RESUMO
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.
RESUMO
Objective To observe the expression of cellular Fas-associated death domain-like interleukin-1β converting enzyme inhibit protein (c-FLIP) in sepsis mice with acute kidney injury (SAKI) and explore its significance. Methods Thirty male ICR mice were divided into the normal control group (Normal group), sham operation group (Sham group) and SAKI group by random number table method, with 10 mice in each group. The SAKI model of mice was established by cecal ligation and puncture (CLP); the Sham group was not ligated and the cecum was not punctured, and other surgical procedures were the same as the SAKI group; the Normal group did not experience any treatment. The serum and renal tissues of mice in each group were harvested 24 hours after CLP model establishment. The levels of serum creatinine (SCr) and blood urea nitrogen (BUN) were detected by enzyme linked immunosorbent assay (ELISA). The renal tissues were stained with hematoxylin-eosin (HE), and the pathological changes of renal tissues were observed under light microscope and the severity of injury was determined. The expression of c-FLIP in renal tissues was detected by immunohistochemistry. The expression of c-FLIP, Bax and caspase-3 protein in renal tissue was detected by Western Blot. The correlation between c-FLIP expression and Bax, caspase-3 protein expressions in renal tissues were analyzed by Pearson test. Results In the Normal group and the Sham group, the renal tubular epithelial cells were regular and intact, and no interstitial inflammatory cell infiltration was observed; the renal injury score was both 1.30±0.48; immunohistochemistry showed a large amount of c-FLIP positive expression in renal tubular epithelial cells (IA: 120.20±3.87, 116.70±3.46); Western Blot showed high expression of c-FLIP in renal tissues (c-FLIP/GAPDH: 0.99±0.01, 0.98±0.02), and low expressions of Bax and caspase-3 (Bax/GAPDH: 0.16±0.04, 0.19±0.03, caspase-3/GAPDH: 0.24±0.04, 0.23±0.05). Compared with the Sham group, in the SAKI group, renal tubular epithelial cells were degenerated and necrosis, and a large number of interstitial inflammatory cells infiltrated, the renal injury score was significantly increased (4.60±0.52 vs. 1.30±0.48, P < 0.01); the levels of SCr and BUN were significantly increased [SCr (μmol/L): 193.90±13.54 vs. 24.50±3.78, BUN (mmol/L): 81.60±7.26 vs. 5.20±0.92, both P < 0.01]; the c-FLIP positive cells in renal tissues was significantly reduced (IA: 17.11±0.82 vs. 116.70±3.46, P < 0.01); the expression of c-FLIP protein in renal tissues was significantly decreased (c-FLIP/GAPDH: 0.29±0.03 vs. 0.98±0.02, P < 0.01), while the expressions of Bax and caspase-3 protein were significantly increased (Bax/GAPDH: 0.87±0.06 vs. 0.19±0.03, caspase-3/GAPDH: 0.88±0.07 vs. 0.23±0.05, both P < 0.01]. The correlation analysis showed that the c-FLIP protein was significantly negatively correlated with Bax (r = -0.468, P = 0.029) and caspase-3 protein expressions (r = -0.663, P = 0.004). Conclusions The expression level of c-FLIP protein was significantly down-regulated in renal tissue of SAKI, and its down-regulation mechanism was associated with increased apoptosis of renal tubular epithelial cells, which could be an effective target for the treatment of SAKI.