Traditional Chinese medicine Pien-Tze-Huang ameliorates LPS-induced sepsis through bile acid-mediated activation of TGR5-STAT3-A20 signalling.

Pien Tze Huang (PZH), a class I nationally protected traditional Chinese medicine (TCM), has been used to treat liver diseases such as hepatitis; however, the effect of PZH on the progression of sepsis is unknown. Here, we reported that PZH attenuated lipopolysaccharide (LPS)-induced sepsis in mice and reduced LPS-induced production of proinflammatory cytokines in macrophages by inhibiting the activation of mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signalling. Mechanistically, PZH stimulated signal transducer and activator of transcription 3 (STAT3) phosphorylation to induce the expression of A20, which could inhibit the activation of NF-κB and MAPK signalling. Knockdown of the bile acid (BA) receptor G protein-coupled bile acid receptor 1 (TGR5) in macrophages abolished the effects of PZH on STAT3 phosphorylation and A20 induction, as well as the LPS-induced inflammatory response, suggesting that BAs in PZH may mediate its anti-inflammatory effects by activating TGR5. Consistently, deprivation of BAs in PZH by cholestyramine resin reduced the effects of PZH on the expression of phosphorylated-STAT3 and A20, the activation of NF-κB and MAPK signalling, and the production of proinflammatory cytokines, whereas the addition of BAs to cholestyramine resin-treated PZH partially restored the inhibitory effects on the production of proinflammatory cytokines. Overall, our study identifies BAs as the effective components in PZH that activate TGR5-STAT3-A20 signalling to ameliorate LPS-induced sepsis.

Anti-inflammatory and anti-rheumatic effects of Phoenix dactylifera L. (date palm) seed by controlling cytokines and inhibiting JAK1/STAT3 pathway on CFA-induced arthritis rat and its phytochemical profiling.

ETHNOPHARMACOLOGICAL RELEVANCE: Phoenix dactylifera L. (date palm) seed is widely used in Arabian traditional medicine to alleviate several health problems including inflammatory conditions. The herbal tea of date palm seed has been consumed by rheumatoid patients to relief their symptoms. AIM OF THE STUDY: The purpose of this study was to investigate the claimed beneficial use of P. dactylifera L. (Sewy variety) seed (PDS) in the treatment of rheumatoid arthritis (RA) and its mechanism of action as well as to study its phytoconstituents. MATERIALS AND METHODS: The anti-inflammatory and anti-oxidative properties of the non-polar and the polar extracts of PDS were studied using Complete Freund's adjuvant (CFA)-induced arthritis rat model. Paw edema, body weight, total nitrate/nitrite NOX content and cytokine markers were evaluated to monitor the progress of arthritis. Also, histological examination and thermal analysis were conducted. The phytoconstituent profiles of non-polar and polar extracts of PDS were investigated using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). The multiple reactions monitoring mode (MRM) of liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) was used to quantify phenolic phytoconstituents in both extracts. RESULTS: According to the findings, the polar and non-polar PDS extracts kept body weight comparable to those of healthy individuals while considerably lowering paw swelling, edema, and neutrophil infiltration. It also reduced the levels of Nuclear Factor Kappa B (NF-κB), Tumor Necrosis Factor Alpha (TNF-α), Interleukin 22, Interleukin 23, Interferon (IFN), Interleukin 17, Interleukin 1ß, Interleukin 6, Interleukin 36, Janus Kinase 1 (JAK1), and Signal Transducer and Activator of Transcription 3 (STAT3). They also reduced the degenerative alterations caused by RA. Thermal research gave additional support for these findings. 83 phytoconstituents were identified in the non-polar PDS extract and 86 phytoconstituents were identified in the polar PDS extract. 74 of the identified phytoconstituents were common in both extracts. 33 phytoconstituents were identified here from P. dactylifera for the first time as far as we know. In MRM-LC-ESI-MS/MS analysis, the major phenolics in both extracts were chlorogenic acid, naringenin, and vanillin. Catechin was only detected in the non-polar PDS extract. On the other hand, apigenin, kaempferol, and hesperetin were only detected in the polar PDS extract. Generally, the polar PDS extract showed higher concentrations of the identified phenolics than the non-polar extract. CONCLUSIONS: The PDS extracts especially the non-polar extract showed significant anti-inflammatory and anti-oxidative properties in the CFA-induced arthritis rat model. PDS might be used to produce RA medicines.

Zhen-Wu-Tang ameliorates lupus nephritis by diminishing renal tissue-resident memory CD8+ T cells via suppressing IL-15/STAT3 pathway.

Zhen-Wu-Tang (ZWT), a conventional herbal mixture, has been recommended for treating lupus nephritis (LN) in clinic. However, its mechanisms of action remain unknown. Here we aimed to define the immunological mechanisms underlying the effects of ZWT on LN and to determine whether it affects renal tissue-resident memory T (TRM) cells. Murine LN was induced by a single injection of pristane, while in vitro TRM cells differentiated with IL-15/TGF-ß. We found that ZWT or mycophenolate mofetil treatment significantly ameliorated kidney injury in LN mice by decreasing 24-h urine protein, Scr and anti-dsDNA Ab. ZWT also improved renal pathology and decreased IgG and C3 depositions. In addition, ZWT down-regulated renal Desmin expression. Moreover, it lowered the numbers of CD8+ TRM cells in kidney of mice with LN while decreasing their expression of TNF-α and IFN-γ. Consistent with in vivo results, ZWT-containing serum inhibited TRM cell differentiation induced by IL-15/TGF-ß in vitro. Mechanistically, it suppressed phosphorylation of STAT3 and CD122 （IL2/IL-15Rß）expression in CD8+ TRM cells. Importantly, ZWT reduced the number of total F4/80+CD11b+ and CD86+, but not CD206+, macrophages in the kidney of LN mice. Interestingly, ZWT suppressed IL-15 protein expression in macrophages in vivo and in vitro. Thus, we have provided the first evidence that ZWT decoction can be used to improve the outcome of LN by reducing CD8+ TRM cells via inhibition of IL-15/IL-15R /STAT3 signaling.

Repurposing diacerein to suppress colorectal cancer growth by inhibiting the DCLK1/STAT3 signaling pathway.

Double cortin-like kinase 1 (DCLK1) exhibits high expression levels across various cancers, notably in human colorectal cancer (CRC). Diacerein, a clinically approved interleukin (IL)-1ß inhibitor for osteoarthritis treatment, was evaluated for its impact on CRC proliferation and migration, alongside its underlying mechanisms, through both in vitro and in vivo analyses. The study employed MTT assay, colony formation, wound healing, transwell assays, flow cytometry, and Hoechst 33342 staining to assess cell proliferation, migration, and apoptosis. Additionally, proteome microarray assay and western blotting analyses were conducted to elucidate diacerein's specific mechanism of action. Our findings indicate that diacerein significantly inhibits DCLK1-dependent CRC growth in vitro and in vivo. Through high-throughput proteomics microarray and molecular docking studies, we identified that diacerein directly interacts with DCLK1. Mechanistically, the suppression of p-STAT3 expression following DCLK1 inhibition by diacerein or specific DCLK1 siRNA was observed. Furthermore, diacerein effectively disrupted the DCLK1/STAT3 signaling pathway and its downstream targets, including MCL-1, VEGF, and survivin, thereby inhibiting CRC progression in a mouse model, thereby inhibiting CRC progression in a mouse model.

Rosmarinic acid alleviates toosendanin-induced liver injury through restoration of autophagic flux and lysosomal function by activating JAK2/STAT3/CTSC pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Rosmarinic acid (RA), a natural polyphenol abundant in numerous herbal remedies, has been attracting growing interest owing to its exceptional ability to protect the liver. Toosendanin (TSN), a prominent bioactive compound derived from Melia toosendan Siebold & Zucc., boasts diverse pharmacological properties. Nevertheless, TSN possesses remarkable hepatotoxicity. Intriguingly, the potential of RA to counteract TSN-induced liver damage and its probable mechanisms remain unexplored. AIM OF THE STUDY: This study is aimed at exploring whether RA can alleviate TSN-induced liver injury and the potential mechanisms involved autophagy. MATERIALS AND METHODS: CCK-8 and LDH leakage rate assay were used to evaluate cytotoxicity. Balb/c mice were intraperitoneally administered TSN (20 mg/kg) for 24 h after pretreatment with RA (0, 40, 80 mg/kg) by gavage for 5 days. The autophagic proteins P62 and LC3B expressions were detected using western blot and immunohistochemistry. RFP-GFP-LC3B and transmission electron microscopy were applied to observe the accumulation levels of autophagosomes and autolysosomes. LysoTracker Red and DQ-BSA staining were used to evaluate the lysosomal acidity and degradation ability respectively. Western blot, immunohistochemistry and immunofluorescence staining were employed to measure the expressions of JAK2/STAT3/CTSC pathway proteins. Dual-luciferase reporter gene was used to measure the transcriptional activity of CTSC and RT-PCR was used to detect its mRNA level. H&E staining and serum biochemical assay were employed to determine the degree of damage to the liver. RESULTS: TSN-induced damage to hepatocytes and livers was significantly alleviated by RA. RA markedly diminished the autophagic flux blockade and lysosomal dysfunction caused by TSN. Mechanically, RA alleviated TSN-induced down-regulation of CTSC by activating JAK2/STAT3 signaling pathway. CONCLUSION: RA could protect against TSN-induced liver injury by activating the JAK2/STAT3/CTSC pathway-mediated autophagy and lysosomal function.

Unveiling the neuroprotection effects of Volvalerenic acid A: Mitochondrial fusion induction via IDO1-mediated Stat3-Opa1 signaling pathway.

BACKGROUND: Ischemic stroke is a leading cause of death and long-term disability worldwide. Studies have suggested that cerebral ischemia induces massive mitochondrial damage. Valerianic acid A (VaA) is the main active ingredient of valerianic acid with neuroprotective activity. PURPOSE: This study aimed to investigate the neuroprotective effects of VaA with ischemic stroke and explore the underlying mechanisms. METHOD: In this study, we established the oxygen-glucose deprivation and reperfusion (OGD/R) cell model and the middle cerebral artery occlusion and reperfusion (MCAO/R) animal model in vitro and in vivo. Neurological behavior score, 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining and Hematoxylin and Eosin (HE) Staining were used to detect the neuroprotection of VaA in MCAO/R rats. Also, the levels of ROS, mitochondrial membrane potential (MMP), and activities of NAD+ were detected to reflect mitochondrial function. Mechanistically, gene knockout experiments, transfection experiments, immunofluorescence, DARTS, and molecular dynamics simulation experiments showed that VaA bound to IDO1 regulated the kynurenine pathway of tryptophan metabolism and prevented Stat3 dephosphorylation, promoting Stat3 activation and subsequent transcription of the mitochondrial fusion-related gene Opa1. RESULTS: We showed that VaA decreased the infarct volume in a dose-dependent manner and exerted neuroprotective effects against reperfusion injury. Furthermore, VaA promoted Opa1-related mitochondrial fusion and reversed neuronal mitochondrial damage and loss after reperfusion injury. In SH-SY5Y cells, VaA (5, 10, 20 µM) exerted similar protective effects against OGD/R-induced injury. We then examined the expression of significant enzymes regulating the kynurenine (Kyn) pathway of the ipsilateral brain tissue of the ischemic stroke rat model, and these enzymes may play essential roles in ischemic stroke. Furthermore, we found that VaA can bind to the initial rate-limiting enzyme IDO1 in the Kyn pathway and prevent Stat3 phosphorylation, promoting Stat3 activation and subsequent transcription of the mitochondrial fusion-related gene Opa1. Using in vivo IDO1 knockdown and in vitro IDO1 overexpressing models, we demonstrated that the promoted mitochondrial fusion and neuroprotective effects of VaA were IDO1-dependent. CONCLUSION: VaA administration improved neurological function by promoting mitochondrial fusion through the IDO1-mediated Stat3-Opa1 pathway, indicating its potential as a therapeutic drug for ischemic stroke.

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

OBJECTIVE: This study explores the mechanism of action of Danhongqing formula (DHQ), a compound-based Chinese medicine formula, in the treatment of cholestatic liver fibrosis. METHODS: In 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. RESULTS: DHQ 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. CONCLUSION: DHQ 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.

Qingkailing granule alleviates pulmonary fibrosis by inhibiting PI3K/AKT and SRC/STAT3 signaling pathways.

Pulmonary fibrosis (PF) poses a significant challenge with limited treatment options and a high mortality rate of approximately 45 %. Qingkailing Granule (QKL), derived from the Angong Niuhuang Pill, shows promise in addressing pulmonary conditions. Using a comprehensive approach, combining network pharmacology analysis with experimental validation, this study explores the therapeutic effects and mechanisms of QKL against PF for the first time. In vivo, QKL reduced collagen deposition and suppressed proinflammatory cytokines in a bleomycin-induced PF mouse model. In vitro studies demonstrated QKL's efficacy in protecting cells from bleomycin-induced injury and reducing collagen accumulation and cell migration in TGF-ß1-induced pulmonary fibrosis cell models. Network pharmacology analysis revealed potential mechanisms, confirmed by western blotting, involving the modulation of PI3K/AKT and SRC/STAT3 signaling pathways. Molecular docking simulations highlighted interactions between QKL's active compounds and key proteins, showing inhibitory effects on epithelial damage and fibrosis. Collectively, these findings underscore the therapeutic potential of QKL in alleviating pulmonary inflammation and fibrosis through the downregulation of PI3K/AKT and SRC/STAT3 signaling pathways, with a pivotal role attributed to its active compounds.

Epigallocatechin gallate suppresses mitotic clonal expansion and adipogenic differentiation of preadipocytes through impeding JAK2/STAT3-mediated transcriptional cascades.

BACKGROUND: Mitotic clonal expansion (MCE) is a prerequisite for preadipocyte differentiation and adipogenesis. Epigallocatechin gallate (EGCG) has been shown to inhibit preadipocyte differentiation. However, the exact molecular mechanisms are still elusive. PURPOSE: This study investigated whether EGCG could inhibit adipogenesis and lipid accumulation by regulating the cell cycle in the MCE phase of adipogenesis and its underlying molecular mechanisms. METHOD: 3T3-L1 preadipocytes were induced to differentiate by a differentiation cocktail (DMI) and were treated with EGCG (25-100 µM) for 9, 18, and 24 h to examine the effect on MCE, or eight days to examine the effect on terminal differentiation. C57BL/6 mice were fed a high-fat diet (HFD) for three months to induce obesity and were given EGCG (50 or 100 mg/kg) daily by gavage. RESULTS: We showed that EGCG significantly inhibited terminal adipogenesis and lipid accumulation in 3T3-L1 cells and decreased expressions of PPARγ, C/EBPα, and FASN. Notably, at the MCE phase, EGCG regulated the cell cycle in sequential order, induced G0/G1 arrest at 18 h, and inhibited the G2/M phase at 24 h upon DMI treatment. Meanwhile, EGCG regulated the expressions of cell cycle regulators (cyclin D1, cyclin E1, CDK4, CDK6, cyclin B1, cyclin B2, p16, and p27), and decreased C/EBPß, PPARγ, and C/EBPα expressions at MCE. Mechanistic studies using STAT3 agonist Colivelin and antagonist C188-9 revealed that EGCG-induced cell cycle arrest in the MCE phase and terminal adipocyte differentiation was mediated by the inhibition of JAK2/STAT3 signaling cascades and STAT3 (Tyr705) nuclear translocation. Furthermore, EGCG significantly protected mice from HFD-induced obesity, reduced body weight and lipid accumulations in adipose tissues, reduced hyperlipidemia and leptin levels, and improved glucose intolerance and insulin sensitivity. Moreover, RNA sequencing (RNA-seq) analysis showed that the cell cycle changes in epididymal white adipose tissue (eWAT) were significantly enriched upon EGCG treatment. We further verified that EGCG treatment significantly reduced expressions of adipogenic factors, cell cycle regulators, and p-STAT3 in eWAT. CONCLUSION: EGCG inhibits MCE, resulting in the inhibition of early and terminal adipocyte differentiation and lipid accumulation, which were mediated by inhibiting p-STAT3 nucleus translocation and activation.

Pterostilbene suppresses gastric cancer proliferation and metastasis by inhibiting oncogenic JAK2/STAT3 signaling: In vitro and in vivo therapeutic intervention.

BACKGROUND: Gastric cancer (GC) represents a significant health burden with dire prognostic implications upon metastasis and recurrence. Pterostilbene (PTE) has been proven to have a strong ability to inhibit proliferation and metastasis in other cancers, while whether PTE exhibits anti-GC activity and its potential mechanism remain unclear. PURPOSE: To explore the efficacy and potential mechanism of PTE in treating GC. METHODS: We employed a comprehensive set of assays, including CCK-8, EdU staining, colony formation, flow cytometry, cell migration, and invasion assays, to detect the effect of PTE on the biological function of GC cells in vitro. The xenograft tumor model was established to evaluate the in vivo anti-GC activity of PTE. Network pharmacology was employed to predict PTE's potential targets and pathways within GC. Subsequently, Western blotting, immunofluorescence, and immunohistochemistry were utilized to analyze protein levels related to the cell cycle, EMT, and the JAK2/STAT3 pathway. RESULTS: Our study demonstrated strong inhibitory effects of PTE on GC cells both in vitro and in vivo. In vitro, PTE significantly induced cell cycle arrest at G0/G1 and S phases and suppressed proliferation, migration, and invasion of GC cells. In vivo, PTE led to a dose-dependent reduction in tumor volume and weight. Importantly, PTE exhibited notable safety, leaving mouse weight, liver function, and kidney function unaffected. The involvement of the JAK2/STAT3 pathway in PTE's anti-GC effect was predicted utilizing network pharmacology. PTE suppressed JAK2 kinase activity by binding to the JH1 kinase structural domain and inhibited the downstream STAT3 signaling pathway. Western blotting confirmed PTE's inhibition of the JAK2/STAT3 pathway and EMT-associated protein levels. The anti-GC effect was partially reversed upon STAT3 activation, validating the pivotal role of the JAK2/STAT3 signaling pathway in PTE's activity. CONCLUSION: Our investigation validates the potent inhibitory effects of PTE on the proliferation and metastasis of GC cells. Importantly, we present novel evidence implicating the JAK2/STAT3 pathway as the key mechanism through which PTE exerts its anti-GC activity. These findings not only establish the basis for considering PTE as a promising lead compound for GC therapeutics but also contribute significantly to our comprehension of the intricate molecular mechanisms underlying its exceptional anti-cancer properties.

Exploring the role and mechanism of hyperoside against cardiomyocyte injury in mice with myocardial infarction based on JAK2/STAT3 signaling pathway.

BACKGROUND: Myocardial infarction (MI) is one of the most deadly diseases in the world. Hyperoside (Hyp) has been shown to have a protective effect on cardiovascular function through various signaling pathways, but whether it can protect myocardial infarction by regulating JAK2/STAT3 signaling pathway is unknown. AIM OF THE STUDY: To investigate whether Hyp could protect the heart against myocardial infarction injury in mice by modulating JAK2/STAT3 signaling pathway and its potential mechanism. METHODS: In vivo experiments, the myocardial infarction model was established by ligating the left anterior descending coronary artery (LAD) of male C57BL/6 mice permanently. The mice were divided into seven groups: sham group, MI group, MI+Hyp (9 mg/kg), MI+Hyp (18 mg/kg) group, MI+Hyp (36 mg/kg) group, MI+Captopril group (15 mg/kg) group and MI+Hyp (36 mg/kg)+AG490 (7.5 mg/kg) group. Each group of animals were given different concentrations of hyperoside, positive control drug or inhibitor of JAK2/STAT3 singaling. After 14 days of administration, the electrocardiogram (ECG), echocardiography and serum myocardial injury markers were examined; Slices of mouse myocardial tissue were assessed for histopathological changes by HE, Masson and Sirius Red staining. TTC and TUNEL staining were used to evaluate the myocardial infarction area and cardiomyocytes apoptosis respectively. The expression of JAK2/STAT3 signaling pathway, apoptosis and autophagy-related proteins were detected by western blot. In vitro experiments, rat H9c2 cardiomyocytes were deprived of oxygen and glucose (OGD) to stimulate myocardial ischemia. The experiment was divided into seven groups: Control group, OGD group, OGD+Hyp (20 µM) group, OGD+Hyp (40 µM) group, OGD+Hyp (80 µM), OGD+Captopril (10 µM) group and OGD+Hyp (80 µM)+AG490 (100 µM) group. Myocardial cell damage and redox index were measured 12 h after OGD treatment. ROS content in cardiomyocytes was detected by immunofluorescence. Cardiomyocytes apoptosis was detected by flow cytometry. The expressions of JAK2/STAT3 signaling pathway-related proteins, apoptosis and autophagy related proteins were detected by western blot. RESULTS: In vivo, hyperoside could ameolirate ECG abnormality, increase cardiac function, reduce myocardial infarction size and significantly reduce myocardial fibrosis level and oxidation level. The experimental results in vitro showed that Hyp could reduce the ROS content in cardiomyocytes, decrease the level of oxidative stress and counteract the apoptosis induced by OGD injury . Both in vivo and in vitro experiments showed that hyperoside could increase phosphorylated JAK2 and STAT3, indicating that hyperoside could play a cardioprotective role by activating JAK2/STAT3 signaling pathway. It was also shown that hyperoside could increase the autophagy level of cardiomyocytes in vivo and in vitro. However the cardiomyocyte-protective effect of Hyp was abolished in combination with JAK2/ STAT3 signaling pathway inhibitor AG490. These results indicated that the protective effect of Hyp on cardiomyocyte injury was at least partially achieved through the activation of the JAK2/STAT3 signaling pathway. CONCLUSION: Hyp can significantly improve cardiac function, ameliorate myocardial hypertrophy and myocardial remodeling in MI mice. The mechanism may be related to improving mitochondrial autophagy of cardiomyocytes to maintain the advantage of autophagy, and blocking apoptosis pathway through phagocytosis, thus suppressing apoptosis level of cardiomyocytes. These effects of Hyp are achieved, at least in part, by activating the JAK2/STAT3 signaling pathway.

Jiangu formula: A novel osteoclast-osteoblast coupling agent for effective osteoporosis treatment.

BACKGROUND: The discovering of an osteoclast (OC) coupling active agent, capable of suppressing OC-mediated bone resorption while concurrently stimulating osteoblast (OB)-mediated bone formation, presents a promising strategy to overcome limitations associated with existing antiresorptive agents. However, there is a lack of research on active OC coupling agents. PURPOSE: This study aims to investigate the potential of Jiangu Formula (JGF) in inhibiting OCs while maintaining the OCOB coupling function. METHODS: The anti-osteoporosis efficacy of JGF was evaluated in osteoporosis models induced by ovariectomy in C57BL/6 mouse and SD rats. The effect of JGF on OCs was evaluated by detecting its capacity to inhibit OC differentiation and bone resorption in an in vitro osteoclastogenesis model induced by RANKL. The OCOB coupling activity of JGF was evaluated by measuring the secretion levels of OC-derived coupling factors, OB differentiation activity of MC3T3-E1 interfered with conditioned medium, and the effect of JGF on OC inhibition and OB differentiation in a C3H10T1/2-RAW264.7 co-culture system. The mechanism of JGF was studied by network pharmacology and validated using western blot, immunofluorescence (IF), and ELISA. Following that, the active ingredients of JGF were explored through a chemotype-assembly approach, activity evaluation, and LC-MS/MS analysis. RESULTS: JGF inhibited bone resorption in murine osteoporosis without compromising the OCOB coupling effect on bone formation. In vitro assays showed that JGF preserved the coupling effect of OC on OB differentiation by maintaining the secretion of OC-derived coupling factors. Network analysis predicted STAT3 as a key regulation point for JGF to exert anti-osteoporosis effect. Further validation assays confirmed that JGF upregulated p-STAT3(Ser727) and its regulatory factors IL-2 in RANKL-induced RAW264.7 cells. Moreover, 23 components in JGF with anti-OC activity identified by chemotype-assembly approach and verification experiments. Notably, six compounds, including ophiopogonin D, ginsenoside Re, ginsenoside Rf, ginsenoside Rg3, ginsenoside Ro, and ononin were identified as OC-coupling compounds. CONCLUSION: This study first reported JGF as an agent that suppresses bone loss without affecting bone formation. The potential coupling mechanism of JGF involves the upregulation of STAT3 by its regulators IL-2. Additionally, the chemotype-assembly approach elucidated the activity compounds present in JGF, offering a novel strategy for developing an anti-resorption agent that preserves bone formation.

Moscatilin inhibits vascular calcification by activating IL13RA2-dependent inhibition of STAT3 and attenuating the WNT3/ß-catenin signalling pathway.

INTRODUCTION: Vascular calcification, a devastating vascular complication accompanying atherosclerotic cardiovascular disease and chronic kidney disease, increases the incidence of adverse cardiovascular events and compromises the efficacy of vascular interventions. However, effective therapeutic drugs and treatments to delay or prevent vascular calcification are lacking. OBJECTIVES: This study was designed to test the therapeutic effects and mechanism of Moscatilin (also known as dendrophenol) from Dendrobium huoshanense (an eminent traditional Chinese medicine) in suppressing vascular calcification in vitro, ex vivo and in vivo. METHODS: Male C57BL/6J mice (25-week-old) were subjected to nicotine and vitamin D3 (VD3) treatment to induce vascular calcification. In vitro, we established the cellular model of osteogenesis of human aortic smooth muscle cells (HASMCs) under phosphate conditions. RESULTS: By utilizing an in-house drug screening strategy, we identified Moscatilin as a new naturally-occurring chemical entity to reduce HASMC calcium accumulation. The protective effects of Moscatilin against vascular calcification were verified in cultured HASMCs. Unbiased transcriptional profiling analysis and cellular thermal shift assay suggested that Moscatilin suppresses vascular calcification via binding to interleukin 13 receptor subunit A2 (IL13RA2) and augmenting its expression. Furthermore, IL13RA2 was reduced during HASMC osteogenesis, thus promoting the secretion of inflammatory factors via STAT3. We further validated the participation of Moscatilin-inhibited vascular calcification by the classical WNT/ß-catenin pathway, among which WNT3 played a key role in this process. Moscatilin mitigated the crosstalk between WNT3/ß-catenin and IL13RA2/STAT3 to reduce osteogenic differentiation of HASMCs. CONCLUSION: This study supports the potential of Moscatilin as a new naturally-occurring candidate drug for treating vascular calcification via regulating the IL13RA2/STAT3 and WNT3/ß-catenin signalling pathways.

Network pharmacology and experimental insights into STAT3 inhibition by novel isoxazole derivatives of piperic acid in triple negative breast cancer.

STAT3 is a crucial member within a family of seven essential transcription factors. Elevated STAT3 levels have been identified in various cancer types, notably in breast cancer (BC). Consequently, inhibiting STAT3 is recognized as a promising and effective strategy for therapeutic intervention against breast cancer. We herein synthesize a library of isoxazole (PAIs) from piperic acid [2E, 4E)-5-(2H-1,3-Benzodioxol-5-yl) penta-2,4-dienoic acid] on treatment with propargyl bromide followed by oxime under prescribed reaction conditions. Piperic acid was obtained by hydrolysis of piperine extracted from Piper nigrum. First, we checked the binding potential of isoxazole derivatives with breast cancer target proteins by network pharmacology, molecular docking, molecular dynamic (MD) simulation and cytotoxicity analysis as potential anti-breast cancer (BC) agents. The multi-source databases were used to identify possible targets for isoxazole derivatives. A network of protein-protein interactions (PPIs) was generated by obtaining 877 target genes that overlapped gene symbols associated with isoxazole derivatives and BC. Molecular docking and MD modelling demonstrated a strong affinity between isoxazole derivatives and essential target genes. Further, the cell viability studies of isoxazole derivatives on the human breast carcinoma cell lines showed toxicity in all breast cancer cell lines. In summary, our study indicated that the isoxazole derivative showed the significant anticancer activity. The results highlight the prospective utility of isoxazole derivatives as new drug candidates for anticancer chemotherapy, suggesting route for the continued exploration and development of drugs suitable for clinical applications.

Argel's stemmoside C as a novel natural remedy for mice with alcohol-induced gastric ulcer based on its molecular mechanistic pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Solenostemma argel is widely distributed in Africa & Asia with traditional usage in alleviating abdominal colic, aches, & cramps. This plant is rich in phytochemicals, which must be explored for its pharmacological effects. PURPOSE: Peptic Ulcer Disease (PUD) is the digestion of the digestive tube. PUD not only interferes with food digestion & nutrient absorption, damages one of the largest defensive barriers against pathogenic micro-organisms, but also impedes drug absorption & bioavailability, rendering the oral route, the most convenient way, ineffective. Omeprazole, one of the indispensable cost-effective proton-pump inhibitors (PPIs) extensively prescribed to control PUD, is showing growing apprehensions toward multiple drug interactions & side effects. Hence, finding a natural alternative with Omeprazole-like activity & limited side effects is a medical concern. STUDY DESIGN: Therefore, we present Stemmoside C as a new gastroprotective phytochemical agent isolated from Solenostemma argel to be tested in upgrading doses against ethanol-induced gastric ulcers in mice compared to negative, positive, & reference Omeprazole groups. METHODS: We carried out in-depth pharmacological & histopathological studies to determine the possible mechanistic pathway. RESULTS: Our results showed that Stemmoside C protected the stomach against ethanol-induced gastric ulcers parallel to Omeprazole. Furthermore, the mechanistic studies revealed that Stemmoside C produced its effect using an orchestrated array of different mechanisms. Stemmoside C stimulates stomach defense by increasing COX-2, PGE-2, NO, & TFF-1 healing factors, IL-10 anti-inflammatory cytokine, & Nrf-2 & HO-1 anti-oxidant pathways. It also suppresses stomach ulceration by inhibiting leucocyte recruitment, especially neutrophils, leading to subsequent inhibition of NF-κBp65, TNF-α, IL-1ß, & iNOS pro-inflammatory cytokines & JAK-1/STAT-3 inflammation-induced carcinogenicity cascade in addition to MMP-9 responsible for tissue degradation. CONCLUSION: These findings cast light on Stemmoside C's clinical application against gastric ulcer progression, recurrence, & tumorigenicity & concurrently with chemotherapy.

Isoalantolactone exerts anti-melanoma effects via inhibiting PI3K/AKT/mTOR and STAT3 signaling in cell and mouse models.

BACKGROUND AND AIM: Although the anti-cancer activity of isoalantolactone (IATL) has been extensively studied, the anti-melanoma effects of IATL are still unknown. Here, we have investigated the anti-melanoma effects and mechanism of action of IATL. MTT and crystal violet staining assays were performed to detect the inhibitory effect of IATL on melanoma cell viability. Apoptosis and cell cycle arrest induced by IATL were examined using flow cytometry. The molecular mechanism of IATL was explored by Western blotting, confocal microscope analysis, molecular docking, and cellular thermal shift assay (CETSA). A B16F10 allograft mouse model was constructed to determine the anti-melanoma effects of IATL in vivo. The results showed that IATL exerted anti-melanoma effects in vitro and in vivo. IATL induced cytoprotective autophagy in melanoma cells by inhibiting the PI3K/AKT/mTOR signaling. Moreover, IATL inhibited STAT3 activation both in melanoma cells and allograft tumors not only by binding to the SH2 domain of STAT3 but also by suppressing the activity of its upstream kinase Src. These findings demonstrate that IATL exerts anti-melanoma effects via inhibiting the STAT3 and PI3K/AKT/mTOR signaling pathways, and provides a pharmacological basis for developing IATL as a novel phytotherapeutic agent for treating melanoma clinically.

Natural compound Byakangelicin suppresses breast tumor growth and motility by regulating SHP-1/JAK2/STAT3 signal pathway.

Byakangelicin mostly obtained from the root of Angelica dahurica and has protective effect on liver injury and fibrosis. In addition, Byakangelicin, as a traditional medicine, is also used to treat colds, headache and toothache. Recent studies have shown that Byakangelicin exhibits anti-tumor function; however, the role of Byakangelicin in breast tumor progression and related mechanism has not yet been elucidated. Our study aims to investigate the role of Byakangelicin in breast tumor progression and the underlying mechanism. To measure the effect of Byakangelicin on JAK2/STAT3 signaling, a dual luciferase reporter assay and a Western blot assay were performed. CCK8, colony formation, apoptosis and cell invasion assays were used to examine the inhibitory potential of Byakangelicin on breast cancer cells. Additionally, SHP-1 was silenced by specific siRNA duplex and the function of SHP-1 on Byakangelicin-mediated inhibition of JAK2/STAT3 signaling was evaluated. Byakangelicin treatment significantly inhibited STAT3 transcriptional activity. In addition, Byakangelicin treatment blocked JAK2/STAT3 signaling in a dose-dependent manner. Byakangelicin-treated tumor cells showed a dramatically reduced proliferation, colony formation and invasion ability. Moreover, Byakangelicin remarkedly induced breast cancer cell apoptosis. Furthermore, Byakangelicin regulated the expression of SHP1.In conclusion, our current study indicated that Byakangelicin, a natural compound, inhibits SHP-1/JAK2/STAT3 signaling and thus blocks tumor growth and motility.

Endogenous sulfur dioxide deficiency as a driver of cardiomyocyte senescence through abolishing sulphenylation of STAT3 at cysteine 259.

OBJECTIVE: Cardiomyocyte senescence is an important contributor to cardiovascular diseases and can be induced by stressors including DNA damage, oxidative stress, mitochondrial dysfunction, epigenetic regulation, etc. However, the underlying mechanisms for the development of cardiomyocyte senescence remain largely unknown. Sulfur dioxide (SO2) is produced endogenously by aspartate aminotransferase 2 (AAT2) catalysis and plays an important regulatory role in the development of cardiovascular diseases. The present study aimed to explore the effect of endogenous SO2 on cardiomyocyte senescence and the underlying molecular mechanisms. APPROACH AND RESULTS: We interestingly found a substantial reduction in the expression of AAT2 in the heart of aged mice in comparison to young mice. AAT2-knockdowned cardiomyocytes exhibited reduced SO2 content, elevated expression levels of Tp53, p21Cip/Waf, and p16INk4a, enhanced SA-ß-Gal activity, and elevated level of γ-H2AX foci. Notably, supplementation with a SO2 donor ameliorated the spontaneous senescence phenotype and DNA damage caused by AAT2 deficiency in cardiomyocytes. Mechanistically, AAT2 deficiency suppressed the sulphenylation of signal transducer and activator of transcription 3 (STAT3) facilitated its nuclear translocation and DNA-binding capacity. Conversely, a mutation in the cysteine (Cys) 259 residue of STAT3 blocked SO2-induced STAT3 sulphenylation and subsequently prevented the inhibitory effect of SO2 on STAT3-DNA-binding capacity, DNA damage, and cardiomyocyte senescence. Additionally, cardiomyocyte (cm)-specific AAT2 knockout (AAT2cmKO) mice exhibited a deterioration in cardiac function, cardiomegaly, and cardiac aging, whereas supplementation with SO2 donors mitigated the cardiac aging and remodeling phenotypes in AAT2cmKO mice. CONCLUSION: Downregulation of the endogenous SO2/AAT2 pathway is a crucial pathogenic mechanism underlying cardiomyocyte senescence. Endogenous SO2 modifies STAT3 by sulphenylating Cys259, leading to the inhibition of DNA damage and the protection against cardiomyocyte senescence.

Npy transcription is regulated by noncanonical STAT3 signaling in hypothalamic neurons: Implication with lipotoxicity and obesity.

Neuropeptide Y (Npy) is an abundant neuropeptide expressed in the central and peripheral nervous systems. NPY-secreting neurons in the hypothalamic arcuate nucleus regulate energy homeostasis, and Npy mRNA expression is regulated by peripheral nutrient and hormonal signals like leptin, interleukin-6 (IL-6), and fatty acids. This study demonstrates that IL-6, which phosphorylates tyrosine 705 (Y705) of STAT3, decreased Npy mRNA in arcuate immortalized hypothalamic neurons. In parallel, inhibitors of STAT3-Y705 phosphorylation, stattic and cucurbitacin I, robustly upregulated Npy mRNA. Chromatin-immunoprecipitation showed high baseline total STAT3 binding to multiple regulatory regions of the Npy gene, which are decreased by IL-6 exposure. The STAT3-Npy interaction was further examined in obesity-related pathologies. Notably, in four different hypothalamic neuronal models where palmitate potently stimulated Npy mRNA, Socs3, a specific STAT3 activity marker, was downregulated and was negatively correlated with Npy mRNA levels (R2 = 0.40, p < 0.001), suggesting that disrupted STAT3 signaling is involved in lipotoxicity-mediated dysregulation of Npy. Finally, human NPY SNPs that map to human obesity or body mass index were investigated for potential STAT3 binding sites. Although none of the SNPs were linked to direct STAT3 binding, analysis show that rs17149106 (-602 G > T) is located on an upstream enhancer element of NPY, where the variant is predicted to disrupt validated binding of KLF4, a known inhibitory cofactor of STAT3 and downstream effector of leptin signaling. Collectively, this study demonstrates that STAT3 signaling negatively regulates Npy transcription, and that disruption of this interaction may contribute to metabolic disorders.

Terpenoid-Mediated Targeting of STAT3 Signaling in Cancer: An Overview of Preclinical Studies.

Cancer has become one of the most multifaceted and widespread illnesses affecting human health, causing substantial mortality at an alarming rate. After cardiovascular problems, the condition has a high occurrence rate and ranks second in terms of mortality. The development of new drugs has been facilitated by increased research and a deeper understanding of the mechanisms behind the emergence and advancement of the disease. Numerous preclinical and clinical studies have repeatedly demonstrated the protective effects of natural terpenoids against a range of malignancies. Numerous potential bioactive terpenoids have been investigated in natural sources for their chemopreventive and chemoprotective properties. In practically all body cells, the signaling molecule referred to as signal transducer and activator of transcription 3 (STAT3) is widely expressed. Numerous studies have demonstrated that STAT3 regulates its downstream target genes, including Bcl-2, Bcl-xL, cyclin D1, c-Myc, and survivin, to promote the growth of cells, differentiation, cell cycle progression, angiogenesis, and immune suppression in addition to chemotherapy resistance. Researchers viewed STAT3 as a primary target for cancer therapy because of its crucial involvement in cancer formation. This therapy primarily focuses on directly and indirectly preventing the expression of STAT3 in tumor cells. By explicitly targeting STAT3 in both in vitro and in vivo settings, it has been possible to explain the protective effect of terpenoids against malignant cells. In this study, we provide a complete overview of STAT3 signal transduction processes, the involvement of STAT3 in carcinogenesis, and mechanisms related to STAT3 persistent activation. The article also thoroughly summarizes the inhibition of STAT3 signaling by certain terpenoid phytochemicals, which have demonstrated strong efficacy in several preclinical cancer models.