Human trypsin inhibitor reduces the apoptosis oflipopolysaccharide-induced human kidney-2 cellsby promoting mitochondrial fusion

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.