Hepatoprotective effect of calculus bovis sativus on nonalcoholic fatty liver disease in mice by inhibiting oxidative stress and apoptosis of hepatocytes

Abstract

Calculus bovis (CB, niu-huang) is a high-class therapeutic drug that is often used in traditional Chinese medicine. CB helps to eliminate heat and toxic components, and prevents the accumulation of phlegm and blood stasis in the liver. In Asian countries, CB Sativus (CBS), an ideal substitute for natural CB, is presently extensively used for long-term treatment of chronic liver diseases. The present study aimed to evaluate the effects and potential mechanism(s) of action of CBS on mice with fructose-induced nonalcoholic fatty liver disease (NAFLD). The NAFLD model was established in C57BL/6 mice by exclusively feeding fluids containing 30% fructose for 8 consecutive weeks. After these 8 weeks, mice were given CBS (50 mg/kg/day or 100 mg/kg/day) for 2 consecutive weeks. Treatment with CBS reversed the fructose-induced impaired glucose tolerance. Compared with the model group, in which mice received 8 weeks of high-fructose diet and 2 weeks of 0.5% sodium carboxymethyl cellulose, CBS treatment significantly decreased the levels of fasting serum glucose, fasting insulin, triglyceride, and total cholesterol, and increased levels of high-density lipoprotein-cholesterol. CBS treatment also significantly decreased the levels of triglyceride, total cholesterol, and free fatty acid in the liver. The activity of superoxide dismutase in the liver was increased after treatment with CBS, however, levels of malondialdehyde and reactive oxygen species decreased. Histopathological examination showed that liver steatosis and injury were significantly reduced in CBS-treated mice. The expression of fatty acid synthase, nuclear factor kappa-light-chain-enhancer of activated B cells, Cysteinyl aspartate-specific proteinase-3, and synonyms B-cell leukemia/lymphoma-2 gene-associated X protein were downregulated after treatment with CBS, whereas the expression of nuclear factor erythroid-2-related factor 2 was upregulated. In conclusion, CBS treatment exerted therapeutic effects in the liver of mice with NAFLD, which may be associated with amelioration of metabolic disorders, enhanced antioxidant effects, and alleviation of apoptosis.

Keywords: antioxidant; fructose; lipid metabolism; liver.

Figure 1

Multiple reaction monitoring (MRM) chromatogram of a representative sample of CBS. Notes: The numbers represent peak sequence and identify which substance the peak represents. Abbreviations: CA, cholic acid; CBS, calculus bovine sativus; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; GCA, glycocholic acid; GCDCA, glycochenodeoxycholic acid; GDCA, glycodeoxycholic acid; HDCA, hyodeoxycholic acid; IS, internal standard; TCA, taurocholic acid; TCDCA, taurochenodeoxycholic acid; TDCA, taurodeoxycholic acid; TUDCA, tauroursodeoxycholic acid; UDCA, ursodeoxycholic acid.

Figure 2

Effects of CBS (50 and 100 mg/kg) on OGTT of NAFLD mice. Notes: (A) Curve showing the change in blood glucose levels during OGTT. (B) Bar chart showing AUC of OGTT. Data are presented as the mean ± SD of 3 mice per group. ^#P<0.05 vs the control group, *P<0.05 vs the model group. Control: non-NAFLD healthy mice; model: high-fructose induced NAFLD mice; CBS (50 mg/kg): NAFLD mice treated with CBS at 50 mg/kg body weight; CBS (100 mg/kg): NAFLD mice treated with CBS at 100 mg/kg body weight. Abbreviations: AUC, area under the curve; BGL, blood glucose level; CBS, calculus bovis sativus; NAFLD, nonalcoholic fatty liver disease; OGTT, oral glucose tolerance test.

Figure 3

ORO and H&E staining of mouse livers. Notes: Effects of CBS (50 and 100 mg/kg) on fructose-induced lipid accumulation and macrovesicular steatosis. Images were captured at 200× magnification. Control: non-NAFLD healthy mice; model: high-fructose induced NAFLD mice; CBS (50 mg/kg): NAFLD mice treated with CBS at 50 mg/kg body weight; CBS (100 mg/kg): NAFLD mice treated with CBS at 100 mg/kg body weight. Abbreviations: CBS, calculus bovis sativus; H&E, hematoxylin and eosin; NAFLD, nonalcoholic fatty liver disease; ORO, Oil-Red-O.

Figure 4

Effects of CBS (50 and 100 mg/kg) on TG and TC (A) and FFA (B) of NAFLD mice. Notes: Data are presented as mean ± SD of 8 mice per group. ^##P<0.01 vs the control group, ^###P<0.001 vs the control group, *P<0.05 vs the model group, ***P<0.001 vs the model group. Control: non-NAFLD healthy mice; model: high-fructose induced NAFLD mice; CBS (50 mg/kg): NAFLD mice treated with CBS at 50 mg/kg body weight; CBS (100 mg/kg): NAFLD mice treated with CBS at 100 mg/kg body weight. Abbreviations: CBS, calculus bovis sativus; FFA, free fatty acid; NAFLD, nonalcoholic fatty liver disease; prot, protein; TC, total cholesterol; TG, triglyceride.

Figure 5

Effects of CBS (50 and 100 mg/kg) on TG and TC (A), LDL-C and HDL-C (B). Notes: Data are presented as mean ± SD of 8 mice per group. ^###P<0.001 vs the control group, *P<0.05 vs the model group, **P<0.01 vs the model group. Control: non-NAFLD healthy mice; model: high-fructose induced NAFLD mice; CBS (50 mg/kg): NAFLD mice treated with CBS at 50 mg/kg body weight; CBS (100 mg/kg): NAFLD mice treated with CBS at 100 mg/kg body weight. Abbreviations: CBS, calculus bovis sativus; HDL-C, high density lipoprotein-cholesterol; LDL-C, low density lipoprotein-cholesterol; NAFLD, nonalcoholic fatty liver disease; TG, triglyceride; TC, total cholesterol.

Figure 6

Effects of CBS (50 and 100 mg/kg) on the protein expression levels of FAS (A) and HSL (B). Notes: Data are presented as mean ± SD of 3 mice per group. ^#P<0.05 vs the control group, ^###P<0.001 vs the control group, *P<0.05 vs the model group, **P<0.01 vs the model group. Control: non-NAFLD healthy mice; model: high-fructose induced NAFLD mice; CBS (50 mg/kg): NAFLD mice treated with CBS at 50 mg/kg body weight; CBS (100 mg/kg): NAFLD mice treated with CBS at 100 mg/kg body weight. Abbreviations: CBS, calculus bovis sativus; FAS, fatty acid synthase; HSL, hormone-sensitive lipase; NAFLD, nonalcoholic fatty liver disease.

Figure 7

Effects of CBS (50 and 100 mg/kg) on SOD (A), MDA (B), and ROS (C) of NAFLD mice. Notes: Data are presented as the mean ± SD of 8 mice per group. ^#P<0.05 vs the control group, ^##P<0.01 vs the control group, ^###P<0.001 vs the control group, *P<0.05 vs the model group, **P<0.01 vs the model group. Control: non-NAFLD healthy mice; model: high-fructose induced NAFLD mice; CBS (50 mg/kg): NAFLD mice treated with CBS at 50 mg/kg body weight; CBS (100 mg/kg): NAFLD mice treated with CBS at 100 mg/kg body weight. Abbreviations: CBS, calculus bovis sativus; MDA, malondialdehyde; NAFLD, nonalcoholic fatty liver disease; prot, protein; ROS, reactive oxygen species; SOD, superoxide dismutase.

Figure 8

Effect of CBS (50 and 100 mg/kg) on the protein expression level of Nrf2. Notes: Data are presented as the mean ± SD of 3 mice per group. ^##P<0.01 vs the control group, *P<0.05 vs the model group, **P<0.01 vs the model group. Control: non-NAFLD healthy mice; model: high-fructose induced NAFLD mice; CBS (50 mg/kg): NAFLD mice treated with CBS at 50 mg/kg body weight; CBS (100 mg/kg): NAFLD mice treated with CBS at 100 mg/kg body weight. Abbreviations: CBS, calculus bovis sativus; NAFLD, nonalcoholic fatty liver disease; Nrf2, nuclear factor erythroid-2 related factor 2.

Figure 9

Effects of CBS (50 and 100 mg/kg) on the hepatocyte apoptosis. Notes: (A) TUNEL stain of liver tissue, (B) the percentages of apoptotic cells, (C) NF-κB, (D) Caspase-3, (E) Bax, and (F) Bcl-2. Data are presented as the mean ± SD of 3 mice per group. ^##P<0.01 vs the control group, ^###P<0.001 vs the control group, *P<0.05 vs the model group, **P<0.01 vs the model group, ***P<0.001 vs the model group. Control: non-NAFLD healthy mice; model: high-fructose induced NAFLD mice; CBS (50 mg/kg): NAFLD mice treated with CBS at 50 mg/kg body weight; CBS (100 mg/kg): NAFLD mice treated with CBS at 100 mg/kg body weight. Abbreviations: Bax, Bcl-2 associated X protein; Bcl-2, B-cell leukemia/lymphoma-2; Caspase-3, cysteinyl aspartate specific proteinase-3; CBS, calculus bovis sativus; NAFLD, nonalcoholic fatty liver disease; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.