Fig 1: NW is protective against acute kidney ischemia–reperfusion injury. 1 h after kidney ischemia and 24 h after reperfusion, H2S production in the kidney was detected (A). The modified biotin switch assay determined sulfhydrated ß-actin (SHY-ß-actin) expression (B). Western blot analysis of CSE protein levels (C). Blood urea nitrogen (D) and serum Cr (creatinine) (E) contents in sham, I/R, and I/R + NW groups. H&E staining showed changes of morphology in the kidney (F). Upper panel: scale bar = 200 µm; lower panel: scale bar = 50 µm. DHE staining indicated the oxidative stress level (G), scale bar = 200 µm. Immunohistochemical staining of cleaved caspase 3 in the kidney (H). Upper panel: scale bar = 200 µm; lower panel: scale bar = 100 µm. Black arrows point out the positive cells. N = 6/group. *Versus sham, # versus I/R.
Fig 2: NW plays a long-term protective role in kidney ischemia–reperfusion injury. 1 h after kidney ischemia and 2 weeks after reperfusion, western blot analyzes the CSE protein level (A). Blood urea nitrogen (B) and serum Cr (creatinine) (C) contents in sham, I/R, and I/R + NW groups. H&E staining of the kidney 1 h after kidney ischemia and 2 weeks after reperfusion (D), scale bar = 100 µm. H&E staining of the kidney 1 h after kidney ischemia and 4 weeks after reperfusion (E), scale bar = 100 µm. N = 6/group. *p < 0.05, **p < 0.01. ns: no significant difference.
Fig 3: NW improved CSE/H2S production. L-Cysteine and pyridoxal phosphate and indicated concentration of NW are added to tissue homogenates. H2S production in the heart (A), aorta (B), kidney (C), and liver (D) was measured, N = 4. SD rats were subcutaneously injected with NW 4.4 mg/kg/day for 1 week. H2S production in the heart and kidney was detected (E), N = 5. In primary adipocytes, the effect of 100 µM NW treatment on H2S production was detected (F), N = 5. In cultured HepG2 cells, the effect of different concentrations of NW on the CSE protein level was detected. The left panel is representing graph, and the right panel is statistical graph (G), N = 3. **p < 0.01.
Fig 4: NW binding to CSE. Kinetics of NW binding to CSE assayed by microscale thermophoresis (MST) (A). CSE inhibitor PPG, as a positive control. Binding kinetics of NW with CSE mutation (Leu68 site and Asp164 site) (B).
Fig 5: Schematic diagram of CSE (A) and NW (B) structures. Schematic diagram of CSE and NW interactions (C, D) by computer simulation and dynamics testing.
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