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Background/Aims: Evidence suggests that IL-6 affects bone mass by modulating osteocyte communication towards osteoclasts. However, the mechanism by which IL-6 enhances osteocyte-mediated osteoclastogenesis is unclear. We aimed to investigate the inflammatory factors in serum after orthodontic surgery and their relationship between osteocytes and osteoclasts. Methods: Serum was obtained from 10 orthognathic surgery patients, and inflammatory factors were detected by ELISA. We treated the osteocyte-like cell line MLO-Y4 with recombinant mouse IL-6 and IL-6 receptor (IL-6R), and used quantitative RT-PCR and Western blotting to explore Receptor activator of nuclear factor-κB ligand (RANKL) expression at both the mRNA and protein level. MLO-Y4 cells were co-cultured with osteoclast precursor cells, and the formation of osteoclasts was detected by tartrate-resistant acid phosphatase (TRAP) staining. To explore the role of JAK2 in the osteocyte-mediated osteoclastogenesis, AG490, a JAK2 inhibitor, was used to inhibit the JAK2-STAT3 pathway in osteocytes. Results: In our study, we found that IL-6 and RANKL were stimulated in serum 3-7 days after orthognathic surgery. Therefore, IL-6 and IL-6 receptor enhanced the expression of RANKL at both the mRNA and protein level in MLO-Y4. Furthermore, when MLO-Y4 cells were co-cultured with osteoclast precursor cells, it significantly stimulated osteoclastogenesis. Our study indicated that osteocytes could promote osteoclastic differentiation and the formation of TRAP-positive multinucleated cells after stimulation with IL-6 and IL-6R. Our results also indicated that treatment with IL-6 and IL-6R increased RANKL mRNA expression and the RANKL/OPG expression ratio. Meanwhile, the phosphorylation of Janus kinase 2 (JAK2) and Signal transducer and activator of transcription (STAT3) also correlated with RANKL levels. Furthermore, we investigated the effects of a specific JAK2 inhibitor, AG490, on the expression of RANKL in osteocyte-like MLO-Y4 cells and osteocyte-mediated osteoclastogenesis. The results showed that AG490 inhibited (p)-JAK2 and RANKL expression. Osteoclastic differentiation was decreased after pretreatment in MLO-Y4 with mouse IL-6/IL-6R and AG490; therefore, we concluded that IL-6 increased osteocyte-mediated osteoclastic differentiation by activating JAK2 and RANKL. Conclusion: The effects of IL-6/il-6R and AG490 on osteocyte-mediated osteoclastogenesis contribute to our understanding of the role of inflammatory factors in the interaction between osteocytes and osteoclast precursors. IL-6 and RANKL are key factors for bone remodelling after the orthodontic surgery, and their roles in bone remodelling may be fundamental mechanisms accelerating tooth movement by orthodontic surgery.

AG490 is a selective inhibitor of the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway. The present study examined its effects on the abnormal behavior of human keloid fibroblasts (HKFs) and evaluated its potential use in the treatment of keloids. Human normal fibroblasts (HNFs) and HKFs were treated with increasing concentrations of AG490. The proliferation of HNFs and HKFs was inhibited by AG490 in both a time- and concentration-dependent manner by increasing apoptosis and inducing G1 cell cycle arrest. The down-regulation of cyclin D1 and connective tissue growth factor (CTGF) expression was associated with a decrease in STAT3 expression in response to AG490. The effects of AG490 on TGF-β-stimulated fibroblasts, including HNFs, HKFs and hypertrophic scar fibroblasts (HSFs) were also evaluated. The TGF-β1-stimulated excessive proliferation and CTGF production were markedly inhibited by the application of AG490 in the HNFs, HSFs and HKFs. In addition, the STAT3-specific decoy oligodeoxynucleotides (SODNs) were transfected into HKFs. The invasive ability of the SODN-transfected HKFs was determined and the expression of extracellular matrix components was quantified. Similarly, SODNs blocked the constitutive activation of STAT3. SODNs inhibited the invasion and progression of HKFs, possibly via the upregulation of the expression of tissue inhibitor of metalloproteinase-2 (TIMP-2), and the downregulation of the expression of matrix metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF). On the whole, the findings of the present study demonstrate that STAT3-specific elimination, such as the application of AG490 and decoy ODNs, may serve as promising therapeutic strategies for the treatment of keloids.

Aim： Berberine （BBR）, an isoquinoline-derived alkaloid isolated from Rhizoma coptidis, exerts cardioprotective effects. Because endoplasmic reticulum （ER） stress plays a pivotal role in myocardial ischemia/reperfusion （MI/R）-induced apoptosis, it was interesting to examine whether the protective effects of BBR resulted from modulating ER stress levels during MI/R injury, and to define the signaling mechanisms in this process. Methods： Male rats were treated with BBR （200 mg.k-1.d-1, ig） for 2 weeks, and then subjected to MI/R surgery. Cardiac dimensions and function were assessed using echocardiography. Myocardial infarct size and apoptosis was examined. Total serum LDH levels and CK activities, superoxide production, MDA levels and the antioxidant SOD activities in heart tissue were determined. An in vitro study was performed on cultured rat embryonic myocardium-derived cells H9C2 exposed to simulated ischemia/reperfusion （SIR）. The expression of apoptotic, ER stress-related and signaling proteins were assessed using Western blot analyses.Results： Pretreatment with BBR significantly reduced MI/R-induced myocardial infarct size, improved cardiac function, and suppressed myocardial apoptosis and oxidative damage. Furthermore, pretreatment with BBR suppressed MI/R-induced ER stress, evidenced by down-regulating the phosphorylation levels of myocardial PERK and elF2a and the expression of ATF4 and CHOP in heart tissues. Pretreatment with BBR also activated the JAK2/STAT3 signaling pathway in heart tissues, and co-treatment with AG490, a specific JAK2/STAT3 inhibitor, blocked not only the protective effects of BBR, but also the inhibition of BBR on MI/R-induced ER stress. In H9C2 cells, treatment with BBR （50 pmol/L） markedly reduced SIR-induced cell apoptosis, oxidative stress and ER stress, which were abolished by transfection with JAK2 siRNA. Conclusion： BBR ameliorates MI/R injury in rats by activating the AK2/STAT3 signaling pathway and attenuating ER stress-induced apoptosis.

Vasculogenic mimicry refers to the process by which highly invasive cancer cells mimic endothelial cells by forming blood channels. Vasculogenic mimicry is important for the invasion and metastasis of tumor cells in colorectal cancer. STAT3 was initially identified as a mediator of the inflammation-associated acute phase response. The phosphorylation of Signal Transducers and Activators of Transcription 3 (p-STAT3) is closely related to tumor invasion and migration. We analyzed the relationship between p-STAT3 and vasculogenic mimicry formation in 65 human colorectal cancer samples, and the results showed that the expression of p-STAT3 is significantly correlated with vasculogenic mimicry, tumor metastasis, Tumor, Lymph Node and Metastasis Stage (TNM Stage), and poor prognosis. It is known that interleukin 6 can induce the phosphorylation of STAT3. We found that using interleukin 6 to induce p-STAT3 activation in colorectal cancer cell lines can result in vasculogenic mimicry and using AG490 to suppress p-STAT3 activation restrained vasculogenic mimicry. Furthermore, the state of p-STAT3 activation can affect epithelial-to-mesenchymal transition. By immunofluorescence double staining, we discovered that p-STAT3 expression is more directly correlated with the epithelial-to-mesenchymal transition marker vimentin than with the vasculogenic mimicry-related protein VE-cadherin. These data show that activated p-STAT3 upregulates epithelial-to-mesenchymal transition–related proteins and promotes vasculogenic mimicry.

Saikosaponin-d (SSd) is an active extract from , the dried root from the plant used in China for thousands of years to treat liver diseases. The SSd extract possesses valuable pharmacological activities including anti-cancer and anti-inflammatory effects; however, the mechanism underlying the anti-cancer activity of SSd is largely unknown. Here, we explored the mechanism of action of SSd as an anti-cancer agent for liver cancer in two human hepatocellular carcinoma cell lines. Using MTT and annexin-V-FITC/PI assays, Western blots, immunohistochemistry, qRT-PCR, luciferase reporter assay, and a JAK2-specific inhibitor (AG490), we demonstrated that the anti-tumorigenic effects of SSd act through the intermediatory p-STAT3/C/EBPβ signaling pathway to suppress cyclooxygenase (COX)-2. SSd effectively inhibited cell proliferation in a dose-dependent manner. Apoptosis was significantly increased in cells treated with SSd (2.5-15 µg/ml) with concurrent increase and decrease in pro- and anti-apoptosis proteins, respectively. COX-2, C/EBPβ, and p-STAT3 were significantly decreased, at both the translational and transcriptional levels, by SSd treatment. AG490 produced similar inhibitory effects on STAT3, p-STAT3, C/EBPβ, and COX-2. In conclusion, our data suggest that SSd controls liver cancer proliferation through suppression of the p-STAT3/C/EBPβ signaling pathway inhibiting COX2 expression. These findings further our understanding of the pharmacological action of SSd, providing new information on SSd mechanism of action and showing potential for SSd as a novel therapy for liver cancer.

Diminished regeneration or healing capacity of tendon occurs during aging. It has been well demonstrated that tendon stem/progenitor cells (TSPCs) play a vital role in tendon maintenance and repair. Here, we identified an accumulation of senescent TSPCs in tendon tissue with aging. In aged TSPCs, the activity of JAK-STAT signaling pathway was increased. Besides, genetic knockdown of JAK2 or STAT3 significantly attenuated TSPC senescence in aged TSPCs. Pharmacological inhibition of JAK-STAT signaling pathway with AG490 similarly attenuated cellular senescence and senescence-associated secretory phenotype (SASP) of aged TSPCs. In addition, inhibition of JAK-STAT signaling pathway also restored the age-related dysfunctions of TSPCs, including self-renewal, migration, actin dynamics, and stemness. Together, our findings reveal the critical role of JAK-STAT signaling pathway in the regulation of TSPC aging and suggest an ideal therapeutic target for the age-related tendon disorders.

Parkinson’s disease (PD) is characterized by motor impairment and dopaminergic neuronal loss. There is no cure for the disease, and treatments have several limitations. The transient receptor potential melastatin 2 (TRPM2), a calcium-permeable non-selective cation channel, has been reported to be upregulated in neuronal death. However, there are no in vivo studies evaluating TRPM2’s role and neuroprotective effects in PD. Here, we test the hypothesis that TRPM2 is upregulated in the 6-hydroxydopamine (6-OHDA) mouse model of PD and that its inhibition, by the AG490, is neuroprotective. For that, AG490 or vehicle were intraperitoneally administered into C57BL/6 mice. Mice then received 6-OHDA into the right striatum. Motor behavior assessments were evaluated 6, 13, and 20 days after surgery using the cylinder and apomorphine-induced rotational testes, and 7, 14, and 21 days after surgery using rotarod test. Brain samples of substantia nigra (SNc) and striatum (CPu) were collected for immunohistochemistry and immunoblotting on days 7 and 21. We showed that TRPM2 protein expression was upregulated in 6-OHDA-treated animals. In addition, AG490 prevented dopaminergic neuron loss, microglial activation, and astrocyte reactivity in 6-OHDA-treated animals. The compound improved motor behaviors and Akt/GSK-3β/caspase-3 signaling. We conclude that TRPM2 inhibition by AG490 is neuroprotective in the 6-OHDA model and that the TRPM2 channel may represent a potential therapeutic target for PD.

Background The cell division cycle associated 4 (CDCA4) plays a crucial role in various biological processes and is implicated in the progression of several tumors, however, the mechanisms by which it operates in bladder cancer remain unclear. Methods Utilizing data from the TCGA and GEO datasets of bladder cancer patients, we analyzed the expression of CDCA4 and its prognostic significance. We then constructed stable overexpression and knockdown bladder cancer cell lines to investigate the effects of CDCA4 on cell proliferation, migration, and invasion in vitro, employing CCK-8, colony formation, transwell, and wound healing assays. Additionally, we validated the potential downstream pathways of CDCA4 through data analysis and western blot assays. Results Our study found that CDCA4 expression is elevated in bladder cancer cells and correlates with poor prognosis in patients. Inhibition of CDCA4 expression reduces the proliferation, migration, and invasion of bladder cancer cells, as well as inhibit the epithelial-mesenchymal transition (EMT) process. Conversely, promoting CDCA4 expression enhances the malignancy of bladder cancer cells. Investigation into the mechanism of CDCA4 revealed that it promotes bladder cancer progression by activating the JAK/STAT signaling pathway, and the JAK inhibitor AG490 can reverse the promoting effects of CDCA4. Conclusion Our findings suggest that CDCA4 enhances the proliferation, migration, and invasion of bladder cancer cells by positively regulating the JAK/STAT signaling pathway, indicating that CDCA4 may serve as a novel molecular target for bladder cancer treatment.

NSCLC (non-small cell lung cancer) is a leading cause of cancer-related deaths worldwide. Clinical trials showed that Hiltonol, a stable dsRNA representing an advanced form of polyI:C (polyinosinic-polycytidilic acid), is an adjuvant cancer-immunomodulator. However, its mechanisms of action and effect on lung cancer have not been explored pre-clinically. Here, we examined, for the first time, how a novel Hiltonol cocktail kills NSCLC cells. By retrospective analysis of NSCLC patient tissues obtained from the tumor biobank; pre-clinical studies with Hiltonol alone or Hiltonol+++ cocktail [Hiltonol+anti-IL6+AG490 (JAK2 inhibitor)+Stattic (STAT3 inhibitor)]; cytokine analysis; gene knockdown and gain/loss-of-function studies, we uncovered the mechanisms of action of Hiltonol+++. We demonstrated that Hiltonol+++ kills the cancer cells and suppresses the metastatic potential of NSCLC through: (i) upregulation of pro-apoptotic Caspase-9 and Caspase-3, (ii) induction of cytosolic cytochrome c, (iii) modulation of pro-inflammatory cytokines (GRO, MCP-1, IL-8, and IL-6) and anticancer IL-24 in NSCLC subtypes, and (iv) upregulation of tumor suppressors, PKR (protein kinase R) and OAS (2′5′ oligoadenylate synthetase). In silico analysis showed that Lys296 of PKR and Lys66 of OAS interact with Hiltonol. These Lys residues are purportedly involved in the catalytic/signaling activity of the tumor suppressors. Furthermore, knockdown of PKR/OAS abrogated the anticancer action of Hiltonol, provoking survival of cancer cells. Ex vivo analysis of NSCLC patient tissues corroborated that loss of PKR and OAS is associated with cancer advancement. Altogether, our findings unraveled the significance of studying tumor biobank tissues, which suggests PKR and OAS as precision oncological suppressor candidates to be targeted by this novel Hiltonol+++ cocktail which represents a prospective drug for development into a potent and tailored therapy for NSCLC subtypes.

Background Cerebral ischemia/reperfusion injury is a severe problem in patients with brain ischemia. Brain injury caused by the immune response is important in the pathogenesis of cerebral ischemia/reperfusion injury and immune pathways. It is important to investigate potential targets for the treatment of cerebral ischemia/reperfusion injury. Methods In this experiment, we evaluated the effect of an exogenous JAK antagonist AG490 in the cerebral ischemia/reperfusion injury model, which was established by middle cerebral artery occlusion (MCAO). Histology study, TUNEL staining, Western blot, and RT‐PCR were employed to examine the effects of AG490 in cerebral ischemia/reperfusion injury. Results In the brain tissue of MCAO mice, JAK2 was highly expressed. AG490 is an inhibitor of JAK2, which reduced the phosphorylation level of JAK2. AG490 downregulated the phosphorylated activation of JAK3 and their downstream STAT3. The antiapoptotic activity of AG490 on cerebral ischemia/reperfusion injury mice was consistent with in vitro data. It reduced the phosphorylation of JAK2/JAK3/STAT3 and the apoptosis rate in cultured neurons upon apoptosis induction. Besides, we also observed the neuroprotective effects of AG490 on cerebral ischemia/reperfusion injury. Administration of AG490 could further enhance the expression of neurotrophins including BNDF, NT3, and the neurotrophin receptor TrkB. Conclusion Therefore, AG490 is pluripotent for cerebral ischemia/reperfusion injury through both antiapoptosis and neuroprotective activities. The antiapoptosis effect is dependent on its regulation of the JAK‐STAT pathway. AG490 injection could attenuate the lesions indicated by histology study. AG490 exhibited antiapoptotic activity indicated by TUNEL staining. AG490 antiapoptotic activity on CIRI rats was consistent with in vitro data, reducing the phosphorylation of JAK2/JAK3/STAT3 and the apoptotic rate in cultured neuron upon apoptosis induction.