Journal: Pharmaceuticals

Article Title: Wound Healing Efficacy of Cucurbitaceae Seed Oils in Rats: Comprehensive Phytochemical, Pharmacological, and Histological Studies Tackling AGE/RAGE and Nrf2/Ho-1 Cue

doi: 10.3390/ph17060733

Figure Lengend Snippet: Effect of different oils on the skin tissue contents of ( A ) TNF-α, ( B ) NF-κB, and ( C ) NLRP3 as inflammatory markers in addition to serum levels of ( D ) CX-43 as a skin integrity marker in wound-injured rats. Statistical analysis was achieved with one-way ANOVA and subsequent multiple comparisons using Tukey’s test. Data are expressed as the mean ± SD [n = 6] with p -value < 0.05 as compared with the normal control group (α) and the wound injury (β), wound injury + PSO/ST (γ), wound injury + HSO (φ), and wound injury + CSO (ε) groups. [F-value of TNF-α = 198.9, F-value of NF-κB = 139.8, F-value of NLRP3 = 253.5, and F-value of CX-43 = 423.8.] CX-43; connexin-43, CSO; Cantaloupe seed oil, HSO; Honeydew melon seed oil, NLRP3; NLR family pyrin domain containing 3, NF-κB; nuclear transcription factor kappa B, PSO; pumpkin seed oil, ST; standard, TNF-α; tumor necrosis factor-alpha, ZSO; Zucchini seed oil.

Article Snippet: The tissue contents of nuclear factor erythropoietin-2-related factor 2 (Nrf-2, Cat# MBS012148), heme oxygenase-1 (HO-1, Cat# MBS800757), tumor necrosis factor-alpha (TNF-α, Cat# MBS624076), nuclear transcription factor-kappa B (NF-κB, Cat# MBS267737), nod like receptor protein 3 (NLRP3, Cat# MBS645773), skin integral signaling protein, and connexin 43 (CX-43, Cat# MBS008326) were determined using MyBioSource ELISA kits (San Diego, CA, USA) according to manufacturer’s protocols.

Techniques: Marker, Control

Journal: Mediators of Inflammation

Article Title: Anti-Inflammatory Effect of 1,3,5,7-Tetrahydroxy-8-isoprenylxanthone Isolated from Twigs of Garcinia esculenta on Stimulated Macrophage

doi: 10.1155/2015/350564

Figure Lengend Snippet: Effect of TIE on NF- κ B, MAPK activation, and miR155 expression in LPS/IFN γ -stimulated RAW264.7 cells. (a) RAW264.7 cells were transiently cotransfected with a pNF- κ B reporter vector. Cells were incubated with TIE (3.125, 6.25, 12.5, and 25 μ M) and L-NIL (50 μ M) for 1 h before stimulation with LPS and IFN γ for 18 h. Luciferase activities were measured by Dual Luciferase Reporter reagents following manufacturer's instruction. Luciferase activity was normalized to transfection efficiency as monitored by Renilla luciferase expression. (b, c) DNA-binding activity of p65 and p50 proteins in nuclear extracts was assessed using NF- κ Bp50/p65 EZ-TFA transcription factor assay. Absorbance was measured at 450 nm in a microplate spectrophotometer. Results were normalized to absorbance/mg protein. (d) RAW264.7 cells (1 × 10 6 cells/dish) were treated with varying doses of TIE with IFN γ (10 U/mL) plus LPS (100 ng/mL) for 4 h. Total RNA was isolated and subjected to qRT-PCR to determine the level of TBK1 mRNA. (e) RAW264.7 cells were plated at a density of 1 × 10 6 cells/well in 30 mm dish overnight. TIE was added to cells followed by 30 min stimulation of IFN γ (10 U/mL) plus LPS (100 ng/mL). Whole cell lysates were prepared and subjected to Western blotting. The ratios of immunointensity of p-ERK1/2, p-JNK, and p-p38 were calculated, respectively. Total ERK1/2, JNK, and p38 (T-ERK1/2, T-JNK, and T-p38) were used as a control of the protein amount in the same samples. Data shown are the representative of three independent experiments. (f) The cells were stimulated by IFN γ (10 U/mL) plus LPS (100 ng/mL) with or without 12.5 and 25 μ M concentrations of TIE for 24 h. Total RNA was isolated and the expression of miR155 was determined by qRT-PCR. RNU6B was used here as an endogenous control. The data represent the mean ± SD of triplicate experiments. ## P < 0.01, # P < 0.05 versus control group; ∗∗ P < 0.01, ∗ P < 0.05 versus model group.

Article Snippet: Murine recombinant IFN γ and NF- κ B p50/p65 EZ-TFA transcription factor assay kit were purchased from Millipore (Bedford, MA, USA); mouse PGE2 and IL-6 ELISA kit were purchased from Cayman Chemical Company (Ann Arbor, MI, USA); lipopolysaccharide (LPS; Escherichia coli O111:B4), dimethyl sulfonamide (DMSO), N -(1-naphthyl)-enthylendiaminedihydrochloride, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoleum (MTT), and L-N 6 -(1-iminoethyl)lysine hydrochloride (L-NIL, iNOS selective inhibitor) were obtained from Sigma Chemical Co. (St. Louis, MO, USA); RPMI-1640 was purchased from Gibco Invitrogen Corporation (Grand Island, NY, USA); fetal bovine serum (FBS) was purchased from Hyclone (Logan, UT, USA); TRIzol Reagent, lipofectamine TM 2000, Reverse Transcription Kit, and SYBR Green PCR Master Mix regents were obtained from Invitrogen (Carlsbad, CA, USA).

Techniques: Activation Assay, Expressing, Plasmid Preparation, Incubation, Luciferase, Activity Assay, Transfection, Binding Assay, Transcription Factor Assay, Spectrophotometry, Isolation, Quantitative RT-PCR, Western Blot

Journal: Mediators of Inflammation

Article Title: Anti-Inflammatory Effect of 1,3,5,7-Tetrahydroxy-8-isoprenylxanthone Isolated from Twigs of Garcinia esculenta on Stimulated Macrophage

doi: 10.1155/2015/350564

Figure Lengend Snippet: Proposed mechanisms of TIE inhibition of LPS plus IFN γ induced inflammation in RAW264.7 cells. TIE inactivates MAPK and NF- κ B signaling pathways in addition to inhibiting expression of miR155, which may result from TIE downregulation of iNOS, COX-2, and IL-6 expression and their production. Arrows indicate the main inflammatory pathway activated by LPS stimulation. The prohibition signs indicate the inhibitory effects of TIE.

Article Snippet: Murine recombinant IFN γ and NF- κ B p50/p65 EZ-TFA transcription factor assay kit were purchased from Millipore (Bedford, MA, USA); mouse PGE2 and IL-6 ELISA kit were purchased from Cayman Chemical Company (Ann Arbor, MI, USA); lipopolysaccharide (LPS; Escherichia coli O111:B4), dimethyl sulfonamide (DMSO), N -(1-naphthyl)-enthylendiaminedihydrochloride, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoleum (MTT), and L-N 6 -(1-iminoethyl)lysine hydrochloride (L-NIL, iNOS selective inhibitor) were obtained from Sigma Chemical Co. (St. Louis, MO, USA); RPMI-1640 was purchased from Gibco Invitrogen Corporation (Grand Island, NY, USA); fetal bovine serum (FBS) was purchased from Hyclone (Logan, UT, USA); TRIzol Reagent, lipofectamine TM 2000, Reverse Transcription Kit, and SYBR Green PCR Master Mix regents were obtained from Invitrogen (Carlsbad, CA, USA).

Techniques: Inhibition, Expressing

Journal: International Journal of Inflammation

Article Title: Neurokinin-1 Receptor Antagonist Treatment in Polymicrobial Sepsis: Molecular Insights

doi: 10.4061/2010/601098

Figure Lengend Snippet: Effect of SR140333 administration, either 30 minutes before or 1 hour after CLP, on lung NF- κ B DNA-binding activity. Mice ( n = 6–9 in each group) were divided into CLP-operated and sham-operated groups. CLP-operated mice received vehicle (DMSO in PBS, 0.25% v/v) or SR140333 (1 mg/kg; 0.25 mg/mL) s.c. either 30 minutes before (pretreatment) or 1 hour after (posttreatment) the CLP. Same surgical procedure as the CLP-operated animals except the cecal ligation and puncture was performed on sham-operated animals. 8 hours after the CLP procedure, mice were sacrificed, and lung (a) NF- κ B DNA-binding activity and (b) I κ B- α level (representative I κ B- α and HPRT control bands shown on the upper panel) were determined. Results shown are the mean ±S.E.M. “Vehicle + CLP” and “SR140333 + CLP” represent the groups that received vehicle and SR140333 treatment, respectively, commencing 30 minutes prior to CLP. “CLP + vehicle” and “CLP + SR140333” represent the groups that received vehicle and SR140333 treatment, respectively, 1 hour after CLP. * P < .001 when vehicle-treated CLP animals were compared with sham group animals; ** P < .001 when SR140333-treated CLP animals were compared with vehicle-treated CLP animals; ∞ P < .01 when vehicle-treated CLP animals were compared with sham group animals; ≠ P < .05 when SR140333-treated CLP animals were compared with vehicle-treated CLP animals. CLP: cecal ligation and puncture; HPRT: Hypoxanthine guanine phosphoribosyl transferase; IOD: integrated optical density.

Article Snippet: ELISA-based TransAM NF- κ B p65 transcription factor assay kit (Active Motif, Carlsbad, CA, USA) was used to measure NF- κ B binding to DNA and activation, as per the manufacturer's instruction.

Techniques: Binding Assay, Activity Assay, Ligation

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Demethoxycurcumin Retards Cell Growth and Induces Apoptosis in Human Brain Malignant Glioma GBM 8401 Cells

doi: 10.1155/2012/396573

Figure Lengend Snippet: DMC inhibits nuclear NF- κ B transcription factor activity in GBM 8401 cells. (a) The NoShift II transcription factor assay kit was used to identify the activity of NF- κ B transcription factor in the cells after exposure to DMC followed by examination with microplate luminometer. (b) Diminished NF- κ B activity in DMC-treated GBM8401 cells. The cells were examined for their NF- κ B activity 6 hours after DMC stimulation by confocal microscopy of NF- κ B subunit p50/52 localization. The cells were stained for p50/52 (red). DAPI (blue) indicates nucleus, where an active form of NF- κ B subunit p50/52 is found. All data were reported as the means (±SEM) of at least three separate experiments. Statistical analysis was as cited above.

Article Snippet: The NF- κ B transcription factor was assessed by the NoShift II NF- κ B transcription factor assay kit (NOvagen, USA) and confocal microscopy.

Techniques: Activity Assay, Transcription Factor Assay, Confocal Microscopy, Staining

Journal: BioMed Research International

Article Title: Trichinella spiralis Excretory-Secretory Products Protect against Polymicrobial Sepsis by Suppressing MyD88 via Mannose Receptor

doi: 10.1155/2014/898646

Figure Lengend Snippet: The effects of TsES on peritoneal macrophages in septic patients. Macrophages were collected from peritoneal lavage or abdominal cavity flushing fluid in 27 septic patients and 16 control patients and then treated with TsES (5 μ g/mL) or the same volume of PBS. Membrane protein fractions were extracted after 6 h in culture. The expressions of TLR2 and TLR4 were detected by western blot (a). The cytoplasmic proteins were extracted after 6 h in culture. MyD88 expression in macrophages was detected by western blot (b). NF- κ B activity in nuclear extractions was determined using the NF- κ B p50/p65 Transcription Factor Assay Kit (c). Culture supernatants were collected after 6 h in culture. Supernatant TNF- α , IL-1 β , IL-10, and TGF β 1 levels were detected by ELISA (d). Data are mean ± SD of three independent experiments, analyzed in triplicate. * P < 0.05 compared to the control group. # P < 0.05 compared to the sepsis group.

Article Snippet: NF- κ B activity was determined in nuclear extracts using the NF- κ B p50/p65 Transcription Factor Assay Kit (Chemicon, Temecula, CA) according to the manufacturer's instructions.

Techniques: Western Blot, Expressing, Activity Assay, Transcription Factor Assay, Enzyme-linked Immunosorbent Assay

Journal: BioMed Research International

Article Title: Trichinella spiralis Excretory-Secretory Products Protect against Polymicrobial Sepsis by Suppressing MyD88 via Mannose Receptor

doi: 10.1155/2014/898646

Figure Lengend Snippet: TsES suppressed MyD88/NF- κ B signaling via MR on peritoneal macrophages from septic patients. Macrophages from peritoneal lavage or abdominal cavity flushing fluid were collected from septic patients and the control group. Peritoneal macrophages were treated with TsES (5 μ g/mL) for 6 h. Membrane protein fractions were extracted. MR expression was detected by western blot. * P < 0.05 compared to the control group. # P < 0.05 compared to the sepsis group (a). Peritoneal macrophages from septic patients were treated with a specific blocking antibody to MR (5 μ g/mL) for 2 h and then with TsES for 6 h. Supernatant TNF- α , IL-1 β , IL-10, and TGF β 1 levels were detected by ELISA (b). MyD88 expression in macrophages was detected by western blot (c). NF- κ B activity was determined using the NF- κ B p50/p65 Transcription Factor Assay Kit (d). Data are mean ± SD of three independent experiments, analyzed in triplicate. * P < 0.05 with respect to macrophages in medium alone. # P < 0.05 with respect to macrophages stimulated with TsES.

Article Snippet: NF- κ B activity was determined in nuclear extracts using the NF- κ B p50/p65 Transcription Factor Assay Kit (Chemicon, Temecula, CA) according to the manufacturer's instructions.

Techniques: Expressing, Western Blot, Blocking Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Transcription Factor Assay

Journal: BioMed Research International

Article Title: Trichinella spiralis Excretory-Secretory Products Protect against Polymicrobial Sepsis by Suppressing MyD88 via Mannose Receptor

doi: 10.1155/2014/898646

Figure Lengend Snippet: TsES protected mice from sepsis via MR. Mice were pretreated with MR siRNA and then underwent the CLP operation. On the day of the operation, MR expression on peritoneal cells was detected by western blot. * P < 0.05 compared to the normal group (a). The survival rate was assessed in six groups of mice ( n = 10 mice/group): (1) PBS + CLP, (2) con siRNA + SLP, (3) MR siRNA + CLP, (4) MR siRNA + TsES + CLP, (5) con siRNA + TsES + CLP, and (6) TsES + CLP. The survival rate was analyzed 4 days after the CLP operation using the log-rank test (b). Twenty-four hours after CLP liver MyD88 expression (c), NF- κ B activity (d), and serum cytokine levels (TNF- α , IL-1 β , IL-10, and TGF β 1) (e) were determined. Triplicate independent experiments were analyzed. * P < 0.05 compared with the PBS + CLP group. # P < 0.05 compared to the TsES + CLP group.

Article Snippet: NF- κ B activity was determined in nuclear extracts using the NF- κ B p50/p65 Transcription Factor Assay Kit (Chemicon, Temecula, CA) according to the manufacturer's instructions.

Techniques: Expressing, Western Blot, Activity Assay