Introduction

Lead (Pb) is a metal element that can be toxic to living organisms, and expose it to nature could induce a wide range of biochemical and physiological dysfunctions in humans and laboratory animals [1, 2], such as negative effects on the birds’ general health, reproduction, and behavior, potentially leading to death [3]. Most previous studies documented that Pb could circulate in the body via blood and accumulate in bones and other vital organs (liver, kidneys, etc.) [4]. Clinical study indicated that exposure to Pb can lead to osteopenia, osteoporosis, impaired bone healing, and even osteoarthritis [5]. Pb toxicity also could cause inhibition of growth plate development in rats, increased cartilage-lead levels in ducks (higher than that of bone), and increased cartilage formation with delayed maturation and calcification and increased formation of fibrous tissue during bone repair in mouse [6, 7]. It had also been reported that Pb could induce oxidative stress by generating free radicals and reactive oxygen species (ROS) [8]. However, the toxic role of Pb is unclear, and the exact mechanism of Pb toxicity still needs to be further studied.

Inflammation was an important indicator of organism damage due to excess heavy metals. Inducible nitric oxide synthase (iNOS) could prevent autoimmunity to a certain degree and could function as an intracellular and intercellular signaling molecule shaping the immune response [9]. Nuclear factor-kappa B (NF-κB) was one of most important transcription factors in terms of inflammatory responses and could control the expression of numerous genes, such as iNOS, cyclooxygenase-2 (COX-2), and tumor necrosis factor-a (TNF-α) [10]. TNF-α was a proinflammatory cytokine that was produced by macrophages [11] and activated NF-κB. COX was the key enzyme in the biosynthesis of prostaglandins. COX-2 expression was usually induced by various cytokines, mitogens, and stresses [12]. Some of the interactions between heavy metals and inflammatory factors have already been reported in chickens. Arsenic exposure increased the messenger RNA (mRNA) level of TNF-α in mice liver [13] and NF-κB, TNF-α, COX-2, and iNOS in chicken gastrointestinal tract [14]. Excess Mn exposure resulted in inflammatory injury to chicken testis tissue through NF-κB/iNOS-COX-2 signaling pathway [15].

Heat shock proteins (HSPs) were major molecular chaperones that perform important functions in the folding/unfolding and translocation of proteins as well as in the assembly/disassembly of protein complexes [16], and HSPs possessed the ability to modulate the cellular antistress responses and played key roles in protecting organisms from metal stress [17]. Some previous studies indicated that heavy metal stresses could cause an increase in the mRNA level of HSPs in different tissues of western painted turtles [18] and chromium could upregulate the expressions of Hsp27 and Hsp70 in mice liver [19].

Selenium (Se) is an essential micronutrient for living organisms and is a well-established antioxidant [20], which can prevent or decrease the harmful effects of metal on the antioxidant system in different tissues [21–23]. The protective effect of Se against the different heavy metal toxicity in biological systems appears to have been studied [8, 24]. Se could protect the effect against cadmium-induced cytotoxicity in chicken splenic lymphocytes [25]. Se also had protective effects on the impairments of spatial learning, memory, and synaptic structural plasticity induced by Pb exposure in weaned rats [26]. Se deficiency could cause exudative diathesis and skeletal myodegeneration in birds which would be rapidly recovered by Se supplementation [27].

In summary, there was some research on Pb toxicity and Se on the antagonism against Pb. However, too little is known about the effect of Pb on the mRNA level of inflammatory injury and HSPs and the antagonistic effect of Se on Pb toxicity in chicken cartilage tissues. Herein, we established a model of Se and Pb interactions in chicken meniscus cartilage and sword cartilage and detected the effect of Se and Pb on the gene expression of inflammatory cytokines and HSPs.

Materials and Methods

Birds and Diets

All procedures used in this study were approved by the Institutional Animal Care and Use Committee of Northeast Agricultural University. A total of 360 male broiler chickens (1 day old; Weiwei Co. Ltd., Harbin, China) were randomly divided into four groups (90 chickens per group). Each group was separated into six pens with 15 chickens per pen. Throughout the entire experimental period, chickens were allowed ad libitum consumption of food and water. In this present study, we fed chickens a supplemental diet of Pb, Se, and Pb + Se to control Pb-induced toxicity and the antagonistic functions of Se in chicken muscle tissues. The Se group was fed a Se-adequate (sodium selenite) diet that contained 1 mg/kg Se and 0.5 mg/kg Pb. The Pb group was fed a Pb-supplemented (Pb acetate) diet containing 0.2 mg/kg Se and 350 mg/kg Pb. The Se + Pb group was fed a Se and Pb compound diet containing 1 mg/kg Se and 350 mg/kg Pb. Finally, the control group was fed a basic diet containing 0.2 mg/kg Se and 0.5 mg/kg Pb. Chickens were euthanized when they were 90 days old. The meniscus cartilage and sword cartilage tissues were quickly removed and rinsed with ice-cold sterile deionized water, frozen immediately in liquid nitrogen, and stored at −80 °C until required for subsequent experiments.

Quantification of Inflammatory Factor and HSP Gene mRNA

The extraction of total RNA (30-mg tissue; n = 6/diet group) was performed using TRIzol reagent according to the manufacturer’s instructions (Invitrogen, China). Then, the dried RNA pellets were resuspended in 50 μl of diethyl-pyrocarbonate-treated water. After determining the concentration and purity of the total RNA at 260/280 nm, first-strand complementary DNA (cDNA) was synthesized from 5 μg of total RNA using oligo dT primers and Superscript II reverse transcriptase according to the manufacturer’s instructions (Invitrogen, China). Finally, synthesized cDNA was diluted five times with sterile water and stored at −80 °C before use.

After quantification, the expression levels of HSPs and inflammatory genes were determined by real-time quantitative reverse transcription PCR with SYBR Premix ExTaq TM (Takara, China), and the real-time PCR was performed with ABI PRISM 7500 (Applied Biosystems). The PCR primers (Table 1) were designed using Oligo Primer Analysis software (version 6.0) and synthesized by Invitrogen (Shanghai, China).

Table 1 Primers used for quantitative real-time PCR
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Reactions consisted of the following: 10 μl of 2× SYBR Green I PCR Master Mix (TaKaRa, China), 0.4 μl of 50× ROX reference Dye II, 0.4 μl of each primer (10 μM), 2 μl of diluted cDNA, and 6.8 μl of PCR-grade water. The PCR procedure for HSPs, inflammatory genes, and GAPDH consisted of 95 °C for 30 s followed by 40 cycles at 95 °C for 15 s and 60 °C for 30 s. Results (fold changes) were expressed as 2−ΔΔCt in which ΔΔCt = (CtHSPs − CtGADPH)B/C/D − (CtHSPs − CtGADPH)A. A is the control group, B is the +Pb group, C is the +Se group, and D is the Se + Pb group. Inflammatory genes use the same formula to calculate the 2−ΔΔCt.

Western Blot Analysis

Protein extracts were subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions on 12 % gels. Separated proteins were then transferred to nitrocellulose membranes using a tank transfer for 2 h at 200 mA in Tris glycine buffer containing 20 % methanol. Membranes were blocked with 5 % skim milk for 16–24 h and incubated overnight with diluted primary chicken antibody Hsp70 (1:500) and Hsp90 (1:500; Hsp90 and Hsp70 production of polyclonal antibody by our lab) followed by a horseradish peroxidase (HRP)-conjugated secondary antibody against rabbit IgG (1:1500, Santa Cruz, CA, USA). To verify equal loading of samples, the membrane was incubated with monoclonal β-actin antibody (1:1000, Santa Cruz, CA, USA), followed by a HRP-conjugated goat antimouse IgG (1:1000). The signal was detected by X-ray films (Trans Gen Biotech Co., China). The optical density (OD) of each band was determined by Image VCD gel imaging system, and the Hsp70 and Hsp90 expressions were detected as the ratio of OD of Hsp70 and Hsp90 and OD of β-actin, respectively.

Statistical Analyses

Statistical analyses of all the data were performed using the SPSS for Windows software (version 13; SPSS Inc., Chicago, IL, USA). The measurements were considered significantly different when P < 0.05 was obtained by a one-way analysis of variance. All the data displayed a normal distribution and passed equal variance testing. Differences between means were assessed using Tukey’s honest significant difference test for post hoc multiple comparisons. The data were expressed as the mean ± standard deviation. In addition, a principal component analysis (PCA) was used to define the most important parameters, which could be used as key factors for individual variations using the Statistics 6.0 program.

Results

Effects of Se, Pb, and Se + Pb on mRNA Levels of iNOS, NF-κB, TNF-α, and COX-2 in Chicken Meniscus Cartilage and Sword Cartilage

The mRNA levels of inflammatory cytokine genes in broiler chickens’ meniscus cartilage were detected. As shown in Fig. 1, Pb increased the mRNA levels of iNOS, NF-κB, TNF-α, and COX-2 in the +Pb group of the meniscus cartilage tissue (P < 0.05), and there was no significant difference between the +Se group and control group in the mRNA levels of iNOS, NF-κB, and COX-2 (P > 0.05), except the mRNA level of TNF-α. In the Se + Pb group, the mRNA level of NF-κB was increased, but the increased degree was less than that in the +Pb group compared with the control group (P < 0.05). Other three genes were not influenced (P > 0.05).

Fig. 1
figure 1

Effects of dietary Se against Pb toxicity on mRNA levels of inflammatory cytokine genes in the meniscus cartilage of chickens. Bars with different lowercase letters are statistically significantly different (P < 0.05) in the same gene. Each value was expressed as the mean ± SD (n = 6)

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Likewise in Fig. 2, Pb increased the mRNA levels of iNOS, NF-κB, TNF-α, and COX-2 in the +Pb group of the sword cartilage tissue (P < 0.05), and there was no significant difference between the +Se group and control group in the mRNA levels of iNOS, NF-κB, TNF-α, and COX-2 (P > 0.05). In the Se + Pb group, the mRNA levels of iNOS, TNF-α, and COX-2 were increased but the increased degree was less than that in the +Pb group compared with the control group (P < 0.05), except NF-κB (P > 0.05).

Fig. 2
figure 2

Effects of dietary Se against Pb toxicity on mRNA levels of inflammatory cytokine genes in the sword cartilage of chickens. Bars with different lowercase letters were statistically significantly different (P < 0.05) in the same gene. Each value was expressed as the mean ± SD (n = 6)

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Effects of Se, Pb, and Se + Pb on mRNA Levels of Hsp27, Hsp40, Hsp60, Hsp70, and Hsp90 in Chicken Meniscus Cartilage and Sword Cartilage

The mRNA levels of HSP genes in broiler chickens’ meniscus cartilage were detected. As shown in Fig. 3, Pb increased the mRNA levels of Hsp27, Hsp40, Hsp60, Hsp70, and Hsp90 in the +Pb group of the meniscus cartilage tissue (P < 0.05), and there was no significant difference between the +Se group and control group in the mRNA levels of Hsp27, Hsp40, Hsp60, Hsp70, and Hsp90 (P > 0.05). In the Se + Pb group, the mRNA levels of Hsp27, Hsp40, Hsp60, Hsp70, and Hsp90 were not influenced compared with the control group (P > 0.05).

Fig. 3
figure 3

Effects of dietary Se against Pb toxicity on mRNA levels of HSP genes in the meniscus cartilage of chickens. Bars with different lowercase letters were statistically significantly different (P < 0.05) in the same gene. Each value was expressed as the mean ± SD (n = 6)

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Likewise in Fig. 4, Pb increased the mRNA levels of Hsp27, Hsp40, Hsp60, Hsp70, and Hsp90 in the +Pb group of the sword cartilage tissue (P < 0.05), and there was no significant difference between the +Se group and control group in the mRNA levels of Hsp27, Hsp40, Hsp60, Hsp70, and Hsp90 (P > 0.05). In the Se + Pb group, the mRNA levels of Hsp27, Hsp60, Hsp70, and Hsp90 were increased but the increased degree was less than that in the +Pb group compared with the control group (P < 0.05), except Hsp40 (P > 0.05).

Fig. 4
figure 4

Effects of dietary Se against Pb toxicity on mRNA levels of HSP genes in the sword cartilage of chickens. Bars with different lowercase letters were statistically significantly different (P < 0.05) in the same gene as well. Each value was expressed as the mean ± SD (n = 6)

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Effects of Se, Pb, and Se + Pb on Western Bolt Analysis of Hsp70 and Hsp90 Levels in Chicken Meniscus Cartilage and Sword Cartilage

The protein expression levels of Hsp70 and Hsp90 were examined by western blots. The results (Figs. 5 and 6) revealed that the Hsp70 and Hsp90 protein expressions in +Pb group were gradually increased in meniscus cartilage and sword cartilage of chickens compared with control group. Also, western blots of Hsp70 and Hsp90 results were consistent with Hsp70 and Hsp90 mRNA responses to Pb-induced toxicity.

Fig. 5
figure 5

Effects of dietary Se against Pb toxicity on protein levels of HSP genes in the meniscus cartilage of chickens. Bars with different lowercase letters were statistically significantly different (P < 0.05) in the same gene as well. Each value was expressed as the mean ± SD (n = 6)

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Fig. 6
figure 6

Effects of dietary Se against Pb toxicity on protein levels of HSP genes in the sword cartilage of chickens. Bars with different lowercase letters were statistically significantly different (P < 0.05) in the same gene as well. Each value was expressed as the mean ± SD (n = 6)

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Principal Component Analysis

Using PCA, all the parameters that were measured in the present study were distinguished on the ordination plots that corresponded to the first and second principal components (95.109 and 4.891 %, respectively) in chicken’s meniscus cartilage (Fig. 7a) and (95.602 and 4.398 %, respectively; Fig. 7b) in sword cartilage. Furthermore, the observed correlations among the parameters were confirmed and quantified according to Spearman’s test (Tables 2 and 3). These results indicated the correlation of all the biomarkers suggesting a possibly positive or negative relationship. The results revealed that Hsp40, Hsp60, Hsp70, and Hsp90 had a close positive correlation with each other in meniscus cartilage. Similar tendency had been observed in iNOS and COX-2 as well. In addition, Hsp40 and Hsp60, TNF-α, and COX-2 presented a high correlation in sword cartilage of chicken. Similar phenomenon has been tested in the sword cartilage of chicken as well.

Fig. 7
figure 7

Ordination diagram of the principal component analysis (PCA) of parameters that were measured in the chicken meniscus cartilage and sword cartilage

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Table 2 Correlation matrix among the genes detected in the meniscus cartilage of chicken
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Table 3 Correlation matrix among the genes detected in the sword cartilage of chicken
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Discussion

A previous study revealed that a complex antagonistic interaction existed between Se and other heavy metals. Se can reduce the content of certain heavy metals, such as Pb, Cd, and Hg [25, 28, 29]. Wang et al. reported that Se alleviated Pb-induced increase of inflammation factor and HSP expression in chicken livers [30]. The chicken cartilage and bone was the main accumulation tissue. Gao et al. reported that Se and Pb influenced the expression of selenoprotein genes in chicken cartilage tissue [31]. However, the effects of Se against Pb toxicity on mRNA levels of inflammatory cytokines and HSP genes have been little reported in chickens. In this current study, we detected the effects of dietary Se against Pb toxicity on mRNA levels of inflammatory cytokine (iNOS, NF-κB, TNF-α, and COX-2) and HSP (Hsp27, Hsp40, Hsp60, Hsp70, and Hsp90) genes in the cartilage tissue of broiler chickens. The results showed that the treatment of Se and Pb influenced the gene expression of inflammatory cytokines and HSPs in chicken cartilage tissues.

Pb is one of high toxic heavy metals and has been known that it is not essential in living organisms. Pb exposure also could cause rat liver inflammatory injury [26]. NF-κB was a heterodimeric transcription factor that translocates to the nucleus and mediated the transcription of a vast array of proteins involved in inflammatory response [32]. NF-κB was involved in regulation of COX-2 and iNOS expressions, which were important enzymes that mediate inflammatory processes [33]. Several previous studies indicated that Pb exposure increased the levels of TNF-α and COX-2 proinflammatory enzymes in mouse microglial cell line [34]. Pb was responsible for a significant morphological alteration and a decline in cell function and TNF-α release which increased inflammation in mice testicular macrophages [35]. In the current study, we found that in the Pb group, the mRNA levels of NF-κB, TNF-α, COX-2, and iNOS were significantly higher than that in the control group. In accordance with the above research results, our results suggested that excess Pb could cause inflammation of chicken cartilage tissues.

As molecular chaperones, HSPs are highly conserved cellular stress proteins present in every organism from bacteria to humans. HSPs could protect organisms from a number of stress conditions including heavy metal stress [36]. Several previous studies presented that the mRNA levels of HSPs (Hsp27, Hsp40, Hsp60, Hsp70, and Hsp90) in chicken livers were increased when induced by dietary Se deficiency [37]. LcHsp27 expression showed a dramatic upregulation after a combined stress of temperature and cadmium [38]. The levels of HSP mRNA (Hsp90, Hsp70, Hsp60, Hsp40, and Hsp27) and protein (Hsp70 and Hsp60) were significantly increased (P < 0.05 or P < 0.01) with the As2O3 treatment [39]. Continuous Pb treatment elevated Hsp70 level in oribatid mite [40]. In this study, we found that Hsp27, Hsp40, Hsp60, Hsp70, and Hsp90 mRNA levels of chicken cartilage tissues were significantly increased (P < 0.05) in the +Pb group. It implied that HSPs may be the biomarkers of Pb poisoning.

Se is one of the essential micronutrients for living organisms, closely related to human and animal health. One of its main functions is an antioxidant action involved in protection against damage caused by free radicals and oxidative stress [41]. Some previous studies demonstrated that the antagonistic effect of Se on other heavy metals, such as Se, could protect the nitric oxide and the gene expression of inflammatory cytokines induced by cadmium in chicken splenic lymphocytes [42]. Se could ameliorate cadmium (Cd)-induced brain damage in chickens [43] and provide protective effects on the chronic poisoning of arsenic and decreased Hsp70 mRNA level which arsenic induced in rat livers [44]. Se also could provide certain protective actions against Pb-induced toxicity in chicken livers [45]. In our study, most of the mRNA levels of NF-κB, TNF-α, iNOS, COX-2, Hsp27, Hsp40, Hsp60, Hsp70, and Hsp90 decreased in the Se + Pb group compared to those in the Pb group. This finding is consistent with previous studies that indicated that Pb accumulation could be alleviated by Se treatment. Therefore, we hypothesized that Se may have a negative effect on Pb and could alleviate Pb-induced toxicity in chicken cartilage tissues.

In conclusion, we demonstrated that Se can alleviate Pb-dependent toxicity in chicken cartilage tissues. Moreover, changes in the mRNA expression of inflammatory cytokines and HSPs are indeed associated with the antagonistic function of Se that protects against Pb-induced toxicity.