Effects of schisandrin B pretreatment on tumor necrosis factor-alpha induced apoptosis and Hsp70 expression in mouse liver

Abstract

Tumor necrosis factor-alpha (TNFalpha) could cause apoptosis in hepatic tissue of D-galactosamine sensitized mice, as evidenced by the increase in the extent of DNA fragmentation. The hepatic apoptosis induced by TNFalpha was associated with hepatocellular damage as assessed by plasma alanine aminotransferase activity. Schisandrin B (Sch B) pretreatment at daily doses ranging from 0.5 to 2 mmol/kg for 3 days caused a dose-dependent protection against TNFalpha-induced apoptosis in mice. The hepatoprotection was accompanied by a parallel reduction in the extent of hepatocellular damage. The same Sch B pretreatment regimens increased hepatic Hsp70 level in a dose-dependent manner. The relevance of Sch B-induced increase in Hsp70 expression to the prevention of TNFalpha-triggered hepatic apoptosis remains to be elucidated.

Fig 1. Chemical structure of Sch B. Sch B was purified from the petroleum ether extract of Fructus schisandrae by silica gel column chromatography as previously described (Ko et al 1995). The chemical structure of Sch B was confirmed by comparing the silica gel TLC and spectral characteristics (¹H- and ¹³C-NMR and mass spectra) with authentic standard obtained from the Institute of Materia Medica, Chinese Academy of Sciences, Beijing. The purity of the compounds, as assessed by HPLC, was found to be higher than 95% (w/w)

Fig 2. Time course of Sch B pretreatment on TNFα-induced hepatic apoptosis and hepatocellular damage in mice: The effect of Sch B pretreatment. Female Balb/c mice (20–22 g) were randomly assigned into groups of 5 individuals. In the pretreatment groups, animals were treated intragastrically with Sch B (suspended in olive oil, 4% [w/v]) for 3 days at daily doses ranging from 0.5 to 2 mmol/ kg. Control animals were administered with the olive oil. Twenty-four hours after the last dosing, animals will be treated intraperitoneally with d-gal (700 mg/kg) followed by an intravenous injection of mouse recombinant TNFα at 5 μg/kg. Animals were sacrificed 4, 6, or 8 hours after the intoxication. (A) The extent of DNA fragmentation, a molecular marker of apoptosis, was quantitated by measuring hepatic cytosolic oligonucleosome-bound DNA using Cell Death Detection Kit (Roche, Germany) and expressed in percentage of untreated control (ie, non-Sch B and non-TNFα). (B) The extent of hepatocellular damage was assessed by measuring plasma alanine aminotransferase (ALT) activity using an assay kit from Sigma. Data were analyzed by one-way ANOVA followed by Duncan's multiple range test in order to detect intergroup differences. Significant difference was determined when P < 0.05. Values given are the mean ± SEM, with n = 5. * Significantly different from the untreated control. # Significantly different from the TNFα-treated control at the same time period

Fig 3. Time course of hepatic Hsp70 expression in Sch B–pretreated mice. (A) Female Balb/c mice (20–22 g) were randomly assigned into groups of 3 individuals. In the pretreatment groups, animals were treated intragastrically with Sch B (suspended in olive oil) at a daily dose of 1 mmol/kg for 1 day (lane 3), 2 days (lane 4), and 3 days (lane 5), respectively. Control animals (lane 2) were administered with the olive oil only. Animals were sacrificed 24 hours after the last dosing. Hsp70 standard (lane 1) was purchased from StressGen (Victoria, BC, Canada). Hepatic cytosolic fraction were separated by SDS-PAGE with 7.5% gel using Mini-PROTEAN II Electrophoresis Cell (Bio-Rad, USA). Separated proteins were transferred electrophoretically onto the nitrocellulose paper using Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad). The nitrocellulose membrane was probed with anti-Hsp70 antibody purchased from StressGen. Subsequently, the Hsp70 band was visualized using Immun-Bot Colorimetric Assay Kit (Bio-Rad). The intensity of the immunostained band was quantitated by an imaging densitometer (GS-670, Bio-Rad). (B) Hepatic Hsp70 level in Sch B–treated mice. Values given are the mean ± SEM, with n = 3. * Significantly different from the control

Fig 4. Dose response of Sch B on hepatic Hsp70 expression in mice. (A) Mice were treated intragastrically with Sch B at a daily dose of 0.5 (lane 3), 1.0 (lane 4), and 2.0 (lane 5) mmol/kg for 3 days, respectively. Control animals (lane 2) were administered with the olive oil only. Hsp70 level in hepatic cytosolic fractions and Hsp70 standard (lane 1) were detected by Western blotting as described in Figure 3. (B) Hepatic Hsp70 level in Sch B–treated mice. Values given are the mean ± SEM, with n = 3. * Significantly different from the control