Inhibited Signal Transducer And Activator Of Transcription 3 Phosphorylation Research Articles

Sevoflurane postconditioning mitigates neuronal hypoxic-ischemic injury via regulating reactive astrocytic STAT3 protein modification

Astrocyte activation plays a pivotal role in accelerating the cascade of neuroinflammation associated with the development of hypoxic-ischemic brain injury. This study aimed to investigate the mechanism by which sevoflurane postconditioning mitigates neuronal damage through astrocytes by regulating reactive astrocytic Signal Transducer and Activator of Transcription 3 (STAT3) modifications. A modified Rice‒Vannucci model in rats and a conditioned culture system established by subjecting primary astrocytes to oxygen glucose deprivation, followed by using the conditioned medium to culture the neuron cell line SH-SY5Y were used to simulate HI insult in vivo and in vitro, respectively. These models were followed by 30 min of 2.5 % sevoflurane treatment. Stattic was used to inhibit STAT3 phosphorylation, and (Z)-PUGNAc or OSMI-1 was added to regulate O-linked-β-N-acetylglucosamine modification (O-GlcNAcylation) in primary astrocytes in vitro. Neurobehavioral tests, Nissl staining, CCK8 assay, and flow cytometry for apoptosis were used to assess neuronal function. Immunofluorescence staining was used to detect astrocyte reactivity and the intracellular distribution of STAT3. Immunoprecipitation combined with Western blotting was used to evaluate the O-GlcNAcylation of STAT3. Protein expression and phosphorylation levels were detected by Western blotting. ELISA was conducted to detect the detrimental cytokines IL-6 and IL-1β in astrocyte-conditioned medium. Sevoflurane postconditioning enhanced the O-GlcNAcylation of astrocytic STAT3 following HI insult via the manner of OGT. Crosstalk between O-GlcNAcylation and phosphorylation of STAT3 showed that O-GlcNAcylation inhibited STAT3 phosphorylation. The inhibitory effect on astrocytes suppressed STAT3 nuclear translocation, reduced astrocyte reactivity, decreased the release of the inflammatory cytokines IL6 and IL-1β, attenuated neuronal apoptosis following HI insult, and improved neuron viability. Sevoflurane postconditioning increased astrocytic STAT3 O-GlcNAcylation level to competitively inhibit STAT3 phosphorylation. This deactivated downstream inflammation pathways and reduced astrocyte reactivity, thereby mitigating HI insult in neurons both in vivo and in vitro.

JAK2/STAT3 signaling pathway mediates methylmercury toxicity in mouse astrocyte neuronal C8-D1A cell line.

Methylmercury (MeHg) is an environmental pollutant. Consumption of contaminated fish is the main exposure route in humans, leading to severe neurological disorders. Upon ingestion MeHg reaches the brain and selectively accumulates in astrocytes disrupting glutamate and calcium homeostasis and increasing oxidative stress. Despite extensive research, the molecular mechanisms underlying MeHg neurotoxicity remain incompletely understood. The induction of nuclear factor erythroid 2-related factor 2 (Nrf2) and its role activating antioxidant responses during MeHg-induced oxidative injury have garnered significant attention as a potential therapeutic target against MeHg toxicity. However, recent studies indicate that the Nrf2 signaling pathway alone may not be sufficient to mitigate MeHg-induced damage, suggesting the existence of other protective mechanisms. The signal transducer and activator of transcription 3 (STAT3) plays a crucial role in cell growth and survival. Several studies have also highlighted its involvement in regulating redox homeostasis, thereby preventing oxidative stress through mechanisms that involve modulation of nuclear genes that encode electron transport complexes (ETC) and antioxidant enzymes. These characteristics suggest that STAT3 could serve as a viable mechanism to mitigate MeHg toxicity, either in conjunction with or as an alternative to Nrf2 signaling. Our previous findings demonstrated that MeHg activates the STAT3 signaling pathway in the GT1-7 hypothalamic neuronal cell line, suggesting its potential role in promoting neuroprotection. Here, to elucidate the role of the STAT3 signaling pathway in MeHg neurotoxicity, we pharmacologically inhibited STAT3 using AG490 in the C8D1A astrocytic cell line exposed to 10 µM MeHg. Our data demonstrated that pharmacological inhibition of STAT3 phosphorylation exacerbates MeHg-induced mortality, antioxidant responses, and ROS production, suggesting that STAT3 may contribute to neuroprotection against MeHg exposure in astrocytes.

Biological evaluation of signal transducer and activator of transcription 3 (STAT3) targeting by phaeosphaeride A and its analogs

The inhibitory activities of phaeosphaeride A (PPA), phaeosphaeride B, and four synthetic derivatives against phosphorylation of signal transducer and activator of transcription 3 (STAT3) and cell proliferation in cervical (HeLa) and breast (MDA-MB-231) cancer cells were evaluated. PPA inhibited IL-6-induced STAT3 phosphorylation and cell proliferation at similar concentrations. The structure–activity relationship studies revealed that the enantiomer of PPA was the most potent of the evaluated phaeosphaerides in both inhibiting STAT3 phosphorylation and cell growth. PPA clearly inhibited the IL-6-activated STAT3 signaling pathway. However, the presence or absence of activation of the STAT3 signaling pathway in cells showed no relationship to the antiproliferative activity. Notably, the possible covalent bond-forming ability of PPA was critical for its biological activities.

Alantolactone alleviates epithelial-mesenchymal transition by regulating the TGF-β/STAT3 signaling pathway in renal fibrosis

ObjectiveThe epithelial-to-mesenchymal transition (EMT) of renal tubular epithelial cells (RTECs) plays a crucial role in renal interstitial fibrosis and inflammation, which are key components of chronic kidney disease (CKD). Alantolactone, a selective inhibitor of signal transducer and activator of transcription 3 (STAT3), is used in Chinese herbal medicine. Despite its use, the effects of alnatolactone on EMT of RTECs has not been fully elucidated. MethodsIn this study, we investigated the potential of alantolactone to EMT in vivo and in vitro. Our experiments were performed using a unilateral ureteral obstruction (UUO) models and HK-2 cells, RTECs, treated with transforming growth factor (TGF-β). ResultsAlantolactone decreased tubular injury and reduced the expression of vimentin, a key EMT marker, while increasing E-cadherin expression in UUO kidneys. Similarly, in RTECs, alantolactone inhibited TGF-β-induced EMT and its markers. Furthermore, alantolactone attenuated UUO- and TGF-β-induced STAT3 phosphorylation both in vivo and in vitro, and inhibited the expression of TWIST, an EMT transcription factor, in both models. ConclusionAlantolactone improves EMT in RTECs by inhibiting STAT3 phosphorylation and Twist expression, suggesting its potential as a therapeutic agent for kidney fibrosis.

Isoliquiritigenin diminishes invasiveness of human nasopharyngeal carcinoma cells associating with inhibition of MMP-2 expression and STAT3 signalling.

Nasopharyngeal carcinoma (NPC) is prevalent in Asia and exhibits highly metastatic characteristics, leading to uncontrolled disease progression. Isoliquiritigenin (ISL) have attracted attention due to their diverse biological and pharmacological properties, including anticancer activities. However, the impact of ISL on the invasive and migratory ability of NPC remains poorly understood. Hence, this study aimed to investigate the invitro anti-metastatic effects of ISL on NPC cells and elucidate the underlying signalling pathways. Human NPC cell NPC-39 and NPC-BM were utilized as cell models. Migratory and invasive capabilities were evaluated through wound healing and invasion assays, respectively. Gelatin zymography was employed to demonstrate matrix metalloproteinase-2 (MMP-2) activity, while western blotting was conducted to analyse protein expression levels and explore signalling cascades. Overexpression of signal transducer and activator of transcription 3 (STAT3) was carried out by transduction of STAT3-expressing vector. Our findings revealed that ISL effectively suppressed the migration and invasion of NPC cells. Gelatin zymography and Western blotting assays demonstrated that ISL treatment led to a reduction in MMP-2 enzyme activity and protein expression. Investigation of signalling cascades revealed that ISL treatment resulted in the inhibition of STAT3 phosphorylation. Moreover, overexpression of STAT3 restored the migratory ability of NPC cells in the presence of ISL. Collectively, these findings indicate that ISL inhibits the migration and invasion of NPC cells associating with MMP-2 downregulation through suppressing STAT3 activation. This suggests that ISL has an anti-metastatic effect on NPC cells and has potential therapeutic benefit for NPC treatment.

Selective STAT3 inhibitor STX-0119 alleviates osteoarthritis progression by modulating the STAT3/PPARγ signaling pathway

Osteoarthritis (OA), characterized by chronic pain, significantly affects the quality of life of affected individuals. Key factors in OA pathogenesis include cartilage degradation and inflammation. Signal transducer and activator of transcription 3 (STAT3), a member of the STAT protein family, plays a pivotal role in mediating inflammation. STX-0119 has been verified as a small molecular compound that can specifically inhibit STAT3. However, the efficacy of STX-0119 in the treatment of OA remains to be evaluated. Therefore, the aim of this study was to explore the therapeutic effects and molecular mechanisms of STX-0119 in the treatment of OA. We found that the expression of phosphorylated STAT3 is upregulated in human OA cartilage as well as in the cartilage of a mouse model of OA. In vivo, joint injection of STX-0119 into OA mice alleviated cartilage degeneration without affecting the subchondral bone. Additionally, STX-0119 could inhibit the phosphorylation of STAT3 in the cartilage. In vitro, STX-0119 suppressed inflammatory responses in chondrocytes and promoted anabolic metabolism in an interleukin-1β-induced chondrocyte inflammation model. Additionally, the results of transcriptome sequencing and lentiviral infection assays demonstrated that in chondrocytes, STX-0119 induces the upregulation of peroxisome proliferators-activated receptor gamma (PPARγ) expression by inhibiting STAT3 phosphorylation. Finally, in ex vivo cultures of human cartilage samples, STX-0119 was reaffirmed to inhibit cartilage degeneration via the STAT3/PPARγ signaling pathway. Together, our findings support the potential of STX-0119 for development as a therapeutic agent targeting STAT3 for the treatment of OA.

Design, synthesis and biological evaluation of novel naphthoquinothiazole derivatives as potent antitumor agents through inhibiting STAT3

The signal transducer and activator of transcription 3 (STAT3) has been established as a crucial drug target in the development of antitumor agents. In this study, a series of 21 derivatives of the STAT3 inhibitor napabucasin were designed and synthesized. Through preliminary screening against tumor cell lines, SZ6 emerged as the most potent compound with half maximal inhibitory concentration (IC50) values of 46.3 nM, 66.4 nM, and 53.8 nM against HCT116, HepG2, and Hela cells respectively. Furthermore, SZ6 effectively suppressed tumor invasion and migration in HCT116 cell assays by inducing S-phase arrest and apoptosis through inhibition of Protein Kinase B (PKB/AKT) activity and induction of reactive oxygen species (ROS). The mechanism underlying SZ6′s action involves inhibition of STAT3 phosphorylation, which was confirmed by western blotting analysis. Additionally, surface plasmon resonance (SPR) and cellular thermal shift assay (CETSA) demonstrated direct binding between SZ6 and STAT3. Notably, in vivo studies revealed that SZ6 significantly inhibited tumor growth without any observed organ toxicity. Collectively, these findings identify SZ6 as a promising STAT3 inhibitor for colorectal cancer treatment.

Exploration of novel isoxazole-fused quinone derivatives as anti-colorectal cancer agents through inhibiting STAT3 and elevating ROS level

Colorectal cancer (CRC) is trending to be a major health problem throughout the world. Therapeutics with dual modes of action have shown latent capacity to create ideal anti-tumor activity. Signal transducer and activator of transcription 3 (STAT3) has been proved to be a potential target for the development of anti-colon cancer drug. In addition, modulation of tumor redox homeostasis through deploying exogenous reactive oxygen species (ROS)-enhancing agents has been widely applied as anti-tumor strategy. Thus, simultaneously targeting STAT3 and modulation ROS balance would offer a fresh avenue to combat CRC. In this work, we designed and synthesized a novel series of isoxazole-fused quinones, which were evaluated for their preliminary anti-proliferative activity against HCT116 cells. Among these quinones, compound 41 exerted excellent in vitro anti-tumor effect against HCT116 cell line with an IC50 value of 10.18 ± 0.4 nM. Compound 41 was proved to bind to STAT3 by using Bio-Layer Interferometry (BLI) assay, and can significantly inhibit phosphorylation of STAT3. It also elevated ROS of HCT116 cells by acting as a substrate of NQO1. Mitochondrial dysfunction, apoptosis, and cell cycle arrest, which was caused by compound 41, might be partially due to the inhibition of STAT3 phosphorylation and ROS production induced by 41. Moreover, it exhibited ideal anti-tumor activity in human colorectal cancer xenograft model and good safety profiles in vivo. Overall, this study provided a novel quinone derivative 41 with excellent anti-tumor activity by inhibiting STAT3 and elevating ROS level, and gave insights into designing novel anti-tumor therapeutics by simultaneously modulation of STAT3 and ROS.

Danhongqing formula alleviates cholestatic liver fibrosis by downregulating long non-coding RNA H19 derived from cholangiocytes and inhibiting hepatic stellate cell activation

ObjectiveThis study explores the mechanism of action of Danhongqing formula (DHQ), a compound-based Chinese medicine formula, in the treatment of cholestatic liver fibrosis. MethodsIn vivo experiments were conducted using 8-week-old multidrug resistance protein 2 knockout (Mdr2-/-) mice as an animal model of cholestatic liver fibrosis. DHQ was administered orally for 8 weeks, and its impact on cholestatic liver fibrosis was evaluated by assessing liver function, liver histopathology, and the expression of liver fibrosis-related proteins. Real-time polymerase chain reaction, Western blot, immunohistochemistry and other methods were used to observe the effects of DHQ on long non-coding RNA H19 (H19) and signal transducer and activator of transcription 3 (STAT3) phosphorylation in the liver tissue of Mdr2-/- mice. In addition, cholangiocytes and hepatic stellate cells (HSCs) were cultured in vitro to measure the effects of bile acids on cholangiocyte injury and H19 expression. Cholangiocytes overexpressing H19 were constructed, and a conditioned medium containing H19 was collected to measure its effects on STAT3 protein expression and cell activation. The intervention effect of DHQ on these processes was also investigated. HSCs overexpressing H19 were constructed to measure the impact of H19 on cell activation and assess the intervention effect of DHQ. ResultsDHQ alleviated liver injury, ductular reaction, and fibrosis in Mdr2-/- mice, and inhibited H19 expression, STAT3 expression and STAT3 phosphorylation. This formula also reduced hydrophobic bile acid-induced cholangiocyte injury and the upregulation of H19, inhibited the activation of HSCs induced by cholangiocyte-derived conditioned medium, and decreased the expression of activation markers in HSCs. The overexpression of H19 in a human HSC line confirmed that H19 promoted STAT3 phosphorylation and HSC activation, and DHQ was able to successfully inhibit these effects. ConclusionDHQ effectively alleviated spontaneous cholestatic liver fibrosis in Mdr2-/- mice by inhibiting H19 upregulation in cholangiocytes and preventing the inhibition of STAT3 phosphorylation in HSC, thereby suppressing cell activation.Please cite this article as: Li M, Zhou Y, Zhu H, Xu LM, Ping J. Danhongqing formula alleviates cholestatic liver fibrosis by downregulating long non-coding RNA H19 derived from cholangiocytes and inhibiting hepatic stellate cell activation. J Integr Med. 2024; 22(2): 188–198.

Repurposing TAK875 as a novel STAT3 inhibitor for treating inflammatory bowel disease

Inflammatory bowel disease (IBD) is a chronic immune-mediated disease associated with a high recurrence rate and an elevated risk of colon cancer. In this study, we screened a bioactive compound library using a luciferase reporter assay and identified the compound TAK875 as a novel inhibitor of signal transducer and activator of transcription 3 (STAT3). Surface plasmon resonance analysis, differential scanning fluorimetry, and isothermal titration calorimetry demonstrated that TAK875 directly bound to recombinant STAT3. TAK875 suppressed the lipopolysaccharide (LPS)-induced release of nitric oxide, inducible nitric oxide synthase, and inflammatory factors in RAW264.7 cells, likely by inhibiting STAT3 phosphorylation. In addition, TAK875 inhibited the differentiation of CD4+ T cells into T-helper 17 cells, which may partially account for its anti-inflammatory effect. TAK875 also alleviated the LPS-induced accumulation of intracellular reactive oxygen species, thus displaying its antioxidant effects. Finally, we demonstrated its satisfactory anti-inflammatory effect in a dextran sulfate sodium-induced mouse model of ulcerative colitis. In conclusion, this study presented TAK875 as a novel STAT3 inhibitor and demonstrated its anti-inflammatory and antioxidant effects both in vitro and in vivo.

(E)-2-methoxy-4-(3-(4-methoxyphenyl)prop-1-en-1-yl)phenol alleviates inflammatory responses in LPS-induced mice liver sepsis through inhibition of STAT3 phosphorylation

Sepsis is a life-threatening disease with limited treatment options, and the inflammatory process represents an important factor affecting its progression. Many studies have demonstrated the critical roles of signal transducer and activator of transcription 3 (STAT3) in sepsis pathophysiology and pro-inflammatory responses. Inhibition of STAT3 activity may therefore represent a promising treatment option for sepsis. We here used a mouse model to demonstrate that (E)-2-methoxy-4-(3-(4-methoxyphenyl)prop-1-en-1-yl)phenol (MMPP) treatment prevented the liver sepsis-related mortality induced by 30 mg/kg lipopolysaccharide (LPS) treatment and reduced LPS-induced increase in alanine transaminase, aspartate transaminase, and lactate dehydrogenase levels, all of which are markers of liver sepsis progression. These recovery effects were associated with decreased LPS-induced STAT3, p65, and JAK1 phosphorylation and proinflammatory cytokine (interleukin 1 beta, interleukin 6, and tumor necrosis factor alpha) level; expression of cyclooxygenase-2 and induced nitric oxide synthase were also reduced by MMPP. In an in vitro study using the normal liver cell line THLE-2, MMPP treatment prevented the LPS-induced increase of STAT3, p65, and JAK1 phosphorylation and inflammatory protein expression in a dose-dependent manner, and this effect was enhanced by combination treatment with MMPP and STAT3 inhibitor. The results clearly indicate that MMPP treatment prevents LPS-induced mortality by inhibiting the inflammatory response via STAT3 activity inhibition. Thus, MMPP represents a novel agent for alleviating LPS-induced liver sepsis.

The roles of JAK2/STAT3 signaling in fusion of the secondary palate.

Cleft palate has a multifactorial etiology. In palatal fusion, the contacting medial edge epithelium (MEE) forms the epithelial seam, which is subsequently removed with the reduction of p63. Failure in this process results in a cleft palate. We herein report the involvement of janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling in palatal fusion and that folic acid rescues the fusing defect by reactivating JAK2/STAT3. In closure of bilateral palatal shelves, STAT3 phosphorylation was activated at the fusing MEE and mesenchyme underlying the MEE. JAK2 inhibition by AG490 inhibited STAT3 phosphorylation and resulted in palatal fusion failure without removal of the epithelial seam, in which p63 and keratin 17 (K17) periderm markers were retained. Folic acid application restored STAT3 phosphorylation in AG490-treated palatal explants and rescued the fusion defect, in which the p63- and K17-positive epithelial seam were removed. The AG490-induced palatal defect was also rescued in p63 haploinsufficient explants. These findings suggest that JAK2/STAT3 signaling is involved in palatal fusion by suppressing p63 expression in MEE and that folate restores the fusion defect by reactivating JAK2/STAT3.

Melatonin attenuates cellular senescence and apoptosis in diabetic nephropathy by regulating STAT3 phosphorylation

AimsMelatonin is an endogenous hormone related to the regulation of biorhythm. Previous researchers have found that melatonin can ameliorate diabetic nephropathy (DN), but the mechanism remains to be elucidated. To discover the possible mechanism by which melatonin prevents DN, we investigated the potential effects of melatonin on signal transducer and activator of transcription 3 (STAT3) on the progression of cellular senescence and apoptosis. Main methodsCellular senescence, apoptosis and the underlying mechanism of melatonin were investigated both in vivo and in vitro. C57BL/6 mice were intraperitoneally injected with streptozotocin (STZ) to establish DN. For an in vitro model of DN, human renal cortex proximal epithelial tubule (HK-2) cells were exposed to high glucose conditions. Key findingsMelatonin inhibited the phosphorylation of STAT3, decreased the expression of senescence proteins p53, p21 and p16INK4A. Melatonin also downregulated the expression of apoptotic proteins, including cleaved PARP1, cleaved caspase-9 and -3. Melatonin treatment decreased the positive area of senescence-associated galactosidase (SA-β-gal) staining and the number of TUNEL-positive cells in kidneys of DN mice. In vitro, melatonin inhibited STAT3 phosphorylation and lowered cellular senescence and apoptosis markers, in a manner similar to the STAT3 inhibitor S3I-201. In addition, the inhibition effect of melatonin on cellular senescence and apoptosis in HK-2 cells was reversed by the usage of recombinant IL-6 (rIL-6), which can induce STAT3 phosphorylation. SignificanceWe, for the first time, demonstrate that melatonin inhibits STAT3 phosphorylation, which is involved in alleviating the cellular senescence and apoptosis in DN.

Discovery, Optimization, and Evaluation of Novel N-(Benzimidazol-5-yl)-1,3,4-thiadiazol-2-amine Analogues as Potent STAT3 Inhibitors for Cancer Treatment.

Signal transducer and activator of transcription 3 (STAT3) is an attractive target for cancer therapy. However, identifying potent and selective STAT3 small-molecule inhibitors with drug-like properties remains challenging. Based on a scaffold combination strategy, compounds with a novel N-(benzimidazol-5-yl)-1,3,4-thiadiazol-2-amine scaffold were designed and their inhibition of the interleukin-6 (IL-6)/JAK/STAT3 pathway was tested in HEK-Blue IL-6 reporter cells. After optimization of lead compound 12, compound 40 was identified as a selective STAT3 inhibitor that directly binds the SH2 domain to inhibit STAT3 phosphorylation, translocation, and downstream gene transcription. Compound 40 exhibited antiproliferative activities against STAT3-overactivated DU145 (IC50 value = 2.97 μM) and MDA-MB-231 (IC50 value = 3.26 μM) cancer cells and induced cell cycle arrest and apoptosis. In the DU145 xenograft model, compound 40 showed in vivo antitumor efficacy following intraperitoneal administration, with a tumor growth inhibition rate of 65.3% at 50 mg/kg, indicating promise for further development.

Lyn attenuates sepsis-associated acute kidney injury by inhibition of phospho-STAT3 and apoptosis

Sepsis-associated acute kidney injury (SA-AKI) is a life-threatening condition associated with high mortality and morbidity. However, the underlying pathogenesis of SA-AKI is still unclear. Lyn belongs to Src family kinases (SFKs), which exert numerous biological functions including modulation in receptor-mediated intracellular signaling and intercellular communication. Previous studies demonstrated that Lyn gene deletion obviously aggravates LPS-induced lung inflammation, but the role and possible mechanism of Lyn in SA-AKI have not been reported yet. Here, we found that Lyn protected against renal tubular injury in cecal ligation and puncture (CLP) induced AKI mouse model by inhibition of signal transducer and activator of transcription 3 (STAT3) phosphorylation and cell apoptosis. Moreover, Lyn agonist MLR-1023 pretreatment improved renal function, inhibited STAT3 phosphorylation and decreased cell apoptosis. Thus, Lyn appears to play a crucial role in orchestrating STAT3-mediated inflammation and cell apoptosis in SA-AKI. Hence, Lyn kinase may be a promising therapeutic target for SA-AKI.

STAT3 inhibitor Stattic and its analogues inhibit STAT3 phosphorylation and modulate cytokine secretion in senescent tumour cells.

Signal transducer and activator of transcription 3 (STAT3) signalling serves an important role in carcinogenesis and cellular senescence, and its inhibition in tumour cells represents an attractive therapeutic target. Premature cellular senescence, a process of permanent proliferative arrest of cells in response to various inducers, such as cytostatic drugs or ionizing radiation, is accompanied by morphological and secretory changes, and by altered susceptibility to chemotherapeutic agents, which can thereby complicate their eradication by cancer therapies. In the present study, the responsiveness of proliferating and docetaxel (DTX)‑induced senescent cancer cells to small molecule STAT3 inhibitor Stattic and its analogues was evaluated using tumour cell lines. These agents displayed cytotoxic effects in cell viability assays on both proliferating and senescent murine TRAMP‑C2 and TC‑1 cells; however, senescent cells were markedly more resistant. Western blot analysis revealed that Stattic and its analogues effectively inhibited constitutive STAT3 phosphorylation in both proliferating and senescent cells. Furthermore, whether the Stattic‑derived inhibitor K1836 could affect senescence induction or modulate the phenotype of senescent cells was evaluated. K1836 treatment demonstrated no effect on senescence induction by DTX. However, the K1836 compound significantly modulated secretion of certain cytokines (interleukin‑6, growth‑regulated oncogene α and monocyte chemoattractant protein‑1). In summary, the present study demonstrated differences between proliferating and senescent tumour cells in terms of their susceptibility to STAT3 inhibitors and demonstrated the ability of the new STAT3 inhibitor K1836 to affect the secretion of essential components of the senescence‑associated secretory phenotype. The present study may be useful for further development of STAT3 inhibitor‑based therapy of cancer or age‑related diseases.

High‑throughput screening identification of a small‑molecule compound that induces ferroptosis and attenuates the invasion and migration of hepatocellular carcinoma cells by targeting the STAT3/GPX4 axis.

Hepatocellular carcinoma (HCC) is a lethal malignancy. Although considerable efforts have been made in recent years regarding treatments, effective therapeutic drugs for HCC remain insufficient. In the present study, polyphyllin VI was identified as a potential therapeutic drug for HCC by screening natural herbal compounds. The therapeutic effects of polyphyllin VI were assessed using Cell Counting Kit‑8, lactate dehydrogenase release and colony formation assays. The occurrence of ferroptosis was determined by assessing lipid peroxidation by reactive oxygen species, malondialdehyde levels, intracellular ferrous iron levels, and the mRNA and protein levels of glutathione peroxidase 4 (GPX4). The migratory and invasive abilities of HCC cells were examined using wound healing and Transwell assays. The results revealed that polyphyllin VI inhibited the proliferation, invasion and metastasis of HCC cells (HCCLM3 and Huh7 cells) by inducing ferroptosis. In addition, through a network pharmacology‑based approach and molecular docking analyses, it was found that polyphyllin VI may target the signal transducer and activator of transcription 3 (STAT3). HCC cells were treated with polyphyllin VI or a STAT3 inhibitor (Stattic), both of which exerted similar inhibitory effects on protein expression. Furthermore, immunofluorescence staining revealed that polyphyllin VI significantly inhibited the nuclear translocation of p‑STAT3 in HCC cells. Mechanistically, by the overexpression of STAT3, it was confirmed that STAT3 binds to GPX4 and promotes its protein expression and transcription, whereas polyphyllin VI induces ferroptosis by inhibiting the STAT3/GPX4 axis. Subsequently, invivo experiments revealed that polyphyllin VI inhibited the growth of subcutaneously transplanted tumors. On the whole, findings of the present study suggest that polyphyllin VI inhibits STAT3 phosphorylation, which inhibits GPX4 expression and induces the ferroptosis of HCC cells, eventually inhibiting their invasion and metastasis. These data suggest that polyphyllin VI may be a candidate for the prevention and treatment of HCC.

PLD2 deletion alleviates disruption of tight junctions in sepsis-induced ALI by regulating PA/STAT3 phosphorylation pathway.

Increased inflammatory exudation caused by endothelium and endothelial junction damage is a typical pathological feature of acute respiratory distress syndrome/acute lung injury (ARDS/ALI). Previous studies have shown that phospholipase D2 (PLD2) can increase the inflammatory response and has a close relationship with the severity of sepsis-induced ALI and the mortality of sepsis, but its mechanism is unknown. This study explored the effect and mechanism of PLD2 deletion on the structure and function of endothelial tight junction (TJ) in lipopolysaccharide (LPS)-induced ALI. We used C57BL/6 mice (wild-type and PLD2 knockout (PLD2-/-)) and human umbilical vein endothelial cell (HUVEC) models of sepsis-ALI. The pathological changes were evaluated by hematoxylin-eosin staining. Pulmonary vascular permeability was detected using wet-dry ratio, fluorescein isothiocyanate (FITC)-dextran, FITC-albumin, and immunoglobulin M concentration of bronchoalveolar lavage fluid. FITC-dextran and trans-endothelial electrical resistance assay were used to evaluate endothelial permeability on LPS-stimulated HUVECs. The mRNA expressions of TJ proteins were detected by real-time quantitative polymerase chain reaction. Then, protein levels were detected through Western blot analysis and immunofluorescence. The content of phosphatidic acid (PA), a downstream product of PLD2, was detected using an enzyme-linked immunosorbent assay kit. PLD2 deficiency not only alleviated lung histopathological changes and improved pulmonary vascular permeability but also increased the survival rate of ALI mice. Knockout of PLD2 or treatment with the PLD2 inhibitor can reduce the damage of endothelial TJ proteins, namely, claudin5, occludin and zonula occludens protein-1, in sepsis-ALI mice and LPS-stimulated HUVECs. The level of the PLD2 catalytic product PA increased in LPS-stimulated HUVECs, and exogenous PA can reduce the TJ protein expression and increase signal transducer and activator of transcription 3 (STAT3) phosphorylation in vitro. Inhibition of STAT3 phosphorylation attenuated PA-induced degradation of endothelial TJs. PLD2 knockout or inhibition may protect against LPS-induced lung injury by regulating the PA/STAT3 phosphorylation/endothelial TJ axis.

Exercise maintains bone homeostasis by promoting osteogenesis through STAT3

Bone exhibits changes in density, strength, and microarchitecture in relation to mechanical loading mediated by exercise. Appropriate exercise maintains bone homeostasis, while the absence of exercise leads to disuse bone loss. However, the acting mechanism of mechanotransduction in bone remains unclear. We performed the running-wheel exercise and tail suspension model to study the effects of exercise on bone metabolism, and found that osteoblastic Signal transducer and activator of transcription 3 (STAT3) activity was closely related to exercise-induced bone mass and metabolism changes. With the Flexcell tension-loading system in vitro, mechanical force promoted STAT3 activity, which was accompanied by increased osteoblastic differentiation of the bone marrow mesenchymal stem cells (BMSCs). In contrast, the inhibition of STAT3 phosphorylation blocked force-induced osteoblastic differentiation. Furthermore, pharmacological inactivation of STAT3 impaired the increase in exercise-induced bone mass and osteogenesis. With an inducible conditional deletion mouse model, we found that the osteoblast lineage-specific Stat3 deletion could also block force-induced osteoblastic differentiation in vitro and impair exercise-promoted bone mass and osteogenesis in vivo. This confirmed the crucial role of osteoblastic STAT3 in exercise-mediated bone metabolism. Finally, colivelin, a STAT3 agonist, promoted osteoblastic differentiation in vitro and partly rescued exercise loss-induced disuse bone loss by improving osteogenesis in the tail suspension model. Taken together, our study revealed the essential role of STAT3 in maintaining exercise-mediated bone homeostasis. In addition, STAT3 might act as a potential target for osteoporosis caused by exercise loss.

KPNA2 promotes angiogenesis by regulating STAT3 phosphorylation

PurposeAngiogenesis is involved in many pathological and physiological processes and is mainly driven by hypoxia. Karyopherin subunit alpha 2 (KPNA2), a member of the nuclear transport protein family, was recently shown to be induced by hypoxia in various types of tumours, so we aimed to investigate the role and mechanism of KPNA2 in angiogenesis under hypoxia.Materials and methodsAfter overexpression or knockdown of KPNA2 in human umbilical vein endothelial cells (HUVEC) by adenovirus vector infection, the tube formation, proliferation and migration of HUVEC under hypoxia were detected by tubule formation assay, 5-ethynyl-2′-deoxyuridine (EdU) staining and Transwell assay, respectively. After overexpression or knockdown of KPNA2 in a murine hindlimb ischemia model by local injection of purified adenovirus vector into the gastrocnemius muscle, blood flow changes were examined with a laser Doppler system. Changes in KPNA2-binding proteins under hypoxia were detected by immunoprecipitation-mass spectrometry (IP-MS) and co-immunoprecipitation (Co-IP). The effect of KPNA2 on signal transducer and activator of transcription 3 (STAT3) was detected by Western blotting and quantitative RT‒PCR.ResultsKPNA2 was upregulated in the HUVEC hypoxia model and murine hindlimb ischemia model. Overexpression of KPNA2 increased the proliferation, migration and tube formation of HUVEC under hypoxia, while knockdown of KPNA2 reduced the proliferation, migration and tube formation of HUVEC. Overexpression of KPNA2 promoted the restoration of blood flow in the murine hindlimb ischemia model, while knockout of KPNA2 inhibited the restoration of blood flow in the murine hindlimb ischemia model. Mechanistically, hypoxia promoted the binding of STAT3 to KPNA2. Overexpression of KPNA2 promoted STAT3 phosphorylation and then upregulated vascular endothelial growth factor (VEGF) and angiopoietin 2(ANGPT2), whereas knockdown of KPNA2 inhibited STAT3 phosphorylation and then downregulated VEGF and ANGPT2.ConclusionOur study demonstrates that hypoxia promotes the binding of STAT3 to KPNA2 and KPNA2 promotes angiogenesis under hypoxia by promoting the binding of STAT3 and JAK1 and regulating STAT3 phosphorylation.