Cinnamic Acid: A Shield Against High-Fat-Diet-Induced Liver Injury-Exploring Nrf2's Protective Mechanisms

Abstract

This study investigated the hepatoprotective effects of cinnamic acid (CA) against liver injury and fat accumulation induced by a high-fat diet (HFD), focusing on the role of the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling. Male Wistar rats were divided into six groups: a control group receiving carboxymethylcellulose; a CA control group (40 mg/kg); an HFD group; two HFD groups treated with CA (20 mg/kg or 40 mg/kg); and a HFD group co-treated with CA (40 mg/kg) and brusatol (2 mg/kg, i.p.), a selective Nrf2 inhibitor. CA was administered orally, and brusatol intraperitoneally, both twice per week for twelve weeks. CA had no effect on serum glucose or insulin but improved serum and hepatic profiles in HFD rats. It also attenuated liver vacuolization and normalized serum levels of ALT, AST, and γ-GT. CA also reduced hepatic apoptosis by increasing Bcl2 and reducing Bax and caspase-3 levels. CA mitigated oxidative stress by reducing MDA and enhancing SOD and GSH levels. It suppressed inflammatory mediators, including TNF-α, IL-6, and NF-κB. CA also downregulated SREBP1, FAS, ACC-1, and Keap1 while increasing mRNA and nuclear translocation of Nrf2. All these effects were dose-dependent. Similar molecular effects of CA were also seen in control rats while CA protection in HFD rats was abolished with brusatol indicating Nrf2-dependency. Such findings highlight CA as a promising nutraceutical candidate for preventing HFD-induced liver injury. Further studies are warranted to explore its clinical applicability in metabolic liver diseases.

Keywords: Nrf2; cinnamic acid; hepatoprotection; high-fat diet; oxidative stress.

Figure 1

(A,B) Fasting plasma glucose during the intraperitoneal glucose tolerance test (IPGTT) and their corresponding area under the curve, respectively, in all experimental groups. (C,D): Fasting plasma glucose during the intraperitoneal insulin tolerance test (IPITT) and their corresponding area under the curve, respectively, in all experimental groups. (A): significantly different as compared to control rats; (B): significantly different as compared with control + CA (40 mg). Normality was tested using the Shapiro–Wilk test. In (A,C), data were analyzed by 2-way ANOVA with multiple measures. In (C), data were analyzed using 2-way ANOVA followed by Tukey’s t-test as post hoc. All data are presented as means + SD for n = 8 rats/groups. a: significantly different vs. control; b: significantly different vs. control + CA (20 mg/kg).

Figure 2

Levels of some oxidative stress (A–C) and inflammation (D–F) in the livers of all experimental groups. Normality was tested using the Shapiro–Wilk test. Data were analyzed by one-way followed by Tukey’s t-test as post hoc. Data are given as the means ± SD of 8 rats/group. Data are given as the means ± SD of 8 rats/group. The level of significance was shown as p < 0.05. a: significantly different vs. control, b: significantly different vs. control + CA (20 mg/kg), c: significantly different vs. HFD, d: significantly different vs. HFD + CA (20 mg/kg), and e: significantly different vs. HFD + CA (40 mg/kg).

Figure 3

The effect of all treatments on hepatic mRNA of Nrf2 (A), Keap1 (B), and NF-κB (D), as well as nuclear protein levels of Nrf2 (C) and NF-κB (E). Normality was tested using the Shapiro–Wilk test. Data were analyzed by one-way followed by Tukey’s t-test as post hoc. Data are given as the means ± SD of 8 rats/group. Data are given as the means ± SD of 8 rats/group. The level of significance was shown as p < 0.05. a: significantly different vs. control, b: significantly different vs. control + CA (20 mg/kg), c: significantly different vs. HFD, d: significantly different vs. HFD + CA (20 mg/kg), and e: significantly different vs. HFD + CA (40 mg/kg).

Figure 4

Expression levels of mRNA of some lipogenic genes (A–D) in the livers of all experimental groups. Normality was tested using the Shapiro–Wilk test. Data were analyzed by one-way followed by Tukey’s t-test as post hoc. Data are given as the means ± SD of 8 rats/group. Data are given as the means ± SD of 8 rats/group. The level of significance was shown as p < 0.05. a: significantly different vs. control, b: significantly different vs. control + CA (20 mg/kg), c: significantly different vs. HFD, d: significantly different vs. HFD + CA (20 mg/kg), and e: significantly different vs. HFD + CA (40 mg/kg).

Figure 5

Histological images of the livers from control (A), control + CA (40 mg/kg) (B), and HFD rats (C,D). (A,B) show normal histological images with well-defined and normally appearing central veins, normally sized sinusoids, and intact hepatocytes radiating from the central vein with round intact nuclei. (C,D) show several abnormalities, including the presence of cytoplasmic vacuoles in the majority of the hepatocytes that were concomitant with an increased number of fat vacuoles, pyknotic nuclei, immune cell infiltration, and hemorrhage.

Figure 6

Histological images of the livers from the HFD + CA (20 mg/kg) (A,B), HFD + CA (40 mg/kg) (C), and HFD + CA (40 mg/kg) + brusatol rats (D). (A,B) show much improvement in the livers of these rats with an increased number of normal hepatocytes with intact nuclei and reductions in the cytoplasmic vacuolization and absence of fat vacuoles. However, some damage to the central vein with immune infiltration is still visible; (C) shows an almost normal liver structure with intact central veins, hepatocytes, and nuclei, as well as normally sized sinusoids and very few cytoplasmic vacuolizations. (D) demonstrates obvious damage in the central vein and the swelling of hepatocytes with increased numbers of fat vacuoles, where vacuolization was seen in the majority of cells. Pyknotic nuclei were also dominant in a large number of cells.

Figure 7

A graphical abstract shows the therapeutic effects of cinnamic acid against high-fat-diet-induced liver injury.