p-STAT3 Levels Research Articles

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 90 mins 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, 24 hours 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 down-regulated SYN, GAP43, PSD95 expression and phosphorylation expression of JAK2 and STAT3 following MCAO and 24 hours of reperfusion. Collectively, these results indicates XXMD may play a neuroprotective role in reducing synaptic damage via JAK2/STAT3 signaling pathway.

Serum level of IFN-λ is elevated in idiopathic inflammatory myopathies.

Idiopathic inflammatory myopathies (IIM) are a heterogeneous group of autoimmune disorders with uncertain pathogenesis. Interferon (IFN)-λ has recently been described as an important mediator of immune responses. The main purpose of this study is to find out whether IFN-λ is involved in IIM. The published RNA-seq data of dermatomyositis (DM) muscle and IIM double negative (DN) B cells were analyzed. Serum IFN-λ1, IFN-λ2, and IFN-λ3 levels of 59 IIM patients and 29 healthy persons were determined by enzyme-linked immunosorbent assay (ELISA). The proportion of DN B cells and subsets as well as phosphorylated STAT1 (p-STAT1) levels in peripheral B cells was measured by flow cytometry. Ex vivo induction of double negative 2 (DN2) B cells were performed for validation. Type III IFN signaling pathway was enriched in muscle tissue from DM patients. Serum IFN-λ1, IFN-λ2, and IFN-λ3 levels were significantly higher in the IIM patients, especially patients with interstitial lung disease (ILD) and skin manifestations. Moreover, serum IFN-λ1 level was positively correlated with the disease activity. In addition, IIM patients with positive anti-melanoma differentiation-associated gene 5 (anti-MDA5) antibodies exhibited higher serum IFN-λ1, IFN-λ2, and IFN-λ3 levels. The frequency of DN2 B cells were elevated in IIM patients' blood. Interestingly, the type III IFN signaling pathway was enriched in circulating DN2 B cells from IIM patients, which could induce T-bet+CD19hi DN2 B cell differentiation ex vivo. IFN-λ may participate in the pathogenesis of IIM by acting on DN2 B cells and serve as a disease biomarker for IIM patients. Key Points • Type III IFN signaling pathway is enriched in the involved muscles of DM patients. • Serum IFN-λ in IIM patients is correlated with disease activity and is higher in patients with ILD, skin lesions, and positive anti-MDA5 antibodies. • DN2 B cells with enriched type III IFN signaling pathway are accumulated in the peripheral blood of IIM patients.

Plumbagin protect against sepsis-induced myocardial injury in mice by inhibiting the JAK2/STAT3 signaling pathway to reduce cardiomyocyte pyroptosis

To explore the mechanism of plumbagin for protecting against sepsis-induced myocardial injury in mice. Network pharmacology analysis was used to obtain the key targets of plumbagin and diseases, which were subjected to GO and KEGG analysis, and the binding energy was verified using molecular docking. In a mouse model of cecal ligation and puncture (CLP), the protective effect of plumbagin treatment prior to CLP against sepsis-induced myocardial injury was evaluated by examination of myocardial function and pathology using echocardiography and HE staining. Serum levels of CK-MB, LDH, MDA, IL-1β and IL-18 and myocardial ROS level in the mice were detected, and Western blotting was used to determine the protein expression levels of STAT3, GSDMD, caspase-11, JAK2, P-STAT3, P-JAK2, GSDMD-N and HMGB1 in the myocardial tissues. Five core targets were screened from the 10 intersecting genes. Molecular docking showed strong binding affinity of plumbagin to STAT3, p-STAT3, and JAK2. Compared with the sham-operated mice, the mouse models of CLP-induced sepsis had significantly decreased CO, LVEF, LVFS and SV and increased serum levels of CK-MB, LDH, MDA and myocardial inflammatory factors and ROS. HE staining and Western blotting showed obvious myocardial injury in the septic mice with increased expressions of JAK2/STAT3 signaling pathway and pyroptosis-related proteins (P < 0.05). Pretreatment with plumbagin significantly improved cardiac functions of CLP mice, lowered serum levels of CK-MB, LDH, MDA, inflammatory factors and myocardial ROS, and decreased the expression levels of JAK2/STAT3 signaling pathway and pyroptosis-related proteins. Plumbagin pretreatment alleviates myocardial injury in septic mice possibly by inhibiting the STAT3 signaling pathway to reduce cardiomyocyte pyroptosis.

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.

Evaluation of Xihuang Pill in inducing pyroptosis in glioma cells through modulation of miR-21-5p.

This study aims to elucidate the mechanism by which Xihuang Pill induces pyroptosis in glioma cells via the regulation of miR-21-5p. Human glioma cell lines U-87 and LN-229 were used as experimental models to assess the effects of Xihuang Pill on glioma pyroptosis. Cells were incubated with Xihuang Pill extract at concentrations of 7.5, 15, and 30µg/mL for 24h, alongside transfection with miR-21-5p mimic, an overexpression vector for STAT3, or incubation with 50µg/mL of the STAT3 activator Colivelin for 4h. Cell viability was measured using the CCK-8 assay, apoptosis was detected by flow cytometry, and expression levels of p-STAT3/STAT3 and pyroptosis-related proteins were determined by Western Blot. Additionally, cleaved caspase-1 was assessed by immunofluorescence, miR-21-5p expression by qRT-PCR, and STAT3 binding to the miR-21-5p promoter region by ChIP and dual-luciferase reporter assays. Results showed that Xihuang Pill significantly reduced cell viability, increased apoptosis, and upregulated the expression of pyroptosis-related proteins such as NLRP3, IL-1β, cleaved caspase-1, and GSDMD-N, while reducing p-STAT3/STAT3 and miR-21-5p levels (P < 0.05). Xihuang Pill inhibited STAT3 activation, which modulated miR-21-5p expression by binding to its promoter region. Co-transfection with miR-21-5p mimic reversed the effect of Xihuang Pill on glioma pyroptosis (P < 0.05). In conclusion, Xihuang Pill promotes glioma cell pyroptosis through the STAT3/miR-21-5p pathway.

Allicin Ameliorated High-glucose Peritoneal Dialysis Solution-induced Peritoneal Fibrosis in Rats via the JAK2/STAT3 Signaling Pathway.

Peritoneal fibrosis (PF) is one of the most serious complications of peritoneal dialysis (PD) and is the greatest obstacle to the clinical application of PD. Chinese herbal monomers have been effective in the prevention and treatment of PF. The aim of this study was to observe the effect of allicin on PF in rats induced by high glucose and to investigate its molecular mechanism of action. A rat model of PF was established by using a 4.25% glucose-based standard peritoneal dialysis solution. The degree of peritoneal pathological damage was assessed by Hematoxylin and eosin (H&E) staining. Peritoneal collagen deposition was detected by Masson's trichrome staining. The levels of Interleukin-6 (IL-6), Tumor necrosis factor-α (TNF-α), Interleukin-1β (IL-1β) and monocyte chemoattractant protein-1 (MCP-1) in the serum were measured by Enzyme Linked Immunosorbent Assay (ELISA). The expression levels of TGF-β, α-smooth muscle actin (α-SMA) and collagen I were examined by western blotting and immunohistochemistry. The protein expression levels and mRNA levels of E-cadherin, N-cadherin, vimentin, janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) in peritoneal tissue were determined by western blotting and qRT-PCR. TGF-β1 stimulated human peritoneal mesothelial cells (HPMCs), and the cells were treated with allicin and the JAK2/STAT3 pathway activator colivelin alone or in combination. A cell counting kit-8 (CCK-8) assay was used to measure cell viability. The role of JAK2/STAT3 in the effects of allicin was confirmed via in vitro mechanistic research by western blotting, wound healing assays and Transwell assays. Allicin relieves the inflammatory response by downregulating the levels of IL-1β, IL-6, MCP-1 and TNF-α. Furthermore, allicin decreased the expression of TGF-β, α-SMA and collagen I. Allicin also alleviated epithelial-to-mesenchymal transition (EMT), as specifically manifested by increased E-cadherin and reduced N-cadherin and vimentin. Further studies revealed that allicin reduced the protein levels of JAK2, STAT3, p-JAK2, and p-STAT3. The results of the cellular experiments verified the above results. The ability of allicin to inhibit fibrosis and the EMT process was significantly attenuated after HPMCs were treated with colivelin. Taken together, these findings suggest that allicin inhibits inflammation and EMT, thereby improving PF, and this protective effect may be achieved by inhibiting the JAK2/STAT3 signaling pathway.

Grilled nux vomica alleviates myasthenia gravis by inhibiting the JAK2/STAT3 signaling pathway: a study in a mice model

BackgroundGrilled Nux Vomica (GNV) is a promising traditional Chinese medicine to treat myasthenia gravis (MG), but its effects and mechanisms need further exploration.MethodsExperimental autoimmune MG (EAMG) model was established by muscle-specific kinase (MuSK) induction on C57BL/6 J mice. Mice were treated with GNV and/or ruxolitinib (JAK2 inhibitor) or AG490 (STAT3 inhibitor) for 30 days via gavage after modeling and randomized into 7 groups: control, model, low-dose GNV, middle-dose GNV, high-dose GNV, GNV + ruxolitinib, GNV + AG490. Body weight, muscle strength, clinical score, MuSK level, neuromuscular junction integrity (agrin and acetylcholine receptor [AChR] levels), inflammatory factors (IL-2 and IL-6), and the activation of the JAK2/STAT3 pathway were measured and compared between groups.ResultsGNV significantly improved body weight and muscle strength, as well as reduced clinical scores, MuSK levels, and inflammatory markers (IL-2 and IL-6) levels compared with untreated EAMG mice. GNV also protected the neuromuscular junction and increased agrin and AChR co-expression in a dose-dependent manner. In addition, GNV attenuated the levels of p-JAK2 and p-STAT3, which are aberrantly upregulated in EAMG mice. After co-treatment with ruxolitinib or AG490, the effect of GNV on body weight, muscle strength, clinical score, MuSK level, neuromuscular junction integrity, levels of inflammatory factors, and JAK2/STAT3 pathway was further amplified in EAMG mice.ConclusionsGNV improves MG by inhibiting the JAK2/STAT3 pathway, which might be an effective therapeutic strategy for MG.

Contribution of the Activated mTOR-STAT3 Pathway to the Pathogenesis of Focal Cortical Dysplasia Type IIIa in Pediatric Patients through Astrocyte Proliferation Mediation.

The aim of this study was to detect the association between the mTOR-STAT3 pathway and focal cortical dysplasia type IIIa (FCD IIIa) in children. A retrospective review was conducted based on 26 pediatric patients diagnosed with FCD IIIa who underwent surgical intervention. These patients were selected from a cohort of 157 individuals presenting with temporal lobe epilepsy. For comparative analysis, a control group consisting of 5 children who underwent intracranial decompression was established. Immunohistochemistry, immunofluorescence, and western blot techniques were used to assess the expression levels of mTOR, P-mTOR, P-70s6k, STAT3, P-STAT3, and GFAP in brain tissue specimens obtained from the two groups. The mTOR-STAT3 pathway exhibited activation in the FCD IIIa group (all P < 0.01). Additionally, immunofluorescence analysis revealed that cells positive for PSTAT3 were identified as astrocytes. Moreover, within the FCD IIIa group, there was a marked elevation in the expression of the mTOR-STAT3 pathway in the hippocampus compared to the brain cortex tissue. The mTOR-STAT3 pathway was demonstrated to be substantially associated with FCD IIIa in pediatric patients. The activation of the mTOR-STAT3 signaling pathway may contribute to the pathogenesis of FCD IIIa in pediatric patients by modulating the proliferation of astrocytes.

Neobavaisoflavone Ameliorates Memory Deficits and Brain Damage in Aβ25-35-Induced Mice by Regulating SIRT1.

Alzheimer's disease (AD) is a common chronic neurodegenerative disease in older people, and there is no specific treatment that can stop or reverse its progression. Neobavaisoflavone (NBIF) is a flavonoid that has been shown to have neuroprotective effects, but its role in AD has not been revealed. The present study investigated the role and mechanism of NBIF on Aβ25-35-induced brain injury. In this experiment, the AD mouse model was established by injection of Aβ25-35 peptides (200 μM, icv), and Donepezil (Don, 10 mg/kg/days), NBIF-L (15 mg/kg/days), and NBIF-H (30 mg/kg/days) were administered orally for 4 weeks. Learning memory, hippocampal pathological changes, pathological markers, apoptosis, oxidative stress, inflammation, immune cells were measured in mice. Network pharmacology combined with the GEO database led to the identification of SIRT1, a key target for NBIF intervention in AD, and levels of SIRT1, p-STAT3 and FOXO1 were measured. In addition, the antagonistic activity of SIRT1 transfection silencing against NBIF in Aβ25-35-induced in N9 cells and N2a-APP69 cells was investigated to assess whether the effects caused by NBIF were mediated by SIRT1. The results showed that NBIF ameliorated learning memory and hippocampal neuronal damage, reduced pathological markers, apoptosis, oxidative stress and neuroinflammation, and modulated immune cells. SIRT1 is a key target for NBIF intervention in AD, and NBIF upregulates SIRT1 and reduces the expression levels of p-STAT3 and FOXO1. Furthermore, silencing SIRT1 effectively reduced the protective effect of NBIF on Aβ25-35-induced N9 cells and N2a-APP69 cells, which indicated that the protective effect of NBIF on AD is related to SIRT1. NBIF ameliorated Aβ25-35-induced brain injury by inhibiting apoptosis, oxidative stress, and neuroinflammation, which may be mediated through SIRT1 signaling. These findings provide a rationale for NBIF in the treatment of AD and help facilitate the development of clinical therapeutic agents for AD.

Increased Frequency of Circulating Activated FOXP3+ Regulatory T Cell Subset in Patients with Chronic Lymphocytic Leukemia Is Associated with the Estimate of the Size of the Tumor Mass, STAT5 Signaling and Disease Course during Follow-Up of Patients on Therapy.

Advanced chronic lymphocytic leukemia (CLL) is accompanied by increased circulating regulatory T cells (Tregs) and increased susceptibility to severe infections, which were also shown to entail a striking induction of FOXP3 expression in Tregs. As homeostasis of the most suppressive CD45RA-FOXP3high activated Treg (aTreg) subset differs, it is critical to analyse homeostatic signalling in Treg subsets. In this study, by using conventional and imaging flow cytometry, we monitored STAT5 signalling/phosphorylation (pSTAT5) and investigated Treg subsets in relation to the Binet stage, the total tumor mass score (TTM) and the disease course during a follow-up of 37 patients with CLL. The aTreg percentage was significantly increased among CD4+ T cells from patients with advanced disease and significantly correlated with the TTM. A subgroup of patients with higher aTreg percentages among CD4+FOXP3+ T cells at the start of therapy was characterised by more frequent episodes of severe infections during follow-up. The results suggesting that an aTreg fraction could represent a possible marker of a severe disease course with infectious complications. Augmented homeostatic STAT5 signalling could support aTreg expansion, as higher pSTAT5 levels were significantly correlated with an increased aTreg frequency among CD4+FOXP3+ T cells during the follow-up of patients on therapy, as well as following SARS-CoV-2 antigen-specific stimulation in vitro.

Enhanced anti-tumor efficacy of S3I-201 in breast cancer mouse model through Wharton jelly- exosome

ObjectiveExosomes, membrane-enveloped vesicles found in various cell types, including Wharton’s jelly mesenchymal stem cells, play a crucial role in intercellular communication and regulation. Their use as a cell-free nanotechnology and drug delivery system has attracted attention. Triple-negative breast cancer (TNBC) is a major global health problem and is characterized by a high mortality rate. This study investigates the potential of Wharton’s Jelly mesenchymal stem cell-derived exosomes (WJ-Exo) as carriers of S3I-201 and their effects on STAT3 expression in breast cancer cell lines, and evaluates whether these exosomes can enhance the anti-tumor effect of S3I-201.MethodsThe filtered WJ-Exos were analyzed by Transmission Electron Microscopy (TEM), Scanning electron microscopy (SEM), Dynamic Light Scattering (DLS), flow cytometry, and Western blotting. These exosomes were then used for loading with S3I-201, resulting in the nano-formulation WJ-Exo(S3I-201). The effect of WJ-Exo(S3I-201) on 4T1 cancer cells was investigated in vitro using MTT assay, flow cytometry, wound healing assay, Western blotting and Quantitative Real-Time Polymerase chain reaction (qPCR) analysis. Finally, the therapeutic efficacy of the nano-formulation was investigated in vivo using a tumor-bearing mouse model.ResultsIn vitro experiments showed that co-incubation of 4T1 cells with the nano-formulation resulted in a significant reduction in p-STAT3 levels, induction of apoptosis, modulation of Bcl-2, Bax and caspase-3 protein and gene expression, and inhibition of migration. In vivo, treatment of tumor-bearing mice with WJ-Exo(S3I-201) showed a strong antitumor effect that exceeded the efficacy observed in the S3I-201 group.ConclusionOur results demonstrate that WJ-Exo is an effective carrier for targeting S3I-201 to tumor cells and enhances the therapeutic efficacy of S3I-201 in tumor-bearing mice.

Interleukin-9 promotes EMT-mediated PM2.5-induced pulmonary fibrosis by activating the STAT3 pathway.

This study investigated the impact of PM2.5 on promoting EMT in PM2.5-induced pulmonary fibrosis (PF) development and explored molecular mechanisms of the IL-9/STAT3/Snail/TWIST1 signaling pathway in PF owing to PM2.5. Four groups of male SD rats were formed: control (0mg/kg.bw), low (1mg/kg.bw), medium (5mg/kg.bw), and high-dose (25mg/kg.bw) PM2.5 groups. Experimental rats were subjected to PM2.5 exposure via intratracheal instillation, given once weekly for 16weeks. 24 h after the final exposure, blood, BALF, and lung tissues were collected. Pulmonary epithelial cells underwent cultivation and exposure to varying PM2.5 concentrations with/without inhibitors for 24h, after which total protein was extracted for relevant protein assays. The findings demonstrated that PM2.5 damaged lung tissue to different degrees and led to PF in rats. Rats subjected to PM2.5 exposure exhibited elevated concentrations of IL-9 protein in both serum and BALF, and elevated levels of IL-9 and its receptor, IL-9R, in lung tissues, compared to control counterparts. Furthermore, PM2.5-exposed groups demonstrated significantly augmented protein levels of p-STAT3, Snail, TWIST1, Vimentin, COL-I, and α-SMA, while displaying notably diminished levels of E-Cadherin compared to control group. The same findings were observed in PM2.5-treated cells. In BEAS-2B cells co-treated with Stattic (STAT3 inhibitor) and PM2.5, the opposite results occurred. Similar results were obtained for cells co-treated with IL-9-neutralizing antibody and PM2.5. Our findings suggest PM2.5 mediates PF development by promoting IL-9 expression, leading to STAT3 phosphorylation and upregulation of Snail and TWIST1 expression, triggering EMT occurrence and progression in lung epithelial cells.

CRISPR-mediated WNK4 point mutation aggravates tumor progression and weakens chemotherapy sensitivity in gastric cancer.

Gastric cancer (GC) is the fifth most common malignancy, the molecular targets of which have been increasingly explored in recent years. As a serine/threonine protein kinase, the role of WNK lysine deficient protein kinase 4 (WNK4) in GC was clarified in this study. Human GC lines AGS and MKN45 were stably transfected with a WNK4 mutant constructed by the CRISPR/Cas9 method and treated with cis-dichlorodiammine platinum (CDDP, 2 μg/mL) and 5-fluorouracil (5-FU, 5 μg/mL) for 48h. Tumor-bearing mice were established with 5×106 mutant-type AGS cells, and injected with 40 mg/kg WP1066, the inhibitor of signal transducer and activator of transcription 3 (STAT3), for 21 days. Cell malignant potential and tumor growth were assessed. STAT3 activation was identified by western blot and immunohistochemistry. The interaction between WNK4 and STAT3 was determined using co-immunoprecipitation and immunofluorescence co-localization. WNK4 mutation promoted proliferation and invasion, and upregulated the p-STAT3/STAT3 value in GC cells with/without 5-FU and CDDP treatments, while inhibiting apoptosis of GC cells without drug treatment. In tumor-bearing mice, WNK4 mutation accelerated tumor growth, increased levels of p-STAT3, STAT3, and p-STAT3/STAT3, and strengthened the co-immunoprecipitation and co-localizing with STAT3; however, these effects were reversed by WP1066 treatment. Through activating STAT3, WNK4 mutation impacts both the natural and drug-treated growth of GC cells or tumors, suggesting a new avenue for preclinical research.

MSU crystallization promotes fibroblast proliferation and renal fibrosis in diabetic nephropathy via the ROS/SHP2/TGFβ pathway

Monosodium urate (MSU) crystallisation deposited in local tissues and organs induce inflammatory reactions, resulting in diseases such as gout. MSU has been recognized as a common and prevalent pathology in various clinical conditions. In this study, we investigated the role of MSU in the pathogenesis of diabetic kidney disease (DKD). We induced renal injury in diabetic kidney disease mice using streptozotocin (STZ) and assessed renal histopathological damage using Masson's trichrome staining and Collagen III immunofluorescence staining. We measured the levels of malondialdehyde (MDA), superoxide dismutase (SOD), and uric acid (UA) using ELISA. Protein expression levels of NLRP3, p-NF-κB, SHP2, p-STAT3, and p-ERK1/2 were analyzed by Western blot. To further investigate the role of MSU in diabetic kidney disease, we conducted in vitro experiments. In our in vivo experiments, we found that compared to the Model group, there was a significant increase in interstitial fibrosis in the kidneys of mice after treatment with MSU, accompanied by elevated levels of MDA, SOD, and UA. Furthermore, the protein expression of NLRP3, p-NF-NB, SHP2, p-STAT3, and p-ERK1/2 was upregulated. In our subsequent studies on mouse fibroblasts (L929 cells), we discovered that high glucose, MSU, and TGF-β could promote the expression of P22, GP91, NLRP3, NF-κB, p-NF-κB, p-SHP2, p-EGFR, p-STAT3, and Collagen-III proteins. Additionally, we found that SHP2 could counteract the upregulation trend induced by MSU on the expression of p-SHP2, p-EGFR, p-STAT3, and Collagen-III proteins, and inhibitors YQ128, NAC, and Cetuximab exhibited similar effects. Furthermore, immunofluorescence results indicated that SHP2 could inhibit the expression of the fibrosis marker α-SMA in L929 cells. These findings suggest that MSU can promote renal fibroblast SHP2 expression, induce oxidative stress, activate the NLRP3/NF-κB pathway, and enhance diabetic kidney disease fibroblast proliferation through the TGFβ/STAT3/ERK1/2 signaling pathway, leading to renal fibrosis.

C188-9 reduces patient-specific primary breast cancer cells proliferation at the low, clinic-relevant concentration

ObjectivesSTAT3 is a transcriptional activator of breast cancer oncogenes, suggesting that it could be a potential therapeutic target for breast cancer. Therefore, this study investigated the potential application of C188-9, a STAT3 signal pathway inhibitor, in the treatment of breast cancer through a novel pre-clinical platform with patient-specific primary cells (PSPCs).MethodsPSPCs were isolated from breast cancer samples obtained via biopsy or surgery from fifteen patient donors with their full acknowledgements. PSPCs were treated with C188-9 or other chemotherapeutic agents, and then analyzed with cell viability assay. Western blot assay and real-time quantitative PCR were also used to determine the expression and activity of STAT3 signaling pathway of corresponding PSPCs.ResultsC188-9 treatment at normal (experimental) concentration had valid inhibition on PSPCs proliferation. Meanwhile, treatment at a low (clinic-relevant) concentration of C188-9 for an extended period reduced cell viability of PSPCs still more than some of other traditional chemotherapy drugs. In addition, C188-9 decreased expression level of pSTAT3 in PSPCs from some, but not all patient samples. The treatment of C188-9 reduced cell viability of the breast cancer samples through inhibiting the STAT3 to C-myc signaling pathway.ConclusionsIn this study, we tested a novel drug C188-9 at a low, clinic-relevant concentration, together with several traditional chemotherapy agents. PSPCs from ten out of fifteen patient donors were sensitive to C188-9, while some of traditional chemotherapy agents failed. This finding suggested that C188-9 could have treatment effects only on those ten PSPC patient donors, indicating the future personalized utilization of PSPCs.

Irisin alleviates the pyroptosis of β cells in T2DM by inhibiting NLRP3 inflammasome through regulating miR-19b-3p/SOCS3/STAT3 axis mediated autophagy.

The purpose of this study was to analyze the mechanism by which irisin affects β-cell pyroptosis in type 2 diabetes mellitus (T2DM). The in vivo T2DM model was established by raised with high-fat diet and intraperitoneally injection of streptozocin. Min6 cells were divided into four groups: negative control (NC), high glucose (HG), HG + irisin, and HG + irisin+3-MA. The cell viability was determined by CCK-8 assay. Dual-luciferase gene reporter assay was conducted to confirm the binding between miR-19b-3p and SOCS3. The expression level of FNDC5 and GSDMD was visualized using the immunofluorescence assay. The protein level of FNDC5, Beclin1, LC3II/I, NLRP3, cleaved-caspase-1, GSDMD-N, STAT3, p-STAT3, and SOCS3 was determined by Western blotting. The secretion of irisin, lactate dehydrogenase (LDH), and insulin was checked by ELISA. In vivo results showed that pathological changes in islet tissues with declined number of β cells, elevated FBG value, decreased FIN and HOMA-β value, elevated autophagy-associated proteins expressions, and activated NLRP3 signaling in T2DM mice, which were dramatically reversed by FNDC5 overexpression. Furthermore, the declined level of miR-19b-3p and p-STAT3, as well as the upregulation of SOCS3, was greatly rescued by FNDC5 overexpression. The in vitro data confirmed the binding site between SOCS3 and miR-19b-3p. SOCS3 was downregulated and p-STAT3 was upregulated in miR-19b-3p mimic-treated Min6 cells. In HG-stimulated Min6 cells, the elevated cell viability, increased production of insulin, decreased release of LDH, and inactivated NLRP3 signaling induced by irisin were abolished by miR-19b-3p inhibitor and STAT3 inhibitor. The increased level of autophagy-related proteins and activated SOCS3/STAT3 axis induced by irisin in HG-stimulated Min6 cells were abolished by miR-19b-3p inhibitor. The inhibitory effect of irisin against NLRP3 signaling in HG-stimulated Min6 cells was abrogated by 3-MA. In conclusion, irisin alleviated the pyroptosis of β cells in T2DM by inhibiting NLRP3 signaling through miR-19b-3p/SOCS3/STAT3 axis mediated autophagy.

Histones Methyltransferase NSD3 Inhibits Lung Adenocarcinoma Glycolysis Through Interacting with PPP1CB to Decrease STAT3 Signaling Pathway.

Histones methyltransferase NSD3 targeting H3K36 is frequently disordered and mutant in various cancers, while the function of NSD3 during cancer initiation and progression remains unclear. In this study, it is proved that downregulated level of NSD3 is linked to clinical features and poor survival in lung adenocarcinoma. In vivo, NSD3 inhibited the proliferation, immigration, and invasion ability of lung adenocarcinoma. Meanwhile, NSD3 suppressed glycolysis by inhibiting HK2 translation, transcription, glucose uptake, and lactate production in lung adenocarcinoma. Mechanistically, as an intermediary, NSD3 binds to PPP1CB and p-STAT3 in protein levels, thus forming a trimer to dephosphorylate the level of p-STAT3 by PPP1CB, leading to the suppression of HK2 transcription. Interestingly, the phosphorylation function of PPP1CB is related to the concentration of carbon dioxide and pH value in the culture environment. Together, this study revealed the critical non-epigenetic role of NSD3 in the regulation of STAT3-dependent glycolysis, providing a piece of compelling evidence for targeting the NSD3/PPP1CB/p-STAT3 in lung adenocarcinoma.

Overexpression of MiR-188-5p Downregulates IL6ST/STAT3/ NLRP3 Pathway to Ameliorate Neuron Injury in Oxygen-glucose Deprivation/Reoxygenation.

CI/R, characterized by ischemic injury following abrupt reestablishment of blood flow, can cause oxidative stress, mitochondrial dysfunction, and apoptosis. We used oxygen-glucose deprivation/reoxygenation (OGD/R) induced injury in HT22 and primary mouse cortical neurons (MCN) as a model for CI/R. This study investigates the role of miR-188-5p in hippocampal neuron cell injury associated with Cerebral Ischemia-Reperfusion (CI/R). HT22 and MCN cells were induced by OGD/R to construct an in vitro model of CI/R. Cell apoptosis and proliferation were assessed using flow cytometry and the Cell Counting Kit-8 (CCK8). ELISA was conducted to measure the levels of IL-1β, IL-6, and TNF-α. Moreover, the interaction between miR-188-5p and IL6ST was investigated using dual luciferase assay, the expression of miR-188-5p, Bax, cleaved-caspase3, IL-6, Bcl-2, IL-1β, TNF-α, IL6ST, NFκB, NLRP3 and STAT3 was evaluated using RT-qPCR or Western blot, and immunofluorescence was used to analyze the co-expression of p-STAT3 and NLRP3 in neuronal cells. OGD/R reduced proliferation and miR-188-5p levels and increased IL6ST expression, inflammation, and apoptosis in HT22 and MCN cells. Moreover, miR-188-5p was found to bind to IL6ST. Mimics of miR-188-5p reduced apoptosis, lowered the expression of cleaved-caspase3 and Bax proteins, and elevated Bcl-2 protein expression in cells treated with OGD/R. Overexpression of miR-188-5p decreased the levels of NLRP3 and p-STAT3 in the OGD/R group. Furthermore, the overexpression of miR-188-5p reduced IL6ST, p- NFκB/NFκB, p-STAT3/STAT3, and NLRP3 proteins in OGD/R, and these effects could be reversed by IL6ST overexpression. Mimics of miR-188-5p were found to inhibit inflammation and the STAT3/NLRP3 pathway via IL6ST, thereby ameliorating injury in HT22 and MCN cells treated with OGD/R in the context of CI/R.

Baicalein Enhances Radiosensitivity in Colorectal Cancer via JAK2/STAT3 Pathway Inhibition.

Radiation resistance is a crucial factor influencing therapeutic outcomes in colorectal cancer (CRC). Baicalein (BE), primarily derived from Scutellaria baicalensis, has demonstrated anti-CRC properties. However, the impact of BE on the radiosensitivity of CRC remains unclear. This study aimed to evaluate the radiosensitization effects of BE and elucidate its mechanism in CRC radiotherapy. We established an invitro radioresistant cell model (CT26-R) using parental CRC cells (CT26) subjected to ionizing radiation (IR). CT26-R cells were pretreated with or without BE, followed by transfection with pcDNA-NC and pcDNA-JAK2. The proliferation of CT26-R cells treated with BE and IR was assessed using a colony formation assay. A CRC animal model was developed in BALB/c mice via CT26-R cell transplantation. The radiosensitizing effect of BE on CRC was evaluated invivo. TUNEL assay was conducted to detect apoptosis in tumor tissue. The expression levels of p-STAT3, JAK2, PD-L1, and SOCS3 invitro and invivo were measured by western blotting. Our results demonstrated that BE significantly increased radiosensitivity invitro and invivo and enhanced apoptosis in tumor tissues. Additionally, BE significantly downregulated the expression of p-STAT3, JAK2, and PD-L1, and significantly upregulated SOCS3 expression. These invivo effects were reversed by pcDNA-JAK2. In summary, our data suggest that BE enhances CRC radiosensitivity by inhibiting the JAK2/STAT3 pathway.

Jiawei Taohe Chengqi Decoction attenuates CCl4 induced hepatic fibrosis by inhibiting HSCs activation via TGF-β1/CUGBP1 and IFN-γ/Smad7 pathway

BackgroundHepatic fibrosis (HF) is an essential stage in the progression of different chronic liver conditions to cirrhosis and even hepatocellular carcinoma. The activation of hepatic stellate cells (HSCs) plays a crucial role in the progression of HF. IFN- γ/Smad7 pathway can inhibit HSCs activation, while TGF-β1/CUGBP1 pathway can inhibit IFN-γ/Smad7 pathway transduction and promote HSCs activation. Thus, inhibiting the TGF-β1/CUGBP1 pathway and activating the IFN-γ/Smad7 pathway reverses HSCs activation and inhibits HF. Jiawei Taohe Chengqi Decoction (JTCD) was derived from the Taohe Chengqi Tang in the ancient Chinese medical text titled “Treatise on Febrile Diseases”. We found several anti-HF components in JTCD including ginsenoside Rb1 and others, but the specific mechanism of anti-HF in JTCD is not clear. PurposeTo elucidate the specific mechanism by which JTCD reverses HF by inhibiting the activation of HSCs, and to establish a scientific foundation for treating HF with Traditional Chinese medicine (TCM). MethodsWe constructed a CCl4-induced mice HF model in vivo and activated human hepatic stellate cell line (LX-2) with TGF-β1 in vitro, after which they were treated with JTCD and the corresponding inhibitors. We examined the expression of pivotal molecules in the two pathways mentioned above by immunofluorescence staining, Western blotting and RT-PCR. ResultsJTCD attenuated liver injury and reduced serum ALT and AST levels in mice. In addition, JTCD attenuated CCl4-induced HF by decreasing the expression of α-SMA, COL1A1 and other markers of HSCs activation in mice liver tissue. Moreover, JTCD effectively suppressed the levels of TGF-β1, p-Smad3, p-p38MAPK, p-ATF2, and CUGBP1 in vivo and in vitro and upregulated the levels of IFN-γ, p-STAT1, and Smad7. Mechanically, after using the inhibitors of both pathways in vitro, we found that JTCD inhibited the activation of HSCs by restoring the balance of the TGF-β1/CUGBP1 and IFN-γ/Smad7 pathways. ConclusionWe demonstrated that JTCD inhibited HSCs activation and reversed HF by inhibiting the TGF-β1/CUGBP1 signalling pathway and upregulating the IFN-γ/Smad7 signalling pathway. Moreover, we have identified specific links where JTCD interferes with both pathways to inhibit HSCs activation. JTCD is an effective candidate for the clinical treatment of HF.