recombinant mouse il 38  (Sino Biological)

Journal: Bone & Joint Research

Article Title: Long noncoding RNA H19 alleviates inflammation in osteoarthritis through interactions between TP53, IL-38, and IL-36 receptor

doi: 10.1302/2046-3758.118.BJR-2021-0188.R1

Figure Lengend Snippet: Expression of interleukin (IL)-38 is upregulated in osteoarthritis (OA). a) Representative images of cartilage damage in OA mice assessed by safranin O-fast green staining (n = 10; 200×); b) Osteoarthritis Research Society International (OARSI) scores of cartilage damage in OA mice, *p < 0.05 versus sham-operated mice (n = 10). c) IL-38 level in synovial fluid from OA mice detected by enzyme-linked immunosorbent assay (ELISA). *p < 0.05 versus sham-operated mice at different timepoints (n = 5). d) IL-38 level in the supernatant of chondrocytes after IL-1β treatment detected by ELISA. *p < 0.05 versus control chondrocytes. e) Messenger RNA (mRNA) and protein expressions of IL-38 in cartilage tissues from OA mice (n = 10). *p < 0.05 versus sham-operated mice (n = 10). f) and g) mRNA (f) and protein (g) expression of IL-38 in chondrocytes after IL-1β treatment measured by quantitative reverse transcription polymerase chain reaction and western blot analysis. h) The expression of IL-38 in chondrocytes determined via immunofluorescence (400×). *p < 0.05 versus control chondrocytes. The measurement data were expressed as mean (standard deviation). Comparison between two groups was conducted by independent-samples t -test. The cell experiment was repeated three times independently. Ctr, control.

Article Snippet: Further, recombinant mouse IL-38 (R&D Systems) was diluted with PBS containing 0.5% BSA and 0.05% Tween-20, and then incubated in a coated/blocked ELISA plate at 4°C overnight.

Techniques: Expressing, Staining, Enzyme-linked Immunosorbent Assay, Control, Reverse Transcription, Polymerase Chain Reaction, Western Blot, Immunofluorescence, Standard Deviation, Comparison

Journal: Bone & Joint Research

Article Title: Long noncoding RNA H19 alleviates inflammation in osteoarthritis through interactions between TP53, IL-38, and IL-36 receptor

doi: 10.1302/2046-3758.118.BJR-2021-0188.R1

Figure Lengend Snippet: Exogenous overexpression of interleukin (IL)-38 attenuates inflammatory response of osteoarthritis (OA) mice. a) Messenger RNA (mRNA) expression of IL-38 in knee joint cartilage tissues from OA mice and sham-operated mice in response to lentivirus vector (LV)-IL-38 or LV-negative control (NC), as determined by quantitative reverse transcription polymerase chain reaction (RT-qPCR). *p < 0.05 versus sham-operated mice injected with LV-NC; #p < 0.05 versus OA mice injected with LV-NC. b) IL-38 protein level in the knee joint cartilage tissues (left) and synovial fluid (right) from OA mice and sham-operated mice in response to LV-IL-38 or LV-NC as measured using western blot analysis and enzyme-linked immunosorbent assay (ELISA). *p < 0.05 versus sham-operated mice injected with LV-NC; #p < 0.05 versus OA mice injected with LV-NC. c) Levels of inflammation-related factors (IL-6, IL-8, IL-17, IL-22, tumour necrosis factor (TNF)-α, interferon (IFN)-γ, and cartilage oligomeric matrix protein (COMP)) in synovial fluid from OA mice and sham-operated mice in response to LV-IL-38 or LV-NC, tested using ELISA. d) The Osteoarthritis Research Society International (OARSI) score of cartilage damage in OA mice and sham-operated mice treated with LV-IL-38 or LV-NC identified by safranin O-fast green staining. e) Chondrocyte apoptosis in OA mice and sham-operated mice treated with LV-IL-38 or LV-NC detected using Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) assay. In panels c) to f), *p < 0.05 versus OA mice with injection of LV-NC. The measurement data were expressed as mean (standard deviation). The cell experiment was repeated three times independently. Comparison between two groups was conducted by independent-samples t -test; n = 10.

Article Snippet: Further, recombinant mouse IL-38 (R&D Systems) was diluted with PBS containing 0.5% BSA and 0.05% Tween-20, and then incubated in a coated/blocked ELISA plate at 4°C overnight.

Techniques: Over Expression, Expressing, Plasmid Preparation, Negative Control, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Injection, Western Blot, Enzyme-linked Immunosorbent Assay, Staining, TUNEL Assay, Standard Deviation, Comparison

Journal: Bone & Joint Research

Article Title: Long noncoding RNA H19 alleviates inflammation in osteoarthritis through interactions between TP53, IL-38, and IL-36 receptor

doi: 10.1302/2046-3758.118.BJR-2021-0188.R1

Figure Lengend Snippet: Long noncoding RNA (lncRNA) H19 increases interleukin (IL)-38 expression through transcription factor TP53. a) Prediction of potential transcription factors regulating IL-38 by PROMO and CIS-BP databases. b) Messenger RNA (mRNA) expression of ETS1, TP53, GATA2, GATA1, ELK1, MAZ, and ETS2 in osteoarthritis (OA) mice and chondrocytes. *p < 0.05 versus sham-operated mice or control chondrocytes. c) TP53 enrichment of lncRNAs GAS5, MEG3, TUG1, MALAT1, and H19 determined by RNA binding protein immunoprecipitation (RIP) assay. Results are normalized to immunoglobulin G (IgG). *p < 0.05 versus anti-IgG. d) mRNA expression of lncRNA H19 in OA mice (n = 10) and chondrocytes. *p < 0.05 versus sham-operated mice or control chondrocytes. e) Relative luciferase activity of IL-38 promoter in OA chondrocytes after various treatments. *p < 0.05 versus OA chondrocytes treated with oe-negative control (NC), #p < 0.05 versus OA chondrocytes treated with sh-NC. f) Relative enrichment of lncRNA H19 by TP53 determined by RIP assay. *p < 0.05 versus anti-IgG. g) Relative enrichment of IL-38 by TP53 in OA chondrocytes determined by chromatin immunoprecipitation (ChIP) assay. *p < 0.05 versus anti-IgG. h) Transfection efficiency in OA chondrocytes detected by quantitative reverse transcription polymerase chain reaction (RT-qPCR). *p < 0.05 versus OA chondrocytes treated with both oe-NC and sh-NC. i) IL-38 level in OA chondrocytes after various treatments. *p < 0.05 versus OA chondrocytes co-treated with oe-NC and sh-NC. j) Transfection efficiency in OA mice detected by RT-qPCR. *p < 0.05 versus OA mice treated with lentivirus vector (LV)-NC. k) IL-38 levels in OA mice after various treatments. *p < 0.05 versus OA mice treated with LV-NC. l) Pearson analysis for the expression of lncRNA H19 and IL-38 in knee joint cartilage tissues from OA mice. The measurement data were expressed as mean (standard deviation). Comparison between two groups was conducted by independent-samples t -test. Comparison among multiple groups was conducted by one-way analysis of variance, followed by Tukey’s post hoc test; n = 10. The cell experiment was repeated three times independently. Ctr, control.

Article Snippet: Further, recombinant mouse IL-38 (R&D Systems) was diluted with PBS containing 0.5% BSA and 0.05% Tween-20, and then incubated in a coated/blocked ELISA plate at 4°C overnight.

Techniques: Expressing, Control, RNA Binding Assay, Immunoprecipitation, Luciferase, Activity Assay, Negative Control, Chromatin Immunoprecipitation, Transfection, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Plasmid Preparation, Standard Deviation, Comparison

Journal: Bone & Joint Research

Article Title: Long noncoding RNA H19 alleviates inflammation in osteoarthritis through interactions between TP53, IL-38, and IL-36 receptor

doi: 10.1302/2046-3758.118.BJR-2021-0188.R1

Figure Lengend Snippet: Elevated long noncoding RNA (lncRNA) H19 exerts anti-inflammation effects on osteoarthritis (OA) by promoting interleukin (IL)-38, tumour protein p53 (TP53), and IL-36 receptor (IL-36R). a) Transfection efficiency in knee joint cartilage tissues from OA mice detected by quantitative reverse transcription polymerase chain reaction (RT-qPCR). b) Levels of inflammation-related factors (IL-6, IL-8, IL-17, IL-22, TNF-α, interferon (IFN)-γ, and cartilage oligomeric matrix protein (COMP)) in synovial fluid from OA mice with various treatments. c) The Osteoarthritis Research Society International (OARSI) score of cartilage damage in OA mice with various treatments identified by safranin O-fast green staining. d) Chondrocyte apoptosis after various treatments determined by Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) assay. *p < 0.05 versus OA mice treated with lentivirus vector (LV)-negative control (NC); #p < 0.05 versus OA mice treated with LV-oe-H19. The cell experiment was repeated three times independently. The measurement data were expressed as mean (standard deviation). One-way analysis of variance was used for data comparison among multiple groups, followed by Tukey’s post hoc test; n = 10.

Article Snippet: Further, recombinant mouse IL-38 (R&D Systems) was diluted with PBS containing 0.5% BSA and 0.05% Tween-20, and then incubated in a coated/blocked ELISA plate at 4°C overnight.

Techniques: Transfection, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Staining, TUNEL Assay, Plasmid Preparation, Negative Control, Standard Deviation, Comparison

Journal: Bone & Joint Research

Article Title: Long noncoding RNA H19 alleviates inflammation in osteoarthritis through interactions between TP53, IL-38, and IL-36 receptor

doi: 10.1302/2046-3758.118.BJR-2021-0188.R1

Figure Lengend Snippet: Long noncoding RNA (lncRNA) H19/interleukin (IL)-38 axis involving IL-36 receptor (IL-36R) harbours the anti-inflammatory property in osteoarthritis (OA) in vitro. a) Binding between mouse IL-38 and IL-36R in peripheral blood mononuclear cells (PBMCs) analyzed by receptor-binding assay. b) Binding between IL-38 and IL-36R in PBMCs detected by co-immunoprecipitation assay. c) Transfection efficiency detected by quantitative reverse transcription polymerase chain reaction. d) Levels of pro-inflammatory factors in PBMCs after various treatments. In panels c) and d), *p < 0.05 versus co-treatment of oe-negative control (NC) and sh-NC; #p < 0.05 versus co-treatment of oe-H19 and sh-NC. The measurement data were expressed as mean (standard deviation). One-way analysis of variance was used for data comparison among multiple groups, followed by Tukey’s post hoc test. Comparison of IL-38 expression among multiple groups was analyzed by two-way analysis of variance, followed by Bonferroni correction. The cell experiment was repeated three times independently. GFP, green fluorescence protein; IFN, interferon; OD, optical density.

Article Snippet: Further, recombinant mouse IL-38 (R&D Systems) was diluted with PBS containing 0.5% BSA and 0.05% Tween-20, and then incubated in a coated/blocked ELISA plate at 4°C overnight.

Techniques: In Vitro, Binding Assay, Reporter Assay, Co-Immunoprecipitation Assay, Transfection, Reverse Transcription, Polymerase Chain Reaction, Negative Control, Standard Deviation, Comparison, Expressing, Fluorescence

Journal: Bone & Joint Research

Article Title: Long noncoding RNA H19 alleviates inflammation in osteoarthritis through interactions between TP53, IL-38, and IL-36 receptor

doi: 10.1302/2046-3758.118.BJR-2021-0188.R1

Figure Lengend Snippet: The regulatory mechanism of the long noncoding RNA (lncRNA) H19/tumour protein p53 (TP53)/interleukin (IL)-38 axis involved in osteoarthritis (OA). lncRNA H19 was upregulated in OA. H19 upregulated the expression of IL-38 by recruiting TP53 to the IL-38 promoter region, which promoted binding of IL-38 with IL-36 receptor and inhibited the inflammatory response in OA. IL-36R, interleukin-36 receptor; mRNA, messenger RNA.

Article Snippet: Further, recombinant mouse IL-38 (R&D Systems) was diluted with PBS containing 0.5% BSA and 0.05% Tween-20, and then incubated in a coated/blocked ELISA plate at 4°C overnight.

Techniques: Expressing, Binding Assay