Data in the activities of caspases and the levels of reactive oxygen species and cytochrome c in the •OH-induced fish erythrocytes treated with alanine, citrulline, proline and their combination

The present study explored the effects of alanine (Ala), citrulline (Cit), proline (Pro) and their combination (Ala10Pro4Cit1) on the activities of caspases and levels of reactive oxygen species (ROS) and cytochrome c in hydroxyl radicals (•OH)-induced carp erythrocytes. The data displayed that •OH induced the increases in the activities of caspase−3, caspase−8 and caspase−9 and the levels of ROS and cytochrome c in carp erythrocytes. However, Ala, Cit, Pro and Ala10Pro4Cit1 effectively suppressed the •OH-induced increases in the activities of caspase−3, caspase−8 and caspase−9 and the levels of ROS and cytochrome c in carp erythrocytes. Furthermore, the activities of caspase−3, caspase−8 and caspase−9 and the levels of ROS and cytochrome c were gradually decreased with increasing concentrations of Ala, Cit, Pro and Ala10Pro4Cit1 (0.175−1.400 mM) in the •OH-induced carp erythrocytes. These data demonstrated that the 50% inhibitory doses (ID50) of Ala10Pro4Cit1 on the activities of caspase−8, caspase−9 and caspase−3 and levels of ROS and cytochrome c were respectively estimated to be the minimum values among amino acids examined so far. The 5% inhibitory doses (ID5) of Ala, Cit, Pro and Ala10Pro4Cit1 on the activities of caspase−8, caspase−9 and caspase−3 and levels of ROS and cytochrome c were estimated to be at their physiological concentrations in mammalian. Our research article for further interpretation and discussion from these data in Li et al. (2016) [1].

and Ala 10 Pro 4 Cit 1 (0.175 À 1.400 mM) in the OH-induced carp erythrocytes. These data demonstrated that the 50% inhibitory doses (ID 50 ) of Ala 10 Pro 4 Cit 1 on the activities of caspase À 8, caspase À 9 and caspase À 3 and levels of ROS and cytochrome c were respectively estimated to be the minimum values among amino acids examined so far. The 5% inhibitory doses (ID 5 ) of Ala, Cit, Pro and Ala 10 Pro 4 Cit 1 on the activities of caspase À 8, caspase À 9 and caspase À 3 and levels of ROS and cytochrome c were estimated to be at their physiological concentrations in mammalian. Our research article for further interpretation and discussion from these data in Li et al. (2016) [1].
& 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Subject area
Biology More specific subject area

Nutritional Medicine
Type of data The measurement of the activities of caspase À 3, caspase À8 and caspase À9 and the levels of reactive oxygen species and cytochrome c were performed using the commercial kits.

Data
In the data, the effects of alanine (Ala), citrulline (Cit), proline (Pro) and their combination (Ala 10 Pro 4 Cit 1 ) at different concentrations on caspases activities and reactive oxygen species (ROS) and cytochrome c levels in OH-induced carp erythrocytes were presented in Figs. 1-5. Compared with the control, the activities of caspase À 3, caspase À 8 and caspase À 9 and levels of ROS and cytochrome c was significantly increased in carp erythrocytes exposed to OH. However, Ala, Cit, Pro and Ala 10 Pro 4 Cit 1 significantly inhibited the OH-induced increase in the activities of caspases and levels of ROS and cytochrome c in carp erythrocytes. The activities of caspases and levels of ROS and cytochrome c were gradually decreased with increasing concentrations of Ala, Cit, Pro and Ala 10 Pro 4 Cit 1 (0.175 À 1.400 mM) in the OH-induced carp erythrocytes [1].
The data in Table 1 showed that when the erythrocytes were treated with Ala 10 Pro 4 Cit 1 in the presence of OH, the 50% inhibitory doses (ID 50 ) on the activities of caspase À 3, caspase À 8 and caspase À 9 and levels of ROS and cytochrome c were estimated to be 0.35, 0.60, 0.61, 0.53 and 1.01 mM that are the minimum values among amino acids examined so far [1].
The data in Table 2 showed that the 5% inhibitory doses (ID 5 ) of Ala 10 Pro 4 Cit 1 on the activity of caspase À 8 and level of cytochrome c were respectively estimated to be 0.026 and 0.028 mM, the ID 5 of Ala 10 Pro 4 Cit 1 and Cit on the activity of caspase À 3 or level of ROS were respectively estimated to be 0.024 and 0.025 mM or 0.041 and 0.047 mM, the ID 5 of Cit on the activity of caspase À 9 was estimated to be 0.021 mM, which are the minimum values among amino acids examined so far [1].

Chemicals
L-Ala, L-Cit and Pro were purchased from Sigma (St. Louis, MO, USA). The aqueous solution of H 2 O 2 (30%) and FeSO 4 (analytical pure) were purchased from Shanghai Chemical Reagent Factory (Shanghai, China). All other chemicals were analytical grade. All water used was Milli-Q grade. Physiological carp saline (PCS) (contained 141.1 mM NaCl, 1.43 mM KC1, 0.99 mM CaCl 2 , 2.64 mM NaHCO 3 and 6.16 mM glucose) modified to give a total osmolarity of 280 mosm L À 1 and pH 7.9 were prepared in our laboratory.

Erythrocyte isolation
The procedures of cell isolation were based on that described by Phillips et al. (2000) [2] with slight modifications. Individual healthy carp (Cyprinus carpio var. Jian) (100-110 g) was anaesthetized, and the blood (approximately 1 ml) was drawn into a syringe via caudal puncture. The blood was placed in a chilled centrifuge tube containing heparinized (40 i.u.ml À 1 ) PCS. The blood from three different carp was pooled and centrifugated (Thermo, Waltham, MA, USA) at 900g and 4°C for 10 min. Then, the plasma and buffy coat of pooled blood were removed in the centrifuge tube. After washing three times with PCS, the isolated erythrocytes were resuspended in PCS at a 1% hematocrit in following experiments. All procedures were approved by the Institutional Animal Care and Use Committee of Sichuan Agricultural University in accordance with the Institutional Ethics Committee of the Chinese Institute of Chemical Biology guidelines.

Erythrocyte treatments
Erythrocyte treatments were based on the method described by Li et al. (2013) [3] with slight modifications. Alanine, Cit and Pro were combined in the molar ratio 10:4:1 (Ala 10 Pro 4 Cit 1 ) for investigating their mixture effects in the erythrocytes. Briefly, the isolated erythrocytes were suspended in PCS (control) and PCS containing 0.000, 0.175, 0.350, 0.700 or 1.400 mM of Ala, Cit, Pro or Ala 10 Pro 4 Cit 1 at a 1% hematocrit, respectively. All erythrocyte suspensions above were pre-incubated at 37°C for 1.5 h. FeSO 4 /H 2 O 2 was then added at a final concentration of 40 μM/20 μM, except in the control. After incubation at 37°C for 9 h, the erythrocytes were collected for measurement of the activities of caspase À 3, caspase À 8 and caspase À 9 and the levels of ROS and cytochrome c. The experiment was performed with 4 replicates per treatment and the control.

Measurement of ROS
ROS levels in the erythrocytes were determined by measuring the oxidative conversion of 2 0 , 7 0dichlorofluorescin diacetate (DCFH-DA) to fluorescent compound DCF using a detection kit (Beyotime, Nantong, China) as described previously [6][7][8]. DCF fluorescence in the erythrocytes was determined at 485 nm excitation and 520 nm emission using a fluorescence spectrophotometer (Thermo, Waltham, USA).

Measurement of cytochrome c
The cytosolic proteins and mitochondria in fish erythrocytes were isolated by a commercial extraction kit (Beyotime, Nantong, China) as described previously [9]. The cytochrome c release from mitochondria in cytosol was evaluated by an enzyme-linked immunosorbent assay (ELISA) kit (ElabScience, Wuhan, China) [10]. Absorbance value in the samples was measured with the Microplate reader at 450 nm.

Protein measurements and statistical analysis
Protein concentration in the hemolysate was measured by Darbkin method [11]. All data are presented as mean7S.D. (nZ4). The data were subjected to one-way analysis of variance (ANOVA). If significant differences were found (Po0.05), Duncan's multiple range tests were used to rank the groups [12]. The 50% and 5% inhibitory doses (ID 50 and ID 5 ) were determined by the probit analysis [3]. Table 2 ID 5 of Ala, Cit, Pro and Ala 10 Pro 4 Cit 1 on the activities of caspase À 8, caspase À 9 and caspase À 3 and the levels of cytochrome c and ROS in the OH-induced carp erythrocytes.