γ-Synuclein promotes proliferation and inhibits apoptosis of oral squamous cell carcinoma via JAK2/STAT5b signaling pathway.

γ-Synuclein (SNCG) has various biological functions associated with tumorigenesis. However, the role of SNCG in oral squamous cell carcinoma (OSCC) remains unknown. In this study, we found that SNCG expression is associated with the malignancy of OSCC. We showed that SNCG promotes cell proliferation and inhibits apoptosis in OSCC. Mechanistically, we demonstrated for the first time, that SNCG interacts with ERK1/2 and promotes its phosphorylation leading to activation of the JAK2/STAT5b signaling pathway. Subsequent experiments with STAT5b interference and ERK1/2 inhibitor treatment reversed the effects of SNCG on OSCC cell proliferation, apoptosis and cell cycle progression. Our findings suggest that SNCG functions as an oncogene in OSCC by targeting the JAK2/STAT5b axis and thus may be a potential new prognostic marker and therapeutic target in OSCC.

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.

Anticancer Effects of 6-Gingerol through Downregulating Iron Transport and PD-L1 Expression in Non-Small Cell Lung Cancer Cells.

Iron homeostasis is considered a key factor in human metabolism, and abrogation in the system could create adverse effects, including cancer. Moreover, 6-gingerol is a widely used bioactive phenolic compound with anticancer activity, and studies on its exact mechanisms on non-small cell lung cancer (NSCLC) cells are still undergoing. This study aimed to find the mechanism of cell death induction by 6-gingerol in NSCLC cells. Western blotting, real-time polymerase chain reaction, and flow cytometry were used for molecular signaling studies, and invasion and tumorsphere formation assay were also used with comet assay for cellular processes. Our results show that 6-gingerol inhibited cancer cell proliferation and induced DNA damage response, cell cycle arrest, and apoptosis in NSCLC cells, and cell death induction was found to be the mitochondrial-dependent intrinsic apoptosis pathway. The role of iron homeostasis in the cell death induction of 6-gingerol was also investigated, and iron metabolism played a vital role in the anticancer ability of 6-gingerol by downregulating EGFR/JAK2/STAT5b signaling or upregulating p53 and downregulating PD-L1 expression. Also, 6-gingerol induced miR-34a and miR-200c expression, which may indicate regulation of PD-L1 expression by 6-gingerol. These results suggest that 6-gingerol could be a candidate drug against NSCLC cells and that 6-gingerol could play a vital role in cancer immunotherapy.

Abnormal Changes of IL3/IL3R and Its Downstream Signaling Pathways in the Prion-Infected Cell Line and in the Brains of Scrapie-Infected Rodents.

Interleukin 3 (IL-3) plays an important role in hematopoiesis and immune regulation, brain IL-3/IL-3R signaling has been shown to involve in the physiological and pathological processes of a variety of neurodegenerative diseases, but its role in prion diseases is rarely described. Here, the changes of IL-3/IL-3R and its downstream signaling pathways in a scrapie-infected cell line and in the brains of several scrapie-infected rodent models were evaluated by various methods. Markedly decreased IL-3Rα were observed in the brains of scrapie-infected rodents at terminal stage and in the prion-infected cell model, which showed increased in the brain samples collected at early and middle stage of infection. The IL-3 levels were almost unchanged in the brains of scrapie-infected mice and in the prion-infected cell line. Morphological assays identified close co-localization of the increased IL-3Rα signals with NeuN- and Iba1-positive cells, whereas co-localization of IL-3 signals with NeuN- and GFAP-positive cells in the scrapie-infected brain tissues. Some downstream components of IL-3/IL-3R pathways, including JAK2-STAT5 and PI3K/AKT/mTOR pathways, were downregulated in the brains of scrapie-infected rodents at terminal stage and in the prion-infected cells. Stimulation of recombinant IL-3 on the cultured cells showed prion that the prion-infected cells displayed markedly more reluctant responses of JAK2-STAT5 and PI3K/AKT/mTOR pathways than the normal partner cells. These data suggest that although prion infection or PrPSc accumulation in brain tissues does not affect IL-3 expression, it significantly downregulates IL-3R levels, thereby inhibiting the downstream pathways of IL-3/IL-3R and blocking the neuroregulatory and neuroprotective activities of IL-3.

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.

Effect of different cooking methods on saponin content and hematopoietic effects of Panax notoginseng-steamed chicken on mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Panax notoginseng-steamed chicken (PNSC) is a medicinal food with ethnic characteristics developed by the Miao ethnic group in the southeast of Yunnan Province, China. PNSC has been eaten for hundreds of years, and its tonic effect has been widely recognized by the people. However, its cooking conditions and scientific connotation of its effect of toning blood and supplementing deficiency are also lack of in-depth analysis. AIM OF THE STUDY: To optimize the cooking conditions of Panax notoginseng-steamed chicken (PNSC) and to explore its anemia-improving effects. MATERIALS AND METHODS: The ratio of P. notoginseng (PN) to chicken and the steaming time were systematically altered, and the PNSC cooking conditions was optimized with the response surface method. By establishing animal models of postpartum blood-deficiency anemia, acute hemorrhagic anemia and myelosuppressive anemia, the blood replenishing effect of PNSC was explored, and the blood replenishing mechanism of PNSC on myelosuppressive anemia was revealed by immunoblotting analyses and histopathological sectioning. RESULTS: The optimal processing conditions included a ratio of chicken to P. notoginseng of 100:5 and a steaming time of 5.5 h. The amounts of P. notoginseng polysaccharides (PNPS), total protein and blood-enriching P. notoginseng saponins were 44.3 mg/g, 2.48% and 2.04%, respectively. Freeze-dried powder of P. notoginseng steamed chicken soup (FPSC) was found to promote the recovery of routine blood factors and organ indexes in the three models of anemia and to activate the JAK2-STAT5 signaling pathway, induce phosphorylation of JAK2 and STAT5 and normalize the secretion of hematopoietic regulators EPO, IL-3, and TNF-α. CONCLUSION: FPSC improves the symptoms of anemia in mice, and it plays a role in tonifying blood by activating the JAK2-STAT5 signaling pathway and altering the expression of hematopoiesis-related factors.

5-Hydroxytryptophan inhibits ß-casein biosynthesis and promotes goat mammary epithelial cell apoptosis through the JAK2/STAT5a axis and the HTR7.

5-Hydroxytryptamine (5-HT) is an amine produced in both the mammary gland and the central nervous system. Tryptophan hydroxylase 1 (TPH1) catalyzes the conversion of 5-hydroxytryptophan (5-HTP) into l-tryptophan, which is then converted into 5-HT by monoamine-oxidase (MAO-A). In the mammary gland, 5-HT has been shown to have a variety of paracrine-autocrine actions, including suppressing lactation, controlling the destiny of mammary epithelial cells, and maintaining calcium homeostasis throughout the transition from pregnancy to lactation. To examine the effects of 5-HT on the composition of colostrum and milk, a total of 30 transition Guan Zhong dairy goats were intramuscularly injected with 5-HTP (1.0 mg/kg) every morning before feeding from 10 d before the projected parturition date to the day of parturition. The average number of days animals received injections was 8.2 ± 3.2 d. 5-HTP treatment increased serum 5-HT concentration from days 5 to 2 relative to parturition (P < 0.05), and decreased the casein concentration of colostrum (P < 0.05). In the in vitro experiment, mammary epithelial cells isolated from three individual goats' mammary glands were separately treated with 200 µM 5-HTP, 30 µM PCPA (the specific inhibitor of TPH1), or 200 µM 5-HTP + 50 µM SB269970 (the selective antagonist of 5-HTR7). The results showed that 200 µM 5-HTP inhibited the expression of ß-casein, downregulated the activity of the JAK2/ STAT5a signaling pathway, and promoted the apoptosis of goat mammary epithelial cells (GMECs) (P < 0.05). When GMECs were treated with 30 µM Four-chloro-dl-phenylalanine (PCPA), a specific inhibitor of 5-HT synthesis, the mRNA expression of STAT5a and the phosphorylated STAT5a protein level were upregulated. The 50 µM SB269970 treatment rescued the effects of 5-HTP on GMECs (P < 0.05). Taken together, the results indicated that 5-HTP exerted an inhibitory effect on ß-casein synthesis and a proapoptotic effect in GMECs via HTR7 and the JAK2/STAT5a axis.

5-Hydroxytryptamine (5-HT), which is produced in both the mammary gland and the central nervous system, is a recognized important regulator of mammary gland homeostasis. Casein is the major protein in the milk of mammals including cows, goats, and humans, and is a crucial source of high-quality amino acids for humans. In this study, prenatal intramuscular injection of 5-hydroxytryptophan (5-HTP), the precursor of 5-HT, not only increased the level of 5-HT in the serum of goats before delivery but also decreased the concentration of casein in colostrum. Furthermore, in goat mammary epithelial cells which are responsible for milk synthesis, it was found that 5-HTP blocked genes and signal pathways related to casein synthesis, and also promoted cell apoptosis. Additional results demonstrated that the type 7 5-HT receptor (HTR7) mediated the impacts of 5-HT, which provided a potential reliable target for improving milk quality.

Combined treatment with ruxolitinib and MK-2206 inhibits the JAK2/STAT5 and PI3K/AKT pathways via apoptosis in MDA-MB-231 breast cancer cell line.

BACKGROUND: Due to deficiencies in the expression of hormone receptors, such as PR, ER and HER2, it is challenging to treat triple-negative breast cancer, which does not respond to single targeted therapy. Ruxolitinib is a Janus kinase (JAK)1/JAK2 inhibitor. MK-2206 is an allosteric AKT inhibitor. Due to the limited activities of ruxolitinib and MK-2206 for monotherapy, the need for cotreatment with other drugs has emerged. This study is the first to examine the effects of ruxolitinib and MK-2206 cotreatment on apoptosis and JAK2/STAT5 and PI3K/AKT signaling in MDA-MB-231 breast cancer cells. Additionally, this work aimed to decrease the side effects of ruxolitinib and increase its anticancer effects with MK-2206 cotreatment. METHODS AND RESULTS: Cell viability was reduced in a dose- and time-dependent manner after exposure to ruxolitinib, MK-2206 or both for 48 h, as shown by MTT assay. Ruxolitinib had a synergistic antiproliferative effect, as demonstrated by colony formation and wound healing assays. The effects of ruxolitinib, MK-2206 and their combination on apoptosis, as well as PI3K/AKT and JAK/STAT signaling, were examined by western blot analyses. Cotreatment with ruxolitinib and MK-2206 reduced proliferation with the dual inhibition of JAK2/STAT5 and PI3K/AKT signaling by decreasing PI3K, AKT, JAK2, STAT5, Caspase-9, Caspase-7, PARP, c-Myc, and Bcl-2 and increasing P53 and PTEN protein expression. CONCLUSIONS: Our results revealed the roles of P53 and PTEN in the regulation of apoptosis and the PI3K/AKT and JAK2/STAT5 signaling pathways. The dual inhibition of JAK2/STAT5 and PI3K/AKT may reduce metastasis by decreasing tumor cell survival.

Proteomics reveals that Di Dang decoction can regulate the Jak2/Stat5 signaling pathway and inhibit apoptosis by reducing the oxidative stress response in rats with acute intracerebral hemorrhagic stroke.

ETHNOPHARMACOLOGICAL RELEVANCE: Di Dang decoction (DDD) is a prescription used for the treatment of cerebral hemorrhage. Its use is derived from the theory of typhoid fever, it has an obvious clinical effect and it has been used in the clinic for a long time. The results of early quantitative proteomics and targeted proteomics studies showed that the administration of high-dose DDD 7 days may regulate the expression of the proteins S100A8, S100A9, Col1a1 and Col1a2. The first 3 days after bleeding begins is the critical period for intervention, what occurs within approximately 3 days after AICH is unclear. AIM OF THE STUDY: To explore the effects of Di Dang decoction (DDD) on the Jak2/Stat5 signaling pathway and apoptosis-related gene expression in rats with acute hemorrhagic stroke via the oxidative stress response by proteomics to reveal its neuroprotective mechanism. MATERIALS AND METHODS: Ninety healthy Sprague-Dawley (SD) rats were randomly divided into the control, model, and low-, medium-, and high-dose DDD groups, with 18 rats in each group. An acute intracerebral hemorrhage (AICH) model was established by injecting autologous blood into the caudate nucleus. The low-, medium- and high-dose groups were intragastrically administered 0.15625 g/mL, 0.3125 g/mL and 0.625 g/mL DDD, respectively, for 1 or 3 days. The control and model groups were given the same amount of normal saline. Neurological deficits were evaluated by the modified neurological severity score (mNSS) test, brain water content was measured to assess brain tissue damage, and pathological changes in the lesion site were observed by hematoxylin and eosin (HE) staining. The cerebral cortex was selected for quantitative proteomics, and >1.2/1 and <1/1.2 were used as the thresholds for upregulated and downregulated proteins, respectively. KEGG pathway and Gene Ontology (GO) enrichment analyses of the differentially expressed proteins were conducted. The levels of the oxidative stress markers malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) were measured by enzyme-linked immunosorbent assay (ELISA). Western blotting was used to assess p-Jak2, Jak2, p-Stat5, Stat5, Bax, Bcl-2, and Caspase-3 protein expression. RESULTS: Compared with the model group, the group treated with high-dose DDD for 3 days exhibited significant improvements in neurological defects, brain histopathology, and brain edema; reduced the level of MDA and significantly increased the levels of CAT and SOD; significantly decreased p-Jak2 and p-Stat5 protein expression and expression of the pro-apoptotic genes Bax and c-Caspase-3; and significantly increased expression of the anti-apoptotic gene Bcl-2 (all p＜0.05). CONCLUSIONS: High-dose DDD administration for 3 days reduces the oxidative stress response, regulates the Jak2/Stat5 signaling pathway and inhibits apoptosis to exert a neuroprotective effect in rats with acute hemorrhagic stroke.

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.

Ruxolitinib inhibits cytokine production by human lung macrophages without impairing phagocytic ability.

Background: The Janus kinase (JAK) 1/2 inhibitor ruxolitinib has been approved in an indication of myelofibrosis and is a candidate for the treatment of a number of inflammatory or autoimmune diseases. We assessed the effects of ruxolitinib on lipopolysaccharide (LPS)- and poly (I:C)-induced cytokine production by human lung macrophages (LMs) and on the LMs' phagocytic activity. Methods: Human LMs were isolated from patients operated on for lung carcinoma. The LMs were cultured with ruxolitinib (0.5 × 10-7 M to 10-5 M) or budesonide (10-11 to 10-8 M) and then stimulated with LPS (10 ng·ml-1) or poly (I:C) (10 µg·ml-1) for 24 h. Cytokines released by the LMs into the supernatants were measured using ELISAs. The phagocytosis of labelled bioparticles was assessed using flow cytometry. Results: Ruxolitinib inhibited both the LPS- and poly (I:C)-stimulated production of tumor necrosis factor alpha, interleukin (IL)-6, IL-10, chemokines CCL2, and CXCL10 in a concentration-dependent manner. Ruxolitinib also inhibited the poly (I:C)- induced (but not the LPS-induced) production of IL-1ß. Budesonide inhibited cytokine production more strongly than ruxolitinib but failed to mitigate the production of CXCL10. The LMs' phagocytic activity was not impaired by the highest tested concentration (10-5 M) of ruxolitinib. Conclusion: Clinically relevant concentrations of ruxolitinib inhibited the LPS- and poly (I:C)-stimulated production of cytokines by human LMs but did not impair their phagocytic activity. Overall, ruxolitinib's anti-inflammatory activities are less intense than (but somewhat different from) those of budesonide-particularly with regard to the production of the corticosteroid-resistant chemokine CXCL-10. Our results indicate that treatment with a JAK inhibitor might be a valuable anti-inflammatory strategy in chronic obstructive pulmonary disease, Th1-high asthma, and both viral and non-viral acute respiratory distress syndromes (including coronavirus disease 2019).

Breast Cancer and Prolactin - New Mechanisms and Models.

The pathogenesis of breast cancer is driven by multiple hormones and growth factors. One of these, prolactin (PRL), contributes to both mammary differentiation and oncogenesis, and yet the basis for these disparate effects has remained unclear. The focus of this review is to examine and place into context 2 recent studies that have provided insight into the roles of PRL receptors and PRL in tumorigenesis and tumor progression. One study provides novel evidence for opposing actions of PRL in the breast being mediated in part by differential PRL receptor (PRLr) isoform utilization. Briefly, homomeric complexes of the long isoform of the PRLr (PRLrL-PRLrL) promotes mammary differentiation, while heteromeric complexes of the intermediate and long PRLr (PRLrI-PRLrL) isoforms trigger mammary oncogenesis. Another study describes an immunodeficient, prolactin-humanized mouse model, NSG-Pro, that facilitates growth of PRL receptor-expressing patient-derived breast cancer xenografts. Evidence obtained with this model supports the interactions of physiological levels of PRL with estrogen and ERBB2 gene networks, the modulatory effects of PRL on drug responsiveness, and the pro-metastatic effects of PRL on breast cancer. This recent progress provides novel concepts, mechanisms and experimental models expected to renew interest in harnessing/exploiting PRLr signaling for therapeutic effects in breast cancer.

Methylsulfonylmethane relieves cobalt chloride-induced hypoxic toxicity in C2C12 myoblasts.

AIMS: In biology and medicine, hypoxia refers to reduced oxygen tension or oxygen starvation resulting from various environmental or pathological conditions. Prolonged hypoxia may lead to an imbalance in protein production and a loss of muscle mass in animals. The physiological response to hypoxia includes oxidative stress-induced activation of complex cell-signaling networks such as hypoxia-inducible factor (HIF), phosphoinositide 3-kinase (PI3K), and Janus kinase/signal transducer and activator of transcription (JAK-STAT). Methylsulfonylmethane (MSM) is a natural sulfur compound that regulates HIF-1α expression and provides cytoprotection from oxidative stress. In this study, we explored the anti-hypoxic activity and cytoprotective effect of MSM in cobalt chloride (CoCl2)-induced hypoxic C2C12 mouse myoblast culture. MATERIALS AND METHODS: We used western blotting, real time PCR, flow cytometry for molecular signaling studies and we also used MTT assay and ChIP assay along with comet assay for cellular processes. KEY FINDINGS: MSM prevented the CoCl2 induced cytotoxicity. Molecular markers of hypoxia, induced by CoCl2, were normalized or reduced by MSM, which also inhibited the effect of CoCl2-induced JAK2/STAT5b/Cyclin D1 and PI3K/AKT signaling. CoCl2-induced oxidative stress results in activation of the NRF2/HO-1-mediated cell survival pathway and inhibition of DNA repair, both of which were prevented by MSM. SIGNIFICANCE: We suggest MSM can be considered as a candidate drug for reducing the effects of hypoxia in both animals and humans.

Nicotinamide mononucleotide ameliorates DNFB-induced atopic dermatitis-like symptoms in mice by blocking activation of ROS-mediated JAK2/STAT5 signaling pathway.

BACKGROUND AND PURPOSE: Atopic dermatitis (AD) is a chronic inflammatory skin disease, characterized by pruritus and impaired skin barrier function. The pathology of AD involves in immune dysfunction and epidermal barrier disruption. Reactive oxygen species (ROS) are found to be associated with AD, and play a role in the immunological abnormalities and dysfunctional skin barrier. Nicotinamide mononucleotide (NMN) plays an important role in oxidative stress related diseases, but its role in AD is unclear. METHODS: KM mice were treated with DNFB to induce AD-like lesion and typical applied with NMN for two weeks. The dermatitis score, the degree of itching and TEWL were evaluated during modeling. Epidermal thickness of skin lesions and histopathological changes were detected. Further, inflammatory factors, epidermal differentiation-related genes, oxidative stress indicators and JAK2/STAT5 signaling pathway were evaluated. NHEK cells were stimulated by TNF-α/IFN-γ after pre-treatment with NMN, then ROS levels, inflammatory factors and JAK2/STAT5 signaling pathway were detected. RESULTS: NMN exhibited potent anti-atopic activities, shown by alleviated AD-like symptoms, inhibited the increased expression of inflammatory cytokines and restored proteins and mRNA level of skin barrier genes. In addition, NMN inhibited TNF-α/IFN-γ-stimulated elevation of inflammatory chemokines, which was associated with blocking the activation of ROS-mediated JAK2/STAT5 pathway. CONCLUSION: NMN may have a positive effect on relieving symptoms of AD.

Mercury Chloride Impacts on the Development of Erythrocytes and Megakaryocytes in Mice.

Inorganic mercury (Hg2+) is a highly toxic heavy metal. The aim of this study was to investigate the impact of Hg2+ on the development of erythrocytes and megakaryocytes. B10.S mice (H-2s) and DBA/2 mice (H-2d) were administrated with 10 µM HgCl2 or 50 µM HgCl2 via drinking water for four weeks, and erythro-megakaryopoiesis was evaluated thereafter. The administration of 50 µM HgCl2 increased the number of erythrocytes and platelets in B10.S mice, which was not due to a reduced clearance for mature erythrocytes. The administration of 50 µM HgCl2, but not 10 µM HgCl2, increased the number of progenitors for erythrocytes and megakaryocytes in the bone marrow (BM) of B10.S mice, including erythroid-megakaryocyte progenitors (EMPs), burst-forming unit-erythroid progenitors (BFU-Es), colony-forming unit-erythroid progenitors (CFU-Es), and megakaryocyte progenitors (MkPs). Moreover, 50 µM HgCl2 caused EMPs to be more proliferative and possess an increased potential for differentiation into committed progenies in B10.S mice. Mechanistically, 50 µM HgCl2 increased the expression of the erythropoietin receptor (EPOR) in EMPs, thus enhancing the Jak2/STAT5 signaling pathway to promote erythro-megakaryopoiesis in B10.S mice. Conversely, 50 µM HgCl2 did not impact erythro-megakaryopoiesis in DBA/2 mice. This study may extend our current understanding for hematopoietic toxicology of Hg.

Biological Functions of Let-7e-5p in Promoting the Differentiation of MC3T3-E1 Cells.

MicroRNAs let-7c and let-7f, two members of the let-7 family, were involved in regulating osteoblast differentiation and have an important role in bone formation. Let-7e-5p, which also belonged to the let-7 family, presented in the differentiation of adipose-derived stem cells and mouse embryonic stem cells. However, the role of let-7e-5p in osteoblast differentiation was unclear. Thus, this study aimed to elucidate the function of let-7e-5p in osteoblast differentiation and its mechanism. Firstly, we found that the let-7e-5p mimic promoted osteoblast differentiation but not the proliferation of MC3T3-E1 cells by positively regulating the expression levels of osteogenic-associated genes (RUNX2, OCN, OPN, and OSX), the activity of ALP, and formation of mineralized nodules. Moreover, we ascertained that the let-7e-5p mimic downregulated the post-transcriptional expression of SOCS1 by specifically binding to the 3' untranslated region of SOCS1 mRNA. Also, let-7e-5p-induced SOCS1 downregulation increased the protein levels of p-STAT5 and IGF-1, which were both modulated by SOCS1 molecules. Furthermore, let-7e-5p abrogated the inhibition of osteogenic differentiation mediated by SOCS1 overexpression. Therefore, these results suggested that let-7e-5p regulated the differentiation of MC3T3-E1 cells through the JAK2/STAT5 pathway to upregulate IGF-1 gene expression by inhibiting SOCS1. These findings may provide a new insight into the regulatory role of let-7e-5p in osteogenic differentiation and imply the existence of a novel mechanism underlying let-7e-5p-mediated osteogenic differentiation.

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.

Silibinin Regulates Tumor Progression and Tumorsphere Formation by Suppressing PD-L1 Expression in Non-Small Cell Lung Cancer (NSCLC) Cells.

Recently, natural compounds have been used globally for cancer treatment studies. Silibinin is a natural compound extracted from Silybum marianum (milk thistle), which has been suggested as an anticancer drug through various studies. Studies on its activity in various cancers are undergoing. This study demonstrated the molecular signaling behind the anticancer activity of silibinin in non-small cell lung cancer (NSCLC). Quantitative real-time polymerase chain reaction and Western blotting analysis were performed for molecular signaling analysis. Wound healing assay, invasion assay, and in vitro angiogenesis were performed for the anticancer activity of silibinin. The results indicated that silibinin inhibited A549, H292, and H460 cell proliferation in a concentration-dependent manner, as confirmed by the induction of G0/G1 cell cycle arrest and apoptosis and the inhibition of tumor angiogenesis, migration, and invasion. This study also assessed the role of silibinin in suppressing tumorsphere formation using the tumorsphere formation assay. By binding to the epidermal growth factor receptor (EGFR), silibinin downregulated phosphorylated EGFR expression, which then inhibited its downstream targets, the JAK2/STAT5 and PI3K/AKT pathways, and thereby reduced matrix metalloproteinase, PD-L1, and vascular endothelial growth factor expression. Binding analysis demonstrated that STAT5 binds to the PD-L1 promoter region in the nucleus and silibinin inhibited the STAT5/PD-L1 complex. Altogether, silibinin could be considered as a candidate for tumor immunotherapy and cancer stem cell-targeted therapy.