Expression Of p-STAT3 Research Articles

A nomogram model to predict recurrence of early-onset endometrial cancer after resection based on clinical parameters and immunohistochemical markers: a multi-institutional study

ObjectiveThis study aimed to investigate the prognosis value of the clinical parameters and immunohistochemical markers of patients with early-onset endometrial cancer (EC) and establish a nomogram to accurately predict recurrence-free survival (RFS) of early-onset EC after resection.MethodsA training dataset containing 458 patients and an independent testing dataset consisting of 170 patients were employed in this retrospective study. The independent risk factors related to RFS were confirmed using Cox regression models. A nomogram model was established to predict RFS at 3 and 5 years post-hysterectomy. The C-index, area under the curve (AUC) of the receiver operating characteristic (ROC) curve, and calibration curve were calculated to assess the predictive accuracy of the nomogram.ResultsIn all early-onset EC patients, more than half (368/628, 58.6%) were diagnosed in the age range of 45-49 years. Meanwhile, the recurrence rate of early-onset EC is approximately 10.8%. Multivariate Cox regression analyses showed that histological subtype, FIGO stage, myometrial invasion, lymphovascular space invasion (LVSI), P53 expression, and MMR status were independent prognostic factors related to RFS (all P < 0.05) and established the nomogram predicting 3- and 5-year RFS. The C-index and calibration curves of the nomogram demonstrated a close correlation between predicted and actual RFS. Patients were divided into high- and low-risk groups according to the model of RFS.ConclusionsCombining clinical parameters and immunohistochemical markers, we developed a robust nomogram to predict RFS after surgery for early-onset EC patients. This nomogram can predict prognosis well and guide treatment decisions.

Impairment of regulatory T cell stability in axial spondyloarthritis: role of EZH2 and pSTAT5

Background and objectivesAxial spondyloarthritis (axSpA) is a chronic inflammatory disease involving the spine, peripheral joints, and entheses. Functional impairment of regulatory T cells (Treg) is linked to inflammatory diseases, but limited data is available regarding Treg involvement in axSpA. Treg stability refers to their ability to maintain their functions and characteristics in pro-inflammatory environments. EZH2 and phosphorylated STAT5 (pSTAT5) play a critical role in maintaining Treg stability. We aimed to characterize Treg stability in patients with axSpA.MethodsPeripheral blood mononuclear cells (PBMCs) from axSpA patients, either naïve from targeted therapy or treated by TNF inhibitors (TNFi), and from healthy donors (HD), were freshly isolated. Expression of stability (EZH2, pSTAT5) and suppressive (TNFR2 and CD39) markers by Treg was analyzed by flow cytometry.ResultsEZH2 expression by Treg was decreased in axSpA patients as compared to HD (p<0.01). Mechanistic study showed that inhibition of EZH2 attenuated Treg differentiation and suppressive phenotype in vitro. EZH2 was predominantly expressed by highly suppressive TNFR2+ and CD39+ Treg. Additionally, axSpA patients also exhibited a reduced frequency of pSTAT5+ Treg compared to HD (p<0.05), and pSTAT5+ Treg frequency increased at 3 months of TNFi treatment compared to baseline (p<0.05). This last result suggested a restoration of Treg stability upon TNFi treatment.ConclusionBy highlighting a deficient expression of EZH2 and pSTAT5 by Treg, we revealed an impaired Treg stability in axSpA. Deciphering the pathways influenced by these molecules is necessary to assess the potential therapeutic benefits of restoring Treg stability in axSpA.

Nicotine promotes M2 macrophage polarization through α5-nAChR/SOX2/CSF-1 axis in lung adenocarcinoma

α5-nicotinic acetylcholine receptor (α5-nAChR) plays a vital part in lung adenocarcinoma (LUAD). However, it is not comprehensively understood that how the α5-nAChR affects LUAD. Through diverse bioinformatics analyses and immunohistochemistry, the expressions of α5-nAChR and SOX2 as well as their relations were dissected. α5-nAChR regulated the differentiation of monocytes into M2 macrophages by targeting the STAT3/SOX2/CSF-1 signaling in the coculture system by western blotting and ChIP. α5-nAChR-mediated macrophage-mediated LUAD cell migration via SOX2/CSF-1 signaling in the cocultured medium. Correlations of α5-nAChR, SOX2 and M2 phenotype tumor-associated macrophages (TAMs) were validated in mouse LUAD models and clinical samples. α5-nAChR expression was connected to SOX2 expression, smoking and bad prognosis of LUAD among clinical samples. Nicotine-induced SOX2 expression was mediated by α5-nAChR via STAT3. Additionally, SOX2-mediated macrophage colony-stimulating factor (CSF-1) expression contributed to LUAD progression in vitro. Furthermore, α5-nAChR expression was strongly linked to pSTAT3, SOX2 and M2 macrophage marker CD206 expression and negatively correlated with M1 macrophage marker CD86 expression in vivo. It is indicated that M2 macrophages are mediated by the new α5-nAChR /SOX2/CSF-1 axis in nicotine-related LUAD, which is a potential therapeutic strategy for cancer.

Atractylodes macrocephala Rhizoma alleviates blood hyperviscosity induced by high-fat, high-sugar, and high-salt diet via inhibition of gut-liver inflammatory activation and fibrinogen synthesis

Ethnopharmacological relevanceUnhealthy dietary patterns and lifestyle changes have been linked to increased blood viscosity, which is recognized as an important pathogenic factor in cardiovascular and cerebrovascular diseases. The underlying mechanism may involve chronic inflammation resulting from intestinal barrier disruption induced by unhealthy diets. The rhizome of Atractylodes macrocephala Koidz. (Called Baizhu in China), is a well-used “spleen-reinforcing” traditional Chinese medicinal herb used for thousands of years. Previous research has demonstrated its multiple gastrointestinal health benefits and its ability to regulate metabolic disorders. However, the effects of Baizhu on blood hyperviscosity induced by long-term unhealthy diets remain unclear. Aim of the studyThis study aimed to investigate the effects of the aqueous extract of Baizhu on blood hyperviscosity induced by unhealthy diet and to explore the possible mechanisms. Materials and methodsThe blood hyperviscosity model in SD rats was established utilizing a high-fat, high-sugar, and high-salt diet (HFSSD). Subsequently, the rats underwent a twelve-week intervention with varying doses of Baizhu and a positive control. To evaluate the efficacy of Baizhu on blood hyperviscosity in model rats, we measured behavioral index, hemorheological parameters, inflammatory cytokines, hematology, adhesion molecules, as well as biochemical indicators in serum and liver. We also assessed the pathological states of the colon and liver. Furthermore, Western blotting, ELISA, IHC, and qRT-PCR were used to determine the effect of Baizhu on the IL-6/STAT3/ESRRG signaling pathway and FIB synthesis. ResultsThe intervention of Baizhu showed evident attenuating effects on blood viscosity and microcirculation disorders, and exhibit the capacity to moderately modulate parameters including grip, autonomous activities, vertigo time, TC, TG, LDL-c, inflammatory factors, adhesion factors, hematological indicators, etc. At the same time, it reduces liver lipid droplet deposition, restores intestinal integrity, and lowers LPS level in the serum. Subsequent experimental results showed that Baizhu downregulated the expression of TLR4 and NF-κB in colon tissue, as well as the expression of IL-6, TLR4, p-JAK2, p-STAT3, and ESRRG in liver tissue. Finally, we also found that Baizhu could regulate the levels of FIB in plasma and liver. ConclusionBaizhu protects HFSSD-induced rats from blood hyperviscosity, likely through repairing the intestinal barrier and inhibiting LPS/TLR4-associated liver inflammatory activation, thus suppressing FIB synthesis through the downregulation of IL-6/STAT3/ESRRG pathway.

Cyclanoline Reverses Cisplatin Resistance in Bladder Cancer Cells by Inhibiting the JAK2/STAT3 Pathway.

Cisplatin is a key therapeutic agent for bladder cancer, yet the emergence of cisplatin resistance presents a significant clinical challenge. This study aims to investigate the potential and mechanisms of cyclanoline (Cyc) in overcoming cisplatin resistance. Cisplatin-resistant T24 and BIU-87 cell models (T24/DR and BIU-87/DR) were established by increasing gradual concentration. Western Blot (WB) assessed the phosphorylation of STAT3, JAK2, and JAK3. T24/DR and BIU-87/DR cell lines were treated with selective STAT3 phosphorylation modulators, and cell viability was evaluated by CCK-8. Cells were subjected to cisplatin, Cyc, or their combination. Immunofluorescence (IHC) examined p-STAT3 expression. Protein and mRNA levels of apoptosis-related and cell cycle-related factors were measured. Changes in proliferation, invasion, migration, apoptosis, and cell cycle were monitored. In vivo, subcutaneous tumor transplantation models in nude mice were established, assessing tumor volume and weight. Changes in bladder cancer tissues were observed through HE staining, and the p-STAT3 was assessed via WB and IHC. Cisplatin-resistant cell lines were successfully established, demonstrating increased phosphorylation of STAT3, JAK2, and JAK3. Cisplatin or Cyc treatment decreased p-STAT3, inhibited invasion and migration, and induced apoptosis and cell cycle arrest in the G0/G1 phase in vitro. In vivo, tumor growth was significantly suppressed, with extensive tumor cell death. IHC and WB consistently showed a substantial downregulation of STAT3 phosphorylation. These changes were more pronounced when cisplatin and Cyc were administered in combination. Cyc reverses cisplatin resistance via JAK/STAT3 inhibition in bladder cancer, offering a potential clinical strategy to enhance cisplatin efficacy in treating bladder cancer.

Colorectal Cancer Cell-Derived Extracellular Vesicles Promote Angiogenesis Through JAK/STAT3/VEGFA Signaling

Background: Angiogenesis plays a crucial role in the growth of colorectal cancer (CRC). Recent studies have identified extracellular vesicles (EVs) in the tumor microenvironment as important mediators of cell-to-cell communication. However, the specific role and mechanisms of CRC-derived EVs in regulating tumor angiogenesis remain to be further investigated. Methods: EVs were isolated from the conditioned medium of the CRC cells using ultracentrifugation. We investigated the effects of HT-29-derived EVs on tumor growth and angiogenesis in a subcutaneous HT-29 CRC tumor model in mice. Additionally, we evaluated the impact of HT-29-derived EVs on the proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs). Subsequently, bioinformatics analysis was performed to identify relevant signaling pathways, and pathway inhibitors were used to block the activation of these pathways, aiming to elucidate their roles in angiogenesis. Results: We found that HT-29-derived EVs can promote tumor growth and angiogenesis in vivo, as well as significantly enhance the proliferation, migration, and tube formation of HUVECs. Bioinformatics analysis revealed that HT-29-derived EVs may regulate angiogenesis through the JAK/STAT3 signaling pathway. Specifically, we observed that CRC-derived EVs promoted the phosphorylation of STAT3 (p-STAT3) and the expression of VEGFA in the nucleus of HUVECs. Treatment with the STAT3 inhibitor Stattic reduced the nuclear expression of p-STAT3, which impaired its function as a transcription factor, thereby inhibiting VEGFA expression and the pro-angiogenic effects of CRC-derived EVs. Conclusions: EVs derived from CRC cells promote CRC tumor angiogenesis by regulating VEGFA through the JAK/STAT3 pathway in endothelial cells.

Effects of Thyroid Hormones on Cellular Development in Human Ovarian Granulosa Tumor Cells (KGN).

Ovarian cancer is a common malignant tumor in the female reproductive system, and Granulosa cell tumor (GCT) of the ovary is a rare type of ovarian cancer, which significantly threatens women's reproductive health. It has been reported that dysregulation of thyroid hormones (THs) may be closely related to the progression and prognosis of ovarian cancer. Moreover, THs regulate phosphorylation of signal transducer and activator of transcription (STAT3) and Octamer-binding transcription factor 4 (OCT4) expression. It has been reported that STAT3 and OCT4 play important roles in cellular development and tumorigenesis. However, the mechanisms by which THs affect the development of GCT are still remained unclear. To evaluate the effect of THs on human ovarian granulosa tumor cells (KGN), cells were treated with 3,5,3' -triiodothyronine (T3). Oct4 small interfering (Oct4 siRNA) or STAT3 inhibitor C188-9 was also co-cultured with cells in some experiments, respectively. The cell viability, proliferation, and proteins content were detected by CCK-8, EdU, and Western Blotting, respectively. The results showed that T3 enhanced cell viability and proliferation. Moreover, T3 also increased the expression of thyroid hormone receptor (TR), p-STAT3, and OCT4 proteins. The effects of T3 on both p-STAT3 and OCT4 expression were blocked by TR antagonist 1-850. Meanwhile, C188-9, an inhibitor of STAT3, decreased T3-induced cellular viability, proliferation, and OCT4 expression, highlighting that p-STAT3 can regulate the expression of OCT4 and affect cellular viability, and proliferation. Furthermore, T3-induced cellular growth was reduced by Oct4 siRNA, which indicates that T3 regulates cellular development through OCT4. These findings suggest that T3 increases cellular development via OCT4, which is mediated by phosphorylation of STAT3, and TR is also involved in these processes.

DAMPs Drive Fibroinflammatory Changes in the Glaucomatous ONH.

The optic nerve head (ONH) is well known to be the initial site of glaucomatous damage; however, the molecular mechanisms initiating this pathology are not fully understood. To further understand the initiating factors in glaucomatous damage we utilized a novel mouse model of glaucoma, B6.EDA+/+ mice, which constitutively express fibronectin containing the extra domain A (FN+EDA). FN+EDA is a known damage-associated molecular pattern (DAMP) that activates Toll-like receptor 4 and elicits a fibro-inflammatory response. Eyes from B6.EDA+/+ and C57BL/6J mice were evaluated for retinal ganglion cell (RGC) death, retinal nerve fiber layer (RNFL) thickness, and optic nerve (ON) damage at 12 months and 22 months of age. ONH sections were isolated using laser capture microdissection for subsequent RNA-sequencing and Gene Set Enrichment Analysis (GSEA). GSEA results were confirmed using immunohistochemical (IHC) staining. B6.EDA+/+ mice exhibit significantly higher intraocular pressure, loss of RGCs, thinning of the RNFL, and progressive levels of ON damage at 12 months and 22 months of age compared to C57BL/6J controls. Protein expression of DAMPs FN+EDA and biglycan was significantly increased in B6.EDA+/+ mice compared to C57BL/6J controls. GSEA analysis identified significantly up- and downregulated gene groupings at both 12 months and 22 months of age, and IHC staining at 12 and 18 months of age demonstrated significant increases of IFNα, IFNβ, and pSTAT1 expression in B6.EDA+/+ mice compared to C57BL/6J controls. Our study characterizes glaucomatous changes to the retina, ON, and ONH over the course of 2 years and identifies novel molecular pathways associated with these pathophysiological changes. These data illustrate the effects of FN+EDA on the fibro-inflammatory response in the aging ONH in a novel mouse model of glaucoma.

Induction of immunogenic cell death and enhancement of the radiation-induced immunogenicity by chrysin in melanoma cancer cells

Chrysin is a natural flavonoid with anti-cancer effects. Despite its beneficial effects, little information is available regarding its immunogenic cell death (ICD) properties. In this work, we hypothesized that chrysin can potentiate radiotherapy(RT)-induced immunogenicity in melanoma cell line (B16-F10). We examined the effects of chrysin alone and in combination with radiation on ICD induction in B16-F10 cells. Cell viability was assessed using an MTT assay. Cell apoptosis and calreticulin (CRT) exposure were determined using flow cytometry. Western blotting and ELISA assay were employed to examine changes in protein expression. Combination therapy exhibited a synergistic effect, with an optimum combination index of 0.66. The synergistic anti-cancer effect correlated with increased cell apoptosis in cancer cells. Compared to the untreated control, chrysin alone and in combination with RT induced higher levels of DAMPs, such as CRT, HSP70, HMGB1, and ATP. The protein expression of p-STAT3/STAT3 and PD-L1 was reduced in B16-F10 cells exposed to chrysin alone and in combination with RT. Conditioned media from B16-F10 cells exposed to mono-and combination treatments elicited IL-12 secretion in dendritic cells (DCs), inducing a Th1 response. Our findings revealed that chrysin could induce ICD and intensify the RT-induced immunogenicity.

STAT3 blockade ameliorates LPS-induced kidney injury through macrophage-driven inflammation

BackgroundSignal transducer and activator of transcription 3 (STAT3), a multifaceted transcription factor, modulates host immune responses by activating cellular response to signaling ligands. STAT3 has a pivotal role in the pathophysiology of kidney injury by counterbalancing resident macrophage phenotypes under inflammation conditions. However, STAT3’s role in acute kidney injury (AKI), particularly in macrophage migration, and in chronic kidney disease (CKD) through fibrosis development, remains unclear.MethodsStattic (a JAK2/STAT3 inhibitor, 5 mg/kg or 10 mg/kg) was administered to evaluate the therapeutic effect on LPS-induced AKI (L-AKI) and LPS-induced CKD (L-CKD), with animals sacrificed 6–24 h and 14 days post-LPS induction, respectively. The immune mechanisms of STAT3 blockade were determined by comparing the macrophage phenotypes and correlated with renal function parameters. Also, the transcriptomic analysis was used to confirm the anti-inflammatory effect of L-AKI, and the anti-fibrotic role was further evaluated in the L-CKD model.ResultsIn the L-AKI model, sequential increases in BUN and blood creatinine levels were time-dependent, with a marked elevation of 0–6 h after LPS injection. Notably, two newly identified macrophage subpopulations (CD11bhighF4/80low and CD11blowF4/80high), exhibited population changes, with an increase in the CD11bhighF4/80low population and a decrease in the CD11blowF4/80high macrophages. Corresponding to the FACS results, the tubular injury score, NGAL, F4/80, and p-STAT3 expression in the tubular regions were elevated. STAT3 inhibitor injection in L-AKI and L-CKD mice reduced renal injury and fibrosis. M2-type subpopulation with CD206 in CD11blowF4/80high population increased in the Stattic-treated group compared with that in the LPS-alone group in the L-AKI model. Additionally, STAT3 inhibitor reduced inflammation driven by LPS-stimulated macrophages and epithelial cells injury in the co-culture system. Transcriptomic profiling identified 3 common genes in the JAK-STAT, TLR, and TNF signaling pathways and 11 common genes in the LPS with macrophage response. The PI3K-AKT (IL-6, Akt3, and Pik3r1) and JAK-STAT pathways were determined as potential Stattic targets. Further confirmation through mRNA and protein expressions analyses showed that Stattic treatment reduced inflammation in the L-AKI and fibrosis in the L-CKD mice.ConclusionsSTAT3 blockade effectively mitigated inflammation by retrieving the CD11blowF4/80high population, further emphasizing the role of STAT3-associated macrophage-driven inflammation in kidney injury.

STAT3/p-STAT3 expression is correlated with clinicopathological characteristics and prognosis in non-small cell lung cancer.

Signal transducer and activator of transcription factor 3 (STAT3)/phosphorylated STAT3 (p-STAT) play a critical role in tumorigenesis, however, there is limited information on its prognostic value in non-small cell lung cancer (NSCLC). To address this question, 239 lung cancer and 71 normal lung tissue samples were obtained in this study. Immunohistochemistry was applied to detect STAT3/p-STAT3 expression. Pearson's Chi-squared test and the Kaplan-Meier method were conducted to evaluate associations with patients' clinical characteristics and survival. According to our results, STAT3/p-STAT3 was significantly upregulated in lung cancer tissue (p<0.001). Moreover, p-STAT3 expression was significantly correlated with age (p=0.046) and pathological types (p=0.037). In survival analysis, STAT3 positivity was negatively associated with survival in patients older than 60 years (p=0.043) but failed to be an independent prognostic factor in multivariate analysis (p=0.083). Therefore, STAT3/p-STAT3 may serve as a critical biomarker in NSCLC.

The combination of p16 and Rb expression pattern is helpful to predict high-risk HPV infection and the primary site in lymph node metastases of squamous cell carcinoma

Identifying the primary site of metastatic squamous cell carcinoma in lymph nodes can be challenging. An immunohistochemistry (IHC) analysis recently revealed that high-risk human papillomavirus (HR-HPV)-associated oropharyngeal squamous cell carcinomas (OPSCCs) typically show overexpression of p16 protein and a partial loss pattern of Rb. Nevertheless, the status of these markers in metastatic lesions is still unclear. In this study, we examined p16 and Rb expression status by IHC and transcriptionally active HR-HPV infection by mRNA in situ hybridization in paired primary and metastatic SCC lesions. A total of 50 patients with OPSCCs (n=17), hypopharyngeal SCCs (n=16), laryngeal SCCs (n=6), or uterine cervical SCCs (n=11) were enrolled. HR-HPV and p16 were positive in 21/50 (42 %) and 23/50 (46 %) patients, respectively. Primary and metastatic lesions showed concordant results for those three markers in individual patients. Among the p16-positive patients (n=23), HPV-positive cases typically showed a partial loss of Rb (n=20) and, rarely, a complete loss of Rb (n=1), whereas HPV-negative cases showed preserved Rb expression (n=2). All 27 p16-negative cases lacked HPV infection, while preserved expression and complete loss of Rb were observed in 26 and 1 of the p16-negative cases, respectively. Compared to standalone p16, the combination of p16 overexpression and Rb-partial/complete loss showed equally excellent sensitivity and negative predictive value (each 100 %) as well as improved specificity (100 % versus 93.1 %) and positive predictive value (100 % versus 91.3 %). Our results suggest that combining p16 and Rb expression patterns may be helpful in screening for HR-HPV infection in metastatic lymph nodes and in estimating the primary site of SCC.

Clinical prognosticators and targets in the immune microenvironment of intrahepatic cholangiocarcinoma

ABSTRACT Background Intrahepatic cholangiocarcinoma (ICC) is a disease with poor prognosis and limited therapeutic options. We investigated the tumor immune microenvironment (TIME) to identify predictors of disease outcome and to explore targets for therapeutic modulation. Methods Liver tissue samples were collected during 2008–2019 from patients (n = 139) diagnosed with ICC who underwent curative intent surgery without neoadjuvant chemotherapy. Samples from the discovery cohort (n = 86) were immunohistochemically analyzed on tissue microarrays (TMAs) for the expression of CD68, CD3, CD4, CD8, Foxp3, PD-L1, STAT1, and p-STAT1 in tumor core and stroma areas. Results were digitally analyzed using QuPath software and correlated with clinicopathological characteristics. For validation of TIME-related biomarkers, we performed multiplex imaging mass cytometry (IMC) in a validation cohort (n = 53). Results CD68+ cells were the predominant immune cell type in the TIME of ICC. CD4+high T cell density correlated with better overall survival (OS). Prediction modeling together with validation cohort confirmed relevance of CD4+ cells, PD-L1 expression by immune cells in the stroma and N-stage on overall disease outcome. In turn, IMC analyses revealed that silent CD3+CD4+ clusters inversely impacted survival. Among annotated immune cell clusters, PD-L1 was most relevantly expressed by CD4+FoxP3+ cells. A subset of tumors with high density of immune cells (“hot” cluster) correlated with PD-L1 expression and could identify a group of candidates for immune checkpoint inhibition (ICI). Ultimately, higher levels of STAT1 expression were associated with higher lymphocyte infiltration and PD-L1 expression. Conclusions These results highlight the importance of CD4+ T cells in immune response against ICC. Secondly, a subset of tumors with “hot” TIME represents potential candidates for ICI, while stimulation of STAT1 pathway could be a potential target to turn “cold” into “hot” TIME in ICC.

Yi-Fei-San-Jie Chinese medicine formula reverses immune escape by regulating deoxycholic acid metabolism to inhibit TGR5/STAT3/PD-L1 axis in lung cancer

BackgroundYi-Fei-San-Jie Formula (YFSJF), a proprietary medicine of the First Affiliated Hospital of Guangzhou University of Chinese Medicine, has been widely used in clinical practice for several years and is currently being tested in randomized controlled trials for early-stage lung cancer in China. However, the mechanisms by which YFSJF affects lung cancer biology, particularly the immune microenvironment and metabolic processes, remain poorly understood. PurposeThis study aims to explore how YFSJF modulates the immune microenvironment and metabolism in lung cancer, specifically its unique role in inhibiting immune evasion by targeting the TGR5/STAT3/PD-L1 pathway, which has not previously been reported. MethodsComputed Tomography (CT) scan was used to assess YFSJF efficacy in patients with lung cancer and a mouse model of urethane-induced lung cancer. Histopathological evaluation, flow cytometry, and metabolomic analysis were used to assess lung tissue structure, immune cell subset changes, and metabolism modulation, respectively. Western blotting and immunohistochemistry were used to detect Ki67, TTF-1, TGR5, STAT3, p-STAT3, and PD-L1 protein expression. Serum cytokines were detected by ELISA. ResultsYFSJF effectively reduced the size of human lung cancer lesions and decreased the tumor burden and improved survival rates in mice. Lung tissue structure was also improved after YFSJF treatment. YFSJF regulated T-cell subsets, particularly by downregulating cells with PD-1-positive expression of CD3+, CD4+, and CD8+, and elevated serum TNF-α, IFN-γ, and GzmB levels. In addition, YFSJF modulated bile acid metabolism, particularly by inhibiting deoxycholic acid metabolism, which participates in immune regulation in lung cancer by acting on the G protein-coupled bile acid receptor TGR5. ConclusionFinally, YFSJF inhibited immune evasion by blocking the TGR5-mediated STAT3/PD-L1 pathway, weakening PD-L1 and PD-1 binding and reviving T-cell immune activity, thereby countering lung cancer immune evasion and exerting anti-tumor effects.

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, 1,756 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.

Proteomic Analysis Reveals Oxidative Phosphorylation and JAK-STAT Pathways Mediated Pathogenesis of Pemphigus Vulgaris.

Pemphigus vulgaris (PV) stands as a rare autoimmune bullous disease, while the precise underlying mechanism remains incompletely elucidated. High-throughput proteomic methodologies, such as LC-MS/MS, have facilitated the quantification and characterisation of proteomes from clinical skin samples, enhancing our comprehension of PV pathogenesis. The objective of this study is to elucidate the signalling mechanisms underlying PV through proteomic analysis. Proteins and cell suspension were extracted from skin biopsies obtained from both PV patients and healthy volunteers and subsequently analysed using LC-MS/MS and scRNA-seq. Cultured keratinocytes were treated with PV serum, followed by an assessment of protein expression levels using immunofluorescence and western blotting. A total of 880, 605, and 586 differentially expressed proteins (DEPs) were identified between the lesion vs. control, non-lesion vs. control, and lesion vs. non-lesion groups, respectively. The oxidative phosphorylation (OXPHOS) pathway showed activation in PV. Keratinocytes are the major cell population in the epidermis and highly expressed ATP5PF, ATP6V1G1, COX6B1, COX6A1, and NDUFA9. In the cellular model, there was a notable increase in the expression levels of OXPHOS-related proteins (V-ATP5A, III-UQCRC2, II-SDHB, I-NDUFB8), along with STAT1, p-STAT1, and p-JAK1. Furthermore, both the OXPHOS inhibitor metformin and the JAK1 inhibitor tofacitinib demonstrated therapeutic effects on PV serum-induced cell separation, attenuating cell detachment. Metformin notably reduced the expression of V-ATP5A, III-UQCRC2, II-SDHB, I-NDUFB8, p-STAT1, p-JAK1, whereas tofacitinib decreased the expression of p-STAT1 and p-JAK1, with minimal impact on the expression of V-ATP5A, III-UQCRC2, II-SDHB, and I-NDUFB8. Our results indicate a potential involvement of the OXPHOS and JAK-STAT1 pathways in the pathogenesis of PV.

Nebulization of Hypoxic hUCMSC-EVs Attenuates Airway Epithelial Barrier Defects in Chronic Asthma Mice by Transferring CAV-1.

Nebulization of hypoxic human umbilical cord mesenchymal stem cell-derived extracellular vesicles (Hypo-EVs) can suppress airway inflammation and remodeling in a chronic asthmatic mouse; however, the exact mechanism remains unclear. Recently, airway epithelial barrier defects have been regarded as crucial therapeutic targets in asthma. The aim of this study was to investigate whether and how Hypo-EVs protect against the disruption of the airway epithelial barrier under asthmatic conditions. The therapeutic effects of Hypo-EVs on airway epithelial barrier defects were evaluated in ovalbumin (OVA)-induced asthmatic mice and in IL-4 and IL-13-induced HBE135-E6E7 cell models by detecting cell monolayer leakage and junctional protein expression. The protein levels in Hypo-EVs were determined by Western blotting, and a gene knockdown approach was used to investigate the biofactors in Hypo-EVs. Nebulization of Hypo-EVs directly alleviated airway epithelial barrier defects in asthmatic mice, as evidenced by colocalization with bronchial epithelial cells, decreased albumin concentration, and increased ZO-1 and E-cadherin expression. In vitro, Hypo-EV treatment dramatically rescued the increase in airway cell permeability, and upregulated the ZO-1 and E-cadherin protein expressions. Based on WB analysis, we found that caveolin-1 (CAV-1) was strongly enriched in Hypo-EVs. The knockdown of CAV-1 protein levels in Hypo-EVs significantly impaired Hypo-EV-mediated barrier protection in vitro and in vivo. Moreover, CAV-1 knockdown significantly abolished the beneficial effects of Hypo-EVs on airway inflammation and remodeling in asthmatic mice. In addition, we showed that IL-4/IL-13-induced airway epithelial barrier defects were mainly related to activation of STAT6 phosphorylation (p-STAT6), and overexpression of CAV-1 or Hypo-EV treatment inhibited the levels of p-STAT6 in IL-4/IL-13-induced HBE135-E6E7 cells. Nebulization of Hypo-EVs can attenuate airway epithelial barrier defects in asthma by delivering CAV-1 to inhibit p-STAT6 expression and may be used to treat other barrier defect diseases.

Naringenin alleviates intestinal ischemia/reperfusion injury by inhibiting ferroptosis via targeting YAP/STAT3 signaling axis

BackgroundIntestinal ischemia/reperfusion injury (IRI) is a significant clinical emergency, and investigating novel therapeutic approaches and understanding their underlying mechanisms is essential for improving patient outcomes. Naringenin (Nar), a flavanone present in tomatoes and citrus fruits, is frequently consumed in the human diet and recognized for having immunomodulatory, anti-inflammatory, and antioxidant properties. Despite Nar being able to alleviate intestinal IRI, the exact molecular mechanisms remain elusive. PurposeTo investigate Nar's protective properties on intestinal IRI and elucidate the mechanisms, a comprehensive approach that combines network pharmacology analysis with experimental verification in vitro and in vivo was adopted. MethodsThe oxygen-glucose deprivation/reoxygenation (OGD/R) model in IEC-6 cells and a murine model of intestinal IRI were used. Nar's effects on intestinal IRI were assessed through histological analysis using H&E staining and tight junction (TJ) protein expression. Ferroptosis-related parameters, including iron levels, superoxide dismutase (SOD), glutathione (GSH), reactive oxygen species (ROS), malondialdehyde (MDA), and mitochondrial morphology, were analyzed. Network pharmacology was utilized to predict the pathways through which Nar exerts its anti-ferroptosis effects. Further mechanistic insights were obtained through si-RNA transfection, YAP inhibitor (verteporfin, VP) treatment, ferroptosis inhibitor (Ferrostatin-1) and ferroptosis inducer (Erastin) application, co-immunoprecipitation (Co-IP) and Western blotting. ResultsOur results revealed that pretreatment with Nar significantly mitigated intestinal tissue damage and improved gut barrier function, as evidenced by increased TJ proteins (ZO-1 and Occludin). Nar reduced iron, MDA, and ROS, while it increased GSH and SOD levels. Additionally, Nar alleviated mitochondrial damage in mice. Nar treatment increased GPX4 and SLC7A11, while decreasing ACSL4 levels both in vivo and in vitro. Network pharmacology analysis suggested that Nar may target the Hippo signaling pathway. Notably, YAP, a key transcriptional co-activator within the Hippo pathway, was downregulated in intestinal IRI mice and OGD/R-induced IEC-6 cells. Nar pretreatment activated YAP, thereby augmenting anti-ferroptosis effects. The inhibition of YAP activation by VP or YAP knockdown increased p-STAT3 expression, thereby diminishing Nar's efficacy. Co-IP and immunofluorescence studies confirmed the interaction between YAP and STAT3. ConclusionThis study shows that Nar can inhibit ferroptosis in intestinal IRI via activating YAP, which in turn suppresses STAT3 phosphorylation, thereby unveiling a novel mechanism and supporting Nar's potential to be a promising therapeutic agent for intestinal IRI.

Prognostic Importance of PTEN, p53, and MDM2 Expressions in Endometrioid and Serous-Type Endometrial Carcinomas

Aim: Endometrial carcinomas (ECs) are neoplasms with the highest rate of change in the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway. In this study, the relationship among PTEN, MDM2, and p53 protein expression in the PI3K/AKT/mTOR pathway with clinicopathological data in endometrioid endometrial carcinomas (EECs) and serous-type endometrial carcinomas (SECs) was evaluated. Material and Method: A hundred and twenty cases of patients who underwent hysterectomy for EC between 2009 and 2021 were included in the study. Thirty cases of SEC and 90 cases of EEC were evaluated. EEC cases consist of grades 1-3 tumors, and each group includes 30 patients. p53 was examined in two groups as normal/wild type and abnormal/mutant type. PTEN and MDM2 were examined in two groups: positive and negative. The relationship among p53, PTEN, and MDM2 immunohistochemical expression status with histological grade, myometrial invasion, cervical invasion, lymphovascular invasion (LVI), metastatic lymph nodes, presence of tumor in peritoneal fluid, tumor stage, and overall and progression-free survival was evaluated. Results: Loss of PTEN was associated with EEC compared to SEC (p

The mechanism of TGF-β mediating BRD4/STAT3 signaling pathway to promote fibroblast proliferation and thus promote keloid progression

ObjectiveThe purpose of this study was to investigate the effect of TGF-β on keloid and its molecular mechanism in fibroblasts. Methods: The difference between normal tissue and keloid tissue can be detected using HE staining. Fibroblasts were treated with TGF-β, and then treated with the BRD4 inhibitor JQ1 and the STAT3 activator Colivelin TFA. Western blot was used to measure the relative protein expression of TGF-β, BRD4, p-STAT3, p-EZH2, C-myc, KLF2, KLF4, α-SMA, and Collagen-I. Immunofluorescence staining was used to measure the relative fluorescence intensity of BRD4, p-STAT3, α-SMA, and Collagen-I. Cell proliferation ability was evaluated by CCK-8 assay and colony formation assay. Results:The expression of TGF-β and BRD4 was significantly higher in keloid tissue compared to normal tissue. TGF-β mediated the BRD4/STAT3 signaling pathway to inhibit p-EZH2 and promote the expression of C-myc, KLF2, KLF4, α-SMA, and Collagen-I. Additionally, TGF-β mediated the BRD4/STAT3 signaling pathway to enhance fibroblast proliferation. Conclusion:TGF-β mediates the BRD4/STAT3 signaling pathway to promote fibroblast proliferation and contribute to the progression of keloid.