Journal: Nature Communications

Article Title: An injectable liposome-anchored teriparatide incorporated gallic acid-grafted gelatin hydrogel for osteoarthritis treatment

doi: 10.1038/s41467-023-38597-0

Figure Lengend Snippet: a , d Live/dead stained fluorescent images and statistical analysis for cells’ cytotoxicity, green stand for the live cells and red is the dead cells. b , e EdU/DAPI stained fluorescent images to evaluate the cell proliferation (red represents the newly proliferated cells (EdU staining), blue represents all cells (DAPI staining)). c , f Flow cytometry (FCM) results and G2/M rate calculated from FCM results. g – l Immunofluorescent images for Ki-67, c-Fos, and PTH1R expression for the ATDC5 cells cultured with hydrogels and the statistical stained positively area for the expression of such genes. m , n Western Blot results for the expression of proliferation proteins (PTH1R, SOX9, MMP13, ADAMTS5) of ATDC5 cells when cultured with hydrogels and the corresponding statistical analysis results. Data are presented as means ± SD of at least three replicate experiments. Unpaired two-tailed Student’s t tests was used to calculate significant difference, * p < 0.05, ** p < 0.01. GGA gallic acid-grafted gelatin, GLP GGA@Lipo@PTH (1–34).

Article Snippet: The immunohistochemical stainings including PTH1R (Cat#BS2710, 1:400, Bioword Technology, US), SOX9 (Cat#ab185966, 1:1000, abcam, UK), ACAN (Cat#13880-1-AP, 1:500, Proteintech, China), COLIIA1 (Cat#28459-1-AP, 1:500, Proteintech, China), MMP13 (Cat#18165-1-AP, 1:500, Proteintech, China); ADAMTS5 (Cat#bs-3573R, 1:200, Bioss, China), Bcl-2 (Cat#68103-1-Ig, 1:500, Proteintech, China), and BAX (Cat#AF0057, 1:200, Beyotime Biotechnology, China). were performed with 4 μm in thick sections.

Techniques: Staining, Flow Cytometry, Expressing, Cell Culture, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: An injectable liposome-anchored teriparatide incorporated gallic acid-grafted gelatin hydrogel for osteoarthritis treatment

doi: 10.1038/s41467-023-38597-0

Figure Lengend Snippet: a , b The FCM results and statistical analysis results for the percentage of apoptosis. c – h Immunofluorescent images for SOX9, Bcl-2, and BAX protein expression as well as the statistical stained positively area from the images. Sox9 is the characteristic gene to indicate the ECM expression, Bcl-2 and Bax genes are the anti-apoptosis and the apoptosis-promoting genes of the cells. i – n The RT-qPCR results of the mRNA expression levels for Sox9, Col2a1, Acan, Adamts5, iNOS, and COX2. o , p The ELISA results for inflammatory mediators, IL-6 and TNF-α. q , r The Western Blot results for the expression of proteins (p-PI3K, PI3K, p-AKT/AKT, ADAMTS5) of IL-1β-induced ATDC5 cells cultured with hydrogels and the corresponding statistical analysis results. Data are presented as means ± SD of at least three replicate experiments. Unpaired two-tailed Student’s t tests was used to calculate significant difference, * p < 0.05, ** p < 0.01. GGA gallic acid-grafted gelatin, GLP GGA@Lipo@PTH (1–34).

Article Snippet: The immunohistochemical stainings including PTH1R (Cat#BS2710, 1:400, Bioword Technology, US), SOX9 (Cat#ab185966, 1:1000, abcam, UK), ACAN (Cat#13880-1-AP, 1:500, Proteintech, China), COLIIA1 (Cat#28459-1-AP, 1:500, Proteintech, China), MMP13 (Cat#18165-1-AP, 1:500, Proteintech, China); ADAMTS5 (Cat#bs-3573R, 1:200, Bioss, China), Bcl-2 (Cat#68103-1-Ig, 1:500, Proteintech, China), and BAX (Cat#AF0057, 1:200, Beyotime Biotechnology, China). were performed with 4 μm in thick sections.

Techniques: Expressing, Staining, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Cell Culture, Two Tailed Test

Journal: Nature Communications

Article Title: An injectable liposome-anchored teriparatide incorporated gallic acid-grafted gelatin hydrogel for osteoarthritis treatment

doi: 10.1038/s41467-023-38597-0

Figure Lengend Snippet: The GLP hydrogel would promote ATDC5 cells proliferation by up-regulating the expression of cell proliferation and anti-apoptosis genes (Ki-67, c-Fos), and this hydrogel would protect IL-1β-induced ATDC5 cells from further progression by up-regulating the expression of key anabolic genes (Sox9, Bcl-2, Col2a1, Acan) and down-regulating the expression of key catabolic genes (Bax, Adamts5), which potentially suggested regulating the PI3K/AKT signaling pathway. GLP GGA@Lipo@PTH (1–34).

Article Snippet: The immunohistochemical stainings including PTH1R (Cat#BS2710, 1:400, Bioword Technology, US), SOX9 (Cat#ab185966, 1:1000, abcam, UK), ACAN (Cat#13880-1-AP, 1:500, Proteintech, China), COLIIA1 (Cat#28459-1-AP, 1:500, Proteintech, China), MMP13 (Cat#18165-1-AP, 1:500, Proteintech, China); ADAMTS5 (Cat#bs-3573R, 1:200, Bioss, China), Bcl-2 (Cat#68103-1-Ig, 1:500, Proteintech, China), and BAX (Cat#AF0057, 1:200, Beyotime Biotechnology, China). were performed with 4 μm in thick sections.

Techniques: Expressing

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Flavokawain A alleviates the progression of mouse osteoarthritis: An in vitro and in vivo study

doi: 10.3389/fbioe.2022.1071776

Figure Lengend Snippet: Protective effects of FKA against IL-1β-induced inflammation and ECM destruction in mouse chondrocytes. Chondrocyte cell treatment with 5 ng/ml of IL-1β only or with FKA (20 and 40 μM) for a period of 24 h. Western blot analysis of inflammatory cytokines (COX2 and iNOS), catabolic (ADAMTS5, MMP3, and MMP13), and anabolic markers (Col2, Aggrecan, and Sox9) (A,C and E) . Quantitative analysis of protein expression (B,D and F) . (G and H) 5 ng/ml of IL-1β was added for treating the cells for 24 h in the absence or presence of 40 µM FKA. Confocal microscopy showing the expression of MMP13 and Aggrecan in immunofluorescence experiments (scale bar 10 μm). (I) Relative quantification of fluorescence intensity. The values are presented as means ± SD ( n = 3). # p < 0.05 vs the control group; * p < 0.05 and ** p < 0.01 vs the IL-1β group.

Article Snippet: ADAMTS5 primary antibody, secondary antibody, phosphate-buffered saline (PBS), trypsin, collagenase type II, the CCK8 assay kit, bovine serum albumin (BSA), and protein extraction kit were ordered and acquired from Boster Biological Technology (Wuhan, Hubei, China).

Techniques: Western Blot, Expressing, Confocal Microscopy, Immunofluorescence, Quantitative Proteomics, Fluorescence, Control

Journal: Frontiers in Pharmacology

Article Title: Physalin A Inhibits MAPK and NF-κB Signal Transduction Through Integrin αVβ3 and Exerts Chondroprotective Effect

doi: 10.3389/fphar.2021.761922

Figure Lengend Snippet: PA suppressed excess expression of the catabolic indicators of chondrocytes induced by IL-1β, including ADAMTS5, MMP1, MMP3, and MMP13. Mice chondrocytes were treated with 5 ng/ml of IL-1β, alone or with PA (2.5, 5, and 10 μM) for 24 h (A) Western blotting results and (B–E) quantitative analysis of ADAMTS5, MMP1, MMP3, and MMP13. (F) MMP13 expression was observed by immunofluorescence staining when chondrocytes were treated with 5 ng/ml of IL-1β, alone or with 10 μM of PA (scale bar 200 μm). (G–I) Relative mRNA levels of ADAMTS5, MMP3, and MMP13 in chondrocytes stimulated with 5 ng/ml of IL-1β, alone or with PA (2.5, 5, and 10 μM) for 24 h. GAPDH was used as an internal reference. Data are presented as means ± SD ( n = 3). The exact p value was marked in the corresponding figure and p < 0.05 was considered statistically significant.

Article Snippet: Primary antibody against ADAMTS5 (A02802-1) was got from Boster (Wuhan, Hubei, China) and was applied at a 1:500 dilution.

Techniques: Expressing, Western Blot, Immunofluorescence, Staining

Journal: Frontiers in Pharmacology

Article Title: Physalin A Inhibits MAPK and NF-κB Signal Transduction Through Integrin αVβ3 and Exerts Chondroprotective Effect

doi: 10.3389/fphar.2021.761922

Figure Lengend Snippet: Knockdown of integrin αVβ3 weakened the anti-inflammatory, anabolism enhancing, and catabolism inhibiting effect of PA on IL-1β-induced chondrocytes. (A,B) Relative mRNA levels of integrin αV (Itg αV) and integrin β3 (Itg β3) in chondrocytes stimulated with 5 ng/ml of IL-1β, alone or with PA (2.5, 5, and 10 μM) for 24 h (C,D) Itg αV and Itg β3 were knocked down by siRNA transfection, and the knockdown efficiency was verified by RT-PCR. (E,F) Inflammatory markers (COX2, iNOS) were detected by western blotting and the band density of protein levels were quantified after mice chondrocytes were added with or without 5 ng/ml of IL-1β, 10 μM of PA, and Itg αVβ3 siRNA. (G–I) Western blotting was applied to measure the anabolic (aggrecan, collagen II) and catabolic markers (MMP1, MMP3, MMP13, and ADAMTS5) in the Itg αVβ3-deficiency mice chondrocytes along with or without the administration of 5 ng/ml of IL-1β and 10 μM of PA, and the band density of these protein levels were quantified in the histogram. GAPDH was used as an internal reference. Data are presented as means ± SD ( n = 3). The exact p value was marked in the corresponding figure and p < 0.05 was considered statistically significant.

Article Snippet: Primary antibody against ADAMTS5 (A02802-1) was got from Boster (Wuhan, Hubei, China) and was applied at a 1:500 dilution.

Techniques: Knockdown, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Physalin A Inhibits MAPK and NF-κB Signal Transduction Through Integrin αVβ3 and Exerts Chondroprotective Effect

doi: 10.3389/fphar.2021.761922

Figure Lengend Snippet: Primer sequence used in the RT-qPCR experiment.

Article Snippet: Primary antibody against ADAMTS5 (A02802-1) was got from Boster (Wuhan, Hubei, China) and was applied at a 1:500 dilution.

Techniques: Sequencing

Journal: Annals of Translational Medicine

Article Title: MicroRNA-25-3p therapy for intervertebral disc degeneration by targeting the IL-1β/ZIP8/MTF1 signaling pathway with a novel thermo-responsive vector

doi: 10.21037/atm-20-6595

Figure Lengend Snippet: The influence of IL-1β on ECM-degrading enzyme (MMP3, MMP13 and ADAMTS5) and ECM protein (collagen type II and aggrecan) expression. (A) Relative expression levels of MMP3 mRNA, MMP13 mRNA and ADAMTS5 mRNA in IL-1β-stimulated nucleus pulposus (NP) cells (10 ng/mL) and control cells via qRT-PCR. (B) Relative protein expression levels of MMP3, MMP13 and ADAMTS5 were examined in IL-1β-stimulated NP cells (10 ng/mL) and control cells via western blotting. (C) Relative protein expression levels of collagen type II and aggrecan were detected in IL-1β-stimulated NP cells (10 ng/mL) and control cells via immunocytochemistry (ICC). The bar is 50 µm. Data are the mean ± standard deviation (SD) of triplicate results. *, P<0.05.

Article Snippet: Membranes were incubated overnight at 4 °C with anti-IL-1β antibody (12703, 1:1,000, CST, USA), anti-MTF1 antibody (ab184119, 1:1,000, Abcam, Cambridge, UK), anti-ZIP8 antibody (NBP1-76505, 1:2,000, Novus, Biologicals, Colorado, USA), anti-MMP3 antibody (NB100-91878, 1:1,000, Novus Biologicals, Colorado, USA), anti-MMP13 antibody (NBP2-45887, 1:1,000, Novus, Biologicals, Colorado, USA), anti-ADAMTS5 antibody (NBP2-15286, 1:1,000, Novus, Biologicals, Colorado,USA), anti-β-actin antibody (abs118937, 1:3,000, Absin, Shanghai, China), and anti-PCNA antibody (abs13075, 1:2,000, Absin, Shanghai, China).

Techniques: Expressing, Control, Quantitative RT-PCR, Western Blot, Immunocytochemistry, Standard Deviation

Journal: Annals of Translational Medicine

Article Title: MicroRNA-25-3p therapy for intervertebral disc degeneration by targeting the IL-1β/ZIP8/MTF1 signaling pathway with a novel thermo-responsive vector

doi: 10.21037/atm-20-6595

Figure Lengend Snippet: The effects of miRNA-25-3p modulation on IL-1β-stimulated ECM-degrading enzyme (MMP3, MMP13 and ADAMTS5) and ECM protein (collagen type II and aggrecan) expression. (A) Relative mRNA expression levels of MMP3, MMP13 and ADAMTS5 in the miRNA-25-3p mimic group and the control group determined via qRT-PCR. (B) Relative protein expression levels of MMP3, MMP13 and ADAMTS5 were examined in the miRNA-25-3p mimic group and the control group via western blotting. (C) Relative protein expression levels of collagen type II and aggrecan were detected in the miRNA-25-3p mimic group and the control group via immunocytochemistry (ICC). The bar is 50 µm. Data are the mean ± standard deviation (SD) of triplicate results. *, P<0.05.

Article Snippet: Membranes were incubated overnight at 4 °C with anti-IL-1β antibody (12703, 1:1,000, CST, USA), anti-MTF1 antibody (ab184119, 1:1,000, Abcam, Cambridge, UK), anti-ZIP8 antibody (NBP1-76505, 1:2,000, Novus, Biologicals, Colorado, USA), anti-MMP3 antibody (NB100-91878, 1:1,000, Novus Biologicals, Colorado, USA), anti-MMP13 antibody (NBP2-45887, 1:1,000, Novus, Biologicals, Colorado, USA), anti-ADAMTS5 antibody (NBP2-15286, 1:1,000, Novus, Biologicals, Colorado,USA), anti-β-actin antibody (abs118937, 1:3,000, Absin, Shanghai, China), and anti-PCNA antibody (abs13075, 1:2,000, Absin, Shanghai, China).

Techniques: Expressing, Control, Quantitative RT-PCR, Western Blot, Immunocytochemistry, Standard Deviation