TCA Cycle Intermediate Mitigates Di(2-ethylhexyl) Phthalate-Induced Cholestatic Liver Injury Through Modulation of the Nrf2/NQO1 Signalling Axis

Abstract

As a commonly used phthalate compound, di(2-ethylhexyl) phthalate (DEHP) has been shown to disrupt the tricarboxylic acid (TCA) cycle and aggravate tissue damage. However, whether the TCA cycle is involved in cholestatic liver injury (CLI) induced by DEHP and the protective effect of dimethyl fumarate (DMF), which is used to supplement TCA intermediate metabolites, remained unclear. Here, mice were randomized into five groups (n = 6/group): (1) Control, (2) DEHP (200 mg/kg/day), (3) DMF (100 mg/kg/day), (4) DEHP + DMF (30 mg/kg/day) and (5) DEHP + DMF (100 mg/kg/day). Our data demonstrated that DEHP exposure upregulated total bile acid (TBA) levels and broke the TCA cycle, resulting in reduced fumaric acid and malic acid. However, we further supplemented fumaric acid with DMF and found that DMF effectively reversed the high levels of TBA, alkaline phosphatase (ALP) and glutamyl transpeptidase (GGT) induced by DEHP in mice. Meanwhile, pathological results in the liver showed that DMF improved bile duct cell damage, inflammatory cell infiltration, collagen deposition and necrosis caused by DEHP. In addition, we found that DEHP elevated the level of interleukin (IL)-1β, IL-6, TNF-α and MDA and decreased the level of SOD in the mouse liver, which was effectively reversed by DMF treatment. Besides, DMF upregulated the expression of Nrf2 and NQO1 in the liver of DEHP-exposed mice. For in vitro validation, AML-12 cells were treated with (1) Control, (2) DEHP (250 μM), (3) DEHP + DMF (10 μM), (4) DEHP + DMF (25 μM) and (5) DEHP + DMF (50 μM). DEHP exposure increased the expression of IL-1β, IL-6 and TNF-α, which was mitigated by DMF, while ML385, an Nrf2 inhibitor, could counteract the anti-inflammatory effects of DMF. These findings indicate that DEHP broke the TCA cycle of the mouse liver, and DMF supplementation protects against DEHP-induced CLI by activating the Nrf2/NQO1 pathway.