Oxidative stress, apoptosis and inflammatory responses involved in copper-induced pulmonary toxicity in mice

At present, there are few studies focused on the relationship between copper (Cu) and oxidative stress, apoptosis, or inflammatory responses in animal and human lungs. This study was conducted to explore the effects of Cu on pulmonary oxidative stress, apoptosis and inflammatory responses in mice orally administered with 0 mg/kg (control), 10 mg/kg, 20 mg/kg, and 40 mg/kg of CuSO4 for 42 days. The results showed that CuSO4 increased ROS production, and MDA, 8-OHdG and NO contents as well as iNOS activities and mRNA expression levels. Meanwhile, CuSO4 reduced the activities and mRNA expression levels of antioxidant enzymes (GSH-Px, CAT, and SOD) and GSH contents, and ASA and AHR abilities. Also, CuSO4 induced apoptosis, which was accompanied by decreasing Bcl-2, Bcl-xL mRNA expression levels and protein expression levels, and increasing Bax, Bak, cleaved-caspase-3, cleaved-caspase-9 mRNA, and protein expression levels, and Bax/Bcl-2 ratio. Concurrently, CuSO4 caused inflammation by increasing MPO activities and activating the NF-κB signalling pathway, and down-regulating the mRNA and protein expression levels of anti-inflammatory cytokines (IL-2, IL-4, IL-10). In conclusion, the abovementioned findings demonstrated that over 10 mg/kg CuSO4 can cause oxidative stress, apoptosis, and inflammatory responses, which contribute to pulmonary lesions and dysfunction in mice.

AGING species (ROS) [15]. It has been identified that high Cu levels have toxic effects including ROS generation leading to DNA damage [16], oxidative damage to biological molecules [17], and peroxidation of cell membrane lipids [18]. In addition, studies have shown that Cu overload leads to oxidative stress, and subsequently damages proteins, lipids and nucleic acids [19][20][21][22][23]. In vivo studies, high dietary copper can inhibit the serum, hepatic [24][25][26][27] and nephritic [28] antioxidase activities in ducklings, and induce oxidative stress in the brain [29,30] and spleen [31] of chickens. Also, it has been reported that intradermal injection of 2% CuSO4 solution in the chicken can increase oxygen-derived free radicals, result in tissue damage and increase vascular permeability [32]. In in vitro studies, Cu and Cu compounds can cause oxidative stress via the production of ROS in human epithelial lung (A549) cells [33] and brain microvascular endothelial cells [34].
Additionally, increased intracellular ROS as a signal of oxidative stress, can break single or double strands of DNA and activate DNA-dependent protein kinase, leading to apoptosis [35,36]. Reports of in vivo studies have shown that high Cu levels induce the higher percentage of apoptotic cells of the lymphoid organs [37] and kidney [38] in ducklings and lymphoid organs [39], kidney [40], and liver [41] in chickens. The findings of Guo et al. (2017) indicated that Cu can induce oxidative stress and apoptosis in the White Shrimp (Litopenaeus vannamei) [42]. Besides, it has been reported that Cu induces apoptosis by increasing the expression levels of cysteine aspartate specific protease-3(caspase-3), cysteinyl aspartate-specific protease9 (caspase-9), and Bcl-2 associated X protein(Bax) in the rat kidney and liver [43,44]. In in vitro studies, the findings have indicated that Cu can induce mitochondrial dysfunction in primary culture of chicken hepatocytes [45] and apoptosis in human skin melanoma A-375 cell line [46] and Aedes C6/36 cells [47].
Inflammation is a defensive response to stimulation and usually is beneficial for animals and human beings [48]: however, continued inflammation can cause damage to the body [49]. Inflammation can be triggered by a variety of factors, such as pathogens, damaged cells, toxic compounds, and irradiation [50]. In addition, oxidative damage can lead to cell metabolism disorders, which accelerates the production of inflammation [51]. In in vivo studies, the study has investigated the potential synergistic effects of chronic exposure to Cu in promoting inflammatory and oxidative events in mouse brains [52]. Brand et al. (2019) has found that inhalation of fumes containing Cu can induce asymptomatic systemic inflammation [53]. Pharyngeal aspiration of CuO nanoparticles causes lung inflammation and lung injuries in Wistar rats [54], however, no reports are focused on CuSO4-induced inflammation in the human and animal lung.
The aforementioned studies suggest that oxidative stress, apoptosis, and inflammatory responses are the three main mechanisms involved in Cu-induced toxic effects on organs, tissues and cells, however, there are few studies focused on the relationship between Cu and oxidative stress, apoptosis, inflammatory responses in animal and human lungs. Therefore, the present study aims to assess pulmonary toxicity via oxidative stress, apoptosis and inflammatory responses after CuSO4 intake in mice by using methods of histopathology, qRT-PCR, and ELISA, flow cytometry, and western blot assay. Figure 1 shows the dose-and time-dependent lesions in lungs at the three CuSO4-treated groups. Histopathologically, alveolar walls were thickened in varying degrees, mainly due to inflammatory cells infiltration and capillary congestion. The aforementioned histopathological lesions were not observed in the control group.

Changes of ROS production levels and Cu contents in the lung
The ROS production levels were increased (p < 0.05 or p < 0.01) in the 20 and 40 mg/kg groups at day 21 of the experiment and in the three CuSO4-treated groups at day 42 of the experiment when compared to the control group. The Cu contents in the lung were increased (p < 0.05 or p < 0.01) in the 20 and 40 mg/kg group at day 42 of the experiment when compared to the control group. The results are illustrated in Figure 2.

Changes of factors related to ROS production in the lung
The contents of malondialdehyde (MDA) and 8hydroxy-2'-deoxyguanosine (8-OHdG) were significantly higher (p < 0.05 or p < 0.01) in the three CuSO4treated groups at day 21 and 42 of the experiment than in the control group.
The anti-superoxide anion (ASA) and anti-hydroxyl radical (AHR) abilities were significantly decreased (p < 0.05 or p < 0.01) in the 20 and 40 mg/kg groups at day 21 of the experiment and in the three CuSO4-treated groups at day 42 of the experiment when compared to the control group. AGING The nitric oxide (NO) content was significantly higher (p < 0.05 or p < 0.01) in the 40 mg/kg group at day 21 of the experiment and in the 20 and 40 mg/kg groups at day 42 of the experiment than in the control group. The inducible nitric oxide synthase (iNOS) activities and mRNA expression levels were significantly increased (p < 0.05 or p < 0.01) in the 20 and 40 mg/kg groups at day 21 and 42 of experiment in comparison to the control group. The results are shown in Figure 3.

Changes of oxidative damage parameters in the lung
The superoxide dismutase (SOD) activities were significantly reduced (p < 0.05 or p < 0.01) in the 40 mg/kg group at 21 and 42 days of the experiment and in the 20 mg/kg group at 42 days in comparison to the control group. The activities of Catalase (CAT), glutathione peroxidase (GSH-Px), Glutathione (GSH) contents, and the GSH/GSSG ratio were significantly decreased (p < 0.01) in the three CuSO4-treated groups at 21 and 42 days. The oxidised glutathione (GSSG) content was significantly increased (p < 0.05 or p < 0.01) in the 20 and 40 mg/kg groups at days 21 and 42. The mRNA expression levels of antioxidant enzymes (GSH-Px, CAT, MnSOD, and ZnSOD) were significantly lower (p < 0.05 or p < 0.01) in the 40 mg/kg group at day 21 and in the three CuSO4-treated groups at day 42 than in the control group. The results are shown in Figure 4.

Changes of apoptosis percentages in the lung
The apoptotic percentages were elevated (p < 0.05 or p < 0.01) in the three CuSO4-treated groups at days 21 and 42 of the experiment when compared to the control group, as shown in Figure 5.

Changes of mRNA and protein expression levels of apoptosis-related mediators in the lung
The mRNA expression levels of caspase 3, Bax, Bcl-2 antagonist killer (Bak), and Bax/BcL-2, and the protein expression levels of cleaved-caspase 3, cleaved-caspase 9, Bax, Bak, and Bax/BcL-2 were significantly higher (p < 0.01 or p <0.05) in the three CuSO4-treated groups at 21 and 42 days of the experiment than in the control group. The mRNA expression levels of the B-cell lymphoma-2 (Bcl-2) and Bcl-extra-large (Bcl-xL) were significantly decreased (p < 0.01 or p < 0.05) in the three CuSO4-treated groups at 21 and 42 days when compared to the control group. Also, protein expression levels of Bcl-2 and Bcl-xL were significantly decreased (p < 0.01) in the 40 mg/kg group at 21 days and in the 20 and 40 mg/kg groups at 42 days in comparison to the control group, as shown in Figure 6.

Changes of MPO activities and PGE2 contents in the lung
The myeloperoxidase (MPO) activities and prostaglandin E2 (PGE2) contents were significantly AGING  higher (p < 0.01) in the 20 and 40 mg/kg groups at days 21 and 42 of the experiment than in the control group, as shown in Figure 7.

Changes of mRNA expression and protein expression levels of NF-κB and IκB in the lung
The mRNA and protein expression levels of nuclear factor-kappa B (NF-κB) were significantly higher (p < 0.01 or p < 0.05) in the 20 and 40 mg/kg groups at 21 and 42 days of the experiment than in the control group. The mRNA and protein expression levels of inhibitory kappa B (IκB) were significantly decreased (p < 0.01 or p < 0.05) in the 20 and 40 mg/kg groups at 21 and 42 days when compared to the control group. The results are shown in Figure 8.

Changes in mRNA expression and protein expression levels of inflammatory cytokines in the lung
The mRNA expression levels and protein expression levels of cyclooxygenase-2 (COX-2), interleukin-1β (IL-1β), interleukin-8 (IL-8), and interleukin-6 (IL-6) were significantly increased (p < 0.01 or p < 0.05) in the 20 and 40 mg/kg groups in comparison to the control group at 21 and 42 days of the experiment.
The mRNA and protein expression levels of tumour necrosis factor-α (TNF-α) were significantly higher (p < 0.01) in the 40 mg/kg group at 21 days of the experiment and in the 20 and 40 mg/kg groups at 42 days than in the control group. The mRNA expression levels and protein expression levels of interleukin-2 (IL-2), interleukin-4 (IL-4), and interleukin-10 (IL-10) were significantly decreased (p < 0.01 or p < 0.05) in the 20 and 40 mg/kg groups at 21 days and in the three CuSO4-treated groups at 42 days when compared to in the control group. The results are shown in Figures 9 and 10.

DISCUSSION
In summary, this study firstly found that CuSO4 could induce oxidative stress, apoptosis, and inflammatory responses in the mouse lung.
We observed histopathological changes induced by CuSO4, alveolar walls were thickened in varying degrees with the dose-and time-dependent lesions, which mainly due to inflammatory cells infiltration and capillary congestion. Also, the pulmonary injuries were strongly consistent with the accumulation of Cu in the lung. It has been suggested that one possible molecular mechanism involved in the Cu toxicity is the formation of ROS, which leads to lesions via oxidative damage [55]. Our results showed that CuSO4 induced the production of ROS in the pulmonary tissue, and then excessive ROS caused oxidative damage to DNA (increased 8-OHdG contents) and lipid peroxidation (increased MDA contents). To reveal causes of the increased ROS, we tested the ASA and AHR abilities. The results showed that ASA and AHR abilities were reduced, which indicated that CuSO4 decreased the capacity of the lung to scavenge ROS and disturbed the dynamic balance between ROS production and elimination of ROS. Once the antioxidant function is impaired, free radicals (including oxygen free radicals, hydroxyl free radicals and nitrogen free radicals, etc.) are accumulated, which then causes oxidative stress in the body [56]. Kubbw et al. has found that rats consuming the zinc-deficient (0.5 mg/kg), high-copper (over 15 mg/kg) diet have the lowest weight gain and have an increase in endogenous free radical production in lung [57]. We also found an increase in the NO contents, iNOS enzyme activities, and mRNA expression levels, leading to oxidative damage in the lung. NO is an important nitrogen free radical in the body, which can also induce inflammation [58]. The increased ROS may indicate that the functioning of the antioxidant defence system is weakened [59], including decreased antioxidant enzymes (SOD, CAT, and GSH-PX) activities and non-enzymatic scavenger (GSH) content. SOD and CAT are important anti-oxidant enzymes that play a major role in ROS clearance [60]. GSH is an important intracellular antioxidant in alveolar epithelial cells [61]. GSHdependent enzymes such as GSH-PX can remove hydrogen peroxide by oxidising GSH to GSSG, but   ROS can oxidise GSH into GSSG, too much GSSG will weaken the protective effect of GSH, and the dysfunction of GSH will also aggravate the damage to organs [62,63]. In the present study, the results showed that CuSO4 decreased CAT, SOD, and GSH-Px activities, GSH contents, and also the GSH/GSSG ratio was decreased in the lung, which were consistent with the report that copper can enhance oxidative stress, reduce the activities of antioxidant enzymes (SOD and CAT) and decrease the contents of GSH in brain tissues of chicken [51]. Cinar et al. (2014) has found that oral intake of CuSO4 can induce oxidative stress by reducing the activity of enzymes such as CAT and SOD in broiler serum [64]. Other studies have shown that the reduced intracellular GSH is associated with activation of NF-κB [65]. The further to explore the molecular mechanism of CuSO4-decreased antioxidant enzyme activities, we also detected mRNA expression levels of antioxidant enzymes in the lung. The results indicated that the mRNA expression levels of CuZnSOD, MnSOD, CAT, and GSH-PX were lower in the CuSO4treated groups than in the control group, which were consistent with the reduction of these antioxidant enzyme activities. Decreased activities and mRNA expression levels of antioxidant enzymes promote oxidative stress, which plays critical roles in the pathogenesis of various diseases [66,67].
In conclusion, our results showed that CuSO4 induces oxidative stress in the lung by promoting excessive ROS production and increased NO contents, and reducing antioxidant enzyme activities, GSH contents, and the GSH/GSSG ratio, which contribute to pulmonary lesions and dysfunction. Also, this study is the first to be focused on CuSO4-induced oxidative stress in the lung. It has been known that ROS and oxidative stress play important roles in the early stages of apoptosis [68]. Next, we explored whether, or not, apoptosis was also involved in the mechanism of CuSO4-cuased toxicology in the lung. After CuSO4 treatment, the results indicated that the percentage of apoptosis was increased in the CuSO4-treated groups at 21 and 42 days. Apoptosis is a process controlled by multiple genes, such as bcl-2 family and caspase family, which are conserved among species [69]. As a group of important apoptotic regulators, the bcl-2 family can indirectly regulate caspases in relevant apoptotic pathways [70,71]. Results in the present study showed that CuSO4 could cause a significant decrease in the protein expression levels and mRNA expression levels of anti-apoptotic proteins (Bcl-2 and Bcl-xL), while the protein and mRNA expression levels of pro-apoptotic proteins (Bax and Bak) were significantly increased in the lung. Hsien et al. (2008) has also observed that Cu-induced apoptosis was accompanied by the increased Bax and Bak expression levels and a decreased Bcl-2 expression level in neuroblastoma cells [72]. We also observed an increased Bax/Bcl-2 ratio in this research. Lee et al. (2008) has indicated that the increase of the ratio between Bax and Bcl-2 is the key point in the occurrence of apoptosis [73]. This increased Bax/Bcl-2 ratio leads to changes in mitochondrial membrane permeability and the release of apoptotic proteins, such as Cyt c [74]. In addition, excessive ROS production with decreasing concomitant in GSH contents also leads to mitochondrial membrane permeability and induction of apoptotic cell death in cultured cells [75]. Cyt c can cleave and activate caspase-9, which then activates downstream caspase-3 and cell apoptosis [76]. In this study, CuSO4 increased mRNA expression levels of caspase-9 and -3 and protein expression levels of cleaved-caspase-9 and -3, which contributed to apoptotic occurrence. The reports have also found that apoptosis is caused by copper-induced ROS formation in MCF7 cells [77], rat liver [78], and chicken intestine [79]. In summary, the present study was the first in which CuSO4-induced apoptosis was observed in the lung, and this was accompanied by decreasing Bcl-2, Bcl-xL mRNA expression levels and protein expression levels, and increasing Bax, Bak, caspase-3, and caspase-9 mRNA expression levels and protein expression levels, and the Bax/Bcl-2 ratio.
Inflammation is also a crucial toxicological mechanism of copper [80]. Myeloperoxidase (MPO) activity is used to measure the inflammatory degree in tissues and organs, and our results suggested that MPO activities were increased, which was consistent with the increased MPO arising from excessive accumulation of ROS [81]. This study is the first to investigate the mechanism of NF-κB activation in pulmonary inflammation by CuSO4 intake in mice. NF-κB is considered to be a major cellular transcription factor in inflammatory processes, and NF-κB activation has been identified as an important feature of inflammatory pulmonary diseases [82,83]. Normally, NF-κB and IκB combine to form an inactive NF-κB-IκB complex [84]. In this study, The expression levels of IκB mRNA and protein were decreased, and the expression levels of NF-κB mRNA and NF-κB protein were increased, which demonstrated that CuSO4-induced IκB degradation promoted the activation of NF-κB. Concurrently, we observed that CuSO4 increased mRNA and protein expression levels of pro-inflammatory cytokines, such as TNF-α, COX-2, IL-6, IL-1β, and IL-8, which was in line with activation of the transcription factor NF-κB pathway. Our results are consistent with the report that copper can increase the expression levels of NF-κB, COX-2, IL-1β, TNF-α, IL-1β, and IL-8 mRNA and protein and exacerbate the damage and oxidative stress in zebrafish larvae tissues [85,86].
It is well known that COX-2 is a classic proinflammatory cytokine, and plays an important role in the regulation of pulmonary inflammation [87]. As a downstream product of COX-2, PGE2 can not only induce the aggregation of inflammatory cells but also accelerate the process of peripheral inflammatory response induced by harmful stimuli [88]. In the present study, we observed that the PGE2 contents as well as the COX-2 mRNA and protein expression levels were significantly increased. Lu et al. (2008) has reported that intake of copper can up-regulate the expression of inflammation-related genes in the mouse brain, such as COX-2 and TNF-α [89]. iNOS, a proinflammatory marker in lung tissue, plays an essential role in exacerbating inflammation and can catalyse the production of NO, while excessive NO can increase the permeability of blood vessels and promote the infiltration of inflammatory cells [90][91][92][93]. In the present study, we found that CuSO4 increased the activities and mRNA expression levels of iNOS, and NO contents, which promoted lung inflammation.
On the contrary, IL-2, IL-4, and IL-10 are recognised as cytokines able to mediate immune suppression, which can inhibit the production of Th1 cells and reduce the release of pro-inflammatory cytokine [94,95]. In the stage of acute pulmonary inflammation, IL-10 has significant anti-lymphocytes and neutrophilic infiltration [96]. Our results showed that CuSO4 decreased the mRNA and protein expression levels of IL-2, IL-4, and IL-10.
In conclusion, CuSO4 can increase MPO activities, activate the NF-κB pathway, and down-regulate antiinflammatory cytokines, indicating that imbalance AGING between pro-inflammatory and anti-inflammatory cytokine induces inflammatory responses in the lung. CuSO4-induced inflammatory responses contribute to pulmonary lesions and dysfunction.

Experimental animals and diets
A total of 240 four-week-old ICR mice (half male and half female) obtained from the Chengdu Dossy Experimental Animals were used in the present study. The animals were housed in separate polypropylene cages, and diet and water were provided ad libitum throughout the experiment. The mice were fed a fullprice diet provided by Dossy. After a week of rest and acclimatisation, mice were equally divided into four different groups (each with n = 60). The control group was given orally distilled water only, groups I, II, and III were given CuSO4 orally at the dose of 10, 20, and 40 mg/kg body mass, respectively. Mice were administered their respective doses daily by gavage for 42 consecutive days, and the gavage volume was 1 ml/100 g body mass.
The animal protocols and all procedures of the experiment were performed in compliance with the laws and guidelines of Sichuan Agricultural University Animal Care and Use Committee.

Histopathological observation of the lung
At 21 and 42 days, eight mice (male female half) in each group were humanely killed and their lungs were removed, fixed in 4% paraformaldehyde solution, dehydrated with increasing concentrations of ethanol, cleared with xylene and embedded in paraffin. And then lungs were serial sectioned at 5μm thickness, stained with hematoxylin and eosin (H&E), and observed by optical microscopy.

Determination of pulmonary Cu contents
At 42 days of the experiment, eight mice (male female half) in each group were humanely killed and their lungs were removed, weighed, dried, and collected for the determination of the Cu contents. The Cu contents in the lung were measured according to a reference method [97].

Determination of the oxidative and anti-oxidative parameters in the lung
At 21and 42 days of age during the experiment, eight mice (male female half) in each group were humanely sacrificed, and the lungs were immediately stored at 4 °C cold phosphate buffer saline in a chilled homogeniser, and centrifuged at 3000 rpm for 15 min. Thereafter, the supernatant was transferred into new Eppendorf tubes. The commercial kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) and used to detect ASA (Cat. No. A052-1) and AHR (Cat.

Detection of MPO, iNOS activities, and NO and PGE2 contents
At 21 and 42 days into the experiment, eight mice (half male, half female) in each group were humanely sacrificed, and the lungs were immediately stored at 4 °C cold phosphate buffer saline in a chilled homogeniser, and centrifuged at 3500 rpm for 10 min. Thereafter, the supernatant was transferred into new Eppendorf tubes. The commercial kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) and used to detect MPO (Cat. No. A044), iNOS (Cat. No. A014-1), and NO (Cat. No. A013-2) according to the manufacturer's instructions. Pulmonary PGE2 levels were measured using ELISA according to the manufacturer's instructions.

Apoptosis and ROS analysis by flow cytometry
At 21and 42 days, eight mice (half male, half female) in each group were humanely sacrificed, and the lungs were immediately stored at 4 °C phosphate buffer saline, and then were cut up to make a cell suspension, which was filtered through a 350-mesh nylon screen. The cells were washed twice with ice-cold phosphate buffer saline (PBS, pH 7.2-7.4), and then suspended in PBS at a concentration of 1 × 10 6 cells/ml. Thereafter, a total of 100 µL cell suspension were transfer into a 5 mL culture tube and centrifuged at 800g for 5 min for apoptosis testing. PE Annexin V and 7-aminoactinomycin (7-AAd) staining were used to dye the specimens. The mixture was gently shaken and then left in the dark for 15 minutes. Then 400 µl binding buffer was added to the tube, and the apoptosis rate of lung cells was detected and analysed by flow cytometry (FACS Calibur, BD, USA). The results were analysed using the Mod Fit LT for Mac V3.0 computer program.
Some 300 μL of the aforementioned cell suspensions were taken and transferred to another centrifuge tube, and stained with 10 μM DCFH-DA for 20 min at 37 °C for ROS testing. Then the cells were washed with PBS  and centrifuged (600g, 5 min) once more. The supernatant was discarded, and cells were resuspended in 0.5 ml PBS and counted by flow cytometry (FACS Calibur, BD, USA).

Determination of mRNA expression levels of antioxidant enzymes, inflammatory cytokines, and apoptotic proteins in the lung by qRT-PCR
At 21 and 42 days, lungs of eight mice in each group (half male, half female) were removed, stored in liquid nitrogen, and then Total RNA was extracted with the RNAiso Plus, and reverse transcribed into cDNA by using the Prim-Script™ RT reagent Kit as per the manufacture's specification. The gene sequences of CuZnSOD, MnSOD, CAT, GSH-PX, Caspase 3, Caspase 9, Bax, Bak, Bcl-2, Bcl-xL, NF-κB, IκB, COX-2, IL-1β, IL-8, IL-6, and TNF-α IL-2, IL-4 and IL-10 were retrieved from NCBI, and the primers of these genes (Tables 1-3) were synthesised by Sangon Biotech (Shanghai, China). β-actin of mice was chosen as the reference gene. qRT-PCR reaction conducted on a C1000 Thermal Cycler (BIO RAD, USA) by using the SYBR ® Premix Ex TaqII (Takara, China) in accordance with the standard steps. All data output from the qRT-PCR experiments were analysed using the 2 -ΔΔCT method.

Determination of protein expression levels of apoptotic proteins and inflammatory cytokines in the lung by Western blot assay
At 21 and 42 days, lungs of eight mice in each group (half male, half female) were removed, stored in liquid nitrogen, and then homogenised in liquid nitrogen by using a mortar and pestle. The total protein in each sample was carried out using RIPA lysis buffer, and protein contents of lungs were measured by using the BCA Protein Assay kit. Then the protein samples were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (10%-15% gels), and protein standards were used as molecular weight marker. After electrophoresis, proteins were transferred to nitrocellulose filter membranes. The membranes were blocked with 5 % non-fat-dried milk in phosphatebuffered saline with 0.1 % Tween 20 (PBST) for 1 h. After primary antibodies were incubated overnight at 4 °C, the membranes were washed three times with PBS for 10 min and incubated with biotin-conjugated secondary antibodies for 1 h with gentle shaking and washed again with PBST. Blots were visualised by ECLTM (Bio-Rad, Hercules, CA, USA) and X-ray film. The detected indicators were: cleaved-caspase-3, cleaved-caspase-9, Bax, Bak, Bcl-2, Bcl-xL, NF-κB, IκB, COX-2, IL-1β, IL-8, IL-6, TNF-α, IL-2, IL-4, and IL-10.

Statistical analysis
The significance of difference was analyzed by the SPSS version 17.0. The results were shown as means ± standard deviation. The analysis was performed with the one-way analysis of variance (ANOVA). The differences between control and experimental group(s) at p < 0.05 were considered significant.