Dexamethasone Pretreatment Alleviates Isoniazid/Lipopolysaccharide Hepatotoxicity: Inhibition of Inflammatory and Oxidative Stress

Abstract

Isoniazid (INH) remains a cornerstone key constitute of the current tuberculosis management strategy, but its hepatotoxic potentiality remains a significant clinical problem. Our previous findings succeed to establish a rat model of INH hepatotoxicity employing the inflammatory stress theory in which non-injurious doses of inflammatory-mediating agent bacterial lipopolysaccharides (LPS) augmented the toxicity of INH that assist to uncover the mechanisms behind INH hepatotoxicity. Following LPS exposure, several inflammatory cells are activated and it is likely that the consequences of this activation rather than direct hepatocellular effects of LPS underlie the ability of LPS to augment toxic responses. In this study, we investigated the potential protective role of the anti-inflammatory agent dexamethasone (DEX), a potent synthetic glucocorticoid, in INH/LPS hepatotoxic rat model. DEX pre-treatment successfully eliminates the components of the inflammatory stress as shown through analysis of blood biochemistry and liver histopathology. DEX potentiated hepatic anti-oxidant mechanisms while serum and hepatic lipid profiles were reduced. However, DEX administration was not able to revoke the principal effects of cytochrome P450 2E1 (CYP2E1) in INH/LPS-induced liver damage. In conclusion, this study illustrated the DEX-preventive capabilities on INH/LPS-induced hepatotoxicity model through DEX-induced potent anti-inflammatory activity whereas the partial toxicity seen in the model could be attributed to the expression of hepatic CYP2E1. These findings potentiate the clinical applications of DEX co-administration with INH therapy in order to reduce the potential incidences of hepatotoxicity.

Keywords: CYP2E1; dexamethasone; hepatotoxicity; inflammatory stress; isoniazid; lipopolysaccharide; oxidative stress.

FIGURE 1

Effects of DEX administration on liver injury parameters induced by INH/LPS co-administration. Rats were treated with INH 200 or 400 mg/kg for 14 consecutive days, at day 14 they were given 4 mg/kg DEX 1 h earlier before they received 2 mg/kg LPS dose followed by INH 2 h later. (A) Effects on body weight. (B) Liver weight variation. (C) Impact on serum TBA and TBil levels. (D) Alterations in serum ALT, AST, and GGT levels. Data were represented as mean ± SD, n = 8 for each bar. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 versus control, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus INH/LPS combination.

FIGURE 2

DEX protects against INH/LPS-induced hepatic damage as indicated by liver histopathological examination. (A) Liver slices were collected and subjected to staining with hematoxylin and eosin. Control group showed normal hepatocyte architecture, meanwhile INH/LPS co-treated animals liver showed sever toxicity symptoms. Inflammatory cells and inflammatory infiltration (green arrow), micro- and macrovesicular steatosis (white arrow), massive necrosis (black arrow), and hepatocellular structure loss (red arrow). On the other hand, DEX pre-treatment minimized these liver injury indicators (B) INH/LPS hepatotoxicity score in absence or presence of DEX. Data were represented as mean ± SD, n = 8 for each bar. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 versus control, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus INH/LPS combination.

FIGURE 3

Lipid profile analysis. (A) Lipid staining in rat livers. Frozen liver sections were stained with red oil O staining; massive steatosis seen in rats received INH/LPS co-treatment whereas DEX addition decreased hepatic lipid accumulation. (B) Variation in both serum and hepatic lipid profile. Data were represented as mean ± SD, n = 8 for each bar. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 versus control, ^##P < 0.01, ^###P < 0.001 versus INH/LPS combination.

FIGURE 4

INH/LPS caused modifications in oxidative stress and hepatic anti-oxidant defense mechanisms while DEX pre-administration helped in elevation of liver anti-oxidant defense. Changes in SOD, T-AOC, MDA, and GSH levels were measured by their relevant kits. Data were represented as mean ± SD, n = 8 for each bar. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 versus control, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus INH/LPS combination.

FIGURE 5

Effects of INH/LPS combination on different targeted genes and proteins profile in absence or presence of DEX. (A) Bile acid regulators FXR and SHP expression. (B) Expression of bile acid synthesis enzymes CYP7A1, CYP27A1, and CYP8B1. (C) Bile acid transporters BSEP, NTCP, MRP₂, and OATP1 expression. (D) Inflammatory mediators IL-6, TNFα, IL-1α, IL-1β, and INFγ expression. (E) Expression of CYP2E1. (F) Lipid profile regulators PPARα, FAS, and HMGCS expression. Data were represented as mean ± SD, n = 8 for each bar. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 versus control, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus INH/LPS combination, GAPDH was set as reference control gene. (G) Immunoblotting analysis of different targeted proteins following INH/LPS with or without DEX treatment, β-actin considered as loading control.

FIGURE 6

Variations in hepatic CYP2E1 protein expression. (A) INH/LPS treatment elevated CYP2E1 expression, especially in rats received INH 400 mg/kg (deep brown color). Moreover, DEX administration marginally reduced CYP2E1 expression probably through elimination of LPS-enhancing effects, as indicated by the decreased color intensity. (B) CYP2E1 immunoblotting analysis, β-actin used as internal control.

FIGURE 7

Alterations of PPARα expression in rat liver. (A) Photomicrograph of PPARα in rat liver immunohistochemistry, control group showed brown staining (color indicating PPARα) while INH/LPS-treated rats their PPARα expression was significantly reduce as indicated by color fading. DEX pre-treatment ameliorate PPARα expression after INH/LPS treatment. (B) Total liver protein was probed for PPARα, β-actin considered as loading control.

FIGURE 8

INH/LPS treatment caused apoptosis in hepatic tissues, meanwhile DEX prevented hepatocellular apoptosis in INH/LPS. (A) TUNEL assay was conducted on liver slices; INH/LPS co-administration caused obvious positive TUNEL results (intensity of brown color indicating apoptotic action). In the meantime, DEX restrained INH/LPS tendency to induce apoptosis (absence of brown color). (B) Representative western blot analysis of cleaved caspase-3 (β-actin used as internal control).

FIGURE 9

Schematic presentation highlighting the proposed mechanisms by which INH/LPS-induced hepatotoxicity and the DEX mechanistic intervention for liver protection in INH/LPS liver injury model.