Anti-apoptotic Effects Of Glutamine In Carp Erythrocytes And The Involved Mechanism

This study was conducted to investigate the effects of glutamine (Gin) on growth performance, antioxidant status, erythrocytes function and metabolites in juvenile Jian carp (Cyprinus carpio var. Jian) and the involved mechanism. Moreover, we employed carp erythrocytes as an experimental model to evaluate the protective effects of Gin and its metabolites [alanine (Ala), citrulline (Cit) and proline (Pro)] on hydroxyl radical (·OH)-induced apoptosis.The experimental methods and main results are as follows:1. Effects of glutamine on biochemical indicator of blood and erythrocytes function in juvenile Jian carpIn this experiment, SGR, feed intake, feed efficiency, whole-body crude protein and crude lipid content, hepatopancreas weight, intestinal weight and intestinal length, the content of glutathione (GSH), malondialdehyde (MDA) and protein carbonyl (PC) in serum, erythrocytes, hepatopancreas and intestine, the haematocrit (Hct). Hb concentration, the erythrocytes count (RBC), lactate dehydrogenase (LDH) and Na+, K+-ATPase activities and the concentration of Gin, Ala, Cit and Pro in serum were investigated to study the effects of Gin on growth performance, antioxidant status, erythrocytes function and metabolites in juvenile Jian carp and the possible mechanism.The trail was divided into two treatments with four replicates, each of which contained50fish. Fish were fed semi-purified isonitrogenous diets with0and1.23%Gin for56days, respectively. The initial fish weight was6.08±0.03g. The results showed that1.23%Gin significantly increased SGR, feed intake (Fl), feed efficiency, whole-body crude protein and crude lipid content, protein efficiency ratio, protein and lipid retention rate, hepatopancreas weight and index, intestinal weight, intestinal length and index as compared with the control (P<0.05). Compared with the control,1.23%Gin significantly increased the content of GSH, and decreased the concentration of MDA and PC in serum, hepatopancreas and intestine (P<0.05). The results also indicated that1.23%Gin resulted in improvement of Hct, Hb concentration, RBC, mean corpuscular volume (MCV), mean corpuscular haemoglobin content (MCH), LDH and Na+, K+-ATPase activities as well as the content of GSH, MDA and PC in carp erythrocytes as compared with the control (P<0.05). In addition,1.23%Gin significantly increased the concentration of Gin, Ala, Cit and Pro in serum of Jian carp (P<0.05).In conclusion, Gin can enhance growth performance, antioxidative defense and erythrocytes function of juvenile Jian carp. Dietary Gin may be converted to Ala, Cit and Pro, which released into the circulation blood in fish. The antioxidant effects of Gin may be in part dependent on the functions of its metabolites (Ala, Cit and Pro). The enhancement of erythrocytes function may be related to the antioxidant effects of Gin on erythrocytes.2. Establishment of apoptotic model of Jian carp erythrocytes induced by·OHIn order to select an optimal·OH concentration to induce apoptosis, we investigated·OH and hemoglobin (Hb) concentration in culture medium, necrosis and apoptosis level and micronuclei (MN) frequencies in·OH-induced carp erythrocytes.The erythrocytes of healthy carp weighed about100-110g were used as study model in vitro. Single-factor experimental design was used in this study. The trail was divided into six treatments with four replicates. Cells were cultured with different FeSO4/H2O2concentrations of0μM/0μM,10μM/5μM,20μM/10μM,30μM/15μM,40μM/20μM and50μM/25μM for36hours. The morphology of erythrocytes was recorded before cells and culture medium were collected for analysis. The results showed that the concentrations of·OH in the culture medium was significantly increased with increasing concentrations of FeS4/H2O2(0/0-50μM/25μM)(r=0.9995, P<0.05). Exposure to FeSO4/H2O2(0/0-50μM/25μM) significantly increased hemolysis and necrosis level in a dose-dependent manner (r1=0.8554, P<0.05; r2=0.8976, P<0.05). MN frequencies and apoptosis level were positively correlated with FeSO4/H2O2(0/0-40μM/20μM) concentration (r1=0.9314, P<0.05; r2=0.9245, P<0.05). The MN frequencies and apoptosis level were significantly higher at40μM FeSO4/20μM H2O2and significantly lower at10μM FeSO4/5μM H2O2than that at other concentrations of FeSO4/H2O2(The MN frequencies and apoptosis level were the maximum at40μM FeSO4/20μM H2O2)(P<0.05). Thus,40μM/20μM was an appropriate concentration of FeSO4/H2O2for inducing MN and apoptosis. Carp erythrocytes suspension (1%v/v) was treated with40μM FeSO4/20μM H2O2for different time periods (6-11hour). Agarose-gel electrophoresis of chromosomal DNA in the erythrocytes treated by·OH for9hour showed a ladder-like pattern of DNA fragments consisting of multiples of approximately180-200base pairs. Thus,9hour was an appropriate time for inducing apoptosis.These results indicated that incubation with40μM Fe2+/20μM H2O2for9hour was appropriate condition for inducing apoptosis in carp erythrocytes.3. The underlying mechanisms of apoptosis in carp erythrocytes induced by·OHIn this experiment, ROS and Ca2+content, caspase-3and calpain activities and apoptosis level in different developmental stages were examined to study the underlying mechanisms of apoptosis in carp erythrocytes induced by·OH.The erythrocytes were separated into three different developmental stages using fixed-angle centrifugation. Carp erythrocytes of three different developmental stages were treated with caspase inhibitors (zVAD-fmk) or Ca2+-free PCS in the presence of40μM FeSO4/20μM H2O2for9hour, respectively. Positive control was treated with only40μM FeSO4/20μM H2O2. Control group was treated with only culture medium without FeSO4/H2JO2 Results showed that positive control significantly increased erythrocytes ROS and Ca2+content, caspase-3and calpain activity and apoptosis level in the early erythroblasts, the intermediate cells and the matured cells as compared with the control (P<0.05). In positive control, ROS content and caspase-3activity of the early erythroblasts are higher than that of the intermediate cells and the matured cells. Inhibition of zVAD-fmk on erythrocytes ROS, caspase-3activity and apoptosis level of the early erythroblasts are stronger than on that of the intermediate cells and the matured cells. In positive control, Ca2+content and calpain activity of the matured cells are higher than that of the early erythroblasts and the intermediate cells. Inhibition of Ca2+-free PCS on Ca2+content, calpain activity and apoptosis level of the matured cells is stronger than that of the early erythroblasts and the intermediate cells.These results indicated that there might be two apoptosis path in fish erythrocytes. One is caspase-dependent apoptosis mechanism in the early erythroblasts, the other is a Ca2+-involved apoptosis mechanism in the matured cells.4. The cytoprotective effects of Gln, Ala, Cit and Pro on·OH-induced apoptosis in carp erythrocytesIn this experiment, we examined the cytoprotective effects of Gln, Ala, Cit, Pro and Ala10Pro4Cit1on the·OH-induced apoptosis in carp erythrocytes. Cells were treated with the substances listed above in the presence of40μM Fe2+/20μM H2O2for9hour. The control erythrocytes were kept in the Gln, Ala, Cit, Pro-free culture medium only.Results showed that Gln, Ala, Cit, Pro and Ala10Pro4Cit1decreased annexin V binding level, volume shrinkage and DNA fragmentation in the·OH-induced carp erythrocytes. Gln, Ala, Cit, Pro and Ala10Pro4Cit1effectively blocked·OH-stimulated hemolysis, reduced the increase in the concentrations of superoxide anion (O2’) and hydrogen peroxide (H2O2) and the decrease in anti-hydroxyl radical (AHR) activity, and inhibited the formation of MDA, PC and Met-Hb, and prevented the decrease in the activities of SOD, CAT, GPx, glutathione S-transferase (GST) and glutathione reductase (GR) as well as GSH concentration in carp erythrocytes induced by·OH. Furthermore, the present results indicated that AlaioPro4Cit1as the combination of Ala, Cit and Pro produces an antioxidant and antiapoptotic effect greater than their individual effects at same concentrations. Meanwhile. Ala. Cit. Pro and Ala10Pro4Cit1as the metabolites of Gin showed the protective effects equivalent to or stronger than Gin in carp erythrocytes induced by·OH.These results indicated that Gin and its metabolites play an effectively protective role against·OH-induced apoptosis in carp erythrocytes. Moreover. Gin and its metabolites inhibit oxidative stress under conditions of apoptosis. So, inhibition of Gin and its metabolites on apoptosis may be due to their inhibition on·OH-induced oxidative stress in carp erythrocytes.5. The effects of Gin, Ala, Cit and Pro on the apoptosis pathways in carp erythrocytes induced by·OHIn this experiment, ROS and Ca2+content, caspase-3and calpain activity and apoptosis level were examined to study the effects of Gin, Ala, Cit and Pro on the mechanisms of apoptosis in carp erythrocytes induced by·OH.The results indicate that Gin, Ala, Cit, Pro and Ala]oPro4Cit1effectively blocked the increase in the levels of ROS and Ca2+and the activities of caspase-3and calpain in carp erythrocytes induced by·OH. Ala10Pro4Cit1as the combination of Ala. Cit and Pro produces a greater inhibition effect on the increase of ROS and Ca2+content and caspase-3and calpain activities than their individual effects at same concentrations. The protective effects of Ala, Cit, Pro and Ala10Pro4Cit1, the metabolites of Gin, were equivalent to or stronger than Gin against the increase in the content of ROS and Ca2+and the activity of caspase-3and calpain in carp erythrocytes induced by·OH. The present results also indicated that the inhibition effects of Gin and Ala10Pro4Cit1on the increase in the content of ROS and the activity of caspase-3were equivalent to that of zVAD-fmk. The inhibition effects of Gln and Ala10Pro4Cit1on the increase of Ca2+content and calpain activity were equivalent to that of Ca2+-free PCS. Moreover, the inhibition effects of Gln and Ala10Pro4Cit] on apoptosis equivalent to that of zVAD-fmk and Ca2+-free PCS in carp erythrocytes induced by·OH.These results demonstrate that Gin and its metabolites can inhibit·OH-induced apoptosis by preventing from the increase in the content of ROS and Ca2+and the activities of caspase-3and calpain in carp erythrocytes.In conclusion, firstly, dietary Gin can improve growth performance, antioxidant status and erythrocytes function in fish. Dietary Gin may be converted to Ala, Cit and Pro, which released into the circulation blood in fish. Gin and its metabolites (Ala, Cit, Pro and Ala10Pro4Cit1) play an effectively protective role against apoptosis in carp erythrocytes induced by·OH. Ala, Cit, Pro and Ala10Pro4Cit1as metabolites of Gin showed equivalent to or stronger anti-apoptotic effect than Gin in·OH-induced carp erythrocytes. So, metabolites of Gin may play an important role in manifestations of the beneficial effects of Gin as a nutritional supplementation. Secondly, Gin and its metabolites inhibited oxidative stress under conditions of apoptosis. Gin and its metabolites prevented from the increase of ROS and Ca2+content and caspase-3and calpain activities. The anti-apoptotic properties of Gin and its metabolites may be ascribed to their anti-oxidative effect and the ability to inhibit the key factor in the apoptosis pathway. Thirdly, this study suggested that Ala10Pro4Cit1as the combination of Ala, Cit and Pro produces a greater protective effect than their individual effects on oxidative stress and key factor in the apoptosis pathway in carp erythrocytes. The stronger effects of metabolites of Gin than that of Gin on apoptosis may be due to their synergistic effects.