Fig 2: AT III ameliorates DSS-induced inflammation and oxidative stress. (a, b) H&E staining of colon tissues and quantification of histological scores. Scale bar: 200 µm. (c) Immunohistochemical detection of MPO in colons. Scale bar: 50 µm. (d–g) qPCR results of TNF-a, IL-6, COX-2, and iNOS mRNA in colons. (h–j) ELISA measurements of MDA and GSH content, as well as SOD activity. $p < 0.05, compared to control; &p < 0.05, compared to DSS.

Fig 3: MK increased inflammatory cytokine secretion and NETosis in vitro. (A) Neutrophils were treated with 10, 50, 100, and 200 ng/mL MK at 0, 24, 48 and 72 h. Next, the CCK-8 kit was used to examine the cell viability of neutrophils. (B) The levels of IL-1 and IL-6 in the cell supernatant was examined using ELISA. (C) The levels of NE in the cell supernatant was detected using ELISA. (D,E) The protein expression of H3Cit and MPO was determined by western blot normalized against ß-actin. The means ± standard deviations of 3 independent samples are shown. &P<0.05 vs. NG group, *P<0.05 vs. MG group, #P<0.05 vs. NG/MG/HG group. NG, normal-glucose; MG, mannitol group; HG, high-glucose; CCK-8, cell counting kit-8; MK, midkine; ELISA, enzyme-linked immunosorbent assay; NE, neutrophil elastase; CitH3, citrullinated histones; MPO, myeloperoxidase; IL-1, interleukin-1; IL-6, interleukin-6; ns, no significant.

Fig 4: MK expression and NETosis were elevated in the diseased kidneys of the DKD patients. Kidney sections were stained with H&E (A), Masson (B), and PAS (C,D). The magnification is ×200 or ×400. (A) Green arrow indicates glomerular necrosis; yellow arrow indicates thinning of tubular epithelial cells; blue arrow indicates glomerular stromal hyperplasia. (B) Yellow arrows indicate fibrosis with blue staining. (C) Yellow arrows heads point to PAS positivity. (E,F) The expression of MK in kidneys was determined by IHC (yellow arrows, positive IHC). The magnification is ×400. (G) The protein expression of H3Cit and MPO was determined by IF. The magnification is ×400. The means ± standard deviations of 6 independent samples are shown. **P<0.01. MK, midkine; H&E, hematoxylin and eosin; PAS, periodic acid-Schiff; IHC, immunohistochemistry; IF, immunofluorescence.

Fig 5: Inhibitory effect of 9-PH on the inflammatory response after TBI. (A, B) Representative immunofluorescence images and quantitative analysis of IBA-1- and CD68-positive cells in surrounding tissue at 24 h after TBI (n = 3). Nuclei are stained with DAPI (blue). Scale bars: 50 μm. (C, D) Western blotting and quantitative analysis of GFAP expression in surrounding tissue 24 h after TBI (n = 5). (E, F) Images of GFAP immunostaining and quantitative analysis of GFAP-positive astrocytes (n = 3). DAPI (blue) was used to label nuclei. Scale bar: 50 μm. (G, H) Immunofluorescence staining with MPO and quantitative analysis of infiltrated neutrophils (MPO positive) in surrounding tissue at 24 h after TBI (n = 3). DAPI (blue) represents nuclei. Scale bars: 50 μm. (I, J) Western blotting and quantitative analysis of TNF-α and IL-6 in surrounding tissue 24 h after TBI (n = 5). *p<0.05, **p<0.01, and ***p<0.001.