*Corresponding author: Meejung Ahn Department of Animal Science, College of Life Science, Sangji University, 83 Sangjidae-gil, Wonju 26339, Korea E-mail: 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
Animals
All experiments used 6- to 8-week-old male Sprague-Dawley rats, weighing 200 g to 300 g, sourced from Orient Bio (Korea). The rats were housed under a controlled temperature of 22°C to 26°C and a 12-hour light/dark cycle, with unrestricted access to standard diet and water. All experimental procedures conformed to the guidelines for the Care and Use of Laboratory Animals at Sangji University (2022-0016).
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.
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).
Histological evaluation
Liver tissues were promptly fixed in 10% neutral buffered formalin solution for 2 days and then processed with routine paraffin wax embedding. The tissues were sectioned into slices 5 µm thick. Following deparaffinization, the sections were stained with hematoxylin and eosin solution (Sigma-Aldrich) and prepared for immunohistochemistry. Histological evaluation was conducted using the modified non-alcoholic fatty liver disease (NAFLD) activity score histological feature scoring system for liver lesions, specifically assessing lobular inflammation on a scale from 0 to 4 [10].
Immunohistochemistry
To assess Kupffer cell and macrophage activation in hepatic tissue, we performed immunohistochemistry using rabbit anti-ionized calcium binding protein-1 (Iba-1, 1 μg/mL; cat. 019-19741, Lot. LKH4161; Wako Pure Chemical Industries, Japan) and rabbit anti-inducible nitric oxide synthase (iNOS, 1 μg/mL; cat. A3774, Lot. 4000000832; ABclonal, USA). The procedure was performed with the ABC Elite Kit (Vector Laboratories) [5]. Quantitative analysis of Iba-1-immunostained areas (centrilobular regions) (n = 3 animals per group) was performed using ImageJ software (NIH, USA). We photographed at least 5 regions of each liver section to ensure thorough data collection. We quantified the iNOS- and Iba-1-positive area in 5 different centrilobular regions of each liver section (n = 3 animals per group).
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.
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.
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).
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.
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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