Mechanism of Dihuangyin in Treatment of Mice with Atopic Dermatitis by Regulating JAK1/STAT3 Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the effect and potential mechanism of Dihuangyin on 2， 4-dinitrochlorobenzene （DNCB） -induced model mice with atopic dermatitis （AD）. MethodA mouse model with AD was established by repeatedly stimulating the back skin of mice with DNCB. After successful modeling， the mice were randomly divided into model group， Runzao group （0.78 g·kg-1）， and high， medium， and low dose （40.30， 20.15， and 10.08 g·kg-1） groups of Dihuangyin， with 12 mice in each group， and the blank group consisted of 12 mice， 72 in total. The administration groups were given the corresponding liquid by dose， and the blank group and model group were given the same dose of pure water by intragastric administration， once a day. The skin lesions and scratching times of mice were observed after continuous administration for two weeks. The back skin lesions of mice were stained with hematoxylin-eosin （HE） and toluidine blue to observe the pathology. The contents of serum immunoglobulin E （IgE）， interleukin-4 （IL-4）， interleukin-6 （IL-6）， and interferon-γ （IFN-γ） were detected by enzyme-linked immunosorbent assay （ELISA）. The mRNA expression levels of IFN-γ， IL-4， IL-6， Janus kinase 1 （JAK1）， and transcriptional activator 3 （STAT3） in skin lesion tissue were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. The expressions of JAK1， phosphorylation（p）-JAK1， STAT3， and p-STAT3 proteins in skin lesion tissue were detected by Western blot. ResultCompared with the blank group， the back skin of the model group showed large-scale scab， dryness， erosion， hypertrophy with scratching， epidermal hyperplasia with hyperkeratosis and parakeratosis， hyperacanthosis with edema， and a large number of mast cell infiltration in the dermis， some of which were degranulated. The contents of IgE， IL-4， IL-6， and IFN-γ in the serum of mice were significantly increased （P<0.01）， and the protein expression levels of p-JAK1， STAT3， and p-STAT3 and mRNA expressions of IL-4， IL-6， IFN-γ， JAK1， and STAT3 in skin lesion tissue were significantly increased （P<0.01）. Compared with the model group， only a small amount of dryness and desquamation were observed in the back skin of mice in each administration group， and cell edema was reduced. The inflammatory infiltration was significantly reduced， and the number of mast cell infiltration was significantly decreased. The serum IgE， IL-4， IL-6， and IFN-γ of mice were decreased to varying degrees （P<0.05， P<0.01）. The protein expression levels of p-JAK1， STAT3， and p-STAT3 and mRNA expressions of IL-4， IL-6， IFN-γ， JAK1， and STAT3 in skin lesion tissue were significantly decreased， and the effect of high dose group of Dihuangyin was the best （P<0.01）. ConclusionDihuangyin can improve skin lesions and pruritus in mice with AD， and its mechanism may be related to the effective regulation of cytokines on the helper T cells （Th1）/Th2 axis by interfering with the JAK1/STAT3 signaling pathway and affecting skin barrier function.

Curcumin Induces Cycle Arrest of Colon Cancer HCT116 Cells via JAK1/STAT1/p21 Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the effect of curcumin on the cycle arrest of human colon cancer HCT116 cells and decipher the possible molecular mechanism. MethodThe methyl thiazolyl tetrazolium （MTT） method was employed to examine the effects of curcumin （0， 12.5， 25， 50， 75， 100 μmol·L-1） and 5-fluorouracil （5-FU， 600 μmol·L-1） on the proliferation of HCT116 cells at different time points （24， 48， 72 h）. Flow cytometry was employed to examine the cycle of HCT116 cells treated with curcumin （0， 25， 50， 75 μmol·L-1） and 5-FU. Western blot was employed to determine the expression of proteins in the Janus kinase 1 （JAK1）/signal transducer and activator of transcription 1 （STAT1） /cyclin-dependent kinase inhibitor 1A （p21） pathway in HCT116 cells. The binding of STAT1 to p21 promoter region was detected by chromatin immunoprecipitation （ChIP）. Small interfering RNA （siRNA） was employed to measure the role of STAT1 in regulating the expression of p21 and that of JAK1 in regulating the activation of STAT1 by Western blot and cellular immunofluorescence， respectively. ResultCompared with the blank group， the HCT-116 cells treated with curcumin and 5-FU showed decreased viability （P<0.05）， increased proportions of cells in the G0/G1 phase （P<0.05）， decreased proportions of cells in the S phase and G2/M phase （P<0.05）， down-regulated protein level of phosphorylated p21 （P<0.05）， and up-regulated protein level of p21 （P<0.05）. Compared with the curcumin group， the p21 siRNA+ curcumin group presented decreased proportion of cells in G0/G1 phase （P<0.05）. Compared with the blank group， curcumin elevated the level of phosphorylated STAT1 （p-STAT1） （P<0.05）. Compared with the curcumin group， the curcumin + STAT1 siRNA group showcased up-regulated protein level of p21 in HCT116 cells （P<0.05）. The mechanism study showed that curcumin treatment enhanced the enrichment of STAT1 in the p21 promoter region （P<0.05） compared with the blank group. Compared with the blank group， curcumin up-regulated the level of phosphorylated JAK1 （p-JAK1） （P <0.05）. Compared with the curcumin group， the curcumin + STAT1 siRNA group demonstrated up-regulated protein levels of p-STAT1 and p21 in HCT116 cells （P<0.05）. ConclusionCurcumin may induce the cycle arrest of human colon cancer HCT116 cells by activating the JAK1/STAT1/p21 signaling pathway.

Ameliorative Effect and Mechanism of Qingwen Baiduyin on Lipopolysaccharide-induced Acute Lung Injury / 中国实验方剂学杂志

To investigate the therapeutic effect and mechanism of Qingwen Baiduyin on acute lung injury （ALI） in mice induced by lipopolysaccharide （LPS）. MethodA total of 144 C57BL/6 mice were randomly divided into the following groups： a normal group， a model group （LPS， 5 mg·kg-1）， a dexamethasone group （5 mg·kg-1）， and low-， medium-， and high-dose Qingwen Baiduyin groups （14.105， 28.21， 56.42 g·kg-1）. The mice were treated once daily for 5 days. One hour after the final administration， the ALI model was established by intratracheal instillation of LPS， and samples were collected at 6 h and 24 h after modeling. The arterial blood gas index of mice was analyzed. The total protein content， total cell count， Evans blue dye （EBD） content， and lung tissue wet-to-dry weight ratio （W/D） of bronchoalveolar lavage fluid （BALF） were measured. Hematoxylin-eosin （HE） staining was performed to assess the pathological changes in mouse lung tissue. Western blot was used to detect the expression levels of key proteins in the Janus kinase 1/signal transducer and activator of transcription 1/interferon regulatory factor 1 （JAK1/STAT1/IRF1） signaling pathway in lung tissue. ResultCompared with the normal group， the model group showed reduced arterial oxygen pressure （pO2）， oxygen saturation （SO2）， and lung tissue W/D （P<0.05， P<0.01）， increased carbon dioxide pressure （pCO2）， total protein content， total cell count， EBD content， interferon-γ （IFN-γ）， tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， chemokine CXC ligand 1 （CXCL1）， chemokine CXC ligand 2 （CXCL2）， chemokine CXC ligand 9 （CXCL9）， and chemokine CXC ligand 10 （CXCL10） content （P<0.05， P<0.01）， thickening of the alveolar walls， fusion of alveolar cavities， and infiltration of inflammatory cells in lung tissue， increased proportion of M1 macrophage polarization and lung cell apoptosis （P<0.05）， and increased protein expression levels of JAK1， phosphorylated JAK1 （p-JAK1）， inducible nitric oxide synthase （iNOS）， STAT1， phosphorylated STAT1 （p-STAT1）， IRF1， gasdermin D （GSDMD）， and mixed lineage kinase domain-like protein （MLKL） （P<0.05， P<0.01）. Compared with the model group， Qingwen Baiduyin significantly increased pO2， SO2， and lung tissue W/D （P<0.05， P<0.01）， improved the pathological changes in lung tissue， and reduced pCO2， total protein content， total cell count， EBD content， IFN-γ， TNF-α， IL-1β， CXCL1， CXCL2， CXCL9， and CXCL10 content， proportion of M1 macrophage polarization， and protein expression levels of JAK1， p-JAK1， iNOS， STAT1， p-STAT1， IRF1， GSDMD， and MLKL （P<0.05， P<0.01）. ConclusionQingwen Baiduyin can improve the lung inflammatory response and reduce lung cell apoptosis in mice with ALI by inhibiting the JAK1/STAT1/IRF1 signaling pathway， thereby exerting a lung-protective effect.

Leptin plays a role in the multiplication of and inflammation in ovarian granulosa cells in polycystic ovary syndrome through the JAK1/STAT3 pathway

Abstract Objectives The authors determined the level of Expression of Leptin (LEP) in Polycystic Ovary Syndrome (PCOS) patients with or without obesity and in GCs treated with insulin. Methods LEP expression was first assessed in ovary cortex specimens collected from women with PCOS with or without obesity as well as from healthy controls. Ovarian Granulosa Cells (OGCs) induced by insulin extracted from a mouse model were used in further functional research. Results Real-time PCR and western blotting indicated that LEP expression was upregulated in GCs induced by insulin, in comparison with that in GCs not induced by insulin. Furthermore, the knockdown of LEP resulted in a reduction in growth and multiplication and an increase in apoptosis and inflammation in GCs induced by insulin. Next, the authors evaluated the effect of LEP on three key pathways of inflammation (MAPK, NF-kB, and JAK1/STAT3); results showed that the JAK1/STAT3 pathway was induced by LEP knockdown, as evidenced by the upregulation of phosphor-JAK1, phosphor-STAT3, and nuclear STAT3 expression. Administration of curcumin, a specific inhibitor of STAT3, counteracted the effect of LEP knockdown on cell inflammation and apoptosis. Conclusion The present data suggest that upregulation of LEP expression in the PCOS granulosa cell model is essential for reducing apoptosis and inflammation by modulating the JAK1/STAT3 pathway axis.

Inhibition of abnormal GRK2 transmembrane transfer to reduce maturation of dendritic cells in CIA mice by regulating JAK1-STAT1 signaling pathway / 中国药理学通报

Institute of Clinical Pharmacology of Anhui Medical University, Key Laboratory of And-inflammatory and Immune Medicine, Ministry of Education .Anhui Collaborative Innovation Center of And-Inflammatory and Immune Medicine, Rheumatoid Arthritis Research Center of Anhui Medical University, Jlefei ,230032, China,Aim To reveal the role of the abnormal activation of G protein-coupled receptor kinase 2(GRK2)and abnormal signal transduction of JAK1-STAT1 in the abnormal immune response of rheumatoid arthritis(RA)by exploring the effects of GRK2 on the JAK1-STAT1 signaling pathway in dendritic cells(DCs)of collagen-induced arthritis(CIA)mice and the undelying mechanisms,so as to provide a basis for revealing the new mechanism of RA.Methods The CIA model was established,and the co-stimulatory molecular level of DCs was detected by flow cytometry,the cytokine levels of plasma in mice were detected by ELISA,and the expression of p-JAK1,p-STAT1 and GRK2 in spleen tissues was detected by immunohistochemistry.Bone marrow cells were induced into DCs in vitro and stimulated with IFN-α and PGE2 for 48 h.Flow cytometry was used to detect the level of co-stimulatory molecules and phagocytosis of DCs,and ELISA to detect the level of cytokines in cell supernatant.CO-IP was employed to detect the co-localization of GRK2 and JAK1 in DCs.Western blotting was used to detect the expression of JAK1-STAT1 and the cell membrane expression of GRK2.Imaging flow cytometry was applied to detect the nucleation rate of p-STAT1.Results In vivo the level of co-stimulatory molecules of dendritic cells of CIA mouse increased,and the expression of GRK2 and p-JAK1,p-STAT1 in spleen was positively correlated.The co-localization of GRK2 and JAK1 in spleen of the CIA group decreased significantly.In vitro GRK2 inhibitors reduced the level of costimulatory molecules,cytokines IL-6 and TNF-α,the expression of JAK1 and STAT1,the expression of GRK2 in the cell membrane,and the rate of p-STAT1 nuclear translocation,and increased the Ag uptake capacity of DCs and the co-localization rate of GRK2 and JAK1.Conclusions The abnormal GRK2 transfer to the cell membrane in DCs mediates the maturation of DCs and the activation of the JAK1-STAT1 signaling pathway.Inhibition of GRK2 transfer membrane can restore its control of the JAK1-STAT1 signal transduction of DCs,reduce the maturation of DCs,and play an important role in improving mouse CIA.

Molecular Mechanism of "Transmission Between Lung and Brain" of Influenza and Intervention Effect of Maxing Shigantang Based on JAK1/STAT1 Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo explore the molecular mechanism of "transmission between the lung and brain" of influenza based on Janus kinase 1/signal transducer and activator of transcription 1（JAK1/STAT1） signaling pathway and further investigate the intervention effect of Maxing Shigantang （MXSGT）. MethodA total of 100 SPF BALB/c mice were randomly divided into a normal group，a model group，an oseltamivir group （21.63 mg·kg-1·d-1），an antiviral granules group（3.9 g·kg-1·d-1）， and an MXSGT group（6.05 g·kg-1·d-1）， with 20 mice in each group. The pneumonia model was induced in mice except for those in the normal group by intranasal infection of influenza A virus（IAV）. Twenty-four hours after modeling，mice were treated with corresponding drugs， while those in the normal group and the model group received the same amount of normal saline by gavage， once a day for 3 and 7 days. The pathological changes in the lung and brain were observed by hematoxylin-eosin（HE）staining. The mRNA expression of IAV nucleoprotein（NP），JAK1， and STAT1 in the lung and brain was detected by real-time quantitative polymerase chain reaction（Real-time PCR）， and the protein expression of JAK1 and STAT1 in the lung and brain was detected by Western blot. Immunohistochemical method was used to detect the expression of phosphorylated（p）-STAT1 in the lung and brain tissues， and enzyme-linked immunosorbent assay（ELISA） was used to detect the serum levels of interleukin-1β（IL-1β） and interleukin-10（IL-10）. ResultCompared with the normal group， the model group showed obvious pathological changes in the lung tissues and cerebral cortex， increased relative mRNA expression of IAV NP in the lung （P<0.01）， elevated mRNA and protein expression of JAK1 and STAT1 in the lung and brain tissues （P<0.05，P<0.01），up-regulated expression level of p-STAT1 in lung tissues and cerebral cortex （P<0.05，P<0.01）， and increased serum level of IL-1β （P<0.05）. Compared with the model group， the MXSGT group showed alleviated pathological damage to lung tissues and cerebral cortex， decreased relative mRNA expression of IAV NP in lung tissues（P<0.01），reduced mRNA and protein expression levels of JAK1 and STAT1 in lung tissues and brain tissues（P<0.05，P<0.01）， and increased serum level of IL-10（P<0.01）. ConclusionThe abnormal activation of the JAK1-STAT1 signaling pathway may be one of the molecular mechanisms of "transmission between the lung and brain" of influenza. As an effective compound prescription against the influenza virus，MXSGT can alleviate the pathological damage of brain tissues in mice infected with IAV by regulating the level of cytokines mediated by this pathway.

Effect of paeoniflorin-6'-O benzene sulfonate on JAK1/STAT3 signaling pathway in rat macrophages with collagen-induced arthritis through regulating G protein coupled receptor kinase 2 / 中国药理学通报

Aim To investigate the inhibitory effect of peonia-6'-o benzene sulfonate (CP-25) on the JAK1/STAT3 signaling pathway in collagen-induced arthritis (CIA) rats macrophages through regulating G protein coupled receptor kinase 2 (GRK2). Methods SD rats were randomly divided into four groups; normal group, model group, CP-25 (50 mg · kg

The anti-neoplastic activities of aloperine in HeLa cervical cancer cells are associated with inhibition of the IL-6-JAK1-STAT3 feedback loop / 中国天然药物

Cervical cancer (CC) is recognized as the most common neoplasm in the female reproductive system worldwide. The lack of chemotherapeutic agents with outstanding effectiveness and safety severely compromises the anti-cipated prognosis of patients. Aloperine (ALO) is a natural quinolizidine alkaloid with marked anti-cancer effects on multiple malignancies as well as favorable activity in relieving inflammation, allergies and infection. However, its therapeutic efficacy and underlying mechanism in CC are still unclear. In the current study, MTT assay was employed to evaluate the viability of HeLa cells exposed to ALO to preliminarily estimate the effectiveness of ALO in CC. Then, the effects of ALO on the proliferation and apoptosis of HeLa cells were further investigated by plate colony formation and flow cytometry, respectively, while the migration and invasion of ALO-treated HeLa cells were evaluated using Transwell assay. Moreover, nude mice were subcutaneously inoculated with HeLa cells to demonstrate the anti-CC properties of ALO in vivo. The molecular mechanisms underlying these effects of ALO were evaluated by Western blot and immunohistochemical analysis. This study experimentally demonstrated that ALO inhibited the proliferation of HeLa cells via G2 phase cell cycle arrest. Simultaneously, ALO promoted an increase in the percentage of apoptotic HeLa cells by increasing the Bax/Bcl-2 ratio. Additionally, the migration and invasion of HeLa cells were attenuated by ALO treatment, which was considered to result from inhibition of epithelial-to-mesenchymal transition. For molecular mechanisms, the expression and activation of the IL-6-JAK1-STAT3 feedback loop were markedly suppressed by ALO treatment. This study indicated that ALO markedly suppresses the proliferation, migration and invasion and enhances the apoptosis of HeLa cells. In addition, these prominent anti-CC properties of ALO are associated with repression of the IL-6-JAK1-STAT3 feedback loop.

miR-140-3p inhibits progression of non-small cell lung cancer by targeting Janus kinase 1

microRNAs (miRNAs) have gained more attention due to the biological functions in many cancers, includingnon-small cell lung cancer (NSCLC). However, the roles and the mechanism of miR-140-3p in NSCLCprogression remain poorly understood. In this study, the expression levels of miR-140-3p and Janus kinase 1(JAK1) were measured in NSCLC tissues and cells by quantitative real-time PCR. Cell viability, apoptosis,migration and invasion were detected by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-trtrazolium bromide, flowcytometry, Western blot or trans-well assay, respectively. Murine xenograft model was conducted to analyzethe anti-tumor effect of miR-140-3p in vivo. Interaction between miR-140-3p and JAK1 was probed byluciferase reporter activity and Western blot. We found that miR-140-3p expression was down-regulated andJAK1 expression was increased in NSCLC tissues and cells compared with those in corresponding controls.Moreover, overexpression of miR-140-3p inhibited cell viability, migration and invasion while promoted cellapoptosis in NSCLC cells and suppressed NSCLC xenograft tumor growth in vivo. Besides, JAK1 was provedas a target of miR-140-3p and its restoration reversed miR-140-3p-mediated regulatory effect on progression ofNSCLC. We concluded that miR-140-3p inhibited NSCLC progression by targeting JAK1, providing a novelavenue for treatment of NSCLC.

Paeonol inhibits macrophage M1 polarization by down-regulating miR-155/JAK1-STAT1 pathway / 中国中药杂志

The aim of this paper was to investigate the effect and mechanism of paeonol on peritoneal macrophage M1 polarization in mice, explore whether the intervention action is related to the down-regulation of miR-155 and the inhibition of downstream JAK1-STAT1 pathway, and provide a new idea for the molecular mechanism of paeonol against atherosclerosis(AS). Lipopolysaccharide(LPS) and interferon-γ(IFN-γ) were used to stimulate macrophages for 24 hours to establish the M1 polarization model, and paeonol was given 24 hours before co-stimulation to provide a pre-protective effect on cells. CCK-8 assay was used to detect the cells damage induced by LPS and IFN-γ co-stimulation; flow cytometry was used to detect the expression of M1 surface markers F4/80 and CD86. ELISA was used to detect the secretion of interleukin 6(IL-6) and tumor necrosis factor-α(TNF-α) in supernatant. RT-qPCR was used to detect the expression of miR-155, and Western blot was used to detect the protein expression at JAK1-STAT1-SOCS1 pathway. The results showed that LPS and IFN-γ had no obvious damage to the cells at the optimal concentration, but they induced macrophages polarized to M1, resulted in high expression of M1 type marker factors F4/80 and CD86 on the cell surface, and increased secretion of IL-6 and TNF-α on the cell surface(P<0.05 or P<0.01). Paeonol significantly reduced the LPS and IFN-γ-induced high expression of F4/80 and CD86, the secretion of inflammatory factors IL-6 and TNF-α(P<0.05 or P<0.01), decreased the expression level of miR-155, significantly down-regulated the protein phosphorylation level of JAK1-STAT1 and up-regulated the protein expression of SOCS1(P<0.01) in RAW264.7 cells. The results showed that paeonol could inhibit M1 polarization of macrophages by down-regulating cell surface marker factors and inflammatory factors secreted by cells, which may be related to the down-regulation of miR-155 expression and the inhibition JAK1-STAT1 pathway activation.

Electroacupuncture protects septic rats from acute lung injury through the JAK1/STAT3 pathway / 南方医科大学学报

OBJECTIVE@#To explore the protective effect of electroacupuncture against acute lung injury (ALI) in septic rats and explore the mechanism.@*METHODS@#Sixty male SD rats were randomly divided into cecal ligation and puncture (CLP)-induced sepsis group (@*RESULTS@#Compared with those in the sham operation group, the rats in ALI group showed obvious lung pathologies with significantly increased lung W/D ratio (@*CONCLUSIONS@#Electroacupuncture can inhibit the release of inflammatory mediators and cell apoptosis via the JAK1/STAT3 pathway to reduce lung injuries in septic rats.

The anti-proliferative and anti-inflammatory mechanisms of JAK1 inhibitor SHR0302 versus Ruxolitinib in SET2 cell line and primary cells / 中华血液学杂志

Objective: To explore the effects and molecular mechanism of the selective JAK1inhibitor SHR0302 and Ruxolitinib on myeloproliterative neoplasms (MPN) cell line SET2 and primary cells in vitro. Methods: Cell proliferation was detected by CCK8 kit. Colony forming experiment was conducted to evaluate erythroid burst colony formation unit (BFU-E) of primary cells from MPN patients. Multi-factor kits were used to detect six inflammatory cytokines. Phosphorylated proteins of Jak-Stat signaling pathway were tested by Western blot. Results: At different time points after treated with SHR0302 and Ruxolitinib, the inhibition of cell proliferation was dose dependent by both drugs (P<0.01) . The inhibitory rates of 2.5 μmol/L SHR0302 and 0.1 μmol/L Ruxolitinib on SET2 cells for 72 h were comparable, i.e. (59.94±0.60) % and (64.00±0.66) %, respectively, suggesting that the inhibitory effect of SHR0302 was weaker than that of Ruxolitinib. Similarly, both SHR0302 and Ruxolitinib inhibited BFU-E in primary marrow cells from MPN patients in a dose-dependent manner. SHR0302 1.0 μmol/L produced similar degree of inhibition compared to Ruxolitinib 0.2 μmol/L. Except IL-12, the expression of other 5 cytokines (IL-6, TNF-α, IL-1β, IL-2, IL-8) was significantly inhibited by 1.6 μmol/L SHR0302 in SET2 cells at 24 h (P<0.01) , while Ruxolitinib 1.0 μmol/L had the same effect. Several phosphorylated molecules of Jak-Stat signaling pathway were significantly inhibited by SHR0302 in SET2 cells only for 3 h. P-stat1 (Tyr701) , p-stat3 (Tyr705) were down-regulated when treated with SHR0302 1.0 μmol/L (P<0.05) , p-jak1 (tyr1022/1023) and p-stat5 (Tyr694) were inhibited at 5.0 μmol/L (P<0.05) . Ruxolitinib significantly inhibited the downstream STAT protein at 0.1 μmol/L. Again, the inhibitory effect of SHR0302 on protein expression was weaker than that of Ruxolitinib. Conclusion: SHR0302 can effectively inhibit the proliferation of MPN cell line and patients' primary cells, as well as the expression of inflammatory factors. The molecular mechanism is possibly related to the down-regulation of phosphorylated proteins of Jak-Stat signaling pathway. Overall, the anti-proliferative and anti-inflammatory effects of SHR0302 are weaker than those of Ruxolitinib.

The anti-proliferative and anti-inflammatory mechanisms of JAK1 inhibitor SHR0302 versus Ruxolitinib in SET2 cell line and primary cells / 中华血液学杂志

Objective@#To explore the effects and molecular mechanism of the selective JAK1inhibitor SHR0302 and Ruxolitinib on myeloproliterative neoplasms (MPN) cell line SET2 and primary cells in vitro.@*Methods@#Cell proliferation was detected by CCK8 kit. Colony forming experiment was conducted to evaluate erythroid burst colony formation unit (BFU-E) of primary cells from MPN patients. Multi-factor kits were used to detect six inflammatory cytokines. Phosphorylated proteins of Jak-Stat signaling pathway were tested by Western blot.@*Results@#At different time points after treated with SHR0302 and Ruxolitinib, the inhibition of cell proliferation was dose dependent by both drugs (P<0.01) . The inhibitory rates of 2.5 μmol/L SHR0302 and 0.1 μmol/L Ruxolitinib on SET2 cells for 72 h were comparable, i.e. (59.94±0.60) % and (64.00±0.66) %, respectively, suggesting that the inhibitory effect of SHR0302 was weaker than that of Ruxolitinib. Similarly, both SHR0302 and Ruxolitinib inhibited BFU-E in primary marrow cells from MPN patients in a dose-dependent manner. SHR0302 1.0 μmol/L produced similar degree of inhibition compared to Ruxolitinib 0.2 μmol/L. Except IL-12, the expression of other 5 cytokines (IL-6, TNF-α, IL-1β, IL-2, IL-8) was significantly inhibited by 1.6 μmol/L SHR0302 in SET2 cells at 24 h (P<0.01) , while Ruxolitinib 1.0 μmol/L had the same effect. Several phosphorylated molecules of Jak-Stat signaling pathway were significantly inhibited by SHR0302 in SET2 cells only for 3 h. P-stat1 (Tyr701) , p-stat3 (Tyr705) were down-regulated when treated with SHR0302 1.0 μmol/L (P<0.05) , p-jak1 (tyr1022/1023) and p-stat5 (Tyr694) were inhibited at 5.0 μmol/L (P<0.05) . Ruxolitinib significantly inhibited the downstream STAT protein at 0.1 μmol/L. Again, the inhibitory effect of SHR0302 on protein expression was weaker than that of Ruxolitinib.@*Conclusion@#SHR0302 can effectively inhibit the proliferation of MPN cell line and patients' primary cells, as well as the expression of inflammatory factors. The molecular mechanism is possibly related to the down-regulation of phosphorylated proteins of Jak-Stat signaling pathway. Overall, the anti-proliferative and anti-inflammatory effects of SHR0302 are weaker than those of Ruxolitinib.

Calenduloside E inhibits lipopolysaccharide-induced inflammatory response by inhibiting activation of ROS-mediated JAK1-stat3 signaling pathway in RAW264.7 cells / 南方医科大学学报

OBJECTIVE@#To investigate the effect of calenduloside E on lipopolysaccharide (LPS)-induced inflammatory response in RAW264.7 cells and explore the underlying molecular mechanism.@*METHODS@#CCK-8 assay was used to examine the effect of different concentrations of calenduloside E (0-30 μg/mL) on the viability of RAW264.7 cells. The release of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in RAW264.7 cells in response to pretreatment with 6, 8, and 10 μg/mL calenduloside E for 2 h followed by stimulation with 100 ng/mL LPS was detected using enzyme-linked immunosorbent assay (ELISA). The expression levels of iNOS and COX-2 and the activation of JAK-stats, MAPKs and NF-кB signaling pathways in the treated cells were determined using Western blotting. A reactive oxygen species (ROS) detection kit was used to detect ROS production in the cells, and the nuclear translocation of the transcription factor stat3 was observed by laser confocal microscopy.@*RESULTS@#Calenduloside E below 20 μg/mL did not significantly affect the viability of RAW264.7 cells. Calenduloside E dose-dependently decreased the expression levels of iNOS and COX-2 induced by LPS, inhibited LPS-induced release of TNF-α and IL-1β, and suppressed LPS-induced JAK1-stat3 signaling pathway activation and stat3 nuclear translocation. Calenduloside E also significantly reduced ROS production induced by LPS in RAW264.7 cells.@*CONCLUSIONS@#Calenduloside E inhibits LPS-induced inflammatory response by blocking ROS-mediated activation of JAK1-stat3 signaling pathway in RAW264.7 cells.

Effect of lncRNA HULC knockdown on rat secreting pituitary adenoma GH3 cells

Pituitary adenoma is one of the most common tumors in the neuroendocrine system. This study investigated the effects of long non-coding RNAs (lncRNAs) highly up-regulated in liver cancer (HULC) on rat secreting pituitary adenoma GH3 cell viability, migration, invasion, apoptosis, and hormone secretion, as well as the underlying potential mechanisms. Cell transfection and qRT-PCR were used to change and measure the expression levels of HULC, miR-130b, and FOXM1. Cell viability, migration, invasion, and apoptosis were assessed using trypan blue staining assay, MTT assay, two-chamber transwell assay, Guava Nexin assay, and western blotting. The concentrations of prolactin (PRL) and growth hormone (GH) in culture supernatant of GH3 cells were assessed using ELISA. The targeting relationship between miR-130b and FOXM1 was verified using dual luciferase activity. Finally, the expression levels of key factors involved in PI3K/AKT/mTOR and JAK1/STAT3 pathways were evaluated using western blotting. We found that HULC was highly expressed in GH3 cells. Overexpression of HULC promoted GH3 cell viability, migration, invasion, PRL and GH secretion, as well as activated PI3K/AKT/mTOR and JAK1/STAT3 pathways. Knockdown of HULC had opposite effects and induced cell apoptosis. HULC negatively regulated the expression of miR-130b, and miR-130b participated in the effects of HULC on GH3 cells. FOXM1 was a target gene of miR-130b, which was involved in the regulation of GH3 cell viability, migration, invasion, and apoptosis, as well as PI3K/AKT/mTOR and JAK1/STAT3 pathways. In conclusion, HULC tumor-promoting roles in secreting pituitary adenoma might be via down-regulating miR-130b, up-regulating FOXM1, and activating PI3K/AKT/mTOR and JAK1/STAT3 pathways.

Down-regulation of JAK1/STAT3 signal pathway by miR-34a in PAX6 affects invasion and migration of retinoblastoma / 中国免疫学杂志

Objective:To investigate the effect of miR-34a targeting PAX6 on JAK/STAT signaling pathway on invasion and metastasis of retinoblastoma.Methods:The expression of PAX6 in retinoblastoma tissues was detected by immunohistochemistry.The expression of miR-34a in retinoblastoma cell line was detected by PCR.The effect of miR-34a on the expression of PAX6 was examined by the dual luciferase gene system.Transwell invasion assay and scratch test was used to detect the ability of invasion and migration in the retinoblastoma cell line Rb44 after overexpression miR-34a.Western blot was used to detect the protein expression of JAK/STAT signal pathway after overexpression miR-34a.Results:The expression of PAX6 was significantly higher in retinoblastoma tissues than that in normal tissues.The miR-34a was lower in retinoblastoma tissues than that in normal tissues.Western blot analysis showed the lowest level of PAX6 in Rb44 retinoblastoma.The dual luciferase reporter gene system showed that miR-34a could directly regulate the transcriptional activity of PAX6.The ability of invasion and migration was inhibited after overexpression miR-34a.The expression level of PAX6 was down-regulated and the expression of JAK1/STAT3 protein were down-regulated after overexpression the miR-34a.Conclusion:miR-34a targets the expression of PAX6 and regulates the invasion and migration of retinoblastoma cells by JAK1/STAT3 signal pathway.

Study on the effect of small molecule inhibitors LDYS-14007 on JAK1-STAT3 signaling pathway / 中国生化药物杂志

Objective To study the effect of LDYS-14007 on JAK1-STAT3 signaling pathways.Methods MDA-MB-231 cells were treated with 10μmol,1 nmol LDYS-14007, and 10 μmol Tofacitinib,respectively.Western blot assay was used to determine the expression of JAK1,Phospho-JAK1, STAT3 and Phospho-STAT3.Results The absorbance value was linearly related to the concentration of protein C, The linear equation is A=0.0075C+0.0029, r=0.9976, The linear range of 1.08-5.08 mg/mL, With the increased concentration of LDYS-14007, the amount of Phospho-JAK1, Phospho-STAT3 were all gradually decreased.Conclusion LDYS-14007 leads to the levels of Phospho-JAK1 and Phospho-STAT3 decrease, which inhibits JAK1-STAT3 signaling pathway.LDYS-14007 may play an important role in the treatment of rheumatoid arthritis.

Jak1/Stat3 Is an Upstream Signaling of NF-kappaB Activation in Helicobacter pylori-Induced IL-8 Production in Gastric Epithelial AGS Cells

Helicobacter pylori (H. pylori) induces the activation of nuclear factor-kB (NF-kappaB) and cytokine expression in gastric epithelial cells. The Janus kinase/signal transducers and activators of transcription (Jak/Stat) cascade is the inflammatory signaling in various cells. The purpose of the present study is to determine whether H. pylori-induced activation of NF-kappaB and the expression of interleukin-8 (IL-8) are mediated by the activation of Jak1/Stat3 in gastric epithelial (AGS) cells. Thus, gastric epithelial AGS cells were infected with H. pylori in Korean isolates (HP99) at bacterium/cell ratio of 300:1, and the level of IL-8 in the medium was determined by enzyme-linked immonosorbent assay. Phospho-specific and total forms of Jak1/Stat3 and IkappaBalpha were assessed by Western blot analysis, and NF-kappaB activation was determined by electrophoretic mobility shift assay. The results showed that H. pylori induced the activation of Jak1/Stat3 and IL-8 production, which was inhibited by a Jak/Stat3 specific inhibitor AG490 in AGS cells in a dose-dependent manner. H. pylori-induced activation of NF-kappaB, determined by phosphorylation of IkappaBalpha and NF-kappaB-DNA binding activity, were inhibited by AG490. In conclusion, Jak1/Stat3 activation may mediate the activation of NF-kappaB and the expression of IL-8 in H. pylori-infected AGS cells. Inhibition of Jak1/Stat3 may be beneficial for the treatment of H. pylori-induced gastric inflammation, since the activation of NF-kappaB is inhibited and inflammatory cytokine expression is suppressed.

Jak1/Stat3 Is an Upstream Signaling of NF-kappaB Activation in Helicobacter pylori-Induced IL-8 Production in Gastric Epithelial AGS Cells

Helicobacter pylori (H. pylori) induces the activation of nuclear factor-kB (NF-kappaB) and cytokine expression in gastric epithelial cells. The Janus kinase/signal transducers and activators of transcription (Jak/Stat) cascade is the inflammatory signaling in various cells. The purpose of the present study is to determine whether H. pylori-induced activation of NF-kappaB and the expression of interleukin-8 (IL-8) are mediated by the activation of Jak1/Stat3 in gastric epithelial (AGS) cells. Thus, gastric epithelial AGS cells were infected with H. pylori in Korean isolates (HP99) at bacterium/cell ratio of 300:1, and the level of IL-8 in the medium was determined by enzyme-linked immonosorbent assay. Phospho-specific and total forms of Jak1/Stat3 and IkappaBalpha were assessed by Western blot analysis, and NF-kappaB activation was determined by electrophoretic mobility shift assay. The results showed that H. pylori induced the activation of Jak1/Stat3 and IL-8 production, which was inhibited by a Jak/Stat3 specific inhibitor AG490 in AGS cells in a dose-dependent manner. H. pylori-induced activation of NF-kappaB, determined by phosphorylation of IkappaBalpha and NF-kappaB-DNA binding activity, were inhibited by AG490. In conclusion, Jak1/Stat3 activation may mediate the activation of NF-kappaB and the expression of IL-8 in H. pylori-infected AGS cells. Inhibition of Jak1/Stat3 may be beneficial for the treatment of H. pylori-induced gastric inflammation, since the activation of NF-kappaB is inhibited and inflammatory cytokine expression is suppressed.

Protection of salvianolic acid B on isoproterenol-induced cardiomyocyte hypertrophy of neonatal rats / 中草药

Objective: To observe the effects of salvianolic acid B (Sal B) on isoproterenol (ISO)-induced cardiomyocyte hypertrophy of neonatal rats and clarify the underlying mechanisms. Methods: Hypertrophy in neonatal rat ventricular myocytes was induced by ISO. The effect of Sal B on the myocardial viability of neonatal rats was measured by MTT. The mRNA expression levels of ANP and BNP were detected by RT-PCR. Colorimetric method was employed to measure SOD activity and MDA content. The expression levels of JAK1 and STAT3 were assessed by Western blotting. Results: Sal B at different concentration had no effect on the myocardial viability of neonatal rats. Compared with the model group, Sal B at 10 and 20 μmol/L could obviously down-regulate the gene expression levels of ANP and BNP (P < 0.01, 0.05), significantly increase SOD activity, and decrease MDA content. The protein expression levels of JAK1/STAT3 were down-regulated (P < 0.01, 0.05). Conclusion: Sal B could effectively inhibit ISO-induced cardiomyocyte hypertrophy of neonatal rats and the mechanism may be related with the anti-oxidative stress and the inhibition of JAK1/STAT3 signaling pathway.