International Journal of
Molecular Sciences Article
Intervention of Grape Seed Proanthocyanidin Extract on the Subchronic Immune Injury in Mice Induced by Aflatoxin B1 Miao Long † , Yi Zhang † , Peng Li, Shu-Hua Yang, Wen-Kui Zhang, Jian-Xin Han, Yuan Wang and Jian-Bin He * Key Laboratory of Zoonosis of Liaoning Province, College of Animal Science & Veterinary Medicine, Shenyang Agricultural University, Shenyang 110866, China; [email protected] (M.L.); [email protected] (Y.Z.); [email protected] (P.L.); [email protected] (S.-H.Y.); [email protected] (W.-K.Z.); [email protected] (J.-X.H.); [email protected] (Y.W.) * Correspondence: [email protected]; Tel./Fax: +86-24-8848-7156 † These authors contributed equally to this study. Academic Editor: Vladimír Kˇren Received: 7 March 2016; Accepted: 31 March 2016; Published: 8 April 2016
Abstract: The aim was to investigate the prevention of grape seed proanthocyanidin extract (GSPE) on the subchronic immune injury induced by aflatoxin B1 (AFB1) and the possible ameliorating effect of GSPE in mice. The subchronic AFB1-induced immune injury mice model was set up with the continuous administration of 100 µg/kg body weight (BW) AFB1 for six weeks by intragastric administration. Then, intervention with different doses (50 and 100 mg/kg BW) of GSPE was conducted on mice to analyze the changes of body weight, immune organ index, antioxidant capability of spleen, serum immunoglobulin content, and the expression levels of inflammatory cytokines. The prevention of GSPE on the immune injury induced by AFB1 was studied. The GSPE could relieve the AFB1-induced reduction of body weight gain and the atrophy of the immune organ. The malondialdehyde (MDA) level of the spleen in the AFB1 model group significantly increased, but levels of catalase (CAT), glutathione (GSH), glutathione peroxidase (GSH-PX ), and superoxide dismutase (SOD) significantly decreased. The GSPE could significantly inhibit the oxidative stress injury of the spleen induced by AFB1. AFB1 exposure could not significantly change the contents of IgA, IgG, or IgM. AFB1 significantly improved the expression of interleukin 1β (IL-1β), IL-6, tumor necrosis factor α (TNF-α), and interferon γ (IFN-γ). Additionally, GSPE could decrease the expression of these four proinflammatory factors to different degrees and inhibit the inflammatory reaction of mice. The results suggest that GSPE alleviates AFB1-induced oxidative stress and significantly improves the immune injury of mice induced by AFB1. Keywords: grape seed proanthocyanidin extract; AFB1; immune injury; alleviate; mice
1. Introduction Aflatoxins are a group of secondary metabolites produced by strains of some Aspergillums species, mainly A. flavus and A. parasiticus. Among the aflatoxins, aflatoxin B1 (AFB1) is the most biologically active form, and it causes poor performance, liver lesions, immunosuppression, alters blood profiles, and gut morphology, and partially damages internal organ in many animals [1–3]. Although in vivo effects of AFB1 toxicity vary considerably with the animal species, extent and duration of exposure, age, and nutritional status [4], AFB1 is harmful to animal and human health, mainly due to effects such as increasing free radical production, leading to oxidative damage and lipid peroxidation, which might ultimately lead to cell damage and death [5,6]. As oxidative stress plays a key role in the toxicity
Int. J. Mol. Sci. 2016, 17, 516; doi:10.3390/ijms17040516
www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2016, 17, 516 Int. J. Mol. Sci. 2016, 17, 516
2 of 10 2 of 10
plays a key role in the toxicity mechanism of AFB1; therefore, some antioxidants might be useful in preventing or the detrimental effects ofmight chronic 1 toxicity in animals [7,8]. mechanism of attenuating AFB1; therefore, some antioxidants beAFB useful in preventing or attenuating the Grape effects seed proanthocyanindin extract (GSPE)[7,8]. is derived from grape seeds. They have detrimental of chronic AFB1 toxicity in animals demonstrated a marked spectrum of biological, pharmacological, chemoprotective Grape seed proanthocyanindin extract (GSPE) is derived therapeutic, from grapeand seeds. They have properties against oxygen free radicals and oxidative stress, and they also have shown the ability to demonstrated a marked spectrum of biological, pharmacological, therapeutic, and chemoprotective mediate anti-inflammatory [9,10]. Many data have shown that they the ability of GSPE improve properties against oxygen free radicals and oxidative stress, and also have showntothe ability antioxidant defenses for protecting the main organ function, such as preventing liver injury in the to mediate anti-inflammatory [9,10]. Many data have shown that the ability of GSPE to improve carbon tetrachlorideinduced and ischemia/reperfusion-induced [11,12], alleviating Arsenic-induced antioxidant defenses for protecting the main organ function, such as preventing liver injury in the oxidative reproductive toxicity [13], and protecting the renal function from Cisplatin-induced carbon tetrachloride- induced and ischemia/reperfusion-induced [11,12], alleviating Arsenic-induced nephrotoxicity [14]. Recent studies have protecting also shown has from anti-inflammatory and oxidative reproductive toxicity [13], and the that renalGSPE function Cisplatin-induced immunodulatory properties [15–19]. nephrotoxicity [14]. Recent studies have also shown that GSPE has anti-inflammatory and However, it has not been clear immunodulatory properties [15–19].whether GSPE could reverse the inflammatory status induced by AFB1. In this study, thebeen effects of whether AFB1 onGSPE oxidative and thestatus expression of However, it has not clear could status, reverseimmunity, the inflammatory induced inflammation-related genes spleens in mice investigated, and whether with by AFB1. In this study, the of effects of AFB1 onwere oxidative status, immunity, andthe thetreatment expression of GSPE was able to counteract its negative effects was also studied. inflammation-related genes of spleens in mice were investigated, and whether the treatment with GSPE was able to counteract its negative effects was also studied. 2. Results 2. Results 2.1. Effects on Body Weight and Organ Index 2.1. Effects on Body Weight and Organ Index From Figure 1, it can be seen that the body weight in low doses of the AFB1 group was From Figure 1, itthan can be seen thatcontrol the body weight low doses of the AFB1 group was significantly significantly lower that in the group at in the end of the five-week experiment (p < 0.05; lower than that in the control group at the end of the five-week experiment (p < 0.05; Figure 1). However, Figure 1). However, compared with AFB1 group, the body weight of mice both in the high-dose compared with the AFB1 group, the body weight of group mice both in the high-dose GSPE + AFB1 group GSPE + AFB1 group and the low-dose GSPE + AFB1 significantly increased (p < 0.05; Figure 1). and theFigure low-dose AFB1that group (p < index 0.05; Figure 1). From Figure 2, in it can From 2, it GSPE can be+seen the significantly spleen indexincreased and thymus significantly decreased the be seengroup that the spleen index andcontrol thymusgroup index(psignificantly decreased in thethese AFB1indexes group compared AFB1 compared with the < 0.05; Figure 2). However, increased with control (p 0.05). row, meanssuperscript superscript with areare notnot significantly different (p > 0.05).
2.3. Effect Effect on on Antioxidant AntioxidantofofSpleen Spleen 2.3. Spleenoxidant oxidant antioxidant parameters mice treatment with GSPE AFB1 are Spleen andand antioxidant parameters of miceoftreatment with GSPE or AFB1 areor summarized summarized in Table 2. The administration of AFB1 resulted in a significant increase in spleen in Table 2. The administration of AFB1 resulted in a significant increase in spleen malondialdehyde malondialdehyde (MDA) contentwith when with the group (p < with 0.05).GSPE Treatment with (MDA) content when compared thecompared control group (p 0.05). Activities of catalase (CAT), glutathione high dose and low dose GSPE groups (p > 0.05). Activities of catalase (CAT), glutathione (GSH), (GSH), glutathione peroxidase (GSH-P X), and superoxide all decreased AFB1 glutathione peroxidase (GSH-PX ), and superoxide dismutasedismutase (SOD) all (SOD) decreased with AFB1with treatment treatment compared with the control group (p < 0.05). However, GSPE improved these antioxidant compared with the control group (p < 0.05). However, GSPE improved these antioxidant enzyme enzyme activities < 0.05). These resultsthat indicate GSPE significantly the antioxidant activities (p < 0.05).(pThese results indicate GSPEthat significantly improves improves the antioxidant activities activities of the spleen and reduces the serious oxidative damage induced by AFB1. of the spleen and reduces the serious oxidative damage induced by AFB1.
Int. J. Mol. Sci. 2016, 17, 516 Int. J. Mol. Sci. 2016, 17, 516
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Table 2. Effect of GSPE on spleen antioxidant parameters in mice induced by AFB1. Table 2. Effect of GSPE on spleen antioxidant parameters in mice induced by AFB1. Group Group
Control Control GSPE (100 mg/kg) AFB1 GSPE(100 (100 μg/kg) mg/kg) GSPE (50 mg/kg) AFB1 (100 µg/kg)+ AFB1 100 μg/kg GSPE (50 mg/kg) + GSPE AFB1(100 100mg/kg) µg/kg + AFB1 100 μg/kg
MDA CAT GSH MDA CAT nmol/mgprot U/mgprot U/mgprot GSH U/mgprot nmol/mgprot U/mgprot 2.82 ± 0.36 c 56.32 ± 3.16 c 279.64 ± 18.14 c c c 2.82 0.36 cc 56.32 ˘ ±3.16 2.15 ˘ ± 0.31 75.14 2.96 d 279.64 396.74˘±18.14 20.63 d d a 396.74 9.87 ˘ ± 0.42 28.17 2.14 196.66˘±20.63 15.43d a 2.15 0.31 ca 75.14 ˘ ±2.96
GSH-PX GSH-PX U/mgprot U/mgprot 14.60 ± 1.725 b b 14.60 19.74˘± 1.725 1.654 c 8.842˘± 1.654 0.475ca 19.74
SOD U/mgprot SOD U/mgprot 156.34 ± 23.16 b b 156.34 214.96˘±23.16 22.34 d d 84.36˘± 22.34 20.47 a 214.96 a 84.36 109.14˘±20.47 19.21 bc
9.87 0.42 ab 5.34 ˘ ± 0.38
a 28.17 ˘ ±2.14 38.75 2.66 b
a 196.66 234.28˘±15.43 18.64 b
a 8.842 12.11˘± 0.475 0.362 b
5.34 ˘ 0.38 b
38.75 ˘ 2.66 b
234.28 ˘ 18.64 b
12.11 ˘ 0.362 b
4.12 ± 0.29 b
43.23 ± 3.41 b
256.54 ± 19.87 b
13.46 ± 0.330 b
109.14 ˘ 19.21 bc
123.45 ± 18.36 b
GSPE (100 mg/kg) + b a,b,c,d Means within 4.12 0.29 b with 43.23different ˘ 3.41 b letters 256.54are ˘ 19.87 13.46different, ˘ 0.330 b p
Molecular Sciences Article
Intervention of Grape Seed Proanthocyanidin Extract on the Subchronic Immune Injury in Mice Induced by Aflatoxin B1 Miao Long † , Yi Zhang † , Peng Li, Shu-Hua Yang, Wen-Kui Zhang, Jian-Xin Han, Yuan Wang and Jian-Bin He * Key Laboratory of Zoonosis of Liaoning Province, College of Animal Science & Veterinary Medicine, Shenyang Agricultural University, Shenyang 110866, China; [email protected] (M.L.); [email protected] (Y.Z.); [email protected] (P.L.); [email protected] (S.-H.Y.); [email protected] (W.-K.Z.); [email protected] (J.-X.H.); [email protected] (Y.W.) * Correspondence: [email protected]; Tel./Fax: +86-24-8848-7156 † These authors contributed equally to this study. Academic Editor: Vladimír Kˇren Received: 7 March 2016; Accepted: 31 March 2016; Published: 8 April 2016
Abstract: The aim was to investigate the prevention of grape seed proanthocyanidin extract (GSPE) on the subchronic immune injury induced by aflatoxin B1 (AFB1) and the possible ameliorating effect of GSPE in mice. The subchronic AFB1-induced immune injury mice model was set up with the continuous administration of 100 µg/kg body weight (BW) AFB1 for six weeks by intragastric administration. Then, intervention with different doses (50 and 100 mg/kg BW) of GSPE was conducted on mice to analyze the changes of body weight, immune organ index, antioxidant capability of spleen, serum immunoglobulin content, and the expression levels of inflammatory cytokines. The prevention of GSPE on the immune injury induced by AFB1 was studied. The GSPE could relieve the AFB1-induced reduction of body weight gain and the atrophy of the immune organ. The malondialdehyde (MDA) level of the spleen in the AFB1 model group significantly increased, but levels of catalase (CAT), glutathione (GSH), glutathione peroxidase (GSH-PX ), and superoxide dismutase (SOD) significantly decreased. The GSPE could significantly inhibit the oxidative stress injury of the spleen induced by AFB1. AFB1 exposure could not significantly change the contents of IgA, IgG, or IgM. AFB1 significantly improved the expression of interleukin 1β (IL-1β), IL-6, tumor necrosis factor α (TNF-α), and interferon γ (IFN-γ). Additionally, GSPE could decrease the expression of these four proinflammatory factors to different degrees and inhibit the inflammatory reaction of mice. The results suggest that GSPE alleviates AFB1-induced oxidative stress and significantly improves the immune injury of mice induced by AFB1. Keywords: grape seed proanthocyanidin extract; AFB1; immune injury; alleviate; mice
1. Introduction Aflatoxins are a group of secondary metabolites produced by strains of some Aspergillums species, mainly A. flavus and A. parasiticus. Among the aflatoxins, aflatoxin B1 (AFB1) is the most biologically active form, and it causes poor performance, liver lesions, immunosuppression, alters blood profiles, and gut morphology, and partially damages internal organ in many animals [1–3]. Although in vivo effects of AFB1 toxicity vary considerably with the animal species, extent and duration of exposure, age, and nutritional status [4], AFB1 is harmful to animal and human health, mainly due to effects such as increasing free radical production, leading to oxidative damage and lipid peroxidation, which might ultimately lead to cell damage and death [5,6]. As oxidative stress plays a key role in the toxicity
Int. J. Mol. Sci. 2016, 17, 516; doi:10.3390/ijms17040516
www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2016, 17, 516 Int. J. Mol. Sci. 2016, 17, 516
2 of 10 2 of 10
plays a key role in the toxicity mechanism of AFB1; therefore, some antioxidants might be useful in preventing or the detrimental effects ofmight chronic 1 toxicity in animals [7,8]. mechanism of attenuating AFB1; therefore, some antioxidants beAFB useful in preventing or attenuating the Grape effects seed proanthocyanindin extract (GSPE)[7,8]. is derived from grape seeds. They have detrimental of chronic AFB1 toxicity in animals demonstrated a marked spectrum of biological, pharmacological, chemoprotective Grape seed proanthocyanindin extract (GSPE) is derived therapeutic, from grapeand seeds. They have properties against oxygen free radicals and oxidative stress, and they also have shown the ability to demonstrated a marked spectrum of biological, pharmacological, therapeutic, and chemoprotective mediate anti-inflammatory [9,10]. Many data have shown that they the ability of GSPE improve properties against oxygen free radicals and oxidative stress, and also have showntothe ability antioxidant defenses for protecting the main organ function, such as preventing liver injury in the to mediate anti-inflammatory [9,10]. Many data have shown that the ability of GSPE to improve carbon tetrachlorideinduced and ischemia/reperfusion-induced [11,12], alleviating Arsenic-induced antioxidant defenses for protecting the main organ function, such as preventing liver injury in the oxidative reproductive toxicity [13], and protecting the renal function from Cisplatin-induced carbon tetrachloride- induced and ischemia/reperfusion-induced [11,12], alleviating Arsenic-induced nephrotoxicity [14]. Recent studies have protecting also shown has from anti-inflammatory and oxidative reproductive toxicity [13], and the that renalGSPE function Cisplatin-induced immunodulatory properties [15–19]. nephrotoxicity [14]. Recent studies have also shown that GSPE has anti-inflammatory and However, it has not been clear immunodulatory properties [15–19].whether GSPE could reverse the inflammatory status induced by AFB1. In this study, thebeen effects of whether AFB1 onGSPE oxidative and thestatus expression of However, it has not clear could status, reverseimmunity, the inflammatory induced inflammation-related genes spleens in mice investigated, and whether with by AFB1. In this study, the of effects of AFB1 onwere oxidative status, immunity, andthe thetreatment expression of GSPE was able to counteract its negative effects was also studied. inflammation-related genes of spleens in mice were investigated, and whether the treatment with GSPE was able to counteract its negative effects was also studied. 2. Results 2. Results 2.1. Effects on Body Weight and Organ Index 2.1. Effects on Body Weight and Organ Index From Figure 1, it can be seen that the body weight in low doses of the AFB1 group was From Figure 1, itthan can be seen thatcontrol the body weight low doses of the AFB1 group was significantly significantly lower that in the group at in the end of the five-week experiment (p < 0.05; lower than that in the control group at the end of the five-week experiment (p < 0.05; Figure 1). However, Figure 1). However, compared with AFB1 group, the body weight of mice both in the high-dose compared with the AFB1 group, the body weight of group mice both in the high-dose GSPE + AFB1 group GSPE + AFB1 group and the low-dose GSPE + AFB1 significantly increased (p < 0.05; Figure 1). and theFigure low-dose AFB1that group (p < index 0.05; Figure 1). From Figure 2, in it can From 2, it GSPE can be+seen the significantly spleen indexincreased and thymus significantly decreased the be seengroup that the spleen index andcontrol thymusgroup index(psignificantly decreased in thethese AFB1indexes group compared AFB1 compared with the < 0.05; Figure 2). However, increased with control (p 0.05). row, meanssuperscript superscript with areare notnot significantly different (p > 0.05).
2.3. Effect Effect on on Antioxidant AntioxidantofofSpleen Spleen 2.3. Spleenoxidant oxidant antioxidant parameters mice treatment with GSPE AFB1 are Spleen andand antioxidant parameters of miceoftreatment with GSPE or AFB1 areor summarized summarized in Table 2. The administration of AFB1 resulted in a significant increase in spleen in Table 2. The administration of AFB1 resulted in a significant increase in spleen malondialdehyde malondialdehyde (MDA) contentwith when with the group (p < with 0.05).GSPE Treatment with (MDA) content when compared thecompared control group (p 0.05). Activities of catalase (CAT), glutathione high dose and low dose GSPE groups (p > 0.05). Activities of catalase (CAT), glutathione (GSH), (GSH), glutathione peroxidase (GSH-P X), and superoxide all decreased AFB1 glutathione peroxidase (GSH-PX ), and superoxide dismutasedismutase (SOD) all (SOD) decreased with AFB1with treatment treatment compared with the control group (p < 0.05). However, GSPE improved these antioxidant compared with the control group (p < 0.05). However, GSPE improved these antioxidant enzyme enzyme activities < 0.05). These resultsthat indicate GSPE significantly the antioxidant activities (p < 0.05).(pThese results indicate GSPEthat significantly improves improves the antioxidant activities activities of the spleen and reduces the serious oxidative damage induced by AFB1. of the spleen and reduces the serious oxidative damage induced by AFB1.
Int. J. Mol. Sci. 2016, 17, 516 Int. J. Mol. Sci. 2016, 17, 516
4 of 10 4 of 10
Table 2. Effect of GSPE on spleen antioxidant parameters in mice induced by AFB1. Table 2. Effect of GSPE on spleen antioxidant parameters in mice induced by AFB1. Group Group
Control Control GSPE (100 mg/kg) AFB1 GSPE(100 (100 μg/kg) mg/kg) GSPE (50 mg/kg) AFB1 (100 µg/kg)+ AFB1 100 μg/kg GSPE (50 mg/kg) + GSPE AFB1(100 100mg/kg) µg/kg + AFB1 100 μg/kg
MDA CAT GSH MDA CAT nmol/mgprot U/mgprot U/mgprot GSH U/mgprot nmol/mgprot U/mgprot 2.82 ± 0.36 c 56.32 ± 3.16 c 279.64 ± 18.14 c c c 2.82 0.36 cc 56.32 ˘ ±3.16 2.15 ˘ ± 0.31 75.14 2.96 d 279.64 396.74˘±18.14 20.63 d d a 396.74 9.87 ˘ ± 0.42 28.17 2.14 196.66˘±20.63 15.43d a 2.15 0.31 ca 75.14 ˘ ±2.96
GSH-PX GSH-PX U/mgprot U/mgprot 14.60 ± 1.725 b b 14.60 19.74˘± 1.725 1.654 c 8.842˘± 1.654 0.475ca 19.74
SOD U/mgprot SOD U/mgprot 156.34 ± 23.16 b b 156.34 214.96˘±23.16 22.34 d d 84.36˘± 22.34 20.47 a 214.96 a 84.36 109.14˘±20.47 19.21 bc
9.87 0.42 ab 5.34 ˘ ± 0.38
a 28.17 ˘ ±2.14 38.75 2.66 b
a 196.66 234.28˘±15.43 18.64 b
a 8.842 12.11˘± 0.475 0.362 b
5.34 ˘ 0.38 b
38.75 ˘ 2.66 b
234.28 ˘ 18.64 b
12.11 ˘ 0.362 b
4.12 ± 0.29 b
43.23 ± 3.41 b
256.54 ± 19.87 b
13.46 ± 0.330 b
109.14 ˘ 19.21 bc
123.45 ± 18.36 b
GSPE (100 mg/kg) + b a,b,c,d Means within 4.12 0.29 b with 43.23different ˘ 3.41 b letters 256.54are ˘ 19.87 13.46different, ˘ 0.330 b p
