Protein Expression Levels Of STAT3 Research Articles

Abstract 7579: Dual inhibition of enhancer of zeste homolog 1 2 induces pyroptosis, modulates oncogenic signaling pathways & sensitizes hepatocellular carcinoma to valemetostat & docetaxel treatments

Abstract HCC is a global health concern, with chemotherapy resistance and patient recurrence posing significant challenges to liver cancer treatment. EZH2 is a crucial part of the polycomb repressive complex 2 (PRC2). It, along with its homolog EZH1, is a focus for cancer therapeutic targets. Dysregulation of EZH1/2 can cause cancer development due to significant changes in geneexpression. The current study aims to investigate the interaction between EZH1/2 and oncogenic signals responsible for HCC progression and pyroptosis induction. Two cell lines, HepG2 and SK-Hep1, were subjected to two FDA-approved drugs, Valemetostat (Val) and Docetaxel (Doc), to determine the impact of EZH1/2 on tumorigenicity and drug resistance. The WST-1 assay was utilized to gauge cell proliferation. The results indisputablydemonstrated a synergistic effect of the drugs after 48 h of treatment. The inhibition of viability was attributed to apoptosis induction in both tested cell lines, and the maximum effect was observed in the combined treatment as compared to either the control or each drug alone. Furthermore, our datashowed activation of caspase-3, upregulation of BAX, indicating apoptotic induction, and downregulation of Bcl-2, Bcl-XL, AKT, S6k, oncogenic β-catenin, and Stat3 protein expression levels, suggesting a modulation of oncogenic signaling pathways at the protein level. After 24 h of Val/Doc treatment, pyroptosis was observed, characterized by cell swelling and bubbles on themembrane and a significant release of lactate dehydrogenase, indicating pyroptotic cell cytotoxicity. The antitumor effects and pyroptosis were enhanced by using a dual inhibitor, UNC1999, for EZH1/2, resulting in a marked increase in the sensitivity of tested cells to the Val/Doc treatment invitro. The present study unequivocally demonstrated that dual inhibition of EZH1/2 acts as a potent sensitizer of HCC cells to Val/Doc treatment, which might be a promising therapeutic approach for inducing cellular pyroptosis in HCC cell lines. Citation Format: Asmaa Elkhder Harira, Adham Maher, Ahmed Sultan. Dual inhibition of enhancer of zeste homolog 1 2 induces pyroptosis, modulates oncogenic signaling pathways & sensitizes hepatocellular carcinoma to valemetostat & docetaxel treatments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7579.

Exploring the Mechanism of Chang-An-Kang on Active Ulcerative Colitis Rats Based on NF-κB/STAT3 Signaling Pathway

Objectives The objective of this study was to investigate the therapeutic effect and mechanism of Chang-An-Kang on ulcerative colitis rats. Materials and Methods The rat model of ulcerative colitis induced by 6% glacial acetic acid was established. The corresponding therapeutic drugs were given 24 h after the model was established. The pathological morphology of colon tissue was observed after 21 days of continuous administration. The levels of interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-10 (IL-10), and tumor necrosis factor-alpha (TNF-α) were measured by ELISA kit. The expression levels of NF-κB and STAT3 proteins in colon tissue were detected by IHC. Results The results showed that the changes in the pathological and biochemical indexes of the rat colon tissue indicate that the model was successfully established. Chang-An-Kang could significantly reduce rat colon tissue damage, decrease the content of IL-6, IL-7, and TNF-α, and increase the content of IL-10. The NF-κB and STAT3 protein expression levels were decreased. Conclusion By increasing the content of anti-inflammatory factor IL-10, decreasing the content of pro-inflammatory factors IL-6 and TNF-α, and inhibiting the expression of NF-κB and STAT3 proteins, Chang-An-Kang can reduce inflammatory infiltration. It has an obvious therapeutic effect on acetic acid ulcerative colitis in rats.

Centella triterpenes cream as a potential drug for the treatment of hypertrophic scar through inhibiting the phosphorylation of STAT3: A network pharmacology analysis and in vitro experiments.

Hypertrophic scars (HS) often affect the normal function and appearance of the skin and bring adverse effects to the body and mind of patients, being a challenge in the fields of burns and plastic surgery as well as rehabilitation. Despite significant efficacy of centella triterpenes cream for treating HS clinically, its pharmacodynamics and molecular targets are still unclear. Therefore, the network pharmacology analysis combined with in vitro cell molecular biology experiments was used to explore the mechanism and targets of centella triterpenes cream treating HS in this study. First, target genes of asiaticoside (AC) were obtained from the databases including the Comparative Toxicogenomics Database, similarity ensemble approach, SwissTargetPrediction and TargetNet, and HS targets were acquired from the databases like Disgenet, GeneCards, and Online Mendelian Inheritance in Man. The common targets of AC-HS were obtained through plotting a Venn diagram. Subsequently, STRING 11.0 was employed for analyzing the protein-protein interaction (PPI) network of the common targets, and cytoscape 3.9.0 for analyzing the connectivity of PPI and plotting the network diagram of "drug-component-target". Additionally, a modified tissue culture method was applied to separate primary normal fibroblasts (NFs) in human skin and hypertrophic scar fibroblasts (HSFs). HSFs after 24-h AC treatment were subjected to MTT assay to detect cell viability, scratch assay to assess cell migration ability, and western blot to test the protein expression levels of STAT3, p-STAT3, transforming growth factor-β1 (TGF-β1), collagen I (COL 1), fibronectin 1 (FN1), and alpha-smooth muscle actin (α-SMA). In network pharmacology analysis, 134 pharmacodynamic targets of AC and 2333 HS targets were obtained after retrieving the database, 50 AC-HS common targets were obtained by a Venn diagram, and a total of 178 edges and 13 core genes such as JUN and STAT3 were acquired by PPI analysis. In vitro experiments showed that the phosphorylation level of STAT3 (p-STAT3) was increased in HSFs. In addition to reducing p-STAT3 in HSFs, AC significantly inhibited the cell viability and migration of HSFs and downregulated the protein levels of TGF-β1, COL 1, FN 1, and α-SMA. STAT3 can be activated in HS. AC may exert its pharmacological effects of inhibiting TGF-β1 signal transduction and regulating extracellular matrix remodeling in HS by inhibiting STAT3 phosphorylation. However, the specific molecular mechanism of AC remains to be verified through further experiments.

Salvianolic acids from Salvia miltiorrhiza Bunge and their anti-inflammatory effects through the activation of α7nAchR signaling

Ethnopharmacological relevanceCardiovascular disease (CVD) is a serious disease with a high incidence rate and mortality. Inflammation is closely related to the occurrence of CVDs. As an essential medicine of promoting blood circulation and removing blood stasis in China, Salvia miltiorrhiza Bunge (Danshen) is widely used to treat CVDs due to its anti-inflammatory and cardiovascular protective effects. Salvianolic acids are the most abundant component in the water extract of S. miltiorrhiza, which has a significant effect on the treatment of CVDs. However, due to the complex composition of salvianolic acids, the active molecules and their underlying mechanisms have not been fully explored. Aim of this studyThe present study aims to isolate and identify salvianolic acids from Danshen with anti-inflammatory activity and explore the potential mechanisms of isolates. MethodsThe structures of isolated salvianolic acids were elucidated by UV, IR, NMR, MS and electronic circular dichroism (ECD) calculations. Then anti-inflammatory activities of isolates were screened out by the zebrafish inflammation models. The most active compound was further used to explore the anti-inflammatory mechanisms on LPS-stimulated RAW 264.7 cells. The key inflammatory cytokines IL-6 and TNF-α were measured by enzyme-linked immunosorbent assay (ELISA). The protein expression levels of STAT3, p-STAT3 (Tyr705), NF-κB p65, IκBα, p-IκBα (Ser32) and α7nAchR were determined by Western blotting. The nuclear translocation of p-STAT3 (Tyr705) and NF-κB p65 was evaluated by immunofluorescence assays. Finally, the in vivo anti-inflammatory mechanisms were investigated by observation of neutrophil migration, H&E staining, survival analysis and quantitative PCR (Q-PCR) in LPS-microinjected zebrafish. ResultsTwo new and four known compounds were isolated from Danshen. Among them, isosalvianolic acid A-1 (C1) and ethyl lithospermate (C5) inhibited neutrophil migrations in three zebrafish inflammation models and C1 with the best activities decreased the secretion of IL-6 and TNF-α and inhibited the expression level of p-IκBα (Ser32) in LPS stimulated RAW 264.7 cells. In addition, C1 also reduced the nuclear translocation of NF-κB p65 and p-STAT3 (Tyr705). Moreover, C1 significantly upregulated the protein expression of α7nAchR, and the knockdown of α7nAchR counteracted the effects of C1 on the production of IL-6 and TNF-α and the expression levels of p-STAT3 (Tyr705), NF-κB p65 and p-IκBα (Ser32). In vivo experiments, C1 decreased the migration and infiltration of inflammatory cells, increased the survival ratio and inhibited the mRNA level of IL-6, TNF-α, STAT3, NF-κB and IκBα in LPS-microinjected zebrafish. ConclusionTwo new and four known compounds were isolated from Danshen. Among them, C1 exerted anti-inflammatory activities by activating α7nAchR signaling and subsequently inhibiting STAT3 and NF-κB pathways. This study provided evidence for the clinical application of Danshen and contributed to the development of C1 as a novel in the treatment of cardiovascular disease.

Jing-Fang n-butanol extract and its isolated JFNE-C inhibit ferroptosis and inflammation in LPS induced RAW264.7 macrophages via STAT3/p53/SLC7A11 signaling pathway

Ethnopharmacological relevanceTraditional Chinese medicine has accumulated valuable experience in the treatment of inflammatory diseases caused by Ferroptosis. Jing Jie and Fang Feng are two warm acrid exterior-resolving medicinal herbs that play an important role in the prevention and treatment of inflammatory diseases. The pairing of the two forms a drug pair (Jing-Fang) that shows significant advantages in fighting oxidative stress and inflammation. Whereas, the underlying mechanism needs to be further improved. Aim of the studyIn this study, the anti-inflammatory effect of Jing-Fang n-butanol extract (JFNE) and its isolate C (JFNE-C) on LPS-induced RAW264.7 cells and the regulation effect on ferroptosis were investigated, and also the mechanism of STAT3/p53/SLC7A11 signal pathway-related to ferroptosis. Materials and methodsJing-Fang n-butanol extract (JFNE) and its active isolate (JFNE-C) were extracted and isolated. LPS-induced inflammation model in RAW264.7 cells was established to assess the anti-inflammatory effect and ferroptosis mechanism of JFNE and JFNE-C. The levels of interleukin 6 (IL-6), interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) were measured. The activity levels of antioxidant substances such as glutathione (GSH), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) were measured. Flow cytometry, immunofluorescence and transmission electron microscopy were used to assess ROS level, ferrous iron content and mitochondrial morphological changes. Through administration of Ferrostatin-1 (Fer-1), an ferroptosis inhibitor, to verify the role of JFNE and JFNE-C in regulating ferroptosis in resistance to the inflammatory response. Western blotting was used to determine whether the JFNE and JFNE-C exerted effectiveness by modulating the STAT3/p53/SLC7A11 signaling pathway. In addition, the important role of STAT3/p53/SLC7A11 signaling pathway in drug regulation of ferroptosis and inflammatory response was further validated by administration of S3I-201 (STAT3 inhibitor). Finally, high performance liquid chromatography-mass spectrometry (HPLC-MS) was used to determine the major active components of JFNE and JFNE-C. ResultsThe results showed that treated with JFNE-C significantly reduced the contents of interleukin 6 (IL-6), interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) in the supernatant of LPS-induced RAW264.7 cells. The pretreatment with JFNE and JFNE-C significantly decreased intracellular oxidative stress levels, including reductions of ROS and MDA levels, and increases of GSH-Px, SOD and GSH levels. In addition, JFNE and JFNE-C obviously reduced intracellular ferrous iron level, and JFNE-C was effective in alleviating mitochondrial damage which includes mitochondrial shrinkage, increase of mitochondrial membrane density and reduction and absence of cristae. Further results indicated that JFNE-C showed a reduction of p53 and p-p53 protein levels in LPS-induced RAW264.7 cells, while significantly increasing the protein expression levels of STAT3, p-STAT3, SLC7A11 and GPX4. Besides, JFNE-C contains key active substances such as 5-O-Methylvisammioside, Hesperidin and Luteolin. Remarkably, this is different from JFNE, which is rich in nutrients such as sucrose, choline and various amino acids. ConclusionThese results suggest that JFNE and JFNE-C may exert anti-inflammatory effect through activating the STAT3/p53/SLC7A11 signaling pathway to inhibit ferroptosis.

Role of NF-κB/ICAM-1, JAK/STAT-3, and apoptosis signaling in the anticancer effect of tangeretin against urethane-induced lung cancer in BALB/c mice

Lung carcinoma is one of the most prevalent and deadly neoplasia worldwide. Numerous synthetic medications have been used in the treatment of cancer. However, there are several drawbacks, such as side effects and inefficiency. The current study focused on the potential anti-cancer effectiveness of tangeretin, an antioxidant flavonoid, on lung cancer induced experimentally in BALB/c mice and explored the involvement of NF-κB/ICAM-1, JAK/STAT-3, and caspase-3 signaling in its anti-cancer effect. BALB/c mice were injected with urethane (1.5 mg/kg) twice; on the first day and on the 60th day of the experiment, then treated with 200 mg/kg tangeretin orally once daily for the last 4 weeks of the experiment. Compared with urethane group, tangeretin normalized oxidative stress markers; MDA, GSH, and SOD activity. Moreover, it had an anti-inflammatory effect by decreasing lung MPO activity, ICAM-1, IL-6, NF-қB, and TNF-α expressions. Interestingly, tangeretin decreased cancer metastasis by reducing p-JAK, JAK, p-STAT-3, and STAT-3 protein expression levels. Furthermore, it increased the apoptotic marker, caspase-3, indicating enhanced apoptosis of cancer cells. Finally, histopathology confirmed the anti-cancer effect of tangeretin. In conclusion, tangeretin could have a promising effect in counteracting lung cancer via modulation of NF-κB/ICAM-1, JAK/STAT-3, and caspase-3 signaling.

Isophysalin A Inhibits the Stemness of Breast Cancer Cells Possibly via Stat3 and IL-6 Signaling.

Breast cancer stem cells (BCSCs) are involved in carcinogenesis of the breast and contribute to therapeutic resistance. In the present study, we found that isophysalin A acts as a potent cancer stem cell inhibitor and investigated the anti-CSC mechanism of action of isophysalin A on breast cancer. The effect of isophysalin A on BCSCs was examined using a mammosphere formation, a colony formation and a cell migration assay, as well as CD44 (Cluster of differentiation 44)high/CD24 (Cluster of differentiation 24)low analysis, an apoptosis assay, quantitative real-time PCR, western blotting, an electrophoretic mobility shift assay, and a cytokine profiling assay. Isophysalin A inhibited cell proliferation, colony formation, cell migration, CSC formation, and mammosphere proliferation and increased BCSC apoptosis. The subpopulation of CD44high/CD24low was decreased by isophysalin A, which also reduced the DNA binding of Stat3 and the total and nuclear protein expression levels of Stat3 and phosphorylated Stat3. Furthermore, the mRNA and media IL-6/IL-8 levels of the mammosphere were also reduced by isophysalin A. Isophysalin A inhibited the Stat3 and IL-6 signaling pathways and induced CSC death; thus, isophysalin A may be a potential natural inhibitor of BCSCs.

Network Pharmacology and In Vitro Experimental Verification Reveal the Mechanism of the Hirudin in Suppressing Myocardial Hypertrophy.

Background: Myocardial hypertrophy is a complex pathological process, which is a common manifestation during the development of various cardiovascular diseases. Hirudin has been shown to have therapeutic effects on a variety of cardiovascular diseases, however, its therapeutic effect on myocardial hypertrophy is still unknown, and its chemical and pharmacological characteristics remain to be elucidated. Methods: In this study, the network pharmacology method was used to characterize the mechanism of hirudin on myocardial hypertrophy. The potential protein targets of hirudin and myocardial hypertrophy were both obtained from the Genecards database, and potential pathways associated with genes were identified by Gene Ontology and pathway enrichment analysis, and the data were displayed in a visual manner. Subsequently, the potential mechanism of action of hirudin on myocardial hypertrophy predicted by network pharmacology analysis was verified by molecular docking, and finally, the main findings were further verified by in vitro experiments by molecular biology techniques. Based on the results obtained from the study of H9c2 cell line, the inhibitory effect of hirudin on myocardial hypertrophy was further proved in the primary rat cardiomyocytes. Results: A total of 250 targets of hirudin, and 5,376 targets related to myocardial hypertrophy after deduplication were collected. The drug-disease network showed the relationship between hirudin, myocardial hypertrophy, and the targets. Further, systematic analysis from the PPI network indicated that blood coagulation, vesicle lumen, and signaling receptor activator activity may be the potential mechanisms of hirudin in the treatment of myocardial hypertrophy, and the PI3K/AKT signaling pathway may be the most relevant to the therapeutic effect of hirudin. Then, three therapeutic targets that were highly related to myocardial hypertrophy were extracted. Hirudin can be highly bound to STAT3, IL-6, and MAPK1 and found by molecular docking, which may be the basis for its inhibitory effect on myocardial hypertrophy. In addition, in vitro experiments showed that hirudin could inhibit AngII-induced hypertrophy and death of H9c2 cells, and significantly reduce the mRNA and protein expression levels of STAT3, MAPK1, and IL-6. The above conclusions were verified in primary rat cardiomyocytes. Conclusion: Hirudin can be used to treat myocardial hypertrophy through a complex mechanism. The application of network pharmacology and experimental validation can promote the application of hirudin in cardiovascular diseases and the interpretation and understanding of molecular biological mechanisms.

Effects of Thymosin β4 on Myocardial Apoptosis in Burned Rats.

The aim of this study was to investigate the effects of thymosin β4 on myocardial apoptosis following burns. Fifty healthy Sprague Dawley (SD) rats were randomly divided into the normal control group, resuscitation group the low-dose Tβ4 (thymosin β4) group (2g), the medium-dose Tβ4 group (6g), and the high-dose Tβ4 group (18g). The rats were immersed in 95°C hot water for 18 seconds, and then the model of 30% body surface area (TBSA) III° scald was established. The resuscated rats were injected with lactate Ringer's solution for antishock rehydration, while the Tβ4 treatment group was injected with lactate Ringer's solution for antishock rehydration, and the animals were sacrificed 6 h after scald. The degree of histopathological damage was observed by HE (hematoxylin and eosin) staining. Western blot was used to detect STAT1 and STAT3 protein expression levels. Real-time PCR was used to detect mRNA expressions of STAT1 and STAT3. The results showed that the apoptosis rate of the resuscitation group was significantly higher than that of the control group (P < 0.01). Compared with the resuscitation group, the apoptosis rate of thymosin β4 in the treatment group was significantly reduced (P < 0.01). Compared with the normal control group, the expression of STAT1 protein was increased and the expression of STAT3 protein was decreased in model group rats after ischemia and reperfusion. Compared with the model group, the expression of STAT1 protein decreased and the expression of STAT3 protein increased after ischemia-reperfusion in the thymosin β4 treatment group. Thymosin β4 may protect the myocardium by downregulating STAT1 and upregulating STAT3 expression and inhibiting myocardial apoptosis induced by ischemia and reperfusion after severe scald injury.

Propofol postconditioning alleviates diabetic myocardial ischemia‑reperfusion injury via the miR‑200c‑3p/AdipoR2/STAT3 signaling pathway.

Myocardial ischemia/reperfusion (MI/RI) syndrome is one of the leading causes of mortality and disability. Propofol postconditioning is known to improve myocardial ischemia/reperfusion injury (MI/RI). The present study aimed to explore the mechanism of propofol postconditioning in diabetic MI/RI. Diabetic MI/RI rat models were established and the rats were treated via propofol postconditioning. Staining with 2,3,5-triphenyl-2H-tetrazolium chloride, H&E staining, TUNEL staining and ELISA were applied to detect infarct size, pathological changes, apoptosis and oxidative stress-related factor and apoptotic factor levels, respectively. Subsequently, the effect of propofol on H9C2 cells was also assessed using the Cell Counting Kit-8 assay. High-glucose hypoxia/reperfusion (H/R) models of H9C2 cardiomyocytes were established. miR-200c-3p overexpression or AdipoR2 silencing combined with propofol postconditioning was performed in H/R-induced H9C2 cells and STAT3 protein expression levels were determined. Propofol postconditioning significantly reduced myocardial infarct size, oxidative stress and apoptosis in diabetic MI/RI models. Furthermore, propofol postconditioning significantly reduced the oxidative stress and apoptosis of H9C2 cells in high-glucose H/R models. Propofol postconditioning also significantly downregulated miR-200c-3p expression levels and promoted AdipoR2 expression levels. miR-200c-3p overexpression or AdipoR2 downregulation significantly reversed the effects of propofol postconditioning on its antioxidation and anti-apoptotic effects in H9C2 cells and on decreasing STAT3 phosphorylation levels. Together, the results of the present study demonstrated that propofol postconditioning inhibited miR-200c-3p, upregulated AdipoR2 and activated the STAT3 signaling pathway, thus alleviating diabetic MI/RI and therefore highlighting its potential as a treatment of diabetic MI/RI.

Role of STAT3 in hepatocyte regeneration after acetaminophen-induced hepatocellular injury in mice

Objective To investigate the role of STAT3 in hepatocyte proliferation after acetaminophen (APAP)-induced hepatocellular injury in mice. Methods Normal mouse AML12 hepatocytes were cultured in vitro and were stimulated by APAP (1, 2.5, 5, 10, and 20 mmol/L) for 12, 24 or 48 hours, and the hepatocytes treated with an equal volume of phosphate buffered saline were established as control group. After the optimal stimulation concentration and duration of action were screened out, AML12 hepatocytes were treated with AG490 (10, 50, and 100 μmol/L). The CCK-8 assay was used to measure the viability of AML12 hepatocytes; RT-PCR was used to measure the mRNA expression levels of PCNA, CyclinD1, and Ki67 in AML12 hepatocytes, and Western blot was used to measure the protein expression levels of STAT3, p-STAT3, PCNA, and CyclinD1. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. Results After 24 and 48 hours of APAP treatment, compared with the control group, all concentration groups had a significant reduction in the viability of AML12 hepatocytes (all P Conclusion STAT3 participates in hepatocyte proliferation after APAP-induced hepatocyte injury in mice, while AG490, as an STAT3 inhibitor, can inhibit hepatocyte proliferation after APAP-induced hepatocyte injury by inhibiting the phosphorylation of STAT3.

NEAT1/miR‑124/STAT3 feedback loop promotes breast cancer progression.

The long non‑coding RNA nuclear enriched abundant transcript1 (NEAT1) has important roles in the regulation of multiple cell functions, such as proliferation, apoptosis and migration. However, the mechanism by which NEAT1 regulates breast cancer progression is not well elucidated. In the present study, NEAT1 and microRNA‑124 (miR‑124) levels were detected by reverse transcription‑quantitative PCR in breast cancer tissues and cell lines. STAT3 protein levels were detected by western blot analysis. Cell proliferation and cell cycle distribution were determined using MTT and colony formation assays, and flow cytometry, respectively. The results demonstrated that NEAT1 and STAT3 expression levels were increased in breast cancer tissues compared with normal breast tissues, whereas miR‑124 expression was significantly decreased. Functional analyses revealed that NEAT1 promoted cell proliferation and cell cycle progression in breast cancer cells. Additionally, NEAT1 and STAT3 expression levels were negatively correlated with miR‑124 levels in breast cancer tissues. A direct interaction between miR‑124, and NEAT1 and STAT3, was predicted by bioinformatics analysis and confirmed using a luciferase activity assay. NEAT1 overexpression markedly increased STAT3 protein expression levels, and this effect was reversed by miR‑124 overexpression in breast cancer cells. Furthermore, miR‑124 overexpression partially attenuated the effects of NEAT1 on breast cancer cell proliferation and cell cycle progression. The inhibitory effects of miR‑124 overexpression on the proliferation rate and cell cycle progression were abolished by STAT3 overexpression. In turn, STAT3 silencing inhibited NEAT1 transcription in breast cancer cells. In summary, the present findings revealed that NEAT1 and STAT3 formed a feedback loop via sponging miR‑124 to promote breast cancer progression.

Effect of Miao medicine, Jinwujiangu decoction, on IL- 17/IL-23 inflammatory axis of fibroblast-like synoviocytes in rheumatoid arthritis

Purpose: To explore the influence of the Miao medicine, Jinwujiangu decoction, on the interleukin (IL)- 17/IL-23 inflammatory axis of fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA).Methods: Synovial tissue samples were randomly divided into a blank control group, high-dose (0.06mg/mL), medium-dose (0.6mg/mL), and low-dose (6.0mg/mL) groups of Jinwujiangu decoction, a leflunomide group, and a tripterygium glycosides group. Proliferation of RA synovial cells was detected by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Enzyme-linked immunosorbent assay (ELISA) was used to determine the secretion of IL-6, transforming growth factor beta (TGF-β), and IL-17. Real-time polymerase chain reaction was used to evaluate the expression of IL-23R, IL-17R, RAR-related orphan receptor alpha (RORα), RORγt, and signal transducer and activator of transcription (STAT3) mRNA. The protein activities of IL-17R, STAT3 and pSTAT3 were assessed by Western blot assay.Results: Jinwujiangu decoction inhibited the proliferation of RA synovial cells. Treatment with different drug concentrations resulted in downregulation of IL-6, TGF-β, and IL-17 secretion. The expression levels of IL-23R, IL-17R, RORα, RORγt, and STAT3 mRNA in RA-FLS were significantly reduced after intervention with different drugs. Protein expression levels of STAT3, pSTAT3, and IL-17 in the different drug treatment groups were significantly decreased.Conclusion: Jinwujiangu decoction inhibits the secretion of IL-6 and TGF-β in RA-FLS, and intervenes to regulate gene expression of IL-23/IL-17 inflammation axis and suppress immune inflammation. The results of this study provide new evidence for the study of anti-inflammatory mechanism of TCM compound prescription.Keywords: Jinwujiangu decoction, IL-17/IL-23, Fibroblast-like synoviocytes, Rheumatoid arthritis, Ethnomedicine

Subcutaneous Inoculation of Echinococcus multilocularis Induces Delayed Regeneration after Partial Hepatectomy

Alveolar echinococcosis (AE) is caused by the larval stage of echinococcus multilocularis (E. multilocularis), and hepatectomy is the main modality in hepatic AE patients. Liver regeneration after partial hepatectomy (PHx) in such patients is challenging, and further investigation is needed. Thus far, knowledge regarding the possible impact of E. multilocularis on liver regeneration after PHx is limited. Herein, a subcutaneous infection model of E. multilocularis was developed in C57 BL/6 mice, and after 3 months, PHx was performed. Plasma and liver samples were harvested under inhalational isofluorane (2%) anaesthesia at designated post-PHx time points (0, 24, 48, 96 and 168 h). The parameters included the future remnant liver/body weight ratio (FLR/BW), liver function tests (AST and ALT) and related cytokines (TNF-α, IL-6, Factor V, HMGB1, TGF-β, TSP-1, and TLR4) and proteins (MyD88 and STAT3). To assess the proliferation intensity of hepatocytes, BrdU, Ki67 and PAS staining were carried out in regenerated liver tissue. The FLR/BW in the infected group from 48 h after surgery was lower than that in the control group. The BrdU positive hepatocyte proportions reached their peak at 48 h in the control group and 96 h in the infected group and then gradually decreased. During the first 48 h after surgery, both the AST and ALT levels in the infected group were lower; however, these levels were altered from 96 h after surgery. In the infected group, the concentrations and mRNA expression levels of the pre-inflammatory cytokines TNF-α and IL-6 demonstrated a delayed peak. Moreover, post-operatively, the TGF-β and TSP-1 levels showed high levels in the infected group at each different time-point compared to those in the control group; however, high levels of TGF-β were observed at 96 h in the control group. The MyD88 and STAT3 protein expression levels in the infected group were markedly higher than those in the control group 96 h after surgery. Delayed liver regeneration after PHx was observed in the C57 BL/6 mice with the subcutaneous infection of E. multilocularis in the current study. This phenomenon could be partially explained by the alteration in the pro-inflammatory cytokines in the immunotolerant milieu induced by chronic E. multilocularis infection.

STAT3 promotes tumour progression in glioma by inducing FOXP1 transcription.

ObjectiveThis paper investigated the effects of STAT3 through promoting FOXP1 transcription on proliferation, apoptosis and invasion in glioma cells.MethodsQuantitative real‐time PCR (qRT‐PCR) and Western blot assay were administered to assess the mRNA and protein expression levels of STAT3 and FOXP1 in glioma tissues and cells, respectively. Luciferase reporter and Chromatin Immunoprecipitation (ChIP) assays were implemented to determine the correlation between STAT3 and FOXP1. MTT and colony formation assays were conducted to identify cell growth. Flow cytometry was run to detect the cell apoptosis rate of glioma cells. Transwell assays were conducted to reveal cell invasion ability.ResultsThe mRNA and protein expression levels of STAT3 were highly expressed in glioma tissues and cells. After cells transfected with siRNA of STAT3, both STAT3 and FOXP1 were simultaneously downregulated. STAT3 directly regulated FOXP1 transcription. STAT3 promoted cell proliferation, inhibited cell apoptosis and enhanced cell invasion through promoting FOXP1 transcription in glioma cells.ConclusionIn summary, STAT3 gene was a transcriptional regulator of FOXP1. Depleted STAT3 restrained cell proliferation and invasion, promoted cell apoptosis in glioma cells. This molecular mechanism between STAT3 and FOXP1 can serve as a therapeutic target for glioma treatment.

Effect of JAK2/STAT3 signaling pathway on liver injury associated with severe acute pancreatitis in rats.

Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling constitutes one of the major pathways for cytokine signal transduction. However, the role of the JAK2/STAT3 pathway in liver injury during severe acute pancreatitis (SAP) remains unclear. The aim of this study was to investigate the role of the JAK2/STAT3 signaling pathway in liver injury after SAP. In the present study 64 male Sprague-Dawley rats were randomly divided into four groups: Control, AG490 (inhibition of JAK2), SAP and SAP with AG490. SAP was induced by retrograde infusion of 4% sodium taurocholate into the biliopancreatic duct. The activities of amylase (AMY) and liver enzymes were measured in serum. Livers and pancreas were isolated for measurements of histological damage. Blood and liver samples were taken for the measurement of TNF-α, IL-6 and IL-18 concentrations. The expression levels of JAK2 and STAT3 in liver tissue were detected by immunohistochemical staining and western blotting. The results demonstrated that amylase and liver enzymes were higher in the SAP groups compared with the control, AG490 and AG490-treated groups. The serum levels of TNF-α, IL-6 and IL-18 were effectively increased in the SAP groups, whereas they were reduced by AG490. Interestingly, JAK2 and STAT3 protein expression levels were significantly increased following induction of SAP and were significantly decreased in the AG490-pretreated groups. Administration of AG490 decreased the activity of pro-inflammatory cytokines and significantly attenuated SAP associated-liver injury in the rats. These results suggested that the mechanism may relate to the inhibition of TNF-α, IL-6 and IL-18, and inhibiting excessive JAK2 and STAT3 activation, and may play a crucial role in the liver injury associated with SAP.

Effects of miR-126 on the STAT3 signaling pathway and the regulation of malignant behavior in lung cancer cells.

The functions of miR-126-mediated signal transducers and activators of the transcription 3 (STAT3) signal pathway were investigated in regulating the behavior of cells in non-small cell lung cancer (NSCLC). Cultured NSCLC A549 cells were transfected with empty, miR-126 overexpression or miR-126 knocked-down expression plasmids. After transfection efficiency verification by reverse transcription polymerase chain reaction (RT-PCR) and culture for 24 h, methyl thiazolyl tetrazolium (MTT) was applied to detect cell proliferation rate, migration distance was measured in scratch assays, cell cycle was determined through flow cytometry, the mRNA expression level of caspase-3 in cells was detected using RT-PCR and protein expression levels of STAT3 were detected using western blotting. Our results showed the cell proliferation rate was significantly higher in cells of the overexpression group than that in those of the control group (p<0.05) and the rate in the cells of the low-expression group was the lowest among the three groups (p<0.05). The migration distance of the overexpression group cells was significantly longer than that in the control group cells and the shortest migration distance was found in the low-expression group cells (p<0.05). The amount of cells in mitotic phase in the overexpression group was significantly higher than that in the control group and the same amount in the low-expression group was the lowest (p<0.05). The mRNA expression level of caspase-3 of cells in the overexpression group was significantly lower than that of cells in the control group and the highest expression level was found in the low-expression group (p<0.05). Finally, the protein expression levels of STAT3 in cells in the overexpression group were significantly lower than those in the control group and the highest expression levels were identified in the low-expression group (p<0.05). Based on our findings, the cancer-promoting miR-126 can mediate the activation of the STAT3 signal pathway to regulate the malignant biological behavior of NSCLC cells affecting their proliferation, migration, cycle and apoptosis susceptibility.

P38 Expression and Modulation of STAT3 Signaling in Oral Cancer.

p38 protein belongs to Mitogen-activated protein kinases family that link extracellular stimuli with intracellular responses participating in numerous of fundamental cell processes. Persistent activation of STAT3 has been associated with cell proliferation, differentiation and apoptosis in oral squamous cell carcinoma (OSCC). This study examines the effects of p38 modulation on STAT3 signaling and cellular activities in OSCC cells and investigates possible correlation of p38 expression with tumor degree of differentiation. Phospho-p38 immunostaining was performed in 60 OSCC including well, moderately and poorly differentiated tumors. Semiquantitative analysis was used, by calculating intensity, percentage and combined scores. Protein expression levels of STAT3 (total, tyrosine and serine phosphorylated), p38 and cyclin D1 were assessed in two OSCC cell lines. p38 inhibition was achieved by pharmacological agent(SB2023580). Cell proliferation and viability rates were also evaluated. Phospho-p38 immunoexpression was intense in almost all tumor specimens, nevertheless did not correlate with tumor differentiation. Inhibition of p38 with SB203580 did not appear to affect tyrosine or serine phosphorylated STAT3 as well as cyclin D1 levels in both cell lines. Moreover, p38 inhibition resulted in mild dose-dependent decreases in cell growth and viability in both cell lines. p38 is highly expressed in OSCC but does not seem to mediate the oncogenic STAT3 pathway. However, changes found in proliferation and viability may suggest that p38 functions as potent regulator of HNSCC. Understanding the complexity of p38 signaling and cross-talk between other major molecules, may guide the development of novel pharmacologic therapies for cancer treatment and prevention.

Effect of modified Xiaochaihu decoction‑containing serum on HepG2.2.15 cells via the JAK2/STAT3 signaling pathway.

The present study aimed to investigate the possible mechanisms underlying the effect of modified Xiaochaihu decoction (mXCHD) in the treatment of chronic hepatitis B (CHB). Patients with CHB, in addition to liver stagnation and spleen deficiency syndrome were randomly assigned to receive either Chinese (mXCHD) or western (entecavir) treatment, with 30 cases in each group. Serum was collected following treatment with mXCHD or entecavir for 7 days. A healthy group of 30 individuals was also included. HepG2.2.15 cells were cultured in vitro and randomly divided into four groups: Healthy; entecavir-treated; 10% mXCHD-treated; and 20% mXCHD-treated. The HepG2.2.15 cells in the four groups were treated with either serum from the healthy volunteers, entecavir-containing serum, or mXCHD-containing serum at different concentrations (10 or 20%, respectively). Following treatment with the corresponding serum, cell proliferation was examined using an MTT assay, and the expression of hepatitis B surface antigen (HBsAg) in the cell supernatant was detected using an enzyme-linked immunosorbent assay. The mRNA and protein expression levels of Janus kinase (JAK)2 and signal transducer and activator of transcription (STAT)3 were measured using reverse transcription-quantitative polymerase chain reaction and western blot analyses, respectively. The results indicated that the most effective treatment for the promotion of HepG2.2.15 cell proliferation was a 20% concentration of mXCHD serum. The expression of HBsAg was significantly decreased in the groups treated with 10 and 20% mXCHD 48 h following intervention (P<0.01). The mRNA and protein expression levels of STAT3 in the 20% mXCHD serum group were significantly increased, compared with those in the healthy group (P<0.01 and P<0.05, respectively), whereas no significant difference was observed in the expression of JAK2 among the four groups. These results indicated that mXCHD suppressed the hepatitis B virus, and treatment of the cells with mXCHD-containing serum promoted HepG2.2.15 cell proliferation via modulating the expression of STAT3, which may contribute to the clinical efficacy of mXCHD against CHB.

Promoter methylation attenuates SHP1 expression and function in patients with primary central nervous system lymphoma.

The Src homology region2 domain-containing phosphatase-1 (SHP1) is a critical negative regulator involved in the JAK/STAT signaling pathway. The SHP1 gene has been proposed as a candidate tumor suppressor in solid and hematological malignancies and promoter methylation is an important biological process in controlling tumorigenesis. However, the detailed roles of SHP1 promoter methylation in the pathogenesis of primary central nervous system lymphoma (PCNSL) is largely unknown. In the present study, we evaluated the correlation between SHP1 expression and promoter methylation in patients with PCNSL. Thirty-three patients with PCNSL were included. We evaluated SHP1 protein expression levels by immunohistochemistry and the SHP1 promoter methylation profile by pyrosequencing analysis. For cases (n=8) with a good yield of total protein, SHP1 phosphorylation (pSHP1) and STAT3 protein expression levels were further analyzed by western blot analysis to uncover the molecular impact of SHP1 promoter methylation on downstream signaling pathways. In this study, a lower expression of SHP1 protein level was observed in 16/33 cases (48.5%) of PCNSL. SHP1 promoter methylation was predominant in 29/33 cases (87.9%) with a mean methylation level of 31.7±36.5%. The mean methylation level of the SHP1 promoter was significantly elevated in patients with a lower SHP1 protein expression, compared with those showing a higher SHP1 protein expression (50.3±38.9 vs.14.2±24.0%, p=0.004). Further analysis showed that SHP1 protein expression was significantly decreased in patients with a higher SHP1 promoter methylation status (p=0.001), and such attenuation was correlated with a downregulation of pSHP1 (p=0.005) and an upregulation of STAT3 protein expression (p=0.020). Our data demonstrated that epigenetic alterations in the promoter region downregulated SHP1 expression in PCNSL patients. SHP1 promoter methylation was correlated with tyrosine phosphorylation and activation of transcription factor STAT3, which may contribute to the pathogenesis of PCNSL. Therapeutical regimens with epigenetic modifiers may be a potential option for patients with PCNSL.