Catechin reduces liver inflammation by regulating Kupffer cell activation in rats

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Korean J Vet Res. 2024;64.e29

Publication date (electronic) : 2024 September 30

doi : https://doi.org/10.14405/kjvr.20240053

Meejung Ahn^,

Department of Animal Science, College of Life Science, Sangji University, Wonju 26339, Korea

^*Corresponding author: Meejung Ahn Department of Animal Science, College of Life Science, Sangji University, 83 Sangjidae-gil, Wonju 26339, Korea E-mail: meeahn20@sangji.ac.kr

Received 2024 August 5; Revised 2024 September 1; Accepted 2024 September 4.

Abstract

Introduction

The liver protects against infections due to its strategic position between the gastrointestinal and systemic circulations [1]. Kupffer cells, tissue macrophages that make up about 35% of the nonparenchymal cells in the liver, play a key role in preventing both hepatic and systemic infections [2].

Carbon tetrachloride (CCl₄) is a potent hepatotoxic agent that induces centrilobular hepatic necrosis in experimental animals after a single injection, while concurrently activating Kupffer cells and triggering subsequent fibrosis [3]. It is widely used as a model to test antioxidants and plant extracts containing antioxidants [4,5], especially for the ethnopharmacological assessment of the therapeutic potential of plant extracts [6] and for studying liver injury mechanisms that resemble human and animal liver disease morphologically and in terms of cellular damage [5].

In dogs and cats, infectious hepatitis encompasses a range of contagious diseases that primarily target the liver or involve it as part of a systemic infection. These can include bacterial, mycobacterial, viral, fungal, protozoal, parasitic, and rickettsial infections [1].

Catechins, primary phenolic compounds in green tea, wine, and cocoa-based products, have various pharmacological effects, including antioxidant, anti-diabetic, anti-inflammatory, anti-mutagenic, anti-carcinogenic, and antimicrobial activities [7,8]. Catechins play a hepatoprotective role via antioxidant mechanisms in acute CCl₄-injured mice [2] and Wister rat [6] models. However, their role in hepatic inflammation, a significant outcome of liver damage, remains poorly understood. This study investigated whether daily administration of catechins influences the progression of hepatic inflammation induced by acute oxidative stress in a CCl₄ injury model.

Materials and Methods

Chemicals and reagents

Unless otherwise noted, all chemicals, including catechin (154234), silymarin (65666-07-1), and reagents, were of analytical grade and purchased from Sigma-Aldrich (USA). Vector Laboratories (USA) provided the commercial reagent kits for immunohistochemistry. To measure catalase (CAT) activity, we purchased colorimetric assay kits from Abcam (UK). The chemical structures of the natural compound, catechin, are shown in Fig. 1.

Fig. 1.

Chemical structure of (+)-catechin.

Experimental design

The rats were divided into 5 groups (n = 5 animals per group): normal control, vehicle and CCl₄ (VCCl₄), catechin (50 mg/kg) and CCl₄ (C50), catechin (100 mg/kg) and CCl₄ (C100), and silymarin (100 mg/kg) and CCl₄ (S100). The catechin and silymarin doses were based on a previous study [9]. Catechin, dissolved in phosphate-buffered saline (pH 7.4), was administered to rats daily for 7 days. To induce liver injury, a 1:1 (v/v) mixture of CCl₄ and sterile olive oil was intraperitoneally injected at a dose of 1.5 mL/kg, in accordance with a published protocol [4]. Rats were fasted for 24 hours after final administration of catechin or vehicle, then anesthetization of the mice by isoflurane inhalation (Hana Pharm Co., Ltd., Korea). Liver tissues were collected from euthanized rats for examination (Fig. 2).

Fig. 2.

Overview of experimental design for the assessment of catechin and silymarin effects on carbon tetrachloride (CCl₄)-induced hepatic injury in rats. PBS, phosphate-buffered saline; P.O., per os; IP, intraperitoneal.

Results

Catechin reduces gross abnormalities in the liver

Fig. 3 shows representative examples of the gross findings across different treatment groups. The findings included significant enlargement, edema, and darker tissue in the vehicle group, along with spotty discolorations on the surface compared with untreated controls. In contrast, treatment with catechin and silymarin reduced these abnormalities.

Fig. 3.

Effects of catechin (Cate) and betaine on carbon tetrachloride (CCl₄)-induced hepatic injury in rats. Representative images of CCl₄-induced hepatic injury in a rat liver from each group: (A) normal control, (B) vehicle: CCl₄ injury with phosphate-buffered saline treatment, (C) C50: CCl₄ injury with 50 mg/kg catechin treatment, (D) C100: CCl₄ injury with 100 mg/kg catechin treatment, and (E) S100: CCl₄ injury with 100 mg/kg silymarin (Sily) treatment. Scale bars: (A-E) 1 cm.

Catechin improves histopathological changes in the liver

In the normal control group, liver tissue displayed a regular arrangement of hepatocytes with no cytopathogenic changes (Fig. 4). By contrast, the livers of rats in the vehicle group exhibited extensive hydropic degeneration, vacuolar degeneration, necrosis, and infiltration of inflammatory cells. Compared with the VCCl₄ group, hepatic damage was reduced in the catechin groups. Additionally, pathophysiological evaluation using the NAFLD activity scoring system indicated that pretreatment with 50 or 100 mg/kg catechin significantly inhibited inflammation compared with vehicle. Similar effects were observed in the silymarin-treated group.

Fig. 4.

Histopathological examination of the livers of normal control rats and carbon tetrachloride (CCl₄)-treated rats pretreated or not with catechin. Livers were sectioned and stained with hematoxylin–eosin (H&E). Representative images of CCl₄-induced hepatic injury in a rat liver from each group: (A) normal control, (B) VCCl₄: CCl₄ injury with phosphate-buffered saline treatment, (C) C50: CCl₄ injury with 50 mg/kg catechin treatment, (D) C100: CCl₄ injury with 100 mg/kg catechin treatment, and (E) S100: CCl₄ injury with 100 mg/kg silymarin treatment. (F) Histological score (0–4). Values represent mean ± standard error of the mean, n = 5 per group. ***p < 0.001 vs. normal control group; ^##p < 0.01 vs. CCl₄-induced liver injury with vehicle treatment. Scale bars: (A–E) 50 μm.

Catechin treatment inhibits inflammation in the liver

The activities of Kupffer cells and inflammatory cells in the liver tissue of CCl₄-intoxicated rats, key indicators of liver toxicity [4], were evaluated. Immunostaining with Iba-1 antibody revealed intriguing findings (Fig. 5). In the hepatic tissues of the VCCl₄ group, there was a significant increase in Iba-1-positive cells around the central vein compared with the normal control group; staining intensity decreased after catechin treatment. The positive area in the VCCl₄ group was significantly increased compared with the normal control group. However, the areas were significantly decreased in the C100 and S100 groups compared with the VCCl₄ group. Furthermore, the number of Iba-1-positive cells was significantly higher in the VCCl₄ group than in the normal control group. The pattern of iNOS expression was consistent with that of Iba-1 expression (Fig. 6).

Fig. 5.

Immunohistochemical staining of ionized calcium-binding adapter molecule 1 (Iba-1) -positive macrophages in CCl₄-treated rat livers. (A–E) Representative immunomicrographs of Iba-1 and (F) quantitative analyses of Iba-1-positive area and number of Iba-1-positive cells in rat liver. ***p < 0.001 vs. normal control group. ^#p < 0.05, ^##p < 0.01 vs. VCCl₄ group. Scale bars: (A–E) 50 μm.

Fig. 6.

Immunohistochemical staining of inducible nitric oxide synthase (iNOS)-positive macrophages in CCl₄-treated rat livers. (A–E) Representative immunomicrographs of iNOS and (F) quantitative analyses of iNOS-positive area and number of iNOS-positive cells in rat liver. **p < 0.01 vs. normal control group. ^##p < 0.01 vs. VCCl₄ group. (A–E) Arrows: iNOS-positive cells. Scale bars = 50 μm.

Evaluation of antioxidant enzymes in liver tissue

The activities of antioxidant enzymes, including CAT, were carefully measured in liver tissues, as illustrated in Fig. 7. In CCl₄-injured rats, CAT activities showed a significant decrease compared with normal controls. However, pretreatment with catechin before CCl₄ injury led to substantial upregulation of the levels of this enzyme compared with the vehicle group. This effect was particularly pronounced in both the C50 and C100 groups. These findings provide new insights regarding the hepatoprotective effect of catechin, which is derived from its potent antioxidant activity.

Fig. 7.

Catalase analysis of carbon tetrachloride (CCl₄)-treated rat liver with catechin treatment. Values represent mean ± standard error of the mean, n = 5 per group. ^**p < 0.01 vs. normal control group; ^#p < 0.05 vs. CCl₄-induced liver injury with vehicle treatment.

Discussion

We examined the effects of catechin on a CCl₄-induced acute liver injury model in rats to determine whether catechin has preventive effects in acute liver damage. This model is characterized by excessive accumulation of reactive oxygen species (ROS) in hepatocytes or extracellular regions, increased activation of inflammatory cells, hepatocellular necrosis, liver fibrosis, and abnormal levels of liver enzymes in the blood [5]. We found that administering catechin before CCl₄ induction in rats led to decreased hepatocellular necrosis, reduced inflammation, and increased activities of reactive oxidative enzymes such as CAT. These results suggest that catechin has a protective effect on hepatocytes in the CCl₄-induced acute liver injury model.

Peroxide and CCl₄ generate free radicals, as indicated by the increased levels of intracellular oxidation; these free radicals attack membranes, resulting in lipid peroxidation [11]. In our study, catechin increased tolerance to all oxidative conditions, demonstrating antioxidant efficacy through both direct and indirect mechanisms. The direct mechanisms include scavenging of ROS and chelation of metal ions. The indirect mechanisms involve induction of antioxidant enzymes, inhibition of pro-oxidant enzymes, and production of phase II detoxification enzymes and antioxidant enzymes [7]. In a prior study, catechins were also found to exert anti-inflammatory effects by regulating the levels of inflammation-associated molecules, including tumor necrosis factor-α, nuclear factor-kappa B, Interleukin (IL)-1β, IL-6, as well as nuclear factor-kappa B/p65, cyclooxygenase-2, and inducible nitric oxide synthase [12]. Catechins have the potential to enhance antioxidant levels, such as glutathione, CAT, and superoxide dismutase, while also reducing lipid peroxidation in brain tissue by upregulating Nrf2-responsive antioxidant protein expression [13]. Although we only compared changes in CAT levels, the antioxidant properties of catechin are well-known [7,14]. Thus, it is likely that catechin alleviated liver damage through its antioxidant mechanisms. In addition, our research demonstrated that catechin reduced the expression of inducible nitric oxide synthase, an inflammatory mediator, in CCl₄-induced hepatic injury models. These findings are largely consistent with the anti-inflammatory effects observed in previous studies [5,12].

Kupffer cell and macrophage activation accompany CCl₄-induced liver injury [4,5,15] and are detected by Iba-1 immunostaining [16]. In a previous study of acute CCl₄-induced liver injury, catechin pretreatment inhibited inflammatory responses by suppressing Kupffer cell and macrophage activation and reducing the expression of pro-inflammatory cytokines [4]. Another study demonstrated that (-)-epigallocatechin-3-gallate (EGCG), a polyphenol monomer extracted from green tea and a key catechin in green tea, exhibits neuroprotective effects. EGCG protects against experimental autoimmune thyroiditis in model rats through anti-inflammatory properties, anti-apoptotic effects, and modulation of the tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL) signaling pathway [17]. In previous studies, we demonstrated that natural compounds such as betaine and norgalanthamine derived from Lycium chinense Miller fruit and Crinum asiaticum var. japonicum, respectively, effectively inhibited inflammatory cells including Kupffer cells and activated macrophages through suppressed expression of iNOS in CCl₄-induced hepatotoxicity [5,18]. In this study, we observed that catechin reduced Kupffer cell activation and the inflammatory response, as indicated by a decrease in the Iba-1-positive area in the liver. These findings suggest that catechin also alleviates inflammation in the CCl₄-induced liver injury model.

In conclusion, catechin treatment reduces liver inflammation in rats exposed to CCl₄, showing effects comparable to those of silymarin treatment. These findings are expected to lead to potential treatment for liver inflammation in veterinary practice.

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