Scutellarin prevents acute alcohol-induced liver injury via inhibiting oxidative stress by regulating the Nrf2/HO-1 pathway and inhibiting inflammation by regulating the AKT, p38 MAPK/NF-κB pathways

Abstract

Alcoholic liver disease (ALD) is the most frequent liver disease worldwide, resulting in severe harm to personal health and posing a serious burden to public health. Based on the reported antioxidant and anti-inflammatory capacities of scutellarin (SCU), this study investigated its protective role in male BALB/c mice with acute alcoholic liver injury after oral administration (10, 25, and 50 mg/kg). The results indicated that SCU could lessen serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels and improve the histopathological changes in acute alcoholic liver; it reduced alcohol-induced malondialdehyde (MDA) content and increased glutathione peroxidase (GSH-Px), catalase (CAT), and superoxide dismutase (SOD) activity. Furthermore, SCU decreased tumor necrosis factor-‍α (TNF-‍α), interleukin-6 (IL-6), and IL-‍1β messenger RNA (mRNA) expression levels, weakened inducible nitric oxide synthase (iNOS) activity, and inhibited nucleotide-binding oligomerization domain (NOD)‍-like receptor protein 3 (NLRP3) inflammasome activation. Mechanistically, SCU suppressed cytochrome P450 family 2 subfamily E member 1 (CYP2E1) upregulation triggered by alcohol, increased the expression of oxidative stress-related nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) pathways, and suppressed the inflammation-related degradation of inhibitor of nuclear factor-‍κB (NF-‍κB)‍-‍α (IκBα) as well as activation of NF‍-‍κB by mediating the protein kinase B (AKT) and p38 mitogen-activated protein kinase (MAPK) pathways. These findings demonstrate that SCU protects against acute alcoholic liver injury via inhibiting oxidative stress by regulating the Nrf2/HO-1 pathway and suppressing inflammation by regulating the AKT, p38 MAPK/NF-κB pathways.

Keywords: Alcoholic liver disease; Inflammation; Oxidative stress; Scutellarin.

Fig. 1. Protection of SCU against acute alcoholic liver injury. (a) Experimental protocol for alcoholic liver injury model. (b) ALT level in serum. (c) AST level in serum. (d) Histological analysis of the liver performed using H&E staining (scale bar: 100 µm; black arrow: necrosis). All values ( n=5) are demonstrated as mean±standard deviation (SD). ^** P<0.01, ^*** P<0.001 versus control; ^# P<0.05 versus model. SCU: scutellarin; ALT: alanine aminotransferase; AST: aspartate aminotransferase.

Fig. 2. Effects of SCU on CYP2E1 expression and oxidative stress in the liver of mice. (a) Protein expression of CYP2E1 in the liver analyzed by western blot. (b) Quantification of CYP2E1 protein expression in (a). GSH-Px (c), SOD (d), CAT (e), and MDA (f) levels in the liver with different treatments. All values ( n=5) are demonstrated as mean±standard deviation (SD). ^** P<0.01, ^*** P<0.001 versus control; ^# P<0.05, ^## P<0.01, ^### P<0.001 versus model. SCU: scutellarin; CYP2E1: cytochrome P450 family 2 subfamily E member 1; GSH-Px: glutathione peroxidase; SOD: superoxide dismutase; CAT: catalase; MDA: malondialdehyde; prot: protein.

Fig. 3. Effects of SCU on the Nrf2/HO-1 pathway of mice liver both in vivo and in vitro. (a) Effects of SCU on the cell viability of HepG2 cells. (b) Effects of ethanol on the cell viability of HepG2 cells. (c) Protection of SCU against ethanol-induced HepG2 cells injury. (d) ROS level in HepG2 cells after SCU (0, 20, 40, and 80 μmol/L) pretreatments for 1 h and 600 mmol/L ethanol treatment for 24 h. (e) The protein levels of HO-1, cytosolic Nrf2, and nuclear Nrf2 in HepG2 cells analyzed by western blot. (f) Quantification analyses of HO-1, cytosolic Nrf2, and nuclear Nrf2 protein expression in (e). (g) ROS level in liver tissues. (h) Protein levels of HO-1, cytosolic Nrf2, and nuclear Nrf2 in the liver tissues analyzed by western blot. (i) Quantification analyses of HO-1, cytosolic Nrf2, and nuclear Nrf2 protein expression in (h). All values ( n=3) are demonstrated as mean±standard deviation (SD). ^* P<0.05, ^** P<0.01, ^*** P<0.001 versus control; ^# P<0.05, ^## P<0.01, ^### P<0.001 versus model. SCU: scutellarin; Nrf2: nuclear factor erythroid 2-related factor 2; HO-1: heme oxygenase-1; ROS: reactive oxygen species; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Fig. 4. Effects of SCU on acute ethanol-induced inflammatory mediators, the activation of NLRP3 inflammasome, and NF-κB pathway. (a‒c) mRNA expression of IL-1β, IL-6, and TNF-α in the liver. (d) iNOS activity in the liver. (e) NLRP3, pro-caspase-1, caspase-1 p20, and ASC expression in the liver determined by western blot. (f‒‍ h) Quantification of NLRP3, caspase-1 p20, and ASC protein expression in (e). (i) Protein levels of p-NF-κB p65 and IκBα analyzed by western blot in HepG2 cells after SCU (0, 20, 40, and 80 μmol/L) pretreatments for 1 h and ethanol (600 mmol/L) treatment for 24 h. (j, k) Quantification analyses of p-NF-κB p65 and IκBα protein expression in HepG2 cells in (i). (l) The nuclear translocation of NF-κB p65 detected by immunofluorescence (scale bar: 10 μm). (m) Protein levels of p-NF-κB p65 and IκBα in the liver analyzed by western blot. (n, o) Quantification analyses of liver p-NF-κB p65 and IκBα protein expression in (m). All values ( n=3) were demonstrated as mean±standard deviation (SD). ^** P<0.01, ^*** P<0.001 versus control; ^# P<0.05, ^## P<0.01, ^### P<0.001 versus model. SCU: scutellarin; NLRP3: nucleotide-binding oligomerization domain (NOD)-like receptor protein 3; NF-κB: nuclear factor-κB; IL: interleukin; TNF-α: tumor necrosis factor-α; iNOS: inducible nitric oxide synthase; p-NF-‍ κB: phosphorylated NF-‍ κB; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; ASC: apoptosis-associated speck-like protein containing a caspase recruitment domain; IκBα: inhibitor of NF-κB-α; mRNA: messenger RNA; prot: protein.

Fig. 5. Effects of SCU on AKT and MAPK pathways in HepG2 cells. (a) Protein levels of p-AKT, p-p38, p-ERK1/2, and p-JNK analyzed by western blot in HepG2 cells after SCU (0, 20, 40, and 80 μmol/L) pretreatments for 1 h and ethanol (600 mmol/L) treatment for 24 h. (b) Quantification analyses of p-AKT, p-p38, p-ERK1/2, and p-JNK protein expression in (a). All values ( n=3) are demonstrated as mean±standard deviation (SD). ^*** P<0.001 versus control; ^### P<0.001 versus model. SCU: scutellarin; AKT: protein kinase B; MAPK: mitogen-activated protein kinase; p-AKT: phosphorylated AKT; ERK1/2: extracellular signal-regulated kinase 1/2; p-ERK1/2: phosphorylated ERK1/2; JNK: c-Jun N-terminal kinase; p-JNK: phosphorylated JNK; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Fig. 6. Inhibition of p-AKT and p-p38 by SCU involved in NF-κB and Nrf2 signaling pathways. (a) Protein levels of p-NF-κB p65 and IκBα analyzed by western blot in HepG2 cells with SCU (80 μmol/L), SB 203580 (10 μmol/L), or MK-2206 2HCl (10 μmol/L) treatment for 1 h and ethanol (600 mmol/L) treatment for 24 h. (b) Quantification analyses of p-NF-‍ κB p65 and IκBα protein expression in HepG2 cells in (a). (c) Protein level of nuclear Nrf2 analyzed by western blot in HepG2 cells with SCU (80 μmol/L), SB 203580 (10 μmol/L), or MK-2206 2HCl (10 μmol/L) treatment for 1 h and ethanol (600 mmol/L) treatment for 24 h. (d) Quantification analysis of nuclear Nrf2 protein expression in HepG2 cells in (c). All values ( n=3) are demonstrated as mean±standard deviation (SD). ^*** P<0.001 versus control; ^### P<0.001, ^$$$ P<0.001 versus model; ^&&& P<0.001 versus ethanol+SCU. SCU: scutellarin; NF‍ -‍ κB: nuclear factor-‍ κB; Nrf2: nuclear factor erythroid 2-related factor 2; p-NF‍ -‍ κB: phosphorylated NF-κB; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IκBα: inhibitor of NF-κB-α; p-AKT: phosphorylated protein kinase B.

Fig. 7. Schematic diagram of the protective mechanisms of scutellarin in acute alcoholic liver damage. Nrf2: nuclear factor erythroid 2-related factor 2; CAT: catalase; SOD: superoxide dismutase; GSH-Px: glutathione peroxidase; HO-1: heme oxygenase-1; ROS: reactive oxygen species; AKT: protein kinase B; MAPK: mitogen-activated protein kinase; NF-‍ κB: nuclear factor-‍ κB; IκBα: inhibitor of NF-κB-‍ α; IL: interleukin; TNF- ‍ α: tumor necrosis factor-‍ α; iNOS: inducible nitric oxide synthase.