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.

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.

[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.

Chinese chestnut shell polyphenol extract regulates the JAK2/STAT3 pathway to alleviate high-fat diet-induced, leptin-resistant obesity in mice.

Chinese chestnut shell is a by-product of chestnut food processing and is rich in polyphenols. This study sought to investigate the effect of chestnut shell polyphenol extract (CSP) on weight loss and lipid reduction in a 12-week high-fat diet (HFD)-induced murine obesity model. CSP (300 mg per kg body weight) was administered intragastrically daily. AG490, a JAK2 protein tyrosine kinase inhibitor, was also intraperitoneally injected. The results showed that an HFD induced leptin resistance (LR). Compared to corresponding values in the HFD group, CSP treatment improved blood lipid levels, weight, and leptin levels in obese mice (p < 0.01). Additionally, CSP treatment enhanced enzyme activity by improving total antioxidant capacity, attenuating oxidative stress, and reducing fat droplet accumulation and inflammation in the liver, epididymal, and retroperitoneal adipose tissue. CSP also activated the LEPR-JAK2/STAT3-PTP1B-SOCS-3 signal transduction pathway in hypothalamus tissue and improved LR while regulating the expression of proteins related to lipid metabolism (PPARγ, FAS, and LPL) in white adipose tissue in the retroperitoneal cavity. However, the amelioration of lipid metabolism by CSP was dependent on JAK2. Molecular docking simulation further demonstrated the strong binding affinity of procyanidin C1 (-10.3983297 kcal mol-1) and procyanidin B1 (-9.12686729 kcal mol-1) to the crystal structure of JAK2. These results suggest that CSP may be used to reduce HFD-induced obesity with potential application as a functional food additive.

[Jingfang Mixture regulates balance of spleen T lymphocyte subsets in urticaria mice by inhibiting JAK2-STAT3 signaling pathway].

Urticaria is an immune-mediated allergic disease. This study explored the effect of Jingfang Mixture on spleen T lymphocyte subsets of urticaria mice. A total of 50 Kunming mice were randomized into normal group(C), model group(V), and low-(JF-L, 0.5 g·kg~(-1)), medium-(JF-M, 1 g·kg~(-1)) and high-dose(JF-H, 2 g·kg~(-1)) Jingfang Mixture groups, with 10 mice in each group. The mixture of ovalbumin and aluminum hydroxide(0.1 mg + 0.1 mL) was used(intraperitoneal injection) to induce urticaria in mice. The administration began 6 days after the first immunization, and the second immunization was carried out 10 days after the first immunization. The pruritus index was detected within 30 min after the second immunization. The administration lasted 21 days. After 21 days, the serum was taken to detect the total IgE level. Based on hematoxylin and eosin(HE) staining, the pathological changes of skin tissue were observed, and Western blot was used to detect the levels of p-Janus kinase 2(JAK2)/JAK2 and p-signal transducer and activator of transcription 3(STAT3)/STAT3 in skin tissue. The spleen was taken to detect the spleen index, and flow cytometry was employed to determine the expression of lymphocyte subsets. The results showed that group V had obvious pathological changes in skin tissue compared with group C. Moreover, group V showed more scratches, higher spleen index, and higher level of total serum IgE than group C. In addition, higher levels of p-JAK2 and p-STAT3, lower proportions of CD4~+T, Th1, and Treg, higher proportions of CD8~+T, Th2, and Th17, and lower ratios of CD4~+/CD8~+, Th1/Th2, and Terg/Th17 were observed in group V than in group C. Compared with group V, each administration group showed alleviation of the pathological morphology of skin tissue, obvious epidermal thickening, relatively intact collagen fiber structure of dermal reticular layer, alleviated edema, and relief of vasodilation and peripheral inflammatory cell infiltration. Moreover, less scratching, lower spleen index, lower p-JAK2/JAK2 and p-STAT3/STAT3 were observed in the administration groups than in group V. JF-M group and JF-H group demonstrated lower levels of total IgE, larger proportions of CD4~+T, Th1, and Treg, smaller proportions of CD8~+ T, Th2, and Th17, and higher ratios of CD4~+/CD8~+, Th1/Th2, and Terg/Th17. In conclusion, Jingfang Mixture may improve the symptoms of urticaria mice by regulating the balance of spleen T lymphocyte subsets through JAK2-STAT3 signaling pathway.

[Therapeutic effect of Shengjiang Powder on experimental autoimmune encephalomyelitis mice through IL-6/JAK2/STAT3 signaling pathway:based on HPLC].

A high performance liquid chromatography(HPLC) method was established to analyze the components in Shengjiang Powder(SJP) such as emodin and curcumin and explore its therapeutic effect on experimental autoimmune encephalomyelitis(EAE) mice. To be specific, HPLC was performed to determine the content of compounds in SJP such as emodin and curcumin. A total of 72 female SPF C57 BL/6 mice were randomized into control group(equivalent volume of ultrapure water, ig), model group(equivalent volume of ultrapure water, ig), low-, medium-, and high-dose SJP groups(SJP, ig), and positive control group(prednisone acetate, ig), 12 each group. EAE was induced in mice except the control group. Administration began from the first day after immunization. The general conditions, symptom score, and body weight of the mice were recorded. On the 21 st day, mouse brain tissues were separrated. Then hematoxylin-eosin(HE) staining and Luxol Fast Blue(LFB) staining were used to detect the pathological changes of brain tissues. Immunohistochemistry(IHC) was employed to determine the myelin basic protein(MBP) level, and Western blot the expression of occludin and claudin-5, as well as the levels of interleukin-6(IL-6) and proteins in the Janus kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3) pathway and their phosphorylation levels. The mRNA expression of IL-6, JAK2, and STAT3 was detected by real-time quantitative polymerase chain reaction(qPCR). Finally, molecular docking of six main active components in SJP, including emodin and curcumin, with IL-6, JAK2 and STAT3 was performed, and the binding affinity was evaluated. The results showed that the established HPLC method demonstrated high precision, reproducibility, stability, and high recovery of samples. Compared with the model group, SJP reduced the clinical symptom score and alleviate the inflammatory infiltration of brain white matter and demyelination of EAE mice. At the same time, SJP increased the expression of occludin and claudin-5, down-regulated the mRNA expression of IL-6, JAK2, and STAT3, as well as the levels of IL-6/JAK/STAT3 proteins and the phosphorylation levels, with significant difference. Molecular docking suggested that the six active components in SJP had high binding energy with IL-6, JAK2, and STAT3 proteins. The established HPLC method is simple, accurate, and highly sensitive, which can simultaneously determine the content of emodin and curcumin in SJP. SJP may alleviate the clinical symptoms of EAE by inhibiting IL-6/JAK2/STAT3 signaling pathway, protecting the blood-brain barrier, and relieving the inflammatory response and demyelinization of brain tissue.

RAPID LOW-COST DETECTION OF TYPE 2CALR MUTATION BY ALLELE-SPECIFIC RT-PCR FOR DIAGNOSIS OF MYELOPROLIFERATIVE NEOPLASMS.

BACKGROUND: Approximately 15% to 24% of essential thrombocythemia (ET) and 25-35% of primary myelofibrosis cases carry a mutation in the calreticulin (CALR) gene. Sanger sequencing, qPCR, high resolution melt or targeted next generation sequencing usually used to detect these mutations are expensive and require costly equipment. Nevertheless, type 1 CALR mutations are detectable by using polymerase chain reaction (PCR) and agarose gel electrophoresis. AIM: To offer the use of the allele-specific reverse transcription (RT) PCR for rapid low-cost detection of the type 2 mutation in the CALR gene. MATERIALS AND METHODS: Allele-specific primers designed for detecting type 2 mutation (5-bp insertion; c.1154_1155 ins TTGTC) of the CALR gene were used for allele-specific RT-PCR analysis of cDNA of the patient with JAK2-, MPL-negative ET, whose mutation in CALR gene has been identified by Sanger sequencing. RT-PCR samples were analyzed by agarose gel electrophoresis. RESULTS: The type 2 mutation (K385fs*47 ins5) in CALR gene was detected by Sanger sequencing in JAK2- and MPL-negative ET patient. The cDNA obtained was then re-analyzed by using allele-specific RT-PCR with newly designed primers. Normal and type 2 mutation alleles of the CALR gene were detected by gel electrophoresis. The results of allele-specific RT-PCR were consistent with the data of Sanger sequencing. CONCLUSION: Allele-specific RT-PCR analysis may be used for the fast low-cost detection of the major type 2 mutation (ins 5) of the CALR gene in patients with MPNs.

Curcumol Attenuates Endometriosis by Inhibiting the JAK2/STAT3 Signaling Pathway.

BACKGROUND Curcumol is a hydrogenated austenitic compound with hemiketal. In this study we evaluated the effects of curcumol on local inflammatory response, cell proliferation, and metastasis in endometriosis, and elucidated the underlying mechanisms. MATERIAL AND METHODS Ectopic endometrial stromal cells were treated with increasing doses of curcumol. The MTT assay was used to assess cell viability. FITC-labeled annexin-V/PI double-staining method and flow cytometry were used to determine cell apoptosis. Cell migration was evaluated using a wound healing assay. ELISA kits were used to detect the levels of TNF-alpha, IL-6, and IL-1ß. Western blot assay was used to examine the phosphorylation degree of JAK2 and STAT3 and the expression of Bax, Bcl2, and caspase-3 proteins. Autologous endometrial transplantation was used to establish a rat model to assess the anti-EMS effect of curcumol in vivo. RESULTS Curcumol can inhibit the proliferation of ectopic endometrial stromal cells, promote cell apoptosis, and weaken cell migration ability. Curcumol can reduce the expression of Bax and caspase-3 protein and increase the expression of Bcl2 protein. Curcumol also can inhibit the secretion of inflammatory cytokines, including tumor necrosis cytokines (TNF)-alpha, interleukin (IL)-6, and IL-1ß, by ectopic endometrial stromal cells. In addition, curcumol can also inhibit the phosphorylation of JAK2 and STAT3. In vivo experiments also proved that curcumol could inhibit the growth of ectopic lesions in EMS model rats. CONCLUSIONS Curcumol can inhibit the JAK2/STAT3 pathway, reduce the inflammatory cytokines secreted by ectopic endometrial stromal cells, inhibit cell proliferation and migration, and reduce the volume of ectopic lesions.

Bu-Shen-Zhu-Yun decoction induces PRLR deubiquitination and JAK2/STAT5 activation via CSN5 in vitro.

PURPOSE: To determine the effect of Bu-Shen-Zhu-Yun Decoction (BSZY-D) on the kisspeptin through JAK2/STAT5 signaling pathway in hyperprolactinemia (HPRL) infertility. METHOD: SD rats were treated with BSZY-D for cerebrospinal fluid (CSF) extraction. GT1-7 cells were subjected to different treatments. The phosphorylation levels of JAK2 and STAT5, and the expressions of PRLR and kisspeptin of GT1-7 cells in different groups were detected by western blot, RT-qPCR and immunofluorescence. The expressions of CSN5 and GATA1 and other molecular features were checked by western blot, RT-PCR, co-immunoprecipitation and renilla luciferase activity. RESULTS: The phosphorylation levels of JAK2 and STAT5, and the expressions of PRLR and kisspeptin in the HPRL group were significantly decreased, and these changes could be reversed after BSZY-D treatment. In addition, the presence of PRLR deubiquitination was detected in the HPRL group, which could be reversed by shRNA-CSN5, suggesting that BSZY-D played a role through targeting CSN5. The binding level of GATA1 and CSN5 promoter in the HPRL group was significantly decreased, but elevated in the HPRL (BSZY-D/CSF) group (P < 0.05). CONCLUSION: BSZY-D improved the transcription activity of GATA1 and increased the binding of GATA1 and CSN5. BSZY-D was involved in the deubiquitination of PRLR, which contributes to alleviating the symptoms of HPRL infertility.

Inferring the dynamics of mutated hematopoietic stem and progenitor cells induced by IFNα in myeloproliferative neoplasms.

Classical BCR-ABL-negative myeloproliferative neoplasms (MPNs) are clonal disorders of hematopoietic stem cells (HSCs) caused mainly by recurrent mutations in genes encoding JAK2 (JAK2), calreticulin (CALR), or the thrombopoietin receptor (MPL). Interferon α (IFNα) has demonstrated some efficacy in inducing molecular remission in MPNs. To determine factors that influence molecular response rate, we evaluated the long-term molecular efficacy of IFNα in patients with MPN by monitoring the fate of cells carrying driver mutations in a prospective observational and longitudinal study of 48 patients over more than 5 years. We measured the clonal architecture of early and late hematopoietic progenitors (84 845 measurements) and the global variant allele frequency in mature cells (409 measurements) several times per year. Using mathematical modeling and hierarchical Bayesian inference, we further inferred the dynamics of IFNα-targeted mutated HSCs. Our data support the hypothesis that IFNα targets JAK2V617F HSCs by inducing their exit from quiescence and differentiation into progenitors. Our observations indicate that treatment efficacy is higher in homozygous than heterozygous JAK2V617F HSCs and increases with high IFNα dose in heterozygous JAK2V617F HSCs. We also found that the molecular responses of CALRm HSCs to IFNα were heterogeneous, varying between type 1 and type 2 CALRm, and a high dose of IFNα correlates with worse outcomes. Our work indicates that the long-term molecular efficacy of IFNα implies an HSC exhaustion mechanism and depends on both the driver mutation type and IFNα dose.

Natural flavonol fisetin attenuated hyperuricemic nephropathy via inhibiting IL-6/JAK2/STAT3 and TGF-ß/SMAD3 signaling.

BACKGROUND: The naturally occurring flavonol fisetin (3,3',4',7-tetrahydroxyflavone), widely dispersed in fruits, vegetables and nuts, has been reported to exert anti-inflammatory, antioxidant and anti-angiogenic effects. Our previous study indicated fisetin ameliorated inflammation and apoptosis in septic kidneys. However, the potential nephroprotective effect of fisetin in hyperuricemic mice remains unknown. PURPOSE: The current study was designed to investigate the effect of fisetin on hyperuricemic nephropathy (HN) and explore the underlying mechanisms. METHODS: The HN was induced in mice by mixing of potassium oxonate (2400 mg/kg) and adenine (160 mg/kg) in male C57BL/6J mice. Fisetin (50 or 100 mg/kg) was orally administrated either simultaneously with the establishment of HN or after HN was induced. As a positive control, allopurinol of 10 mg/kg was included. Uric acid levels in the serum and urine as well as renal function parameters were measured. Renal histological changes were measured by periodic acid-Schiff (PAS) and Masson's trichrome stainings. The expression of gene/protein in relation to inflammation, fibrosis, and uric acid excretion in the kidneys of HN mice or uric acid-treated mouse tubular epithelial (TCMK-1) cells were measured by RNA-seq, RT-PCR, western blot and immunohistochemical analysis. RESULTS: Treatment with fisetin, regardless of administration regimen, dose-dependently attenuated hyperuricemia-induced kidney injury as indicated by the improved renal function, preserved tissue architecture, and decreased urinary albumin-to-creatinine ratio. Additionally, fisetin lowered uricemia by modulating the expression of kidney urate transporters including urate transporter 1(URAT1), organic anion transporter 1 (OAT1), organic anion transporter 3 (OAT3) and ATP binding cassette subfamily G member 2 (ABCG2). Moreover, hyperuricemia-induced secretions of proinflammatory factors including tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6) and monocyte chemoattractant protein-1(MCP-1) in HN mice and uric acid-stimulated TCMK-1 cells were mitigated by fisetin treatment. Meanwhile, fisetin attenuated kidney fibrosis in HN mice with restored expressions of alpha-smooth muscle actin (α-SMA), collagen I and fibronectin. Mechanistically, fisetin regulated the aberrant activation of signal transducer and activator of transcription-3 (STAT3) signaling and transforming growth factor-ß (TGF-ß) signaling in the HN kidneys and uric acid-stimulated TCMK-1 cells. CONCLUSION: Fisetin lowered uricemia, suppressed renal inflammatory response, and improved kidney fibrosis to protect against hyperuricemic nephropathy via modulation of STAT3 and TGF-ß signaling pathways. The results highlighted that fisetin might represent a potential therapeutic strategy against hyperuricemic nephropathy.

Phlorizin from sweet tea inhibits the progress of esophageal cancer by antagonizing the JAK2/STAT3 signaling pathway.

Phlorizin, an important member of the dihydrochalcone family, has been widely used as a Chinese Traditional Medicine for treatment of numerous diseases. The present study aimed to investigate the potential therapeutic effects of phlorizin on esophageal cancer. Phlorizin, extracted from sweet tea, was used to treat esophageal cancer cells. Cell proliferation, migration and invasion were determined using Cell Counting Kit­8 and colony formation assays, and wound healing and Transwell assays, respectively. RNA sequencing and bioinformatics analysis was used to investigate the potential mechanism of phlorizin in the development of esophageal cancer. Fluorescent staining and flow cytometry was used to measure the level of apoptosis. The expression level of the proteins, P62/SQSTM1 and LC3 Ð†/II, and the effect of phlorizin on the JAK2/STAT3 signaling pathway was detected using western blot analysis. The results demonstrated that phlorizin could inhibit cell proliferation, migration and invasion. Bioinformatics analysis showed that phlorizin might be involved in pleiotropic effects, such as the 'JAK/STAT signaling pathway' (hsa04630), 'MAPK signaling pathway'(hsa04010) and 'apoptosis' (hsa04210). It was also confirmed that phlorizin promoted apoptosis and inhibited autophagy in the esophageal cancer cells. Notably, phlorizin might inhibit the proteins in the JAK/STAT signaling pathway, which would affect cancer cells. Taken together, the present data showed that phlorizin inhibited the progression of esophageal cancer by antagonizing the JAK2/STAT3 signaling pathway.

Shengxuening Extracted from Silkworm Excrement Mitigates Myelosuppression via SCF-Mediated JAK2/STAT3 Signaling.

Shengxuening (SXN) is a Chinese patent medicine with main ingredients (including chlorophyll derivatives and sodium iron chlorophyllin) extracted from silkworm excrement. SXN exhibited efficacy in clinical trials of renal anemia and iron deficiency anemia; however, the specific mechanisms remain unclear. This study found that SXN increased the number of peripheral blood cells and improved the bone marrow morphology in myelosuppressed mouse model, reversed the reduction in body weight and spleen indices, and increased the serum levels of erythropoietin and granulocyte-macrophage colony-stimulating factor. Quantitative real-time PCR array and Western blot analysis showed the enhanced expression of stem cell factor (SCF), JAK2, and STAT3 in the liver. These results suggested that SXN promoted the recovery of hemopoietic function in myelosuppressed models by increasing the secretion of hematopoietic factors and activating the JAK2/STAT3 pathway. Therefore, this medicine may be applied as therapeutic pharmaceutical drug to mitigate myelosuppression.

Blockage of the JAK/STAT3 signaling pathway in multiple myeloma by leelamine.

BACKGROUND: Leelamine (LEE) is a lipophilic diterpene amine phytochemical, which can be naturally extracted from pine's bark trees. It has been extensively studied recently for its promising chemopreventive and anti-cancer effects against various cancers such as that of prostate and breast. HYPOTHESIS: We examined the potential impact of LEE in affecting the activation of signal transducer and activator of transcription 3 (STAT3) and promoting apoptosis in human multiple myeloma (MM) cells. METHODS: We evaluated the effect of LEE on STAT3 signaling pathway in MM cells by using Western blot analysis and reverse transcription polymerase chain reaction (RT-PCR). Thereafter, apoptosis was evaluated using cell cycle analysis and Annexin V assay. RESULTS: We noted that LEE could attenuate the phosphorylation of STAT3 and other up-stream signaling molecules such as JAK1, JAK2, and Src activation in U266 and MM.1S cells. It also diminished STAT3 translocation into the nucleus and enhanced the expression of protein-tyrosine phosphatase epsilon (PTPε). Additionally, LEE caused cell cycle arrest and synergistically augmented the apoptotic actions of bortezomib against MM cells. CONCLUSIONS: Our data indicates that LEE could block STAT3 signaling cascade linked to tumorigenesis and can be used in combination with approved anti-cancer agents in attenuating MM growth and survival.

Preclinical evaluation of JAK1/2 inhibition by ruxolitinib in a murine model of chronic graft-versus-host disease.

The objective of this study was to examine the therapeutic effect of ruxolitinib, an orally administered selective Janus kinase (JAK) 1/2 inhibitor, on chronic graft-versus-host disease (cGVHD) using a murine model of sclerodermatous GVHD (scl-GVHD). Compared with scl-GVHD controls, ruxolitinib-treated recipients had scl-GVHD of significantly attenuated clinical and pathological severity in the skin and decreased frequencies of effector cells, CD4+ T cells, and CD11b+ macrophage/monocytes. Regulatory CD4+ Foxp3+ T cells were expanded whereas interferon-γ (IFN-γ)-producing CD4+ T cells were significantly decreased in ruxolitinib-treated recipients. Ruxolitinib suppressed not only the production of IFN-γ from CD4+ T cells and monocyte chemoattractant protein 1 (MCP-1) from CD11b+ macrophage/monocytes, but also the proliferation of these cells in vitro. Levels of both cytokines (IFN-γ and MCP-1) were also reduced in the spleen and skin of ruxolitinib-treated recipients in vivo. IFN-γ-induced MCP-1 production and migration of RAW 264.7 cells, a macrophage cell line, were inhibited by ruxolitinib. However, supplementation with MCP-1 restored this effect of ruxolitinib. In addition, blocking JAK-STAT signaling using ruxolitinib reduced the activation of STAT1 in stimulated immune effector cells. Taken together, these results suggest that ruxolitinib can prevent scl-GVHD by suppressing IFN-γ produced by T cells and MCP-1 expression in macrophage/monocytes via inhibition of JAK-STAT signaling.

Tibetan medicine Ershiwuwei Lvxue Pill attenuates collagen-induced arthritis via inhibition of JAK2/STAT3 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Ershiwuwei Lvxue Pill (ELP, མགྲིན་མཚལ་ཉེར་ལྔ།), a traditional Tibetan medicine preparation, has been used hundreds of years for the clinical treatment of rheumatoid arthritis (RA) in the highland region of Tibet, China. However, the underlying mechanism of its therapeutic effect remains unclear. AIM OF THE STUDY: The present study aimed to investigate the potential pharmacological mechanisms of anti-arthritic effect of ELP. MATERIALS AND METHODS: The main chemical constituents of ELP were analyzed by ultra-performance liquid chromatography quadrupole-time-flight mass spectrometry (UPLC-Q-TOF-MS). Forty-eight male Wistar rats (220 ± 20 g) were randomly divided into six groups: normal group, collagen-induced arthritis (CIA) group, methotrexate group (1.05 mg/kg), ELP groups (115, 230 and 460 mg/kg). CIA rat models were assigned to evaluate the anti-RA activity of ELP by determining the paws swelling, arthritis score, organ coefficients of spleen and thymus, and histopathological analysis of knee joints of synovial tissues. The levels of TNF-α, IL-10, IL-6 and IL-17 in serum were measured by ELISA. In addition, mRNA and protein expression levels associated with JAK2/STAT3 signaling pathway in synovial tissues of CIA rats were detected by qRT-PCR, immunohistochemistry and Western blot analyses. RESULTS: Fourteen main chemical constituents of ELP were quantitatively determined by UPLC-Q-TOF-MS analysis. Treatment with ELP reduced the paw swelling, arthritis score and organ coefficients of spleen and thymus. Histopathological examination revealed the protective effects of ELP on CIA rats with knee joint injury. The levels of serum pro-inflammatory cytokines (TNF-α, IL-6 and IL-17) were markedly reduced while the anti-inflammatory cytokine IL-10 was significantly increased with the treatment of ELP. Further investigations showed ELP down-regulated the mRNA and protein expression levels of Bcl-2, whereas up-regulated Bax, SOCS1 and SOCS3. Meanwhile, the ratios of p-JAK2/JAK2 and p-STAT3/STAT3 proteins from synovial tissues were dramatically decreased with the treatment of ELP, whereas no changes of the mRNA and protein expression levels of JAK2 and STAT3 were observed. CONCLUSION: These results indicated that ELP reduced the severity of arthritis and joint swelling, suggesting an antirheumatic effect on CIA rats. The possible mechanism is related to inhibiting inflammatory response and inducing apoptosis in synovial tissues by regulating JAK2/STAT3 signaling pathway. However, further in vivo and in vitro investigations are still needed to clarify the underlying mechanism of ELP in treating RA.

Hepatoprotective effect and possible mechanism of phytoestrogen calycosin on carbon tetrachloride-induced liver fibrosis in mice.

The study was to explore the hepatoprotective effect and possible mechanism of calycosin on carbon tetrachloride (CCl4)-induced liver fibrosis in mice. Hepatic fibrosis was induced by intraperitoneal injection of CCl4 in C57BL/6 male mice. Serum alanine aminotransferase (ALT) and aspartate transaminase (AST) activity, superoxide dismutase (SOD) activity, and hydroxyproline (Hyp) and malondialdehyde (MDA) levels were determined by biochemical assays. Liver histopathology was assessed by H&E and Masson trichrome staining. The mRNA expressions of α-smooth muscle actin (α-SMA), collagen-I (Col-I), Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) were determined using qRT-PCR. The protein levels of α-SMA, Col-I, estrogen receptor α (ERα), estrogen receptor ß (ERß), tissue inhibitor of metalloproteinase-1 (TIMP-1), matrix metalloproteinase-1 (MMP-1), JAK2, phospho-JAK2 (p-JAK2), STAT3, and phospho-STAT3 (p-STAT3) were detected by Western blotting. The levels of α-SMA and ERß were measured by immunohistochemistry. Calycosin significantly reduced liver index, MDA level, and ALT and AST activity and increased SOD activity. The α-SMA, Col-I, and Hyp of the calycosin group were significantly lower than those of the model group. Calycosin increased MMP-1 and inhibited TIMP-1 expression resulting in the improvement of MMP-1/TIMP-1 ratio. Importantly, calycosin improved ERß protein expression, JAK2 and STAT3 mRNA expressions, p-JAK2/JAK2, and p-STAT3/STAT3 relative protein expressions. However, ERα, JAK2, and STAT3 protein expressions were relatively unchanged. Calycosin significantly inhibits liver fibrosis in mice, and its mechanism may involve the following: calycosin inhibits oxidative stress; calycosin inhibits collagen synthesis and balances MMP-1/TIMP-1 system; calycosin increases ERß expression and activates JAK2-STAT3 pathway.

Electroacupuncture ameliorates mechanical hypersensitivity by down-regulating spinal Janus kinase 2/signal transducer and activation of transcription 3 and interleukin 6 in rats with spared nerve injury.

BACKGROUND: Evidence shows that the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway participates in the pathogenesis of neuropathic pain. Our previous study revealed that electroacupuncture (EA) attenuated neuropathic pain via activation of alpha-7 nicotinic acetylcholine receptor (α7nAChR) in the spinal cord. However, whether 2 Hz EA alleviates neuropathic pain by regulating the downstream molecules JAK2/STAT3 has not been fully clarified. METHODS: Paw withdrawal threshold (PWT) was used as a marker of mechanical allodynia in rats with spared nerve injury (SNI). After applying 2 Hz EA on day 3, 7, 14 and 21 post-surgery, spinal expression of JAK2, STAT3 and pro-inflammatory cytokine interleukin (IL)-6 was examined using quantitative reverse transcription and real-time polymerase chain reaction (qRT-PCR) and Western blot analysis. Intrathecal injection of the α7nAChR antagonist alpha-bungarotoxin (α-Bgtx) was used to further explore the mechanism underlying the effects of 2 Hz EA on expression of JAK2/STAT3 in SNI rats. RESULTS: It was found that levels of spinal STAT3 and IL-6 mRNA, as well as levels of phosphorylated (p)-JAK2, p-STAT3 and IL-6 protein, were markedly increased in SNI rats. 2 Hz EA attenuated the SNI-induced up-regulation of p-JAK2, p-STAT3 and IL-6 expression in the spinal cord. Furthermore, intrathecal injection of α-Bgtx (1.0 µg/kg) not only inhibited the effect of 2 Hz EA on mechanical hypersensitivity but also ameliorated the down-regulation of p-JAK2, p-STAT3 and IL-6 expression induced by 2 Hz EA. CONCLUSION: This study revealed that 2 Hz EA attenuated SNI-induced mechanical hypersensitivity and the concomitant up-regulation of spinal JAK2, STAT3 and IL-6 in SNI rats, suggesting that suppression of the JAK2/STAT3 signaling pathway might be the mechanism underlying the therapeutic effect of 2 Hz EA on neuropathic pain.

Ethyl acetate extract of Tibetan medicine Rhamnella gilgitica ameliorated type II collagen-induced arthritis in rats via regulating JAK-STAT signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhamnella gilgitica Mansf. et Melch. (སེང་ལྡེང་།, RG) is a traditional Tibetan medicinal plant that is currently grown throughout Tibet. According to the theory of Tibetan medicine, RG is efficient for removing rheumatism, reducing swelling, and relieving pain. Hence, it has been used for the treatment of rheumatoid arthritis (RA) in Tibet for many years. However, there are no previous reports on the anti-RA activities of ethyl acetate extract of RG (RGEA). AIM OF THE STUDY: This study aimed to explore the anti-RA effect and mechanism of RGEA on collagen-induced arthritis (CIA) in rats. MATERIALS AND METHODS: The CIA model was established in male Wister rats by intradermal injection of bovine type II collagen and Complete Freund's Adjuvant at the base of the tail and left sole, respectively. The rats were orally administered with RGEA (9.71, 19.43, or 38.85 mg/kg) for 23 days. The body weight, swelling volume, arthritis index score, thymus and spleen indices, and pathological changes were observed to evaluate the effect of RGEA on RA. Furthermore, the inflammatory cytokines in serum, such as interleukin1 beta (IL-1ß), tumor necrosis factor alpha (TNF-α), interleukin6 (IL-6), interleukin17 (IL-17), interferon-γ (INF-γ), interleukin4 (IL-4), and interleukin10 (IL-10) were measured by enzyme linked immunosorbent assay (ELISA) to explore the anti-inflammatory effects of RGEA. The terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) staining was used to examine apoptosis. Finally, the protein and gene expression of B-cell lymphoma-2-associated X (Bax), B-cell lymphoma 2 (Bcl-2), Caspase3, janus-activated kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3), suppressor of cytokine signaling1 (SOCS1), and 3 (SOCS3) in synovial tissue were detected using immunohistochemistry and real-time quantitative polymerase chain reaction (RT-qPCR). RESULTS: After the treatment with RGEA, the body weight of rats was restored, both the arthritis index and paw swelling were suppressed, and spleen and thymus indices were decreased. RGEA reduced the inflammatory cells and synovial hyperplasia in the synovial tissue of the knee joint, and suppressed bone erosion. Meanwhile, RGEA decreased the levels of IL-1ß, IL-6, IL-17, TNF-α, and INF-γ, while increased the levels of IL-4 and IL-10. TUNEL fluorescence apoptosis results confirmed that RGEA obviously promoted the apoptosis of synovial cells. Further studies showed that RGEA inhibited the proteins and mRNAs expression of JAK2 and STAT3 as well as increased the proteins and mRNAs expression of SOCS1 and SOCS3. In addition, RGEA upregulated the expression of Bax and Caspase3, and downregulated the expression of Bcl-2. CONCLUSION: The anti-RA effectof RGEA might be related to the promotion of apoptosis and inhibition of inflammation, which regulated the JAK-STAT pathway.