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Ginsenoside Rg1 enhanced immune responses to infectious bursal disease vaccine in chickens with oxidative stress induced by cyclophosphamide
Ginsenoside Rg1 enhanced immune responses to infectious bursal disease vaccine in chickens with oxidative stress induced by cyclophosphamide S. Bi, X. Chi, Y. Zhang, X. Ma, S. Liang, Y. Wang, and S. H. Hu1 Department of Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou 310058, Zhejiang, P. R. China cytokines, and oxidative parameters. Splenocytes were prepared for lymphocyte proliferation assay. The results showed that oral administration of ginsenoside Rg1 significantly enhanced specific antibody, IFN-γ , and IL-6 responses, and lymphocyte proliferation induced by concanavalin A and lipopolysaccharide in chickens injected with cyclophosphamide. Antioxidant activity of ginsenoside Rg1 was also observed in chickens by increased total antioxidant capacity, total superoxide dismutase, catalase, glutathione peroxidase, glutathione, ascorbic acid, and α-tocopherol, as well as decreased malondialdehyde and protein carbonyl. Therefore, oral administration of Rg1 was shown to improve the immune responses to infectious bursal disease vaccine in chickens suffering from oxidative stress.
ABSTRACT This study was designed to evaluate the effect of oral administration of ginsenoside Rg1 on oxidative stress induced by cyclophosphamide in chickens. Ninety-six chickens were randomly divided into 4 groups, each consisting of 24 birds. Groups 2 and 3 received intramuscular injection of cyclophosphamide at 100 mg/kg body weight for 3 d to induce oxidative stress and immune suppression. Groups 1 and 4 were injected with saline in the same way as groups 2 and 3. Then chickens in group 3 were orally administrated Rg1 of 1 mg/kg body weight in drinking water for 7 d. After that, groups 1 to 3 were orally vaccinated with attenuated infectious bursal disease vaccine (Strain B87). Blood samples were collected for determination of infectious bursal disease virus-specific antibodies,
Key words: Ginsenoside Rg1, immunosuppression, oxidative stress, infectious bursal disease vaccine 2018 Poultry Science 0:1–10 http://dx.doi.org/10.3382/ps/pey132
INTRODUCTION
population, inappropriate management, foodborne mycotoxins, and environmental ammonia (Liu et al., 2017). During oxidative stress, accumulations of reactive oxygen species (ROS) lead to an imbalance in the oxidant system and suppress the immune function (Cunnick et al., 1990). Saponins isolated from the stem and leaf of Panax ginseng C.A. Meyer (GSLS) have been found effective to improve immune responses to vaccines (Zhai et al., 2011a; Li et al., 2016; Ni et al., 2016). Oral administration of GSLS has been found to improve vaccination against Newcastle disease, avian influenza, and infectious bursal disease in chickens (Zhai et al., 2011b; Zhai et al., 2014). In addition, oral administration of GSLS has been observed to enhance the immune responses to Newcastle disease and avian influenza in chickens of immunosuppression and oxidative stress induced by cyclophosphamide (Cy) (Yu et al., 2015a; Yu et al., 2015b). According to the Chinese Veterinary Pharmacopoeia, ginsenoside Rg1 (Rg1) is one of the major constituents in GSLS. In the present study, Rg1 was investigated for its effect on the immune responses to IBDV vaccine and antioxidative activities in chickens of immunosuppression and oxidative stress induced by injection of Cy.
Infectious bursal disease (IBD) is a highly communicated disease of chickens caused by infectious bursal disease virus (IBDV) and is endangering poultry industry by increasing susceptibility to secondary infections and suboptimal responses to vaccinations (Bwana et al., 2017; Kurukulasuriya et al., 2017). The disease increases morbidity and mortality, resulting in huge economic losses in poultry production (Heidari et al., 2010; Shini et al., 2010). Vaccination is one of the important approaches to protection of chickens against infection as IBDV is highly resistant to physical and chemical inactivation (Enterradossi and Saif, 2008). Yet, the efficacy of vaccination is often unexpected because of poor response to IBDV vaccine caused by immunosuppression and many other factors. Oxidative stress is a common reason for the immunosuppression in poultry, which may be caused by highly condensed
C 2018 Poultry Science Association Inc. Received October 24, 2017. Accepted March 19, 2018. 1 Corresponding author: [email protected]
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BI ET AL. Table 1. Experimental design. Group
n
Cy injection
Rg1
Vaccination
1 2 3 4
24 24 24 24
+ +
+
+ + +
(VC), α-tocopherol (VE), malondialdehyde (MDA), protein carbonyl (Carbonyl) were purchased from the Institute of Nanjing Jiancheng Bioengineering (Nanjing, China). All other chemicals were analytic grade.
Figure 1. Chemical structure of ginsenoside Rg1 (C42 H72 O14 ; molecular weight, 801.02).
MATERIALS AND METHODS Animals One-day-old specific-pathogen-free White Leghorn chickens (female) were purchased from Zhejiang Shennong Stock Breeding Inc. (Ningbo, China) and acclimated for 14 d before experiment. The animals were housed into wire cages and the room was set at 35◦ C for the first 3 d and then gradually reduced to 26◦ C. Feed and water were supplied ad libitum. The bird experiment was approved by the Zhejiang University Committee on Animal Care and Use.
Vaccine Attenuated IBDV vaccine (Strain B87) was purchased from Zhejiang EBVAC Bioengineering Co. Ltd. (Hangzhou, China).
Experimental Design Ninety-six chickens were randomly divided into 4 groups, each consisting of 24 birds (Table 1). Groups 2 and 3 received intramuscular injection of Cy at 100 mg/kg body weight (BW) for 3 d to induce oxidative stress and immunosuppression. Groups 1 and 4 were injected with saline in the same way as groups 2 and 3. Then chickens in group 3 were orally administrated Rg1 of 1 mg/kg BW in drinking water for 7 d. After that, groups 2 to 4 were orally vaccinated with IBDV vaccine. Blood samples were collected from 12 chickens before Cy treatment and 7 and 14 d after immunization or 6 chickens 21 d after immunization of each group for determination of IBDV specific titers, cytokines test, and measurement of oxidative parameters. Splenocytes were obtained from 6 chickens of each group and prepared for lymphocyte proliferation assay. Data were compared between groups for their antiIBDV antibody, serum cytokines, lymphocyte proliferations, serum enzymatic, and non-enzymatic antioxidant activities.
Serum IBDV-Specific Antibody Assay Ginseng Saponins Ginsenosides Rg1 (98%) with the chemical structure as shown in Figure 1 was purchased from Chengdu Purify Biotechnology Co. Ltd. (Chengdu, China).
Reagents The infectious bursa disease virus antibody test kit was purchased from IDEXX Laboratories Inc. (Westbrook, ME, USA). Chicken IFN-γ and IL-6 test kit were purchased from Shanghai Lengton Bioscience Co. Ltd. (Shanghai, China). Cyclophosphamide was purchased from Pude Pharmaceutical Co. Ltd. (Shanghai, China). Concanavalin A (ConA), lipopolysaccharides (LPS), and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). Detection kits for test of total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), catalase (CAT), glutathione peroxidase (GSH-PX), glutathione (GSH), ascorbic acid
Serum antibodies to IBDV were tested using ELISA kits according to the manufacturers’ instructions. The relative level of antibody titer in the samples was determined by calculating the sample to positive (S/P) ratio as [(mean of sample optimal density)—(mean of negative control optimal density)]/[(mean of positive control optimal density)—(mean of negative control optimal density). Endpoint titers were calculated with the equation: log10 titer = 1.09 (log 10 S/P) + 3.36 (Flock Check program, IDEXX).
Analysis of Serum IFN-γ and IL-6 Serum gamma interferon (IFN-γ ) levels were measured using an ELISA kit (Shanghai Lengton Bioscience Co. Ltd. Shanghai, China), and serum IL-6 levels were determined by an ELISA kit (Shanghai Lengton Bioscience Co. Ltd. Shanghai, China). Serum cytokine levels were evaluated by interpolation of cytokine standard curves.
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Lymphocyte Proliferation Assay The method was performed as previously reported with minor modifications (Li et al., 2009). Briefly, spleens collected into Hank’s balance salt solution were minced and passed through a fine steel mesh to obtain the cell suspension under aseptic conditions. The cells were washed in Hank’s balance salt solution 3 times by centrifugation (500 × g at 4◦ C for 10 min), and suspended in cold RPMI 1640 supplemented with 0.05 mM 2-mercaptoethanol, 100 IU/mL penicillin, 100 μg/mL streptomycin and 10% heat-inactivated fetal calf serum. Cell viability was assessed with trypan blue exclusion stain, and the cell suspension was adjusted to 5.0 × 106 /mL. Mitogen-induced proliferation was performed by incubating 100 μL of lymphocyte suspension with 100 μL of either ConA (40 μg/mL) or LPS (35 μg/mL) in each well for the MTT assay. RPMI 1640 medium (100 μL) was added to the cell suspension in control wells. Cells were added in triplicate, and incubated in a humidified atmosphere at 5% CO2 at 40◦ C for 44 h. After that, 50 μL MTT (2 mg/mL) was added to each well and incubated for an additional 4 h. The plates were centrifuged (1,000 × g for 10 min) and the supernatant was carefully removed. One hundred and fifty μL of acidic dimethyl sulfoxide (0.04 N HCl) was added to each well and mixed thoroughly by slightly shaking to solubilize the MTT formazan. The mean optical density (OD) was read at 570 nm. The stimulation index (SI) was calculated based on the formula: SI = OD value of mitogen-stimulated cells divided by OD value of non-stimulated cells.
Biochemical Determination The activity of T-AOC, T-SOD, CAT, and GSHPX, and the levels of GSH, VC, VE, MDA, and protein carbonyl content were determined by using test kits according to the manufacturers’ protocols. T-AOC was measured by using the ferric reducing antioxidant ability assay (Benzie and Szeto, 1999). The assay for T-SOD activity involved inhibition of nitroblue tetrazolium reduction with xanthine oxidase used as a superoxide generator (Sun et al., 1988). Catalase activity was measured by evaluation of hydrogen peroxide based on the formation of stable complex with ammonium molybdate (Aebi, 1984). Glutathione peroxidase was measured by monitoring the reduction of t-butyl hydroperoxide (Wheeler et al., 1990). Glutathione was estimated by using 5,5’-dithiobis-(2-nitrobenzoic acid) reagent (Abegg et al., 2012). Ascorbic acid was determined by turning ferric iron to ferrous iron (Zannoni et al., 1974). α-tocopherol was analyzed by the reducing properties of tocopherol (Sloan and Lappin, 1982). Lipid peroxidation in serum was evaluated by the spectrophotometric method based on the reaction between MDA and thiobarbituric acid (Mihara and Uchiyama, 1978). The oxidative damage to proteins was measured by quantification of carbonyl groups based on
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their reaction with 2,4-dinitro-phenylhydrazine to form hydrazones (Pirinccioglu et al., 2010).
Statistical Analysis Analysis of data was performed using SPSS software (version 20.0, SPSS Inc., Chicago, IL). One-way analysis of variance (ANOVA) with Duncan post hoc test was used for multiple comparisons between groups. Values were expressed as the mean ± standard error (SE). P-values of less than 0.05 were considered statistically significant.
RESULTS Serum Antibody Response As shown in Figure 2, prior to Cy-treatment, no specific antibody titers were detected in any group. Antibody titers progressively increased at 2 wk after immunization. However, the antibody titers in chickens treated with Cy (Cy + Vaccine) were found significantly lower than the Vaccine group. Interestingly, the antibody titers in chickens administrated with Rg1 (Cy + Rg1 + Vaccine) after treated with Cy were significantly higher than that without Rg1 (Cy + Vaccine) and even recovered to the levels similar to the Vaccine group.
IFN-γ and IL-6 As shown in Figure 3, before Cy-treatment, there was no significant difference between groups. The IFN-γ and IL-6 concentrations in chickens treated with Cy (Cy + Vaccine) were found significantly decreased when compared with the Vaccine group at 2 wk after vaccination. However, the cytokines in chickens administrated Rg1 (Cy + Rg1 + Vaccine) after treated with Cy were found significantly increased than that without Rg1 (Cy + Vaccine).
Lymphocyte Proliferative Responses to ConA and LPS As illustrated in Figure 4, before Cy-treatment, there was no significant difference between groups. At 1 and 2 wk after vaccination, SI was significantly lower in chickens treated with Cy (Cy + Vaccine) than the Vaccine group. However, SI was significantly higher in chickens administrated with Rg1 after treated with Cy than that without Rg1 (Cy + Vaccine).
Serum T-AOC, T-SOD, CAT, and GSH-PX As shown in Figure 5, before Cy-treatment, there was no significant difference between groups. Treatment with Cy (Cy + Vaccine) significantly decreased the activities of T-AOC, T-SOD, CAT, and GSH-Px when
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Figure 2. Serum antibody responses to IBDV vaccination. Chickens (n = 24/group) were intramuscularly (i.m.) injected with Cy at 100 mg/kg for 3 d to induce immunosuppression and oxidative stress, and then orally administrated Rg1 (1 mg/kg) in drinking water for 7 d. After that, chickens were orally immunized with a live attenuated IBDV vaccine (B87 strain). Blood samples were collected from 12 chickens before Cy treatment and 7 and 14 d after immunization or 6 chickens 21 d after immunization of each group for analysis of IFN-γ and IL-6 concentration by ELISA. The values were represented as mean ± SE. Bars with different letters are statistically different (P < 0.05).
compared with the Vaccine group. However, administration of Rg1 (Cy + Rg1 + Vaccine) significantly inhibited Cy-induced decreases in activities of the antioxidant enzymes.
Serum Levels of GSH, VC, VE, MDA, and Carbonyl As shown in Figure 6, before Cy-treatment, there was no significant difference between groups. Treatment with Cy (Cy + Vaccine) significantly reduced the levels of serum GSH, VC, and VE, and enhanced the MDA and Carbonyl content when compared with the Vaccine group. However, administration of Rg1 (Cy + Rg1 + Vaccine) significantly increased the levels of serum GSH, VC, VE, as well as inhibited elevated MDA and Carbonyl content induced by Cy.
DISSCUSSION In this study, Rg1 was demonstrated effectively to enhance the immune responses in chickens of immunosuppression and oxidative stress induced by Cy. After oral administration of Rg1 for 7 d, immunization of an attenuated IBDV vaccine induced significantly higher specific antibody titers, serum IFN-γ and IL-6, and splenocyte proliferative responses to Con A and LPS. In addition, administration of Rg1 significantly increased antioxidant capacity as indicated by T-AOC, T-SOD, CAT, GSH-PX, GSH, VC, and VE and decreased products of lipid and protein peroxidation such as MDA and Carbonyl. Like previous observation (Zhai et al., 2011b; Zhai et al., 2014), the lag period was found for the chickens to respond to a treatment and the major-
ity of the parameters measured only reached statistical significance at d 14 post-immunization. In animals, immune suppression is closely associated with oxidative stress. The various regulatory functions of immune system ultimately depend on an oxidant/antioxidant balance (Knight, 2000). Increased oxidative stress has been found in correlation with decreased various immune responses involving T cellmediated functions and antibody production (Freed et al., 1987; Ercal et al., 2000; Remans et al., 2004). Reactive oxygen species are produced during immune responses as signaling molecules as well as anti-microbial agents (Syed et al., 2015). However, overproduction of ROS can overwhelm host antioxidants and induce oxidative stress. The mechanisms to protect organism from free radical damage are defense systems involving enzymes such as SOD, CAT, and GSH-PX and nonenzymes such as GSH, VC, and VE. Superoxide dismutase is a class of highly conserved enzymes that catalyze the disputations of superoxide into oxygen and hydrogen peroxide, and sustain low concentration of superoxide radicals (Nagi and Almakki, 2009). This reaction is then followed by CAT inactivation of H2 O2 by converting it to water and oxygen (Ince et al., 2014). Similar to CAT, GSH-PX is another important cellular enzyme. In the presence of reduced GSH, GSH-PX can convert hydrogen peroxide to water and oxygen, and convert nitrate (a by-product of NO radicals) to nitrite (Flatow et al., 2013). Total antioxidant capacity is always used as the most important representative marker of in vivo antioxidative activity and may give more biologically relevant information than individual index (Ghiselli et al., 2000). Non-enzymatic antioxidant system including GSH, VC, and VE are also important in cellular antioxidative defense. GSH is oxidized by GSH-PX to
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Figure 3. Serum IFN-γ and IL-6 concentration. Chickens (n = 24/group) were intramuscularly (i.m.) injected with Cy at 100 mg/kg for 3 d to induce immunosuppression and oxidative stress, and then orally administrated Rg1 (1 mg/kg) in drinking water for 7 d. After that, chickens were orally immunized with a live attenuated IBDV vaccine (B87 strain). Blood samples were collected from 12 chickens before Cy treatment and 7 and 14 d after immunization or 6 chickens 21 d after immunization of each group for analysis of IFN-γ and IL-6 concentration by ELISA. The values were represented as mean ± SE. Bars with different letters are statistically different (P < 0.05).
oxidized glutathione. Ascorbic acid can directly scavenge free radicals in extracellular fluids and regenerate vitamin E in cell membranes and maintain low density lipoprotein (LDL) particle integrity (Niki, 1987; Frei et al., 1989; Harats et al., 1998). Vitamin E, acting as an antioxidant, can inhibit lipid peroxidation and maintain the stability of cell membranes. Malondialdehyde is a major product of lipid peroxidation and is supposed to be a marker of tissue damage (Echeverry et al., 2016). Carbonyl is considered as a surrogate marker of protein peroxidation. Cyclophosphamide is a prominent alkylating agent and has been widely used in treatment of some malignant cancers for more than 50 yr (Sikov et al., 2015). It remains one of the most useful antirejection drugs in bone marrow transplant (Slade et al., 2017). The agent is also used to induce immunosuppression and
oxidative stress in animal models (Sevko et al., 2013; Cho et al., 2015; Cheng et al., 2017). Cyclophosphamide is in vivo metabolized to 4-hydroxycyclophosphamide, aldophosphamide, and ultimate phosphoramide mustard, which induces DNA cross-linking and inhibits DNA synthesis especially in S phase of cells (Perini et al., 2007; Goldstein et al., 2008). Cyclophosphamide can also induce apoptosis by activating Fas and caspases pathway (Lee et al., 1997; Wang and Cai, 1999; Chow and Loo, 2003). In addition, Cy treatment has been found to increase MDA and decrease intracellular SOD and GSH, which change the balance between ROS and antioxidants, resulting in oxidative stress (Tripathi and Jena, 2009; Singh et al., 2015). Similar to previous reports, injection of Cy has been demonstrated as effective in inducing immunosuppression and oxidative stress in chickens (Fan et al., 2013; Chi et al., 2017).
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Figure 4. Splenocyte proliferative responses to ConA and LPS. Chickens (n = 24/group) were intramuscularly (i.m.) injected with Cy at 100 mg/kg for 3 d to induce immunosuppression and oxidative stress, and then orally administrated Rg1 (1 mg/kg) in drinking water for 7 d. After that, chickens were orally immunized with a live attenuated IBDV vaccine (B87 strain). Splenocytes were collected from 6 chickens of each group before Cy treatment and 7, 14, and 21 d after immunization for lymphocyte proliferative assay measured by the MTT method. The values were represented as mean ± SE. Bars with different letters are statistically different (P < 0.05).
The present study showed that Cy treatment decreased antibody, IFN-γ , IL-6, and lymphocytes proliferative responses, as well as reduced activities of T-AOC, T-SOD, CAT, and GSH-PX. Improved immunity of animals by oral administration of ginseng stem and leaf saponins has been reported previously. Li et al. (2016) found elevated antibody responses to foot-and-mouth disease and gut mucosal immunity in mice after oral administration of GSLS. Zhai et al. (2011a) reported enhanced specific antibody and lymphocyte proliferative responses in chickens after medication of GSLS in drinking water. In addition, Yu et al. (2015a) observed that oral administration of GSLS significantly enhanced the immune responses and gut mucosal immunity in chickens with immunosuppression induced by Cy when compared to the birds without GSLS. The group also found that oral
administration of GSLS increased antioxidant activities in chickens with oxidative stress induced by Cy (Yu et al., 2015b). In the present study, enhanced antibody responses to IBD vaccine and lymphocyte proliferation as well as elevated antioxidant activities were observed in chickens with immune suppression and oxidative stress after oral administration of Rg1. As Rg1 is one of the constituents in GSLS, this agent is supposed to contribute the immune modulation and antioxidant activities of GSLS as previously reported. Immune modulatory activities of Rg1 were previously reported. Sun et al. (2007, 2008) found enhanced Th1 (IgG1 and IL-5) and Th2 (IgG2a and IFN-γ ) responses when injection of Rg1 with ovalbumin (OVA) in mice. Yuan et al. (2016) observed increased antibody, lymphocyte proliferative, IFN-γ , and IL-4 responses in mice injected with hepatitis B surface antigen in
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Figure 5. Serum enzymatic antioxidant activities. Chickens (n = 24/group) were intramuscularly (i.m.) injected with Cy at 100 mg/kg for 3 d to induce immunosuppression and oxidative stress, and then orally administrated Rg1 (1 mg/kg) in drinking water for 7 d. After that, chickens were orally immunized with a live attenuated IBDV vaccine (B87strain). Splenocytes were collected from 6 chickens of each group before Cy treatment and 7, 14, and 21 d after immunization for lymphocyte proliferative assay measured by the MTT method. The values are represented as mean ± SE. Bars with different letters are statistically different (P < 0.05).
combination with Rg1. Su et al (2012) observed that Rg1 enhanced immune responses to OVA in C3H/HeB but not in C3H/HeJ mice carrying a defective TLR4 gene. Taken together, the present study demonstrated that oral administration of Rg1 at 1 mg/kg BW enhanced immune responses in immunosuppressed chickens, evidenced by recovered antibody response, serum IFN-γ and IL-6, and splenocyte proliferation induced by ConA and LPS. The increased immune responses paralleled
the enhanced T-AOC, T-SOD, CAT, GSH-PX, GSH, VC, and VE, and decreased protein carbonyl content and MDA in oxidative stress induced by Cy. Therefore, oral administration of Rg1 has been shown as effective in immune modulatory and antioxidant property to improve vaccination in chickens with oxidative stress. As Rg1 is an important constituent in GSLS, the content of this agent is suggested to be a useful parameter for the quality control when GSLS is used for immune enhancement in poultry industry.
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Figure 6. Serum non-enzymatic antioxidant activities. Chickens (n = 24/group) were intramuscularly (i.m.) injected with Cy at 100 mg/kg BW for 3 d to induce immunosuppression and oxidative stress, and then orally administrated Rg1 (1 mg/kg BW) in drinking water for 7 d. After that, chickens were orally immunized with a live attenuated IBDV vaccine (B87 strain). Blood samples were collected from 12 chickens before Cy treatment and 7 and 14 d after immunization or 6 chickens 21 d after immunization of each group for determination of serum GSH, VC, VE, MDA, and carbonyl. The values are represented as mean ± SE. Bars with different letters are statistically different (P < 0.05).
ACKNOWLEDGMENTS
FUNDING
The authors are grateful to the students in the Laboratory of Traditional Chinese Veterinary Medicine (TCVM Lab) for their assistance in the present study.
This study was supported by the project (No. 31172356) of the National Natural Scientific Foundation of China and the Special Fund for Agro-research in the Public Interest of China (20130304–3).
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C. A. Hudis, and E. P. Winer. 2015. Impact of the addition of carboplatin and/or bevacizumab to neoadjuvant once-per-week paclitaxel followed by dose-dense doxorubicin and cyclophosphamide on pathologic complete response rates in stage II to III triple-negative breast cancer: CALGB 40603 (Alliance). J. Clin. Oncol. 33:13–21. Singh, A., M. Kaur, A. Choudhary, and B. Kumar. 2015. Effect of Butea monosperma leaf extracts on cyclophosphamide induced clastogenicity and oxidative stress in mice. Phcog. Res. 7:85–91. Slade, M., J. F. DiPersio, P. Westervelt, R. Vij, M. A. Schroeder, and R. Romee. 2017. Haploidentical hematopoietic cell transplant with post-transplant cyclophosphamide and peripheral blood stem cell grafts in older adults with acute myeloid leukemia or myelodysplastic syndrome. Biol. Blood Marrow Transplant. 23:1736–1743. Sloan, J. C., and T. R. Lappin. 1982. Automated colorimetric determination of α-tocopherol. Clin. Chim. Acta. 119:197–207. Su, F., L. Yuan, L. Zhang, and S. Hu. 2012. Ginsenosides Rg1 and Re act as adjuvant via TLR4 signaling pathway. Vaccine 30:4106– 4112. Sun, J., S. Hu, and X. Song. 2007. Adjuvant effects of protopanaxadiol and protopanaxatriol saponins from ginseng roots on the immune responses to ovalbumin in mice. Vaccine 25:1114–1120. Sun, J., X. Song, and S. Hu. 2008. Ginsenoside Rg1 and aluminum hydroxide synergistically promote immune responses to ovalbumin in BALB/c mice. Clinical and Vaccine Immunology. 15:303– 307. Sun, Y., L. W. Oberley, and Y. Li. 1988. A simple method for clinical assay of superoxide dismutase. Clin. Chem. 34:497–500. Syed, M. S., L. Wang, D. Li, and L. H. Jiang. 2015. TRPM2 channelmediated ROS-sensitive Ca(2+ ) signaling mechanisms in immune cells. Front. Immunol. 6:407. Tripathi, D. N., and G. B. Jena. 2009. Intervention of astaxanthin against cyclophosphamide-induced oxidative stress and DNA damage: a study in mice. Chem. Biol. Interact. 180:398–406.
Wang, G. J., and L. Cai. 1999. Relatively low-dose cyclophosphamide is likely to induce apoptotic cell death in rat thymus through Fas/Fas ligand pathway. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis. 427:125–133. Wheeler, C. R., J. A. Salzman, N. M. Elsayed, S. T. Omaye, and D. J. Korte. 1990. Automated assays for superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase activity. Anal. Biochem. 184:193–199. Yu, J., F. S. Shi, and S. Hu. 2015. Improved immune responses to a bivalent vaccine of Newcastle disease and avian influenza in chickens by ginseng stem-leaf saponins. Vet. Immunol. Immunopathol. 167:147–155. Yu, J., Y. Chen, L. Zhai, L. Zhang, Y. Xu, S. Wang, and S. Hu. 2015. Antioxidative effect of ginseng stem-leaf saponins on oxidative stress induced by cyclophosphamide in chickens. Poult. Sci. 94:927–933. Yuan, D., Q. Yuan, Q. Cui, C. Liu, Z. Zhou, H. Zhao, Y. Dun, T. Wang, and C. Zhang. 2016. Vaccine adjuvant ginsenoside Rg1 enhances immune responses against hepatitis B surface antigen in mice. Can. J. Physiol. Pharmacol. 94:676–681. Zannoni, V., M. Lynch, S. Goldstein, and P. Sato. 1974. A rapid micromethod for the determination of ascorbic acid in plasma and tissues. Biochem. Med. 11:41–48. Zhai, L., Y. Li, W. Wang, Y. Wang, and S. Hu. 2011. Effect of oral administration of ginseng stem-and-leaf saponins (GSLS) on the immune responses to Newcastle disease vaccine in chickens. Vaccine 29:5007–5014. Zhai, L., Y. Li, W. Wang, and S. Hu. 2011. Enhancement of humoral immune responses to inactivated Newcastle disease and avian influenza vaccines by oral administration of ginseng stem-and-leaf saponins in chickens. Poult. Sci. 90:1955–1959. Zhai, L., Y. Wang, J. Yu, and S. Hu. 2014. Enhanced immune responses of chickens to oral vaccination against infectious bursal disease by ginseng stem-leaf saponins. Poult. Sci. 93: 2473–2481.
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ABSTRACT This study was designed to evaluate the effect of oral administration of ginsenoside Rg1 on oxidative stress induced by cyclophosphamide in chickens. Ninety-six chickens were randomly divided into 4 groups, each consisting of 24 birds. Groups 2 and 3 received intramuscular injection of cyclophosphamide at 100 mg/kg body weight for 3 d to induce oxidative stress and immune suppression. Groups 1 and 4 were injected with saline in the same way as groups 2 and 3. Then chickens in group 3 were orally administrated Rg1 of 1 mg/kg body weight in drinking water for 7 d. After that, groups 1 to 3 were orally vaccinated with attenuated infectious bursal disease vaccine (Strain B87). Blood samples were collected for determination of infectious bursal disease virus-specific antibodies,
Key words: Ginsenoside Rg1, immunosuppression, oxidative stress, infectious bursal disease vaccine 2018 Poultry Science 0:1–10 http://dx.doi.org/10.3382/ps/pey132
INTRODUCTION
population, inappropriate management, foodborne mycotoxins, and environmental ammonia (Liu et al., 2017). During oxidative stress, accumulations of reactive oxygen species (ROS) lead to an imbalance in the oxidant system and suppress the immune function (Cunnick et al., 1990). Saponins isolated from the stem and leaf of Panax ginseng C.A. Meyer (GSLS) have been found effective to improve immune responses to vaccines (Zhai et al., 2011a; Li et al., 2016; Ni et al., 2016). Oral administration of GSLS has been found to improve vaccination against Newcastle disease, avian influenza, and infectious bursal disease in chickens (Zhai et al., 2011b; Zhai et al., 2014). In addition, oral administration of GSLS has been observed to enhance the immune responses to Newcastle disease and avian influenza in chickens of immunosuppression and oxidative stress induced by cyclophosphamide (Cy) (Yu et al., 2015a; Yu et al., 2015b). According to the Chinese Veterinary Pharmacopoeia, ginsenoside Rg1 (Rg1) is one of the major constituents in GSLS. In the present study, Rg1 was investigated for its effect on the immune responses to IBDV vaccine and antioxidative activities in chickens of immunosuppression and oxidative stress induced by injection of Cy.
Infectious bursal disease (IBD) is a highly communicated disease of chickens caused by infectious bursal disease virus (IBDV) and is endangering poultry industry by increasing susceptibility to secondary infections and suboptimal responses to vaccinations (Bwana et al., 2017; Kurukulasuriya et al., 2017). The disease increases morbidity and mortality, resulting in huge economic losses in poultry production (Heidari et al., 2010; Shini et al., 2010). Vaccination is one of the important approaches to protection of chickens against infection as IBDV is highly resistant to physical and chemical inactivation (Enterradossi and Saif, 2008). Yet, the efficacy of vaccination is often unexpected because of poor response to IBDV vaccine caused by immunosuppression and many other factors. Oxidative stress is a common reason for the immunosuppression in poultry, which may be caused by highly condensed
C 2018 Poultry Science Association Inc. Received October 24, 2017. Accepted March 19, 2018. 1 Corresponding author: [email protected]
1 Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pey132/4962447 by University of Durham user on 10 April 2018
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BI ET AL. Table 1. Experimental design. Group
n
Cy injection
Rg1
Vaccination
1 2 3 4
24 24 24 24
+ +
+
+ + +
(VC), α-tocopherol (VE), malondialdehyde (MDA), protein carbonyl (Carbonyl) were purchased from the Institute of Nanjing Jiancheng Bioengineering (Nanjing, China). All other chemicals were analytic grade.
Figure 1. Chemical structure of ginsenoside Rg1 (C42 H72 O14 ; molecular weight, 801.02).
MATERIALS AND METHODS Animals One-day-old specific-pathogen-free White Leghorn chickens (female) were purchased from Zhejiang Shennong Stock Breeding Inc. (Ningbo, China) and acclimated for 14 d before experiment. The animals were housed into wire cages and the room was set at 35◦ C for the first 3 d and then gradually reduced to 26◦ C. Feed and water were supplied ad libitum. The bird experiment was approved by the Zhejiang University Committee on Animal Care and Use.
Vaccine Attenuated IBDV vaccine (Strain B87) was purchased from Zhejiang EBVAC Bioengineering Co. Ltd. (Hangzhou, China).
Experimental Design Ninety-six chickens were randomly divided into 4 groups, each consisting of 24 birds (Table 1). Groups 2 and 3 received intramuscular injection of Cy at 100 mg/kg body weight (BW) for 3 d to induce oxidative stress and immunosuppression. Groups 1 and 4 were injected with saline in the same way as groups 2 and 3. Then chickens in group 3 were orally administrated Rg1 of 1 mg/kg BW in drinking water for 7 d. After that, groups 2 to 4 were orally vaccinated with IBDV vaccine. Blood samples were collected from 12 chickens before Cy treatment and 7 and 14 d after immunization or 6 chickens 21 d after immunization of each group for determination of IBDV specific titers, cytokines test, and measurement of oxidative parameters. Splenocytes were obtained from 6 chickens of each group and prepared for lymphocyte proliferation assay. Data were compared between groups for their antiIBDV antibody, serum cytokines, lymphocyte proliferations, serum enzymatic, and non-enzymatic antioxidant activities.
Serum IBDV-Specific Antibody Assay Ginseng Saponins Ginsenosides Rg1 (98%) with the chemical structure as shown in Figure 1 was purchased from Chengdu Purify Biotechnology Co. Ltd. (Chengdu, China).
Reagents The infectious bursa disease virus antibody test kit was purchased from IDEXX Laboratories Inc. (Westbrook, ME, USA). Chicken IFN-γ and IL-6 test kit were purchased from Shanghai Lengton Bioscience Co. Ltd. (Shanghai, China). Cyclophosphamide was purchased from Pude Pharmaceutical Co. Ltd. (Shanghai, China). Concanavalin A (ConA), lipopolysaccharides (LPS), and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). Detection kits for test of total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), catalase (CAT), glutathione peroxidase (GSH-PX), glutathione (GSH), ascorbic acid
Serum antibodies to IBDV were tested using ELISA kits according to the manufacturers’ instructions. The relative level of antibody titer in the samples was determined by calculating the sample to positive (S/P) ratio as [(mean of sample optimal density)—(mean of negative control optimal density)]/[(mean of positive control optimal density)—(mean of negative control optimal density). Endpoint titers were calculated with the equation: log10 titer = 1.09 (log 10 S/P) + 3.36 (Flock Check program, IDEXX).
Analysis of Serum IFN-γ and IL-6 Serum gamma interferon (IFN-γ ) levels were measured using an ELISA kit (Shanghai Lengton Bioscience Co. Ltd. Shanghai, China), and serum IL-6 levels were determined by an ELISA kit (Shanghai Lengton Bioscience Co. Ltd. Shanghai, China). Serum cytokine levels were evaluated by interpolation of cytokine standard curves.
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Lymphocyte Proliferation Assay The method was performed as previously reported with minor modifications (Li et al., 2009). Briefly, spleens collected into Hank’s balance salt solution were minced and passed through a fine steel mesh to obtain the cell suspension under aseptic conditions. The cells were washed in Hank’s balance salt solution 3 times by centrifugation (500 × g at 4◦ C for 10 min), and suspended in cold RPMI 1640 supplemented with 0.05 mM 2-mercaptoethanol, 100 IU/mL penicillin, 100 μg/mL streptomycin and 10% heat-inactivated fetal calf serum. Cell viability was assessed with trypan blue exclusion stain, and the cell suspension was adjusted to 5.0 × 106 /mL. Mitogen-induced proliferation was performed by incubating 100 μL of lymphocyte suspension with 100 μL of either ConA (40 μg/mL) or LPS (35 μg/mL) in each well for the MTT assay. RPMI 1640 medium (100 μL) was added to the cell suspension in control wells. Cells were added in triplicate, and incubated in a humidified atmosphere at 5% CO2 at 40◦ C for 44 h. After that, 50 μL MTT (2 mg/mL) was added to each well and incubated for an additional 4 h. The plates were centrifuged (1,000 × g for 10 min) and the supernatant was carefully removed. One hundred and fifty μL of acidic dimethyl sulfoxide (0.04 N HCl) was added to each well and mixed thoroughly by slightly shaking to solubilize the MTT formazan. The mean optical density (OD) was read at 570 nm. The stimulation index (SI) was calculated based on the formula: SI = OD value of mitogen-stimulated cells divided by OD value of non-stimulated cells.
Biochemical Determination The activity of T-AOC, T-SOD, CAT, and GSHPX, and the levels of GSH, VC, VE, MDA, and protein carbonyl content were determined by using test kits according to the manufacturers’ protocols. T-AOC was measured by using the ferric reducing antioxidant ability assay (Benzie and Szeto, 1999). The assay for T-SOD activity involved inhibition of nitroblue tetrazolium reduction with xanthine oxidase used as a superoxide generator (Sun et al., 1988). Catalase activity was measured by evaluation of hydrogen peroxide based on the formation of stable complex with ammonium molybdate (Aebi, 1984). Glutathione peroxidase was measured by monitoring the reduction of t-butyl hydroperoxide (Wheeler et al., 1990). Glutathione was estimated by using 5,5’-dithiobis-(2-nitrobenzoic acid) reagent (Abegg et al., 2012). Ascorbic acid was determined by turning ferric iron to ferrous iron (Zannoni et al., 1974). α-tocopherol was analyzed by the reducing properties of tocopherol (Sloan and Lappin, 1982). Lipid peroxidation in serum was evaluated by the spectrophotometric method based on the reaction between MDA and thiobarbituric acid (Mihara and Uchiyama, 1978). The oxidative damage to proteins was measured by quantification of carbonyl groups based on
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their reaction with 2,4-dinitro-phenylhydrazine to form hydrazones (Pirinccioglu et al., 2010).
Statistical Analysis Analysis of data was performed using SPSS software (version 20.0, SPSS Inc., Chicago, IL). One-way analysis of variance (ANOVA) with Duncan post hoc test was used for multiple comparisons between groups. Values were expressed as the mean ± standard error (SE). P-values of less than 0.05 were considered statistically significant.
RESULTS Serum Antibody Response As shown in Figure 2, prior to Cy-treatment, no specific antibody titers were detected in any group. Antibody titers progressively increased at 2 wk after immunization. However, the antibody titers in chickens treated with Cy (Cy + Vaccine) were found significantly lower than the Vaccine group. Interestingly, the antibody titers in chickens administrated with Rg1 (Cy + Rg1 + Vaccine) after treated with Cy were significantly higher than that without Rg1 (Cy + Vaccine) and even recovered to the levels similar to the Vaccine group.
IFN-γ and IL-6 As shown in Figure 3, before Cy-treatment, there was no significant difference between groups. The IFN-γ and IL-6 concentrations in chickens treated with Cy (Cy + Vaccine) were found significantly decreased when compared with the Vaccine group at 2 wk after vaccination. However, the cytokines in chickens administrated Rg1 (Cy + Rg1 + Vaccine) after treated with Cy were found significantly increased than that without Rg1 (Cy + Vaccine).
Lymphocyte Proliferative Responses to ConA and LPS As illustrated in Figure 4, before Cy-treatment, there was no significant difference between groups. At 1 and 2 wk after vaccination, SI was significantly lower in chickens treated with Cy (Cy + Vaccine) than the Vaccine group. However, SI was significantly higher in chickens administrated with Rg1 after treated with Cy than that without Rg1 (Cy + Vaccine).
Serum T-AOC, T-SOD, CAT, and GSH-PX As shown in Figure 5, before Cy-treatment, there was no significant difference between groups. Treatment with Cy (Cy + Vaccine) significantly decreased the activities of T-AOC, T-SOD, CAT, and GSH-Px when
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Figure 2. Serum antibody responses to IBDV vaccination. Chickens (n = 24/group) were intramuscularly (i.m.) injected with Cy at 100 mg/kg for 3 d to induce immunosuppression and oxidative stress, and then orally administrated Rg1 (1 mg/kg) in drinking water for 7 d. After that, chickens were orally immunized with a live attenuated IBDV vaccine (B87 strain). Blood samples were collected from 12 chickens before Cy treatment and 7 and 14 d after immunization or 6 chickens 21 d after immunization of each group for analysis of IFN-γ and IL-6 concentration by ELISA. The values were represented as mean ± SE. Bars with different letters are statistically different (P < 0.05).
compared with the Vaccine group. However, administration of Rg1 (Cy + Rg1 + Vaccine) significantly inhibited Cy-induced decreases in activities of the antioxidant enzymes.
Serum Levels of GSH, VC, VE, MDA, and Carbonyl As shown in Figure 6, before Cy-treatment, there was no significant difference between groups. Treatment with Cy (Cy + Vaccine) significantly reduced the levels of serum GSH, VC, and VE, and enhanced the MDA and Carbonyl content when compared with the Vaccine group. However, administration of Rg1 (Cy + Rg1 + Vaccine) significantly increased the levels of serum GSH, VC, VE, as well as inhibited elevated MDA and Carbonyl content induced by Cy.
DISSCUSSION In this study, Rg1 was demonstrated effectively to enhance the immune responses in chickens of immunosuppression and oxidative stress induced by Cy. After oral administration of Rg1 for 7 d, immunization of an attenuated IBDV vaccine induced significantly higher specific antibody titers, serum IFN-γ and IL-6, and splenocyte proliferative responses to Con A and LPS. In addition, administration of Rg1 significantly increased antioxidant capacity as indicated by T-AOC, T-SOD, CAT, GSH-PX, GSH, VC, and VE and decreased products of lipid and protein peroxidation such as MDA and Carbonyl. Like previous observation (Zhai et al., 2011b; Zhai et al., 2014), the lag period was found for the chickens to respond to a treatment and the major-
ity of the parameters measured only reached statistical significance at d 14 post-immunization. In animals, immune suppression is closely associated with oxidative stress. The various regulatory functions of immune system ultimately depend on an oxidant/antioxidant balance (Knight, 2000). Increased oxidative stress has been found in correlation with decreased various immune responses involving T cellmediated functions and antibody production (Freed et al., 1987; Ercal et al., 2000; Remans et al., 2004). Reactive oxygen species are produced during immune responses as signaling molecules as well as anti-microbial agents (Syed et al., 2015). However, overproduction of ROS can overwhelm host antioxidants and induce oxidative stress. The mechanisms to protect organism from free radical damage are defense systems involving enzymes such as SOD, CAT, and GSH-PX and nonenzymes such as GSH, VC, and VE. Superoxide dismutase is a class of highly conserved enzymes that catalyze the disputations of superoxide into oxygen and hydrogen peroxide, and sustain low concentration of superoxide radicals (Nagi and Almakki, 2009). This reaction is then followed by CAT inactivation of H2 O2 by converting it to water and oxygen (Ince et al., 2014). Similar to CAT, GSH-PX is another important cellular enzyme. In the presence of reduced GSH, GSH-PX can convert hydrogen peroxide to water and oxygen, and convert nitrate (a by-product of NO radicals) to nitrite (Flatow et al., 2013). Total antioxidant capacity is always used as the most important representative marker of in vivo antioxidative activity and may give more biologically relevant information than individual index (Ghiselli et al., 2000). Non-enzymatic antioxidant system including GSH, VC, and VE are also important in cellular antioxidative defense. GSH is oxidized by GSH-PX to
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Figure 3. Serum IFN-γ and IL-6 concentration. Chickens (n = 24/group) were intramuscularly (i.m.) injected with Cy at 100 mg/kg for 3 d to induce immunosuppression and oxidative stress, and then orally administrated Rg1 (1 mg/kg) in drinking water for 7 d. After that, chickens were orally immunized with a live attenuated IBDV vaccine (B87 strain). Blood samples were collected from 12 chickens before Cy treatment and 7 and 14 d after immunization or 6 chickens 21 d after immunization of each group for analysis of IFN-γ and IL-6 concentration by ELISA. The values were represented as mean ± SE. Bars with different letters are statistically different (P < 0.05).
oxidized glutathione. Ascorbic acid can directly scavenge free radicals in extracellular fluids and regenerate vitamin E in cell membranes and maintain low density lipoprotein (LDL) particle integrity (Niki, 1987; Frei et al., 1989; Harats et al., 1998). Vitamin E, acting as an antioxidant, can inhibit lipid peroxidation and maintain the stability of cell membranes. Malondialdehyde is a major product of lipid peroxidation and is supposed to be a marker of tissue damage (Echeverry et al., 2016). Carbonyl is considered as a surrogate marker of protein peroxidation. Cyclophosphamide is a prominent alkylating agent and has been widely used in treatment of some malignant cancers for more than 50 yr (Sikov et al., 2015). It remains one of the most useful antirejection drugs in bone marrow transplant (Slade et al., 2017). The agent is also used to induce immunosuppression and
oxidative stress in animal models (Sevko et al., 2013; Cho et al., 2015; Cheng et al., 2017). Cyclophosphamide is in vivo metabolized to 4-hydroxycyclophosphamide, aldophosphamide, and ultimate phosphoramide mustard, which induces DNA cross-linking and inhibits DNA synthesis especially in S phase of cells (Perini et al., 2007; Goldstein et al., 2008). Cyclophosphamide can also induce apoptosis by activating Fas and caspases pathway (Lee et al., 1997; Wang and Cai, 1999; Chow and Loo, 2003). In addition, Cy treatment has been found to increase MDA and decrease intracellular SOD and GSH, which change the balance between ROS and antioxidants, resulting in oxidative stress (Tripathi and Jena, 2009; Singh et al., 2015). Similar to previous reports, injection of Cy has been demonstrated as effective in inducing immunosuppression and oxidative stress in chickens (Fan et al., 2013; Chi et al., 2017).
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Figure 4. Splenocyte proliferative responses to ConA and LPS. Chickens (n = 24/group) were intramuscularly (i.m.) injected with Cy at 100 mg/kg for 3 d to induce immunosuppression and oxidative stress, and then orally administrated Rg1 (1 mg/kg) in drinking water for 7 d. After that, chickens were orally immunized with a live attenuated IBDV vaccine (B87 strain). Splenocytes were collected from 6 chickens of each group before Cy treatment and 7, 14, and 21 d after immunization for lymphocyte proliferative assay measured by the MTT method. The values were represented as mean ± SE. Bars with different letters are statistically different (P < 0.05).
The present study showed that Cy treatment decreased antibody, IFN-γ , IL-6, and lymphocytes proliferative responses, as well as reduced activities of T-AOC, T-SOD, CAT, and GSH-PX. Improved immunity of animals by oral administration of ginseng stem and leaf saponins has been reported previously. Li et al. (2016) found elevated antibody responses to foot-and-mouth disease and gut mucosal immunity in mice after oral administration of GSLS. Zhai et al. (2011a) reported enhanced specific antibody and lymphocyte proliferative responses in chickens after medication of GSLS in drinking water. In addition, Yu et al. (2015a) observed that oral administration of GSLS significantly enhanced the immune responses and gut mucosal immunity in chickens with immunosuppression induced by Cy when compared to the birds without GSLS. The group also found that oral
administration of GSLS increased antioxidant activities in chickens with oxidative stress induced by Cy (Yu et al., 2015b). In the present study, enhanced antibody responses to IBD vaccine and lymphocyte proliferation as well as elevated antioxidant activities were observed in chickens with immune suppression and oxidative stress after oral administration of Rg1. As Rg1 is one of the constituents in GSLS, this agent is supposed to contribute the immune modulation and antioxidant activities of GSLS as previously reported. Immune modulatory activities of Rg1 were previously reported. Sun et al. (2007, 2008) found enhanced Th1 (IgG1 and IL-5) and Th2 (IgG2a and IFN-γ ) responses when injection of Rg1 with ovalbumin (OVA) in mice. Yuan et al. (2016) observed increased antibody, lymphocyte proliferative, IFN-γ , and IL-4 responses in mice injected with hepatitis B surface antigen in
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Figure 5. Serum enzymatic antioxidant activities. Chickens (n = 24/group) were intramuscularly (i.m.) injected with Cy at 100 mg/kg for 3 d to induce immunosuppression and oxidative stress, and then orally administrated Rg1 (1 mg/kg) in drinking water for 7 d. After that, chickens were orally immunized with a live attenuated IBDV vaccine (B87strain). Splenocytes were collected from 6 chickens of each group before Cy treatment and 7, 14, and 21 d after immunization for lymphocyte proliferative assay measured by the MTT method. The values are represented as mean ± SE. Bars with different letters are statistically different (P < 0.05).
combination with Rg1. Su et al (2012) observed that Rg1 enhanced immune responses to OVA in C3H/HeB but not in C3H/HeJ mice carrying a defective TLR4 gene. Taken together, the present study demonstrated that oral administration of Rg1 at 1 mg/kg BW enhanced immune responses in immunosuppressed chickens, evidenced by recovered antibody response, serum IFN-γ and IL-6, and splenocyte proliferation induced by ConA and LPS. The increased immune responses paralleled
the enhanced T-AOC, T-SOD, CAT, GSH-PX, GSH, VC, and VE, and decreased protein carbonyl content and MDA in oxidative stress induced by Cy. Therefore, oral administration of Rg1 has been shown as effective in immune modulatory and antioxidant property to improve vaccination in chickens with oxidative stress. As Rg1 is an important constituent in GSLS, the content of this agent is suggested to be a useful parameter for the quality control when GSLS is used for immune enhancement in poultry industry.
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Figure 6. Serum non-enzymatic antioxidant activities. Chickens (n = 24/group) were intramuscularly (i.m.) injected with Cy at 100 mg/kg BW for 3 d to induce immunosuppression and oxidative stress, and then orally administrated Rg1 (1 mg/kg BW) in drinking water for 7 d. After that, chickens were orally immunized with a live attenuated IBDV vaccine (B87 strain). Blood samples were collected from 12 chickens before Cy treatment and 7 and 14 d after immunization or 6 chickens 21 d after immunization of each group for determination of serum GSH, VC, VE, MDA, and carbonyl. The values are represented as mean ± SE. Bars with different letters are statistically different (P < 0.05).
ACKNOWLEDGMENTS
FUNDING
The authors are grateful to the students in the Laboratory of Traditional Chinese Veterinary Medicine (TCVM Lab) for their assistance in the present study.
This study was supported by the project (No. 31172356) of the National Natural Scientific Foundation of China and the Special Fund for Agro-research in the Public Interest of China (20130304–3).
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