Hepatoprotective and Anti-Oxidative Effects of Total Flavonoids From Qu Zhi Qiao (Fruit of Citrus Paradisi cv.Changshanhuyou) on Nonalcoholic Steatohepatitis In Vivo and In Vitro Through Nrf2-ARE Signaling Pathway

Abstract

Nonalcoholic steatohepatitis (NASH) is a liver disease defined as the dynamic condition of hepatocellular injury during the progress of nonalcoholic fatty liver disease (NAFLD). Total flavonoids from the dry and immature fruits of Citrus Paradisi cv.Changshanhuyou (accepted species name: Citrus × aurantium L) (Qu Zhi Qiao, QZQ) are purified and named TFCH. This study was purposed to investigate and analyze the effect of TFCH on NASH model through Nuclear factor erythroid 2-related factor 2 (Nrf2)- antioxidant response elements pathway in vivo and in vitro. In vivo study was performed using male C57BL/6 mice fed with high fat diet 16 weeks for NASH model. After 7-week modeling, mice in TFCH-treated group were daily treated with intragastric administration of TFCH at 25 mg/kg, 50 mg/kg, 200 mg/kg, respectively, for successive 8 weeks. Histopathological and immunohistochemical analyses were conducted for evaluating severity of NASH-mice model and the effect of TFCH treatment. In vitro experiment was performed by using human LX-2 cells and cultured with Free fatty acid (FFA) (Oleic acid: palmitic: l: 0.5 mmol/L) for 24 h and then treated with TFCH at different concentrations (0, 25, 50, 100, 200 mg/ml) for 6 h,12 h, and 24 h. Anti-apoptosis effect of TFCH on LX-2 cells cultured with FFA was revealed by the CCK-8 assay. Lipid parameters and oxidative stress markers were measured in vivo and in vitro, results showed that TFCH dose-dependently and greatly increased the antioxidant ability and reduced the oxidative damage in NASH model. The protein expression of Nrf2 and the downstream target genes in mice liver and human LX-2 cells were tested by Western blot analysis to investigate the possible molecular mechanisms of TFCH. Our results indicated that TFCH up-regulated protein expression of these genes and have the significant influence in activating the Nrf2-ARE signaling pathway. This study shows Nrf2-ARE signaling pathway may provide novel therapeutic opportunities for NASH therapy in the future.

Keywords: Nrf2; Nrf2-ARE signaling pathway; anti-oxidative; nonalcoholic steatohepatitis; total flavonoids from Citrus.

Figure 1

High-performance liquid chromatography (HPLC) chromatographic profile of TFCH and standard substances of narirutin, naringin, neohesperidin, and hesperidin.

Figure 2

Levels of biochemical parameters and oxidative stress markers in the serum and livers of mice. Values are shown as mean ± SD (n = 8). Data (mean ± SD) are statistically different with each other at LSD multiple comparisons, ^#p < 0.05 or ^##p < 0.01 vs. NC group; *p < 0.05 or **p < 0.01 vs. model group.

Figure 3

(A) Histopathological observation of HE staining on livers of mice. Images were obtained at 400× magnification (scale bar=50μm). (B) Histopathological observation of oil red O staining on livers of mice. Images were obtained at 400× magnification (scale bar=50μm).

Figure 4

Immunohistochemistry observation of nuclear factor erythroid 2-related factor 2 (Nrf2) expressions in livers of mice. Images were obtained at 400× magnification (scale bar=50μm).

Figure 5

Expression of the targeted proteins in mice liver. Values are shown as mean ± SD (n = 8). Data (mean ± SD) with different case letters are statistically different with each other at LSD multiple comparisons, which with red letters differed significantly as compared to model group level. ^{uppercase letter} p < 0.01 vs. another group.

Figure 6

Cell viabilities of LX-2 cells were assayed by CCK-8. (A) Cell viability of LX-2 cells in each group (normal group; free fatty acid [FFA] group; FFA-100 mg/L TFCH group) were measured at 6 h, 12 h, and 24 h. (B) Cell viability of FFA-induced LX-2 cells in different TFCH group were measured at 24 h. Values are shown as mean ± SD (n =3). Data (mean ± SD) with different case letters are statistically different with each other at LSD multiple comparisons, which with red letters differed significantly as compared to model group level; ^{uppercase lette r} p <0.01 vs. another group; ^{lowercase letter} p < 0.05 vs. another group.

Figure 7

Levels of biochemical parameters and oxidative stress markers in the LX-2 cells. Values are shown as mean ± SD (n =3). Data (mean ± SD) are statistically different with each other at LSD multiple comparisons, ^#p < 0.05 or ^##p < 0.01 vs. NC group; *p < 0.05 or **p < 0.01 vs. model group.

Figure 8

Effect of TFCH on Nrf2 and the downstream target genes in the LX-2 cells cultured with free fatty acid (FFA). Values are shown as mean ± SD (n =3). Data (mean ± SD) with different case letters are statistically different with each other at LSD multiple comparisons, which with red letters differed significantly as compared to model group level. ^{uppercase letter} p < 0.01 vs. another group.

Figure 9

Proposed mechanism of TFCH against nonalcoholic steatohepatitis (NASH).