Korean J Physiol Pharmacol.  2020 Sep;24(5):373-384. 10.4196/kjpp.2020.24.5.373.

Four active monomers from Moutan Cortex exert inhibitoryeffects against oxidative stress by activating Nrf2/Keap1signaling pathway

• Zhang B
• Yu D
• Luo N
• Yang C
• Zhu Y

• KMID: 2505772
• DOI: http://doi.org/10.4196/kjpp.2020.24.5.373

Abstract

Paeonol, quercetin, -sitosterol, and gallic acid extracted from MoutanCortex had been reported to possess anti-oxidative, anti-inflammatory, and antitumoractivities. This work aimed to illustrate the potential anti-oxidative mechanismof monomers in human liver hepatocellular carcinoma (HepG2) cells-induced byhydrogen peroxide (H₂O₂) and to evaluate whether the hepatoprotective effect ofmonomers was independence or synergy in mice stimulated by carbon tetrachloride(CCl₄). Monomers protected against oxidative stress in HepG2 cells in a doseresponsemanner by inhibiting the generation of reactive oxygen species, increasingtotal antioxidant capacity, catalase and superoxide dismutase (SOD) activities, andactivating the antioxidative pathway of nuclear factor E2-related factor 2/KelchlikeECH-associated protein 1 (Nrf2/Keap1) signaling pathway. We found that thein vitro antioxidant capacities of paeonol and quercetin were better than those of-sitosterol and gallic acid. Furthermore, paeonol apparently diminished the levelsof alanine transaminase and aspartate aminotransferase, augmented the contentsof glutathione and SOD, promoted the expressions of Nrf2 and heme oxygenase-1proteins in mice stimulated by CCl₄. In HepG2 cells, paeonol, quercetin, -sitosterol,and gallic acid play a defensive role against H₂O₂-induced oxidative stress throughactivating Nrf2/Keap1 pathway, indicating that these monomers have anti-oxidativeproperties. Totally, paeonol and quercetin exerted anti-oxidative and hepatoprotectiveeffects, which is independent rather than synergy.

Keyword

Kelch like ECH-associated protein 1; Liver failure; Nuclear factor E2-related factor 2; Oxidative stress; Signal transduction

Figure

• 

  Fig. 1 Effects of four monomers and hydrogen peroxide (H2O2) on cell viability of human liver hepatocellular carcinoma (HepG2) cells. (A) The effects of monomers (paeonol, quercetin, β-sitosterol, gallic acid, and vitamin E) on HepG2 cell viability. (B) The effect of H2O2 on HepG2 cell viability at different concentration of H2O2. (C) The effects of monomers on HepG2 cell viabilities at the concentration of 7 mM H2O2. The data from three independent experiments are presented as the mean ± standard deviation. *p < 0.05, **p < 0.01, and ***p < 0.001, the H2O2 vs. control group; ##p < 0.01, and ###p < 0.001, the monomers vs. H2O2 group.

• 

  Fig. 2 Effects of four monomers on reactive oxygen species (ROS) generation of human liver hepatocellular carcinoma (HepG2) cells induced by hydrogen peroxide (H2O2). (A, C) ROS generation of HepG2 cells was measured at the different incubated time by fluorescence microscope assay. (B, D) ROS generation of HepG2 cells was inhibited by four monomers at 15 min incubated time through fluorescence microscope assay. The data from three independent experiments are presented as the mean ± standard deviation. **p < 0.01, and ***p < 0.001, the H2O2 vs. control group; ###p < 0.001, the monomers vs. H2O2 group.

• 

  Fig. 3 Activation of four monomers on the Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor E2-related factor 2 (Nrf2) signaling pathway in human liver hepatocellular carcinoma (HepG2) cells induced by hydrogen peroxide (H2O2). (A) The levels of Nrf2, Keap1, NQO1, and HO-1 expressions were measured by western blot assay. (B) Translocation of Nrf2 protein from the cytosol to the nucleus was also evaluated by western blot assay. (C) Effects of four monomers on the binding capacities of Nrf2-ARE complexes in HepG2 cells after H2O2 treatment. The data from three independent experiments are presented as the mean ± standard deviation. NQO1, quinone oxidoreductase 1; HO-1, heme oxygenase-1; ARE, antioxidant-responsive element. *p < 0.05, **p < 0.01, and ***p < 0.001, the H2O2 vs. control group; #p < 0.05, ##p < 0.01, and ###p < 0.001, the monomers vs. H2O2 group.

• 

  Fig. 4 The expressions of Nrf2, NQO1, and HO-1 proteins were determined in the presence or absence of paeonol, quercetin and ML385 in HepG2 cells induced by H2O2. The data from three independent experiments are presented as the mean ± standard deviation. Nrf2, nuclear factor E2-related factor 2; NQO1, quinone oxidoreductase 1; HO-1, heme oxygenase-1; HepG2, human liver hepatocellular carcinoma; H2O2, hydrogen peroxide. **p < 0.01, and ***p < 0.001, the H2O2 vs. control group; ###p < 0.001, the monomers vs. H2O2 group.

• 

  Fig. 5 Effects of separate or combined administration of paeonol and quercetin on liver tissues and serum markers of mice after carbon tetrachloride (CCl4) intraperitoneal injection. (A) Representative histopathological changes in mice livers were observed with H&E staining. Magnification 200× (1, central venous hyperemia; 2, inflammatory infiltration; 3, hepatocyte necrosis). (B–F) Effects of active monomers on the levels of AST, ALT, AST/ALT ratio, GSH, and SOD in mice serums induced by CCl4. Paeonol and quercetin groups were pretreated with 100 mg/kg paeonol and 100 mg/kg quercetin, respectively. L-(paeonol + quercetin) group was pretreated with 50 mg/kg paeonol and 50 mg/kg quercetin. H-(paeonol + quercetin) group was pretreated with 100 mg/kg paeonol and 100 mg/kg quercetin. The data from three independent experiments are presented as the mean ± standard deviation. AST, aspartate aminotransferase; ALT, alanine transaminase; GSH, glutathione; SOD, superoxide dismutase. *p < 0.05, ***p < 0.001, the CCl4 vs. control group; #p < 0.05, ##p < 0.01, and ###p < 0.001, the monomers vs. CCl4 group.

• 

  Fig. 6 Effects of separate or combined administration of paeonol and quercetin on the expressions of nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) proteins in mice livers induced by carbon tetrachloride (CCl4). (A) Representative changes of Nrf2 in mice livers were obtained by immunohistochemical staining assay (magnification 200×). (B) The expressions of Nrf2 and HO-1 proteins were detected by western blot. The data from three independent experiments are presented as the mean ± standard deviation. *p < 0.05, the CCl4 vs. control group; #p < 0.05, ##p < 0.01, and ###p < 0.001, the monomers vs. CCl4 group.

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