Thymic stromal lymphopoietin suppresses markers of neuroinflammation and the JAK2/STAT5 pathway in activated microglia.

Thymic stromal lymphopoietin (TSLP) is highly expressed in the central nervous system in response to inflammation, but its exact function remains unclear. In this study, we used a model of LPS-stimulated microglia to investigate the direct impact of TSLP on microglial activation and the underlying mechanism. We measured oxidative stress, expression of microglial activation markers, and inflammatory indexes. The results show that TSLP treatment increased the expression of TSLP receptors and reduced LPS-induced oxidative stress, inflammation, and the expression of M1-type markers in microglia. Interestingly, TSLP treatment also influenced the differentiation of microglia towards the M2 type, suppressing LPS-induced activation, mediated by the JAK2/STAT5 pathway. Moreover, TSLP also promoted the expression of macrophage markers in the absence of LPS. These findings support the hypothesis that TSLP plays a role in reducing neuroinflammation by blocking the JAK2/STAT5 pathway induced by LPS, thus indicating a regulatory role in the central nervous system. Targeting this cytokine might provide a novel strategy for controlling an inflammatory response in the central nervous system.

Copper (II) complex of salicylate phenanthroline induces the apoptosis of colorectal cancer cells, including oxaliplatin­resistant cells.

Oxaliplatin (Oxa) is one of the most effective chemotherapeutic drugs used in the treatment of colorectal cancer (CRC). However, the use of this drug is associated with severe side­effects and patients eventually develop resistance to Oxa. In recent years, copper complexes have been extensively investigated as substitutes for platinum­based drugs. Therefore, a number of copper complexes have also been developed for cancer therapy, such as copper (II) complex of salicylate phenanthroline [Cu(sal)(phen)]. In the present study, the antitumor activity and the related molecular mechanisms of Cu(sal)(phen) were examined in CRC cells. As compared with the chemotherapeutic drug, Oxa, Cu(sal)(phen) was more effective in inducing apoptosis and reactive oxygen species (ROS) production, and in decreasing mitochondrial membrane potential in the CRC cell lines, HCT116 and SW480. In addition, the expression of the apoptosis­related proteins, Bcl­2 and survivin, and those of the upstream regulators, p­JAK2 and p­STAT5, were significantly decreased in the two cell lines following treatment with Cu(sal)(phen). Furthermore, the efficacy of the complex against CRC was found to be excellent in an animal model. The results of immunohistochemical analysis revealed that the expression levels of Bcl­2, survivin and Ki­67 in tumor tissues were decreased following Cu(sal)(phen) treatment. The antitumor mechanisms underlying Cu(Sal)(phen) treatment were the induction of ROS generation, the inhibition of the JAK2/STAT5 signaling pathway and the downregulation of the expression of anti­apoptotic proteins, such as Bcl­2 and survivin. On the whole, the findings of the present study indicated that Cu(sal)(phen) effectively inhibited the viability and proliferation of HCT116 and SW480 CRC cells; in the future, the authors aim to conduct further experiments in future studies to provide more evidence that supports the development of Cu(sal)(phen) as a therapeutic agent for CRC.

Prolactin Regulates Testicular Gene Expression and Cell Cycle Processes Predominantly via JAK2/STAT5 Pathway in the Male Rat.

Hyperprolactinemia is prevalent in up to 16% of infertile males. Although the prolactin receptor (PRLR) is present on various testicular cells, the physiological role of this receptor in spermatogenesis remains elusive. The aim of this study is to delineate prolactin actions in rat testicular tissue. Serum prolactin, developmental expression of PRLR, signaling pathways associated, and gene transcription regulation in the testes were investigated. Serum prolactin and testicular PRLR expression was found to be significantly increased at pubertal and adult ages as compared to prepubertal. Further, PRLR activated the JAK2/STAT5 pathway, but not the MAPK/ERK and PI3K/AKT pathway in the testicular cells. Gene expression profiling following prolactin treatment in seminiferous tubule culture resulted in a total of 692 differentially expressed genes, of which 405 were upregulated and 287 were downregulated. Enrichment map analysis showed that prolactin target genes are involved in processes such as cell cycle, male reproduction, chromatin remodeling, and cytoskeletal organization. Novel gene targets of prolactin whose role in testes is unexplored were obtained and validated by qPCR. Additionally, 10 genes involved in cell cycle process were also validated; 6 genes (Ccna1, Ccnb1, Ccnb2, Cdc25a, Cdc27, Plk1) were found to be significantly upregulated, whereas 4 genes (Ccar2, Nudc, Tuba1c, Tubb2a) were found to be significantly downregulated in testes after treatment with prolactin. Taken together, the findings from this study suggest a crucial role of prolactin in male reproduction and identified target genes regulated by prolactin in the testes.

Freeze-dried powder of daylily bud improves bromocriptine-induced lactation disorder in rats via JAK2/STAT5 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Milk deficiency is a prevalent problem in the world. Daylily (Hemerocallis citrina Borani), called the Chinese mother flower, is a traditional vegetable and is believed to possess a galactagogue effect in China. Flavonoids and phenols are considered as the active ingredients of daylily to promote lactation and improve depression. AIM OF THE STUDY: The aim of this study was to investigate the prolactin effects of freeze-dried powder of flower buds of H. citrina Baroni in rat and its action mechanisms. MATERIALS AND METHODS: The chemical constituents of flower buds of H. citrina Baroni treated by different drying techniques were analyzed by ultrahigh pressure liquid chromatography-mass spectrometry. Sprague-Dawley (SD) rat model induced by bromocriptine was used to evaluate the effect of freeze-dried powder of daylily buds on promoting lactation. Network pharmacology method, ELISA, qPCR, and Western blot were used to clarify the action mechanisms. RESULTS: We detected 657 compounds in daylily buds. The relative contents of total flavonoids and phenols in freeze-dried samples were higher than those in dried ones. Bromocriptine, as a dopamine receptor agonist, can significantly inhibit prolactin in rats. Daylily buds can restore the levels of prolactin, progesterone and estradiol depressed by bromocriptine, effectively improve the milk production of the rat, and promote the repair of rat mammary gland tissue. We analyzed the relationship between the chemical components of daylily buds and the genes related to lactation with network pharmacology method, revealing that flavonoids and phenols may be the active components that promoted milk production via JAK2/STAT5 pathway, which was confirmed by the results of qPCR and Western blot. Daylily buds can increase the mRNA expression of PRLR, CSN2, LALBA and FASN and the protein expression of PRLR, JAK2 and STAT5. CONCLUSION: Daylily buds can improve the insufficient lactation of rats induced by bromocriptine through PRLR/JAK2/STAT5 pathway, and the freeze-dried processing method may better retain the active components of flavonoids and phenols that promote milk in daylily.

Antibody targeting TSLP suppresses DSS-induced colitis and activation of the JAK2/STAT5 pathway in mice.

There is currently no safe or effective treatment for inflammatory bowel disease (IBD), which is defined as recurrent and persistent intestinal inflammation. Thymic stromal lymphopoietin (TSLP) has been shown to be associated with the pathogenesis of IBD, and the JAK2/STAT5 signalling pathway has demonstrated much promise as a novel therapeutic target for IBD. In this study, we first evaluated levels of TSLP in dextran sodium sulphate (DSS)-induced IBD mice. Second, we applied tezepelumab, an anti-TSLP monoclonal antibody (20 µg per mouse, intraperitoneally), to DSS-induced IBD mice and quantified the signs of histopathological change, intestinal inflammation, and integrity of the mucosal barrier. In addition, the effect of DSS and/or tezepelumab on the phosphorylation of the JAK/STAT pathway was investigated. TSLP expression levels were elevated in DSS-induced IBD mice, whereas TSLP antibody treatment suppressed the pathological features associated with IBD and alleviated intestinal inflammation and mucosal barrier disruption. Moreover, level of phosphorylated JAK2/STAT5 were increased in DSS-induced IBD mice, but were strongly decreased in the presence of tezepelumab. Our findings suggest that targeting TSLP via the JAK2/STAT5 signalling pathway may be an effective approach for the treatment of IBD.

Promoted CD4+ T cell-derived IFN-γ/IL-10 by photobiomodulation therapy modulates neurogenesis to ameliorate cognitive deficits in APP/PS1 and 3xTg-AD mice.

BACKGROUND: The immune system has been implicated in synaptic plasticity, inflammation, and the progression of Alzheimer's disease (AD). However, there were few studies on improving the niche microenvironment of neural stem cells (NSCs) in the brain of AD to promote adult hippocampal neurogenesis (AHN) by regulating the function of non-parenchymal immune cells. METHODS: The lymph nodes of amyloid precursor protein/presenilin 1 (APP/PS1) and 3xTg (APP/PS1/tau) mouse models of AD were treated with photobiomodulation therapy (PBMT) for 10 J/cm2 per day for 1 month (10 min for each day), T lymphocytes isolated from these two AD models were treated with PBMT for 2 J/cm2 (5 min for each time). The NSCs isolated from hippocampus of these two AD models at E14, and the cells were co-cultivated with PBMT-treated T lymphocyte conditioned medium for NSCs differentiation. RESULTS: Our results showed that PBMT treatment could promote AHN and reverse cognitive deficits in AD mouse model. The expression of interferon-γ (IFN-γ) and interleukin-10 (IL-10) was upregulated in the brain of these two AD models after PBMT treated, which was induced by the activation of Janus kinase 2 (JAK2)-mediated signal transducer and activator of transcription 4 (STAT4)/STAT5 signaling pathway in CD4+ T cells. In addition, elevated CD4+ T cell levels and upregulated transforming growth factor-ß1 (TGFß1)/insulin-like growth factors-1 (IGF-1)/brain-derived neurotrophic factor (BDNF) protein expression levels were also detected in the brain. More importantly, co-cultivated the PBMT-treated T lymphocyte conditioned medium with NSCs derived from these two AD models was shown to promote NSCs differentiation, which was reflected in the upregulation of both neuronal class-III ß-tubulin (Tuj1) and postsynaptic density protein 95 (PSD95), but the effects of PBMT was blocked by reactive oxygen species (ROS) scavenger or JAK2 inhibitor. CONCLUSION: Our research suggests that PBMT exerts a beneficial neurogenesis modulatory effect through activating the JAK2/STAT4/STAT5 signaling pathway to promote the expression of IFN-γ/IL-10 in non-parenchymal CD4+ T cells, induction of improvement of brain microenvironmental conditions and alleviation of cognitive deficits in APP/PS1 and 3xTg-AD mouse models.

Alternatively activated macrophages promote airway inflammation through JAK3-STAT5-Fra2 in asthma.

BACKGROUND: Fos-related antigen-2 (Fra-2) is a transcription factor belonging to the activator protein 1 (AP-1) family, which is associated with many chronic airway diseases such as asthma. Alternatively activated (M2) macrophages are associated with Fra2 in airway diseases such as pulmonary fibrosis. However, there is no specific study that explores the relationship between M2 macrophages and Fra2 in asthma. OBJECTIVE: We hypothesized that a potential mechanism of allergic asthma could be that Fra2 is highly expressed in M2 macrophages through JAK3-STAT5 and facilitates the production of downstream T-helper 2 (Th2) cytokines, thus promoting the pathogenesis of asthma. METHODS: Peripheral venous blood and airway tissue samples of patients with asthma and controls were obtained. Moreover, a C57BL/6 mouse model of asthma was established. Fra2 expression was detected using immunohistochemistry and immunofluorescence. Macrophages were obtained by flow sorting, and expression of the JAK3-STAT5-Fra2 signaling pathway was determined using PCR and western blotting. Enzyme-linked immunosorbent assay was used to determine M2 macrophage-associated Th2-type cytokine levels. RESULTS: Fra2 was highly expressed in patients with asthma and asthmatic mice. The JAK3-STAT5 was a signal pathway related to the high expression of Fra2 in M2 macrophages. Moreover, we found that Fra2 could affect the production of Th2 cytokines downstream of M2 macrophages, including interleukin 4 (IL-4) and IL-13. CONCLUSION: M2 macrophages could promote airway inflammation through JAK3-STAT5-Fra2 to induce allergic asthma. Our study offers a new insight to further understand the pathogenesis of asthma and also provides a new direction for targeted treatment.

Aloperine Ameliorates IMQ-Induced Psoriasis by Attenuating Th17 Differentiation and Facilitating Their Conversion to Treg.

Aloperine is an anti-inflammatory compound isolated from the Chinese herb Sophora alopecuroides L. Previously, our group has reported that the generation of induced Treg was promoted by aloperine treatment in a mouse colitis model. However, the effect of aloperine on effector T cell subsets remains unclear. We therefore carefully examined the effect of aloperine on the differentiation of major subsets of T helper cells. Based on our results, psoriasis, a Th17 dominant skin disease, is selected to explore the potential therapeutic effect of aloperine in vivo. Herein, we demonstrated that topical application of aloperine suppressed epidermal proliferation, erythema, and infiltration of inflammatory cells in skin lesions. Mechanistic studies revealed that aloperine suppressed the differentiation of Th17 cells directly through inhibiting the phosphorylation of STAT3 or indirectly through impairing the secretion of Th17-promoting cytokines by dendritic cells. Moreover, aloperine enhanced the conversion of Th17 into Treg via altering the pSTAT3/pSTAT5 ratio. Collectively, our study supported that aloperine possesses the capacity to affect Th17 differentiation and modulates Th17/Treg balance, thereby alleviating imiquimod (IMQ)-induced psoriasis in mice.

A Prognostic Model for Acute Myeloid Leukemia Based on IL-2/STAT5 Pathway-Related Genes.

Accurate prognostic stratification of patients can provide guidance for personalized therapy. Many prognostic models for acute myeloid leukemia (AML) have been reported, but most have considerable inaccuracies due to contained variables with insufficient capacity of predicting survival and lack of adequate verification. Here, 235 genes strongly related to survival in AML were systematically identified through univariate Cox regression analysis of eight independent AML datasets. Pathway enrichment analysis of these 235 genes revealed that the IL-2/STAT5 signaling pathway was the most highly enriched. Through Cox proportional-hazards regression model and stepwise algorithm, we constructed a six-gene STAT5-associated signature based on the most robustly survival-related genes related to the IL-2/STAT5 signaling pathway. Good prognostic performance was observed in the training cohort (GSE37642-GPL96), and the signature was validated in seven other validation cohorts. As an independent prognostic factor, the STAT5-associated signature was positively correlated with patient age and ELN2017 risk levels. An integrated score based on these three prognostic factors had higher prognostic accuracy than the ELN2017 risk category. Characterization of immune cell infiltration indicated that impaired B-cell adaptive immunity, immunosuppressive effects, serious infection, and weakened anti-inflammatory function tended to accompany high-risk patients. Analysis of in-house clinical samples revealed that the STAT5-assocaited signature risk scores of AML patients were significantly higher than those of healthy people. Five chemotherapeutic drugs that were effective in these high-risk patients were screened in silico. Among the five drugs, MS.275, a known HDAC inhibitor, selectively suppressed the proliferation of cancer cells with high STAT5 phosphorylation levels in vitro. Taken together, the data indicate that the STAT5-associated signature is a reliable prognostic model that can be used to optimize prognostic stratification and guide personalized AML treatments.

Prolactin-regulated Pbk is involved in pregnancy-induced ß-cell proliferation in mice.

Gestational diabetes mellitus (GDM) is a condition of diabetes with onset or first recognition in pregnancy. Its incidence is increasing, and GDM deleteriously affects both mother and the fetus during and even after pregnancy. Previous studies in mice have shown that during pregnancy, ß-cell proliferation increases in the middle and late stages of pregnancy and returns to normal levels after delivery. Hormones, such as prolactin, estradiol, and progesterone as well as protein kinases, play important roles in regulating gestation-mediated ß-cell proliferation; however, the regulatory relationship between them is uncertain. We previously found that protein kinase Pbk was crucial for basal proliferation of mouse islet cells. Herein we show that Pbk is upregulated during pregnancy in mice and Pbk kinase activity is required for enhanced ß- cell proliferation during pregnancy. Notably, knock-in (KI) of a kinase-inactivating Pbk mutation leads to impaired glucose tolerance and reduction of ß-cell proliferation and islet mass in mice during pregnancy. Prolactin upregulates the expression of Pbk, but the upregulation is diminished by knockdown of the prolactin receptor and by the inhibitors of JAK and STAT5, which mediate prolactin receptor signaling, in ß-cells. Treatment of ß-cells with prolactin increases STAT5 binding to the Pbk locus, as well as the recruitment of RNA polymerase II, resulting in increased Pbk transcription. These results demonstrate that Pbk is upregulated during pregnancy, at least partly by prolactin-induced and STAT5-mediated enhancement of gene transcription, and Pbk is essential for pregnancy-induced ß-cell proliferation, increase in islet mass, and maintenance of normal blood glucose during pregnancy in preclinical models. These findings provide new insights into the interplay between hormones and protein kinases that ultimately prevent the development of GDM.

Electroacupuncture Improves Cerebral Ischemic Injury by Enhancing the EPO-JAK2-STAT5 Pathway in Rats.

OBJECTIVE: Clinically, electroacupuncture (EA) improves cerebral ischemic injury, but its mechanism remains unknown. The aim of this study was to confirm the protective effects of EA on focal cerebral ischemia (FCI)-induced injury and the possible mechanism. METHODS: Sprague-Dawley (SD) rats served as the FCI model and were divided into the sham, model, EA, AG490 and EA+AG490 groups. Rats in the EA and EA+AG490 groups were acupunctured at the Baihui (GV20) and Dazhui (GV14) acupoints, and those in the AG490 and EA+AG490 groups were administered an intracerebroventricular injection of AG490 (a Janus-tyrosine kinase-2 (JAK-2) phosphorylation inhibitor). Neurological deficits and morphological changes in the ischemic cortex were observed through neurological deficit scoring and HE staining, respectively, and neuronal apoptosis was examined using the TUNEL assay. Transmission electron microscopy was used to observe neuronal ultrastructure, and HIF-1α, erythropoietin (EPO), phosphorylated (p)-JAK2, p-STAT5, HSP70, Bax and Bcl-2 expression was measured by RT-PCR and immunohistochemistry. RESULTS: FCI model rats showed obvious neurological deficits and neuronal apoptosis compared with sham rats. EA alleviated FCI-induced neurological deficits, improved neuronal ultrastructure, reduced neuronal apoptosis, and induced HIF-1α, EPO, p-JAK2, p-STAT5, HSP70 and Bcl-2 expression in a time-dependent manner. In contrast, AG490 treatment impaired the effects of EA on neurological deficits, neuronal apoptosis and HIF-1α, EPO, p-JAK2, p-STAT5, HSP70, Bax and Bcl-2 expression. CONCLUSION: EA at GV20 and GV14 could improve neurological deficits and reduce neuronal apoptosis, thereby improving FCI-induced injury, which may be related to enhancing the EPO-JAK2-STAT5 pathway.

LPS-induced SOCS3 antagonizes the JAK2-STAT5 pathway and inhibits ß-casein synthesis in bovine mammary epithelial cells.

Bovine mammary epithelial cells (BMECs) are essential for lactation in the dairy cow mammary gland, and are often used as a cellular model to study changes in inflammatory responses and lactation functions with exogenous stimuli. Prolactin (PRL) promotes milk protein synthesis by continuously activating the Janus kinase 2 and signal transducer and activator of transcription 5 (JAK2-STAT5) pathway. Lipopolysaccharides (LPS) activates inflammatory responses in cells and inhibits casein synthesis, but the exact mechanism is still unclear. Suppressor of cytokine signaling-3 (SOCS3) is a negative regulator of the JAK-STATs signaling pathway, and regulates a variety of inflammatory responses by inhibiting STAT3. Previous studies also suggested that SOCS3 plays a role in the development and involution of bovine mammary glands. The purpose of this study was to investigate whether LPS activated SOCS3, and whether SOCS3 resisted the regulation of casein synthesis by PRL in a JAK2-STAT5-dependent manner. We treated in vitro BMECs with 125 ng/mL PRL, 10 µg/mL LPS, SOCS3 siRNA (silencing), a SOCS3-GFP adenovirus overexpression vector, or combinations, to determine ß-casein expression. We demonstrated that PRL up-regulated phospho-JAK2, phsopho-STAT5 and ß-casein expression, whereas LPS caused the opposite effects, and activated SOCS3. SOCS3 overexpression interrupted the JAK2-STAT5 pathway in BMECs. With SOCS3 was silenced, LPS could not activate the JAK2-STAT5 pathway, and no inhibition of ß-casein expression was observed. In conclusion, we showed that LPS activated SOCS3 in BMECs, antagonized the JAK2-STAT5 pathway via SOCS3 regulation, and ultimately reduced ß-casein expression in these cells.

LncRNA NEAT1 promotes airway smooth muscle cell inflammation by activating the JAK3/STAT5 pathway through targeting of miR-139.

Background Asthma is a chronic inflammatory heterogeneous respiratory disease. Previous studies showed that the lncRNA NEAT1 (nuclear paraspeckle assembly transcript 1) might play an important role in the pathogenesis of asthma, but its potential mechanism in airway smooth muscle cell (ASMC) inflammation remains largely unknown and needs further investigation.Methods We performed cellular immunofluorescence to identify the features of ASMCs and detected the expression levels of lncRNA NEAT1, miR-139, TNF-α, IL-6, IL-8 and IL-1ß by quantitative real-time PCR (Q-PCR) and ELISA. Western blotting (WB) was used to measure the protein expression of the related genes, and bioinformatics as well as dual luciferase assays were used to validate the interaction between lncRNA NEAT1 and miR-139 and the interaction between miR-139 and the 3'-UTR of JAK3.Results The expression of lncRNA NEAT1 was increased in the ASMCs of asthma patients, but miR-139 was decreased. Overexpression of lncRNA NEAT1 promoted the expression of the inflammatory cytokines such as TNF-α, IL-6, IL-8 and IL-1ß in ASMCs. LncRNA NEAT1 was able to target miR-139 to activate the JAK3/STAT5 signaling pathway and induced the expression of these inflammatory cytokines in ASMCs. Overexpression of miR-139 or suppression of the JAK3/STAT5 signaling pathway reversed the inflammatory effect of lncRNA NEAT1.Conclusion LncRNA NEAT1 played a pivotal role in ASMC inflammation and exerted its function through the miR-139/JAK3/STAT5 signaling network.

Activation of the STAT5 Signaling Pathway by Yiqi Jiedu Formula Induces Regulatory T Cell-Mediated Alleviation of Corneal Immunopathological Damage in Mice With Recurrent Herpes Simplex Keratitis.

This study aimed to investigate the effect of Yiqi Jiedu (YQJD) formula on the repair of corneal lesions in mice with recurrent herpes simplex virus keratitis (HSK). Sixty female BALB/c mice were randomly divided into three groups: a normal control group (Naive), a recurrence model group (Re), and a YQJD group. After inducing recurrence by ultraviolet irradiation, the ocular surfaces of different groups of mice were observed using a slit lamp and photographed, and ocular surface scores were calculated. The abundance of CD4+CD25+Foxp3+ regulatory T (Treg) cells was determined by flow cytometry in peripheral blood and spleen cells. The CD4+Foxp3+ Tregs were assessed by immunofluorescence in the cornea. The levels of the cytokines IL-10 and TGF-ß in serum and splenocyte culture supernatants were detected by enzyme-linked immunosorbent assay. Furthermore, the activation status of the STAT5 signaling pathway was examined by protein blotting, and the effect of YQJD on Treg cells through inhibition of the STAT5 pathway was observed in vitro. YQJD alleviated corneal inflammation by enhancing the STAT5 signaling pathway, thereby promoting the differentiation of CD4+CD25+Foxp3+ Treg cells, increasing the levels of anti-inflammatory cytokines such as IL-10 and TGF-ß, and maintaining immune tolerance. YQJD increased the proportion of CD4+Foxp3+ Treg cells; also, in the cornea, YQJD inhibited the aggregation of macrophages and CD4+ cells and reduced the proportion of Th17 cells and other pro-inflammatory cells. Moreover, YQJD promoted the secretion of IL-4 to protect the cornea, leading to the mitigation of corneal immunopathological damage. YQJD reduced corneal lesions in recurrent HSK mice by stimulating Treg cells, inducing immune tolerance, and inhibiting corneal immunopathological responses via modulation of the STAT5 signaling pathway.

The anti-CXCL4 antibody depletes CD4(+)CD25(+)FOXP3(+) regulatory T cells in CD4+ T cells from chronic osteomyelitis patients by the STAT5 pathway.

BACKGROUND: Chronic osteomyelitis is associated with the immune suppression. CD4(+)CD25(+) FOXP3(+) regulatory T cells (Tregs) play a key role in the peripheral tolerance to prevent immune responses to self-antigens and allergens. Evidence has suggested that the accumulation and activity of Tregs are regulated by chemokine family member CXCL10 and its receptor CXCR3 in human atherosclerotic lesions. This study aimed to investigated the effect of CXCL4, a member of chemokine family, on Tregs, and the underlying mechanisms. METHODS: CD4+ T cells were isolated from peripheral blood of patients with chronic osteomyelitis or healthy controls. Anti-CXCL4 antibody and recombinant CXCL4 protein were used for treatment. The expression of forkhead box P3 (FOXP3), cytotoxic T lymphocyte antigen-4 (CTLA-4) and phosphorylated signal transducer and activator of transcription 5 (STAT5) were measured to assess the mechanism. STAT5 inhibitor (IST5-002) was used to retard STAT5 pathway. RESULTS: We found that serum concentration of CXCL4 in chronic osteomyelitis was significantly enhanced. Through the prevention of STAT5 activity, CXCL4 antibody could inhibit the protein expression of CXCL4, CXCR3, FOXP3, CTLA-4 and phosphorylated-STAT5, as well as decrease the percentage of Tregs in CD4+ T cells. Conversely, recombinant CXCL4 protein resulted in the opposite in CD4+ T cells from healthy controls, obviously enhancing Tregs percentage and promoting STAT5 activation, which were significantly reversed by an STAT5 inhibitor. CONCLUSIONS: CXCL4 antagonism inhibited Tregs percentage and Tregs-associated proteins within CD4+ T cells from chronic osteomyelitis patients via blocking the STAT5 pathway.

MiR-920 and LSP1 co-regulate the growth and migration of glioblastoma cells by modulation of JAK2/STAT5 pathway.

This study probes the function and mechanism of lymphocyte-specific protein 1 (LSP1) in glioblastoma pathogenesis. According to the data acquired from TCGA, Oncomine and GEO databases, the expression and prognostic value of LSP1 and miR-920 in glioblastoma patients were analyzed. The expression levels of LSP1 in U251 and A172 cell lines were analyzed by qRT-PCR and western blotting. CCK8, colony formation and transwell assays were utilized to test glioblastoma cell malignant abilities. Furthermore, the associations between LSP1 and miR-920 were indentified by bioinformatics analysis and rescue assays. Moreover, the protein expression levels of p-JAK2, JAK2, p-STAT5 and STAT5, as the hallmark of JAK/STAT5 signaling, were detected by western blotting. The observations showed that LSP1 was highly augmented in glioblastoma samples. Additionally, up-regulation of LSP1 was associated with a unfavorable prognosis in glioblastoma patients. Biological experiments revealed that depletion of LSP1 significantly suppressed the proliferation, invasion and migration of U251 and A172 cells. MiR-920, as an upstream regulator of LSP1, negatively modulated LSP1 expression and promoted U251 cells malignant behaviors after miR-920 inhibitor treatment. However, together knockdown LSP1 and miR-920 inhibited these effects. Moreover, the expression levels of p-JAK2 and p-STAT5 were increased or decreased in U251 cells after transfection of miR-920 inhibitor or si-LPS1. Taken together, miR-920 might blocked the malignant development of glioblastoma cells, which is possibly realized by targeting LSP1 and modulation of JAK/STAT5 pathway. These findings implied that miR-920/LSP1 was a potential therapeutic target for glioblastoma treatment.

Immunomodulatory activity-guided isolation and characterization of a novel polysaccharide from Atractylodis macrocephalae Koidz.

The Rhizoma of Atractylodis macrocephala Koidz. is a traditional Chinese herbal medicine that has been widely and empirically used for unexplained recurrent spontaneous abortion. In this study, a polysaccharide from the Rhizoma of Atractylodis macrocephala Koidz., designated as RAMP2, with an absolute molecular weight of 4.354 × 103 Da was isolated and found to be composed of mannose, galacturonic acid, glucose, galactose and arabinose. The NMR results displayed that →3-ß-glcp-(1→, →3,6-ß-glcp-(1→, →6-ß-glcp-(1→, T-ß-glcp-(1→, →4-α-galpA-(1→, →4-α-galpA-6-OMe-(1→, →5-α-araf-(1→, →4,6-ß-manp-(1→ and →4-ß-galp-(1→ were the main linkages in RAMP2. TEM and SEM results indicated that RAMP2 was globular in structure. Furthermore, in vitro experiments on murine CD4+ T cells revealed that RAMP2 could increase the percentage of Treg cells, up-regulate Foxp3, IL-10 and IL-2 mRNA expressions and the secretion of IL-10 and IL-2. RAMP2 was further shown to increase STAT5 phosphorylation levels in Treg cells, suggesting that RAMP2 increased the number of Treg cells through IL-2/STAT5 pathway.

Anti-CCL22 increases regulatory T cells in CD4+ T cells of rheumatoid arthritis patients via STAT5 pathway.

Abnormality in the number and function of CD4+CD25+FOXP3+ regulatory T cells (Tregs) in peripheral blood has been linked to the initiation and progression of rheumatoid arthritis (RA). Effect of chemokine CCL22 on the number of Tregs in CD4+ T cells and the underlying mechanism were investigated. Downregulation of peripheral Tregs were observed while upregulation of serum chemokine CCL22 in RA patients. Tregs count and the expression of FOXP3 (Tregs function-related maker) and phosphorylated-signal transducer and activator of transcription 5 (p-STAT5) in CD4+ T cells from RA patients were increased while C-C chemokine receptor 4 (CCR4) was decreased by anti-CCL22 antibody, however, recombinant CCL22 resulted in the opposite effects in CD4+ T cells from the healthy control. STAT5 inhibitor significantly reversed the effects of anti-CCL22 antibody. Similarly, sinomenine, an anti-arthritis drug, which decreased CCL22 and CCR4, showed the same trends as the above events, and was reversed by recombinant CCL22 or STAT5 inhibitor. Collectively, anti-CCL22 induced the number of Tregs via STAT5 pathway, leading to expansion of Tregs and subsequently to control of the autoimmune reaction in RA patients. Our study provides s novel strategy for RA treatment.

Intracellular matrix Gla protein promotes tumor progression by activating JAK2/STAT5 signaling in gastric cancer.

Matrix Gla protein (MGP) has been widely reported as an extracellular matrix protein with abnormal expression in various types of cancer. However, the function of intracellular MGP in gastric cancer (GC) cells remains largely unknown. Here, we demonstrated aberrantly high expression of intracellular MGP in GC as compared to adjacent normal tissues by immunohistochemistry. Moreover, The Cancer Genome Atlas (TCGA) dataset analysis suggested a positive correlation between MGP overexpression and unfavorable prognosis. MGP silencing reduced cell proliferation, migration, invasion, and survival in GC cell lines. Gene set enrichment analysis of TCGA dataset indicated significant enrichment of the IL2-STAT5 signaling in MGP-high GC patients. Immunofluorescence staining and immunoprecipitation showed that MGP binds to p-STAT5 in the nuclei of GC cells. Furthermore, ChIP-qPCR and luciferase reporter assays indicated that MGP acts as a transcriptional co-activator through the enhancement of STAT5 binding to target gene promoters. Use of STAT5 inhibitor revealed that the oncogenic functions of intracellular MGP mainly depend on the JAK2/STAT5 signaling pathway. Taken together, our results indicate that intracellular MGP promotes proliferation and survival of GC cells by acting as a transcriptional co-activator of STAT5. The detected aberrant, high MGP expression in GC tissues highlights MGP as a potential new prognostic biomarker in patients with GC.

You-Gui-Yin improved the reproductive dysfunction of male rats with chronic kidney disease via regulating the HIF1α-STAT5 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: You-Gui-Yin (YGY) is a famous Chinese traditional medicine compound that has been used to treat renal function diseases for more than 300 years. It is recorded in Jing Yue Quanshu, which was written by a famous medical scientist named Jiebing Zhang in the Ming Dynasty. AIM OF THE STUDY: Reproductive dysfunction is one of the most serious complications of chronic kidney disease (CKD). The aim of this study was to observe the effect of You-Gui-Yin (YGY) on reproductive dysfunction of male rats with adenine-induced CKD and to determine if any effects occurred via regulation of the HIF1α-STAT5 pathway. MATERIALS AND METHODS: UPLC-Q-TOF-MS was used to detect the main medicinal components and conduct quality control of YGY. A total of 60 rats were randomly divided into 2 groups: the NC group (10 rats) and the CKD model group (50 rats). The CKD model rats was established by administration of adenine 150 mg kg-1 orally for 14 days. After that, the CKD rats were randomly divided into 5 groups: the CKD group, YGY (10 g kg-1 group, 20 g kg-1 group, 40 g kg-1 group) and the GUI-LU-ER-XIAN-JIAO (GL) 10 g kg-1 group with 10 rats in each group. From the 15th day to the 45th day rats were given 150 mg kg-1 adenine orally every other day to maintain the model (except in the NC group). The YGY groups and the GL group were orally administered the relevant drug once per day for 30 days. The NC group and the CKD group were orally administered an equal volume of normal saline for 30 days. On the 45th day, the rats' sexual behavior index was tested. On the 46th day, the rats were sacrificed. Biochemical indexes, histopathological changes of the kidneys and testes, sperm morphology, sperm abnormality rate, and key proteins in the HIF1α-STAT5 pathway in the kidney and testis were detected. RESULTS: Thirteen components in the YGY extract were identified by UPLC-Q-TOF-MS for quality control of the YGY extract. The results of the biochemical and physiological tests validated the success of inducing CKD accompanied by reproductive dysfunction in rats. YGY significantly retarded the CKD progression and improved the hormone levels of male CKD rats. Sexual behavior tests showed YGY can significantly improve CKD rats' sexual function. In addition, the pathological changes of the kidney and testis, sperm abnormality rate and sperm morphological abnormalities of the CKD rats were reduced by YGY. Furthermore, decreased expression of HIF1α and EPO, and increased expression of p-EPOR (Tyr368), p-JAK2 (Tyr570) and p-STAT5 (Ser725) were observed in the kidney and the testis of the CKD rats. The YGY extract dramatically increased the expression of HIF1α and EPO, and decreased the expression of p-EPOR (Tyr368), p-JAK2 (Tyr570) and p-STAT5 (Ser725) to regulate key proteins in the HIF1α-STAT5 pathway of the kidney and testis. CONCLUSIONS: YGY has obvious reversal effects on the abnormal symptoms of adenine-induced CKD and the abnormal symptoms of rats with hypothyroidism and male reproductive hypotension. Its mechanism is related to its ability to regulate the HIF1α-STAT5 pathway.