Zinc Supplementation with Polaprezinc Protects Mouse Hepatocytes against Acetaminophen-Induced Toxicity via Induction of Heat Shock Protein 70

Abstract

Polaprezinc, a chelate compound consisting of zinc and l-carnosine, is clinically used as a medicine for gastric ulcers. It has been shown that induction of heat shock protein (HSP) is involved in protective effects of polaprezinc against gastric mucosal injury. In the present study, we investigated whether polaprezinc and its components could induce HSP70 and prevent acetaminophen (APAP) toxicity in mouse primary cultured hepatocytes. Hepatocytes were treated with polaprezinc, zinc sulfate or l-carnosine at the concentration of 100 microM for 9 h, and then exposed to 10 mM APAP. Polaprezinc or zinc sulfate increased cellular HSP70 expression. However, l-carnosine had no influence on it. Pretreatment of the cells with polaprezinc or zinc sulfate significantly suppressed cell death as well as cellular lipid peroxidation after APAP treatment. In contrast, pretreatment with polaprezinc did not affect decrease in intracellular glutathione after APAP. Furthermore, treatment with KNK437, an HSP inhibitor, attenuated increase in HSP70 expression induced by polaprezinc, and abolished protective effect of polaprezinc on cell death after APAP. These results suggested that polaprezinc, in particular its zinc component, induces HSP70 expression in mouse primary cultured hepatocytes, and inhibits lipid peroxidation after APAP treatment, resulting in protection against APAP toxicity.

Keywords: acetaminophen; heat shock protein; hepatotoxicity; polaprezinc; zinc.

Fig. 1

Effect of polaprezinc, zinc sulfate or l-carnosine on HSP70 expression. HSP70 expression was analyzed by Western blot as described in materials and methods. (A) Time course of the expression of HSP70 after polaprezinc treatment. Cells were incubated in the presence of 100 µM polaprezinc for 0, 3, 6 or 9 h. (B) HSP70 expression 9 h after treatment with polaprezinc, zinc sulfate or l-carnosine at a concentration of 100 µM. Western blots were quantified with NIH Image. The data are expressed as means ± SE of 3 separate experiments. PZ; polaprezinc, ZS; zinc sulfate, LC; l-carnosine. **p<0.01 vs initial (0 h) time point (in A); ^##p<0.01 vs control (in B).

Fig. 2

Changes in zinc concentrations after polaprezinc or zinc sulfate treatment. Cells were incubated in the presence of polaprezinc or zinc sulfate at a concentration of 100 µM for 0, 1, 3, 6 or 9 h. Zinc concentration was measured by atomic absorption spectrophotometry. The data are expressed as means ± S.E. of 5 separate experiments. PZ; polaprezinc, ZS; zinc sulfate. **p<0.01, *p<0.05 vs initial (0 h) time point of PZ treatment, ^##p<0.01 vs initial (0 h) time point of ZS treatment.

Fig. 3

Effect of polaprezinc, zinc sulfate or l-carnosine on cell viability after APAP exposure. The cell viability was analyzed using WST-8 assay. Cells were treated with polaprezinc, zinc sulfate or l-carnosine at a concentration of 100 µM at 9 h before APAP (10 mM) treatment. The cell viability was measured 0, 6 or 12 h after APAP. The data are expressed as means ± SE of 7 separate experiments. PZ; polaprezinc, ZS; zinc sulfate, LC; l-carnosine. **p<0.01 vs APAP alone.

Fig. 4

Time course of lipid peroxide content after APAP exposure. Cells were incubated in the presence of polaprezinc, zinc sulfate or l-carnosine at a concentration of 100 µM for 9 h before APAP (10 mM) treatment. The lipid peroxide content was measured at 0, 3, 6 or 12 h after APAP. The data are expressed as means ± SE of 3–7 separate experiments. PZ; polaprezinc, ZS; zinc sulfate, LC; l-carnosine. **p<0.01, *p<0.05 vs APAP alone at each time point.

Fig. 5

Effect of polaprezinc on intracellular GSH contents after APAP treatment. Cells were treated with 100 µM polaprezinc (PZ) 9 h before APAP (10 mM) exposure. GSH contents were measured at 3 h after APAP. The data are expressed as means ± SE of 3 separate experiments. **p<0.01, *p<0.05 vs initial (0 h) time point, respectively.

Fig. 6

Inhibition by KNK437 against HSP70 induction and prevention of APAP toxicity by polaprezinc. Cells were treated with KNK437 of 50 µM at 6 h before polaprezinc (PZ) treatment. (A) After 6 h, cells were incubated in the presence of 100 µM polaprezinc for 9 h. HSP70 expression was analyzed by Western blot. (B) After incubation with KNK437 for 6 h followed by polaprezinc for 9 h, cells were treated with 10 mM APAP. The cell viability was measured at 12 h after APAP. The data are expressed as means ± SE of 3–7 separate experiments. *p<0.05 vs PZ alone (in A), ^#p<0.05 vs PZ + APAP (in B).