STAT3 mRNA Expression Research Articles

Nephroprotective activity of Aframomum melegueta seeds extract against diclofenac-induced acute kidney injury: A mechanistic study

Ethnopharmacological relevanceIn Africa, Aframomum species have been traditionally used to treat illnesses such as inflammation, hypertension, diarrhea, stomachache and fever. Moreover, Aframomum melegueta seed extracts (AMSE) are used in traditional medicine to relieve stomachaches and inflammatory diseases. AimChronic administration of diclofenac (DIC) has been reported to cause acute kidney injury (AKI), which is a serious health condition. The nephroprotective effect of AMSE is yet to be elucidated. Accordingly, this study aims to investigate the phytoconstituents of standardized AMSE, evaluate its nephroprotective effects against DIC-induced AKI in rats, and elaborate its underlying molecular mechanisms. Materials and methodsThe quantitative estimation of major AMSE constituents and profiling of its secondary metabolites were conducted via RP-HPLC and LC-ESI/Triple TOF/MS, respectively. Next, DIC (50 mg/kg)-induced AKI was achieved in Sprague-Dawley rats and DIC-challenged rats were administered AMSE (100 and 200 mg/kg) orally. All treatments were administered for five consecutive days. Blood samples were collected and the sera were used for estimating creatinine, urea and, kidney injury molecule (KIM)-1 levels. Kidney specimens were histopathologically assessed and immunohistochemically examined for c-Myc expression. A portion of the kidney tissue was homogenized and examined for levels of oxidative stress markers (MDA and GSH). Heme oxygenase (HO)-1, TNF-α, IL-6, Bax, Bcl2 and caspase-3 renal levels were quantified by ELISA. Moreover, the protein expression levels of NF-ҡB p65 was quantified using Western blot analysis, whereas mRNA expression levels of AMPK, SIRT-1, nuclear factor erythroid-2-related factor (Nrf2) and STAT3 were detected using qRT-PCR in the remaining kidney tissues. ResultsStandardized AMSE was shown to primarily contain 6-gingerol, 6-shogaol and 6-paradol among the 73 compounds that were detected via LC-ESI/Triple TOF/MS including phenolic acids, hydroxyphenylalkanes, diarylheptanoids and fatty acids. Relative to DIC-intoxicated rats, AMSE modulated serum creatinine, urea, KIM-1, renal MDA, TNF-α, IL-6, Bax, and caspase-3 levels. AMSE has also improved renal tissue architecture, enhanced GSH and HO-1 levels, and upregulated renal Nrf2, AMPK, and SIRT-1 mRNA expression levels. Furthermore, AMSE suppressed NF–ҡB p65 protein and STAT3 mRNA expression, and further reduced c-Myc immunohistochemical expression in renal tissues. Overall, our findings revealed that AMSE counteracted DIC-induced AKI via its antioxidant, anti-inflammatory, and antiapoptotic activities. Moreover, AMSE activated Nrf2/HO1 and AMPK/SIRT1, and inhibited NF-ҡB/STAT3 signaling pathways. Therefore, AMSE is a promising agent for inhibiting DIC-induced nephrotoxicity.

Insulin-Like Growth Factor 1 Receptor Drives Hepatocellular Carcinoma Growth and Invasion by Activating Stat3-Midkine-Stat3 Loop.

Activation of the insulin-like growth factor 1 receptor (IGF-1R)-mediated Janus kinase (JAK)1/2-Stat3 pathway contributes to hepatocarcinogenesis. Specifically, a previous study showed that IGF-1R inhibition downregulated Midkine expression in hepatocellular carcinoma (HCC). The present study investigated the role of IGF-1R-JAK1/2-Stat3 and Midkine signaling in HCC, in addition to the molecular link between the IGF-1R-Stat3 pathway and Midkine. The expression levels of IGF-1R, Stat3, and Midkine were measured using reverse transcription-quantitative PCR, following which the association of IGF-1R with Stat3 and Midkine expression was evaluated in HCC. The molecular link between the IGF-1R-Stat3 pathway and Midkine was then investigated in vitro before the effect of IGF-1R-Stat3 and Midkine signaling on HCC growth and invasion was studied in vitroand in vivo. IGF-1R, Stat3, and Midkine mRNA overexpressions were all found in HCC, where the levels of Stat3 and Midkine mRNA correlated positively with those of IGF-1R. In addition, Midkine mRNA level also correlated positively with Stat3 mRNA expression in HCC tissues. IGF-1R promoted Stat3 activation, which in turn led to the upregulation of Midkine expression in Huh7 cells. Similarly, Midkine also promoted Stat3 activation through potentiating JAK1/2 phosphorylation. Persistent activation of this Stat3-Midkine-Stat3 positive feedback signal loop promoted HCC growth and invasion, the inhibition of which resulted in significant antitumor activities both in vitroand in vivo. Constitutive activation of the IGF-1R-mediated Stat3-Midkine-Stat3 positive feedback loop is present in HCC, the inhibition of which can serve as a potential therapeutic intervention strategy for HCC.

Momordica charantia silver nanoparticles modulate SOCS/JAK/STAT and P13K/Akt/PTEN signalling pathways in the kidney of streptozotocin-induced diabetic rats.

Diabetes nephropathy (DN) is one of the complications of diabetes mellitus (DM) marked by gradual progressive loss of renal function. SOCS/JAK/STAT and PI3K/Akt/PTEN signalling pathways are among the chain of interactions implicated in the onset, progression and pathology of DN. Momordica charantia (bitter melon) is often used in folk medicine as therapy for DM due to its hypoglycemic properties. This study was designed to evaluate M. charantia silver nanoparticles' therapeutic effect on DN-induced by streptozotocin (STZ) in Wistar rats. The M. charantia nanoparticles used was synthesized using the filtrate from the plant methanolic extract added to 1mM concentration of aqueous silver nitrate. DM was induced in Wistar rats by intraperitoneal injection of STZ (65mg/kg). The animals' treatment groups were divided into; Diabetic control (65mg/kg STZ), Control, and groups treated with silver nitrate (10mg/kg), M. charantia nanoparticles (50mg/kg), metformin (100mg/kg), and plant extract (100mg/kg). Treatment was terminated after 11days. RT-PCR determined renal mRNA expression of Akt, PI3k, PTEN, TGF-β, JAK2, STAT3, STAT5, SOCS3, SOCS4 and glucokinase (GCK). Consequently, characterized compounds from M. charantia identified from literatures were docked with PI3K, JAK2 and TGF-β and STAT3 to retrieve potential hits. Oral administration of M. charantia nanoparticles (50mg/kg) to STZ-induced diabetic untreated rats significantly ((p<0.05) down-regulated the mRNA expression of Akt, PI3k, TGF-β, JAK2, STAT3 and upregulated the mRNA expression of PTEN, SOCS3 and SOCS4, thus establishing the role of M. charantia nanoparticles in alleviating DN in diabetic rats. Additionally, there was a significant up-regulation of glucose metabolizing gene (glucokinase) upon administering M. charantia nanoparticles. Molecular docking results showed 12 compounds from bitter melon with docking score ranging from -6.114kcal/mol to -8.221kcal/mol that are likely to exert anti-diabetic properties. Observation drawn from this study suggests that M. charantia nanoparticles ameliorate DN through regulation of SOCS/JAK/STAT and PI3K/Akt/PTEN signalling pathways.

Effects of flavonoids from Allium mongolicum Regel on cell apoptosis, cell cycle and STAT1 mRNA expression in peripheral blood lymphocytes of sheep

ABSTRACT Flavonoids from Allium mongolicum Regel (AMR) have immunoregulatory and anti-inflammatory properties. However, the effect of flavonoids from A. mongolicum Regel on sheep peripheral blood lymphocytes is still unknown. In this study, we found that total flavonoids from A. mongolicum Regel reduced the apoptosis of peripheral blood lymphocytes in sheep, increased the cell cycle progression to the G2/M phase, and promoted mitosis. These molecules inhibited mRNA expression of the apoptosis-related gene STAT1, indicating that the flavonoids alter JAK-STAT signalling and through this classical pathway inhibit cell apoptosis. The bioactive effects are predominantly related to the structure of flavonoids from A. mongolicum Regel, and the 55% elution group, which contained rutin, quercetin, and mignonette element -5-0-glucose-4-hydroxybenzoic acid, compounds found in plants, was the most effective. The effects were the strongest at a flavonoid concentration of 100μg/ml. In conclusion, the present study showed that flavonoids from A. mongolicum Regel inhibited the expression of STAT1 mRNA, reduced the apoptosis of sheep peripheral blood lymphocytes and promoted cell cycle progression. They also increased the proportion of cells in the G2/M phase. Our findings confirmed a novel role of flavonoids from A. mongolicum Regel in regulating immune function.

Rubi Fructus Water Extract Alleviates LPS-Stimulated Macrophage Activation via an ER Stress-Induced Calcium/CHOP Signaling Pathway.

Despite the availability of antibiotics and vaccines, many intractable infectious diseases still threaten human health across the globe. Uncontrolled infections can lead to systemic inflammatory response syndrome and the excessive production of inflammatory cytokines, known as a cytokine storm. As cytokines also play necessary and positive roles in fighting infections, it is important to identify nontoxic and anti-inflammatory natural products that can modulate cytokine production caused by infections. Rubi Fructus, the unripe fruits of Rubus coreanus Miquel, are known to possess antioxidative properties. In this study, the effect of the water extract of Rubi Fructus (RF) on the lipopolysaccharide (LPS)-induced inflammatory response in RAW 264.7 macrophages was investigated using biochemical and cell biology techniques. Our data indicated that RF inhibits p38 phosphorylation, intracellular calcium release, and the production of nitric oxide (NO), interleukin (IL)-6, monocyte chemotactic activating factor (MCP)-1, tumor necrosis factor (TNF)-α, leukemia inhibitory factor (LIF), lipopolysaccharide-induced CXC chemokine (LIX), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF), macrophage colony-stimulating factor (M-CSF), macrophage inflammatory protein (MIP)-1α, MIP-1β, MIP-2, and regulated on activation, normal T cell expressed and secreted (RANTES) in LPS-treated macrophages. In addition, we observed decreasing mRNA expression of Chop, Camk2a, Stat1, Stat3, Jak2, Fas, c-Jun, c-Fos, Nos2, and Ptgs2 without cytotoxic effects. We concluded that RF demonstrated immunoregulatory activity on LPS-stimulated macrophages via an endoplasmic reticulum (ER) stress-induced calcium/CCAAT-enhancer-binding protein homologous protein (CHOP) pathway and the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway.

Ghrelin inhibits inflammatory response and apoptosis of myocardial injury in septic rats through JAK/STAT signaling pathway.

The purpose of this study was to explore the influence of Ghrelin on myocardial injury of septic rats through the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway. A total of 36 Sprague-Dawley rats were randomly divided into normal group (n=12), model group (n=12), and Ghrelin group (n=12). The rats in the normal group were fed normally, while those in the model group were intraperitoneally injected with endotoxin to establish the sepsis model. The rats in the Ghrelin group were given intraperitoneal injection of Ghrelin solution to prepare the sepsis model. 9 h later, the specimens were obtained. Then, the expressions of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) were detected via immunohistochemistry, and the protein expressions of phosphorylated JAK (p-JAK) and STAT3 were determined by Western blotting (WB). Next, enzyme-linked immunosorbent assay (ELISA) was performed to measure the content of IL-6 and TNF-α, and quantitative Polymerase Chain Reaction (qPCR) was applied to examine the messenger ribonucleic acid (mRNA) expressions of JAK and STAT3. Finally, the cell apoptosis was detected through terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. The results of immunohistochemistry showed that compared with those in the normal group, the positive expression levels of IL-6 and TNF-α were markedly increased in other groups (p<0.05), while in comparison with those in the model group, the positive expression levels of IL-6 and TNF-α were decreased significantly in the Ghrelin group (p<0.05). The WB results indicated that the model group and Ghrelin group had remarkably higher protein expression levels of p-JAK and STAT3 than the normal group (p<0.05), and Ghrelin group exhibited notably lower protein expression levels of p-JAK and STAT3 than the model group (p<0.05). According to the results of qPCR, the relative mRNA expression levels of JAK and STAT3 were distinctly raised in the model group and Ghrelin group in comparison with those in the normal group (p<0.05), while they were reduced evidently in the Ghrelin group compared with those in the model group (p<0.05). Furthermore, it was manifested in the results of ELISA that the model group and Ghrelin group had prominently elevated content of TNF-α and IL-6 compared with normal group (p<0.05), and Ghrelin group displayed significantly lowered content of TNF-α and IL-6 in comparison with the model group (p<0.05). Moreover, the TUNEL results revealed that the apoptosis rate was remarkably higher in the other two groups than that in the normal group (p<0.05), while it was evidently lower in the Ghrelin group than that in the model group (p<0.05). Ghrelin can inhibit inflammatory response and apoptosis in the process of myocardial injury in septic rats by repressing the JAK/STAT signaling pathway.

Panton-valentine leucocidin carrying Staphylococcus aureus causing necrotizing pneumonia inactivates the JAK/STAT signaling pathway and increases the expression of inflammatory cytokines.

Methicillin-resistant Staphylococcus aureus (MRSA) carrying Panton-Valentine leukocidin, a pore-forming toxin, is a common cause of necrotizing pneumonia. However, the early pulmonary inflammatory response following PVL(+) MRSA infection is unknown. The purpose of this study was to use a murine model to determine the effect of PVL(+) MRSA on lung tissues and the expression of cytokines and JAK and STAT mRNA and protein. Mice were randomly divided into 3 groups and intra-nasally treated with PBS (control group), recombinant PVL (rPVL group), and PVL(+) MRSA (PVL group). At 24 and 48h after inoculation, bronchoalveolar lavage fluid (BALF) was tested for cytokine levels, and lung tissues were tested for JAK and STAT mRNA and protein expression, and examined after hematoxylin and eosin staining. Mice infected with the PVL(+) strain became ill, characterized by impaired mobility, hunched posture, ruffled fur, and labored breathing. Lung tissue exhibited tissue necrosis and hemorrhage. BALF levels of IL-8, TNF-α, IFN-γ, IL-12, sICAM-1, and sVCAM-1 were increased in the rPVL or PVL groups, while levels of IL-10 and IL-4 levels were similar among the groups. JAK1 and STAT1 mRNA expression and protein levels were increased in lung tissue from mice infected with PVL(+) MRSA and rPVL. PVL is a significant S. aureus virulence factor, and upregulates the expression of proinflammatory cytokines but does not affect the expression of anti-inflammatory cytokines. The effect of PVL may be due to JAK/STAT pathway activation. Blockade of the JAK/STAT pathway may decrease the severity of PVL(+) MRSA pneumonia.

DICER1-AS1 Promotes the Malignant Behaviors of Colorectal Cancer Cells by Regulating miR-296-5p/STAT3 Axis.

BackgroundLong non-coding RNA (lncRNA) exerts a regulatory role in the occurrence and progression of tumors. This study aimed at probing into the function and mechanism of lncRNA DICER1 antisense RNA 1 (DICER1-AS1) in colorectal cancer (CRC).MethodsThe expressions of DICER1-AS1, miR-296-5p and STAT3 mRNA were tested by quantitative real-time polymerase chain reaction (qRT-PCR). Cell counting kit-8 (CCK-8) assay was employed to detect cell proliferation, and Transwell was used to detect cell migration and invasion. In addition, the expressions of apoptosis-related proteins Bax and Bcl2 were detected by Western blot. Interactions between DICER1-AS1 and miR-296-5p, and miR-296-5p and STAT3 were predicted and determined by bioinformatics analysis, luciferase reporter assay and RNA binding protein immunoprecipitation (RIP) assay.ResultsThe expressions of DICER1-AS1 and STAT3 mRNA were significantly up-regulated while miR-296-5p expression was remarkably down-regulated in CRC tissues and cell lines. Over-expression of DICER1-AS1 or transfection of miR-296-5p inhibitors could promote the proliferation, migration and invasion and inhibit apoptosis of CRC cells, whereas knockdown of DICER1-AS1 or transfection of miR-296-5p mimics had the opposite effects. Additionally, DICER1-AS1 could down-regulate miR-296-5p expression via sponging it. DICER1-AS1 also enhanced the expression of STAT3, which was identified as a target gene of miR-296-5p.ConclusionDICER1-AS1 acts as an oncogenic lncRNA in CRC via modulating miR-296-5p/STAT3 axis. Our results provide a new direction for the diagnosis and treatment of CRC.

Association of PD-L1 and IDO1 expression with JAK-STAT pathway activation in soft-tissue leiomyosarcoma.

Therapies targeting the immune checkpoint molecules programmed death ligand 1 (PD-L1) and indoleamine 2,3-dioxygenase 1 (IDO1) have been explored in various malignant tumours. In this study, we examined the relationship between PDL-1, IDO1 and JAK2 expression and the roles of these signal pathways in soft tissue leiomyosarcoma (LMS). The next-generation sequencing data of 53 patients with LMS were obtained from an online public database and were used to assess PD-L1, IDO1 and JAK2 gene amplification and mRNA expression. Then, we determined the relationship between JAK-STAT pathway activation and PD-L1 and IDO1 expression in a LMS cell line. In addition, immunohistochemical staining of 69 cases of LMS was performed for PD-L1, IDO1, TDO2 and phosphorylated JAK2 (pJAK2). Comprehensive gene expression analysis using microarray and RNA-Seq data revealed that PD-L1 and IDO1 mRNA expression positively correlated with JAK2 and STAT1 mRNA expression. Two of the 53 cases exhibited PD-L1 and JAK2 gene amplification; however, they were not related to their gene expression. LMS cell line analysis revealed that IFN-γ supplementation induced IDO1 and PD-L1 expression; these effects were suppressed by JAK inhibition. Immunohistochemical analysis of the resected specimens revealed that TDO2 expression positively correlated with pJAK2 (P = 0.0490) and IDO1 expression (P < 0.0001). PD-L1-positive specimens tended to express pJAK2; however, the relationship did not reach statistical significance (P = 0.1477). The results suggest the possible feasibility of the combined inhibition of PD-1/PD-L1 or IDO1 with IFN-γ-JAK-STAT pathway inhibition to treat soft tissue LMS.

Abstract 6077: The role of STAT3 and Snai2 in ovarian cancer metastasis

Abstract Ovarian cancer remains a deadly disease due in part to ovarian cancer metastasis. Recent work has demonstrated that ovarian cancer forms clusters of cells (spheroids) that attach to the peritoneum by clearing the mesothelial cell layer and then invade the underlying matrix to metastasize. Understanding the key pathways and downstream transcription factors that are driving ovarian cancer metastasis may allow us to develop new therapies to treat metastatic ovarian cancer. Importantly, we have found that the STAT3 transcription factor is necessary for mesothelial clearance by ovarian spheroids. To identify key downstream STAT3 target genes involved in ovarian cancer metastasis, we analyzed publicly available STAT3 chromatin immunoprecipitation (ChIP)-seq datasets for genes associated with epithelial-to-mesenchymal transition. One of the genes that we identified was the transcriptional modulator Snai2 (Slug). To verify that Snai2 was downstream of STAT3, we transfected ovarian cancer cells with siRNA to STAT3 and analyzed Snai2 gene expression. We found reduced expression of Snai2 with siRNA to STAT3 suggesting that STAT3 upregulates Snai2 expression. To determine the effects of Snai2 on mesothelial clearance by ovarian spheroids, we transfected ovarian cancer cells with siRNA to Snai2 or control, generated spheroids, and performed a mesothelial clearance assay. We found that reduced expression of Snai2 decreased the ability of the ovarian cancer cells to clear the mesothelial cell layer suggesting that Snai2 plays an important role in the early stages of ovarian cancer metastasis. Recognizing that Snai2 is a transcriptional modulator, we were interested in determining if Snai2 affected STAT3 activity. Analysis of available Snai2 ChIP-seq data identified three putative Snai2 binding sites associated with the STAT3 gene, suggesting that Snai2 may also regulate STAT3 gene expression. To determine if Snai2 affects STAT3 gene expression, ovarian cancer cells were transfected with siRNA to Snai2 and STAT3 mRNA expression was analyzed. From this, we found that reducing Snai2 expression also reduced STAT3 expression. Taken together, our data suggests that STAT3 and Snai2 form a feedforward loop promoting upregulation of each other and together they promote the early stages of ovarian cancer metastasis. Therefore, developing an inhibitor to one of these transcription factors would affect both proteins and may be beneficial to prevent or treat metastatic ovarian cancer. Citation Format: David F. Walker, Sarah J. Lacroix, Yuri Makar, Sarah R. Walker. The role of STAT3 and Snai2 in ovarian cancer metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6077.

Ectopic PD-L1 expression in JAK2 (V617F) myeloproliferative neoplasm patients is mediated via increased activation of STAT3 and STAT5.

Escalated PD-L1 expression has been identified during malignant transformation in a number of cancer types and helps cancer cells escape an effective anti-tumor immune response. The mechanisms underlying escalated production of PD-L1 in many cancers, however, are still far from clear. We studied PD-L1, STAT3 and STAT5 mRNA expression using qRT-PCR in 72 BCR/ABL1 negative myeloproliferative neoplasm (MPN) patients (39 polycythemia vera and 33 essential thrombocythemia). Furthermore, phosphorylation status of STAT3 and STAT5 was studied using immunoblotting in the same patients. All MPN patients were first screened for JAK2 (V617F) mutation by tetra-primer ARMS-PCR, followed by quantification of JAK2 (V617F) mutation burden in all V617F positive MPN patients by ASO-PCR. Patients were screened for BCR/ABL1 fusion gene transcripts to rule out Ph positive status. Our findings showed that mRNA levels of PD-L1 and STAT3 were significantly higher in JAK2 (V617F) MPN patients, while as STAT5 was insignificantly upregulated. STAT3 and STAT5 phosphorylation was seen to be higher in JAK2 (V617F) MPN patients compared to the JAK2 (WT) patients. Upregulation of PD-L1, STAT3 and STAT5 was significantly associated with JAK2 (V617F) percentage in MPN patients. PD-L1, STAT3 and STAT5 expression significantly and positively correlated with JAK2 (V617F) allele burden. In addition, significant coexpression of PD-L1 with STAT3 and STAT5 was observed in MPN patients. In summary, JAK2 (V617F) mutation is accompanied by increased PD-L1 expression and this PD-L1 over expression is mediated by JAK2 (V617F) mainly through STAT3, while as STAT5 may play a minor role.

Effect of MiR-21 on Regulating the Proliferation of Synovial Fibroblasts in Rheumatoid Arthritis

Background: This study investigated whether miR-21 regulates the expression of STAT3 and affects FLS cells. Methods: MiR-21 and STAT3 mRNA level was assessed by qRT-PCR and STAT3 and p-STAT3 level was evaluated by Western blot. Spearman correlation was used to analyze the relationship between miR-21 and STAT3 mRNA expression in synovial tissue of RA patients. FLS cells were treated with IL-17A, and the cells without treatment was included as the control group. Under IL-17A treatment, FLS cells were divided into 2 groups: miR-NC group and miR-21 mimic group. MiR-21, STAT3, p-STAT3 expression were detected and compared. EdU staining was used to detect cell proliferation and flow cytometry was used to measure cell apoptosis. Results: There was a target relationship of miR-21 with STAT3 mRNA. IL-17A treatment significantly downregulated miR-21 in FLS cells, upregulated STAT3 and p-STAT3 and enhanced cell proliferation. Transfection of miR21 mimic significantly downregulated STAT3 and p-STAT3 in FLS cells, reduced cell proliferation and increased cell apoptosis. Conclusion: MiR-21 overexpression down-regulates STAT3, inhibits FLS cell proliferation and promotes apoptosis, indicating that it might be a therapeutic target for treating RA.

Melatonin relieves sepsis-induced myocardial injury via regulating JAK2/STAT3 signaling pathway.

To study the effect of melatonin on myocardial injury of septic rats through regulating the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling pathway. Healthy rats were selected as the samples and divided into blank group, sepsis group and sepsis + melatonin group. The difference in the myocardial tissue structure in the three groups of rats was observed via hematoxylin-eosin (HE) staining, and the messenger ribonucleic acid (mRNA) expression levels of JAK2 and STAT3 in myocardium were compared among groups through fluorescence quantitative polymerase chain reaction (qPCR). Western blotting was applied to detect the protein expressions of JAK2, phosphorylated (p)-JAK2, STAT3 and p-STAT3, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was utilized to determine the cell apoptosis in myocardial tissues. Moreover, the activity of superoxide dismutase (SOD) and content of malondialdehyde (MDA) in myocardial tissues as well as the activity of serum creatine kinase (CK) and lactate dehydrogenase (LDH) were measured and compared. There were inflammatory cells in the myocardium in sepsis group, and the pathological changes were milder in sepsis + melatonin group. The mRNA expressions of JAK2 and STAT3 and the protein expressions of JAK2, STAT3, p-JAK2 and p-STAT3 were raised remarkably in sepsis group compared with those in blank group and sepsis + melatonin group. The apoptosis rate in tissues was significantly different among the three groups, and it was the highest in sepsis group, followed by that in sepsis + melatonin group and blank group. There were significant differences in SOD activity and MDA content in myocardial tissues among the three groups, of which the SOD activity was the strongest in blank group, followed by that in sepsis + melatonin group and sepsis group, and the MDA content was the highest in sepsis group, followed by that in sepsis + melatonin group and blank group. Sepsis group had extremely notably increased activity of CK and LDH compared with blank group, while sepsis + melatonin group exhibited evidently decreased activity of CK and LDH in comparison with sepsis group. The JAK2/STAT3 signaling pathway is associated with sepsis-induced myocardial injury, and melatonin can relieve sepsis-induced myocardial injury by regulating the JAK2/STAT3 signaling pathway.

THU0081 MIR-17-5P REDUCES INFLAMMATION AND BONE EROSIONS IN COLLAGEN INDUCED ARTHRITIS MICE AND DIRECTLY TARGETS THE JAK-STAT PATHWAY IN RHEUMATOID ARTHRITIS FIBROBLAST-LIKE SYNOVIOCYTES.

Background:micro-RNAs (miR) are strong regulators of gene expression. Their involvement in RA key cytokines pathway regulation entitles them as important players in RA pathophysiology. The miR 17-92 cluster has been widely studied in cancer as they regulate cell apoptosis.Objectives:The aims of this study were to screen miR 17-92 cluster’s expression in different RA phenotypes (erosive and non erosive), further elucidate the mechanisms and direct targets involved in miR-17-5p anti-inflammatory role and to investigate miR-17-5p therapeutic effect in arthritis.Methods:A miR array was performed in synovial tissue from naïve erosive and non-erosive RA patients. Intra-articular delivery of miR-17 lipoplex was performed in collagen induced arthritis model in mice. Clinical, histological and structural effects were studied over the course of arthritis. In depth studies of miR-17 mechanisms of action were performed in primary RA-FLS isolated from RA synovial tissue.Results:Among others, miR-17-5p expression was reduced in erosive RA. miR-17 transfection in arthritic paws significantly reduced clinical inflammation. Moreover, synovial B cells, T cells, macrophages and polynuclear neutrophils infiltrates were significantly reduced. Structural damage was also decreased as shown by a reduction in the number of osteoclasts and erosion score by CT analysis. Pro-inflammatory cytokines of the IL-6 family, STAT3 target genes and IL-1β expression were also significantly reduced, but not TNF-alpha. miR17 directly targeted the 3’-untranslated region of STAT3 and JAK1. STAT3 and JAK1 mRNA and protein expression were reduced in RA-FLS following miR-17 transfection. STAT3 and JAK1 mRNA and activation of STAT3 as assessed by immunohistochemistry were also reduced in injected paws.Conclusion:We demonstrate an anti-inflammatory and anti-erosive role of miR-17 in vivo. This effect involves the suppression of the IL-6 family autocrine amplifying loop through the direct targeting of JAK1 and STAT3 as shown in RA-FLS.FigureEffect of miR-17 lipoplex injection in Collagen induced arthritis (CIA) mice’s ankles.Disclosure of Interests:None declared

PTPRD/PTPRT mutations as potential positive predictor for response of immune checkpoint inhibitors in non-small cell lung cancer (NSCLC).

e15112 Background: Immune checkpoint inhibitors (ICIs) have demonstrated positive results in non-small cell lung cancer (NSCLC) patients, with durable responses and prolonged overall survival (OS). Nevertheless, the response rate to immunotherapy is still limited. It is necessary to identify clinically useful biomarkers that can distinguish patients who can respond to ICIs. PTPRD/PTPRT are the phosphatases of JAK-STAT signaling, which may be associated with response to ICIs. Here we aimed to demonstrate the association between PTPRD/PTPRT and ICIs. Methods: Genomic and survival data of NSCLC patients administrated with anti–PD-1/PD-L1 or anti–CTLA-4 antibodies (Rizvi2015; Hellmann2018; Rizvi2018 Samstein2019) were retrieved from publicly accessible data. Genomic, survival and mRNA data of 1007 patients with NSCLC was obtained from The Cancer Genome Atlas (TCGA). Association between PTPRD/PTPRT mutation and progression-free survival (PFS) and overall survival (OS) were analyzed. Gene set enrichment analysis (GSEA) was used to determine potentially relevant gene expression signatures between specific subgroups. Results: PTPRD/PTPRT mutations were significantly associated with better PFS in Rizvi2015 cohort (HR = 0.16; 95% CI, 0.02-1.17; P = 0.03), Hellmann2018 cohort (HR, 0.49; 95% CI, 0.26-0.94; P = 0.03) and Rizvi2018 cohort (HR = 0.64; 95% CI, 0.44-0.92; P = 0.01). PTPRD/PTPRT mutation was also significantly associated with better OS in Samstein2019 cohort (HR, 0.66; 95% CI, 0.45-0.97; P = 0.03). In TCGA, no association between PTPRD/PTPRT mutations and OS was observed (P = 0.91), suggesting that PTPRD/PTPRT mutations were not prognostic factor. PTPRD/PTPRT mutations were associated with increased TMB (P &lt; 0.0001). The mRNA expression of STAT1 and CD4 was higher in patients with PTPRD/PTPRT mutant type than PTPRD/PTPRT wild type. Gene Set Enrichment Analysis revealed prominent enrichment of signatures related to inflammatory response, interferon gamma response and antigen processing and presentation in patients with PTPRD/PTPRT mutation. Conclusions: Our results suggest that PTPRD/PTPRT mutation is associated with better PFS and OS in NSCLC patients receiving ICIs by increasing immune-related gene signatures. The role of PTPRD/PTPRT in immunotherapy is needed to be further studied.

Exploration of the expression and significance of IL-6, STAT3 and PD-L1 in esophageal squamous cell carcinoma.

e16554 Background: Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies worldwide and has a very high morbidity and mortality rate. The IL-6/STAT3 axis can simultaneously promote the expansion of immunosuppressive cells.This study detected IL-6, STAT3 and PD-L1 in ESCC and analyzed the potential clinical application value. Methods: Between July 2018 and April 2019, 70 ESCC patients underwent radical thoracic surgery at Affiliated Hospital of North Sichuan Medical College. RT-PCR was used to detect PD-L1, IL-6, and STAT3 mRNA expression in cancer and adjacent normal tissues. Western blot detection of PD-L1, IL-6, and STAT3 was performed in 40 cancer and adjacent normal tissues. The correlation of PD-L1, IL-6 and STAT3 protein expression with tumor invasion depth, tumor size, lymph node metastasis,clinical stage and other clinicopathological characteristics as well as the correlation between the three proteins were analyzed statistically. Results: The mRNA levels of PD-L1, IL-6, and STAT3 in 70 ESCC patients were significantly different between cancerous and adjacent normal tissues (P &lt; 0.001). In 40 ESCC patients, 28 ESCC tissues showed significantly higher PD-L1 expression than adjacent normal tissue (28/12, P &lt; 0.001), 29 ESCC tissues showed significantly higher STAT3 expression (29/11,P = 0.009 ), and 27 ESCC tissues showed significantly higher IL-6 expression (27/13,P = 0.042). There was no correlation between PD-L1 expression and age, sex, tumor site or maximum tumor diameter, but PD-L1 expression showed a positive correlation with T stage (P = 0.009), lymph node metastasis (P = 0.044), and clinical stage (P = 0.033). There was no correlation between STAT3 expression and age, sex, tumor site, maximum tumor diameter, lymph node metastasis or clinical stage, but STAT3 expression showed a positive correlation with T stage (P = 0.025). IL-6 expression was not correlated with age, sex, maximum tumor diameter, lymph node metastasis or clinical stage, but IL-6 expression showed a positive correlation with T stage ( P = 0.003)and tumor site (P = 0.011). In addition, PD-L1 expression showed a positive correlation with STAT3 (P = 0.013) and IL-6 ( P = 0.008), and STAT3 showed a positive correlation with IL-6 ( P = 0.027). Conclusions: PD-L1, IL-6, and STAT3 are highly expressed in ESCC and their protein levels exhibit a positive correlation. The IL-6/STAT3 signaling pathway proteins may be a potential biomarker predicting the efficacy of immunotherapy by affecting the expression of PD-L1.

Cardioprotective effect of taurine and β-alanine against cardiac disease in myocardial ischemia and reperfusion-induced rats

Abstract Background The present study analyzed the synergistic protective effect of β-alanine and taurine against myocardial ischemia/reperfusion. Myocardial infarct size, lipid peroxidation, and levels of glutathione peroxidase (Gpx), superoxide dismutase (SOD), reduced glutathione (GSH), catalase, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), reactive oxygen species (ROS), apoptosis, and the mRNA and protein expression of Janus kinase 2 (JAK2) and signal transducer and activator 3 of transcription (STAT3) were determined. The molecular docking was carried out by using AutoDock 4.2.1. Results Combined treatment with β-alanine and taurine reduced myocardial infarct size, lipid peroxidation, inflammatory marker, ROS levels, and apoptosis and increased Gpx, SOD activity, GSH, and catalase activity. Furthermore, combined treatment significantly reduced JAK2 and STAT3 mRNA and protein expression compared with the control. The small molecule was docked over the SH2 domain of a STAT3, and binding mode was determined to investigate the inhibitory potential of β-alanine and taurine. β-Alanine bound to SH2 domain with ΔG of -7.34 kcal/mol and KI of 1.91 μM. Taurine bound to SH2 domain with ΔG of -7.38 kcal/mol and KI of 1.95 μM. Conclusion Taken together, these results suggest that the combined supplementation of β-alanine and taurine should be further investigated as an effective therapeutic approach in achieving cardioprotection in myocardial ischemia/reperfusion. How to cite: Hou X, Sun G, Gou L. Cardioprotective effect of taurine and β-alanine against cardiac disease in myocardial ischemia and reperfuson induced rats. Electron J Biotechnol 2020;45. https://doi.org/10.1016/j.ejbt.2020.04.003

The JAK2/STAT3 pathway is involved in dexmedetomidine-induced myocardial protection in rats undergoing cardiopulmonary bypass.

BackgroundMany studies have reported that dexmedetomidine protects organs from ischemia/reperfusion-induced injury. However, the mechanism of this protective effect remains inconclusive.MethodsRats were randomly divided into 6 groups (n=8). Rats in the sham group were not subjected to cardiopulmonary bypass (CPB) while rats in the other groups underwent CPB for 2 h. Groups L and H received a low and a high dose of dexmedetomidine, respectively. Rats in group AG490 received 10 mg/kg of the Janus kinase 2 (JAK2) inhibitor, AG490, 30 min before anesthesia. Plasma levels of the inflammatory cytokines, interleukin (IL)-6 and IL-10, were measured by enzyme-linked immunosorbent (ELISA), and the apoptosis rate of myocardial cells, the expression of JAK2 and signal transducer and activator of transcription (STAT)3 mRNA, and the protein expression of JAK2, STAT3, pJAK2, pSTAT3, and caspase-3 were analyzed in myocardial tissues by real-time quantitative polymerase chain reaction (qRT-PCR), western blotting, and immunohistochemistry.ResultsWe observed that, in both group L and group H, the level of IL-6 decreased (P<0.05), and the apoptosis rate of myocardial cells were reduced (P<0.05) compared to those in the CPB group. Moreover, qRT-PCR results revealed that dexmedetomidine administration reduced the expression of JAK2 and STAT3 mRNA (P<0.05); pJAK2 and pSTAT3 (P<0.05) protein levels were also reduced as assessed by western blotting and immunohistochemistry (P<0.05).ConclusionsDexmedetomidine treatment reduced CPB-related myocardial injury by inhibiting inflammatory reactions and myocardial apoptosis, and can be a potential therapy in CPB-related surgery.

β-Glucan augments IL-1β production by activating the JAK2/STAT3 pathway in cultured rabbit keratinocytes

Trichophyton mentagrophytes (T. mentagrophytes) is the main cause of rabbit dermatophytosis. As the main pathogen-associated molecular pattern of T. mentagrophytes, the role of β-glucan in the pathogenesis of rabbit dermatophytosis remains elusive. Keratinocytes (KC) are the main cellular component and the first defensive line against fungal pathogens in the skin. Therefore, the present study investigated the effects of β-glucan on rabbit KC from dorsal skin. β-glucan was found to inhibit KC proliferation by 10% at 20 ug/ml and this concentration was thus considered as optimal. Next, 20 ug/ml β-glucan stimulation for 24 h significantly increased CXCL8, CXCL11, and IL-1β secretions in KC. Furthermore, β-glucan exposure induced the expressions of JAK2 mRNA, STAT3 mRNA, and p-STAT3 protein. Silencing JAK2 expression inhibited p-STAT3 protein expression and β-glucan-induced IL-1β secretion. And overexpression of JAK2 further promoted β-glucan-mediated p-STAT3 protein and IL-1β productions. These results suggested that β-glucan-induced CXCL8, CXCL11, and IL-1β secretions in rabbit KC might be involved in the inflammatory response of T. mentagrophytes infected rabbit dorsal skin. However, only IL-1β secretion was promoted by the JAK2/STAT3 signaling pathway. In conclusion, this study is a necessary step toward elucidating the mechanisms that underlie skin immune system injury stimulated by β-glucan.

Loss of receptor tyrosine kinase-like orphan receptor 2 impairs the osteogenesis of mBMSCs by inhibiting signal transducer and activator of transcription 3

BackgroundReceptor tyrosine kinase-like orphan receptor 2 (Ror2) plays a key role in bone formation, but its signaling pathway is not completely understood. Signal transducer and activator of transcription 3 (Stat3) takes part in maintaining bone homeostasis. The aim of this study is to reveal the role and mechanism of Ror2 in the osteogenic differentiation from mouse bone marrow mesenchymal stem cells (mBMSCs) and to explore the effect of Stat3 on Ror2-mediated osteogenesis.MethodsRor2 CKO mice were generated via the Cre-loxp recombination system using Prrx1-Cre transgenic mice. Quantitative real-time PCR and western blot were performed to assess the expression of Stat3 and osteogenic markers in Ror2-knockdown mBMSCs (mBMSC-sh-Ror2). After being incubated in osteogenic induction medium for 3 weeks, Alizarin Red staining and western blot were used to examine the calcium deposit and osteogenic markers in Stat3 overexpression in mBMSC-sh-Ror2.ResultsLoss of Ror2 in mesenchymal or osteoblast progenitor cells led to a dwarfism phenotype in vivo. The mRNA expression of osteogenic markers (osteocalcin, osteopontin (OPN), and collagen I) in the ulna proximal epiphysis of Ror2 CKO mice was significantly decreased (P < 0.05). The mRNA and protein expression of Stat3 and osteogenic markers (Runx2, osterix, and OPN) decreased in mBMSC-sh-Ror2 cells (P < 0.05). The overexpression of Stat3 in mBMSC-sh-Ror2 cells rescued the calcium deposit and expression of Runx2, osterix, and OPN to a level comparable to normal mBMSCs.ConclusionsRor2 was essential for skeleton development by regulating mBMSCs’ osteogenesis and osteoblast differentiation. Loss of Ror2 may impair the osteogenesis of mBMSCs by inhibiting Stat3.