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Objective:To study the effect and mechanism of mitochondria-targeted antioxidant peptide SS-31 on sepsis-induced acute kidney injury (AKI).Methods:Sixty adult male C57BL/6 mice were randomly divided into four groups according to the random number table method: sham group (10 mice), positive control group (10 mice), sepsis model group (20 mice), and SS-31 peptide group (20 mice). The sepsis-induced AKI mouse model was reproduced by cecal ligation and puncture (CLP). The sham group only received laparotomy. SS-31 peptide (5 mg/kg) was intraperitoneally injected in SS-31 peptide group and positive control group 30 minutes after the operation, while an equivalent amount of normal saline was given in sham group and sepsis model group for 7 days. The blood samples were collected 24 hours after the operation from orbit, and the serum was collected to test the serum creatinine (SCr) and blood urea nitrogen (BUN). The mice were sacrificed 7 days after surgery. The kidney tissues were collected to observe the pathologic structure changes under the hematoxylin-eosin (HE) staining by light microscope. And the mitochondrial ultrastructure was checked under the transmission electron microscope. Cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method (TUNEL). The expression level of peroxisome proliferator-activated receptorγ coactivator-1α (PGC-1α), adenosine monophosphate-activated protein kinase (AMPK), and cleaved caspase-3 protein were tested by Western blotting.Results:Compared with sham group, the levels of SCr and BUN were significantly increased in sepsis model group [SCr (μmol/L): 93.12±11.80 vs. 32.94±3.37, BUN (mmol/L): 41.36±6.48 vs. 9.49±3.58, both P<0.05]. The expression levels of AMPK, PGC-1α and cleaved caspase-3 protein increased (AMPK/β-actin: 0.30±0.02 vs. 0.12±0.01, PGC-1α/β-actin: 0.38±0.03 vs. 0.16±0.02, cleaved caspase-3/β-actin: 0.20±0.01 vs. 0.11±0.02, all P<0.05). HE staining showed that inflammatory cell was infiltrated, glomerular basement membrane was exposed and vacuole-like transparent casts were found in the lumen. Mitochondria were damaged under electron microscope with swelling, ridge disappearance and ruptured membranes, with increasing of apoptotic cells [cells: 24.00 (18.75, 31.00) vs. 2.00 (0.72, 3.25) , P<0.05]. Meanwhile, compared with sepsis model group, the levels of SCr, BUN and the expressions of AMPK, PGC-1α, cleaved caspase-3 protein were significantly decreased in the SS-31 peptide group [SCr (μmol/L): 71.33±10.14 vs. 93.12±11.80, BUN (mmol/L): 27.00±5.52 vs. 41.36±6.48, AMPK/β-actin: 0.23±0.01 vs. 0.30±0.02, PGC-1α/β-actin: 0.27±0.02 vs. 0.38±0.03, cleaved caspase-3/β-actin: 0.13±0.01 vs. 0.20±0.01, all P < 0.05]. HE staining showed that cell swelling reduced, the mitochondrial structure was intact, the ridge swelling was also reduced, and the membrane structure was relatively intact, the number of apoptotic cells was significantly reduced [cells: 13.00 (9.00, 16.50) vs. 24.00 (18.75, 31.00) , P<0.05]. Conclusion:The protective effect of SS-31 peptide on organ dysfunction induced by sepsis-induced AKI is related to maintaining mitochondrial homeostasis and inhibiting cell apoptosis.
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Aim To explore the effect and mechanism of antioxidant peptide AOP1 on repair of skin burn wound healing in mice.Methods Fluorescence probe DCFH-DA was used to detect the changes of intracellular reactive oxygen species (ROS).Cell proliferation and migration assay were used to detect AOP1 toxicity and its effect on wound healing.Moreover,the skin scald wound was made on the shaved dorsum of the anesthetized mice with a 1.0 cm diameter brass cylinder heated in a water bath at 80 ℃ for 2 min and pressed against the rat skin for 10 s.The effects of AOP1 on the healing of skin burns were observed by HE and Masson staining and the contents of MDA and the activity of SOD in skin tissues were measured.Results The antioxidant peptide AOP1 could significantly reduce the number of ROS in HaCaT and L929 cells,and promote cell migration and proliferation.Compared with the untreated group,the skin healing time of AOP1 group was short,the healing rate was high,the area of scab was small,and inflammation and the content of MDA in burned tissue significantly decreased.The effect of AOP1 on healing of burn wound healing was also confirmed by HE and Masson staining.Conclusion It is suggested that the antioxidant peptide AOP1 with natural activity may promote the healing of skin burns by reducing the oxidative stress caused by burns.
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In order to prepare antioxidant peptide through hydrolyzing low-value protein resources with bacterial extracellular proteases and to discover novel proteases, crude extracellular protease from Pseudoalteromonas sp. SHK1-2 was obtained through fermentation which was used to hydrolyze collagen extracted from Cirrhinus molitorella skin. Small peptide fraction was isolated from hydrolysate by ultrafiltration and Sephadex LH-20 size exclusion chromatography and showed 1, 1-diphenyl-2-picrylhydrazyl radical scavenging activity (35.6%±7%), oxygen radical absorbance capacity and inhibition of DNA oxidation damage. The molecule weight was 776.2 Da, and amino acid sequence was Thr-Ala-Gly-His-Pro- Gly-Thr-His through liquid chromatography mass spectrum. Our findings suggest that peptide obtained from low-value protein of fish waste by hydrolysis with bacterial protease has antioxidant activity.