M1 muscarinic receptors modify oxidative stress response to acetaminophen-induced acute liver injury

Abstract

The role of muscarinic receptor subtypes in modulating acute liver injury is unknown. We detected M1 muscarinic receptors (M1R) expression in human and murine hepatocytes, and investigated the consequences of M1R deficiency on acute liver injury in vivo and inhibiting M1R activation on hepatocyte injury in vitro. Age-matched wild-type (WT) and M1R-deficient (Chrm1−/−) male mice were injected intraperitoneally with 200 mg/kg acetaminophen (APAP) and euthanized 0, 2, 4, 16, 24 and 36 h later. Biochemical and histological parameters indicated that liver injury peaked within 16 h after APAP treatment and resolved by 24 h. Compared to WT, M1R-deficient mice had reduced intrahepatic hemorrhage and hepatocyte necrosis, reflected by an attenuated rise in serum alanine aminotransferase levels. Livers of M1R-deficient mice showed reduced hepatocyte DNA fragmentation and attenuated expression of injury cytokines (Il-1α, Il-1β, Il-6 and Fasl). In all mice hepatic glutathione levels decreased after APAP injection, but they recovered more quickly in M1R-deficient mice. During the course of APAP-induced liver injury in M1R-deficient compared to WT mice, hepatic Nrf-2, Gclc and Nqo1 expression increased and nitrotyrosine generation decreased. APAP metabolic pathways were not altered by M1R deficiency; expression of hepatic Cyp2e1, Cyp1a2, Cyp3a11, Cyp3a13, Car and Pxr were similar in Chrm1−/− and WT mice. Finally, treatment of murine AML12 hepatocytes with a novel M1R antagonist, VU0255035, attenuated H2O2-induced oxidative stress, prevented GSH depletion and enhanced viability. We conclude that M1R modify hepatocyte responses to oxidative stress and that targeting M1R has therapeutic potential for toxic liver injury.

Withaferin-A Reduces Acetaminophen-Induced Liver Injury in Mice

“…indicate that WA has short half-life (1.3 h) and clears rapidly (AUC o- t : 541 ng-h/ml [1.09 μM.h]) [16]. Therefore, to achieve maximal and prolonged effect on necrosis signaling—which follows GSH depletion within 2 h after APAP injection and leads to peak necrosis by 16 h in mice [19, 20]—we administered one log higher dose (40 mg/kg WA i.p.) than used previously [16].…”

“…Semi-quantitative scoring system was used to assess hepatocyte necrosis and intrahepatic hemorrhage (0, none; 1, <10% of the total area; 2, <30% of the total area; 3, <50% of the total area; 4, >50% of the total area) [20] by two investigators blinded to the study.…”

“…The resultant supernatant was further centrifuged at 10000 g for 10 min (4°C) to sediment mitochondrial pellet, which was washed twice with and suspended in isolation buffer, and protein content was quantified using Bradford assay (Sigma-Aldrich, St. Louis, MO). Reduced glutathione (GSH) was measured spectrophotometrically by assessing the reduction of DTNB (dithiobis-2-nitrobenzoic acid) to a yellow anion at 412 nm as described previously [20]. The GSH content was calculated as μg/mg protein using a standard GSH curve.…”

“…The GSH content was calculated as μg/mg protein using a standard GSH curve. Mitochondrial lipid peroxidation was assessed by measuring malondialdehyde (MDA) formation [20, 21]. Commercially available 1, 1, 3, 3-tetraethoxypropane (Sigma-Aldrich, St. Louis, MO) was used as a standard for calculating MDA content and expressed as MDA formed (nmoles/mg protein).…”

“…Mitochondrial fractions were prepared as described previously [20]. Equal amount of protein (40 μg) from each sample was resolved on SDS-PAGE, trans-blotted onto a PVDF membrane (Bio-Rad, USA) and subjected to immunoblot assay by using primary antibodies (1:1000) for JNK, Bax and p-JNK (Cell Signaling, Danvers, MA) followed by secondary anti-rabbit horseradish peroxidase antibody (1:5000; Cell Signaling, Danvers, MA).…”