Journal: PLoS ONE

Article Title: Positive associations between upregulated levels of stress-induced phosphoprotein 1 and matrix metalloproteinase-9 in endometriosis/adenomyosis

doi: 10.1371/journal.pone.0190573

Figure Lengend Snippet: (A) STIP1 knockdown by siRNA in ARK2 cells decreased MMP-9 protein expression without affecting AKT, ERK, and IkB phosphorylation levels. (B) Decreased MMP-9 protein expression induced by siRNA knockdown of STIP1 was unaffected by exposure either to the DNA methylation inhibitor 5-aza-2’-deoxycytidine (AZA; 10 μM) or the histone deacetylase inhibitor trichostatin A (TSA; 20 nM). (C) Suppression of MMP-9 protein levels by siRNA knockdown of STIP1 was unaffected by treatment with either the proteasome inhibitor MG-132 (25 μM) or a JAK2 inhibitor AG490 (35 μM). (D) Treatment with an STIP1 inhibitor, peptide 520, suppressed MMP-9 protein levels in a dose-dependent manner.

Article Snippet: In the functional experiments, cells were pretreated with one of the following inhibitors: the histone deacetylase (HDAC) inhibitor trichostatin A (TSA; Sigma-Aldrich, St. Louds, MO) at a concentration of 20 nM for 48 h; the DNA methyltransferase (DNMT) inhibitor 5-aza-2’-deoxycytidine (5’-AZA; Sigma-Aldrich, St. Louds, MO) at a concentration of 10 μM for 24 h; the proteasome inhibitor MG-132 (Sigma-Aldrich, St. Louds, MO) at a concentration of 25 μM for 5 h; or the JAK2 inhibitor AG490 (Merck, Kenilworth, NJ) at a concentration of 35 μM for 5 h.

Techniques: Expressing, DNA Methylation Assay, Histone Deacetylase Assay

Journal: International Journal of Molecular Medicine

Article Title: Matrine induces the apoptosis of fibroblast-like synoviocytes derived from rats with collagen-induced arthritis by suppressing the activation of the JAK/STAT signaling pathway

doi: 10.3892/ijmm.2016.2843

Figure Lengend Snippet: The effect of matrine on cell cycle progression was determined by propidium iodide (PI) staining and flow cytometry. Fibroblast-like synoviocytes (FLS) were isolated from control or collagen-induced arthritis (CIA) rats, cultured in vitro , and treated with 0.75 mg/ml matrine, 25 µ mol/l AG490, or a combination of matrine and AG490 for 24 h. (A) Cell cycle distribution on FLS after 24 h of drug treatment. (B) Histogram depicts the percentage of cells in G0/G1, S and G2/M phases of the cell cycle. Data are expressed as mean ± SD of three independent experiments. * P<0.05, ** P<0.01 vs. the control group; ## P<0.01 vs. the CIA group; Δ P<0.05, ΔΔ P<0.01 vs. the AG490 group.

Article Snippet: Cell treatments included the JAK2 inhibitor, AG490 (25 μ mol/l; Abmole Bioscience, Houston, TX, USA) or matrine (0.75 mg/ml; Aladdin Reagents Co., Ltd., Shanghai, China).

Techniques: Staining, Flow Cytometry, Isolation, Control, Cell Culture, In Vitro

Journal: International Journal of Molecular Medicine

Article Title: Matrine induces the apoptosis of fibroblast-like synoviocytes derived from rats with collagen-induced arthritis by suppressing the activation of the JAK/STAT signaling pathway

doi: 10.3892/ijmm.2016.2843

Figure Lengend Snippet: The effect of matrine on apoptosis was determined by double staining with Annexin V-FITC/propidium iodide (PI) and flow cytometry. Fibroblast-like synoviocytes (FLS) were isolated from control or collagen-induced arthritis (CIA) rats, cultured in vitro , and treated with 0.75 mg/ml matrine, 25 µ mol/l AG490, or a combination of matrine and AG490 for 24 h. (A) Representative FACS scatter plots for each drug treatment. Q1 indicates necrotic cells; Q2 indicates late apoptotic cells; Q3 indicates early apoptotic cells; and Q4 indicates living cells. (B) Cell apoptosis rates were analyzed via flow cytometry. Data are presented as mean ± SD of three independent experiments. ** P<0.01 vs. the control group; ## P<0.01 vs. the CIA group; Δ P<0.05 vs. the AG490 group.

Article Snippet: Cell treatments included the JAK2 inhibitor, AG490 (25 μ mol/l; Abmole Bioscience, Houston, TX, USA) or matrine (0.75 mg/ml; Aladdin Reagents Co., Ltd., Shanghai, China).

Techniques: Double Staining, Flow Cytometry, Isolation, Control, Cell Culture, In Vitro

Journal: International Journal of Molecular Medicine

Article Title: Matrine induces the apoptosis of fibroblast-like synoviocytes derived from rats with collagen-induced arthritis by suppressing the activation of the JAK/STAT signaling pathway

doi: 10.3892/ijmm.2016.2843

Figure Lengend Snippet: The effects of matrine on the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway were assessed by western blot analysis. Fibroblast-like synoviocytes (FLS) were isolated from control or collagen-induced arthritis (CIA) rats, cultured in vitro , and treated with 0.75 mg/ml matrine, 25 µ mol/l AG490, or a combination of matrine and AG490 for 24 h. (A) Western blot analysis of p-JAK2, p-STAT1 and p-STAT3 levels. β-actin was used as a loading control. (B) Quantitative densitometry of p-JAK2, p-STAT1 and p-STAT3 levels relative to β-actin. Data are presented as mean ± SD of three independent experiments. ** P<0.01 vs. the control group; # P<0.05, ## P<0.01 vs. the CIA group; Δ P<0.05, ΔΔ P<0.05 vs. the AG490 group.

Article Snippet: Cell treatments included the JAK2 inhibitor, AG490 (25 μ mol/l; Abmole Bioscience, Houston, TX, USA) or matrine (0.75 mg/ml; Aladdin Reagents Co., Ltd., Shanghai, China).

Techniques: Western Blot, Isolation, Control, Cell Culture, In Vitro

Journal: International Journal of Molecular Medicine

Article Title: Matrine induces the apoptosis of fibroblast-like synoviocytes derived from rats with collagen-induced arthritis by suppressing the activation of the JAK/STAT signaling pathway

doi: 10.3892/ijmm.2016.2843

Figure Lengend Snippet: The effects of matrine on apoptotic markers were assessed by qRT-PCR. Fibroblast-like synoviocytes (FLS) were isolated from control or collagen-induced arthritis (CIA) rats, cultured in vitro , and treated with 0.75 mg/ml matrine, 25 µ mol/l AG490, or a combination of matrine and AG490 for 24 h. qRT-PCR analysis of relative expression of (A) Bax, (B) Bcl-2 and (C) caspase-3. (D) Bax to Bcl-2 ratio (based on relative intensity). In all experiments, β-actin was used as an internal control. Data are presented as the mean ± SD of three independent experiments. ** P<0.01 vs. the control group; ## P<0.01 vs. the CIA group; Δ P<0.05, ΔΔ P<0.01 vs. the AG490 group.

Article Snippet: Cell treatments included the JAK2 inhibitor, AG490 (25 μ mol/l; Abmole Bioscience, Houston, TX, USA) or matrine (0.75 mg/ml; Aladdin Reagents Co., Ltd., Shanghai, China).

Techniques: Quantitative RT-PCR, Isolation, Control, Cell Culture, In Vitro, Expressing

Journal: Neuropsychopharmacology

Article Title: Ketamine Corrects Stress-Induced Cognitive Dysfunction through JAK2/STAT3 Signaling in the Orbitofrontal Cortex

doi: 10.1038/npp.2016.236

Figure Lengend Snippet: Ketamine corrects the CIC stress-induced cognitive deficit in reversal learning and activates the JAK2/STAT3 pathway in the OFC. (a) Experimental timeline. (b) CIC-stressed rats exhibited a cognitive deficit in reversal learning that was prevented by acute systemic injection of ketamine (10 mg/kg, i.p.) given 24 h prior to testing (*p<0.0001, CIC- stressed rats compared with Non-stress control rats given vehicle; #p<0.0001, CIC-stressed rats given ketamine compared with CIC-stressed rats given vehicle; n=8/group). (c) Local microinjection of ketamine (2 nmol/0.5 μl/side) directly into the OFC 24 h before testing also corrected the CIC stress-induced reversal learning deficit (*p<0.001, CIC-stressed rats compared with Non-stress control rats given vehicle; #p<0.0001, CIC-stressed rats given ketamine compared with CIC-stressed rats given vehicle; n=3–7/group. Acute systemic injection of ketamine (10 mg/kg, i.p.) increased phosphorylation of both JAK2 (d) and STAT3 (e) in the OFC (*p<0.05 compared with baseline; n=5/group). (f) Three days after the end of CIC stress, levels of pJAK2 were reduced in the OFC (*p<0.05). (g) CIC stress did not change pSTAT3 levels. (h) Levels of the synaptic plasticity-related protein Arc were modestly reduced after CIC stress. At 2 h post injection, ketamine increased expression of Arc in control rats (h; *p<0.05), and increased both pJAK2 (f; #p<0.01) and Arc (h; #p<0.01) in CIC-stressed rats, By contrast, ketamine increased pSTAT3 only in controls (*p<0.05), but not in CIC-stressed rats (n=5–6/group). Values in all figures represent mean±SEM.

Article Snippet: In preliminary studies, we established that systemic administration of the JAK2 inhibitor Tryphostin AG490 (10 mg/kg, i.p. Fisher Scientific, Waltham, MA) decreased phospho-JAK2 in the rat OFC for at least 2 h after administration.

Techniques: Injection, Control, Microinjection, Phospho-proteomics, Expressing

Journal: Neuropsychopharmacology

Article Title: Ketamine Corrects Stress-Induced Cognitive Dysfunction through JAK2/STAT3 Signaling in the Orbitofrontal Cortex

doi: 10.1038/npp.2016.236

Figure Lengend Snippet: Pharmacological inhibition of the JAK2/STAT3 pathway in the OFC prevents the beneficial effects of ketamine on reversal learning and induction of the plasticity-related protein Arc. (a) Systemic administration of the JAK2 inhibitor AG490 (10 mg/kg, i.p.) at the time of ketamine administration blocked the beneficial effect of ketamine on reversal learning in CIC-stressed rats, tested 24 h after injection (*p<0.05 compared with non-stressed-vehicle; #p<0.01 compared with CIC vehicle; +p<0.001 compared with CIC-ketamine; n=5/group). (b) Local microinjection of AG490 (1.47 ng/0.5 μl) in the OFC similarly prevented the beneficial effect of ketamine in CIC-stressed animals (*p<0.001 compared with non-stressed vehicle/vehicle; #p<0.001 compared with CIC vehicle/vehicle; +p<0.001 compared with CIC-ketamine-vehicle; n=4–5/group). (c) Ketamine increased the expression of the synaptic plasticity-related protein Arc in the OFC 2 h after administration (*p<0.05 compared with baseline; n=5–8/group). (d) Pre-treatment with AG490 prevented ketamine-mediated induction of Arc protein in the OFC at 2 h (*p<0.05 compared with baseline; #p<0.05 compared with Ket-2h; n=5/group). Values represent mean±SEM.

Article Snippet: In preliminary studies, we established that systemic administration of the JAK2 inhibitor Tryphostin AG490 (10 mg/kg, i.p. Fisher Scientific, Waltham, MA) decreased phospho-JAK2 in the rat OFC for at least 2 h after administration.

Techniques: Inhibition, Injection, Microinjection, Expressing

Journal: Neuropsychopharmacology

Article Title: Ketamine Corrects Stress-Induced Cognitive Dysfunction through JAK2/STAT3 Signaling in the Orbitofrontal Cortex

doi: 10.1038/npp.2016.236

Figure Lengend Snippet: Systemic ketamine administration depresses evoked field potentials in the OFC, and this is prevented by inhibiting JAK2 activity in the OFC. In rats receiving a microinjection of ACSF vehicle (0.5 μl) into OFC, a subsequent systemic saline injection had no effect on evoked LFP responses in the OFC (a), whereas systemic ketamine administration (10 mg/kg, i.p.) induced a depression of the evoked LFP response that emerged 30–60 min after injection and lasted for the duration of recording (b). Local microinjection of the JAK2 inhibitor AG490 into OFC prior to a systemic injection of saline had no effect on the evoked LFP alone (c), but prevented the ketamine-induced depression of the evoked LFP (d). Insets show representative traces recorded at the time points indicated. Data represent mean percent of baseline±SEM (n=5/group).

Article Snippet: In preliminary studies, we established that systemic administration of the JAK2 inhibitor Tryphostin AG490 (10 mg/kg, i.p. Fisher Scientific, Waltham, MA) decreased phospho-JAK2 in the rat OFC for at least 2 h after administration.

Techniques: Activity Assay, Microinjection, Saline, Injection

Journal: Neuropsychopharmacology

Article Title: Ketamine Corrects Stress-Induced Cognitive Dysfunction through JAK2/STAT3 Signaling in the Orbitofrontal Cortex

doi: 10.1038/npp.2016.236

Figure Lengend Snippet: Ketamine administration induces phosphorylation of JAK2 and STAT3 in rat primary cortical neurons in culture, and blocking JAK2 but not STAT3 prevents ketamine-induced Arc expression. Ketamine administration (0.5 μM) induced phosphorylation of both JAK2 (a) and STAT3 (b) within 30 min of drug application (*p<0.01 compared with baseline). (c) Ketamine administration (0.5 μM) increased expression of the synaptic plasticity-related protein Arc 60 min after administration (*p<0.01 compared with baseline). (d) siRNA knockdown of JAK2, but not STAT3, reduced basal Arc expression and prevented the ketamine-induced increase in Arc expression (*p<0.01 compared with baseline; #p<0.001 compared with scrambled-ketamine). Values represent mean±SEM (n=4–7 independent cultures per time point).

Article Snippet: In preliminary studies, we established that systemic administration of the JAK2 inhibitor Tryphostin AG490 (10 mg/kg, i.p. Fisher Scientific, Waltham, MA) decreased phospho-JAK2 in the rat OFC for at least 2 h after administration.

Techniques: Phospho-proteomics, Blocking Assay, Expressing, Knockdown

Journal: Neuropsychopharmacology

Article Title: Ketamine Corrects Stress-Induced Cognitive Dysfunction through JAK2/STAT3 Signaling in the Orbitofrontal Cortex

doi: 10.1038/npp.2016.236

Figure Lengend Snippet: Phosphorylated JAK2 colocalizes with Arc. E18 rat cortical neurons (DIV 18) were stimulated with 0.5 μM ketamine or saline vehicle for 30 min prior to fixation and processing for immunofluorescence. Phosphorylated JAK2 (pJAK2) was colocalized with Arc in a subset of punctate structures in both vehicle- (a) and ketamine-treated (b) neurons. Arrowheads indicate puncta where the two proteins are colocalized. Full arrows indicate non-colocalized Arc or pJAK2. Bar=20 μm. (c) Quantification of colocalization of pJAK2 and Arc in vehicle- and ketamine-treated neurons. After ketamine stimulation, the percentage of pJAK2 signal colocalized with Arc increased (*p<0.01), whereas the percentage of total Arc signal colocalized with pJAK2 decreased (*p<0.001); unpaired Student's t-test; n=9 fields analyzed from three independent cultures. (d) G-actin (visualized with DNAseI stain, green) is also present at sites where pJAK2 (red) and Arc (blue) colocalize. Arrowheads indicate triple-labeled puncta. Bar=20 μm.

Article Snippet: In preliminary studies, we established that systemic administration of the JAK2 inhibitor Tryphostin AG490 (10 mg/kg, i.p. Fisher Scientific, Waltham, MA) decreased phospho-JAK2 in the rat OFC for at least 2 h after administration.

Techniques: Saline, Immunofluorescence, Staining, Labeling