Green tea extract provides extensive Nrf2-independent protection against lipid accumulation and NFκB pro- inflammatory responses during nonalcoholic steatohepatitis in mice fed a high-fat diet

Abstract

Scope: Green tea extract (GTE) reduces liver steatosis and inflammation during nonalcoholic steatohepatitis (NASH). We hypothesized GTE would mitigate NASH in a nuclear factor erythroid-2-related-factor-2 (Nrf2)-dependent manner in a high fat (HF) induced model.

Methods and results: Nrf2-null and wild-type (WT) mice were fed an HF diet containing 0 or 2% GTE for eight weeks prior to assessing parameters of NASH. Compared to WT mice, Nrf2-null mice had increased serum alanine aminotransferase, hepatic triglyceride, expression of free fatty acid uptake and lipogenic genes, malondialdehyde and NFκB phosphorylation and expression of pro-inflammatory genes. In WT mice, GTE increased Nrf2 and NADPH:quinone oxidoreductase-1 mRNA, and lowered hepatic steatosis, lipid uptake and lipogenic gene expression, malondialdehyde, and NFκB-dependent inflammation. In Nrf2-null mice, GTE lowered NFκB phosphorylation and TNF-α and MCP1 mRNA to levels observed in WT mice fed GTE whereas hepatic triglyceride and lipogenic genes were lowered only to those of WT mice fed no GTE. Malondialdehyde was lowered in Nrf2-null mice fed GTE, but not to levels of WT mice, and without improving the hepatic antioxidants α-tocopherol, ascorbic acid and uric acid.

Conclusion: Nrf2 deficiency exacerbates NASH whereas anti-inflammatory and hypolipidemic activities of GTE likely occur largely independent of Nrf2 signaling.

Keywords: Green tea; Inflammation; NASH; Nrf2; Oxidative stress.

Figure 1. Time- and dose-response effects of EGCG on cell cytotoxicity and nuclear accumulation and mRNA expression of Nrf2 in HC-04 human hepatocytes

HC-04 were treated with 0-5 μM EGCG for up to 2 h as described under Methods. A) Representative Western blot of Nrf2 from isolated nuclear fractions of HC-04 cells following 2 h incubation with EGCG or 100 μM t-butylhydroperoxide (positive control). B) HC-04 cells were treated for up to 2 h with 0.1-5 μM EGCG before isolating total RNA using Trizol and reverse transcribing for RT-PCR analysis of Nrf2 mRNA expression. C) LDH leakage and D) cell viability were assessed using spectrophotometric kits as described under Methods following treatment of HC-04 for 2 h with 0-5 μM EGCG. Shown are mean responses of experiments performed in triplicate. Data were analyzed by 1- or 2-way ANOVA as appropriate. There were no statistically significant time- or concentration-dependent effects of EGCG. Abbreviations: EGCG, epigallocatechin gallate; LDH, lactate dehydrogenase; Nrf2, nuclear factor erythroid-2-related factor-2.

Figure 2. Hepatic mRNA expression of Nrf2 (A) and Nqo1 (B) in Nrf2-null and wild-type mice fed a high-fat diet containing GTE at 0 or 2% for 8 wk

RNA was isolated using Trizol and reverse transcribed for RT-PCR analysis using the primers described in Table 1. Data (means ± SEM, n = 10-12 mice per group) were analyzed by 2-way ANOVA with Newman–Keuls post-test to evaluate main and interactive effects. Groups without a common letter are significantly different (P<0.05). Abbreviations: GTE, green tea extract; Nrf2, nuclear factor erythroid-2-related factor-2; Nqo1, NADPH:quinone oxidoreductase 1; WT, wild-type.

Figure 3. Histologic evaluation of livers from Nrf2-null and wild-type mice fed a high-fat diet containing GTE at 0 or 2% for 8 wk

A) Representative hematoxylin and eosin-stained liver sections (original magnification 200x) in WT and Nrf2-null mice fed a high fat with or without GTE. Nrf2-null mice had exacerbated macrovesicular steatosis and hepatocyte ballooning relative to WT mice. GTE reduced these parameters regardless of genotype, but the effects were more pronounced in WT mice. Histologic scoring of liver steatosis (B), hepatocyte ballooning (C), and inflammatory infiltrates. Data were analyzed by 2-way ANOVA with Newman–Keuls post-test to evaluate main and interactive effects. Groups without a common letter are significantly different (P<0.05). Data are means ± SEM, n = 10-12 mice per group). Abbreviations: GTE, green tea extract; Nrf2, nuclear factor erythroid-2-related factor-2; WT, wild-type.

Figure 4. Hepatic mRNA expression of genes involved in free fatty acid uptake and lipid synthesis in Nrf2-null and wild-type mice fed a high-fat diet containing GTE at 0 or 2% for 8 wk

RNA was isolated using Trizol and reverse transcribed for RT-PCR analysis using the primers described in Table 1. Data (means ± SEM, n = 10-12 mice per group) were analyzed by 2-way ANOVA with Newman–Keuls post-test to evaluate main and interactive effects. Groups without a common letter are significantly different (P<0.05). Abbreviations: CD36, cluster of differentiation-36; DGAT, diglyceride acyltransferase; FAS, fatty acid synthase; GTE, green tea extract; Nrf2, nuclear factor erythroid-2-related factor-2; SCD1, stearoyl-CoA desaturase-1; SREBP-1c, sterol regulatory element binding protein-1c; WT, wild-type.

Figure 5. Hepatic lipid peroxidation and antioxidant markers in Nrf2-null and wild-type mice fed a high-fat diet containing GTE at 0 or 2% for 8 wk

A) Hepatic MDA was measured by HPLC-FL following incubation of liver homogenates with thiobarbituric acid and extraction with butanol. B-C) Hepatic ascorbic acid and uric acid were measured by HPLC-ECD following deproteinization of liver homogenates. D) Hepatic α-tocopherol was measured by HPLC-ECD following alcoholic saponification and extraction with hexane. Data (means ± SEM, n = 10-12 mice per group) were analyzed by 2-way ANOVA with Newman-Keuls post-test to evaluate main and interactive effects. Groups without a common letter are significantly different (P<0.05). Abbreviations: GTE, green tea extract; MDA, malondialdehyde; Nrf2, nuclear factor erythroid-2-related factor-2; WT, wild-type.

Figure 6. Hepatic protein of the phosphorylated p65 subunit of NFκB or total p65 in Nrf2-null and wild-type mice fed a high-fat diet containing GTE at 0 or 2% for 8 wk

A) Representative Western blot of phospho-p65 and p65 and the loading control β-actin. B) Quantitative densitometry analysis of target protein accumulation in total cellular extracts. Data (means ± SEM, n = 10-12 mice per group) were analyzed by 2-way ANOVA with Newman-Keuls post-test to evaluate main and interactive effects. Groups without a common letter are significantly different (P<0.05). Abbreviations: GTE, green tea extract; Nrf2, nuclear factor erythroid-2-related factor-2; phospho-p65; phosphorylated p65 subunit of NFκB; WT, wild-type.

Figure 7. Hepatic mRNA expression of pro-inflammatory genes in Nrf2-null and wild-type mice fed a high-fat diet containing GTE at 0 or 2% for 8 wk

RNA was isolated using Trizol and reverse transcribed for RT-PCR analysis for the NFκB-dependent targets TNFα, MCP-1, and iNOS using the primers described in Table 1. Data (means ± SEM, n = 10-12 mice per group) were analyzed by 2-way ANOVA with Newman-Keuls post-test to evaluate main and interactive effects. Groups without a common letter are significantly different (P<0.05). Abbreviations: GTE, green tea extract; iNOS, inducible nitric oxide synthase; MCP-1, monocyte chemoattractant protein-1; Nrf2, nuclear factor erythroid-2-related factor-2; TNFα, tumor necrosis factor-α: phospho-p65; WT, wild-type.

Figure 8. Hepatic mRNA expression of TNFR1 and TLR4 in Nrf2-null and wild-type mice fed a high-fat diet containing GTE at 0 or 2% for 8 wk

RNA was isolated using Trizol and reverse transcribed for RT-PCR analysis for TNFR1 and TLR4 using the primers described in Table 1. Data (means ± SEM, n = 10-12 mice per group) were analyzed by 2-way ANOVA with Newman-Keuls post-test to evaluate main and interactive effects. Groups without a common letter are significantly different (P<0.05). Abbreviations: GTE, green tea extract; Nrf2, nuclear factor erythroid-2-related factor-2; TLR4, toll-like receptor 4; TNFR1, tumor necrosis factor receptor-1; WT, wild-type.