Experimental animals
Fifty healthy, 60-day-old SD rats were provided by the Laboratory Animal Center of College of Medicine of Xi’an Jiaotong University (Xi‘an, Shaanxi Province, Chnia). The weight range of the rats was 180~220g. There were 25 male rats and 25 female rats.
Reagents
Main reagents included: KT (Sigma, K3378, USA), D-galactose (Sigma), Lymphocyte separation medium (Shanghai Huajing Biological Technology Co., Ltd. China), anti-rat CD
3+, CD
4+ and CD
8+ antibody (labeled with PE, FITC and PE respectively) and heparin (Beijing Suolaibao Co.,Ltd. China), annexin V-FITC apoptosis detection kit (Beyotime), cell cycle detection kit (Beyotime).
Instruments
Glass homogenizer(Haimen Hong Cheng Experimental Equipment Manufacturing Co., Ltd.); ELx800 absorbance microplate reader (BioTek Instruments, USA); BD FACSAria flow cytometer (Becton, Dickinson and Company, USA).
Methods
Grouping and process of the experimental animals
The SD rats were randomly divided into five groups which included young control group, aging model group, low-dose KT group, intermediate-dose KT group and high-dose KT group. Each group had 10 rats (half were male and half were female).
The rats were fed for 7 days after being purchased to reduce stress response resulting from environmental change. During the whole experiment, the animals were fed with normal diet and clean water. The room temperature was maintained around 20
oC and humidity was controlled to be 65%~75%. The rats in the young control group were continuously injected subcutaneously with normal saline in the dorsal neck for 45 days (once per day). Rats in the other four groups were continuously injected subcutaneously with D-gal (125 mg/kg•BW, once per day) in the dorsal neck for 45 days to establish the aging model. From the 11
st day of the experiment, rats in the young control group and aging model group were intraperitoneally injected with normal saline until the 45
th day of the experiment, while rats in the KT treatment groups received daily intraperitoneal injections of different doses (5 mg/kg BW, 10 mg/kg BW and 20 mg/kg BW) of KT until the 45
th day of the experiment.
Test of CD3+, CD4+ and CD8+ T lymphocyte subpopulations
Blood samples were gathered at the 45
th day. Heparin was added for anticoagulant and the blood was transferred to centrifuge tubespre-filled with lymphocyte separation liquid. Lymphocytes were isolated by 1000rpm/min centrifugation for 20min and the isolated lymphocytes were then washed with PBS. The lymphocytes were stained with trypan blue dye for 3 seconds. The viability of the lymphocytes was more than 97% observed under the microscope. Then RPMI-1640 medium containing 10% fetal bovine serum was added to adjust the cell concentration to 1~10×10
6 cells/ml. 1ml of lymphocyte suspension was taken out and centrifuged, washed with PBS for three times and stained with anti-rat CD
3+, CD
4+ and CD
8+ monoclonal antibodies according to the instruction of the kit. After half hour of incubation, the lymphocytes were washed with PBS and then tested in the flow cytometry. BD FACSDiva Software was used to analyze CD
3+, CD
4+ and CD
8+ T lymphocyte subpopulations in peripheral blood.
Preparation of thymus lymphocyte suspension
The rats were euthanized after finishing blood sampling in the 45th day of the experiment. Thymus tissues of the rats were sterilely removed; after 1ml of PBS was added, the tissues were grinded and the tissue lysates were collected and transferred to centrifuge tubespre-filled with lymphocyte separation liquid. After 20 min of 1000 rpm/min centrifugation, the lymphocytes were collected and washed with PBS, then stained with trypan blue solution. The viability of the lymphocytes was more than 97% observed under the microscope. Then RPMI-1640 medium containing 10% fetal bovine serum was added to adjust the cell concentration to 1~10×10
6 cells/ml. The cells were cultured in a 5% CO
2 incubator for future use.
Detection of apoptosis
1ml of the prepared thymus lymphocyte suspension was used for the detection of apoptosis in which Annexin V-FITC/PI double staining method was applied. The operation was carried out according to the instruction of the kit and the cells were applied to flow cytometry to test. BD FACSDiva software was used to analyze apoptotic rate of the thymus lymphocytes.
Detection of cell cycle
Each well of a 96-well plate was added 195µl of the prepared thymus lymphocyte suspension. Three replicates were set up for each rat. RPMI 1640 medium containing 10% fetal bovine serum and ConA (5µg/ml) was added to each well and incubated for 72 hours. PI staining was applied and the cells were processed according to the instruction of the kit. ModFit LT software was used to analyze the number of the thymus lymphocytes in resting phase (G0/G1 phase) , DNA synthesis phase (S phase) and mitotic phase (G2/M phase) and then proliferation index (PI) of the thymus lymphocytes was calculated according to the following formula:
Research period and the name of the University
The experiment period was from January 2013 to December 2015. The university where the work was carried out is Henan University of Science and Technology.
Statistical analysis
Statistical analysis was conducted using GraphPad Prism5 was used to perform inter-group comparison. Comparison of experimental groups and control groups was conducted using Duncan’s test. All data was expressed as X ± SE. P>0.05 represented the difference was not significant; 0.01<P<0.05 represented the difference was significant and P<0.01 represented the difference was highly significant.
