Journal: Molecular medicine reports

Article Title: NACHT, LRR and PYD domains-containing protein 3 inflammasome is activated and inhibited by berberine via toll-like receptor 4/myeloid differentiation primary response gene 88/nuclear factor-κB pathway, in phorbol 12-myristate 13-acetate-induced macrophages.

doi: 10.3892/mmr.2017.8189

Figure Lengend Snippet: Figure 2. Berberine inhibits the NLRP3 inflammasome activation and IL-1β synthesis in PMA-induced macrophages. (A) Berberine effects on the secretion of IL-1β in cell supernatant of THP-1 cells using ELISA. (B) Berberine reduces protein expression levels of IL-1β (black arrow), Caspase-1 (black arrow), ASC and NLRP3 in PMA-induced macrophages. (C) Berberine suppresses IL-1β both at the protein and mRNA levels in PMA-induced macrophages. (D) Berberine inhibits NLRP3 both at the protein and mRNA levels in PMA-induced macrophages. Band density of native THP-1 cells were defined as control and set to 1. Data are presented as the mean ± standard deviation. *P<0.05, **P<0.01 vs. PMA group; #P<0.05 vs. control. IL-1β, interleukin-1β; NLRP3, NACHT, LRR and PYD domains-containing protein 3; Ber, berberine; PMA, phorbol 12-myristate 13-acetate; ASC, apoptosis-associated speck-like protein containing a CARD.

Article Snippet: For the determination of IL-1β levels in cell medium, a cytokine-specific ELISA kit (cat. no. DLB50; R&D Systems Europe Ltd., Abingdon, UK) was used according to the manufacturer's protocol.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Expressing, Control, Standard Deviation

Journal: Nature Communications

Article Title: Hyperglycaemia inhibits REG3A expression to exacerbate TLR3-mediated skin inflammation in diabetes

doi: 10.1038/ncomms13393

Figure Lengend Snippet: ( a ) Quantification of REG3A mRNA expression in the skin wounds of normal or diabetic patients with acute injury ( n =5). ( b ) Immunohistochemical analysis of REG3A in the skin wounds from three normal or diabetic patients. The samples from normal patients were taken 1 day (two patients) or 2 days after acute injury, whereas the samples from diabetic patients were taken 5 or 9 or 14 days after acute injury. ( c ) The expression of RegIIIγ mRNA in skin wounds of normal and T1D mice 3 days after aseptic injury ( n =6). ( d ) Immunoblot analysis of RegIIIγ in skin extracts taken from 2 mm surrounding the wound edges of normal and T1D mice. ( e ) Immunohistochemical analysis of RegIIIγ in day-3 skin wounds of normal and T1D mice. ( f ) Quantification of TNF-α and IL-6 mRNA expression in day-3 skin wounds from normal and T1D mice treated with or without 100 μg RegIIIγ ( n =8). ( g ) Quantification of TNF-α and IL-6 production by ELISA in day-3 skin wounds of normal and T1D mice ( n =5). ( h ) Wound healing of normal and T1D mice treated with ( n =7) or without 100 μg RegIIIγ ( n =6). ( i ) Photographs of healing wounds from normal and T1D mice treated as in h . All the mice used in the study without specific notes were C57BL/6 strain, and T1D mice were developed by STZ induction. Long scale bars represent 200 μm. Short scale bars represent 50 μm. Black rectangles designate region of × 400 magnification shown in inset of b and e . The abbreviations used here are normal (Nor), diabetes mellitus (DM), unwounded (unW), wounded (W) and RegIIIγ (R). * P <0.05, ** P <0.01 and *** P <0.001. P values were determined by two-tail t -tests ( a ) or two-way analysis of variance (ANOVA; c , f – h ). Data are means±s.e.m. and representative of two to three independent experiments.

Article Snippet: For gene silencing, human SHP-1 , IL-33 and REG3A specific shRNA (short hairpin RNA; ) were cloned into the pLL3.7 vector (Addgene) as the manufacturer described.

Techniques: Expressing, Immunohistochemical staining, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Hyperglycaemia inhibits REG3A expression to exacerbate TLR3-mediated skin inflammation in diabetes

doi: 10.1038/ncomms13393

Figure Lengend Snippet: ( a ) IL-17 production by ELISA in skin extracts taken from 2 mm skin surrounding the wound edges of normal and T1D mice at indicated times ( n =4). ( b ) Immunoblot of REG3A in NHEKs stimulated by different doses of rhIL-33 for 12 h. ( c ) RegIIIγ and IL-33 production by ELISA in skin extracts taken as in a ( n =4). ( d , e ) Immunohistochemical analyses of IL-33 in day-3 skin wounds of normal and T1D mice ( d ) or skin wounds of normal and diabetic patients taken as in ( e ). Long scale bars represent 200 μm while short scale bars represent 50 μm. Black rectangles designate region of × 400 magnification shown in inset. ( f ) Immunoblot of RegIIIγ in the skin wounds of T1D mice treated with PBS or rmIL-33. The first two samples were day-3 skin wounds of normal and T1D mice. The other three samples were from the skin wounds of T1D mice treated with rmIL-33 a day before wounding (−1) or 1 or 3 days post wounding. ( g ) Wound healing of normal and T1D mice treated with ( n =7) or without rmIL-33 ( n =6). ( h ) IL-33 production in NHEKs induced by different doses of rhIL-17 for 24 h. ( i ) The production of IL-33 and RegIIIγ in day-3 skin wounds of wild-type (WT) and Il17 −/− mice. ( j ) REG3A production in NHEKs induced by 200 ng ml −1 rhIL-17 before and after IL-33 was silenced. ( k ) Immunoblot of RegIIIγ in skin wounds of WT and Il17 −/− mice treated with PBS or 2 μg rmIL-33. ( l ) IL-33 production induced by 200 ng ml −1 rhIL-17 in NHEKs exposed to 20 mM glucose or mannitol for 24 h. ( m ) Immunofluorescent staining of IL-33 in NHEKs treated as in l . Scale bars represent 25 μm. ( n ) IL-33 production in NHEKs treated with 200 ng ml −1 rhIL-17 in the presence or absence of 20 mM glucose before and after AGE was inhibited by 2 mM aminoguanidine. The abbreviations used here are IL-33 shRNA ( IL-33 sh), glucose (Glu), mannitol (Man), aminoguanidine (AG). * P <0.05, ** P <0.01 and *** P <0.001. P values were determined by two-way analysis of variance (ANOVA). Data are means±s.e.m. and representative of two to three independent experiments.

Article Snippet: For gene silencing, human SHP-1 , IL-33 and REG3A specific shRNA (short hairpin RNA; ) were cloned into the pLL3.7 vector (Addgene) as the manufacturer described.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Immunohistochemical staining, Staining, shRNA

Journal: Nature Communications

Article Title: Hyperglycaemia inhibits REG3A expression to exacerbate TLR3-mediated skin inflammation in diabetes

doi: 10.1038/ncomms13393

Figure Lengend Snippet: ( a ) Quantification of Tlr3 mRNA expression in day-3 skin wounds of normal and T1D mice ( n =6). ( b ) Quantification of TNF-α and IL-6 mRNA expression in the skin extracts taken from 2 mm surrounding the wound edges of WT and Tlr3 −/− T1D mice at day 3 ( n =6). ( c ) Wound healing of normal WT ( n =12), normal Tlr3 −/− ( n =8), diabetic WT ( n =12) and diabetic Tlr3 −/− ( n =10) mice. ( d ) Quantification of TNF-α ( n =5) and IL-6 ( n =8) protein by ELISA in the skin extracts taken from 2 mm surrounding the wound edges of WT and Tlr3 −/− T1D mice treated with or without RegIIIγ. ( e ) Quantification of TNF-α and IL-6 protein by ELISA in NHEKs treated with 5 μg ml −1 poly(I:C) in the presence or absence of 30 nM REG3A ( n =3). ( f ) Quantification of TNF-α and IL-6 protein by ELISA in NHEKs treated with 5 μg ml −1 poly(I:C) in the presence or absence of different mutants of REG3A ( n =3). ( g ) Quantification of TNF-α and IL-6 protein by ELISA in adult human epidermal keratinocytes (AHEKs) treated with 10 μg ml −1 poly(I:C) in the presence or absence of 30 nM REG3A or C-REG3A ( n =3). * P <0.05, ** P <0.01 and *** P <0.001. NS, no significance; ND, not detected. P values were analysed by one-way analysis of variance (ANOVA; f , g ) or two-way ANOVA ( a – e ). Data are means±s.e.m. and representative of two to three independent experiments.

Article Snippet: For gene silencing, human SHP-1 , IL-33 and REG3A specific shRNA (short hairpin RNA; ) were cloned into the pLL3.7 vector (Addgene) as the manufacturer described.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Hyperglycaemia inhibits REG3A expression to exacerbate TLR3-mediated skin inflammation in diabetes

doi: 10.1038/ncomms13393

Figure Lengend Snippet: ( a ) Immunoblot of SHP-1 in NHEKs stimulated with 30 nM REG3A for indicated times. ( b ) Immunoblot of SHP-1 in AHEKs stimulated with different doses of REG3A for 24 h. ( c ) Immunoblot of SHP-1 in mouse skin intradermally injected with 100 μg RegIIIγ for indicated times. ( d , e ) Immunoblot of SHP-1 in NHEKs transfected with different doses of pSG5-C-REG3A ( d ) for indicated times ( e ). ( f ) Immunoblot of SHP-1 in NHEKs stimulated with 100 ng ml −1 rhIL-33 before and after REG3A silencing. ( g ) TNF-α and IL-6 mRNA expression in NHEKs transfected with 1 μg pCMV vector or pCMV-SHP-1 in the presence or absence of 5 μg ml −1 poly(I:C) for 24 h ( n =3). ( h ) TNF-α and IL-6 mRNA expression in NHEKs treated with 5 μg ml −1 poly(I:C) and/or 30 nM REG3A before or after SHP-1 silencing ( n =3). ( i ) TNF-α and IL-6 production in AHEKs treated as in h ( n =3). ( j ) TNF-α and IL-6 mRNA expression in day-3 skin wounds of WT normal or T1D mice ( n =5). The mice were intradermally injected with H 2 O or 4 mg of SHP-1 inhibitor SSG before wounding. Three days later, 2 mm skin surrounding the wounds was taken for RNA isolation. The abbreviations used here are SHP-1 shRNA ( SHP-1 sh) and REG3A (R). * P <0.05, ** P <0.01 and *** P <0.001. NS, no significance; ND, not detected. P values were analysed by one-way analysis of variance (ANOVA; g ) or two-way ANOVA ( h – j ). Data are means±s.e.m. and representative of two to three independent experiments.

Article Snippet: For gene silencing, human SHP-1 , IL-33 and REG3A specific shRNA (short hairpin RNA; ) were cloned into the pLL3.7 vector (Addgene) as the manufacturer described.

Techniques: Western Blot, Injection, Transfection, Expressing, Plasmid Preparation, Isolation, shRNA

Journal: Nature Communications

Article Title: Hyperglycaemia inhibits REG3A expression to exacerbate TLR3-mediated skin inflammation in diabetes

doi: 10.1038/ncomms13393

Figure Lengend Snippet: ( a ) Interaction between Flag-tagged different domains of EXTL3 and REG3A assessed by immunoblot analysis after immunoprecipitation with anti-Flag or anti-REG3A. M: mock; F: full-length EXTL3; N: N-EXTL3 (1–548); C: C-EXTL3 (141–919); ΔNΔC: EXTL3 ΔNΔC (141–548). ( b ) Interaction between REG3A or C-REG3A and Flag-tagged N-EXTL3 assessed by immunoblot analysis after immunoprecipitation with anti-REG3A or anti-Flag. F: full-length REG3A; C: CTLD domain of REG3A. ( c ) Quantification of TNF-α and IL-6 mRNA expression in NHEKs stimulated with 5 μg ml −1 poly(I:C) and/or 30 nM REG3A before or after EXTL3 silencing ( n =3). ( d ) Immunoblot of SHP-1 in NHEKs treated with 30 nM REG3A before or after EXTL3 silencing. EXTK3 si: EXTL3 siRNA. ( e ) SHP-1 production in NHEKs treated with 30 nM REG3A in the presence or absence of NF-κB inhibitor (Bay11, 10 μM), Erk inhibitor (PD98059, 20 μM), p38 MAPK inhibitor (SB202190, 5 μM), PI3K inhibitor (LY294002, 50 μM), STAT3 inhibitor (S3I-201,50 μM) and AKT inhibitor (AKT1/2 inhibitor, 8 μM). ( f ) Immunoblot of phosphorylated AKT in NHEKs treated with 30 nM REG3A for 1 h before or after EXTL3 was silenced. ( g ) Immunoblot of p-STAT3 and p-AKT in NHEKs treated with 30 nM REG3A and/or AKT1/2 inhibitor for 1 h. AKT1/2 i: AKT1/2 inhibitor. ( h ) Immunoblot of p-STAT3 and p-AKT in NHEKs treated with 30 nM REG3A and/or STAT3 inhibitor (S3I-201) for 1 h. STAT3 i: STAT3 inhibitor. *** P <0.001. NS, no significance. P values were analysed by two-way analysis of variance (ANOVA). Data are means±s.e.m. and representative of two to three independent experiments.

Article Snippet: For gene silencing, human SHP-1 , IL-33 and REG3A specific shRNA (short hairpin RNA; ) were cloned into the pLL3.7 vector (Addgene) as the manufacturer described.

Techniques: Western Blot, Immunoprecipitation, Expressing

Journal: Nature Communications

Article Title: Hyperglycaemia inhibits REG3A expression to exacerbate TLR3-mediated skin inflammation in diabetes

doi: 10.1038/ncomms13393

Figure Lengend Snippet: ( a , b ) Quantification of TNF-α and IL-6 production by ELISA in NHEKs treated with 5 μg ml −1 poly(I:C) ( a ) or in AHEKs treated with 10 μg ml −1 poly(I:C) ( b ) in the presence or absence of JNK inhibitor (SP600125, 15 μM) for 24 h ( n =3). ( c ) The phosphorylation of JNK and c-Jun in NHEKs stimulated by 5 μg ml −1 poly(I:C) in the presence or absence of JNK inhibitor for 1 h. JNK i: JNK inhibitor. ( d , e ) Normalized luciferase activity in 293T cells co-transfected with the plasmid expressing c-Jun (0, 50, 100, 150, 200 ng) and plasmids containing TNF-α promoter (50 ng; d ), or TNF-α promoter with mutation in c-Jun binding site (50 ng; d ), or IL-6 promoter (100 ng; e ), or IL-6 promoter with the mutation in c-Jun binding site (100 ng; e ) for 24 h. ( f ) The phosphorylation of JNK and c-Jun in WT primary murine keratinocytes stimulated by 10 μg ml −1 poly(I:C) for indicated times. ( g ) The phosphorylation of JNK2 and c-Jun in Jnk1 −/− primary murine keratinocytes stimulated by 10 μg ml −1 poly(I:C) for indicated times. ( h ) The phosphorylation of JNK1 and c-Jun in Jnk2 −/− primary murine keratinocytes stimulated by 10 μg ml −1 poly(I:C) for indicated times. ( i , j ) JNK2 phosphorylation in the skin wounds of WT and Tlr3 −/− normal mice ( i ) or T1D mice ( j ). ( k ) Quantification of TNF-α and IL-6 production by ELISA in day-3 skin wounds of WT and Jnk2 −/− normal and T1D mice ( n =6). ( l ) Wound healing of WT ( n =6), Jnk1 −/− ( n =4) and Jnk2 −/− T1D mice ( n =6). ( m , n ) JNK2 phosphorylation in NHEKs stimulated by 5 μg ml −1 poly(I:C) in the presence or absence of 30 nM REG3A ( m ) or C-REG3A ( n ). ( o ) JNK2 phosphorylation in AHEKs stimulated by 10 μg ml −1 poly(I:C) in the presence or absence of 30 nM REG3A. * P <0.05, ** or ## P <0.01 and ***or ### P <0.001. P value was analysed by one-way analysis of variance (ANOVA; a , b ) or two-way ANOVA ( d , e , k , l ). Data are the means±s.e.m. and representative of two to three independent experiments.

Article Snippet: For gene silencing, human SHP-1 , IL-33 and REG3A specific shRNA (short hairpin RNA; ) were cloned into the pLL3.7 vector (Addgene) as the manufacturer described.

Techniques: Enzyme-linked Immunosorbent Assay, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Expressing, Mutagenesis, Binding Assay

Journal: Nature Communications

Article Title: Hyperglycaemia inhibits REG3A expression to exacerbate TLR3-mediated skin inflammation in diabetes

doi: 10.1038/ncomms13393

Figure Lengend Snippet: ( a ) Phosphorylated JNK2 in NHEKs transfected with pCMV vector, pCMV-muSHP-1(C453S) and pCMV-SHP-1 in the presence or absence of 5 μg ml −1 poly(I:C). ( b ) Phosphorylated JNK2 in NHEKs stimulated with 5 μg ml −1 poly(I:C) and/or 30 nM REG3A before or after SHP-1 silencing. ( c ) Interaction between SHP-1 and JNK2 in NHEKs stimulated with 5 μg ml −1 poly(I:C) and/or 30 nM REG3A assessed by immunoblot analysis after immunoprecipitation with anti-SHP-1 or anti-JNK2. ( d ) Immunoblot of p-JNK2, SHP-1 and RegIIIγ in the skin extracts taken from 2 mm surrounding the wound edges of WT normal and T1D mice at indicated times. ( e ) Immunoblot of p-JNK2, SHP-1 and RegIIIγ in the skin wounds of WT normal mice injected with IgG or RegIIIγ-neutralizing antibody. ( f ) Immunoblot of p-JNK2 and SHP-1 in the skin wounds of WT normal and T1D mice injected with 100 μg RegIIIγ. ( g ) Wound healing in T1D mice treated with or without 100 μg RegIIIγ before or after SHP-1 was inhibited by its inhibitor SSG ( n =9). ( h ) The schematic graph reflects the interaction between REG3A and TLR3 signalling. After skin injury, IL-33 induces REG3A expression in epidermal keratinocytes. REG3A, in turn, acts on keratinocytes to induce the negative regulator SHP-1 to selectively inhibit TLR3-activated JNK2, thus controlling TLR3-induced inflammation in skin wounds. However, in diabetes, hyperglycaemia inhibits IL-33 expression induced by IL-17. The reduction of IL-33 leads to the decrease in REG3A and SHP-1 but increased TLR3-activated JNK2 phosphorylation, thus exacerbating inflammation in diabetic skin wounds. The abbreviations used here are control (Ctrl), poly(I:C) (P), REG3A (R) and Reg Ab (RegIIIγ antibody). * P <0.05, and *** P <0.001. NS, no significance. P value was analysed by two-way analysis of variance (ANOVA). Data are the means±s.e.m. and representative of two independent experiments.

Article Snippet: For gene silencing, human SHP-1 , IL-33 and REG3A specific shRNA (short hairpin RNA; ) were cloned into the pLL3.7 vector (Addgene) as the manufacturer described.

Techniques: Transfection, Plasmid Preparation, Western Blot, Immunoprecipitation, Injection, Expressing