Expression Of p-STAT3 Research Articles

Impact of p. Gingivalis-induced chronic apical periodontitis on systemic iron homeostasis via the hepatic IL-6/STAT3/Hepcidin signaling pathway.

Chronic apical periodontitis (CAP), an inflammatory disease of the oral cavity caused by bacterial infections with Porphyromonas gingivalis (P. gingivalis) as a key pathogen, has been associated with systemic effects, potentially influencing distant organs including liver. The liver plays a key role in iron metabolism and immunity by hepcidin. This study aims to investigate the impact of P. gingivalis-induced CAP on liver and systemic iron metabolism, focusing on the role of the IL-6/STAT3 signaling pathway in hepatic hepcidin synthesis. A murine model of CAP was established by pulp chamber infection with P. gingivalis. Serum levels of IL-6, ferritin, and hepcidin were measured via ELISA. High-throughput sequencing was used to analyze hepatic gene expression, and immunohistochemistry with fluorescent staining was performed to validate protein expression in liver tissues. CAP led to significant changes in serum iron, ferritin, and IL-6. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed enrichment in pathways like JAK/STAT signaling and acute-phase responses, and gene set enrichment analysis (GSEA) also indicated activation of IL-6/JAK/STAT3 signaling pathway. Iimmunofluorescence confirmed increased IL-6, p-STAT3, and hepcidin expression. These levels were alleviated by stattic treatment, mitigating CAP-induced inflammatory and iron-regulatory effects. P. gingivalis-induced CAP triggered systemic inflammation and disrupts iron metabolism via the IL-6/STAT3 signaling pathway, potentially affecting liver function. Targeting this pathway may offer therapeutic strategies for managing iron dysregulation in chronic inflammatory diseases like CAP.

Expression of interleukin-17 in oral tongue squamous cell carcinoma and its effect on biological behavior

Tongue squamous cell carcinoma (TSCC) is a common malignant oral cancer characterized by substantial invasion, a high rate of lymph node and distant metastasis, and a high recurrence rate. This study aims to provide new ideas for the diagnosis and treatment of TSCC patients by exploring the related mechanisms that affect the migration and invasion of TSCC and inhibit the migration and spread of cancer cells. The results indicated the rate of high expression of IL-17 in cancer tissues was greater than that in tongue tissues, and the expression of IL-17 was related to the TNM stage. The expression of IL-17 in Cal-27 cells was greater than that in HOEC. With increasing IL-17 concentration, cell proliferation, migration, and invasion increased, and the apoptosis rate decreased. After adding the IL-17 inhibitor, the cell proliferation, invasion, and migration abilities decreased, the apoptosis rate increased, and the expression of JAK1and p-STAT3 decreased.IL-17 is highly expressed in oral tongue squamous cell carcinoma and is involved in the occurrence and development of TSCC, possibly through the JAK‒Stat signaling pathway. This study provides a new target and theoretical basis for treating tongue squamous cell carcinoma.

Monocyte STAT1 phosphorylation and treatment response of JAK inhibitors in chronic nonbacterial osteomyelitis

BackgroundChronic nonbacterial osteomyelitis (CNO) is a rare autoinflammatory disease of unknown cause, predominantly affecting teens and young adults. The early diagnosis and management are challenging due to the lack of reliable diagnostic markers and the occasional intractable cases despite conventional anti-inflammatory treatments. Janus kinase (JAK) inhibitors have recently shown potential utility; however, reports on their use for pediatric patients with CNO remain limited, and no established biomarkers exist to monitor disease activity. We aimed to investigate the pathophysiology of CNO and explore the rapid testing methods for accurate diagnosis and also assessing the disease activity.MethodsWe assessed intracellular phosphorylation of signal transducer and activator of transcription 1 (pSTAT1) in peripheral blood monocytes or T cells following interferon-gamma (IFNγ) stimulation, using flow cytometry in 9 patients under 15 years old with CNO. The pSTAT1 expression levels were compared with those in patients with STAT1-gain of function (STAT1-GOF) mutations (n = 5), other autoinflammatory diseases (n = 7), and healthy controls. Clinical and immunological data were monitored in 4 patients with intractable CNO treated with adjunctive JAK inhibitors, focusing on scoring scales, imaging data, lymphocyte subsets, cytokine profiles, and pSTAT1 levels.ResultsMonocyte pSTAT1 expression after IFNγ stimulation was elevated at diagnosis or during active CNO, similar to levels observed in STAT1-GOF cases. The pSTAT1 levels in CNO patients were significantly higher than those in other autoinflammatory diseases (p = 0.024) or controls (p < 0.001). Notably, pSTAT1 levels in CNO monocytes fluctuated with disease activity, decreasing in 5 patients during clinical remission following conventional therapies (p = 0.016). In four intractable cases, pSTAT1 levels remained high despite conventional treatments but significantly decreased after initiating JAK inhibitors (p = 0.036). This reduction correlated with improved patient pain visual analog scale (p = 0.008), CNO clinical disease activity score (p = 0.029), and better bone and joint imaging, though cytokine levels remained unchanged.ConclusionsThe monocyte pSTAT1 levels after IFNγ stimulation reflect the activity of CNO, indicating the diagnostic utility as well as the monitoring effect of disease control. Adjunctive JAK inhibitors successfully controlled inflammation in treatment-resistant cases. Rapid pSTAT1 testing may help reduce osteo-articular complications, although the long-term adverse effects and resistance should be further investigated.

DOP074 JAK inhibition to target fibroblasts and IBD-related fibrosis

Abstract Background Intestinal fibrosis is a common complication of inflammatory bowel disease (IBD). Uncontrolled accumulation of extracellular matrix deposited by fibroblasts is thought to be the underlying cause of fibrogenesis. Janus kinases (JAK) inhibitors are a relatively novel therapeutic strategy in IBD which show a robust anti-inflammatory effect. We hypothesize that JAK-inhibitors might not only play a role in IBD by reducing inflammation, but also offer a therapeutic potential to treat intestinal fibrosis. In this study we explored the effects of JAK inhibition on intestinal fibroblasts in vitro and in vivo. Methods In vitro studies were performed with human CCD-18Co and primary colonic fibroblasts. Levels of activated JAKs and STATs were detected by western blot after cytokine stimulation and were inhibited using tofacitinib (10uM) or upadacitinib (10uM). For the T cell transfer colitis model, Rag-/- mice (n=5-12 mice/group) were used. After engraftment of donor T cells the severity of colitis was evaluated with the mouse endoscopy score (MEICS) and mice were treated with upadacitinib (20mg/kg, 4x/week intraperitoneal injection). Intestinal fibroblast subsets were analyzed using anti-CD90, anti-αSMA, anti-podoplanin, anti-FAP, anti-PDGFR-α and -β, by flow cytometry. Sirius red staining was performed to measure collagen deposition. Levels of activated JAKs and downstream STATs were detected by immunofluorescent staining. Results Re-analysis of our previously published single-cell RNA sequencing data from fibrostenotic tissue of Crohns disease patients identified that a pathogenic subset of activated pro-inflammatory FAP+ fibroblasts was driven by the activation of STAT3 [1]. Inflammatory cytokines activated the JAK-STAT pathway in human fibroblasts and both upadacitinib and tofacitinib strongly inhibited pJAK1, pJAK2, pSTAT1 and pSTAT3 in a concentration dependent manner. Mice treated with upadacitinib showed a significant lower MEICS compared to controls (2.4 vs 5.3, Figure 1). Interestingly, specific intestinal fibroblast subsets, e.g. CD90+PDGFR-β+ fibroblasts were less abundant upon JAK-inhibition. pSTAT3 expression in the colon seemed to change after JAK inhibition. Furthermore, upadacitinib was able to restore the amount and distribution of collagen in the colon. Conclusion Our study is the first to describe that the JAK-STAT pathway can be activated in intestinal fibroblasts and JAK-inhibitors including upadacitinib/ tofacitinib suppress this pathway in vitro. Furthermore, human data show activation of the JAK-STAT pathway in FAP+ fibroblasts and in mice, JAK inhibition has an effect on fibroblasts subsets and collagen deposition. This shows the potential of JAK-inhibitors to target more than immune cells.

Quercetin mediates the therapeutic effect of Centella asiatica on psoriasis by regulating STAT3 phosphorylation to inhibit the IL-23/IL-17A axis.

To explore the active components that mediate the therapeutic effect of Centella asiatica on psoriasis and their therapeutic mechanisms. TCMSP, TCMIP, PharmMapper, Swiss Target Prediction, GeneCards, OMIM and TTD databases were searched for the compounds in Centella asiatica and their targets and the disease targets of psoriasis. A drug-active component-target network and the protein-protein interaction network were constructed, and DAVID database was used for pathway enrichment analysis. In a RAW264.7 macrophage model of LPS-induced inflammation, the anti-inflammatory effect of 7.5, 15, 30, and 60 μmol/L quercetin, asiaticoside, and asiatic acid, which were identified as the main active components in Centella asiatica, were tested by measuring cellular production of NO, TNF‑α and IL-6 using Griess method and ELISA and by detecting mRNA expressions of IL-23, IL-17A, TNF-α and IL-6 and protein expressions of p-STAT3 (Tyr705) and p-STAT3 (Ser727) with RT-qPCR and Western blotting. A total of 139 targets of Centella asiatica and 4604 targets of psoriasis were obtained, and among them CASP3, EGFR, PTGS2, and ESR1 were identified as the core targets. KEGG analysis suggested that quercetin, asiaticoside, and asiatic acid in Centella asiatica were involved in cancer and IL-17 and MAPK signaling pathways. In the RAW264.7 macrophage model of inflammation, treatment with quercetin significantly reduced cellular production of NO, TNF‑α and IL-6, and lowered mRNA expressions of IL-23, IL-17A, TNF‑α and IL-6 and protein expressions of p-STAT3 (Tyr705) and p-STAT3 (Ser727). Quercetin, asiaticoside and asiatic acid are the main active components in Centella asiatica to mediate the therapeutic effect against psoriasis, and quercetin in particular is capable of suppressing cellular production of NO, TNF‑α and IL-6 and regulating the IL-23/IL-17A inflammatory axis by mediating STAT3 phosphorylation to inhibit inflammatory response.

The prognostic value of p53 and Ki-67 expression status in penile cancer: a systematic review and meta-analysis

The prognostic value of p53 and Ki-67 expression status in penile cancer: a systematic review and meta-analysis

Total alkaloids in Fritillaria cirrhosa D. Don alleviate OVA-induced allergic asthma by inhibiting M2 macrophage polarization

Fritillaria cirrhosa D. Don (FCD) is a traditional Chinese medicine used to treat respiratory disorders, known for its effects in clearing heat, moistening the lungs, resolving phlegm, and relieving cough. Additionally, the total alkaloids extracted from FCD can alleviate asthma symptoms and reduce airway inflammation. However, no studies have investigated the effects of total alkaloids on lung macrophages. This study explored whether the total alkaloids of FCD (TAs-FCD) reduce M2 macrophage polarization and, consequently, attenuate airway remodeling in asthmatic mice. This study further elucidated its mechanism of action in treating allergic asthma. The extracted TAs-FCD was analyzed for its composition using UPLC-Q-TOF/MS. Network pharmacology was employed to identify the active ingredients and potential mechanisms of TAs-FCD in the treatment of allergic asthma. A mouse model of ovalbumin-induced allergic asthma was established, adopted, and validated through in vivo experiments. Hematoxylin-eosin staining (H&E), immunohistochemistry (IHC), immunofluorescence staining (IF), enzyme-linked immunosorbent assay (ELISA), Western blotting (WB), and real-time fluorescence quantitative polymerase chain reaction (q-PCR) were used to investigate the role of TAs-FCD in inhibiting M2 macrophage polarization in the context of allergic asthma. A total of 66 active ingredients were screened from 116 compounds using SWISSADME. The targets of these 66 compounds were predicted by SwissTargetPrediction, resulting in 808 unique drug targets after excluding duplicates. Additionally, 1756 targets related to allergic asthma were identified from the DisGeNET, Genecard, and OMIM databases. This led to 267 cross-targets between the active ingredient targets and allergic asthma targets, including interleukin (IL)-1β, tumor necrosis factor (TNF), and STAT3. Animal experiments demonstrated that TAs-FCD improved histopathological injury in mouse lungs, reduced peri-airway collagen fiber accumulation, airway mucus secretion, and airway smooth muscle proliferation. TAs-FCD also lowered IL-1β, TNF-α and IL-4 levels in lung tissues and alleviated airway inflammation. Furthermore, TAs-FCD significantly reduced levels of Arg1 and CD206, which are closely associated with M2 macrophages, and downregulated the expression of p-STAT3 and p-JAK2. TAs-FCD may inhibit M2 macrophage polarization by regulating the JAK2/STAT3 pathway, thereby alleviating airway remodeling and inflammation in allergic asthmatic mice.

LCZ696, an angiotensin receptor-neprilysin inhibitor, ameliorates epithelial-mesenchymal transition of peritoneal mesothelial cells and M2 macrophage polarization

Aims To investigate the effects and mechanisms of LCZ696, an angiotensin receptor-neprilysin inhibitor (ARNI), on epithelial-mesenchymal transition (EMT) of peritoneal mesothelial cells and on macrophage M2 polarization. Methods We examined the effects of LCZ696 in a 4.25% high glucose peritoneal dialysis fluid (PDF)-induced peritoneal fibrosis (PF) mouse model, and explored the mechanisms of LCZ696 on human peritoneal mesothelial cells (HPMCs) stimulated by TGF-β1 (5 ng/mL) and on Raw264.7 cells stimulated by IL-4 (10 ng/mL). To further elucidate the mechanism, we treated HPMCs with the conditioned medium of Raw264.7 cells. Results LCZ696 effectively improved PF and inhibited the process of EMT in PDF mice. In vitro, LCZ696 also significantly alleviated the EMT of TGF-β1 induced HPMCs, although there was no statistically significant difference when compared to the Valsartan treatment group. Moreover, LCZ696 ameliorates the increased expression of Snail and Slug, two nuclear transcription factors that drive the EMT. Mechanistically, TGF-β1 increased the expression of TGFβRI, p-Smad3, p-PDGFRβ and p-EGFR, while treatment with LCZ696 abrogated the activation of TGF-β/Smad3, PDGFRβ and EGFR signaling pathways. Additionally, exposure of Raw264.7 to IL-4 results in increasing expression of Arginase-1, CD163 and p-STAT6. Treatment with LCZ696 inhibited IL-4-elicited M2 macrophage polarization by inactivating the STAT6 signaling pathway. Furthermore, we observed that LCZ696 inhibits EMT by blocking TGF-β1 secretion from M2 macrophages. Conclusion Our study demonstrated that LCZ696 improves PF and ameliorates TGF-β1-induced EMT of HPMCs by blocking TGF-β/Smad3, PDGFRβ and EGFR pathways. Meanwhile, LCZ696 also inhibits M2 macrophage polarization by regulating STAT6 pathway.

A MODIFICATION OF THE CHRONIC CONSTRICTION INJURY MODEL OF NEUROPATHIC PAIN: SILK USAGE INSTEAD OF CHROMIC CATGUT

Objective: In the chronic constriction injury (CCI) neuropathic pain rat model, usage of chromic catgut causes intense neuroinflammation and also undesirable conditions such as autotomy. This causes difficulties in the application and interpretation of pain behaviour tests. In this study, it was aimed to investigate the suitability of the use of surgical silk as a suture material in the CCI model, based on behavioural tests and certain biomarkers. Material and Methods: CCI model was created by loosely ligating the right sciatic nerves of rats with surgical silk. Tactile allodynia, thermal and mechanical hyperalgesia were evaluated on the 2nd, 7th, 14th, 21st and 29th postoperative days. Expressions of glial fibrillary acidic protein (GFAP), signal transducer and transcription activator 3 (STAT3) and its phosphorylated form (p-STAT3) were evaluated from the spinal cord tissue by western blotting technique. Results: Sciatic nerve-ligated rats did not lose weight but gained weight on days 14, 21, and 29 like control rats. Statistically significant decreases were found in pain thresholds in three different pain tests after the operation. The decrease in the pain threshold was accompanied by a statistically significant increase in GFAP protein expression in the spinal cord on days 2 and 14 simultaneously. No statistically significant difference was found in the expression of STAT3 or p-STAT3 protein in the spinal cord at any time. Conclusion: The absence of weight loss and autotomy expected from the classical CCI model, the reversal of the decrease in the pain threshold, the concomittant increase in GFAP protein expression in the spinal cord, and the absence of change in STAT3 and p-STAT3 expression in our study suggest that the model we created by tying 3 loose silk knots on the sciatic nerve has a milder course of neuroinflammation.

Xiao-xu-ming decoction improved synaptic damage after acute cerebral ischemia and reperfusion via JAK2/STAT3 pathway in rats.

Ischemic stroke is an important cause of death and disability worldwide. Xiao-xu-ming Decoction (XXMD) is a classic prescription for the treatment of stroke patients, which has been widely used in China and has significant therapeutic effect, but the therapeutic target and mechanism are still unclear. The current study aimed to investigate temporal alternation of synaptic damage and the protective effects of XXMD on synaptic damage following cerebral ischemia and reperfusion in vivo. Adult male Sprague-Dawley (SD) rats subjected to 90min of middle cerebral artery occlusion (MCAO) and subsequent reperfusion at various time points. Neurobehavioral function was assessed using modified neurological severity scores (mNSS), while pro-inflammatory cytokine levels were measured using ELISA kits. Histological assessment involved silver staining and Luxol Fast Blue (LFB) staining, and the ultrastructural alterations in neurons and synapses were examined using a transmission electron microscope (TEM). Golgi-cox staining was used to evaluate the density of dendritic spines. Levels of synapse-related proteins were quantified via immunofluorescence staining and Western blotting. Additionally, JAK2/STAT3 pathway related protein levels were assessed using Western blotting. In the second part, the rats were randomly divided into sham operation group, 24h of reperfusion group, and XXMD group. The ultrastructural alterations and dendrite spine density of synapses were observed by TEM and Golgi-Cox staining respectively, and the expression levels of SYN, PSD95, GAP43, p-JAK2 and p-STAT3 were evaluated by WB. Findings included deteriorated neurobehavioral function, increased release of IL-6, IL-1β, and TNFα, and time-dependent neuronal and synaptic damages during the initial phase of ischemia and reperfusion. At the ultrastructural level, neurons and synapses exhibited structural failure in the peri-infarct cortex. In addition, golgi-cox staining showed dendritic density in ischemic cortex significantly reduced after cerebral ischemia and reperfusion. Moreover, significant reductions in SYN, PSD95, and GAP43 expression levels, along with increases in p-JAK2 and p-STAT3 expression levels, were observed after cerebral ischemia and reperfusion. Meantime, XXMD significantly reduced synaptic impairment and up-regulated SYN, GAP43, PSD95 expression and down-regulated phosphorylation expression of JAK2 and STAT3 following MCAO and 24h of reperfusion. Collectively, these results indicates XXMD may play a neuroprotective role in reducing synaptic damage via JAK2/STAT3 signaling pathway.

Safety, tolerability, pharmacokinetics, and pharmacodynamics of the oral allosteric TYK2 inhibitorESK-001 using a randomized, double-blind, placebo-controlled study design.

ESK-001 is a highly selective allosteric inhibitor of tyrosine kinase 2 (TYK2), which plays an essential role in mediating cytokine signaling in multiple immune-mediated diseases. In 2 phase I studies, a first-in-human single ascending dose (SAD) and multiple ascending dose (MAD) study and a multiple-dose (MD) study, we evaluated the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of orally administered ESK-001 in healthy participants using a randomized, double-blind, placebo-controlled study design. ESK-001 was rapidly absorbed with systemic exposures generally increasing dose-proportionally across all cohorts. The mean terminal half-life ranged from 8 to 13 h with no to minimal accumulation of ESK-001 following q.d. doses and ~2-fold accumulation following Q12 doses. Less than 1% of unchanged ESK-001 was eliminated in urine. ESK-001 inhibited the downstream TYK2 pathway as shown by inhibition of pSTAT1 expression. Transcriptomic analysis of unstimulated whole blood samples confirmed dose-dependent inhibition of Type I IFN-induced genes and SIGLEC1, a novel TYK2-responsive biomarker. By correlating PK exposure data with PD readouts, a strong PK/PD relationship was demonstrated. There were no deaths, serious treatment-emergent adverse events (TEAEs), nor severe TEAEs, and most TEAEs were mild in severity. In conclusion, ESK-001 was generally safe and well-tolerated in healthy participants, showed linear dose-dependent PK characteristics, and maximally inhibited TYK2-dependent pathways with a predictable concentration-dependent PK/PD relationship. These findings were used to select the dose range of ESK-001 for the STRIDE phase II trial in plaque psoriasis and to support further clinical development of ESK-001 in other TYK2-mediated diseases.

Experimental study of the effects of pirfenidone and nintedanib on joint inflammation and pulmonary fibrosis in a rheumatoid arthritis-associated interstitial lung disease mouse model.

Rheumatoid arthritis-associated interstitial lung disease (RA-ILD) is a serious pulmonary complication in rheumatoid arthritis (RA) patients, is one of the leading causes of death in RA patients. This study was designed to determine whether pirfenidone and nintedanib can alleviate joint inflammation and pulmonary fibrosis in a mouse model of RA-ILD. Male DBA/1 mice were injected with bovine type II collagen (bCII) to establish the RA-ILD model. Pirfenidone (20 mg/kg) and nintedanib (60 mg/kg) were administered, and body weight, joint swelling, pathology of the lungs and knees, macrophage polarization in bronchoalveolar lavage fluid (BALF), and the fluorescence intensity of phosphorylated janus kinase 2/phosphorylated signal transducer and activator of transcription 3 (p-Jak2/p-Stat3) in the lungs and knees were determined. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure mRNA, and western blotting was conducted to detect the protein. Macrophage line RAW264.7 were divided into the following groups: the RAW264.7, RAW264.7 + IL-4/IL-13 (IL-4/IL-13, 60 ng/mL), RAW264.7 + IL-4/IL-13 + pirfenidone (0.5 and 1.0 mmol/L), RAW264.7 + IL-4/IL-13 + nintedanib (0.1 and 0.5 µmol/L). Mouse primary fibroblast-like synovial (FLS) cells were divided into the following groups: the FLS, FLS + transforming growth factor-β1 (TGF-β1; 10 µg/L), FLS + TGF-β1 + pirfenidone (0.5 and 1.0 mmol/L), FLS + TGF-β1 + nintedanib (0.1 and 0.5 µmol/L) groups. Proteins in each group were detected. The body weights of the mice in the pirfenidone and nintedanib groups were greater than those in the RA-ILD group (P<0.05), the arthritis scores were also significantly lower (P<0.05). The proportion of M2-type macrophages in the BALF of the nintedanib group significantly decreased (P<0.05). Inflammatory cell infiltration in the lung was reduced in the pirfenidone and nintedanib groups; additionally, decreased levels of synovium, collagen, angiogenesis, and bone destruction of the knee joint and a lower synovitis score were observed (P<0.05). Masson staining revealed that collagen deposition in the lungs in the pirfenidone and nintedanib groups was reduced (P<0.05). P-Jak2/p-Stat3 expression in the lungs and knee joints in the pirfenidone and nintedanib groups was low (P<0.001 in the lung and P<0.005 in the knee joint). The mRNA expression of collagen-IV, Stat3, and Jak2 in the lungs was lower in the pirfenidone and nintedanib (P<0.05); the protein expression levels of p-Jak2/Jak2, p-Stat3/Stat3, p-Smad3/Smad3, and TGF-β receptor 2 (TGF-βR2) in the lungs in the pirfenidone and nintedanib groups decreased (P<0.05). P-Jak2/Jak2, p-Stat3/Stat3, TGF-βR2, cluster of differentiation 206 (CD206), and arginase-1 (ARG-1) were lower in the pirfenidone and nintedanib groups of RAW264.7 cells (at all different concentrations, P<0.05). P-JAK2/JAK2, p-Stat3/Stat3, and TGF-βR2 were lower in the pirfenidone and nintedanib groups of FLS cells (at all different concentrations, P<0.05). Pirfenidone and nintedanib not only reduced the degree of pulmonary fibrosis but also relieved joint symptoms in an RA-ILD mouse model. The mechanisms of action are related to the inhibition of the TGF-β signaling pathway, Jak2/Stat3 signaling pathway, and polarization of macrophages to the M2 phenotype.

Bushen Zhuyun Decoction Enhances Endometrial Receptivity via the IL-6/STAT3 Signaling Pathway in Rats

Background: Reproductive endocrine disorder can impair endometrial receptivity, preventing embryo implantation and increasing miscarriage risk. Impaired endometrial receptivity contributes significantly to female infertility. Inflammatory signaling pathways including the IL-6/STAT3 pathway help embryos implant. Therefore, it is crucial to explore the relationship between the IL-6/STAT3 signaling pathway and endometrial receptivity. Objective: To investigate the mechanism by which Bushen Zhuyun decoction (BSZY) enhances endometrial receptivity in rats through the IL-6/STAT3 signaling pathway. Methods: Mifepristone-induced poor endometrial receptivity models of female SD rats were established, followed by histopathological observation. ELISA was used to measure serum sex hormones and VEGF. Western blotting or IHC was used to measure steroid receptors, IGFBP1, and IL-6/STAT3 pathway activation in the uterus during each estrus cycle and early gestation of normal rats. The Treg/Th17 balance was assessed using flow cytometry. Results: Significant differences were found in the protein expressions of steroid receptors, IL6, STAT3, and p-STAT3 during each estrus cycle and early gestation of normal rats. The protein expressions of STAT3 and PR were strongly correlated with each other. BSZY notably improved uterine morphology increased the expression of implantation markers and raised the serum concentrations of sex hormones and VEGF. BSZY enhanced the expressions of IL-6 and its receptors and restored the expressions of STAT3 and p-STAT3 in the uterus of pregnant rats. In addition, BSZY effectively restored the Treg/Th17 balance in the peripheral blood of pregnant rats. Conclusion: BSZY enhances endometrial receptivity and promotes decidualization in SD rats via the IL-6/STAT3 signaling pathway.

Therapeutic efficacy of jeoryeong-tang in dextran sulfate sodium-induced mouse model of inflammatory bowel disease

BackgroundJeoryeong-tang (JRT) was first recorded in Shanghanlun. It is composed of Polyporus Sclerotium, Poria, Asini Corii Colla, Alisma Rhizome, and Talcum at the same weight ratio. These medicinal materials are known for diuretic and hemostatic effects and have been traditionally used to treat kidney and bladder diseases. However, their potential therapeutic effects on colon diseases, particularly inflammatory bowel disease (IBD), have not been extensively studied. Therefore, this study aimed to investigate the therapeutic efficacy of JRT in IBD and explore its underlying anti-inflammatory mechanisms using a murine model of colitis induced by dextran sulfate sodium (DSS). MethodsMice were treated with 3.0 % or 2.5 % DSS for 6 days to induce colitis and JRT extract was then administered at a low level of 40 mg/kg (JRT-L), a medium level of 120 mg/kg (JRT-M), or a high level of 400 mg/kg (JRT-H) once a day. During the administration period, clinical disease activity index (DAI) reflecting survival rate, diarrhea, bloody stool, and weight loss rate was evaluated. The degree of colonic tissue damage was scored and evaluated through hematoxylin and eosin staining. Cyclooxygenase-2 (COX-2), p-STAT3 and p-ERK expression were examined with immunohistochemistry. Tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and -1β levels were analyzed using a cytokine enzyme-linked immunosorbent assay. ResultsAmong mice treated with 3.0 % DSS, JRT-M significantly improved the survival rate compared to other treatments as a result of observation for a total of 14 days. While, in the 2.5 % DSS-treated model, the average body weights of mice in both of JRT-M and JRT-H groups were significantly higher than that in the DSS group. In addition, the JRT-M group showed significantly lower DAI score than that in the DSS group. As a result of evaluating the extent of colon tissue damage, JRT-M and JRT-H groups both showed significantly lower inflammatory index and thinner muscular externa thickness than the DSS group. The expression of COX-2, p-STAT3 and p-ERK in colon tissue were significantly suppressed in JRT-M and JRT-H groups compared to that in the DSS group. Moreover, serum TNF-α was significantly suppressed in the JRT-H group compared to that in the DSS group. ConclusionsJeoryeong-tang has a promising therapeutic potential for treating IBD through its anti-inflammatory properties. Findings of this study suggest that JRT could be a valuable candidate for further clinical investigations in the treatment of IBD.

Repression of JAK2-STAT1 and PD-L1 by CEP-33779 ameliorates the LPS-induced decline in phagocytic activity of alveolar macrophages and mitigates lung injury in mice.

The role of the JAK2-STAT1/PD-L1 pathway in the phagocytic activity of alveolar macrophages (AMs) during LPS-induced acute lung injury in mice remains poorly understood. This study aims to explore whether the JAK2-STAT1/PD-L1 pathway is upregulated on AMs in LPS-induced mice acute lung injury and to further explore the impact of the JAK2-specific inhibitor CEP-33779 on the LPS-induced impairment of AMs phagocytic activity and lung injury. ALI was induced in mice via intratracheal administration of LPS, followed by intragastric administration of JAK2 inhibitor CEP-33779 suspension. Immunohistochemistry was conducted to assess PD-L1 expression in lung tissue, as well as p-JAK2, p-STAT1, and PD-L1 expression on AMs in bronchoalveolar lavage fluid (BALF) using immunofluorescence. Levels of TNF-α and IL-6, as well as protein concentration in BALF, were measured using enzyme-linked immunosorbent assay and Bicinchoninic acid assays, respectively. Hematoxylin-eosin staining and lung injury score were employed to evaluate pathological changes in mouse lungs. Total cell count in BALF was determined using a cell counter. Furthermore, western blot and immunofluorescence was conducted to assess the effect of JAK2 and STAT1 inhibitor on JAK2-STAT1 pathway activation and PD-L1 expression, while confocal microscopy with latex beads rabbit IgG FITC complex was used to observe MH-S cells phagocytic ability. The study revealed that LPS stimulation triggered the activation of the JAK2-STAT1 pathway and an upregulation of PD-L1 on AMs in both LPS-induced acute lung injury mice and MH-S cell lines. Moreover, treatment with the JAK2 and STAT1 inhibitor effectively reduced the activation of JAK2-STAT1 signaling, downregulated PD-L1 expression on AMs in BALF from LPS-induced ALI mice and LPS-stimulated MH-S cells, and significantly improved the LPS-induced reduction in phagocytic activity in MH-S cells. Most notably, CEP-33779 treatment significantly mitigated the pulmonary inflammatory response and lung injury in mice with LPS-induced ALI. Collectively, these findings imply that the JAK2-STAT1 pathway plays a role in the upregulation of PD-L1, which in turn is associated with the diminished phagocytic activity in LPS-induced AMs as well as lung injury. Furthermore, our study highlights that CEP-33779 treatment can effectively improve the reduced phagocytic activity of AMs and relieve lung injury induced by LPS through suppression of the JAK2-STAT1/PD-L1 pathway.

Microglial cannabinoid receptor type II stimulation improves cognitive impairment and neuroinflammation in Alzheimer's disease mice by controlling astrocyte activation.

Alzheimer's disease (AD) is the most common form of dementia and is characterized by the accumulation of amyloid β (Aβ) and phosphorylated tau. Neuroinflammation, mainly mediated by glial activation, plays an important role in AD progression. Although there is growing evidence for the anti-neuroinflammatory and neuroprotective effects of the cannabinoid system modulation, the detailed mechanism remains unclear. To address these issues, we analyzed the expression levels of cannabinoid receptor type II (Cnr2/Cb2) in AppNL-G-F/NL-G-F mice and human AD precuneus, which is vulnerable to amyloid deposition in AD, and the effects of JWH 133, a selective CB2 agonist, on neuroinflammation in primary glial cells and neuroinflammation and cognitive impairment in AppNL-G-F/NL-G-F mice. The levels of Cnr2/Cb2 were upregulated in microglia isolated from the cerebral cortex of AppNL-G-F/NL-G-F mice. CNR2 expression was also increased in RNAs derived from human precuneus with advanced AD pathology. Chronic oral administration of JWH 133 significantly ameliorated the cognitive impairment of AppNL-G-F/NL-G-F mice without neuropsychiatric side effects. Microglia and astrocyte mRNAs were directly isolated from the mouse cerebral cortex by magnetic-activated cell sorting, and the gene expression was determined by quantitative PCR. JWH 133 administration significantly decreased reactive astrocyte markers and microglial C1q, an inducer for the reactive astrocytes in AppNL-G-F/NL-G-F mice. In addition, JWH133 administration inhibited the expression of p-STAT3 (signal transducer and activator of transcription 3) in astrocytes in AppNL-G-F/NL-G-F mice. Furthermore, JWH 133 administration suppressed dystrophic presynaptic terminals surrounding amyloid plaques. In conclusion, stimulation of microglial CB2 ameliorates cognitive dysfunction in AppNL-G-F/NL-G-F mice by controlling astrocyte activation and inducing beneficial neuroinflammation, and our study has implications that CB2 may represent an attractive therapeutic target for the treatment of AD and perhaps other neurodegenerative diseases involving neuroinflammation.

Fe-capsaicin nanozyme attenuates sepsis-induced acute lung injury by regulating the functions of macrophages.

In sepsis, the lung is one of the worst affected organs, often leading to acute lung injury (ALI). More and more evidence suggests that macrophages are also involved in the pathogenesis of ALI. In our previous study, we successfully synthesized Iron-capsaicin-based nanoparticles (Fe-CAP NPs) and found that it could inhibit the secretion of inflammatory cytokines to alleviate ALI. Here, we further explore the anti-inflammatory mechanism of Fe-CAP NPs. Bone marrow-derived macrophages (BMDM) and C57 mice were divided into four groups: control group, lipopolysaccharide (LPS) group, CAP + LPS group and Fe-CAP + LPS group. Western blot and Immunofluorescence were used to detect the expression of macrophage phenotypic markers CD86 and CD206 in BMDM and lung tissue. Fluorescence microbeads, Transwell and ROS kit were used to detect the phagocytosis, migration and ROS clearing capability of BMDM. Western blot was used to detect the expression of JAK2/STAT3 pathway and apoptosis proteins in BMDM. TUNEL kit and H&E staining were used to evaluate apoptosis and pathological changes in lung tissue. In vitro, CD86 expression was increased in LPS, but decreased after Fe-CAP pretreatment. CD206 expression was the opposite. Fe-CAP reduced phagocytosis, migration and scavenged ROS in LPS-treated BMDM. Fe-CAP inhibited P-JAK2 and P-STAT3 expression and reduced apoptosis. In vivo, Fe-CAP improved lung histopathology and reduced apoptosis in lung tissue of LPS group. CD86 expression was increased in lung tissue of LPS group, but decreased in Fe-CAP preconditioning, and CD206 expression was reversed. Fe-CAP NPs could alleviate sepsis-induced ALI by regulating the polarization and function of macrophages, reducing ROS level and apoptosis.

PSTAT3 Expression is Increased in Advanced Prostate Cancer in Post-Initiation of Androgen Deprivation Therapy.

The transcription factor Signal Transducer and Activator of Transcription 3 (STAT3) plays a role in carcinogenesis and is involved in processes, such as proliferation, differentiation, drug resistance and immunosuppression. STAT3 can be activated by phosphorylation of tyrosine at position 705 (pSTAT3Tyr705) or serine at 727 (pSTAT3Ser727). High expression levels of pSTAT3 are implicated in advanced stages of prostate cancer (PCa) and are known to interact with the androgen receptor signaling pathway. However, not much is known about how androgen deprivation therapy (ADT) in advanced disease affects pSTAT3 expression. The aim of this study was to determine the influence of ADT on pSTAT3 expression in PCa tissue. The study cohort came from a PCa tissue microarray resource containing prostate specimens from patients before and post-initiation of ADT. Tissue samples from 111 patients were immunostained for pSTAT3Tyr705 and pSTAT3Ser727. H-score was used to evaluate the intensity and the percentage of pSTAT3 expression in malignant epithelial and stromal compartments. Univariate and multivariable Cox regression analyses were used to assess pSTAT3Tyr705 and pSTAT3Ser727 as biomarkers of oncological outcome in patients undergoing ADT. Post-ADT PCa samples demonstrated increased nuclear and cytoplasmic levels of pSTAT3Ser727 in the stroma compared to pre-ADT samples, whereas pSTAT3Tyr705 expression was increased significantly in both stromal and malignant epithelial compartments except for stromal cytoplasm. High cytoplasmic pSTAT3Ser727 in stromal compartments correlated with reduced overall survival, shorter time to castration-resistant PCa development, and decreased metastasis-free survival. An increase in nuclear and cytoplasmic pSTAT3Ser727 expression within the stromal compartment of post-ADT samples corresponded to a shorter time to CRPC development, which was not observed for pSTAT3Tyr705. Multivariable survival analysis using Cox's regression identified that high cytoplasmic pSTAT3Ser727 expression in the stroma of post-ADT samples and pT3 or pT4-stage were associated with worse overall survival and 5-year metastasis-free survival (MFS). This study presents novel insights into the impact of ADT on the expression levels of pSTAT3Tyr705 and pSTAT3Ser727 in PCa. Cytoplasmic pSTAT3Ser727 status of cancer-associated stromal cells in post-ADT samples may serve as an independent prognostic marker for OS and 5-year MFS, identifying prostate cancer patients prone to developing resistance to ADT.

TMIC-08. PATTERNS OF PSTAT3 EXPRESSION IN REACTIVE ASTROCYTES OF BRAIN METASTASES FROM NON-SMALL CELL LUNG CANCER AND CORRELATIONS WITH OUTCOME

Abstract BACKGROUND pSTAT3 expression in peritumoral reactive astrocytes (RA) of brain metastases (BM) from breast cancer may favor a pro-metastatic environment and negatively affect the intracranial progression-free survival (i-PFS) (Pellerino, 2023). We aim to evaluate whether pSTAT3 plays the same role also in BM from NSCLC. MATERIAL AND METHODS Seventy-two BM specimens from NSCLC were identified from the biobank of Pathology Unit of University of Turin and Berlin. STAT3 expression was scored in RA of peritumoral tissue according to Priego et al. (Nat Med 2018). Clinical and molecular data, and i-PFS were retrospectively retrieved. RESULTS Median age was 64 years (IC95% 59-67). Immunohistochemistry for GFAP and pSTAT3 was feasible in 66/72 (91,6%). 42/66 (63,6%) had BM with druggable mutations, while 24/66 (36,4%) did not show any actionable mutations. Druggable mutations were as follows: 8/42 (19,1%) KRAS G12C, 6/42 (14,3%) EGFR, 5/42 (11,9%) ALK, 1/42 (2,4%) BRAFv600E, 31/42 (73,8%) PDL1 expression. Overall, 53/66 (80,3%) showed positive staining of pSTAT3: 22/66 (33,3%) with score 3, 21/66 (31,8%) with 2, 10/66 (15,1%) with 1, and 12/66 (19.8%) with 0 (negative). High pSTAT3 expression (score 2-3) was observed in 32/42 (76,2%) BM with actionable mutations and in 12/24 (50%) without actionable mutations (p 0,031). PDL1 expression was significantly correlated with high pSTAT3 expression (29/31, 93,5%), while most of BM without PDL1 (20/35 – 57,1%) had low or absent pSTAT3 (score 0-1) (p&amp;lt;0,0001). Median i-PFS was 12 months (IC95% 8-23): low pSTAT3 BM had a median i-PFS of 18 months (IC95% 14-28) versus 9 months (IC95% 8-12) for high pSTAT3 BM (HR 5,3;p 0,0001). CONCLUSION pSTAT3 overexpression in RA is associated with PDL1 expression and may negatively impact the i-PFS in BM from NSCLC. These data support the investigation of STAT3 inhibitor silibinin in preventing intracranial recurrence in a clinical trial (NCT05689619).

TMIC-27. PDPN ACTIVATION OF CLEC5A IN THE PERI-NECROTIC NICHE DRIVES TAM POLARIZATION AND TME IMMUNOSUPPRESSION IN GLIOBLASTOMA

Abstract Glioblastoma, IDH-wildtype (GBM, WHO grade 4) is the most common malignant glioma of adults and is characterized by a severely hypoxic and immunosuppressive tumor microenvironment (TME). CLEC5A regulates immune responses in inflammatory and infectious diseases. However, its role in GBM immune regulation remain unclear. Here, we found that CLEC5A has the strongest association with poor clinical outcomes among all immune-related genes in GBM. Tumor-associated macrophages (TAMs) account for the majority of immune cells in the GBM and CLEC5A expression in this population is highest in hypoxic, peri-necrotic zones. Both hypoxia and GBM conditioned media cause increased CLEC5A expression by THP-1 cells, and overexpression of CLEC5A enhanced TAM polarization, migration toward glioma cells, and increased secretion of cytokines that mediate immunosuppression. We show that CLEC5A is a cell surface receptor activated by podoplanin (PDPN) and induces TAM polarization through Syk-JAK-STAT3 signaling. Co-IP assay showed that CLEC5A and PDPN could be binded together using antibodies for CLEC5A, Flag-CLEC5A or PDPN when we cocultured control THP-1 cells and THP-1 cells overexpressing Flag-CLEC5A with primary GBM cells (NU02068). PDPN blocking antibody (α-PDPN) reduced the specific binding between PDPN and CLEC5A, as well as inhibited the TAM-IM polarization. Overexpression of CLEC5A significantly upregulated the expression of p-Syk, Jak2, p-Jak2, STAT3, p-STAT3, CD163, CD206 and PD-L1. Moreover, Bay 61-3606, a selective Syk inhibitor, rescued CLEC5A overexpression-mediated TAM-IM polarization and Syk-JAK-STAT3 activation in THP-1 cells. In vivo, both silencing CLEC5A in TAMs and silencing PDPN in GBM cells, as well as pharmacologically targeting Syk delayed glioma growth, prolonged survival and was associated with diminished immunosuppressive TAMs. Collectively, we found that PDPN-CLEC5A-Syk-JAK-STAT3 signaling causes TAM polarization and enhances TME immunosuppression in GBM and can be targeted to suppress growth.