Abstract
Lysosome membrane permeabilization (LMP) has been implicated in cell death. In the present study, we investigated the relationship between cell death and H2O2-/CCl4-induced LMP in hepatocytes in vitro and following acute liver injury in vivo. The key finding was that H2O2 triggered LMP by oxidative stress, as evidenced by a suppression of LAMP1 expression, a reduction in LysoTracker Green and AO staining, and the leakage of proton and cathepsin B/D from the lysosome to the cytoplasm, resulting in cell death. CCl4 also triggered hepatocyte death by decreasing lysosome LAMP1 expression and by inducing the accumulation of products of peroxidative lipids and oxidized proteins. Furthermore, a novel compound 5,8-dimethoxy-6-methyl-7-hydroxy-3-3(2-hydroxy-4-methoxybenzyl) chroman-4-one (58-F) was extracted from Ophiopogon japonicus and served as a potential therapeutic drug. In vivo and in vitro results showed that 58-F effectively rescued hepatocytes by decreasing LMP and by inducing lysosomal enzyme translocation to the cytosol.
Highlights
Our results reveal that LAMP1 protein levels gradually decreased with the increasing duration of H2O2 treatment, reaching its lowest level at 8 h, with a partial restoration of LAMP1 levels observed at 24 h
Our in vivo studies suggest that the levels of lipid peroxidation, protein oxidation, and serum ALT activity in hepatocytes following cell injury or death are increased after CCl4 treatment
The molecular structure of LAMP1 is composed of mainly carbohydrates that form a nearly continuous coating on the inner surface of the lysosome membrane and serve as a barrier to soluble hydrolases, preventing the release of lysosomal enzymes and H+ into the cytoplasm[25]
Summary
58-F Attenuates Lysosomal Damage after CCl4-induced Acute Liver Injury. Haematoxylin and eosin (H&E) staining revealed normal hepatic architecture within the clear hepatic lobule and no dead hepatocytes. Both LAMP1 mRNA and protein levels decreased significantly after CCl4 treatment (p < 0.01), and 58-F pretreatment increased both mRNA and protein levels (p < 0.01) (Fig. 1F–H) These results suggested that lysosomes mediated CCl4-induced hepatocyte injury. In cells pretreated with 58-F, the LAMP1 protein levels significantly increased following a similar time course as the H2O2 treatment (Fig. 5D,E). After treatment with 100 μg/ml NAC after 500 μM of H2O2, 3.26% of the cells were early apoptotic and 13.17% of the cells were late apoptotic after 12 h, whereas in the H2O2 treatment group, 0.58% of the cells were early apoptotic and 5.22% of the cells were late apoptotic(Fig. 8A) Together, these results showed that NAC protected hepatocytes from H2O2-induced apoptosis and death by inhibiting LMP. Statistical analysis was performed using Student’s t test. (**p < 0.01). (E) For the Annexin V/PI assay with FACS, the cells were treated with 100 μM NH4Cl for 2 h
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