Journal: Acta biochimica et biophysica Sinica

Article Title: The Nrf2/HMGB1/NF-κB axis modulates chondrocyte apoptosis and extracellular matrix degradation in osteoarthritis.

doi: 10.3724/abbs.2023078

Figure Lengend Snippet: Figure 2. Nrf2 binds to the HMGB1 promoter to inhibit its transcription (A) Chondrocytes were treated with PBS or IL-1β (10 ng/mL for 24 h) and examined for HMGB1 protein levels by western blot anaysis. (B) Nrf2 knockdown or overexpression was achieved in chondrocytes by transfecting with small interfering RNA targeting Nrf2 (si-Nrf2) or Nrf2 OE; Nrf2 knockdown/overexpression and HMGB2 mRNA expression were confirmed by qRT-PCR. (C) Chondrocytes were transfected with si-Nrf2 or Nrf2 OE and examined for the protein levels of Nrf2 and HMGB1 by western blot analysis. (D) psiCheck-2-based wild- and mutant-type HMGB1 promoter luciferase reporter plasmids were constructed and cotransfected with Nrf2 OE or vector into chondrocytes, and the luciferase activity was determined. (E) ChIP assay was performed with anti-IgG or anti-Nrf2; the abundance of HMGB1 promoter in immunoprecipitated was examined by real time-PCR. **P<0.01 vs si-NC group or IgG group; †P<0.05, ††P<0.01 vs vector group.

Article Snippet: Immunohistochemistry (IHC) staining Following the deparaffinization, hydration and blockage of endogenous peroxidase, the sections were incubated for 20 min with 10% nonfat milk in PBS to block the specific sites, followed by an overnight incubation with anti-Nrf2 (CSB-PA003481; Cusabio) or HMGB1 (CSB-PA01604A0Rb; Cusabio) at 4°C.

Techniques: Western Blot, Knockdown, Over Expression, Small Interfering RNA, Expressing, Quantitative RT-PCR, Transfection, Mutagenesis, Luciferase, Construct, Plasmid Preparation, Activity Assay, Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: Acta biochimica et biophysica Sinica

Article Title: The Nrf2/HMGB1/NF-κB axis modulates chondrocyte apoptosis and extracellular matrix degradation in osteoarthritis.

doi: 10.3724/abbs.2023078

Figure Lengend Snippet: Figure 3. Specific effects of HMGB1 on chondrocyte apoptosis and cellular inflammation (A) HMGB1 knockdown was achieved in chondrocytes by transfecting with small interfering RNA targeting HMGB1 (si-HMGB1), which was confirmed using western blot analysis. Next, chondrocytes were transfected with si-HMGB1 or si-NC (negative control) in the presence or absence of IL-1β (10 ng/mL for 24 h). (B) Cell apoptosis was examined by flow cytometry using an Annexin-V/PI apoptosis kit. (C) The mRNA expressions of IL-6, TNF-α, INOS, and COX2 were examined by qRT-PCR. (D) The protein levels of aggrecan, COL2A1, ADAMTS-5, and MMP13 were examined by western blot analysis. (E) The protein levels of p- p65 and p65 were examined by western blot analysis. *P<0.05, **P<0.01 vs si-NC group; ††P<0.01 vs si-NC+IL-1β group.

Article Snippet: Immunohistochemistry (IHC) staining Following the deparaffinization, hydration and blockage of endogenous peroxidase, the sections were incubated for 20 min with 10% nonfat milk in PBS to block the specific sites, followed by an overnight incubation with anti-Nrf2 (CSB-PA003481; Cusabio) or HMGB1 (CSB-PA01604A0Rb; Cusabio) at 4°C.

Techniques: Knockdown, Small Interfering RNA, Western Blot, Transfection, Negative Control, Flow Cytometry, Quantitative RT-PCR

Journal: Acta biochimica et biophysica Sinica

Article Title: The Nrf2/HMGB1/NF-κB axis modulates chondrocyte apoptosis and extracellular matrix degradation in osteoarthritis.

doi: 10.3724/abbs.2023078

Figure Lengend Snippet: Figure 4. Nrf2 acts on chondrocytes through the HMGB1/NFκB pathway (A) Chondrocytes were cotransfected with Nrf2 OE and HMGB1 OE and examined for the protein levels of Nrf2 and HMGB1 using western blot analysis. (B‒E) In the presence of IL-1β stimulation (10 ng/mL for 24 h), cell apoptosis was examined by flow cytometry using an Annexin-V/PI apoptosis kit (B); the mRNA expression of IL-6, TNF-α, INOS, and COX2 was examined by qRT-PCR (C); the protein levels of aggrecan, COL2A1, ADAMTS-5, and MMP13 were examined by western blot analysis (D); the protein levels of p-p65 and p65 were examined by western blot analysis (E). *P<0.05, **P<0.01 vs vector group; ††P<0.01 vs vector+HMGB1 OE group.

Article Snippet: Immunohistochemistry (IHC) staining Following the deparaffinization, hydration and blockage of endogenous peroxidase, the sections were incubated for 20 min with 10% nonfat milk in PBS to block the specific sites, followed by an overnight incubation with anti-Nrf2 (CSB-PA003481; Cusabio) or HMGB1 (CSB-PA01604A0Rb; Cusabio) at 4°C.

Techniques: Western Blot, Flow Cytometry, Expressing, Quantitative RT-PCR, Plasmid Preparation

Journal: Acta biochimica et biophysica Sinica

Article Title: The Nrf2/HMGB1/NF-κB axis modulates chondrocyte apoptosis and extracellular matrix degradation in osteoarthritis.

doi: 10.3724/abbs.2023078

Figure Lengend Snippet: Figure 5. Effects of Nrf2 and HMGB1 on OA mouse damage in vivo Experimental OA in mice was induced by destabilization of the medial meniscus (DMM) surgery, and mice were assigned into five groups: (i) Sham, (ii) DMM, (iii) DMM+TBHQ, (iv) DMM+rHMGB1 and (v) DMM+TBHQ+rHMGB1. (A) Histopathological alterations in knee articular cartilage from different groups of mice were verified by H&E staining, scale bar: 100 μm. (B) The expression levels of inflammatory factors (IL-6, TNF-α, iNOS, and COX2) in knee articular cartilage from different groups of mice were determined by ELISA. (C) The protein levels of aggrecan, COL2A1, ADAMTS-5, and MMP13 in knee articular cartilage from different groups of mice were examined by western blot analysis. (D) The protein levels of p-p65 and p65 in knee articular cartilage from different groups of mice were examined by western blot analysis. *P<0.05, **P<0.01 vs sham group; ††P<0.01 vs DMM group; ##P<0.01 vs DMM+rHMGB1 group.

Article Snippet: Immunohistochemistry (IHC) staining Following the deparaffinization, hydration and blockage of endogenous peroxidase, the sections were incubated for 20 min with 10% nonfat milk in PBS to block the specific sites, followed by an overnight incubation with anti-Nrf2 (CSB-PA003481; Cusabio) or HMGB1 (CSB-PA01604A0Rb; Cusabio) at 4°C.

Techniques: In Vivo, Staining, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Acta biochimica et biophysica Sinica

Article Title: The Nrf2/HMGB1/NF-κB axis modulates chondrocyte apoptosis and extracellular matrix degradation in osteoarthritis.

doi: 10.3724/abbs.2023078

Figure Lengend Snippet: Figure 6. Nrf2 and HMGB1 levels in OA patient cartilage tissue samples (A) OA and normal cartilage tissues were collected and examined for histopathological features by H&E (left panel) and Safranin O staining (right panel). (B) The levels of Nrf2 and HMGB1 in OA and normal cartilage tissues were examined by immunohistochemical (IHC) staining. The quantitative analysis is shown in the right panel. (C) Apoptosis in OA and normal cartilage tissues was determined by TUNEL staining; quantitative analysis is shown in the right panel. (D) The protein levels of IL-1β, cleaved caspase 3 (C-caspase 3), total caspase 3, BAX, and Bcl-2 in OA and normal cartilage tissues were examined by western blot analysis. (E) The mRNA expression levels of IL-6, TNF-α, INOS, and COX2 in OA and normal cartilage tissues were examined by qRT-PCR. Scale bar: 100 μm. **P<0.01, ***P<0.001 vs the normal group.

Article Snippet: Immunohistochemistry (IHC) staining Following the deparaffinization, hydration and blockage of endogenous peroxidase, the sections were incubated for 20 min with 10% nonfat milk in PBS to block the specific sites, followed by an overnight incubation with anti-Nrf2 (CSB-PA003481; Cusabio) or HMGB1 (CSB-PA01604A0Rb; Cusabio) at 4°C.

Techniques: Staining, Immunohistochemical staining, Immunohistochemistry, TUNEL Assay, Western Blot, Expressing, Quantitative RT-PCR

Journal: Acta biochimica et biophysica Sinica

Article Title: The Nrf2/HMGB1/NF-κB axis modulates chondrocyte apoptosis and extracellular matrix degradation in osteoarthritis.

doi: 10.3724/abbs.2023078

Figure Lengend Snippet: Figure 7. Schematic diagram showing the Nrf2/HMGB1/NF-κB axis affecting OA chondrocytes In OA chondrocytes, downregulated Nrf2 increases HMGB1 transcription and protein expression and subse- quently activates the NF-κB pathway, finally causing ECM degrada- tion, inflammation and cell apoptosis. ↑indicates increase or activation; ↓indicates decrease; ┴indicates inhibition.

Article Snippet: Immunohistochemistry (IHC) staining Following the deparaffinization, hydration and blockage of endogenous peroxidase, the sections were incubated for 20 min with 10% nonfat milk in PBS to block the specific sites, followed by an overnight incubation with anti-Nrf2 (CSB-PA003481; Cusabio) or HMGB1 (CSB-PA01604A0Rb; Cusabio) at 4°C.

Techniques: Expressing, Activation Assay, Inhibition