Indian Journal of Animal Research

  • Chief EditorK.M.L. Pathak

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Indian Journal of Animal Research, volume 57 issue 6 (june 2023) : 777-782

Protective Effect of Naringenin on Cadmium-induced Toxicity in Rat Liver

Ke Wang1, Lulu Ding1, Congying Kou1, Ruxue Huang1, Pengli Zhao1, Zijun Yang1, Jicang Wang1,*
1College of Animal Science and Technology, Henan University of Science and Technology, Luoyang, China.
Cite article:- Wang Ke, Ding Lulu, Kou Congying, Huang Ruxue, Zhao Pengli, Yang Zijun, Wang Jicang (2023). Protective Effect of Naringenin on Cadmium-induced Toxicity in Rat Liver . Indian Journal of Animal Research. 57(6): 777-782. doi: 10.18805/IJAR.BF-1425.
Background: Cadmium (Cd) is a widespread environmental toxic heavy metal. Naringin (Nar) is reported to have a protective effect on Cd-induced liver injury. This study aimed to explore the hepatic injury induced by Cd and the protective effects of Nar on Cd-induced oxidative stress and apoptosis in rat liver.

Methods: In accordance with groups, male SD rats were injected intraperitoneally with Cd and orally with Nar every day. The treatment period was 3 weeks. Body weight, the morphological changes in liver, the activity of antioxidant indices and expression of the apoptotic genes caspase-3 and -9 were assessed.

Result: Results showed that the body weight of Cd-exposed rats decreased, the superoxide dismutase and catalase activities in liver decreased, the glutathione content decreased and the malondialdehyde content increased. Further, Cd-induced hepatic structural damage and cell apoptosis were observed. However, Nar could alleviate liver damage caused by Cd. Therefore, Cd caused oxidative damage and cell apoptosis in rat liver, while Nar had preventive and ameliorative effects on these injuries.
Cadmium (Cd) is a common environmental toxicant that has harmful effects on plants, animals and humans (Nisha et al., 2009). In 1993, the International Agency for Research on Cancer classified Cd as human carcinogen I and the National Toxicology Program identified Cd as human carcinogen (Xiong et al., 2020). Cd is one of the most toxic heavy metals and a common industrial pollutant present in the environment (Adefegha et al., 2015). At present, the main sources of Cd are smoking tobacco and drinking contaminated water pollution (Soisungwan et al., 2013). Other sources include fertilizers, glass and batteries (Claudio et al., 2011). The biological half-life of Cd in the human body is up to 10–30 years (Kazuhiro et al., 2016) and the absorbed Cd mainly accumulates in the liver and kidney of the organism. The toxicity of Cd is mainly attributed to its damage to the redox system of the body (Stohs et al., 2000). Cd exposure leads to the change in mitochondrial membrane permeability, intensifies the occurrence of various apoptotic pathways and leads to apoptosis (Rahman et al., 2017). Naringenin (Nar) is a dihydroflavonol active ingredient found in Ruta plants. It is. Fruits, vegetables, tea and red wine are rich in Nar (Manchope et al., 2017). In addition, Nar has various biological activities, such as antioxidant, anti-inflammatory and liver protection (Zhu et al., 2015). Therefore, some studies have found that Nar is an important ingredient for effective treatment of Cd poisoning (Manchope et al., 2017). However, no relevant report is available about the regulation of Nar on the caspase-dependent apoptosis pathway.
In this study, an acute Cd poisoning rat model was established via intraperitoneal injection of CdCl2 solution. The liver antioxidant index, liver pathological section and mRNA expression of caspase-3 and -9 were detected using real-time quantitative PCR (qRT-PCR) to evaluate the effect of Cd on the structure and function of rat liver and the preventive effect of Nar on liver toxicity caused by acute Cd-exposure. The results showed that subsequent use of plant polyphenol extract Nar could prevent and ameliorate Cd poisoning.
Anhydrous CdCl2 was purchased from Aladdin Industrial Corporation. Nar (purity: > 98%) was obtained from Beijing Bailingwei Technology Co., Ltd (Beijing, China). Superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and malondialdehyde (MDA) kits were from Nanjing Jiancheng Bioengineering Institute, (Nanjing, China). TRIzol isolation kit and PCR primers were obtained from Sangon Biotech Co., Ltd (Shanghai, China). SYBR Premix Taq II kit was purchased from Takara (Beijing, China). All other routine chemicals and solvents were of pure analytical grade.

Animal treatment
The experiment was conducted from August to December 2020 at the Environmental Animal Product Safety Laboratory of Henan University of Science and Technology, Luoyang, China. Adult male Sprague Dawley (SD) rats weighing 160-180 g were housed in polypropylene cages maintained at room temperature (24°C±2°C) on a 12-h light/12-h dark cycle throughout the experiment. They were fed with a commercial standard diet, with free access to drinking water under standard laboratory conditions. The grouping and processing methods is shown in Table 1,2. All animal experimental processes were approved by the Institute of Zoology and Medical Ethics Committee of Henan University of Science and Technology and they were strictly designed under the consideration of animal welfares (approval number HAUST 19013).

Table 1: Grouping and processing methods (n=6).

Table 2: Effects of Nar on Cd-induced changes in body weight (g) (n=6).

Every weekend, the rat’s body weight was recorded. The experiment lasted for 3 weeks. The rats were sacrificed by cervical decapitation 24 h after the last treatment. Liver tissues were removed from the sacrificed rats and washed in PBS. The tissues were fixed with 10% formalin for histopathological studies, frozen for qRT-PCR and crushed, homogenized (10%, w/v) in PBS solution (pH 7.4) and centrifuged (3000 rpm for 10 min) for various biochemical estimations.
Histopathological studies
Tissue samples of the liver was fixed in 10% formalin at 4°C for 24 h. Then, different concentrations of ethanol were used as a dehydrating agent for step-by-step dehydration. The dehydrated liver tissues were embedded in paraffin. The paraffin blocks were sliced into 4-6 μm thick with a microtome. The obtained tissue sections were stained with hematoxylin–eosin and observed using an optical microscope.
Antioxidant analysis
SOD, MDA, CAT and GSH activities were measured using the diagnostic kits in accordance with the manufacturer’s protocol. These results were measured spectrophotometrically.
mRNA expression
Total RNA was extracted from the rat liver tissue by using a TRIzol reagent and the total RNA was reverse transcribed to synthesize cDNA. The primers were designed using Primer Premier 6 (Table 3). The instructions in the SYBR Premix Taq Gamma II Kit for qRT-PCR were followed. β-actin was used as endogenous control and the relative mRNA levels were analyzed using 2-DDCT method.

Table 3: Primer sequences of real-time PCR target genes.

Statistical analysis
The data obtained under different experimental conditions were analyzed by one-way variance method in SPSS version 17. When P<0.05 or P<0.01, the difference was considered significant or highly significant, respectively.
Effect of Nar on Cd-induced changes in body weight
Compared with the control group, the Cd groups decreased showed body weight. The body weight of the 1 mg/kg Cd group decreased significantly (P<0.05), while that of the 2 mg/kg Cd group decreased highly significantly (P<0.01), as shown in Table 2. However, after treatment with Nar (100 mg/kg) and Cd exposure for 2 and 3 weeks, the body weight significantly increased compared with that of the 2 mg/kg Cd group (P<0.05).

Histological assessment of liver tissues
The results showed that the structure of liver tissues in the control group (Fig 1A) was complete and normal, the shape was clear and the arrangement of hepatocytes was orderly. The Cd groups (Fig 1B-D) showed Cd-induced pathological changes in the liver tissue and irregular arrangement of hepatic cords, atrophic and necrotic hepatocytes, indistinct in space and extensively denatured and necrotic and some cell nuclei showed pyknosis. In the Nar group (Fig 1E), the morphology of liver tissue cells was normal and without any pathological phenomenon.

In the Nar + Cd group, a small amount of tissue showed slight hyperplasia or degeneration and the degree of damage was relieved compared with the Cd group (Fig 1F).

Fig 1: Pathological changes in liver tissue were observed by staining (40×). A (control), B (Cd, 0.5 mg/kg b.w.), C (Cd, 1 mg/kg b.w.), D (Cd, 2 mg/kg b.w.), E (Nar, 100 mg/kg b.w.) and F (Nar, 100 mg/kg + Cd, 2 mg/kg b.w.).

Effect of Nar on liver oxidative stress
As shown in Fig 2, a significant (P<0.05) decrease in the activities of enzymatic antioxidants (SOD and CAT) and the GSH content were observed in the Cd-treated rats. However, the MDA content was significantly higher (P<0.05) and the 2 mg/kg Cd group demonstrated a highly significant difference compared with the control group (P<0.01). Compared with the 2 mg/kg Cd group, the Nar + Cd group showed that the activities of SOD and CAT increased highly significantly (P<0.01) and the GSH content significantly increased (P<0.05). However, the MDA content of the Nar + Cd group decreased highly significantly (P<.01). No significant difference was found on these indicators between the Nar group and the control group (P <0.05).

Fig 2: Effect of Nar on liver oxidative stress. (A) SOD, (B) MDA, (C) GSH and (D) CAT. Date are presented as mean ± SEM deviation. *P<0.05 or **P<0.01 represents a significant difference or highly significant difference compared with the control group. #P<0.05 or ##P<0.01 represents a significant difference or highly significant difference compared with the 2 mg/kg Cd group. n=6 in each group.

Effect of Nar on caspase-9 and -3 mRNA expression
The qRT-PCR results showed that Cd exposure induced an increase in the mRNA expression of caspase-9 and -3 and the mRNA expression increased with the increase in Cd concentration (Fig 3). However, the level of caspase-9 and -3 mRNA expression was significantly lower (P<0.05) in the Nar + Cd group than in the 2mg/kg Cd group.

Fig 3: Expression of mRNA of caspase-9 (A) and caspase-3 (B) in the mitochondrial apoptosis pathway. Date are presented as mean ± SEM deviation. *P<0.05 or **P<0.01 represents a significant difference or highly significant difference compared with the control group. #P<0.05 or ##P<0.01 represents a significant difference or highly significant difference compared with the 2 mg/kg Cd group. n=6 in each group.

Cd is one of the most widely used heavy metals in the contemporary world and its effect on the environment and humans are well known. Under Cd exposure, various organs of the body are affected to varying degrees, with liver being the most affected (Dkhil et al., 2014).

Body weight is regarded as an important indicator of animal health and weight gain depends on the supply and absorption of nutrients (Bhattacharya and Haldar, 2012). Cd exposure could lead to intestinal mucosal cell damage, thereby reducing the absorption and retention of nutrients. In addition, heavy-metal exposure may damage the glucocorticoid system and glucocorticoid system disorders could lead to weight changes (Akomolafe et al., 2016). In the present study, the fur of SD rats in the Cd-induced group was messy and dull. As shown in Table 2, the body weight of Cd-induced rats was significantly reduced compared with that of rats in the control group. These results are consistent with those of a previous study (Wang et al., 2019). The weight loss of rats may be due to the damage in the intestinal mucosa and the disorder of glucocorticoid system caused by Cd.

Histopathological changes could directly reflect liver injury. Cd exposure caused significant liver pathological changes and a comparable finding was made by (Gong et al., 2019). This finding may be due to the accumulation of free radicals caused by Cd exposure, which eventually leads to organ damage (Skipper et al., 2016). Oxidative damage is the most representative of Cd-induced liver damage; the Cd in the body induces tissues to produce reactive oxygen species (ROS), which reduces the organ’s ability to respond to oxidative stress (Branca et al., 2018). In the past few years, Nar is one of the most popular antioxidants and it has shown that antioxidants could resist the toxicity of heavy metals (Zhang et al., 2012). In the present study, the levels of SOD, CAT, GSH and MDA were determined to illustrate the toxicity of Cd to the liver and the effect of Nar.

GSH resists oxidation by combining sulfhydryl groups with ROS and inhibiting the formation of ROS is the first line of defense for non-enzymatic antioxidants (Loro et al., 2012). SOD has the function of scavenging oxygen free radicals produced in the body. CAT is an important enzyme in the body’s redox system and it could protect cells from the oxidative damage from H2O2 and OH- (Shahat  et al., 2017). In the present study, the activities of CAT and SOD and the GSH content in the Cd-induced group were lower than those in the control group, indicating that Cd remarkably weakened the antioxidant capacity of the rat liver and the activities of CAT and SOD and the GSH content decreased in a dose-dependent manner in liver treated with Cd. Then, the rats were administrated with Nar and Cd. As shown in Fig 2, the activities of SOD and CAT between the Nar + Cd group and the 2 mg/kg Cd group were highly significantly different (P<0.01). The GSH content was also significantly different (P<0.05). MDA is considered one of the biomarkers of oxidative stress (Zhang et al., 2014). Fig 2 shows that compared with the control group, the Cd-exposed group had significantly increased MDA content and with the increase in Cd concentration, the difference became more significant. After Nar was administered, the MDA content in the Nar + Cd group decreased highly significantly compared with that in the 2 mg/kg Cd group (P<0.01). The above results indicated that Cd could cause oxidative damage to the liver and as an antioxidant, Nar could protect the liver and reduce Cd damage.

Several studies have reported that Cd induced mitochondrial caspase-dependent apoptotic gene expression, leading to cell apoptosis (Banik et al., 2019). However, whether Nar could inhibit Cd-induced liver cell apoptosis was unclear. Therefore, the mRNA expression levels of related apoptotic genes were analyzed in the present study. Cd-induced mitochondrial superoxides preferentially accumulate, leading to mitochondrial dysfunction and rupture of the outer membrane; cytochrome c (cyt-c) is released by the mitochondria and caspase-9 is activated and then cascades to activate caspase-3, causing caspase-dependent apoptosis (Kim et al., 2015). Caspase-9 is an essential executor of the mitochondrial pathway to induce apoptosis and caspase-3 is the key final executive molecule of various apoptotic methods and an important marker of apoptosis (Wu et al., 2020). In the present study, the mRNA expression levels of caspase-9 and -3 in the Cd groups markedly increased compared with those in the control group, while those in the Nar + Cd group were significantly lower than those in the 2 mg/kg Cd group. Therefore, Cd could induce apoptosis in rat liver cells and increase the mRNA expression of caspase-9 and -3, while Nar suppresses Cd-induced apoptosis. In the mitochondrial apoptosis pathway, oxidative stress plays a key role in Cd-induced apoptosis (Wang et al., 2009). Therefore, as an antioxidant, Nar inhibits Cd-induced apoptosis by reducing oxidative stress.
This study was supported by grants from the National Nature Science Foundation of China (No.31972753).

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