Potential protective effect of hesperidin on hypoxia/reoxygenation-induced hepatocyte injury

Abstract

Hesperidin (HDN) has been reported to have hydrogen radical- and hydrogen peroxide-removal activities and to serve an antioxidant role in biological systems. However, whether HDN protects hepatocytes (HCs) against hypoxia/reoxygenation (H/R)-induced injury remains unknown. The present study aimed to explore the role of HDN in H/R-induced injury. HCs were isolated and cultured under H/R conditions with or without HDN treatment. HC damage was markedly induced under H/R, as indicated by cell viability, supernatant lactate dehydrogenase levels and alanine aminotransferase levels; however, HDN treatment significantly reversed HC injury. Oxidative stress markers (malondialdehyde, superoxide dismutase, glutathioneand reactive oxygen species) were increased markedly during H/R in HCs; however, this effect was significantly attenuated after exposure to HDN. Compared with those of the control group, the mRNA expression levels of IL-6 and TNF-α in HCs and the concentrations of IL-6 and TNF-α in the supernatants increased significantly following H/R, and HDN significantly ameliorated these effects. Western blotting demonstrated that microtubule-associated protein 1 light chain 3α (MAP1LC3A, also known as LC3) and Beclin-1 protein expression levels increased, while sequestosome 1 levels decreased during H/R following exposure to HDN. The number of GFP-LC3 puncta in HCs following exposure to HDN was increased compared with that observed in HCs without HDN exposure under the H/R conditions after bafilomycin A1 treatment. In summary, the present study demonstrated that HDN attenuated HC oxidative stress and inflammatory responses while enhancing autophagy during H/R. HDN may have a potential protective effect on HCs during H/R-induced injury.

Keywords: autophagy; hepatocyte; hesperidin; hypoxia/reoxygenation; oxidative stress.

Figure 1

HDN protects HCs against H/R injury. (A) Cell viability was determined by MTT assay for HCs exposed to different doses of HDN under normoxic conditions. (B) Morphology of HCs in control PBS group, control HDN group, H/R PBS group and H/R HDN group under a light microscope (scale bar, 50 µm). (C) Cell viability was determined by MTT assayfor HCs in control PBS group, control HDN group, H/R PBS group and H/R HDN group. (D) Levels of supernatant LDH and (E) ALT in control PBS group, control HDN group, H/R PBS group and H/R HDN group. Results areshown as the mean ± SEM of three independent experiments. ^***P<0.001 with comparisons shown by lines. HDN, hesperidin; HC, hepatocyte; H/R, hypoxia/reoxygenation; LDH, lactate dehydrogenase; ALT, alanine aminotransferase; ns, not significant.

Figure 2

HDN attenuates hepatocyte oxidative stress induced by H/R. (A) MDA, (B) SOD, (C) GSH and (D) ROSlevels in control PBS group, control HDN group, H/R PBS group and H/R HDN group. ROS (green) was detected by fluorescence microscopy (magnification, x40). Results are shown as the mean ± SEM of three independent experiments. ^***P<0.001 with comparisons shown by lines. HDN, hesperidin; H/R, hypoxia/reoxygenation; MDA, malondialdehyde; SOD, superoxide dismutase; GSH, glutathione; ROS, reactive oxygen species; ns, not significant.

Figure 3

HDN ameliorates HC inflammatory responses during H/R. (A) mRNA expression levels of IL-6 in control PBS group, control HDN group, H/R PBS group and H/R HDN group. (B) Levels of IL-6 in supernatants assessed by ELISA. (C) mRNA expression levels of TNF-α. (D) Levels of TNF-α in supernatants assessed by ELISA. Results are shown as the mean ± SEM from three independent experiments. ^***P<0.001 with comparisons shown by lines. HDN, hesperidin; HC, hepatocyte; H/R, hypoxia/reoxygenation; IL, interleukin; TNF, tumor necrosis factor; ns, not significant.

Figure 4

HDN induces autophagy to protect HCs against H/R injury. (A) Representative western blots showing the protein expression levels of LC3, Beclin-1 and P62 in control PBS group, control HDN group, H/R PBS group and H/R HDN group. (B) Representative western blots showing the levels of LC3 in HCs subjected to normoxia with or without bafilomycin A1 (50 nM). (C) Representative western blots showing the levels of LC3 in HCs subjected to H/R with or without bafilomycin A1 (50 nM). (D) Confocal microscopy images of HCs overexpressing GFP-LC3 (green) and subjected to normoxia or H/R, with/without bafilomycin A1 (50 nM) treatment (magnification, x40). Nuclei were counterstained with Hoechst 33342 (blue). The numbers of GFP-LC3 puncta were counted per cell and represented in the bar graph. Results are shown as the mean ± SEM of three independent experiments. ^**P<0.01 with comparisons shown by lines. HDN, hesperidin; HC, hepatocyte; H/R, hypoxia/reoxygenation; LC3, microtubule-associated protein 1 light chain 3α; P62, sequestosome 1; ns, not significant.

Figure 5

Schematic demonstrating the interplay among oxidative stress, inflammatory activity, and autophagy following H/R injury. H/R leads to oxidative stress, inflammatory activity, and autophagy, while HDN exposure inhibits these effects. Both oxidative stress and inflammation induce autophagy and HC injury. Appropriate autophagy activity contributes to HC recovery by reducing oxidative stress and inflammatory activity, while autophagy dysfunction aggravates HC injury. HDN exposure promotes the effects of autophagy, thus reducing HCinjury. H/R, hypoxia/reoxygenation; HDN, hesperidin; HC, hepatocyte.