[0023] Experimental case
[0024] First, the experimental animals
[0025] Sexually mature female C57BL / 6 mice, body weight (20-22g), were purchased from Experimental Animal Center of Shandong Province (animal quality certification: No.37009200018914, license number: SCXK (Lu) 2,014,000). Rearing environment: Mice were housed four per cage, 22 ± 1 ℃ temperature, 50 ± 10% humidity, 12h light - dark cycle, food and water freely. Feeding and operation of experimental animals are to comply with the relevant provisions of Guangdong Ocean University Laboratory Animal breeding and use.
[0026] Thymosin β4: purchased from Guangzhou Shang Bin biotechnology company.
[0027] Mice maintain powder feed: Guangdong Experimental Animal Center.
[0028] Second, the experimental program
[0029] Laboratory animals repurchase after one week of acclimation, packet random numbers, a total of three sets, as shown in Table 1:
[0030] Table 1. Animal Grouping
[0031]
[0032] CUMS CUMS + Tβ4 group and the group exposed to stressors, to model processing; Control group under standard feeding environment, stress is not processed.
[0033] All stressors stochastically independent operation, independent operation of two daily stressors, after 9 weeks continuous stress, measured behavior. Stressors are: lack of food overnight to dry overnight, overnight odor, wet litter overnight, single cage overnight, 200rpm / min shaker 1.5h, 50 mL centrifuge tubes bound 1.5h, 4 ℃ low temperature 1.5h, inclined cage overnight, overnight strobe lights, lights and brightly lit circadian 24h, crowded environment 3h, 18 ℃ swimming 10min.
[0034] Third, behavioral experiments
[0035] Sucrose preference test
[0036] 1. Experimental Procedure
[0037] When the train starts, the animals free access to food and two bottles, one containing 1% sucrose solution bottle, the second bottle containing purified water.
[0038] The first day of the experiment mice were freely fed with 19:00 1% sucrose solution bottles and bottles of feed;
[0039] The next experiment mice were freely fed with 19:00 1% sucrose solution bottle and a bottle of water and feed;
[0040] Experimental third 19:00 feed was removed and the solution bottles;
[0041] Experiment Day 19:00 to mice cages and consisting of 1% sucrose solution bottle intake and a bottle of water and feed, 11 night sucrose preference is determined according to the following formula. Wherein the syrup is calculated as the degree of preference:
[0042] Sucrose consumption of sucrose preference = / (sugar + water consumption amount consumed) * 100%
[0043] Experimental results
[0044] like figure 1 Indicated (shown) * P <0.05vs.Control; ## P <0.01vs.CUMS), results showed that: compared with the control group, mice preference CUMS group reduced sugar 11.73%, indicating the animals anhedonia; administering thymosin β4 after treatment, compared with the group CUMS, sucrose preference degree raised 16.21%, indicating that thymosin β4 for CUMS-induced anhedonia extent have reverse effect.
[0045] IV Determination of monoamine neurotransmitters in the brain tissue of mice
[0046] 1. Experimental Procedure
[0047] 1.1 of brain tissue extracts monoamine neurotransmitters
[0048] Solvent configuration: tissue lysate was 0.6mol / L perchloric acid, 0.5mmol / L disodium edetate, 0.1g / LL- mixed aqueous solution of cysteine. Perchloric acid precipitating agent is 1.2mol / L dipotassium hydrogen phosphate, disodium mixed aqueous solution of 2mmol / L EDTA. Citric acid - sodium acetate buffer solution: 50mmol / L citric acid, 50mmol / L sodium acetate, 0.5mmol / L1- heptane sulfonate, 5mmol / L triethylamine, 0.5mmol / L disodium edetate.
[0049] Extraction Step: 4mL brain was weighed out into a centrifuge tube, tissue lysate was added 150 L, sufficiently homogenized, centrifuged at 15min (14000r / min, 4 ℃), the supernatant was centrifuged again. An equal volume of liquid after centrifugation perchloric acid precipitating agent, centrifuged at ice bath for 10min 15min (14000r / min, 4 ℃), filtered through a 0.45μm membrane, the content of the filtrate was analyzed machine. Determination of the type Monoamine neurotransmitters are: dopamine (dopamine, DA), dihydroxyphenylacetic acid (3,4-dihydroxyphenylacetic acid, DOPAC), 5- hydroxytryptamine (5-HT), 5- hydroxy indole acetic acid (5-HIAA), norepinephrine (NE), 3- methoxy -4-hydroxyphenyl glycol (3-methoxy-4-hydroxyphenylglycol, MHPG).
[0050] 1.2 HPLC - Chromatography with Fluorescence Detector Conditions
[0051] Chromatographic conditions: Column Waters C18 column for the (150mm × 4.6mm, 5μm); mobile phase is citric acid - sodium acetate buffer: methanol (87: 13, v / v); flow rate of 1.0mL / min; Injection in an amount of 20 L; emission wavelength of 330nm, an excitation wavelength of 280nm.
[0052] Qualitative and quantitative methods: Retention time by monoamine neurotransmitters qualitative standards of the respective sample peaks; The content of neurotransmitter performed by known standard sample concentration and peak area and peak area of the sample.
[0053] 2. Experimental results
[0054] 2.1 concentration of monoamine neurotransmitters
[0055] like figure 2 ( * P <0.05, *** P <0.001vs.Control; # P <0.05, ## P <0.01vs.CUMS), the results showed that: compared with the control group, 5-HT concentrations CUMS group reduced 19.93%, 5-HIAA concentrations decreased 24.19%, MHPG reduces the concentration of 30.38%, NE reduces the concentration of 27.09%, DOPAC concentration decreased 47.31%, DA concentration tends to decrease, HVA concentration increased 58.48%; after administration of thymosin β4 treatment, compared with CUMS group, 5-HT concentration raised 26.72%, NE concentration raised 38.88%, DOPAC concentration raised 46.54%, NE, DA, DOPAC and 5-HIAA concentrations were the upward trend; showed thymosin β4 chronic stress-induced neurotransmitter deficiency with improved effect.
[0056] 2.1 monoamine neurotransmitters and its metabolites ratio change
[0057] like image 3 ( * P <0.05, ***. P <0.001vs controls (Control); # P <0.05vs.CUMS), the results showed that: compared with the control group, the CUMS group of 5-HT / 5-HIAA ratio of 24.24% and decreased DA / DOPAC ratio increased by 86.91%; after treatment administered Thymosin β4, compared with CUMS group, 5-HT / 5-HIAA ratio raised 30.42%, DA / DOPAC ratio lowered 30.94%, indicating that thymosin β4 cause of chronic stress neurotransmitters and their metabolites disorders with improved effect.
[0058] In the present embodiment, derivatives of thymosin α1 employed thymosin or thymosin [beta] 4, for example, oxidation β4, Gly-Tβ4, Ala-Tβ4 similar results can be obtained.
