S14G‐humanin attenuates renal ischaemia–reperfusion injury, potentially through activation of STAT3 and ERK1/2 signalling pathways in rats

Figures

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  FIGURE 1. Histopathological damage and apoptosis in renal tissue after I/R injury and HNG treatment. (a) Representative H&E‐stained kidney sections showing tubular necrosis (), tubular dilation (), cast formation () and loss of brush border () in experimental groups. (b) Representative TUNEL‐stained kidney sections showing apoptotic cells (brown‐stained nuclei). (c) Semi‐quantitative histopathological injury scores based on tubular necrosis, tubular dilation, cast formation and brush border loss (n = 8). Sham vs. I/R, P < 0.001; HNG‐Sham vs. HNG‐I/R, P = 0.00691; I/R vs. HNG‐I/R, P = 0.999. (d) Quantification of apoptotic index (% TUNEL‐positive cells) (n = 7). Sham vs. I/R, P < 0.001; I/R vs. HNG‐I/R, P = 0.00276; HNG‐Sham vs. HNG‐I/R, P = 0.0542. Data are presented as means ± SD. Exact P values for post hoc comparisons are shown above the bars. Scale bars: 50 µm. HNG, humanin G; I/R, ischaemia–reperfusion; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labelling.

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  FIGURE 2. Effects of HNG on mitochondrial function in renal tissue following I/R injury. (a) Intracellular ATP levels in renal tissue homogenates (n = 8). Sham vs. I/R, P < 0.001; I/R vs. HNG‐I/R, P = 0.0414; HNG‐Sham vs. HNG‐I/R, P = 0.550. (b) Mitochondrial complex I activity in renal tissue homogenates (n = 7). Sham vs. I/R, P = 0.00129; I/R vs HNG‐I/R, P = 0.0420; HNG‐Sham vs HNG‐I/R, P = 0.944. Data are presented as means ± SD. Exact P‐values for post hoc comparisons are shown above the bars. HNG, humanin G; I/R, ischaemia–reperfusion.

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  FIGURE 3. Effects of HNG on oxidative stress, antioxidant status and inflammation in renal tissue. (a) Malondialdehyde (MDA) levels (n = 11). Sham vs. I/R, P < 0.001; I/R vs. HNG‐I/R, P = 0.0150; HNG‐Sham vs. HNG‐I/R, P = 0.258). (b) Glutathione (GSH) levels (n = 10). Sham vs. I/R, P < 0.001; I/R vs. HNG‐I/R, P = 0.00325; HNG‐Sham vs. HNG‐I/R, P = 0.0484. (c) Superoxide dismutase (SOD) activity (n = 10). Sham vs. I/R, P = 0.0117; I/R vs. HNG‐I/R, P = 0.0228; HNG‐Sham vs. HNG‐I/R, P = 0.552. (d) Catalase (CAT) activity (n = 10). Sham vs. I/R, P < 0.001; HNG‐Sham vs. HNG‐I/R, P < 0.001; I/R vs. HNG‐I/R, P = 0.895. (e) Myeloperoxidase (MPO) activity (n = 8). Sham vs. I/R, P < 0.001; HNG‐Sham vs. HNG‐I/R, P < 0.001; I/R vs. HNG‐I/R, P = 0.0185. Data are presented as means ± SD. Exact P‐values for post hoc comparisons are shown above the bars. CAT, catalase; GSH, glutathione; HNG, humanin G; I/R, ischaemia–reperfusion; MDA, malondialdehyde; MPO, myeloperoxidase; SOD, superoxide dismutase.

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  FIGURE 4. Effects of HNG Treatment on p‐ERK1/2 and p‐STAT3 phosphorylation following I/R injury. (a, b) Representative western blot bands for p‐ERK1/2, p‐STAT3 and β‐actin (45 kDa, loading control) in Sham, I/R, HNG‐Sham and HNG‐I/R groups. (c) Densitometric quantification of p‐ERK1/2 protein levels relative to β‐actin (n = 4 animals per group, 6 homogenates total). Sham vs. I/R, P = 0.772; I/R vs. HNG‐I/R, P < 0.001; HNG‐Sham vs. HNG‐I/R, P < 0.001. (d) Densitometric quantification of p‐STAT3 protein levels relative to β‐actin (n = 4 animals per group, 6 homogenates total). Sham vs. I/R, P = 0.00516; I/R vs. HNG‐I/R, P < 0.001; HNG‐Sham vs. HNG‐I/R, P < 0.001. Data are presented as means ± SD. Exact P‐values for post hoc comparisons are shown above the bars. ERK1/2, extracellular signal‐regulated kinases 1 and 2; HNG, humanin G; I/R, ischaemia–reperfusion; STAT3, signal transducer and activator of transcription 3.

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  FIGURE 5. Schematic representation of the effects of HNG on mitochondrial function and oxidative stress following renal ischaemia–reperfusion injury. HNG treatment increased ATP levels and complex I activity, enhanced antioxidant defences by increasing GSH and SOD levels, and reduced MDA levels and apoptosis. These effects may be associated with activation of the ERK1/2 and STAT3 signalling pathways. CNTF, ciliary neurotrophic factor; ERK1/2, extracellular signal‐regulated kinases 1 and 2; FPRL1/2, formyl peptide receptor‐like 1/2; GSH, glutathione; HNG, humanin G; I/R, ischaemia–reperfusion; MDA, malondialdehyde; SOD, superoxide dismutase; STAT3, signal transducer and activator of transcription 3.