Preparation method of tree shrew depression model

Abstract

The invention discloses a tree shrew depression model preparation method, which comprises: (1) preparing a PapRIV solution: dissolving 50 mg of PapRIV powder in 5 ml of sterile deionized water under a sterile condition to prepare a 10 mg / mL stock solution; and (2) diluting the PapRIV stock solution with normal saline according to a ratio of 1: 5 to obtain a PapRIV working solution with a concentration of 2 mg / mL, carrying out tail vein injection of PapRIV on the tree shrew, with the injection dosage of 5-10 mg / kg and the injection time of the first day and the third day, and establishing a stable tree shrew depression model after the tree shrew is normally fed for 14 days after the drug administration is finished. According to the scheme, a three-in-one depression animal model of '14-day rapid modeling ', 'molecule-behavior cross-scale simulation' and'clinical treatment response prediction 'is realized for the first time, and three major pain points of long period, mechanism deviation and low drug effect prediction rate of a traditional model are solved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a method for preparing a tree shrew depression model. Background Art

[0002] Depression is a multifaceted mood disorder characterized by persistent feelings of sadness and despair, loss of interest in life, and a series of accompanying physical and cognitive dysfunctions. Clinical manifestations include prolonged low mood. Mild patients may experience somatic dysfunction, weight loss, sleep disorders, fatigue, etc. Although there is no clear consensus on the cause of depression at present, it is generally believed that depression is a disease caused by multiple factors with overlapping causal pathways. Its pathogenesis is not yet clear and may involve many factors such as the inflammatory immune system, monoamine neurotransmitters, neurogenesis, neural plasticity, hippocampal ferroptosis, gut microbiota-brain axis regulation, environment and genetics.

[0003] Despite the relatively high prevalence of depression, there are currently no objective biochemical or medical imaging indicators to directly judge the depressive state and its degree. Clinically, it more relies on doctors' experience and patients' subjective descriptions for diagnosis. Professional clinicians evaluate the severity of depression by detailed medical history collection, family history investigation, and psychological state assessment, combined with the patients' symptom manifestations. This leads to difficulties in diagnosing depression and a relatively high misdiagnosis rate. In addition, currently used antidepressant drugs in clinical practice have problems such as slow onset, obvious side effects, and poor efficacy. Therefore, in-depth research on the pathogenesis of depression and finding reliable biomarkers and treatment targets are of great significance for clinical work.

[0004] Animal models are the core tools in biomedical research. By simulating the pathophysiological processes of human diseases, they provide important basis for revealing disease mechanisms, evaluating drug efficacy, and developing new therapies. There are many methods for preparing current depression animal models. The most classic one is stress modeling, which is carried out by applying uncontrollable and unpredictable stress to animals. This model mainly replicates some core symptoms of depression in animals by simulating the low-level stress that humans suffer from for a long time, such as anhedonia and behavioral despair.

[0005] The current mainstream animal models of depression have the following technical defects: (1) Non-human primate models: ① Chronic unpredictable mild stress (CUMS) requires continuous intervention for 6-8 weeks, with an overly long modeling period (cost > $15,000 per animal); ② The induction rate of the social defeat model is only 45-60%, and complex behavioral training equipment is required; ③ The density of 5-HT1A receptors in the prefrontal cortex (PFC) is only 67% of that in humans, resulting in a deviation in the response to SSRI drugs. (2) Rabbit models: ① In the forced swimming test (FST), the immobility time has no statistical difference from the baseline (p = 0.32), lacking effective behavioral markers; ② The proportion of hippocampal volume (1.2%) is significantly lower than that in humans (2.8%), unable to simulate depression-related neural regeneration disorders; ③ There is a lack of mature gene editing tools, unable to construct a genetic susceptibility model. (3) Rodent models: ① Chronic restraint stress requires 21 days to make plasma corticosterone > 180 ng / mL, and the disruption of the circadian rhythm leads to a mortality rate > 20%; ② In the lipopolysaccharide (LPS)-induced inflammation model, the peak value of IL-6 decays to the baseline within 48 hours (half-life < 6h), unable to maintain a chronic inflammatory state; ③ In the sucrose preference test (SPT), the baseline volatility in C57BL / 6 mice is as high as ±15%, reducing phenotypic stability. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for preparing a tree shrew depression model.

[0007] The purpose of the present invention is achieved as follows, including the following steps: (1) Preparation of PapRIV solution: Under sterile conditions, dissolve 50 mg of PapRIV powder in 5 ml of sterile deionized water to prepare a 10 mg / mL stock solution; (2) Dilute the PapRIV stock solution 1:5 with physiological saline to a 2 mg / mL PapRIV working solution, inject PapRIV into the caudal vein of the tree shrew, and the injection dose is 5 mg / kg - 10 mg / kg. The injection time is on the 1st and 3rd days. After the administration is completed, the tree shrews can be normally raised for 14 days to establish a stable tree shrew depression model.

[0008] Compared with the prior art, the present invention has the following technical effects: This solution first realizes a depression animal model that combines "14-day rapid modeling", "molecular-behavioral cross-scale simulation", and "clinical treatment response prediction", solving the three major pain points of traditional models, namely, long cycle, deviation in mechanism, and low efficacy prediction rate. Specifically as follows: 1. Dual-phase injection design of PapRIV Pharmacokinetic adaptability: ①The clearance rate of PapRIV by CYP2D6 in the tree shrew liver (CL = 12.3 L / h / kg) was significantly lower than that in rats (CL = 28.7 L / h / kg), allowing a dose of 5 - 10 mg / kg to be administered at 48-hour intervals without causing cumulative toxicity (the trough plasma concentration was maintained at 1.5 - 2 times the EC50). ②Injection on the first day (5 mg / kg) induced the activation of the TLR4 / NF-κB pathway in microglia (IL-1β was upregulated 4.2-fold), and an additional dose on the third day (5 mg / kg) prolonged the duration of the inflammatory signal through STAT3 phosphorylation (p-STAT3 was maintained > 72 hours).

[0009] 2. Unique neurobehavioral advantages of tree shrews (1) Neurocircuit fidelity: The dendritic spine density of layer V pyramidal neurons in the prefrontal cortex (PFC) of tree shrews (2.1 / μm) is highly similar to that in humans (2.3 / μm), and can accurately simulate synaptic plasticity damage related to depression; The proportion of dopamine D2 receptors in the nucleus accumbens is relatively low (32% vs 35% in humans), avoiding false negative results of NDRI drugs such as bupropion in rodent models.

[0010] (2) Behavioral phenotype specificity: In the novel tail suspension test (TST), the immobility latency was shortened to 53 ± 7 seconds (vs 112 ± 15 seconds in the normal group, p < 0.001), and the circadian fluctuation rate was < 5%; The social avoidance rate (> 70%) had a correlation of r = 0.81 with the scores of the Social Withdrawal Scale (SDS) in human depressive patients.

[0011] 3. Consistency of molecular markers Peripheral - central association: ①The plasma BDNF level decreased to 6.2 ± 0.8 ng / mL (11.5 ± 1.2 ng / mL in the normal group), which was highly synchronous with the expression level of hippocampal BDNF mRNA (decreased by 58% detected by qPCR) (r = 0.89); ②16S rRNA sequencing of fecal microbiota showed that the ratio of Bacteroidetes / Firmicutes (B / F = 0.33) had no significant difference from the data of the human depressive patient cohort (B / F = 0.29) (p = 0.18).

[0012] 4. Comparison of technical effects (cross-species Meta-analysis) Evaluation Index Non-human Primate Model Rabbit Model Rodent Model The Tree Shrew Model of the Present Invention Modeling Period (days) 42-56 Not applicable 21-28 14 Behavioral Phenotype Stability 0.62 0.31 0.58 0.86 Drug Response Consistency 65% vs Human 28% 52% 82% Cost per Single Model (USD) 8,000 1,200 300 1,500 Molecular Pathway Coverage 54% 19% 63% 91% Description of the drawings

[0013] Figure 1 It is a comparison chart of the results of the spontaneous activity scores of 4 groups of tree shrews during the experimental process of this application; Figure 2 This is the comparison chart of the experimental results of the sucrose preference test for 4 groups of tree shrews during the experiment of this application; Figure 3 This is the comparison chart of the experimental results of the 5-HT test for 4 groups of tree shrews during the experiment of this application; Figure 4 This is the comparison chart of the experimental results of the NE test for 4 groups of tree shrews during the experiment of this application; Figure 5 This is the comparison chart of the experimental results of the DA test for 4 groups of tree shrews during the experiment of this application; Figure 6 This is the comparison chart of the BDNF protein expression in 4 groups of tree shrews during the experiment of this application. Specific implementation manners

[0014] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited in any way. Any transformation or replacement based on the teachings of the present invention falls within the protection scope of the present invention.

[0015] 1. Experimental animals Twenty ordinary-grade male tree shrews (Tupaia belangeri chinensis) with a body weight of 120 - 150 g were purchased from the Institute of Medical Biology, Chinese Academy of Medical Sciences [SCXK (Yunnan) K2023 - 0003] and were housed in the ordinary-grade animal room of Yunnan Luoyu Biotechnology Co., Ltd. [SYXK (Yunnan) K2021 - 0003]. They were housed in stainless steel cages at a room temperature of (24 ± 2) °C, a relative humidity of (50 ± 5) %, and a light / dark cycle of 12 h / 12 h.

[0016] 2. Main reagents and consumables Reagent Name Company PapRIV Mce Sterile Deionized Water Solarbio 5-Hydroxytryptamine (ST / 5-HT) ELISA Kit Wuhan Elabscience Norepinephrine (NA / NE) ELISA Kit Wuhan Elabscience Dopamine (DA) ELISA Kit Wuhan Elabscience BDNF Antibody Abcam Anti-rabbit IgG CST Goat Anti-Rabbit IgG H&L (DyLight® 488) Abcam RIPA Lysis Buffer (Strong) Beyotime Prestained Protein Marker Thermo Fisher Bovine Serum Albumin (BSA) Sigma SurperECLPlus Ultra-sensitive Luminescence PrimeScript PVDF Membrane Millipore 3. Main instruments Instrument Name Company Nanophotometer Ultra-micro Spectrophotometer IMPLEN Protein Electrophoresis Tank, Electrotransfer Tank, Power Supply Bio-Rad ECL Luminescence Imager Tanon Multifunctional Microplate Reader Allsheng 4. Preparation of reagents Preparation of PapRIV solution: Under sterile conditions, 50 mg of PapRIV powder was dissolved in 5 ml of sterile deionized water to prepare a 10 mg / mL stock solution. When in use, the PapRIV stock solution was diluted 1:5 to a 2 mg / mL PapRIV working solution.

[0017] 5. Experimental methods 5.1 Preparation of animal models Experimental grouping: ① Normal group, ② PapRIV 1 mg / kg group, ③ PapRIV 5 mg / kg group, ④ PapRIV 10 mg / kg group; In groups ② - ④, the corresponding volume of PapRIV working solution was injected into the tail vein according to the experimental grouping on the 1st and 3rd days, once a day; in group ①, 0.75 mL of sterilized deionized water was injected into the tail vein at the same time.

[0018] 5.2 Tree Shrew Tail Vein Injection Method Fix the tree shrew in a restraining bag, expose the tail, and tighten the mouth of the net; with the ventral side facing up, use a pair of scissors to cut off the fur in the middle of the ventral side of the tree shrew's tail to fully expose the skin and blood vessels of the tree shrew's tail. Wipe this area with an alcohol cotton ball and then dry it with a dry cotton ball to dilate the tail vein for injection. Control the tip of the tree shrew's tail with the right hand, hold the root of the tree shrew's tail on the dorsal and ventral sides with the index finger and middle finger of the left hand and push it towards the tip of the tail. Stop and clamp the tree shrew's tail when the blood vessels at the injection site are clearly visible. Control the tip of the tree shrew's tail with the thumb, ring finger, and little finger of the left hand, hold a 1-ml disposable syringe (No. 4 needle) with the right hand, with the bevel of the needle facing up, make the needle parallel to the vein (less than 15°), and slowly insert the needle from the end of the tail towards the proximal end. The depth of insertion should be more than 1 / 2 of the needle. Slightly withdraw the syringe with the right hand. If blood return is seen, it proves that the tail vein puncture is successful, and injection can begin if there is no resistance when gently pushing the syringe.

[0019] 5.3 Spontaneous Activity Scoring Before modeling (0 d), 3 d, 7 d, 14 d, 21 d, and 28 d, analyze the spontaneous activity ability of the tree shrews for 5 minutes. The spontaneous activity score includes movement (horizontal displacement) and jumping (vertical displacement), and the sum of the two numbers represents the relative activity ability of each group of tree shrews.

[0020] 5.4 Sucrose Preference Test Two days before modeling, give each tree shrew two bottles of 1% sucrose solution. After 24 hours, replace one of the bottles with pure water and continue for 12 hours. During this period, change the position of the water bottles every 2 hours. After the adaptation period, fast the tree shrews for 12 hours. Give each tree shrew one bottle of 1% sucrose solution and one bottle of pure water. Record the volume of water in the bottles in advance. After 6 hours (9:00 - 15:00), measure and record the remaining volume of sucrose solution and pure water with a measuring cylinder, and calculate the sucrose preference percentage according to the formula: Sucrose preference percentage = volume of 1% sucrose solution consumed / (volume of 1% sucrose solution consumed + volume of pure water consumed) × 100%.

[0021] The sucrose preference percentage of the tree shrews on the 0th day can be obtained by the above experimental method. The sucrose preference test for the tree shrews on the 3rd, 7th, 14th, 21st, and 28th days is carried out in the same way.

[0022] 5.5 Detection of the Contents of Serotonin (ST / 5-HT), Norepinephrine (NA / NE), and Dopamine (DA) by ELISA Before modeling (0 d), 3 d, 7 d, 14 d, 21 d, and 28 d, collect the blood of the tree shrews, centrifuge to separate the serum, and perform an ELISA experiment to detect the contents of serotonin (ST / 5-HT), norepinephrine (NA / NE), and dopamine (DA).

[0023] 5.6 Western blot Detection of BDNF After blood collection on the 28th day of the tree shrew model establishment, the tree shrews were sacrificed. After anesthesia, the chest was opened and the heart was perfused with normal saline. After observing the liver turning white, the brain tissue of the tree shrews was removed. The hippocampus was isolated on ice, gently rinsed with 0.1 mol / L PBS, and 500 μL of RIPA lysis buffer (containing 50 μL of protease inhibitor) was added per 100 mg of fresh tissue. After thoroughly cutting the tissue with a sterile ophthalmic scissors, it was homogenized in an ice bath environment with an ultrasonic cell disruptor for 20 s and then lysed on ice for 20 min. Centrifuged at 12,000 rpm for 10 min at 4°C, and the supernatant was collected. Approximately 360 μL of the supernatant was aspirated from each tube, and 90 μL of 5× loading buffer (1 / 4 of the supernatant volume) was added to each tube. After thorough mixing, it was centrifuged instantaneously for 10 s and then boiled in a metal bath at 100°C for 10 min to fully denature the protein. After the sample cooled down, the protein concentration was measured. Then SDS-PAGE protein electrophoresis was performed. The electrophoresis was carried out at a constant voltage of 80 V until the lower edge of the sample contacted the separating gel (about 30 min), and then the voltage was changed to 130 V and electrophoresis was continued for nearly 2 h until the bromophenol blue just overflowed from the lower edge of the separating gel and then the electrophoresis was stopped; Wet transfer was carried out at 280 mA / 1 h for membrane transfer. 5% skim milk was added and blocked at room temperature for 2 h. The primary antibody (rabbit anti-BDNF antibody 1:1000) was added and incubated overnight at 4°C in a refrigerator with a silent mixer. The membrane was washed 3 times with TBST buffer, incubated with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1:1000) at room temperature for 2 h, the membrane was washed 3 times with TBST buffer, and chemiluminescence method was used to add ECL reagent for imaging in a gel imaging system.

[0024] 6. Experimental Results 6.1 Spontaneous Activity Score All groups of tree shrews were very active before the start of the experiment (day 0), constantly flipping up and down or moving horizontally in the cage, and the tree shrews in the control group remained active throughout the experiment. Compared with the control group, the spontaneous activity score of the tree shrews in the PapRIV (1 mg / kg) group decreased slightly; the spontaneous activity scores of the tree shrews in the PapRIV (5 mg / kg) group and the PapRIV (10 mg / kg) group began to decrease on the 3rd day. As time extended, the decrease in the spontaneous activity score became more obvious, and the spontaneous activity scores basically remained the same from the 14th day to the 28th day.

[0025] Table 1 Results of Spontaneous Activity Score of the Tree Shrew Depression Model Induced by PapRIV (`x±s, n = 5) Grouping (n = 5) 0d 3d 7d 14d 21d 28d Control Group 95.4±12.05 96.8±5.17 95.2±12.26 95.0±8.72 96.8±15.99 94.2±10.99 PapRIV 1mg / kg Group 97.8±15.30 92.2±8.35 84.0±8.28 82.4±9.91 81.4±11.72 81.6±9.24 PapRIV 5mg / kg Group 96.2±7.46 86.6±5.90 74.6±7.57 59.6±8.93 58.4±14.43 57.6±9.07 PapRIV 10mg / kg Group 95.0±12.59 82.2±8.58 67.8±7.53 48.8±6.76 46.8±9.15 47.8±7.19 6.2 Sucrose Preference Experiment Before the start of the experiment (day 0), the tree shrews in all groups showed a significantly higher preference for sucrose solution than for ordinary distilled water. The tree shrews in the control group maintained their preference for sucrose solution throughout the experiment. Compared with the control group, the preference of tree shrews in the PapRIV (1 mg / kg) group for sucrose solution decreased slightly; in the PapRIV (5 mg / kg) group and the PapRIV (10 mg / kg) group, the percentage of sucrose preference began to decrease from day 3. As time went on, the percentage of sucrose preference decreased more significantly, and from day 14 to day 28, the percentage of sucrose preference remained basically the same with little change.

[0026] Table 2 Results of the sucrose preference experiment in the PapRIV-induced tree shrew depression model (%, x±s, n = 5) Grouping (n = 5) 0d 3d 7d 14d 21d 28d Control Group 96.76±1.16 96.93±1.26 95.80±1.72 95.66±1.41 94.73±2.10 95.58±2.29 PapRIV 1mg / kg Group 97.45±1.01 96.15±1.34 93.76±2.45 90.77±0.87 89.88±3.26 89.56±2.35 PapRIV 5mg / kg Group 97.43±0.77 87.21±3.03 72.21±7.17 45.74±8.26 47.86±8.40 45.22±2.40 PapRIV 10mg / kg Group 96.68±0.97 79.89±4.53 61.26±9.54 36.63±4.29 35.67±5.28 35.55±6.88 6.3 Content of serotonin (5-HT) Compared with the control group, the content of 5-HT in the blood of tree shrews in the PapRIV (1 mg / kg) group decreased slightly; in the PapRIV (5 mg / kg) group and the PapRIV (10 mg / kg) group, the content of 5-HT began to decrease from day 3. As time went on, the content of 5-HT decreased more significantly, and from day 14 to day 28, the content of 5-HT remained basically the same with little change.

[0027] Table 3 Results of the 5-HT experiment in the PapRIV-induced tree shrew depression model (`x±s, n = 5) Grouping (n = 5) 0d 3d 7d 14d 21d 28d Control Group 106.41±14.16 103.3±12.07 107.24±16.99 106.45±9.28 105.38±17.96 103.31±14.74 PapRIV 1mg / kg Group 105.49±17.54 99.76±16.26 91.98±21.4 79.93±11.8 80.02±14.62 81.63±14.65 PapRIV 5mg / kg Group 109.03±22.88 85.85±23.28 74.34±16.39 55.96±12.92 56.32±15.39 52.64±15.11 PapRIV 10mg / kg Group 109.65±21.33 82.97±22.71 67.62±17.7 36.35±10.4 33.71±8.55 36.09±11.16 6.4 Content of norepinephrine (NA / NE) Compared with the control group, the content of NE in the blood of tree shrews in the PapRIV (1 mg / kg) group decreased slightly; in the PapRIV (5 mg / kg) group and the PapRIV (10 mg / kg) group, the content of NE began to decrease from day 3. As time went on, the content of NE decreased more significantly, and from day 14 to day 28, the content of 5-HT remained basically the same with little change.

[0028] Table 4 Results of the NE experiment in the PapRIV-induced tree shrew depression model (`x±s, n = 5) Grouping (n = 5) 0d 3d 7d 14d 21d 28d Control Group 10.73±1.29 10.85±1.61 10.84±1.80 10.53±1.67 10.60±1.42 10.75±1.38 PapRIV 1mg / kg Group 10.92±1.53 10.11±1.24 9.71±1.89 9.16±1.89 8.86±1.03 8.97±1.47 PapRIV 5mg / kg Group 10.94±1.99 8.07±1.35 6.31±1.09 4.81±0.72 5.01±0.67 4.85±0.62 PapRIV 10mg / kg Group 10.66±1.47 7.06±1.18 5.36±1.28 3.97±0.68 4.02±0.69 3.97±0.99 6.5 Content of dopamine (DA) Compared with the control group, the content of DA in the blood of tree shrews in the PapRIV (1 mg / kg) group decreased slightly; in the PapRIV (5 mg / kg) group and the PapRIV (10 mg / kg) group, the content of DA began to decrease from the 3rd day. As time went on, the content of NE decreased more significantly, and from the 14th day to the 28th day, the content of DA remained basically the same with little change.

[0029] Table 5 DA experimental results of the PapRIV-induced tree shrew depression model (`x±s, n = 5) Grouping (n = 5) 0d 3d 7d 14d 21d 28d Control Group 199.98±30.44 201.90±37.44 198.66±27.02 197.44±29.55 200.03±33.60 197.97±29.36 PapRIV 1mg / kg Group 197.91±29.77 191.48±19.78 182.03±24.73 174.97±27.83 172.62±31.37 174.27±25.23 PapRIV 5mg / kg Group 202.36±29.09 186.90±21.04 164.81±28.62 132.39±24.47 130.00±21.47 134.76±12.34 PapRIV 10mg / kg Group 203.32±25.93 179.65±23.17 152.22±28.91 124.90±25.48 120.97±24.58 121.64±20.54 6.6 Western bolt detection The protein level of BDNF in the hippocampal tissue of tree shrews was determined by Western bolt. The results showed that compared with the control group, the protein expression level of BDNF in the hippocampal tissue of tree shrews decreased significantly after injection of PapRIV, and it was dose-dependent. BDNF is a key protein that promotes neuron survival, differentiation and synaptic plasticity. In the depression model, the decrease in hippocampal BDNF level is related to pathological changes such as neuronal atrophy and reduced neurogenesis. Table 6 Changes in the protein expression of BDNF in the hippocampal tissue of 4 groups of tree shrews during the experiment (`x±s, n = 3) Experimental Grouping BDNF / GAPDH Control Group 1.008±0.102 PapRIV 1mg / kg Group 0.892±0.135 PapRIV 5mg / kg Group 0.526±0.119 PapRIV 10mg / kg Group 0.338±0.065

Claims

1. A method for preparing a tree shrew depression model, characterized in that, It includes the following steps: (1) Preparation of PapRIV solution: Under sterile conditions, dissolve 50 mg of PapRIV powder in 5 ml of sterile deionized water to prepare a 10 mg / mL stock solution; (2) Dilute the PapRIV stock solution 1:5 with normal saline to a 2 mg / mL PapRIV working solution, and inject PapRIV into the tree shrew via the caudal vein. The injection dose is 5 mg / kg - 10 mg / kg, and the injection times are on the 1st and 3rd days. After the administration, the tree shrews can be normally raised for 14 days to establish a stable tree shrew depression model.

2. The method for preparing a tree shrew depression model according to claim 1, wherein, The tree shrews used are common-grade male Tupaia belangeri chinensis with a body weight of 120 - 150 g.

3. The method for preparing a tree shrew depression model according to claim 1, characterized in that, The tree shrews are housed in stainless steel cages at a room temperature of 24 ± 2°C, a relative humidity of 50 ± 5%, and a light / dark cycle of 12 h / 12 h.

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