Ginsenoside Rk1 ameliorates paracetamol-induced hepatotoxicity in mice through inhibition of inflammation, oxidative stress, nitrative stress and apoptosis

Abstract

Background: Frequent overdose of paracetamol (APAP) has become the major cause of acute liver injury. The present study was designed to evaluate the potential protective effects of ginsenoside Rk1 on APAP-induced hepatotoxicity and investigate the underlying mechanisms for the first time.

Methods: Mice were treated with Rk1 (10 mg/kg or 20 mg/kg) by oral gavage once per d for 7 d. On the 7th d, all mice treated with 250 mg/kg APAP exhibited severe liver injury after 24 h, and hepatotoxicity was assessed.

Results: Our results showed that pretreatment with Rk1 significantly decreased the levels of serum alanine aminotransferase, aspartate aminotransferase, tumor necrosis factor, and interleukin-1β compared with the APAP group. Meanwhile, hepatic antioxidants, including superoxide dismutase and glutathione, were elevated compared with the APAP group. In contrast, a significant decrease in levels of the lipid peroxidation product malondialdehyde was observed in the ginsenoside Rk1-treated group compared with the APAP group. These effects were associated with a significant increase of cytochrome P450 E1 and 4-hydroxynonenal levels in liver tissues. Moreover, ginsenoside Rk1 supplementation suppressed activation of apoptotic pathways by increasing Bcl-2 and decreasing Bax protein expression levels, which was shown using western blotting analysis. Histopathological observation also revealed that ginsenoside Rk1 pretreatment significantly reversed APAP-induced necrosis and inflammatory infiltration in liver tissues. Biological indicators of nitrative stress, such as 3-nitrotyrosine, were also inhibited after pretreatment with Rk1 compared with the APAP group.

Conclusion: The results clearly suggest that the underlying molecular mechanisms in the hepatoprotection of ginsenoside Rk1 in APAP-induced hepatotoxicity may be due to its antioxidation, antiapoptosis, anti-inflammation, and antinitrative effects.

Keywords: APAP-induced hepatotoxicity; anti-apoptosis; anti-inflammation; ginsenoside Rk1; oxidative stress.

Fig. 1

Pretreatment with Rk1 protected against APAP-induced liver injury. (A) Experimental design of the hepatoprotective effect of Rk1 on APAP-induced liver injury in mice. (B) The levels of GSH, MDA and SOD in liver tissues of mice (B). All data are expressed as the mean ± standard deviation, n = 8. **p < 0.01 versus normal group; ^#p < 0.05 versus APAP group; ^##p < 0.01. APAP, paracetamol; GSH, glutathione; MDA, malondialdehyde; prot, protein; SOD, superoxide dismutase.

Fig. 2

Pretreatment with Rk1 protected against APAP-induced liver injury. (A) 4-hydroxynonenal (4-HNE), (B) cytochrome P450 E1 (CYP2E1), and (C) 3-nitrotyrosine (3-NT). The expression levels of 4-HNE, CYP2E1 (green) and 3-NT (red) in tissue sections isolated from different groups were assessed by immunofluorescence. Representative immunofluorescence images were taken at 200×. 4′, 6-diamidino-2-phenylindole (DAPI) (blue) was used as a nuclear counterstain. All data are expressed as the mean ± standard deviation, n = 8. **p < 0.01 versus normal group; ^##p < 0.01 versus APAP group. APAP, paracetamol.

Fig. 3

Pretreatment with Rk1 protected against APAP-induced inflammatory cytokines. The levels of (A) tumor necrosis factor-α (TNF)-α and (B) interleukin-1β (IL)-1β in the serum of mice. All data are expressed as the mean ± standard deviation, n = 8. *p < 0.05 versus normal group; **p < 0.01; ^#p < 0.05 versus APAP group; ^##p < 0.01. APAP, paracetamol.

Fig. 4

Pretreatment with Rk1 protected against APAP-induced liver of Rk1 on the expression of: (A, E) nitric oxide synthase (iNOS), (B, F) cyclooxygenase-2 (COX-2), (C, G) Bax, and (D, H) Bcl-2. The protein expression was examined by immunohistochemistry. All data are expressed as the mean ± standard deviation, n = 8. **p < 0.01 versus normal group; ^#p < 0.05 versus APAP group; ^##p < 0.01. APAP, paracetamol.

Fig. 5

Histological examination of morphological changes in liver tissues. Liver tissues stained with (A) hematoxylin–eosin (100×, 400×); (B) Hoechst 33258 (100×, 400×) and (D) the percentage of apoptosis; (C) TUNEL (400×) and (E) the presence of TUNEL-positive cells (E). Arrows show necrotic and apoptotic cells. All data are expressed as the mean ± standard deviation, n = 8. **p < 0.01 versus normal group; ^#p < 0.05 versus APAP group; ^##p < 0.01. APAP, paracetamol; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Fig. 6

Protein expression of Bax and Bcl-2. (A) Effects of Rk1 on the protein expression of Bax and Bcl-2. (B) Results quantified from Bax and Bcl-2 band intensities. The protein expression was examined using western blotting analysis in liver tissues. All data are expressed as the mean ± standard deviation, n = 8. *p < 0.05 versus normal group; **p < 0.01; ^#p < 0.05 versus APAP group; ^##p < 0.01. APAP, paracetamol.