Hepatoprotective activity of Lepidium sativum seeds against D-galactosamine/lipopolysaccharide induced hepatotoxicity in animal model

Mohammad Raish, Ajaz Ahmad, Khalid M Alkharfy, Syed Rizwan Ahamad, Kazi Mohsin, Fahad I Al-Jenoobi, Abdullah M Al-Mohizea, Mushtaq Ahmad Ansari, Mohammad Raish, Ajaz Ahmad, Khalid M Alkharfy, Syed Rizwan Ahamad, Kazi Mohsin, Fahad I Al-Jenoobi, Abdullah M Al-Mohizea, Mushtaq Ahmad Ansari

Abstract

Background: Fulminant hepatic failure (FHF) is clinical syndrome with very poor prognosis and high mortality there is urgent need for the development of safe and non-toxic hepatoprotective agents for the adequate management of hepatitis. Hepatoprotective effect of the Lepidium sativum ethanolic extract (LSEE) was assessed by D-galactosamine-induced/lipopolysaccharide (400 mg/kg and 30 μg/kg) liver damage model in rats.

Methods: Hepatoprotective activity of LSEE (150 and 300 mg/kg) and silymarin on D-GalN/LPS induced FHF in rat was assessed using several liver function enzyme parameters. Antioxidant properties as antioxidant stress enzymes were assessed in hepatic Liver as well as mRNA expression of cytokines genes such as TNF-α, IL-6, and IL-10 and stress related genes iNOS and HO-1 were determined by RT-PCR. Protein expression of apoptotic genes were evaluated through western blot. MPO and NF-κB DNA-binding activity was analyzed by ELISA. The magnitude of hepatic impairment was investigated through histopathological evaluation.

Results: Marked amelioration of hepatic injuries by attenuation of serum and lipid peroxidation has been observed as comparable with silymarin (25 mg/kg p.o). D-GalN/LPS induced significant decrease in oxidative stress markers protein level, and albumin. LSEE significantly down-regulated the D-GalN/LPS induced pro-inflammatory cytokines TNFα and IL-6 mRNA expression in dose dependent fashion about 0.47 and 0.26 fold and up-regulates the IL-10 by 1.9 and 2.8 fold, respectively. While encourages hepatoprotective activity by down-regulating mRNA expression of iNOS and HO-1. MPO activity and NF-κB DNA-binding effect significantly increased and was mitigated by LSEE in a dose-dependent style as paralleled with silymarin.

Conclusion: Our data suggests that pretreatment of LSEE down regulates the caspase 3 and up-regulates the BCl2 protein expression. The above findings revealed that Lepidium sativum has significant hepatoprotective activity.

Keywords: Cytokines; D-galactosamine/lipopolysaccharide; Hepatotoxicity; Lepidium sativum.

Figures

Fig. 1
Fig. 1
Effect of Lepidium sativum extract on mRNA expression of cytokines genes such tumor necrotic factor-α (TNFα), Interluekine 6 (IL-6), and Interluekine 10 (IL-10). All values represent mean ± SEM. *p < 0.05; ANOVA, followed by Dunnett’s multiple comparision test. *compared to GalN/LPS only group
Fig. 2
Fig. 2
Effect of Lepidium sativum extract on mRNA expression of cytokines genes such nitrous oxide synthase (iNOS) and Haemoxygensae1 (HO-1). All values represent mean ± SEM. *p < 0.05; ANOVA, followed by Dunnett’s multiple comparision test. * compared to GalN/LPS only group
Fig. 3
Fig. 3
Effect of LSEE on D-GalN/LPS-induced changes in inflammatory and apoptotic markers in Liver tissues of rats. a Myeloperoxidase (MPO) (b) Nuclear NF-κB (p65) DNA-binding activity determined by using NF-κB (p65) transcription factor ELISA assay kit. c Immunoblot analysis of apoptotic marker cleaved caspase-3 and antiapoptotic marker Bcl-2 protein in comparison with β-actin expression was used as a loading control. All values represent mean ± SEM. *p < 0.05; ANOVA, followed by Dunnett’s multiple comparision test. * compared to GalN/LPS only group
Fig. 4
Fig. 4
Histopathology of liver tissues. a Liver section of normal control rat shows central vein surrounded by hepatic cord of cells (normal architecture), b liver section of D-GalN/LPS treated rats showing massive fatty changes, focal central vein congestion, ballooning formation, necrosis with inflammation and loss of cellular boundaries, c liver section of rats treated D-GalN/LPS and 150 mg/kg of LSEE showing mild central vein congestion (indicated by arrow), ballooning, necrosis with sinusoidal dilatation, d liver section of rats treated D-GalN/LPS and 300 mg/kg of LSEE showing absence of ballooning, inflammatory cells and regeneration of hepatocytes around central vein toward near normal liver architecture but slight congestion in central vein (indicated by arrow), e liver section of rats treated D-GalN/LPS and 10 mg/kg of silymarin showing normal liver architecture

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