Fig 1.
The inhibitory effect of batroxobin on human NETs in the presence of TNF-α with human Fgn.
(a) Upper panels show flow cytometry scattergrams of isolated cells stained with SO and anti-MPO antibody-FITC in each condition. Lower panels show scattergrams of the population stained with anti-MPO antibody–FITC gated from the respective panels in each condition. (b) The bar graphs indicate the percentages of NETs doubly stained with SO and anti-MPO antibody-FITC as mean ± SD. ***P < 0.001, *P < 0.05, n = 3. (c) SEM images show the features of NETs under the respective conditions. Scale bar = 5 μm. (A), depleted rhTNF-α and hFgn; (B) or (C), absence or presence of rhTNF-α with hFgn. rhTNF-α = ng/mL, hFgn = 1 mg/mL.
Fig 2.
Evidence of the anti-inflammatory effects of batroxobin in ischemic ATM.
(a) The protocol of the animal experiment. (b, c) NETs detected by staining with anti-histone H3 (citrulline R2+R8+R17) antibody and DAPI in ischemic tissues on day 2 post ischemia. (b) The representative pictures of H3Ct+/DAPI+ NETs in ischemic ATMs. Scale bar = 50 μm. (c) The bar graph shows the number of H3Ct+/DAPI+ NETs per square millimeter as mean ± SD. *P < 0.05, n = 6. (d, e) The deposited fibrinogen in the tissue of ischemic ATMs on day 2 post ischemia. (d) The representative pictures of tissue-recruited Fgn in ischemic ATMs. The brown area indicates tissue-recruited Fgn. Scale bar = 100 μm. (A) saline control, (B) DF-521-administered group. Scale bar = 100 μm. (e) The bar graph shows the percentage of Fgn-occupied area in ischemic tissue as mean ± SD. *P < 0.05, n = 6.
Fig 3.
The gene expression profiles of ischemic ATMs.
The bar graph shows the fold change of expression of each gene in ischemic ATMs in DF-521-administered mice vs. saline control mice as mean. Clear bar = day 3, gray bar = day 7, **P < 0.01, *P < 0.05, n = 5.
Fig 4.
Sequential features of angiogenesis in ischemic ATMs of each group.
(a) The protocol of the experiment. (b) The representative pictures of angiogenesis on day 4, day 7, and day 14 post ischemia. Angiogenesis was evaluated by fluorescence immunohistochemistry with the Alexa-594-conjugated anti-mouse CD31 antibody. Scale bar = 100 μm. (c) The bar graph shows the capillary density (capillaries /mm2) at each time point as mean ± SD. Clear column = control mice, gray column = DF-521-administered mice. *** P < 0.001, **P < 0.001, *P < 0.05, n = 6.
Fig 5.
Arteriogenesis in ischemic ATMs of each group.
(a) The representative pictures of arteriogenesis in ischemic ATMs on day 14. Arteriogenesis was evaluated by fluorescence immunohistochemistry with the anti-αSMA antibody-Cy3 conjugate and isolectin B4-Alexa488 conjugate. (A, C, E) control group, (B, D, F) DF-521-administered group. (A, B) Isolectin B4-Alexa488 conjugate-stained images, (C, D) anti-αSMA antibody-Cy3 conjugate-stained images, (E, F) merged images. Scale bar = 200 μm. (b, c) The bar graphs indicate the number and total length of doubly stained arterioles as mean ± SD. **P < 0.001, *P < 0.05, n = 6.
Fig 6.
Blood perfusion analysis by laser Doppler imaging in DF-521-administered mice vs. control mice.
(a) The representative pictures of laser Doppler imaging in each mouse. The yellow or white square shows the region of interest to measure flux intensity (arbitrary unit) indicating blood perfusion in the ischemic or healthy toe. (b) The line graph shows the blood perfusion ratio of the ischemic hindlimb vs. healthy hindlimb in each group as mean ± SD. *P < 0.05, n = 10.
Fig 7.
Histological features of ATMs in ischemic hindlimbs.
(a) The images are the features of myofibers in ischemic ATMs on day 14 post ischemia. Scale bar = 200 μm. The bar graphs show the area per myofiber (μm2) (b), the number of myofibers (c) and the distribution of immature and mature myofibers (%) (d) per square millimeter in the tissue cross section as mean ± SD. **P < 0.01, ***P < 0.001, n = 7.