Inhibition of oxidative stress and NLRP3 inflammasome by Saikosaponin-d alleviates acute liver injury in carbon tetrachloride-induced hepatitis in mice

Abstract

NLRP3 inflammasome activation results in severe liver inflammation and injury. Saikosaponin-d (SSd) possesses anti-inflammatory and hepatoprotective effects. This study aimed to determine the protective effects of SSd on carbon tetrachloride (CCl₄)-induced acute liver injury in mice, and whether oxidative stress and NLRP3 inflammasome activation participate in the process.The CCl₄ mice model and controls were induced. The mice were treated with SSd at 1, 1.5, or 2.0 mg/kg in a total volume of 100 µl/25 g of body weight. Liver injury was assessed by histopathology. Oxidative stress was determined using mitochondrial superoxide production (MSP), malondialdehyde (MDA) content, and superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) activities. NLRP3, ASC, and Caspase 1 were determined by real-time PCR and western blot. IL-1β and IL-18 levels were determined by ELISA.Significantly elevated oxidative stress was induced in the liver by CCl₄, as demonstrated by histopathology and increases of MDA and MSP levels and decreases of SOD, GPx, and CAT activities (all P < 0.01). SSd significantly decreased the MDA and MSP levels and increased the activities of SOD, GPx, and CAT (all P < 0.05). The mRNA expression of NLRP3, ASC, and Caspase 1, and the protein expression of Caspase 1-p10, NLRP3, ASC, IL-1β, and IL-18 were significantly increased after CCl₄ induction (all P < 0.01). These changes were reversed by SSd (all P < 0.05).Suppression of the oxidative stress and NLRP3 inflammasome activation were involved in SSd-alleviated acute liver injury in CCl₄-induced hepatitis.

Keywords: NLRP3 inflammasome; acute liver injury; carbon tetrachloride; mitochondrial reactive oxygen species; oxidative stress; saikosaponin-d.

Figure 1.

Effects of saikosaponin-d (SSd) or mito-Tempo (positive control) on CCl₄-induced histopathological damage and hepatic dysfunction. Mice were injected intraperitoneally with the indicated concentrations of SSd or mito-Tempo at 24 and 0.5 h before CCl₄ injection. Livers and serums were harvested after intraperitoneal administration of CCl₄ for 24 h. (a) Representative micrographs of liver sections stained by hematoxylin and eosin (magnification ×100). The yellow areas indicate the necrotic area (arrows). (b) Necrosis areas (%) were calculated by ImagePro Plus. Serum levels of alanine transaminase (ALT) (c), aspartate transaminase (AST) (d), and lactate dehydrogenase (LDH) (e) were measured by routine laboratory methods. Data are expressed as means ± standard error of mean (SEM) (n = 7/group). **P < 0.01 versus the control group. #P < 0.05, ##P < 0.01 versus the CCl₄ group.

Figure 2.

Effect of saikosaponin-d (SSd) or mito-Tempo (positive control) on CCl₄-induced oxidative stress in the liver. Mice were injected intraperitoneally with the indicated concentrations of SSd or mito-Tempo at 24 and 0.5 h before CCl₄ injection. Livers and serums were harvested after intraperitoneal administration of CCl₄ for 24 h. Liver malondialdehyde (MDA) level (a), superoxide dismutase (SOD) activity (b), glutathione peroxidase (GPx) activity (c), and catalase (CAT) activity (d). Mitochondria were isolated from the liver and used for the determination of mitochondrial superoxide production (MSP) levels (e). Data are expressed as means ± SEM (n = 7/group). **P < 0.01 versus the control group. #P < 0.05, ##P < 0.01 versus the CCl₄ group.

Figure 3.

Effects of saikosaponin-d (SSd) or mito-Tempo (positive control) on CCl₄-induced NLRP3 inflammasome activation in the liver. Mice were injected intraperitoneally with the indicated concentrations of SSd or mito-Tempo at 24 and 0.5 h before CCl₄ injection. Livers and serums were harvested after intraperitoneal administration of CCl₄ for 24 h. The mRNA expressions of NLRP3 (a), ASC (b), and Caspase 1 (c) were determined by real-time RT-PCR. (d) The protein expressions of NLRP3, ASC, Caspase 1 p10, and pro-Caspase 1 were determined by western bolt. Data are expressed as means ± SEM (n = 7/group). The content of IL1β (e) and IL-18 (f) were examined by ELISA. Data are expressed as means ± SEM (n = 6/group). **P < 0.01 versus the control group. #P < 0.05, ##P < 0.01 versus the CCl₄ group.

Figure 4.

Measurement of mitochondrial superoxide production using fluorescence microscopy after treatment with saikosaponin-d (SSd) or mito-Tempo (positive control) on CCl₄-induced liver damage in mice. Mice were injected intraperitoneally with the indicated concentrations of SSd or mito-Tempo at 24 and 0.5 h before CCl₄ injection. Livers and serums were harvested after intraperitoneal administration of CCl₄ for 24 h. Fluorescence microscopy of hepatocytes stained with 5 µM MitoROS and DAPI. MitoROS specifically stain the production of mitochondrial reactive oxygen species.