Journal: Molecular Therapy. Nucleic Acids

Article Title: MBD2 Mediates Septic AKI through Activation of PKCη/p38MAPK and the ERK1/2 Axis

doi: 10.1016/j.omtn.2020.09.028

Figure Lengend Snippet: Amelioration of LPS-Induced AKI in MBD2-KO Mice The MBD2-KO and -WT littermate mice were injected with 10 mg/kg LPS for days 0 to 7 or saline as control. (A) Representative of survival cure. (B and C) Blood samples were collected for the measurement of serum nitrogen (BUN) (B) and creatinine (C) concentration at 24 h. (D and E) Representative of TNF-α (Δ) and IL-1β (E). (F) The kidney sections were stained with hematoxylin and eosin (H&E). (G and H) Tubular damage scores of kidney cortex (G) and OSOM (H). Scale bar, 100 μM. For the survival, the animal number is 12; for other experiments, the data are expressed as mean ± SD (n = 6). #p < 0.05 versus the saline group; ∗p < 0.05 versus the MBD2-WT with LPS group. Original magnification, ×200.

Article Snippet: TNF-α, IL-1β, and ICAM1 are from Proteintech North America (Rosemont, IL, USA). p38MAPK activator dehydrocorydaline chloride and pERK1/2 activator honokiol were purchased from MedChemExpress USA (Deer Park, NJ, USA).

Techniques: Injection, Saline, Control, Concentration Assay, Staining

Journal: Molecular Therapy. Nucleic Acids

Article Title: MBD2 Mediates Septic AKI through Activation of PKCη/p38MAPK and the ERK1/2 Axis

doi: 10.1016/j.omtn.2020.09.028

Figure Lengend Snippet: MBD2-Mediated LPS Induced the Expression of TNF-α, IL-1β, and ICAM1 in BUMPT Cells BUMPT cells were transfected with MBD2 plasmid or MBD2 siRNA or primary macrophages from bone marrow of MBD2 and MBD2-KO mice, followed by treatment with 300 μg/mL LPS for 24 h. (A) Real-time PCR analysis of TNF-α, IL-1β, and ICAM1 expression after LPS treatment with or without transfection of MBD2. (B) Immunoblot analysis of TNF-α, IL-1β, ICAM1, or MBD2, and GAPDH after LPS treatment with or without transfection of MBD2. (C)The protein signal was quantified by densitometry and normalized to internal control of GAPDH after LPS treatment with or without transfection of MBD2. (D) Real-time PCR analysis of TNF-α, IL-1β, and ICAM1 expression after LPS treatment with or without transfection of MBD2 siRNA. (E) Immunoblot analysis of TNF-α, IL-1β, ICAM1, or MBD2, and GAPDH after LPS treatment with or without transfection of MBD2 siRNA . (F)The protein signal was quantified by densitometry and normalized to internal control of GAPDH after LPS treatment with or without transfection of MBD2 siRNA. (G) Immunoblot analysis of TNF-α, IL-1β, ICAM1, or MBD2, and GAPDH in MBD2 KO primary tubular cells with LPS treatment. (H) The protein signal was quantified by densitometry and normalized to internal control of GAPDH in MBD2 KO primary tubular cells with LPS treatment. (I) Immunoblot analysis of TNF-α, IL-1β, ICAM1, or MBD2, and GAPDH in MBD2 KO primary macrophage cells with LPS treatment. (J) The protein signal was quantified by densitometry and normalized to internal control of GAPDH in MBD2 KO primary macrophage cells with LPS treatment. Data are expressed as mean ± SD (n = 6). #p < 0.05 versus the saline group; ∗p < 0.05 versus the MBD2 plasmid or MBD2 siRNA with LPS group; Δp > 0.05 versus the LPS group.

Article Snippet: TNF-α, IL-1β, and ICAM1 are from Proteintech North America (Rosemont, IL, USA). p38MAPK activator dehydrocorydaline chloride and pERK1/2 activator honokiol were purchased from MedChemExpress USA (Deer Park, NJ, USA).

Techniques: Expressing, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Western Blot, Control, Saline

Journal: Journal of Cardiovascular Translational Research

Article Title: Bufalin Ameliorates Myocardial Ischemia/Reperfusion Injury by Suppressing Macrophage Pyroptosis via P62 Pathway

doi: 10.1007/s12265-024-10577-9

Figure Lengend Snippet: Bufalin attenuated macrophage pyroptosis and reduced IL-1β release into the myocardium and serum during I/R injury. A-C The RNA sequencing data were extracted from GSE 126772. The mRNA levels of Nlrp3 , Pycard ( ASC ) and Il-1b in macrophages isolated from heart after MI were shown as fold change compared to the Pre group (control group, n = 2,3). D Macrophages were identified and isolated from the heart by flow cytometry. E–F Immunoblotting was performed to assess the expression levels of NLRP3, GSDMD and IL-1β in macrophages after I/R injury with or without bufalin treatment. Each target band was normalized to the β-actin band and the relative expression was calculated as the fold change compared to the Sham + PBS group (control group, n = 3). G Representative IL-1β expression and localization in murine hearts are shown by IHC analysis in longitudinal sections of whole hearts. For each mouse, 5 representative zones in the left ventricle were chosen randomly to determine the percentage of IL-1β-positive ratio by ImageJ ( n = 3). H IL-1β levels in the peripheral blood of mice that were subjected to I/R injury and were treated with or without bufalin were measured by ELISA ( n = 6). IHC (immunohistochemistry). Data are expressed as the mean ± SEM. P values were determined by ordinary one-way ANOVA with Dunnett’s multiple comparisons test ( A - C ) and two-way ANOVA with Tukey’s multiple comparisons test ( F and H )

Article Snippet: For interleukin-1β (IL-1β) treatment, recombinant mouse IL-1β (HY-P7073, MedChemExpress, New Jersey, USA) was added to HL-1 cells at different concentrations (0, 100 pg/ml, 300 pg/ml, 1000 pg/ml) and incubated for 24 h. For anakinra treatment, the recombinant human interleukin-1 receptor (IL-1R) antagonist anakinra (HY-108841, MedChemExpress) was added to cardiomyocytes every 6 h for 24 h when they were cocultured with normoxia-treated or H/R-treated macrophages.

Techniques: RNA Sequencing, Isolation, Control, Flow Cytometry, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemistry

Journal: Journal of Cardiovascular Translational Research

Article Title: Bufalin Ameliorates Myocardial Ischemia/Reperfusion Injury by Suppressing Macrophage Pyroptosis via P62 Pathway

doi: 10.1007/s12265-024-10577-9

Figure Lengend Snippet: Bufalin attenuated H/R-treated macrophage pyroptosis in vitro. Macrophages were subjected to 1 h of hypoxia and 24 h of reoxygenation treated with PBS or bufalin. A-B Proteins were extracted from cells and immunoblotting analysis was performed. The expressions of NLRP3, GSDMD, and IL-1β were normalized to that of β-actin and then compared with the Sham + PBS group (control group, n = 3). C PI staining was used to examine the plasma membrane integrity of macrophages. Positive cells were counted and normalized to the total number of cells ( n = 5). D Cell viability was assessed by measuring the quantity of ATP in macrophages subjected to different treatments ( n = 3, 6). E IL-1β levels in the culture medium of macrophages were measured by ELISA ( n = 3). Data are presented as the mean ± SEM. P values were calculated by two-way ANOVA with Tukey’s multiple comparisons test ( B , C , and E ) or Šídák’s multiple comparisons test ( D )

Article Snippet: For interleukin-1β (IL-1β) treatment, recombinant mouse IL-1β (HY-P7073, MedChemExpress, New Jersey, USA) was added to HL-1 cells at different concentrations (0, 100 pg/ml, 300 pg/ml, 1000 pg/ml) and incubated for 24 h. For anakinra treatment, the recombinant human interleukin-1 receptor (IL-1R) antagonist anakinra (HY-108841, MedChemExpress) was added to cardiomyocytes every 6 h for 24 h when they were cocultured with normoxia-treated or H/R-treated macrophages.

Techniques: In Vitro, Western Blot, Control, Staining, Clinical Proteomics, Membrane, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cardiovascular Translational Research

Article Title: Bufalin Ameliorates Myocardial Ischemia/Reperfusion Injury by Suppressing Macrophage Pyroptosis via P62 Pathway

doi: 10.1007/s12265-024-10577-9

Figure Lengend Snippet: Bufalin restored autophagic flux by suppressing P62 pathway in H/R-induced macrophages. Macrophages were transfected with the p62-overexpression plasmid for 24 h and then subjected to H/R with or without bufalin. A - B The protein levels of P62 were measured by immunoblotting. The bands were normalized to the β-actin band, and the relative expressions were calculated as the fold changes compared to the control group ( n = 3). C The mRNA levels of p62 were measured by qRT‒PCR. The Cq results were normalized to Mus-18 s and then compared with the control group ( n = 3). D-E Proteins were extracted from H/R-treated macrophages that were infected with vector or OE-p62 plasmid in advance and treated with bufalin or PBS. Immunoblotting was performed to measure the expressions of NLRP3, GSDMD and IL-1β. Each band was normalized to the β-actin band and the relative expression was calculated as the fold change compared to the Con + PBS group (control group, n = 3). F PI staining was used to assess pyroptosis in macrophages. Positive ratios were calculated as the percentage of positive cells compared with total number of cells ( n = 5). G IL-1β levels in the culture medium of macrophages were measured by ELISA ( n = 3). Data are presented as the mean ± SEM. P values were calculated by ordinary one-way ANOVA with Tukey’s multiple comparisons test

Article Snippet: For interleukin-1β (IL-1β) treatment, recombinant mouse IL-1β (HY-P7073, MedChemExpress, New Jersey, USA) was added to HL-1 cells at different concentrations (0, 100 pg/ml, 300 pg/ml, 1000 pg/ml) and incubated for 24 h. For anakinra treatment, the recombinant human interleukin-1 receptor (IL-1R) antagonist anakinra (HY-108841, MedChemExpress) was added to cardiomyocytes every 6 h for 24 h when they were cocultured with normoxia-treated or H/R-treated macrophages.

Techniques: Transfection, Over Expression, Plasmid Preparation, Western Blot, Control, Infection, Expressing, Staining, Enzyme-linked Immunosorbent Assay

Journal: Inflammation

Article Title: CXCL12 Promotes Peripheral Nerve Injury Repair by Inhibiting the Ferroptosis-Inflammation Axis via the ERK/Nrf2 Pathway

doi: 10.1007/s10753-026-02453-2

Figure Lengend Snippet: CXCL12 inhibits NF-κB signaling pathway activation and inflammatory factor secretion in SCs. ( A ) Western blot analysis of NF-κB, p-NF-κB, IκBα, and p-IκBα protein expression levels in SCs after CXCL12 treatment. ( B - C ) Quantification of gray values for the NF-κB, p-NF-κB, κBα, and p-IκBα proteins. ( D - E ) Levels of IL-1β and TNF-α in SCs after CXCL12 treatment. The data are presented as the means ± SDs, n=3, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: An enzyme-linked immunosorbent assay was used to measure the concentrations of the inflammatory factors IL-1β (Elabscience, E-HSEL-R0002, China) and TNF-α (Elabscience, E-HSEL-R0001, China) in the cell culture supernatant (centrifuged at 3000×g for 15 min).

Techniques: Activation Assay, Western Blot, Expressing

Journal: Inflammation

Article Title: CXCL12 Promotes Peripheral Nerve Injury Repair by Inhibiting the Ferroptosis-Inflammation Axis via the ERK/Nrf2 Pathway

doi: 10.1007/s10753-026-02453-2

Figure Lengend Snippet: CXCL12 inhibits the inflammatory response in SCs via the NF-κB signaling pathway. ( A ) Western blot analysis of NF-κB, p-NF-κB, IκBα, and p-IκBα protein expression levels in SCs after NF-κB overexpression. ( B - C ) Quantification of the gray values for the NF-κB, p-NF-κB, IκBα, and p-IκBα proteins. ( D - E ) Levels of IL-1β and TNF-α in SCs after NF-κB overexpression. The data are presented as the means ± SDs, n=3, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, not significant

Article Snippet: An enzyme-linked immunosorbent assay was used to measure the concentrations of the inflammatory factors IL-1β (Elabscience, E-HSEL-R0002, China) and TNF-α (Elabscience, E-HSEL-R0001, China) in the cell culture supernatant (centrifuged at 3000×g for 15 min).

Techniques: Western Blot, Expressing, Over Expression

Journal: Inflammation

Article Title: CXCL12 Promotes Peripheral Nerve Injury Repair by Inhibiting the Ferroptosis-Inflammation Axis via the ERK/Nrf2 Pathway

doi: 10.1007/s10753-026-02453-2

Figure Lengend Snippet: CXCL12 inhibits SC ferroptosis, thereby mitigating the inflammatory response. ( A ) Western blot analysis of the expression levels of ferroptosis-related proteins ACSL4, GPX4, and FSP1 in SCs after FAC treatment. ( B - D ) Densitometric analysis of proteins ACSL4, GPX4, and FSP1. ( E , F ) Fluorescence images of ROS and Fe 2+ in SCs after FAC treatment, Scale bar = 50µm. ( G , H ) Quantification of the relative fluorescence intensity of ROS and Fe 2+ . ( I , J ) Expression levels of MDA and GSH in SCs after FAC treatment. ( K ) Western blot analysis of the expression levels of NF-κB, p-NF-κB, IκBα, and p-IκBα in SCs after FAC treatment. ( L , M ) Densitometric analysis of proteins NF-κB, p-NF-κB, IκBα, and p-IκBα. ( N , O ) Expression levels of IL-1β and TNF-α in SCs after FAC treatment. The data are presented as the means ± SDs, n=3, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, not significant

Article Snippet: An enzyme-linked immunosorbent assay was used to measure the concentrations of the inflammatory factors IL-1β (Elabscience, E-HSEL-R0002, China) and TNF-α (Elabscience, E-HSEL-R0001, China) in the cell culture supernatant (centrifuged at 3000×g for 15 min).

Techniques: Western Blot, Expressing, Fluorescence

Journal: Inflammation

Article Title: CXCL12 Promotes Peripheral Nerve Injury Repair by Inhibiting the Ferroptosis-Inflammation Axis via the ERK/Nrf2 Pathway

doi: 10.1007/s10753-026-02453-2

Figure Lengend Snippet: CXCL12 inhibits NF-κB signaling pathway activation and inflammatory factor secretion in the sciatic nerve. ( A ) Western blot analysis of NF-κB, p-NF-κB, IκBα, and p-IκBα protein expression levels in the sciatic nerve after CXCL12 treatment. ( B - C ) Quantification of the gray values for the NF-κB, p-NF-κB, IκBα, and p-IκBα proteins. ( D - E ) Levels of IL-1β and TNF-α in the sciatic nerve after CXCL12 treatment. The data are presented as the means ± SDs, n=3, **p < 0.01, ***p < 0.001, **** p < 0.0001

Article Snippet: An enzyme-linked immunosorbent assay was used to measure the concentrations of the inflammatory factors IL-1β (Elabscience, E-HSEL-R0002, China) and TNF-α (Elabscience, E-HSEL-R0001, China) in the cell culture supernatant (centrifuged at 3000×g for 15 min).

Techniques: Activation Assay, Western Blot, Expressing

Journal: Inflammation

Article Title: CXCL12 Promotes Peripheral Nerve Injury Repair by Inhibiting the Ferroptosis-Inflammation Axis via the ERK/Nrf2 Pathway

doi: 10.1007/s10753-026-02453-2

Figure Lengend Snippet: CXCL12 inhibits sciatic nerve ferroptosis, thereby mitigating the inflammatory response. ( A ) Western blot analysis of ferroptosis-related protein expression levels (ACSL4, GPX4, FSP1) in the sciatic nerve after FAC treatment. ( B - D ) Quantification of the gray values for the ACSL4, GPX4, and FSP1 proteins. ( E ‒ G ) Levels of Fe 2+ , MDA, and GSH in the sciatic nerve after FAC treatment. ( H ) Western blot analysis of NF-κB, p-NF-κB, IκBα and p-IκBα protein expression levels in the sciatic nerve after FAC treatment. ( I , J ) Quantification of the gray values for the NF-κB, p-NF-κB, IκBα and p-IκBα proteins. (K, L) Levels of IL-1β and TNF-α in the sciatic nerve after FAC treatment. The data are presented as the means ± SDs, n=3, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: An enzyme-linked immunosorbent assay was used to measure the concentrations of the inflammatory factors IL-1β (Elabscience, E-HSEL-R0002, China) and TNF-α (Elabscience, E-HSEL-R0001, China) in the cell culture supernatant (centrifuged at 3000×g for 15 min).

Techniques: Western Blot, Expressing

Journal: Journal of Orthopaedic Surgery and Research

Article Title: Pure platelet-rich plasma delays intervertebral disc degeneration by activating SIRT1-mediated autophagy in nucleus pulposus cells

doi: 10.1186/s13018-025-06205-0

Figure Lengend Snippet: Characterization of P-PRP and its effects on inflammatory NPCs. A , Diagram of P-PRP preparation. B - C , Leukocyte and platelet concentration. D - G , ELISA results show the levels of TNF-α, TGF-β, PDGF-A, and IL-1β. H , CCK-8 assay result show cell viability. I - J , Transwell assay evaluate the migration ability of NPCs after different treatments (Bar = 100 μm). K - L , the representative WB images ( K ) quantification data ( L ) of SIRT1 protein levels in NPCs under different treatments. Data are shown as mean ± SD; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ns, no significance

Article Snippet: All groups were cultured for a total of 48 h. The experimental conditions were as follows: (1) the control group, which was treated with complete medium (DMEM/F12: fetal bovine serum = 9:1) for 48 h without any stimulation; (2) the IVDD group, which was treated with IL-1β (10 ng/mL, MedChemExpress, HY- P73150 , USA) for 48 h; (3) the SRT1720 group, which was treated with IL-1β (10 ng/mL) for 24 h, followed by co-incubation with IL-1β (10 ng/mL) and SRT1720 (1 μmol/L, MedChemExpress, HY-10532, USA), a selective SIRT1 activator, for an additional 24 h; (4) the EX527 group, which was treated as in the SRT1720 group but with the addition of EX-527 (5 μmol/L, MedChemExpress, HY-15452, USA), a selective SIRT1 inhibitor, during the second 24 h period; (5) the PRP group, which was treated with IL-1β (10 ng/mL) for 24 h, then co-incubated with IL-1β (10 ng/mL) and 200 μL of P-PRP for another 24 h, achieving a final PRP concentration of 10% (v/v); and (6) the PRP + EX527 group, which was treated identically to the PRP group, with the addition of EX-527 (5 μmol/L) during the final 24 h.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Transwell Assay, Migration