Elsevier

Biomedicine & Pharmacotherapy

Volume 94, October 2017, Pages 140-149
Biomedicine & Pharmacotherapy

Rac2 deficiency attenuates CCl4-induced liver injury through suppressing inflammation and oxidative stress

https://doi.org/10.1016/j.biopha.2017.07.074Get rights and content

Abstract

Oxidative stress is a leading cause to liver injury. Rac2 is a Ras-associated guanosine triphosphatase, an important molecule modulating a large number of cells and involved in the regulation of reactive oxygen species (ROS). For the study described here, we supposed that Rac2 knockout protects mice against CCl4-induced acute liver injury. We found that Rac2 expressed highly in CCl4-induced liver tissues. CCl4-treated Rac2 knockout (Rac2−/−) mice had reduced CD24 levels and steatosis. In addition, CCl4-induced high expression of pro-inflammatory cytokines and chemokine were reversed by Rac2 deficiency compared to CCl4-treated wild type (WT) mice. We also found that fibrosis-related signals of MMP-9, MMP-2 and TGF-β1 were also down-regulated in Rac2 knockout mice induced by CCl4. Significantly, oxidative stress induced by CCl4 was also suppressed owing to the lack of Rac2, evidenced by enhanced superoxide dismutase (SOD) activity, and reduced malondialdehyde (MDA) levels, superoxide radical, H2O2, xanthine oxidase (XO), xanthine dehydrogenase (XDH) and XO/XDH ratio. Moreover, c-Jun N-terminal protein kinase mitogen-activated protein kinases (JNK MAPK) was activated by CCl4, which was reversed in the liver of Rac2−/− mice through western blot and immunohistochemical analysis. In vitro, endotoxin (LPS) was treated to hepatocytes isolated from WT mice and Rac2−/− mice. The data further confirmed the role of Rac2 deficiency suppressed pro-inflammatory cytokines and chemokine, as well as fibrosis-related signals. Of note, production of ROS induced by LPS was reduced in Rac2−/− cells, accompanied with enhanced SOD1, SOD2 and reduced XO and phosphorylated-JNK expressions. Our results indicated that Rac2 played an essential role in acute liver injury induced by CCl4, providing the compelling information of the effects of Rac2 on liver injury, and revealing a novel regulatory mechanism for acute liver injury.

Introduction

The liver, mainly composed of hepatocytes accounting for 80%, is known as the primary organ essential for drug and chemical metabolism [1], [2]. Virus infection, drug abuse, and heavy alcohol consumption etc., were considered as the major contributor to development of liver injury [3]. According to previous studies, carbon-tetrachloride (CCl4) as a common inducer in establishment of animal acute liver injury has been widely confirmed. CCl4 is a well-known hepatotoxin, inducing liver injury through various mechanisms, such as inflammatory response, oxidative stress and apoptosis [4], [5]. CCl4-induced acute liver injury has been well established, resulting in fibrosis, inflammation and apoptotic response in mice [6], [7]. A variety of researches have indicated the possible molecular mechanism of liver toxicity induced by CCl4 [8]. Accumulating studies have indicated that injured hepatocytes could release critical cytokines and chemokine, which are associated with the immune response [9], [10]. Although the cytokines and chemokines have been supposed to be participated in the progression of liver injury, the molecular mechanism by which liver injury is developed is still not fully understood.

Rac2 belongs to a member of the Rho subfamily of ras-related guanosine triphosphatase. It is an important molecule modulating a number of cells functions [11]. Rac2 could shift from a resting, and inactive GDP-bound state to an active GTP-bound state responding to receptor stimulation. The GTP-bound form of Rac2 is critical for the activation of the superoxide-generating NADPH oxidase complex, which is participated in oxidative stress [12], [13]. According to previous studies, suppressing Rac2 could reduce fibrosis-related signals, including MMP-9 and MMP-2, and alleviate acute lung injury induced by LPS [14]. Thus, we supposed that Rac2 might be also involved in acute liver injury caused by CCl4.

Here, in our study, the wild type and Rac2−/− mice were used to explore the role of Rac2 in CCl4-induced acute liver injury. We found that Rac2−/− could attenuate the liver injury triggered by CCl4. And the pro-inflammatory cytokines and chemokines were also reduced in Rac2−/− mice with CCl4 induction. Of note, oxidative stress induced by CCl4 in liver tissues was alleviated due to Rac2 knockout. Next, we confirmed that Rac2 executed its regulatory function through JNK pathway.

Section snippets

Animals and treatments

40 healthy male, 6–8 weeks old C57BL/6 mice (wild type), weighed 20–22 g, were purchased from Shanghai Experimental Animal Center (Shanghai, China). 40 male 6–8 weeks old C57BL/6J male mice (Rac2−/−) weighing 20–22 g purchased from the Jackson Laboratory (Bar Harbor, ME). All animals were kept in a standard condition of temperature (25 ± 2 °C) and humidity-controlled environment (50 ± 10% humidity) with a standard light: dark (12:12 h) cycle with food and water in cages. The experimental protocols of

Rac2 expression in CCl4-induced acute injured liver

First, Rac2 levels in liver tissue of the mouse with acute liver injury induced by intraperitoneal (ip) injection of CCl4 (1 μL/g body weight) were measured using RT-qPCR and western blot analysis. As shown in Fig. 1A and B, we found that Rac2 in the injured liver tissues expressed highly from gene and protein levels were significantly increased from 24 h of CCl4 treatment, which were reach to the maximal up-regulation at 48 h, and then decreased at 72 h. The findings here indicated that Rac2

Discussion

Liver is an essential organ with highly regenerative ability and linked with various biological activities, such as production of blood clotting factors, bile acid excretion, destruction of bacteria in the blood and detoxification [1], [21], [22]. Hence, acute and chronic liver injury can be caused by a variety of factors, including drugs, xenobiotics and microbes, when they were metabolized by liver [23]. Rac2 is a ras-related guanosine triphosphatase. As previously reported, the disruption of

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