Diacerein ameliorates acetaminophen hepatotoxicity in rats via inhibiting HMGB1/TLR4/NF-κB and upregulating PPAR-γ signal

Abstract

Background: Acetaminophen (APAP) is a worldwide antipyretic as well as an analgesic medication. It has been extensively utilized during the outbreak of coronavirus 2019 (COVID-19). APAP misuse would lead to liver injury. Diacerein (DIA), an anthraquinone derivative, has antioxidant and inflammatory properties. Hence, this study attempted to evaluate the impact of DIA treatment on liver injury induced by APAP and its influence on nuclear factor-κB (NF-κB) /toll-like receptor 4 (TLR4)/high mobility group box-1(HMGB-1) signaling as well as the expression of peroxisome proliferator-activated receptor-gamma (PPAR-γ) expression.

Methods: Male albino rats received 25 as well as 50 mg/kg/day DIA orally for seven days. One hour after the last administration, rats received APAP (1gm/kg, orally). For histopathological analysis, liver tissues and blood were collected, immunohistochemical (IHC) assay, biochemical assay, as well as quantitative real-time polymerase chain reaction (qRT-PCR).

Results: DIA markedly reduced liver injury markers and ameliorated histopathological changes. Moreover, DIA dose-dependently alleviated oxidative stress status caused by APAP administration along with inflammatory markers, including the level of interleukin-1 beta (IL-1β), myeloperoxidase (MPO), tumor necrosis factor-alpha (TNF-α), and interleukin 6 (IL-6). Furthermore, DIA downregulated protein levels as well as mRNA of HMGB-1, TLR4, NF-κB p65 expression, and enhanced PPAR-γ expression. Moreover, DIA ameliorated apoptotic (Bax) and caspase-3 expressions and increased the anti-apoptotic (Bcl2) expression.

Conclusions: This study demonstrated that DIA exerts anti-apoptotic, anti-inflammatory, and antioxidant properties against liver injury induced by APAP that is attributed to inhibition of the HMGB1/TLR4/NF-κB pathway, besides upregulation of the expression of PPAR-γ.

Keywords: Acetaminophen (APAP); Bax/Bcl2/caspase-3; Diacerein (DIA); HMGB1/TLR4/NF-κB signaling; Hepatotoxicity; PPAR-γ.

Fig. 1

Effect of DIA pretreatment on histopathological changes in APAP-treated rats. Representative photomicrographs of A control sections show normal hepatocytes and portal area while APAP sections B–D show hepatocytes' diffuse vacuolation along with infiltration of portal mononuclear inflammatory cells (arrow), congestion (star) (B), portal fibroplasia (arrow) (C), and necrosis (D). The DIA 25 + APAP (E and F) sections display periportal hepatocellular vacuolation (E), congestion (star), and portal mononuclear inflammatory cells infiltration (F). The DIA 50 + APAP sections (G and H) show apparently normal hepatocytes normal portal area (G) with mild vacuolation in some hepatocytes (H). Magnifications: × 200 for all slides

Fig. 2

Effect of DIA on hepatic TNF-α (A) IL-1β (B), IL-6 (C), and MPO (D) levels in APAP-treated rats. Results are expressed in the form of means ± S.E.M. (n = 6 per group), whereas data analysis was performed using one-way ANOVA and then the Tukey post hoc test. (^a), (^b), (^c): P < 0.05, as compared to the control group, APAP, DIA 25 + APAP groups respectively

Fig. 3

DIA effect on HMGB-1 (A) and TLR4 (B) gene expression, and their content of protein (C and D), in APAP-treated rats' liver tissues. Gene expression quantitation was performed utilizing real-time PCR. The mRNA levels were normalized to that of β-actin in each group and then expressed as relative quantification in comparison to controls. Gene values in the control cohort were defined as 1. (^a), (^b), (^c): P < 0.05, compared to the control group, APAP, DIA 25 + APAP groups, respectively. Data are expressed as means ± S.E.M. (n = 6 per group), and data analysis was carried out utilizing the one-way ANOVA and then the Tukey post hoc test

Fig. 4

Representative photomicrograph of immunohistochemical staining of hepatic NF-κB (p65) positive cells in the liver (arrows). Control group (A), APAP-treated group (B), DIA 25 + APAP-treated group (C), DIA 50 + APAP-treated group (D) [× 200]

Fig. 5

Representative photomicrograph of immunohistochemical of hepatic PPAR-γ positive cells in the liver (arrows). Control group (A), APAP -treated group (B), (C), DIA 25 + APAP-treated group (D), DIA 50 + APAP-treated group [× 200]

Fig. 6

Representative photomicrograph of immunohistochemical of hepatic BAX, caspase-3, and Bcl2 positive cells in the liver (arrows). Control group (A), APAP-treated group (B), DIA 25 + APAP-treated group (C), and DIA 50 + APAP-treated group (D) [× 200]