Introduction

Cadmium (Cd), a toxic heavy metal found in 1817 by F. Stromeyer, is one of the most common environmental pollutants. It is associated with various human activities, such as the discharge of industrial wastewater and waste gas and the combustion of fossil fuels, and thus, Cd can be found in the air [1], soil [2], and water [3]. The poultry industry has been commonly found to be affected by Cd exposure via feed and water [4, 5]. It is also well known that Cd targets multiple organs and impacts the antioxidant enzyme system, inflammatory reaction, and the expression of heat shock proteins (HSPs) in a similar way to other heavy metals, such as arsenic [6–8], molybdenum [9, 10], and lead [11, 12]. Cd mainly accumulates in the liver and kidney and presents as a complex bound to the metal-binding protein metallothionein (MT). Cd-MT complex is a temporary detoxifying mechanism of Cd demonstrated by humans and other mammals [13]. Alghasham et al. demonstrated that Cd toxicity induced the release of TNF-α and IL-6 in rats, which is associated with systemic oxidative stress and may be involved in the Cd toxicity mechanism [14]. Some researchers have demonstrated that Cd had a negative effect on primary cultures of rat proximal tubular cells by altering antioxidant defense enzyme systems, lipid peroxidation, and apoptosis [15]. However, Cd is incapable of activating the redox reactions directly in biological systems but probably increases the concentration of free redox-active metals, such as Fe and Cu, by replacing them with various metalloproteins or by changing their mitochondrial membrane potential [16]. Cd-treated A549 cells induce reactive oxygen species (ROS) and cause membrane damage with the leakage of lactate dehydrogenase (LDH). Oxidative stress and apoptosis were induced by Cd due to depletion of antioxidant enzymes [17]. Sant’Ana revealed that exposure to Cd for 28 days induced hepatic toxicity in quail by measuring body weight and biochemical parameters [18]. Cao et al. illustrated that the high messenger RNA (mRNA) expression of HSPs and inflammatory cytokines may play a role in the resistance of liver toxicity in ducks induced by Mo and Cd [19]. Sfaxi-Bousbih et al. demonstrated that Cd adversely affected the germination rate and embryo growth of bean seeds [20]. These results illustrated that Cd may damage various organs in various creatures.

A. blazei Murill (ABM) is a Brazilian mushroom, which has received increasing attention in recent years because of its various biological activities, such as antioxidant, anti-inflammatory, and anticancer activities. Hsu proved the potential benefits of ABM extract in normalizing the liver functions of patients with hepatitis B [21]. Al-Dbass suggested that ABM extract can protect the liver against carbon tetrachloride (CCl4)-induced oxidative damage in rats and is an efficient hepatoprotective and antioxidant agent against CCl4-induced liver injury [22]. Zhao demonstrated that purified polysaccharides from Schisandra with a low molecular weight could attenuate the 5-fluorouracil-induced toxicity effect in mice [23]. Cao et al. suggested that Astragalus polysaccharide could ameliorate doxorubicin-mediated cardiotoxicity by regulating the PI3k/Akt and p38 MAPK pathways [24]. Bei illustrated that some polysaccharides and polysaccharide–protein complexes isolated from ABM were bioactive principles responsible for treating and preventing cancer [25]. Wang et al. observed that water-soluble polysaccharide (WSP)-ABM supplementation had anti-inflammatory effects in a model of osteoporosis by remarkably decreasing the serum IL-1β and TNF-α levels and the expressions of ICAM-1, COX-2, iNOS, and NF-κB [26]. Based on these studies, we speculate that ABP may have protective effects against Cd-induced liver damage in chickens.

In the present study, we designed the experiment to evaluate the Cd content, antioxidant system activities, histopathological changes, and expression of inflammatory cytokines and HSPs in chicken livers after the exposure to Cd and the concurrent administration of Cd and ABP by oral gavage. The aim of this study was to investigate the protective roles of ABP against Cd-induced damage in chicken livers.

Materials and Methods

Animals and Experimental Design

All procedures used in the current study were approved by the Institutional Animal Care and Use Committee of Northeast Agricultural University.

The ABP in the current study was isolated from ABM using the water extraction and alcohol precipitation method [27], deproteinized by the Sevag method [28], and dried at 50 °C. Then, the content of ABP in the ABM extract was detected by the phenol-sulfuric acid method using glucose as the standard [29]. The result showed that the content of ABP was 87.5%. The ABP solid was dissolved in saline into a concentration of 30 mg/ml. A total of 80 Hy-line laying chickens (7 days old) were randomly divided into four groups (n = 20). Group I (control) was fed with a basic diet of 0.2 ml saline per day. Group II (Cd-treated group) was fed with a basic diet containing 140 mg/kg CdCl2 and 0.2 ml saline per day. Group III (Cd + ABP-treated group) was fed with a basic diet containing 140 mg/kg CdCl2 and 0.2-ml ABP solution (30 mg/ml) per day via oral gavage. Group IV (ABP-treated group) was fed with 0.2-ml ABP solution (30 mg/ml) per day via oral gavage. The feeding experiment lasted for 60 days, and the experimental animals had free access to food and water. On days 20, 40, and 60, livers of five chickens in each group were collected after euthanasia. The prepared tissues were divided into three portions: the first was for mRNA detection, the second was fixed in 4% paraformaldehyde solution for histological examination, and the third was for protein detection.

The Content of Cd in the Liver

The sample of liver (0.5 g) was pretreated in a Graphite digestion system (Polytech ST60, Polytech Instrument Ltd., Beijing, China). The Cd content in the sample was detected by inductively coupled plasma mass spectrometry (ICP-MS) (Agilent 7800, Agilent Technologies, Beijing, China). The operating conditions are shown in Table 1.

Table 1 ICP-MS operating conditions
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Measurement of Antioxidant Status

Liver samples were homogenized on ice in physiological saline and centrifuged at 3000 rpm to collect supernatants for the biochemical assays. Protein concentrations were measured by total protein quantitative assay kit (A045-2, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Glutathione peroxidase (GSH-PX) and superoxide dismutase (SOD) activities were determined by a GSH-PX assay kit and total superoxide dismutase (T-SOD) assay kit, respectively (A005 and A001-1, Nanjing Jiancheng Bioengineering Institute). The malondialdehyde (MDA) concentration was detected by an MDA assay kit (A003-1, Nanjing Jiancheng Bioengineering Institute).

Inflammatory Cytokines and HSP mRNA Expression Analysis

The total RNA was isolated from the liver tissue samples (0.1 g) with TRIpure reagent according to the manufacturer’s instructions (RP1202, BioTeke, Beijing, China). Then, the total RNA concentration was determined using a NanoDrop-2000 Spectrophotometer (Gene Company Limited, Hong Kong, China) at 260/280 nm. The first-strand complementary DNA (cDNA) was synthesized from 10 μl of total RNA using 2 μl oligo (dT) primers, 4 μl 5× M-MLV buffer, 1 μl dNTP (10 m/μl), 0.5 μl RRI, and 0.5 μl M-MLV reverse transcriptase. The synthesized cDNA was stored at −80 °C ready to use. Real-time quantitative reverse transcription polymerase chain reaction (PCR) was used to detect gene expressions of inflammatory cytokines (TNF-α, IL-1β, and IL-6) and HSPs (HSP27, HSP40, HSP60, HSP70, and HSP90) in chicken livers with the Light Cycler® 480 System (Roche, Basel, Switzerland). Reaction mixtures consisted of the following: 10 μl 2× SYBR premix (PR7011, BioTeke), 0.4 μl each primer (10 μM), 1 μl cDNA, and 8.2 μl tri-distilled water. The PCR program for the eight genes and β-actin was 1 cycle at 95 °C for 30 s, 40 cycles at 95 °C for 15 s, annealing at 60 °C for 30 s, and extension at 72 °C for 15 s. The primers were designed with the Oligo 7 software and are listed in Table 2. General PCRs were first performed to confirm the specificity of the primers. The relative mRNA levels were calculated according to the 2−ΔΔCt method, accounting for gene-specific efficiencies and were normalized to the mean expression of the housekeeping gene β-actin.

Table 2 Gene-specific primers used for qPCR
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Western Blot Analysis of HSPs

Protein extracts were subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. The separated proteins were then transferred to nitrocellulose membranes for 1 h at 15 V in a transfer apparatus (Bio-Rad, Philadelphia USA). Membranes were blocked with 5% skim milk for 2 h at room temperature and incubated overnight with diluted primary antibodies against HSP60, HSP70, and HSP90 separately (1:1000, polyclonal antibody produced by the College of Veterinary Medicine, Northeast Agricultural University), and then, a horseradish peroxidase (HRP)-conjugated secondary antibody against rabbit IgG was added (1:5000, ZSGB-BIO, Beijing, China). To verify equal loading of the samples, the membrane was incubated with a monoclonal β-actin antibody (1:1000, Beyotime Institute of Biotechnology, Shanghai, China), followed by an HRP-conjugated goat anti-mouse IgG (1:5000, ZSGB-BIO) secondary antibody. The signal was detected with a ChemiScope 3100 Mini (Clinx Science Instruments Co., Ltd., Shanghai, China). The integrated density (IntDen) of each band was determined by the ImageJ software, and the HSP60, HSP70, and HSP90 expression levels were expressed as the ratio of the IntDen of HSP60, HSP70, or HSP90 to the IntDen of β-actin.

Histopathological Analysis

The histopathology of the tissue was performed according to the study of Denli [30]. Specimens were fixed in 4% paraformaldehyde solution, dehydrated through a graded series of ethanol from 70 to 100% and acetone, cleared in xylene, and embedded in paraffin. Five-millimeter-thick sections were removed from the paraffin blocks, followed by staining with hematoxylin and eosin (H&E) for light microscopy.

Statistical Analysis

All statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) 17.0 software package, and all data were assessed using the one-way ANOVA. The differences of one group from the others were considered to be significant when P < 0.05.

Results

The Cd Content in Chicken Livers

The Cd content in livers is shown in Fig. 1. The Cd content in group Cd was significantly higher compared with those of groups control and ABP (P < 0.05), whereas group Cd + ABP was significantly lower when compared with group Cd (P < 0.05). The Cd content in group ABP showed no significant difference from group control, but it was a little lower at the same time point (P > 0.05).

Fig. 1
figure 1

Effects of ABP on the Cd content in chicken livers. The values in group control were used as the reference values. Different letters indicate significant differences (P < 0.05) between two groups. Each value is represented in the format of the mean ± SD

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The Oxidant Status

As shown in Fig. 2a, b, the GSH-PX and SOD activities in the chicken livers of group Cd were decreased compared with those in the other three groups, and there were significant differences at 40 and 60 days (P < 0.05). It was obvious that the GSH-PX and SOD activities in the livers of group Cd + ABP had no significant difference but were a little lower compared with group control at 20 and 40 days (P > 0.05) and showed a significant difference at 60 days (P < 0.05). The GSH-PX and SOD activities in group control were lower than those in group ABP, but there were no significant differences (P > 0.05).

Fig. 2
figure 2

Effects of ABP on Cd-induced changes in the antioxidant system in chicken livers. a Effects of ABP administration on the GSH-PX activity. b Effects of ABP administration on the SOD activity. c Effects of ABP administration on the content of MDA. The values in group control were used as the reference values. Different letters indicate significant differences (P < 0.05) between any two groups. Each value is represented in the format of the mean ± SD

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The MDA content in group Cd was increased compared with the other three groups at the same time point, and there were no significant differences at 40 and 60 days in Fig. 2c (P > 0.05). The MDA content in group Cd + ABP was higher than that in group control, but there were no significant differences other than at 60 days (P > 0.05). In addition, the level of MDA in group control showed no significant difference, but it was higher than that in group ABP (P > 0.05).

The mRNA Expression of Inflammatory Cytokines

As shown in Fig. 3, the mRNA expression of inflammatory cytokines, including TNF-α (Fig. 3a), IL-1β (Fig. 3b), and IL-6 (Fig. 3c), in group Cd was significantly higher compared with those in the other three groups at the same time point (P < 0.05). On the other hand, the mRNA levels of inflammatory cytokines (TNF-α, IL-1β, and IL-6) in group Cd + ABP were significantly lower compared with group Cd (P < 0.05), but still higher than those in group control. In addition, the chickens in group ABP showed no significant difference compared with group control in the mRNA levels of inflammatory cytokines (TNF-α, IL-1β, and IL-6) in livers (P > 0.05).

Fig. 3
figure 3

Effects of ABP on Cd-induced changes in the mRNA levels of inflammatory cytokines in chicken livers. a Effects of ABP administration on the mRNA level of TNF-α. b Effects of ABP administration on the mRNA level of IL-1β. c Effects of ABP administration on the mRNA level of IL-6. Relative mRNA levels from group control were used as the reference values; Different letters indicate significant differences (P < 0.05) between any two groups. Each value is represented in the format of the mean ± SD

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The HSP mRNA Expression Analysis

As shown in Fig. 4, the mRNA levels of HSP27 (Fig. 4a), HSP40 (Fig. 4b), HSP60 (Fig. 4c), HSP70 (Fig. 4d), and HSP90 (Fig. 4e) in group Cd were significantly higher compared with those of the other three groups at the same time point (P < 0.05). On the other hand, the mRNA levels of HSPs (HSP27, HSP40, HSP60, HSP70, and HSP90) in group Cd + ABP were significantly lower compared with group Cd (P < 0.05), but still higher than those in group control. In addition, the chickens in group ABP showed no significant difference compared with group control in terms of the mRNA levels of HSPs (HSP27, HSP40, HSP60, HSP70, and HSP90) in livers (P > 0.05).

Fig. 4
figure 4

Effects of ABP on Cd-induced changes in the mRNA levels of HSPs. a Effects of ABP administration on the mRNA level of HSP27. b Effects of ABP administration on the mRNA level of HSP40. c Effects of ABP administration on the mRNA level of HSP60. d Effects of ABP administration on the mRNA level of HSP70. e Effects of ABP administration on the mRNA level of HSP90. Relative mRNA levels from group control were used as the reference values. Different letters indicated that there were significant differences (P < 0.05) between any two groups. Each value was represented in the format of the mean ± SD

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The Protein Productions of HSPs

It is evident from Fig. 5a, b that the relative protein levels of HSP60, HSP70, and HSP90 in group Cd were significantly higher compared with the other three groups (P < 0.05). However, the protein levels of HSPs in group Cd + ABP were significantly lower compared with those in group Cd (P < 0.05), but still higher than group control. In addition, the protein levels of HSPs in group ABP showed no significant difference compared with group control (P > 0.05).

Fig. 5
figure 5

Effects of ABP on Cd-induced changes in the protein production of HSPs. a The effects of ABP on changes in the protein productions of HSP60, HSP70, and HSP90. b The protein levels of HSP60, HSP70, and HSP90 as shown by images of western blot analyses. Relative protein levels in group control were used as the reference values. Different letters indicate that there were significant differences (P < 0.05) between any two groups. Each value is represented in the format of the mean ± SD

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The Histopathology

The liver tissues for pathological sections in this paper were collected at 60 days. Liver sections of group control chickens showed the normal structure with regular morphology in Fig. 6a. Livers in group Cd showed impairment with cell malalignment and tumefaction, vacuolar degeneration, karyolysis, pyknosis, abundance of erythrocytes, and leukocyte infiltration in Fig. 6b. Figure 6c shows liver sections in group Cd + ABP, which indicate minor lesions compared with group Cd, while Fig. 6d indicates that liver sections in group ABP showed no significant difference compared with group control.

Fig. 6
figure 6

Sections of the liver stained with H&E. a Hepatic tissue section of group control chicken showing normal structure, hepatocyte (H) with normal nucleus (N) and central vein (CV), and a few erythrocytes. b Section of group Cd chicken liver showing karyolysis or pyknosis (short arrows), vacuolar degeneration (arrowheads), leukocytic infiltration (arrows), the loss of structure (bold arrows), and erythrocytes (irregular arrows). c Hepatic tissue section of group Cd + ABP chickens showing relatively normal structure with less erythrocytes (irregular arrow) and vacuolar degeneration (arrowhead) than group Cd. d Section of group ABP chicken liver showing normal structure similar to group control

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Discussion

Cd is a heavy metal toxic to many organs, especially the liver. When absorbed into the circulation system, Cd arrives and accumulates rapidly in the liver. The liver is impaired first because it is the body’s main detoxification organ. Many studies have demonstrated that Cd can impair the antioxidant system of both animals and plants [31–33] and cause inflammatory responses [34]. The induction of ROS by Cd leads to oxidative stress within cells by reacting with macromolecules and causing damage, such as mutations in DNA and destruction of protein structure and function [17]. Increased superoxide radicals, lipid peroxidation, and alterations in gene expression may lead to apoptosis [35]. Wu et al. demonstrated that ABM is a natural source of antioxidant compounds and has hepatoprotective functions against CCl4-induced liver damage [36]. Additionally, Padilha et al. illustrated that ABP could decrease the levels of inflammatory cytokines [37]. However, little is known about the protective effects of ABP on the liver damage induced by sub-chronic Cd exposure. In the present study, we demonstrated that Cd accumulated in the liver consistently inhibited the activities of antioxidant enzymes and induced the expressions of inflammatory factors and HSPs. ABP can increase the activities of antioxidant enzymes and decrease the mRNA levels of inflammatory cytokines and HSPs by promoting the metabolism of Cd and alleviating the toxicity of Cd in the liver.

Histopathological sections can directly describe the changes of organ structure and morphology, indicating whether the organ studied is healthy or not. In the research by Binkowski et al., congestion, foamy lipids in the hepatocytes, and leukocytic infiltration close to blood vessels induced by Cd and lead exposure were found in the Mallards Anas platyrhynchos liver lesions [38]. Meanwhile, Tête et al. illustrated hepatocyte degeneration, necrosis, and steatosis in hepatocytes and inflammation and lymphocyte infiltration caused by Cd and lead exposure in mice liver lesions [39]. Cd exposure caused liver cell degeneration, necrosis, erythrocytosis, and leukocyte infiltration in our study, and these pathological changes were less when chickens were given ABP supplementation, indicating that ABP could alleviate the inflammatory reactions induced by Cd by promoting the metabolism of Cd. This result was consistent with Lei’s result that Astragalus polysaccharides could alleviate LPS-induced inflammatory reaction [40] and with Joseph’s result that Ganoderma lucidum polysaccharides had anti-inflammatory properties [41], which might imply that ABP could amend the Cd-induced pathological changes in chicken livers.

It is well known that the biological effect of SOD is catalyzing superoxide anion radical into H2O2 and O2, while GSH-PX could resolve peroxide, clear lipids, and the product of peroxide reaction in the cell to protect the integrity of membrane structure and function, and the content of MDA reflects the level of lipid peroxidation, which indirectly indicates the degree of cell injury. Thus, the detections of SOD, GSH-PX, and MDA could reflect the degree of damage induced by Cd. Cd induces the production of cellular ROS, especially hydroxyl radicals, superoxide radicals, and hydrogen peroxide [42]. Neutralizing the ROS in the cell may induce change in the antioxidant status by enhanced production of nonenzymatic antioxidants [43]. Thus, the continuous exposure of cells/tissues to Cd leads to constant production of free radicals, and that may deplete the enzymatic and nonenzymatic antioxidants and result in diseased status of the tissues or organs [17]. Also, Gong’s results indicated that significant increases in the oxidative stress markers were observed during Cd treatment [44]. In our study, Cd obviously inhibited the activities of antioxidant enzymes, resulting in the promotion of MDA production, but ABP could significantly increase the Cd-inhibited antioxidant ability in chicken livers. Many researchers have demonstrated that plant polysaccharides and fungi polysaccharides have antioxidant bioactivity. For example, Pu et al. observed that polysaccharides from Angelica and Astragalus had antioxidant properties in scavenging free radicals to ameliorate oxidative stress and to inhibit lipid peroxidation [45]. Chen illustrated that polysaccharides from G. lucidum against pulmonary fibrosis in rats could be related to improving the lung tissue antioxidant ability [46]. Our results were consistent with these results, demonstrating that ABP could efficiently improve the antioxidant activities. This may be due to the fact that ABP is able to scavenge Cd-induced ROS, such as hydroxyl radicals, superoxide radicals, and hydrogen peroxide, and enhance the activity of enzymatic antioxidants. However, more studies are necessary to understand the antioxidant mechanism of ABP on Cd toxicity.

Studies have shown that liver injury caused by Cd exposure is associated with the release of inflammatory cytokines, such as TNF-α, IL-1β, and IL-6 [47]. The current results showed that Cd exposure could cause serious leukocyte infiltration and apoptosis and induce the release of TNF-α, IL-1β, and IL-6 in the liver, and these results are consistent with those of Liu et al. [48]. Many researchers have illustrated that polysaccharides, including ABP, could decrease the expressions of inflammatory cytokines. Luo proved that Astragalus polysaccharide could attenuate lipopolysaccharide-induced inflammatory response in microglial cells [49]. Liu et al. demonstrated that G. lucidum polysaccharide could protect hepatocyte injury induced by CCl4 via inhibiting lipid peroxidation, elevating antioxidant enzyme activity, and suppressing apoptosis and inflammatory response [50]. Meanwhile, Silveira et al. illustrated that the β-d-glucan isolated from Pleurotus sajor-caju exhibited significant anti-inflammatory activity [51]. Hetland et al. demonstrated that ABP could be rich in the immunomodulating polysaccharides such as β-glucans [52] and could have anti-inflammatory effects in inflammatory bowel disease patients [53]. In the present study, ABP significantly decreased Cd-induced increase of mRNA levels of inflammatory cytokines (TNF-α, IL-1β, and IL-6), indicating that ABP could efficiently alleviate the inflammatory reaction induced by Cd by improving the antioxidant ability and thus could alleviate the liver damage induced by Cd.

When living organisms are exposed to various stress conditions, such as temperature, virus, and heavy metals, the synthesis of most proteins is inhibited. However, a series of highly conserved proteins, HSPs, is rapidly synthesized to protect cells. Cao et al. reported that Cd exposure may lead to tissue damage, and high expressions of HSPs and inflammatory cytokines may play a role in the resistance to spleen toxicity induced by Cd [54]. Selim et al. demonstrated that Cd chloride caused damage to the DNA of testis cells, and this damage increased the synthesis of HSP70, which has been shown to have an ameliorative effect on the cells for recovery [55]. In the present study, the mRNA levels of HSPs (HSP27, HSP40, HSP60, HSP70, and HSP90) and protein levels of HSPs (HSP60, HSP70, and HSP90) were significantly increased by Cd exposure. The mRNA and protein levels of HSPs in chickens treated with the concurrent administration of Cd and ABP were significantly decreased compared with chickens treated with Cd alone. A tentative inference on this result is that ABP promoted the metabolism of Cd, resulted in the alleviation of the Cd-induced stress, and decreased the levels of HSPs in livers. These results were consistent with Xu’s finding [56] that polysaccharides of Atractylodes macrocephala Koidz could serve as immune regulators under HS conditions, which inhibited the expression of HSPs in chicken immune organs.

In conclusion, ABP may alleviate the liver damage induced by Cd exposure by promoting the metabolism of Cd, enhancing the activities of antioxidant enzymes, decreasing the content of MDA, and reducing the expressions of inflammatory cytokines (TNF-α, IL-1β, and IL-6) and HSPs (HSP27, HSP40, HSP60, HSP70, and HSP90) in chicken livers.