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.

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.

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.

Targeting the JAK2/STAT3 signaling pathway with natural plants and phytochemical ingredients: A novel therapeutic method for combatting cardiovascular diseases.

The aim of this article is to introduce the roles and mechanisms of the JAK2/STAT3 pathway in various cardiovascular diseases, such as myocardial fibrosis, cardiac hypertrophy, atherosclerosis, myocardial infarction, and myocardial ischemiareperfusion. In addition, the effects of phytochemical ingredients and different natural plants, mainly traditional Chinese medicines, on the regulation of different cardiovascular diseases via the JAK2/STAT3 pathway are discussed. Surprisingly, the JAK2 pathway has dual roles in different cardiovascular diseases. Future research should focus on the dual regulatory effects of different phytochemical ingredients and natural plants on JAK2 to pave the way for their use in clinical trials.

Selenomethionine modulates the JAK2 / STAT3 / A20 pathway through oxidative stress to alleviate LPS-induced pyroptosis and inflammation in chicken hearts.

Selenium (Se) is involved in many physiopathologic processes in humans and animals and is strongly associated with the development of heart disease. Lipopolysaccharides (LPS) are cell wall components of gram-negative bacteria that are present in large quantities during environmental pollution. To investigate the mechanism of LPS-induced cardiac injury and the efficacy of the therapeutic effect of SeMet on LPS, a chicken model supplemented with selenomethionine (SeMet) and/or LPS treatment, as well as a primary chicken embryo cardiomyocyte model with the combined effect of SeMet / JAK2 inhibitor (INCB018424) and/or LPS were established in this experiment. CCK8 kit, Trypan blue staining, DCFH-DA staining, oxidative stress kits, immunofluorescence staining, LDH kit, real-time fluorescence quantitative PCR, and western blot were used. The results proved that LPS exposure led to ROS explosion, hindered the antioxidant system, promoted the expression of the JAK2 pathway, and increased the expression of genes involved in the pyroptosis pathway, inflammatory factors, and heat shock proteins (HSPs). Upon co-treatment with SeMet and LPS, SeMet reduced LPS-induced pyroptosis and inflammation and restored the expression of HSPs by inhibiting the ROS burst and modulating the antioxidant capacity. Co-treatment with INCB018424 and LPS resulted in inhibited of the JAK2 pathway, attenuating pyroptosis, inflammation, and high expression of HSPs. Thus, LPS induced pyroptosis, inflammation, and changes in HSPs activity by activating of the JAK2 / STAT3 / A20 signaling axis in chicken hearts. Moreover, SeMet has a positive effect on LPS-induced injury. This work further provides a theoretical basis for treating cardiac injury by SeMet.

Casticin induces apoptosis and cytoprotective autophagy while inhibiting stemness involving Akt/mTOR and JAK2/STAT3 pathways in glioblastoma.

Glioblastoma (GBM) is the most common malignant glioma. However, the current systemic drugs cannot completely cure GBM. Casticin is a methoxylated flavonol compound isolated from a traditional Chinese medicine Vitex rotundifolia L.f. and exhibits a strong antitumor activity in multiple human malignancies. This study was aimed to explore the effects and underlying mechanisms of casticin in GBM. The MTT assay and colony formation was used to evaluate the casticin-induced cell viability in GBM cells. Apoptosis was assessed by ANNEXIV/PI staining assay. Autophagy was analyzed by transmission electron microscopy and immunofluorescence assays. GBM stem cell (GSC) was analyzed by tumor-sphere formation assay and ALDEFLUOR assay. The anti-GBM effect of casticin was also determined by the U87MG xenograft model. Casticin inhibited tumor cell growth in vitro and in vivo, as well as significantly induced apoptosis and autophagy. Autophagy inhibition augmented casticin-induced apoptosis. Casticin also reduced the GSC population by suppressing Oct4, Nanog, and Sox2. Mechanistically, casticin inhibited Akt/mTOR and JAK2/STAT3 signal pathways. The antitumor effect of casticin in GBM was demonstrated by inducing apoptosis, autophagy, and reducing population of GSCs; thus, it may be a potential GBM therapeutic agent for future clinical usage.

Multiomic profiling reveals metabolic alterations mediating aberrant platelet activity and inflammation in myeloproliferative neoplasms.

Platelets from patients with myeloproliferative neoplasms (MPNs) exhibit a hyperreactive phenotype. Here, we found elevated P-selectin exposure and platelet-leukocyte aggregates indicating activation of platelets from essential thrombocythemia (ET) patients. Single-cell RNA-seq analysis of primary samples revealed significant enrichment of transcripts related to platelet activation, mTOR, and oxidative phosphorylation in ET patient platelets. These observations were validated via proteomic profiling. Platelet metabolomics revealed distinct metabolic phenotypes consisting of elevated ATP generation accompanied by increases in the levels of multiple intermediates of the tricarboxylic acid cycle, but lower α-ketoglutarate (α-KG) in MPN patients. Inhibition of PI3K/AKT/mTOR signaling significantly reduced metabolic responses and hyperreactivity in MPN patient platelets, while α-KG supplementation markedly reduced oxygen consumption and ATP generation. Ex vivo incubation of platelets from both MPN patients and Jak2 V617F-knockin mice with α-KG supplementation significantly reduced platelet activation responses. Oral α-KG supplementation of Jak2 V617F mice decreased splenomegaly and reduced hematocrit, monocyte, and platelet counts. Finally, α-KG treatment significantly decreased proinflammatory cytokine secretion from MPN CD14+ monocytes. Our results reveal a previously unrecognized metabolic disorder in conjunction with aberrant PI3K/AKT/mTOR signaling that contributes to platelet hyperreactivity in MPN patients.

Astaxanthin Augmented the Anti-Hepatocellular Carcinoma Efficacy of Sorafenib Through the Inhibition of the JAK2/STAT3 Signaling Pathway and Mitigation of Hypoxia within the Tumor Microenvironment.

SCOPE: The optimization of anti-cancer drug effectiveness through dietary modifications has garnered significant attention among researchers in recent times. Astaxanthin (AST) has been identified as a safe and biologically active dietary supplement. METHODS AND RESULTS: The tumor-bearing mice are treated with sorafenib, along with supplementation of 60 mg kg-1 AST during the treatment. The coadministration of AST and a subclinical dosage of 10 mg kg-1 sorafenib demonstrates a tumor inhibition rate of 76.5%, which is notably superior to the 45% inhibition rate observed with the clinical dosage of 30 mg kg-1 sorafenib (p < 0.05). The administration of AST leads to a tumor inhibition increase of around 25% when combined with the clinical dose of 30 mg kg-1 sorafenib (p <0.05). AST enhances the inhibitory effect of sorafenib on tumor angiogenesis through the JAK2/STAT3 signaling pathway. Furthermore, AST exhibits a reduction in hypoxia within the tumor microenvironment. CONCLUSION: The results suggest that AST supplement enhances the inhibitory effects of sorafenib on hepatocellular carcinoma. This study presents a new dietary management program for oncology patients.

Eupatilin ameliorates postmenopausal osteoporosis via elevating microRNA-211-5p and repressing JAK2/STAT3 pathway.

Postmenopausal osteoporosis (PMOP) poses a significant threat to women's health worldwide. Eupatilin is a key bioactive component of the Chinese herbal medicine Artemisia asiatica Nakai. Recent research reports have proved the inhibitory function of Eupatilin in many diseases. MicroRNAs (miRNAs) are 21-23 nucleotide-long, single-stranded, noncoding RNA molecules generated endogenously, and many studies have indicated that miRNAs are involved in the development of osteoporosis. This study explored the role and potential mechanism of Eupatilin underlying PMOP. First, rats were given intragastric administration of Eupatilin every day and subcutaneous injections of oligonucleotides or plasmids that interfered with miR-211-5p or janus kinase 2 (JAK2) once a week. After 4 weeks, the PMOP rat model was established. Then, serum alkaline phosphatase, calcium, and phosphorus levels, as well as femur bone mineral density and biomechanical parameters, were detected. Hematoxylin-eosin staining and Masson staining were applied for detecting the pathological condition of femur, and immunohistochemical staining was for detecting osteocalcin. MC3T3-E1 cells were transfected with plasmid vectors interfering with miR-211-5p or JAK2; and cell viability, lactate dehydrogenase cytotoxicity, and cell mineralization were subsequently examined. The relationship between miR-211-5p and JAK2/signal transducer and activator of transcription 3 (STAT3) pathway was analyzed. The targeting relation between miR-211-5p and JAK2 was also verified. The experimental results revealed that Eupatilin improved the pathological conditions of PMOP rats by promoting the proliferation and mineralization of osteoblasts. MiR-211-5p was down-regulated and JAK2/STAT3 was upregulated in PMOP rats. Upregulation of miR-211-5p further improved the pathological conditions of PMOP rats based on Eupatilin treatment. MiR-211-5p inhibited the JAK2/STAT3 pathway. JAK2 offset the effects of elevated miR-211-5p on PMOP rats. Overall, Eupatilin attenuates PMOP through elevating miR-211-5p and repressing JAK2/STAT3 pathway, which suggests the utility of Eupatilin as a potential drug for POMP treatment.

Abelmoschus manihot (L.) medik. seeds alleviate rheumatoid arthritis by modulating JAK2/STAT3 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Abelmoschus manihot (L.) Medik. Seeds (AMS, སོ་མ་ར་ཛ།), a Tibetan classical herbal in China, are rich in flavonoids and phenolic glycosides compounds, such as quercetin and its derivatives. Moreover, it has been found to possess anti-rheumatoid arthritis (RA) effects. Nonetheless, its anti-RA mechanism is yet unknown. AIM OF THE STUDY: This research aimed to examine the active ingredients of AMS as well as potential pharmacological mechanisms in AMS on RA. MATERIALS AND METHODS: The ultra-performance liquid chromatography-electrospray ionization-tandem multistage mass spectrometry (UPLC-ESI-IT-MSn) technique was used to determine the primary chemical components of AMS that were responsible for the therapeutic effects on RA. In addition, 36 male Wistar rats weighing between 200 and 220 g were classified at random into six groups [normal control group, collagen-induced arthritis (CIA) group, methotrexate group (positive control, 1.05 mg/kg), AMS group (157.5 mg/kg, 315 mg/kg, 630 mg/kg)]. CIA rats were given AMS extract by intragastric administration for 28 days, and their ankles were photographed to observe the degree of swelling. Further, the arthritis score, paws swelling, and body weight changes of CIA rats were determined to observe whether AMS has any effect on RA, and synovial and cartilage tissue injuries were identified by histopathology. Besides, the levels of IL-10, TNF-α, IL-1ß, INF-γ, etc. in serum were estimated by ELISA. Western blot experiments were implemented to identify the expression levels of protein involved in the JAK2/STAT3 signaling pathway in the CIA rats' synovial tissues. Moreover, the mechanisms and targets of active ingredient therapy of AMS for RA were predicted using network pharmacology and then verified using molecular docking. RESULT: In the present study, 12 compounds were detected by UPLC-ESI-IT-MSn, such as quercetin and its derivative which could be potential active ingredients that contribute to the anti-RA properties of AMS. Our in vivo studies on CIA rats revealed that an AMS-H dose of 630 mg/kg significantly improved joint damage while decreasing the arthritic index and paw swelling. Furthermore, AMS inhibited the INF-γ, IL-6, IL-17, IL-1ß, and TNF-α, levels while upregulating the expression of anti-inflammatory cytokines IL-10 and IL-4 in serum. Besides, AMS inhibited the protein Bcl-2/Bax, STAT3, and JAK2 levels, and promoted the expression of Caspase3, SOCS1, and SOCS3 in the JAK2/STAT3 pathway. Additionally, the JAK/STAT signaling pathway was found to perform a remarkable function in the AMS therapy of RA as evidenced by enrichment in GO terms and KEGG pathways. Meanwhile, data from molecular docking experiments indicated that the core targets of PIK3CA, JAK2, and SRC bound stably to the active ingredients of mimuone, 4'-methoxy-bavachromanol, and quercetin. CONCLUSION: According to these findings, the AMS could improve joint inflammation in CIA rats, and its underlying mechanism could be linked to the regulation of the JAK2/STAT3 pathway. Therefore, AMS might become a promising agent for alleviating inflammation in RA patients.

The role of JAK/STAT signaling pathway in cerebral ischemia-reperfusion injury and the therapeutic effect of traditional Chinese medicine: A narrative review.

Cerebral ischemia is a cerebrovascular disease with symptoms caused by insufficient blood or oxygen supply to the brain. When blood supplied is restored after cerebral ischemia, secondary brain injury may occur, which is called cerebral ischemia-reperfusion injury (CIRI). In this process, the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway plays an important role. It mediates neuroinflammation and participates in the regulation of physiological activities, such as cell proliferation, differentiation, and apoptosis. After CIRI, M1 microglia is activated and recruited by the damaged tissue. The inflammatory factors are produced by M1 microglia through the JAK/STAT pathway, eventually leading to cell apoptosis. Meanwhile, the JAK2/STAT3 signaling pathway and the expression of lipocalin-2 and caspase-3 could increase. In the pathway, phosphorylated JAK2 and phosphorylated STAT3 function of 2 ways. They not only promote the proliferation of neurons, but also affect the differentiation direction of neural stem cells by further acting on the Notch signaling pathway. Recently, traditional Chinese medicine (TCM) is a key player in CIRI, through JAK2, STAT3, STAT1 and their phosphorylation. Therefore, the review focuses on the JAK/STAT signaling pathway and its relationship with CIRI as well as the influence of the TCM on this pathway. It is aimed at providing the basis for future clinical research on the molecular mechanism of TCM in the treatment of CIRI.

Identification of novel inhibitors from Urtica spp against TNBC targeting JAK2 receptor for breast cancer therapy.

Breast cancer is the most prevalent form of cancer in women globally, and TNBC (triple-negative breast cancer) is its aggressive type since it lacks the usual targets. JAK2/STAT3 pathway can be an important lead in anticancer drug discovery, as restraining the downstream signalling of this pathway results in the induction of cell apoptosis. Moreover, various limitations associated with chemotherapy are the reason to find an alternative herbal-based therapy. For this study, we collected Urtica dioica and U. parviflora from different regions of Uttarakhand, followed by preparation of their leaf and stem extracts in different solvents. The GC-MS analysis of these extracts revealed a total of 175 compounds to be present in them. Further, by molecular docking approach, we studied the interaction between these compounds and JAK2, and 12 major compounds with better binding energy than the control Paclitaxel were identified. In addition, the selected hits were also reported to display better pharmacokinetic properties. Moreover, extracts from both the Urtica spp. displayed significant anticancer activity against MDA-MB-231(TNBC cell line) and exhibited lower cytotoxicity in healthy cell lines, i.e. HEK293T, indicating that these extracts were safer to use. Hence, the findings in our study can be crucial in the area of herbal-based target-specific drug development against breast cancer.

High Intensity Interval Training can Ameliorate Hypothalamic Appetite Regulation in Male Rats with Type 2 Diabetes: The Role of Leptin.

Disruption of leptin (LEP) signaling in the hypothalamus caused by type 2 diabetes (T2D) can impair appetite regulation. The aim of this study was to investigate whether the improvement in appetite regulation induced by high-intensity interval training (HIIT) in rats with T2D can be mediated by LEP signaling. In this study, 20 male Wister rats were randomly assigned to one of four groups: CO (non-type 2 diabetes control), T2D (type 2 diabetes), EX (non-type 2 diabetes exercise), and T2D + EX (type 2 diabetes + exercise).To induce T2D, a combination of a high-fat diet for 2 months and a single dose of streptozotocin (35 mg/kg) was administered. Rats in the EX and T2D + EX groups performed 4-10 intervals of treadmill running at 80-100% of their maximum velocity (Vmax). Homeostatic Model Assessment for Insulin Resistance (HOMA-IR), serum levels of insulin (INS) and LEP (LEPS) as well as hypothalamic expression of LEP receptors (LEP-R), Janus kinase 2 (JAK-2), signal transducer and activator of transcription 3 (STAT-3), neuropeptide Y (NPY), agouti-related protein (AGRP), pro-opiomelanocortin cocaine (POMC), amphetamine-related transcript (CART), suppressor of cytokine signaling (SOCS3), forkhead box protein O1 (FOXO1) were assessed. ANOVA and Tukey post hoc tests were used to compare the results between the groups. The levels of LEPS and INS, as well as the levels of LEP-R, JAK-2, STAT-3, POMC, and CART in the hypothalamus were found to be higher in the T2D + EX group compared to the T2D group. On the other hand, the levels of HOMA-IR, NPY, AGRP, SOCS3, and FOXO1 were lower in the T2D + EX group compared to the T2D group (P < 0.0001). The findings of this study suggest that HIIT may improve appetite regulation in rats with T2D, and LEP signaling may play a crucial role in this improvement. Graphical abstract (leptin signaling in the hypothalamus), Leptin (LEP), Leptin receptor (LEP-R), Janus kinase 2 (JAK2), Signal transducer and activator of transcription 3 (STAT3), expressing Neuropeptide Y (NPY), Agouti-related protein (AGRP), anorexigenic neurons (expressing pro-opiomelanocortin cocaine (POMC), Amphetamine-related transcript (CART), suppressor of cytokine signaling (SOCS3), forkhead box protein O1 (FOXO1).

[Effect of transcutaneous auricular vagus nerve stimulation on the splenic α7nAchR/JAK2/STAT3 signaling pathway in LPS-induced depressive-like behavior rats].

OBJECTIVE: To observe the effect of transcutaneous auricular vagus nerve stimulation (taVNS) on the improvement of depressive-like behavior and the splenic α7 nicotinic acetylcholine receptor (α7nAchR) / Janus kinase 2 (JAK2 / signal transducer and activator of transcription 3 (STAT3) signaling pathway in lipopolysaccharide (LPS)-induced depressive-like behavior rats, so as to investigate the antidepressant mechanism of taVNS. METHODS: SD rats were randomly divided into SD control group, SD model group and SD taVNS group, and α7nAchR knockout rats were also randomly divided into α7 control group, α7 model group and α7 taVNS group, with 6 rats in each group. Rat model of depressive-like behavior was established by intraperitoneal injection of LPS (1 mg/kg). Rats in both SD taVNS and α7 taVNS groups received taVNS intervention once a day (2 Hz/15 Hz, 2 mA, 30 min) from 7 days before LPS injection to 2 days after LPS injection, respectively. The mean speed, activity time and side immobility time in the open field test were recorded after taVNS. The contents of interleukin 10 (IL-10) and chemokine (C-X-C motif) ligand 1 (CXCL1) in serum were detected by electrochemiluminescence multifactorial method. The splenic phosphorylated (p)-JAK2 and p-STAT3 protein expressions were detected by Western blot. RESULTS: Compared with their respective control groups, the mean speed and active time were reduced (P<0.01, P<0.05, P<0.001) and the side immobility time was increased (P<0.001) in the open field test, serum IL-10 and CXCL1 levels were up-regulated (P<0.01, P<0.05, P<0.001), and splenic p-JAK2 protein expressions were down-regulated (P<0.05, P<0.01) in SD and α7nAchR knockout rats, and splenic p-STAT3 protein expression were down-regulated (P<0.05) in SD rats after LPS injection. Following taVNS intervention and in comparison with the model group , the mean speed and active time were increased (P<0.01) and the side immobility time was decreased (P<0.001) in the open field test, serum IL-10 and CXCL1 levels down-regulated (P<0.05), while splenic p-JAK2 and p-STAT3 protein expressions were up-regulated (P<0.01, P<0.001) in the SD taVNS group rather than in the α7 taVNS group. Compared with SD taVNS group, the α7 taVNS group showed increased (P<0.001, P<0.05) side immobility time in the open field test and serum IL-10, decreased splenic p-JAK2 and p-STAT3 protein expressions (P<0.01, P<0.05). CONCLUSION: taVNS may exert anti-inflammatory effects through modulating the splenic α7nAchR/JAK2/STAT3 signaling pathway, thereby ameliorating LPS-induced depressive-like behavior in rats.

[Effect of Sparganii Rhizoma-Curcumae Rhizoma-medicated serum on proliferation, apoptosis, and migration of human ectopic endometrial stromal cells: based on JAK2/STAT3 signaling pathway].

Based on the Janus kinase 2/signal transducer and activator of transcription 3(JAK2/STAT3) signaling pathway, this study investigated the effect of medicated serum of Sparganii Rhizoma(SR) and Curcumae Rhizoma(CR) on the proliferation, apoptosis, migration, and secretion of inflammatory factors of ectopic endometrial stromal cells(ESCs). Specifically, human ESCs were primary-cultured. The effect of different concentration(5%, 10%, 20%) of SR-, CR-, and SR-CR combination-medicated serum, and AG490 solution(50 µmol·L~(-1)) on the proliferation of ESCs was detected by methyl thiazolyl tetrazolium(MTT) assay, and the optimal dose was selected accordingly for further experiment. The cells were classified into normal serum(NS) group, SR group(10%), CR group(10%), combination(CM) group(10%), and AG490 group. The apoptosis level of ESCs was detected by flow cytometry, and the migration ability was examined by wound healing assay. The secretion of interleukin(IL)-1ß, IL-6, and tumor necrosis factor(TNF)-α was determined by enzyme-linked immunosorbent assay(ELISA). The protein levels of cysteinyl aspartate specific protei-nase-3(caspase-3), B-cell lymphoma(Bcl-2), and Bcl-2-associated X protein(Bax) and the levels of phosphorylated(p)-JAK2 and p-STAT3 were detected by Western blot. The results showed that the viability of ESCs cells was lowered in the administration groups compared with the blank serum group(P<0.01), especially the 10% drug-medicated serum, which was selected for further experiment. The 10% SR-medicated serum, 10% CR-medicated serum, and 10% CM-medicated serum could increase the apoptosis rate(P<0.01), up-regulate the protein expression of caspase-3 and Bax in cells(P<0.05 or P<0.01), down-regulate the expression of Bcl-2(P<0.01), decrease the cell migration rate(P<0.05 or P<0.01), and reduce the secretion levels of IL-1ß, IL-6, and TNF-α(P<0.05 or P<0.01), and levels of p-JAK2 and p-STAT3(P<0.05 or P<0.01). Compared with the SR and CR groups, CM group showed low cell viability(P<0.01), high protein expression of caspase-3 and Bax(P<0.05 or P<0.01), and low protein expression of Bcl-2 and p-JAK2(P<0.05). After incubation with CM, the apoptosis rate was higher(P<0.05) and the migration rate was lower(P<0.01) than that of the CR group. The p-STAT3 protein level of CM group was lower than that of the RS group(P<0.05). The mechanism of SR, CR, and the combination underlying the improvement of endometriosis may be that they blocked JAK2/STAT3 signaling pathway, inhibited ESC proliferation, promoted apoptosis, weakened cell migration, and reduced the secretion of inflammatory factors. The effect of the combination was better than that of RS alone and CR alone.

Fu-Zheng-Tong-Luo formula promotes autophagy and alleviates idiopathic pulmonary fibrosis by controlling the Janus kinase 2/signal transducer and activator of transcription 3 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Fu-Zheng-Tong-Luo (FZTL) formula is a Chinese herbal prescription which is used to treat idiopathic pulmonary fibrosis (IPF). We previously reported that the FZTL formula could improve IPF injury in rats; however, the mechanism remains unelucidated. AIM OF THE STUDY: To elucidate the effects and mechanisms of the FZTL formula on IPF. MATERIALS AND METHODS: The bleomycin-induced pulmonary fibrosis rat model and transforming growth factor-ß-induced lung fibroblast model were used. Histological changes and fibrosis formation were detected in the rat model after treatment with the FZTL formula. Furthermore, the effects of the FZTL formula on autophagy and lung fibroblast activation were determined. Moreover, the mechanism of FZTL was explored using transcriptomics analysis. RESULTS: We observed that FZTL alleviated IPF injury in rats and inhibited inflammatory responses and fibrosis formation in rats. Moreover, it promoted autophagy and inhibited lung fibroblast activation in vitro. Transcriptomics analysis revealed that FZTL regulates the Janus kinase 2 (JAK)/signal transducer and activator of the transcription 3 (STAT) signaling pathway. The JAK2/STAT3 signaling activator interleukin 6 inhibited the anti-fibroblast activation effect of the FZTL formula. Combined treatment with the JAK2 inhibitor (AZD1480) and autophagy inhibitor (3-methyladenine) did not enhance the antifibrotic effect of FZTL. CONCLUSIONS: The FZTL formula can inhibit IPF injury and lung fibroblast activation. Its effects are mediated via the JAK2/STAT3 signaling pathway. The FZTL formula may be a potential complementary therapy for pulmonary fibrosis.

Zi-Su-Zi decoction improves airway hyperresponsiveness in cough-variant asthma rat model through PI3K/AKT1/mTOR, JAK2/STAT3 and HIF-1α/NF-κB signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Cough-variant asthma (CVA) is one of the most common causes of chronic cough. Its pathogenesis is closely related to chronic airway inflammation and airway hyperresponsiveness. CVA belongs to the category of "wind cough" in Traditional Chinese medicine (TCM). Zi-Su-Zi decoction (ZSD) is a Chinese herbal formula that is clinically used for the treatment of cough and asthma, especially CVA. However, the mechanism of action remains unclear. AIM OF THE STUDY: In this study, we aimed to explore the potential mechanism by which ZSD improves CVA airway hyperresponsiveness. MATERIALS AND METHODS: The targets of ZSD in CVA were studied using a Network pharmacology. The main chemical components of ZSD were detected and analyzed using ultra-high-pressure liquid chromatography (UHPLC-MS/MS). In animal experiments, the rat model of CVA was established using Ovalbumin (OVA)/Aluminum hydroxide (AL(OH)3) sensitization. Moreover, the experiment also evaluated cough symptoms, percentage of eosinophils (EOS%), pulmonary function tests, histopathological sections, blood cytokine levels, mRNA and protein levels. RESULTS: The results showed that Network pharmacology suggested 276 targets of ZSD and CVA and found that ZSD treatment with CVA was closely related to the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway. UHPLC-MS/MS revealed that ZSD contained 52 main chemical components. Compared with the model group, the cough symptoms of the rats in the different ZSD concentration groups were relieved, the EOS% index was lowered, and body weight was increased. HE staining showed that ZSD reduced airway inflammation, edema and hyperplasia, thereby improving the pathological structure of lung tissue, and the effect of high-dose ZSD was especially significant. Our most important finding was that ZSD blocked the entry of hypoxia-inducible factor-1α (HIF-1α), signal transducer and activator of transcription-3 (STAT3) and nuclear factor kappa-B (NF-κB) into the nucleus by interfering with PI3K/AKT1/mechanistic target of rapamycin (mTOR), and janus kinase 2 (JAK2) signaling factors. Consequently, inhibiting the release of cytokines and immunoglobulin-E, thereby reducing airway hyperresponsiveness (AHR) and partially reverses airway remodeling. CONCLUSIONS: This study showed that ZSD can improve airway hyperresponsiveness and partially reverse airway remodeling by inhibiting the PI3K/AKT1/mTOR, JAK2/STAT3 and HIF-1α/NF-κB signaling pathways. Therefore, ZSD is an effective prescription for the treatment of CVA.