR-451-overexpressed RAW264.7 cells. Ex vivo stimulation experiments, performed using alveolar macrophages isolated from miR-144/451 KO mice, confirmed that Rac1 inhibitor alleviated levels of TNF-α and ROS in response to LPS stimulation compared with WT controls. Luciferase reporter assay demonstrated that STAT-3 is a direct target of miR-451. STAT-3 protein levels were elevated in miR-144/451 KO macrophages. LPS treatment also resulted in higher phosphorylation levels of STAT-3 in macrophages from KO mice than in WT cells. Our study identified miR-144/451 as an anti-inflammatory factor in LPS-induced lung inflammation that acts by downregulating Rac1 and STAT-3.

Over Activation of IL-6/STAT3 Signaling Pathway in Juvenile Dermatomyositis.

Juvenile dermatomyositis (JDM) is characterized by persistent non-purulent inflammation in the muscle and skin. The underlying mechanisms still remain uncertain. This study aims to elucidate the mechanism of interleukin-6 (IL-6) activation of Janus kinase/signal transducer and activator of transcription 3 pathway (JAK/STAT3), contributing to the pathogenesis of JDM. Serum IL-6 levels were compared between 72 newly diagnosed patients with JDM and the same patient cohort in treatment remission. Single-cell RNA sequencing (scRNA-seq) was employed to identify differential signaling pathway expression in muscle biopsy samples from two patients with JDM and healthy controls. Immunohistochemistry was used to examine differences in STAT3 phosphorylation between JDM and control muscle tissues. In vitro, skeletal muscle cell lines were stimulated with IL-6, and the transcription levels of genes related to mitochondrial calcium channels were quantified via reverse transcription-polymerase chain reaction (RT-PCR). Reactive oxygen species (ROS) production was measured in both IL-6 treated and untreated groups. ROS levels were then compared between IL-6 receptor antagonist pre-treated skeletal muscle cells and untreated cells. IL-6 levels in newly onset patients with JDM were significantly higher compared to the same patient cohort in remission states (p < 0.0001). Serum IL-6 was significantly increased in patients with negative myositis specific antibody (MSA), positive melanoma differentiation associated protein 5 (MDA5) and positive nuclear matrix protein 2 (NXP2), yet not for JDM with positive transcriptional intermediary factor γ(TIF1γ), based on subgroup analysis. ScRNA-seq analysis of muscle biopsies from patients with MDA5-positive JDM and patients with MSA negative JDM revealed abnormal activation of the JAK/STAT3 pathway in skeletal myocytes, macrophages, and vascular endothelial cells. The phosphorylation levels of STAT3 were elevated in active JDM cases. Transcription of the calcium channel modulation gene sarcolipin (SLN) was significantly higher in JDM primary skeletal muscle cells compared to normal cells. In vitro, IL-6 enhanced SLN transcription and induced ROS production, and blocking the IL-6 receptor resulted in decreased ROS generation in skeletal muscle cells. IL-6/JAK/STAT3 signaling pathway was abnormally activated in patients with JDM. IL-6 may be involved in the pathogenesis of muscle damage by triggering the development of calcium overload and production of ROS. Blockade of the IL-6/JAK/STAT3 pathway can be a potential treatment option for JDM, especially MDA5-positive patients and those who are negative for MSA.

Network pharmacology and phytochemical composition combined with validation in vivo and in vitro reveal the mechanism of platycodonis radix ameliorating PM2.5-induced acute lung injury

Ethnopharmacological relevancePlatycodonis radix (PR), the root of Platycodon grandiflorus (Jacq.) A. DC., is a traditional Chinese medicine recognized for its dual role as both a medicinal and dietary substance, exhibiting significant anti-inflammatory properties. It is frequently utilized in the treatment of lung diseases. However, the molecular mechanisms by which PR exerts its effects in the treatment of acute lung injury (ALI) remain unclear. Aim of the studyThis study presents a novel strategy that integrates network pharmacology, molecular docking, untargeted metabolomics analysis and experimental validation to investigate the molecular mechanisms through which PR treats ALI. Materials and methodInitially, the bioactive components of PR, along with its targets and pathways in the treatment of ALI, were identified using network pharmacology. Following this, preliminary validation was conducted through molecular docking. The active ingredients in the aqueous extract of PR were characterized using HPLC-MS. Finally, in vivo and in vitro experiments were performed to further validate the findings from the network pharmacology. ResultsA total of 14 bioactive components and 156 effective targets were identified using the TCMSP, DisGeNET, Genecard, OMIM databases and Venny 2.1.0. Protein-protein interaction (PPI) analysis revealed 22 core targets including TP53, AKT1, STAT3 and JUN. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses indicated that these targets primarily participate in the regulation of cellular apoptosis, lung cancer and inflammatory pathways. Molecular docking demonstrated that four bioactive components exhibited strong affinities with their respective docking targets. LC-MS analysis confirmed that the aqueous extract of PR contained 87 components, including two active ingredients identified through network pharmacology and molecular docking. Preliminary validation was conducted in mice with ALI induced by acute PM2.5 exposure, revealing that the aqueous extract of PR reduced inflammatory factor levels in bronchoalveolar lavage fluid, enhanced antioxidant capacity in lung tissue, and decreased lung cell apoptosis in PM2.5-exposed mice. Notably, PR alleviated PM2.5-induced ALI through the STAT3, JUN, and AKT1 signaling pathways. Similarly, the results of in vitro intervention experiments further confirmed that the aqueous extract of PR protected pulmonary epithelial cells against PM2.5 exposure through activating AKT1 sinalling pathway, and inhibiting STAT3 and JUN signalling pathways. ConclusionThis study identifies the active components of PR and elucidates the molecular mechanisms by which PR alleviates ALI, specifically by inhibiting the phosphorylation levels of STAT3 and c-JUN, or by activating the phosphorylation level of AKT1. These results provide a foundational basis for the application of PR in the treatment or prevention of lung injuries induced by particulate matter.

Inhibition of CXCR2 as a therapeutic target for chronic post-surgical pain: Insights from animal and cell models.

Studies have shown that chemokines can stimulate the migration and activation of microglia to cause chronic post-surgical pain (CPSP). However, the involvement of C-X-C motif chemokine receptor 2 (CXCR2) as a new chemotactic factor in regulating CPSP and its underlying mechanism remains unclear. This study is to investigate the role of CXCR2 in the development of CPSP and reveal the underlying mechanism. A rat model of skin/muscle incision and retraction was established, and treated with or without SB225002 (a selective inhibitor of CXCR2). In addition, the primary microglia cells induced by lipopolysaccharide were applied as an in vitro model for CPSP and treated individually with si-negative control (NC), si-CXCR2, si-CXCR2+Interleukin (IL)-6 (an agonist of the janus kinase (JAK)/signal transducers and activators of transcription (STAT)3 signaling pathway), si-CXCR2+IL-6+si-NC, or si-CXCR2+IL-6+si-exchange protein 1 directly activated by cAMP (EPAC1). Results from the database analysis showed that CXCR2 and JAK/STAT3 signaling pathway-related genes, including JAK1, STAT3, and EPAC1, were mainly involved in the development of CPSP. Inhibition of CXCR2 expression not only inhibited the reduction of foot pain threshold in CPSP models but also led to a decreased expression of CXCR2 and the phosphorylation levels of JAK and STAT3 in both animal and cell models. Furthermore, inhibition of EPAC1 expression can hinder the regulatory function of CXCR2. This study indicated that the high expression of CXCR2 activates the JAK1/STAT3 signaling pathway, enhances EPAC1 activation in microglial cells, and exacerbates CPSP.

Effects and Mechanisms of the Xianhecao-Huanglian Drug Pair on Autophagy-Mediated Intervention in Acute Inflammatory Bowel Disease via the JAK2/STAT3 Pathway

To explore the effects and mechanisms of the Xianhecao-Huanglian drug pair on autophagy-mediated intervention in acute inflammatory bowel disease (IBD) via the JAK2/STAT3 pathway. The study examined the underlying mechanisms of action of Xianhecao (APL) and Huanglian (CR) using a mouse model of dextran sodium sulfate (DSS)-induced acute inflammatory bowel disease (IBD) and in an in vitro model of IBD induced by lipopolysaccharide (LPS). The assessment of the therapeutic efficacy of the Xianhecao-Huanglian drug combination in a mouse model of IBD caused by DSS included the following parameters: Assessment of weight loss or gain. Measurement of the disease activity index (DAI). Assessment of histological damage. Determination of organ index. Measurement of colon length. Ascertain the levels of inflammatory cytokines in the intestinal tissues and serum of mice. Immunohistochemistry (IHC) for the measurement of tight junction protein concentrations in the colon mucosa, including ZO-1, claudin-1, and occludin. Measurement of mucin levels, specifically Mucin 2 (Muc2). Hematoxylin and eosin (HE) staining for the observation of histopathological alterations in colonic tissues. Examining the effect on goblet cells using periodic acid-Schiff (PAS) labeling. Application of Western blot and immunofluorescence techniques for the detection of autophagy-related markers in colonic tissues and proteins associated with the JAK2/STAT3 pathway. A cell inflammation model of IBD was induced through LPS stimulation, and a serum containing the Xianhecao-Huanglian drug pair (referred to as ACHP-DS) was formulated. Cell viability, anti-proinflammatory cytokines, tight junction proteins, mucins, autophagy-related markers, and the JAK2/STAT3 signaling pathway were assessed. The Xianhecao-Huanglian drug pair significantly ameliorated the symptoms and survival quality of acute IBD mice, reducing the disease activity index score, raising MUC2 secretion and tight junction protein expression to improve the integrity of the intestinal barrier, and preserving goblet cell function; thus, protecting the intestines. It effectively restrained triggering the signaling pathway that involves JAK2 and STAT3, leading to the suppression of inflammation and amelioration of colonic inflammation damage. Additionally, it induced autophagy in mouse colonic tissues.The in vitro experiments demonstrated that the Xianhecao-Huanglian drug combination enhanced the viability of LOVO and NCM460 cells when exposed to LPS stimulation. Furthermore, it suppressed the production of inflammatory cytokines such as IL-6, IL-1β, as well as TNF-α, whilst increasing the production of IL-10, ZO-1, along with MUC2. These effects collectively led to the alleviation of inflammation and the restoration of mucosal integrity. The results were consistent with what was shown in the in vivo trial. Moreover, the medication demonstrated effectiveness in reducing JAK2 along with STAT3 phosphorylation levels in the LPS-induced inflammatory model of IBD cells. The intervention with either the Xianhecao-Huanglian drug combination-containing serum or the JAK2/STAT3 pathway inhibitor AG490 reversed the pro-inflammatory effects and increased autophagy levels in the LPS-stimulated cells. The Xianhecao-Huanglian drug combination modulates the JAK2/STAT3 pathway, leading to the induction of autophagy, which serves as an intervention for IBD.

IFITM10 Enhance Tumor Angiogenesis and Promotes Cancer Progression through STAT3 Activation.

Humankind have been struggling with colorectal cancer (CRC) for long period with its rapid progression and invasive metastasis. By hyperactivating IL-6/STAT3 signaling, CRC facilitates the capacity of angiogenesis to plunder massive nutrients and develops gradually under harsh condition. The Cancer Genome Atlas database was analyzed for acquiring interferon-γ inducible protein 10 (IFITM10) expression levels and their correlation with clinical outcomes. The cell angiogenic ability were assessed by Cell Counting Kit-8 (CCK-8) and tube formation assay. Immunofluorescence, Western blot, and enzyme-linked immunosorbent assay (ELISA) assay were using to assess potential mechanism. In our study, we find that IFITM10 is upregulated in CRC and is positively related with tumor angiogenesis. We also find that IFITM inhibition decreased STAT3 phosphorylation level and IFITM10-mediated angiogenesis depends on STAT3 activation. Furthermore, our data suggests that IFITM10 may be a key prognostic biomarker in colorectal cancer. Together, our study suggests that IFITM10 enhance angiogenesis through STAT3 activation during CRC progression, which highlighting its potency as a therapeutic target for colorectal cancer.

Manuka Honey Inhibits Human Breast Cancer Progression in Preclinical Models.

Manuka honey (MH) exhibits potential antitumor activity in preclinical models of a number of human cancers. Treatment in vitro with MH at concentrations ranging from 0.3 to 5.0% (w/v) led to significant dose-dependent inhibition of proliferation of human breast cancer MCF-7 cells, but anti-proliferative effects of MH were less pronounced in MDA-MB-231 breast cancer cells. Effects of MH were also tested on non-malignant human mammary epithelial cells (HMECs) at 2.5% w/v, and it was found that MH reduced the proliferation of MCF-7 cells but not that of HMECs. Notably, the antitumor activity of MH was in the range of that exerted by treatment of MCF-7 cells with the antiestrogen tamoxifen. Further, MH treatment stimulated apoptosis of MCF-7 cells in vitro, with most cells exhibiting acute and significant levels of apoptosis that correlated with PARP activation. Additionally, the effects of MH induced the activation of AMPK and inhibition of AKT/mTOR downstream signaling. Treatment of MCF7 cells with increased concentrations of MH induced AMPK phosphorylation in a dose-dependent manner that was accompanied by inhibition of phosphorylation of AKT and mTOR downstream effector protein S6. In addition, MH reduced phosphorylated STAT3 levels in vitro, which may correlate with MH and AMPK-mediated anti-inflammatory properties. Further, in vivo, MH administered alone significantly inhibited the growth of established MCF-7 tumors in nude mice by 84%, resulting in an observable reduction in tumor volume. Our findings highlight the need for further research into the use of natural compounds, such as MH, for antitumor efficacy and potential chemoprevention and investigation of molecular pathways underlying these actions.

Wutou decoction alleviates arthritis inflammation in CIA mice by regulating Treg cell stability and Treg/Th17 balance via the JAK2/STAT3 pathway

Ethnopharmacological relevanceWutou Decoction (WTD) is a classic traditional Chinese medicine formula, which has shown clinical efficacy in treating rheumatoid arthritis (RA). The Treg stability and Th17/Treg imbalance is an important immunological mechanism in RA progression. Whether WTD regulates CD4+ T cell subsets has not been thoroughly investigated yet. Aim of the studyThis study aimed to explore the potential role and mechanisms of WTD in regulating the diminished stability of Treg cells and the imbalance of CD4+ T cell subsets via in vivo and in vitro experiments. Materials and methodsFirstly, the therapeutic effects of WTD on the collagen-induced arthritis (CIA) mouse and its potential regulatory function on CD4+ T cell subsets were evaluated in vivo. Animal specimens were collected after 31 days of treatment with WTD. The anti-arthritic and anti-inflammatory effects of WTD were assessed through arthritis scoring, body weight, spleen index, serum IL-6 levels, and micro-PET/CT imaging. Gene enrichment analysis was performed to evaluate the activation T cell-related signaling pathway. Flow cytometry was used to determine the proportions of CD4+ T cell subsets in vitro and in vitro. Additionally, ELISA was used to assess the secretion of IL-10 and TGF-β by Treg cells under inflammatory conditions. The suppressive function of Treg cells on cell proliferation under inflammatory conditions was examined using CFSE labeling. Immunofluorescence staining was performed to detect the phosphorylation levels of STAT3 in CD4+ T cells from mouse spleen tissues. Western blotting was used to evaluate the phosphorylation levels of JAK2/STAT3 in Treg cells. ResultsWTD significantly alleviated joint inflammation in CIA mice. WTD reduced serum IL-6 levels in CIA mice, improved their body weight and spleen index. WTD treatment inhibited the activation of CD4+ T cell subgroup-related signaling in the joint tissues of CIA mice. In vitro and in vitro experiments showed that WTD increased the proportion of Treg cells and decreased the proportion of Th17 cells in CIA mice spleen. Furthermore, WTD promoted the secretion of IL-10 and TGF-β by Treg cells and enhanced the inhibitory capacity of Treg cells on cell proliferation under inflammatory conditions. Immunofluorescence detected decreased STAT3 phosphorylation levels in CD4+ T cells from CIA mice spleen, while western blotting revealed a decrease in JAK2/STAT3 phosphorylation levels in Treg cells in vitro. ConclusionsInhibiting JAK2/STAT3 phosphorylation is a potential mechanism through which WTD improves Treg cell stability, balances CD4+ T cell subsets, and attenuates RA joint inflammation.

GPR160 regulates the self-renewal and pluripotency of mouse embryonic stem cells via JAK1/STAT3 signaling pathway

G-protein-coupled receptors (GPCRs) are the largest family of transmembrane receptors and regulate various physiological and pathological processes. Despite extensive studies, the roles of GPCRs in mouse embryonic stem cells (mESCs) remain poorly understood. Here, we show that GPR160, a class A member of GPCRs, is dramatically downregulated concurrent with mESC differentiation into embryoid bodies in vitro. Knockdown of Gpr160 leads to downregulation of the expression of pluripotency-associated transcription factors and upregulation of the expression of lineage markers, accompanying with the arrest of the mESC cell-cycle in the G0/G1 phase. RNA-seq analysis shows that GPR160 participates in the JAK/STAT signaling pathway crucial for maintaining ESC stemness, and the knockdown of Gpr160 results in the downregulation of STAT3 phosphorylation level, which in turn is partially rescued by colivelin, a STAT3 activator. Consistent with these observations, GPR160 physically interacts with JAK1, and cooperates with leukemia inhibitory factor receptor (LIFR) and gp130 to activate the STAT3 pathway. In summary, our results suggest that GPR160 regulates mESC self-renewal and pluripotency by interacting with the JAK1–LIFR–gp130 complex to mediate the JAK1/STAT3 signaling pathway.

Icariside I reduces breast cancer proliferation, apoptosis, invasion, and metastasis probably through inhibiting IL-6/STAT3 signaling pathway.

Breast cancer is a common malignancy in women. More than 90% of breast cancer deaths are caused by metastasis. Epimedii Folium (EF) is a commonly used herb with anti-tumor benefits, but its underlying mechanisms and active components for breast cancer prevention are little understood. This study assessed the therapeutic role of Icariside I (ICS I) in Epimedium flavonoids (EF) on lung metastasis of breast cancer, including the underlying mechanism. Western blot, RT-qPCR, wound healing assay, colony formation assay, and flow cytometry were used to investigate the inhibition of breast cancer cells growth and migration by EF and ICS I through disrupting the IL-6/STAT3 pathway. Combined with 4T1 breast cancer model in mice, Western blot, RT-qPCR, Hematoxylin and Eosin staining, immunohistochemistry were used to evaluate the therapeutic role of ICS I in proliferation, apoptosis, invasion, and metastasis of breast cancer. EF can inhibit STAT3 phosphorylation and reduce the colony formation and migration of breast cancer cells. Detecting the active ingredients in EF, we found ICS I can reduce the activation of STAT3 in 4T1 breast cancer cells, impair colony formation and migration. Moreover, ICS I induced cells G1 phase arrest and modulated Cyclin D1, CDK4, bcl-2, and bax to inhibit proliferation and survival of breast cancer cells. Similarly, the in vivo studies demonstrated that ICS I significantly suppressed tumor development and lung metastasis in the 4T1 mouse model. Tumor cells in vehicle group were arranged in a spoke-like pattern with obvious heterogeneity, and multinucleated tumor giant cells were seen. But, the tumor cells in the ICS I group were disorganized and necrotic lysis was seen in some areas. In ICS I-treated group, tumors' STAT3 phosphorylation level, IL-6, Cyclin D1, CDK4, bcl-2, and vimentin expression were downregulated, bax and cleaved caspase 3 expression were upregulated. In the lung tissue, we could find less metastasis of breast cancer cells and less lung injury in the ICS I group. Besides, the expression of metastasis-related genes MMP9 and vimentin was decreased in the lung tissue of ICS I group. These findings suggest that ICS I can inhibit breast cancer proliferation, apoptosis, invasion and metastasis probably via targeting IL-6/STAT3 pathway. Therefore, ICS I has the potential to become an innovative therapeutic candidate to breast cancer prevention and treatment.

Amplified STAT3 increases expression of the CD39 ectoenzyme and impairs function of CD8+ T cells.

Abstract Regulation of T cell signaling is paramount to mount an appropriate immune response and protect the host from pathophysiology. Here, we study the impact on CD8+ T cells in monogenic inborn error of immunity patients exhibiting dysregulated signal 3 (cytokine signaling) pathways resulting from STAT3 gain- or loss-of-function variants (STAT3 GOF and STAT3 LOF). Using high dimensional immune profiling, we demonstrate increased expression of the ectoenzyme, CD39, which hydrolyzes extracellular ATP, on STAT3 GOF patient CD8+ T cells. Moreover, CD8+ T cells from two mouse models of STAT3 GOF (STAT3+/G421R and NOD-STAT3+/K392R) recapitulated this increase in CD39. In vitro, STAT3-activating cytokines (e.g., IL-6, IL-21, IL-27, IL-10) augment CD39 induction in a TCR-dependent manner in healthy donor and in STAT3 GOF CD8+ T cells. In contrast, STAT3 LOF patients show diminished CD39 induction. We find a positive correlation between CD39 induction and phosphorylated STAT3 levels while STAT3 inhibition prevents this cytokine-driven augmentation of CD39. Finally, acutely activated, and sorted CD39+CD8+ T cells from healthy donors inhibit bystander CD8+ T cell cytokine production (e.g., IFN-g). Pharmacologic inhibition of CD39 enzymatic activity or genetic ablation via CRISPR/Cas9 editing reversed decreases in cytokine production. Together, our data demonstrate a mechanistic connection between amplified STAT3 signaling and CD39 levels which is capable of impairing CD8+ T cell function.

Dihydroartemisinin inhibits tumor progress via blocking ROR1-induced STAT3-activation in non-small cell lung cancer

In non-small cell lung cancer (NSCLC), identifying a component with certain molecular targets can aid research on cancer treatment. Dihydroartemisinin (DHA) is a semisynthetic derivative of artemisinin which induced the anti-cancer effects via the STAT3 signaling pathway, but the underlying molecular mechanism is still elusive. In this study, we first proved that DHA prohibits the growth of tumors both in vitro and in vivo. Data from transcriptomics showed that DHA reduced the expression level of the genes involved in cell cycle-promoting and anti-apoptosis, and most importantly, DHA restricted the expression level of receptor tyrosine kinase-like orphan receptor 1 (ROR1) which has been reported to have abnormal expression on tumor cells and had close interaction with STAT3 signaling. Then, we performed comprehensive experiments and found that DHA remarkably decreased the expression of ROR1 at both mRNA and protein levels and it also diminished the phosphorylation level of STAT3 in NSCLC cell lines. In addition, our data showed that exogenously introduced ROR1 could significantly enhance the phosphorylation of STAT3 while blocking ROR1 had the opposite effects indicating that ROR1 plays a critical role in promoting the activity of STAT3 signaling. Finally, we found that ROR1 overexpression could partially reverse the decreased activity of STAT3 induced by DHA which indicates that DHA-induced anti-growth signaling is conferred, at least in part, through blocking ROR1-mediated STAT3 activation. In summary, our study indicates that in NSCLC, ROR1 could be one of the critical molecular targets mediating DHA-induced STAT3 retardation.

Protective effect of salidroside on renal damage in diabetic nephropathy mice by regulating RAGE/JAK1/STAT signaling pathway

This study aims to investigate the protective effect of salidroside(SAL) on renal damage in diabetic nephropathy(DN) mice based on the receptor for advanced glycation end products/janus activated kinase 1/signal transduction and activator of transcription 3(RAGE/JAK1/STAT3) signaling pathway. The mouse DN model was established by high-fat/high-sucrose diets combined with intraperitoneal injection of streptozocin(STZ). Mice were randomly divided into normal group, model group, low-dose SAL group(20 mg·kg~(-1)), high-dose SAL group(100 mg·kg~(-1)), and metformin group(140 mg·kg~(-1)), with 12 mice in each group. After establishing the DN model, mice were given drugs or solvent intragastrically, once a day for consecutive 10 weeks. Body weight, daily water intake, and fasting blood glucose(FBG) were measured every two weeks. After the last dose, the glucose tolerance test was performed, and the samples of 24-hour urine, serum, and kidney tissue were collected. The levels of 24 hours urinary total protein(24 h-UTP), serum creatinine(Scr), blood urea nitrogen(BUN), triglyceride(TG), total cholesterol(TC), low density lipoprotein cholesterol(LDL-C), and high density lipoprotein cholesterol(HDL-C) were detected by biochemical tests. Periodic acid-schiff(PAS) staining was used to observe the pathological changes in the kidney tissue. The protein expressions of α-smooth muscle actin(α-SMA), vimentin, and advanced glycation end products(AGEs) in kidneys were detected by immunohistochemical staining. The activities of superoxide dismutase(SOD), catalase(CAT), glutathione peroxidase(GSH-PX), and the level of malondialdehyde(MDA) in kidneys were detected by using a corresponding detection kit. Enzyme-linked immunosorbent assay(ELISA) was used to detect the levels of AGEs, carboxymethyllysine(CML), and carboxyethyllysine(CEL) in serum. The protein expressions of RAGE and the phosphorylation level of JAK1 and STAT3 in kidneys were detected by Western blot. Compared with the normal group, the levels of FBG, the area under the curve of glucose(AUCG), water intake, kidney index, 24 h-UTP, tubular injury score, extracellular matrix deposition ratio of the renal glomerulus, the serum levels of Scr, BUN, TG, LDL-C, AGEs, CEL, and CML, the level of MDA, the protein expressions of α-SMA, vimentin, AGEs, and RAGE, and the phosphorylation level of JAK1 and STAT3 in kidney tissue were increased significantly(P&lt;0.01), while the level of HDL-C in serum and the activity of SOD, CAT, and GSH-PX in kidney tissue were decreased significantly(P&lt;0.01). Compared with the model group, the above indexes of the high-dose SAL group were reversed significantly(P&lt;0.05 or P&lt;0.01). In conclusion, this study suggests that SAL can alleviate oxidative stress and renal fibrosis by inhibiting the activation of AGEs-mediated RAGE/JAK1/STAT3 signaling axis, thus playing a potential role in the treatment of DN.

In vitro effect of diazoxon on cell signaling and second messengers in Nile tilapia (Oreochromis niloticus) leukocytes.

The physiological and molecular responses of leukocytes are altered by organophosphate pesticides (OPs). Some reports have shown that diazinon (DZN) causes immunotoxic effects; diazoxon (DZO), the oxon metabolite of DZN, is attributed to influence the immune response by affecting the leukocyte cholinergic system. In this study, the in vitro effects of DZO on molecules involved in cell signaling (cAMP, IP3, DAG, JAK1, and STAT3), which play a crucial role in the activation, differentiation, and survival of leukocytes, were evaluated. Data indicate that DZO leads to a decrease in cAMP concentration and an increase in basal IP3 levels. However, DZO does not affect basal levels of JAK1 and STAT3 phosphorylation. Instead, DZO inhibits leukocyte responsiveness to PMA and ionomycin, substances that, under normal conditions, enhance JAK/STAT signaling. These findings demonstrate that DZO significantly affects key molecular parameters related to cell signaling.

Abstract 3245: Use of hypomethylating agents to reprogram the epigenome in large granular lymphocyte leukemia

Abstract Large granular lymphocyte (LGL) leukemia is a rare heterogeneous blood cancer that is characterized by the clonal lymphoproliferation of CD8+ T cells (T-LGL leukemia) or, less frequently, natural killer cells (NK-LGL leukemia). These cells evade activation induced cell death, allowing them to accumulate as highly cytotoxic lymphocytes that contribute to autoimmune attacks, neutropenia, and anemia. Frontline treatments are limited to repurposed, nonspecific immunosuppressive agents with limited efficacy and incomplete response rates. As there are no FDA-approved antineoplastic agents for LGL leukemia, our research explores novel therapeutic avenues for this orphan disease. Somatic activating mutations in the transcription factor STAT3 are the most common molecular alteration found in &amp;gt;50% of patients, and STAT3 signaling is dysregulated in almost all LGL leukemia patients. In the largest molecularly profiled LGL leukemia patient cohort to date, we found that STAT3 mutations frequently co-occur with mutations in chromatin and epigenetic modifying genes, suggesting crosstalk and cooperativity between STAT3 activation and epigenetic dysregulation. Moreover, TET2, which promotes DNA demethylation, is frequently inactivated in LGL leukemia via loss-of-function mutations and promoter hypermethylation. As DNA hypermethylation is an emerging hallmark of LGL leukemia, we evaluated the therapeutic potential of two epigenetic agents, azacitidine (AZA) and cladribine (CLAD), that directly and indirectly inhibit DNA hypermethylation, respectively. We hypothesize that AZA and CLAD will 1) exhibit cell intrinsic efficacy, 2) reprogram the epigenome of LGL leukemia through DNA hypomethylation to normalize aberrant gene expression, and 3) induce cytokine-mediated immune responses. AZA and CLAD cytotoxicity was assessed by the Cell Titer Glo viability assay in human LGL leukemia cell lines. Each exhibited potent anti-leukemic effects with IC50 values in the low micromolar and nanomolar ranges. Western blotting analysis demonstrated the decrease in protein expression of DNA methyltransferases and phosphorylated STAT3 levels with treatment in a time- and dose-dependent manner. We also explored the immunomodulatory effects of these hypomethylating agents since LGL leukemia is a pro-inflammatory malignancy with cytotoxic lymphocytes. Preliminary findings using the Isoplexis platform suggest that AZA and CLAD induce production of anti-inflammatory cytokines in LGL leukemia cells. Ongoing studies will identify differential methylation, chromatin accessibility, and gene expression using RRBS, ATAC-seq, and RNA-seq of AZA or CLAD treated samples to define the mechanisms and consequences of DNA demethylation. Uncovering the reprogramming capabilities of AZA and CLAD could allow for the potential repurposing of these inhibitors for treatment of LGL leukemia patients. Citation Format: Ariana Sabzevari, Ipsita Pal, Tess Deddens, Todd Fox, Aakrosh Ratan, David J. Feith, Thomas P. Loughran. Use of hypomethylating agents to reprogram the epigenome in large granular lymphocyte leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3245.