Novel molecular triggers underlie valproate-induced liver injury and its alleviation by the omega-3 fatty acid DHA: role of inflammation and apoptosis

Abstract

Background/aim: Hepatic injury is a hallmark adverse reaction to Valproate (VPA), a common used drug in the management of numerous CNS disorders, including epilepsy. DHA has a myriad of health benefits, including renal- and hepato-protective effects. Unfortunately, however, the underpinnings of such liver-pertinent VPA- and DHA-actions remain largely undefined. Accordingly, this study attempted to unveil the cellular and molecular triggers whereby VPA evokes, while DHA abates, hepatotoxicity.

Methods: We evaluated activity and/or expression of cellular markers of oxidative stress, inflammation, and apoptosis in rat liver, following treatment with VPA (500 mg/kg/day) with and without concurrent treatment with DHA (250 mg/kg/day) for two weeks.

Results and conclusion: VPA promoted hepatic oxidative stress as evidenced by enhancing activity/expression of NADPH-oxidase and its subunits, a ROS-generator, and by accumulation of lipid-peroxides. Moreover, VPA enhanced hepatic phosphorylation/activation of mitogen-activated protein kinase (MAPK), and expression of cyclooxygenase-2(COX-2), as proinflammatory signals. Besides, VPA promoted hepatocellular apoptosis, as attested by enhanced expression of cleaved caspase-9 and increased number of TUNEL-positive hepatocytes. Lastly, VPA upregulated levels of hypoxia-inducible factor-1-alpha (HIF-1α), a multifaceted modulator of hepatocytic biology, and activity of its downstream antioxidant enzyme heme-oxygenase-1(HO-1). These changes were significantly blunted by co-administration of DHA. Our findings demonstrate that VPA activated NADPH-oxidase and HIF-1α to induce oxidative-stress and hypoxia as initiators of hepatic injury. These changes were further aggravated by up-regulation of inflammatory (MAPK and COX-2) and apoptotic cascades, but could be partly lessened by HO-1 activation. Concurrent administration of DHA mitigated all VPA-induced anomalies.

Keywords: Biochemistry; Cell biology; Medicine; Physiology; Systems biology.

Fig. 1

Hepatic TBARs levels (A. NADPH oxidase activity (B), and P67 and gp91 (NOX2) expression levels relative to β-actin (C&D) in control and valproate (VPA)-treated rats (500 mg/kg PO, daily) with or without DHA (VPA. 500 mg/kg PO, daily) for two weeks. VPA increased hepatic TBARs levels, activated NADPH oxidase and up-regulated expression levels of NADPH oxidase subunits P67 and gp91 phox when compared to control. Co-administration of DHA prevented VPA-induced oxidative stress. (*P < 0.05 vs. control. #P < 0.05 vs. VPA-treated rats, n = 6–8/group for TBARs and NADPH oxidase and n = 4 for Western blotting data). Please see Figs. 1S–4S in the Supplementary Material for full blot images 2016.

Fig. 2

Hepatic HIF-1α expression relative to β-actin (A), hemeoxygenase-1 (HO-1) levels (B) in control and VPA-treated rats with or without DHA daily treatment for two weeks. HIF-1α levels and HO-1 activity were enhanced in VPA-treated versus control rats, in a DHA-sensitive manner (*P < 0.05 vs. control and #P < 0.05 vs. VPA-treated rats, n = 6–8/group for HO-1 and n = 4/group for HIF-1α Western blotting). Please see Figs. 5S–6S in the Supplementary Material for full blot images.

Fig. 3

Hepatic P-MAPK relative to MAPK (A) and hepatic COX-2 expression levels relative to β-actin (B) in livers of VPA-treated rats +/− DHA. DHA treatment diminished the VPA-induced inflammation (*P < 0.05 vs. control and #P < 0.05 vs. VPA-treated rats, n = 4/group). Please see Figs. 7S–10S in the Supplementary Material for full blot images.

Fig. 4

Representative images (200X) of liver sections for TUNEL assay for detection of apoptotic cells in the presence of Propidium Iodide (PI) as a nuclear counter stain (A), and hepatic cleaved caspase-9 relative to β-actin as a marker of apoptosis (B) in the liver of control and VPA-treated rats with or without DHA supplementation. DHA significantly reduced number of apoptotic cells and attenuated the elevation in cleaved caspase-9 expression shown with VPA-treated rats (*P < 0.05 vs. control, n = 4/group). Please see Figs. 11S–12S in the Supplementary Material for full blot images.