Human trypsin inhibitor reduces the apoptosis of lipopolysaccharide‑induced human kidney‑2 cells by promoting mitochondrial fusion

Liu,Ning; Jiang,Zhiyi; Liu,Yongjun; Nie,Yao; Chen,Juan; Ouyang,Bin; Guan,Xiangdong; Chen,Minying

doi:10.3892/mmr.2017.6927

Human trypsin inhibitor reduces the apoptosis of lipopolysaccharide‑induced human kidney‑2 cells by promoting mitochondrial fusion

Authors:
- Ning Liu
- Zhiyi Jiang
- Yongjun Liu
- Yao Nie
- Juan Chen
- Bin Ouyang
- Xiangdong Guan
- Minying Chen
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Affiliations: Department of Surgical Intensive Care Unit of The First Affiliated Hospital of Sun Yat‑sen University, Guangzhou, Guangdong 510080, P.R. China
Published online on: July 5, 2017 https://doi.org/10.3892/mmr.2017.6927
Pages: 2899-2906

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Abstract

Imbalance in mitochondrial fusion/fission is one of the mechanisms leading to sepsis‑induced mitochondrial dysfunction and cell apoptosis. The present study examined the effects of human trypsin inhibitor (UTI), a well‑known antioxidant and anti‑inflammatory substance, on mitochondrial dynamics and cell apoptosis in lipopolysaccharide (LPS)‑induced human kidney‑2 (HK‑2) cells. The HK‑2 cells were incubated for 24 h either with LPS (800 ng/ml) or LPS (800 ng/ml) mixed with UTI (250 U/ml). Cell viability was assessed using a3‑(4,5‑dimethyl‑2‑thiazolyl)‑2, 5‑diphenyl‑2‑H‑tetrazolium bromide assay. Oxidative activities (estimated by maleic dialdehyde and superoxide dismutase), levels of inflammatory cytokines interleukin (IL)‑6 and tumor necrosis factor (TNF)‑α, and levels of ATP were measured using an enzyme‑linked immunosorbent assay. The expression levels of the mitochondrial fission protein, death‑associated protein kinase 2 (DAPK‑2), mitofusin (Mfn)1 and Mfn2 mitochondrial fusion proteins, and apoptotic and anti‑apoptotic biomarkers, including cytochrome c, caspase‑3, caspase‑9, B‑cell lymphoma (Bcl)‑2, Bcl‑extra large and poly ADP‑ribose polymerase (PARP), were assessed using western blot analyses. The changes in mitochondrial membrane potential were analyzed following JC‑1 staining. Annexin V/propidium iodide assays were used to evaluate cell apoptosis. The results showed that the balance of mitochondrial dynamics was towards mitochondrial fusion in the UTI group, as a reduced expression of DAPK2, and increased expression levels of Mfn1 and Mfn2 were detected (P<0.05, vs. LPS group). In addition, adecline in the levels of the inflammatory cytokines, TNF‑α and IL‑6, and the oxidative activities, reflected by an increase in SOD and a decrease in MDA (P<0.05, vs. LPS group) were observed. Cell apoptosis was inhibited following co‑treatment with UTI (P<0.05, vs. LPS group). It was concluded that UTI may protect mitochondrial functions by promoting mitochondrial fusion and limiting mitochondrial fission, thus reducing the apoptosis of LPS‑induced HK‑2 cells.

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