Journal: Experimental & Molecular Medicine

Article Title: Disruption of cholesterol homeostasis triggers periodontal inflammation and alveolar bone loss

doi: 10.1038/s12276-023-01122-w

Figure Lengend Snippet: Representative images of filipin staining of gingival tissue from human patients with PD ( a ) and in the maxilla region of ligature-induced PD mice ( b ). Scale bar, 25 μm. Total, free cholesterol, and cholesteryl ester ( n = 6) levels ( c ) and mRNA ( n = 3) ( d ) levels of cholesterol synthesis-related genes in human GFs treated with IL1β or TNFα. e Total cholesterol level in human GFs treated with the cholesterol synthesis inhibitors triparanol (TP) or lovastatin (LV) in the presence of IL1β (2 ng ml −1 ) or TNFα (50 ng ml −1 ) for 24 h ( n = 4). f mRNA levels of MMP1 , MMP3 , IL6 , IL8 , CCL5 , and PTGS2 in human GFs treated with cholesterol synthesis inhibitors (TP and LV) in the presence of IL1β (2 ng ml −1 ) or TNFα (50 ng ml −1 ) for 24 h ( n = 4). n indicates the number of biologically independent samples. Values are presented as the mean ± SEM based on one-way ANOVA with Tukey’s test. (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: The membranes were incubated with the following antibodies: rabbit anti-MMP1 (Abnova, PAB12708), rabbit anti-IL6 (Novus), rabbit anti-IL8 (Biorbyt), rabbit anti-PTGS2 (Cayman), rabbit anti-RORα (Santa Cruz, Sc-28612), and rabbit anti-CH25H (Bioss).

Techniques: Staining

Journal: Experimental & Molecular Medicine

Article Title: Disruption of cholesterol homeostasis triggers periodontal inflammation and alveolar bone loss

doi: 10.1038/s12276-023-01122-w

Figure Lengend Snippet: a Heatmaps of genes involved in cholesterol metabolism and metabolic pathways of bile acid synthesis from the microarray analysis of human GFs treated with IL1β (left square) and TNFα (right circle) ( n = 3). Transcript levels of CH25H and CYP7B1 in human GFs treated with IL1β (5 ng ml −1 ) and TNFα (100 ng ml −1 ) for 24 h ( n = 4) ( b ) and the gingiva of healthy patients and patients with PD ( n = 6) ( c ). d Representative immunostaining images of CH25H and CYP7B1 in human and mouse gingiva. Scale bar, 25 μm. e , f mRNA levels in human GFs infected with empty (Ad-C) or CH25H -encoding adenovirus (Ad- CH25H ) ( n = 3) for 48 h ( n = 3). g Protein levels of CH25H, MMP1, IL6, IL8, and PTGS2 in cell lysates of human GFs infected with CH25H -overexpressing adenovirus (Ad- CH25H ; n = 3). h , i Representative images of CH25H immunostaining, μCT, and H&E staining images of mouse gingiva injected with an empty virus (Ad-C) or Ad- Ch25h (1 × 10 9 PFU per 5 μl). Magnification, ×100. Scale bar, 100 μm (left). Scale bar, 25 μm (right) ( h ). Magnification, ×200. Scale bar, 100 μm ( i ). j BMD analysis and CEJ-ABC distance measurement of the mouse maxillae injected intragingivally with Ad-C and Ad- Ch25h (1 × 10 9 PFU per 5 μl; n = 10). Scale bar, 100 μm. n indicates the number of biologically independent samples and mice per group. Values are presented as the mean ± SEM based on a two-tailed t -test ( c , j ) and one-way ANOVA with Tukey’s test ( b , e , f ). (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: The membranes were incubated with the following antibodies: rabbit anti-MMP1 (Abnova, PAB12708), rabbit anti-IL6 (Novus), rabbit anti-IL8 (Biorbyt), rabbit anti-PTGS2 (Cayman), rabbit anti-RORα (Santa Cruz, Sc-28612), and rabbit anti-CH25H (Bioss).

Techniques: Microarray, Immunostaining, Infection, Staining, Injection, Virus, Two Tailed Test

Journal: Experimental & Molecular Medicine

Article Title: Disruption of cholesterol homeostasis triggers periodontal inflammation and alveolar bone loss

doi: 10.1038/s12276-023-01122-w

Figure Lengend Snippet: mRNA levels of CH25H , MMP1 , IL6 , IL8 , and PTGS2 in human GFs transfected with CH25H siRNA in the presence of IL1β (2 ng ml −1 ) ( a ) or TNFα (50 ng ml −1 ) ( b ) for 24 h ( n ≥ 3). Representative μCT ( c ) and H&E staining images ( d ) for BMD analysis and CEJ-ABC distance measurement of the maxillae of ligature-induced PD wild-type (WT) and CH25H knockout ( Ch25h −/− ) mice ( n ≥ 10). Scale bar, 100 μm. n indicates the number of biologically independent samples and mice per group. Values are presented as the mean ± SEM based on a two-tailed t -test ( c , d ) and one-way ANOVA with Tukey’s test ( a , b ). (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: The membranes were incubated with the following antibodies: rabbit anti-MMP1 (Abnova, PAB12708), rabbit anti-IL6 (Novus), rabbit anti-IL8 (Biorbyt), rabbit anti-PTGS2 (Cayman), rabbit anti-RORα (Santa Cruz, Sc-28612), and rabbit anti-CH25H (Bioss).

Techniques: Transfection, Staining, Knock-Out, Two Tailed Test

Journal: Archives of Medical Science : AMS

Article Title: Urotensin II promotes secretion of LTB 4 through 5-lipoxygenase via the UT-ROS-Akt pathway in RAW264.7 macrophages

doi: 10.5114/aoms.2019.85197

Figure Lengend Snippet: UII promotes 5-lipoxygenase (5-LO) expression in RAW264.7 macrophages. A – Time course of UII-stimulated 5-LO expression determined by real-time PCR. The cells were incubated with 10 –8 M UII for the indicated times. B – Concentration-response relationship of UII with 5-LO expression in RAW264.7 macrophages via UT determined by real-time PCR. The cells were incubated with different concentrations of UII for 24 h or pretreated with urantide (10 μM) for 1 h prior to stimulation with UII (10 –8 M) for 24 h. C – Time course of UII-stimulated 5-LO expression determined by western blot analysis. D – Concentration-response relationship of UII with 5-LO expression in RAW264.7 macrophages via UT determined by western blot analysis UA – urantide. The data are from three independent experiments and are expressed as the mean ± SEM. *P < 0.05 compared with control; **p < 0.01 compared with control; # p < 0.01 compared with the UII (10 –8 M) group.

Article Snippet: The reagents used in the study were purchased from the following suppliers: Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS) and trypsin (Gibco); mouse UII (Phoenix Pharmaceuticals); urantide (Peptides International); Invitrogen TRIZOL reagent (Life Technologies); High-Capacity cDNA Reverse Transcription Kit and SYBR Select Master Mix (Applied Biosystems); mouse LTB 4 ELISA kit (Cayman); antibodies against 5-LO, p-Akt, Akt (CST), and β-actin (Beijing TransGen Biotechnology); LY294002 (CST); 2′,7′-dichlorofluorescein diacetate (DCFH-DA); N-acetylcysteine (NAC) and diphenyliodonium (DPI) (Sigma); goat anti-rabbit secondary antibody and rabbit anti-mouse second antibody (Beijing Zhongshan Golden Bridge Biotechnology), and SuperEnhanced chemiluminescence detection reagents (Millipore).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Incubation, Concentration Assay, Western Blot, Control

Journal: Archives of Medical Science : AMS

Article Title: Urotensin II promotes secretion of LTB 4 through 5-lipoxygenase via the UT-ROS-Akt pathway in RAW264.7 macrophages

doi: 10.5114/aoms.2019.85197

Figure Lengend Snippet: Involvement of ROS in UII-induced 5-LO expression and LTB4 release in RAW264.7 macrophages. A – Effect of the different inhibitors on the UII-stimulated generation of ROS. The cells were stimulated with UII (10 –8 M) for 2 h after pretreatment with urantide (10 μM), NAC (10 mM) and DPI (10 μM) for 1 h and then incubated for 30 min with DCFH-DA (10 μM). B – Effect of NAC and DPI on UII-induced 5-LO mRNA expression determined by real-time PCR. The cells were stimulated with UII (10 –8 M) for 24 h after pretreatment with NAC (10 mM) and DPI (10 μM) for 1 h. C – Effect of NAC and DPI on UII-induced 5-LO protein expression determined by western blot analysis. D – Effect of different inhibitors on LTB 4 release in the presence of UII (10 –8 M) for 24 h UA – urantide. The data are from three independent experiments and are expressed as the mean ± SEM. *P < 0.05 compared with control; **p < 0.01 compared with control; #P < 0.01 compared with the UII (10 –8 M) group.

Article Snippet: The reagents used in the study were purchased from the following suppliers: Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS) and trypsin (Gibco); mouse UII (Phoenix Pharmaceuticals); urantide (Peptides International); Invitrogen TRIZOL reagent (Life Technologies); High-Capacity cDNA Reverse Transcription Kit and SYBR Select Master Mix (Applied Biosystems); mouse LTB 4 ELISA kit (Cayman); antibodies against 5-LO, p-Akt, Akt (CST), and β-actin (Beijing TransGen Biotechnology); LY294002 (CST); 2′,7′-dichlorofluorescein diacetate (DCFH-DA); N-acetylcysteine (NAC) and diphenyliodonium (DPI) (Sigma); goat anti-rabbit secondary antibody and rabbit anti-mouse second antibody (Beijing Zhongshan Golden Bridge Biotechnology), and SuperEnhanced chemiluminescence detection reagents (Millipore).

Techniques: Expressing, Incubation, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Archives of Medical Science : AMS

Article Title: Urotensin II promotes secretion of LTB 4 through 5-lipoxygenase via the UT-ROS-Akt pathway in RAW264.7 macrophages

doi: 10.5114/aoms.2019.85197

Figure Lengend Snippet: Akt mediates UII-induced expression of 5-LO and LTB4 release in RAW264.7 macrophages. A – UII induces Akt phosphorylation in RAW264.7 macrophages at the indicated time points. The cells were exposed to 10 –8 M UII for 0 to 60 min. B, C – Effect of the different inhibitors on UII-induced 5-LO expression. The cells were pretreated with PD98059 (10 μM), SB203580 (10 μM) and LY294002 (10 μM) for 1 h prior to stimulation with UII (10 –8 M) for 24 h. 5-LO mRNA expression ( C ) and protein level ( B ) were then detected by real-time PCR and western blot analysis, respectively. D – Effect of different inhibitors on LTB4 release in the presence of UII (10 –8 M) for 24 h UA – urantide, PD – PD98059, SB – SB203580, LY – LY294002. The data are from three independent experiments and are expressed as the mean ± SEM. *P < 0.01 compared with control; # p < 0.01 compared with the UII (10 –8 M) group.

Article Snippet: The reagents used in the study were purchased from the following suppliers: Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS) and trypsin (Gibco); mouse UII (Phoenix Pharmaceuticals); urantide (Peptides International); Invitrogen TRIZOL reagent (Life Technologies); High-Capacity cDNA Reverse Transcription Kit and SYBR Select Master Mix (Applied Biosystems); mouse LTB 4 ELISA kit (Cayman); antibodies against 5-LO, p-Akt, Akt (CST), and β-actin (Beijing TransGen Biotechnology); LY294002 (CST); 2′,7′-dichlorofluorescein diacetate (DCFH-DA); N-acetylcysteine (NAC) and diphenyliodonium (DPI) (Sigma); goat anti-rabbit secondary antibody and rabbit anti-mouse second antibody (Beijing Zhongshan Golden Bridge Biotechnology), and SuperEnhanced chemiluminescence detection reagents (Millipore).

Techniques: Expressing, Phospho-proteomics, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Archives of Medical Science : AMS

Article Title: Urotensin II promotes secretion of LTB 4 through 5-lipoxygenase via the UT-ROS-Akt pathway in RAW264.7 macrophages

doi: 10.5114/aoms.2019.85197

Figure Lengend Snippet: Effects of different inhibitors on the phosphorylation level of Akt. The cells were stimulated with UII (10 –8 M) for 10 min after pretreatment with urantide (10 μM), NAC (10 mM), DPI (10 μM), and LY294002 (10 μM) for 1 h. Total and phosphorylated Akt levels were determined by western blot analysis UA – urantide, LY – LY294002. The data are from three independent experiments and are expressed as the mean ± SEM. *P < 0.01 compared with control; #P < 0.01 compared with the UII (10 –8 M) group.

Article Snippet: The reagents used in the study were purchased from the following suppliers: Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS) and trypsin (Gibco); mouse UII (Phoenix Pharmaceuticals); urantide (Peptides International); Invitrogen TRIZOL reagent (Life Technologies); High-Capacity cDNA Reverse Transcription Kit and SYBR Select Master Mix (Applied Biosystems); mouse LTB 4 ELISA kit (Cayman); antibodies against 5-LO, p-Akt, Akt (CST), and β-actin (Beijing TransGen Biotechnology); LY294002 (CST); 2′,7′-dichlorofluorescein diacetate (DCFH-DA); N-acetylcysteine (NAC) and diphenyliodonium (DPI) (Sigma); goat anti-rabbit secondary antibody and rabbit anti-mouse second antibody (Beijing Zhongshan Golden Bridge Biotechnology), and SuperEnhanced chemiluminescence detection reagents (Millipore).

Techniques: Phospho-proteomics, Western Blot, Control