Application of ventral hippocampal region transferrin receptor 1 inhibitor in preparation of drug for preventing and / or treating anxiety disorder

Abstract

The invention relates to application of a ventral hippocampal region transferrin receptor 1 inhibitor in preparation of a medicine for preventing and / or treating anxiety disorder, and belongs to the technical field of biological medicine. The application of the inhibitor for interfering with the animal brain ventral hippocampal region transferrin receptor 1 (TfR1) in the drugs for preventing and / or treating anxiety is discovered and proposed for the first time. Animal experiments prove that anxiety disorder caused by social isolation stress is closely related to TfR1 in a brain abdominal side hippocampus area of an animal, and when the animal shows anxiety symptom, the expression level of TfR1 is improved, so that the iron concentration in the brain abdominal side hippocampus area is improved to cause iron disorder. On the basis of the research, an inhibitor for interfering with the TfR1 in the belly hippocampus area of the brain of the animal is injected into an anxiety model animal, social isolation treatment is continued, the anxiety behavior of the animal in a social isolation treatment group is improved, and the effect of treating the anxiety is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of a ventral hippocampal region transferrin receptor 1 inhibitor in the preparation of a drug for preventing and / or treating anxiety. Background Art

[0002] Anxiety disorder is the most common mental disorder in the world. According to the World Health Organization, 301 million people worldwide suffered from anxiety disorders in 2019. In daily life, anxiety can affect people's attention, learning cognitive processes, and information processing abilities. When facing specific situations or objects, people with anxiety disorders may feel extremely uneasy and even have physical reactions such as increased heart rate, rapid breathing, sweating, or muscle tension, which can have a significant negative impact on the individual's quality of life, social function, and work ability. The negative mental health symptoms such as anxiety shown by the growing social isolation group not only affect individuals, but also bring huge public health burdens to society. Therefore, the prevention and treatment of anxiety disorders caused by social isolation is an urgent problem to be solved.

[0003] At present, for the treatment of anxiety, the main clinical recommendations are to use a combination of psychotherapy, drug therapy, or both, which are widely used for various types of anxiety. However, more and more studies have shown that anxiety disorders caused by different factors involve different mechanism pathways, so the corresponding treatment targets should also have different focuses. The above-mentioned recommended treatment methods lack specificity for the types of anxiety disorders caused by social isolation, and may not achieve the expected treatment effect. In addition, these methods themselves have limitations, such as: cognitive behavioral therapy is the most recognized psychotherapy, but it often requires a certain amount of time and energy, some patients may feel resistance or discomfort in the exposure process of fear, and the efficacy is not clear enough; first-line drugs include selective serotonin reuptake inhibitors (SSRIs) and serotonin-norepinephrine reuptake inhibitors (SNRIs). These drugs can quickly relieve symptoms and help patients recover function in the short term, but they may have side effects such as dizziness, drowsiness, dependence, and withdrawal symptoms. Long-term use requires monitoring and dosage adjustment. Therefore, it is of great research value to explore the anxiety mechanism caused by social isolation stress and find new treatment targets. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and use an inhibitor capable of reducing the expression level of transferrin receptor 1 (TfR1) in the ventral hippocampus region of the brain in the preparation of a drug for preventing and / or treating anxiety.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is:

[0006] In a first aspect, the present invention provides the use of a ventral hippocampal region transferrin receptor 1 inhibitor in the preparation of a drug for preventing and / or treating anxiety.

[0007] The present invention first discovered and proposed the use of inhibitors that interfere with transferrin receptor 1 (TfR1) in the ventral hippocampus region of the animal brain in the prevention and / or treatment of anxiety drugs. The present invention has confirmed through animal experiments that anxiety caused by social isolation stress is closely related to TfR1 in the ventral hippocampus region of the animal brain, and when the animal shows anxiety symptoms, the expression level of TfR1 increases, so that the iron concentration in the ventral hippocampus region of the brain increases, causing iron disorders. On the basis of this study, the present invention injects inhibitors that interfere with transferrin receptor 1 (TfR1) in the ventral hippocampus region of the animal brain into anxiety model animals and continues to give social isolation treatment. It is found that the anxiety behavior of animals in the social isolation treatment group is improved, achieving the effect of treating anxiety.

[0008] In addition, the present invention also confirms through animal experiments that anxiety disorder is associated with iron transfer into the ventral hippocampus of the brain, and excludes the possibility of iron metabolism disorder caused by iron transporter protein leading to a decrease in iron export cells and ferroptosis-dependent iron deposition.

[0009] In a second aspect, the present invention provides the use of a ventral hippocampal region transferrin receptor 1 inhibitor in the preparation of a drug for inhibiting the iron transfer capacity of cells in the ventral hippocampal region.

[0010] In a third aspect, the use of transferrin receptor 1 inhibitors in the ventral hippocampus region in the preparation of drugs for preventing and / or treating iron metabolism disorders in the ventral hippocampus region.

[0011] As a preferred embodiment of the use of the present invention, the ventral hippocampal region transferrin receptor 1 inhibitor includes Ferristatin II and / or a virus that interferes with the expression of transferrin receptor 1 in cells of the ventral hippocampal region.

[0012] Ferristatin Ⅱ is a ferritin inhibitor containing two copper binding centers, which can promote the degradation of TfR1. The present invention has confirmed through animal experiments that the anxiety symptoms of animals in the social isolation group after injection of Ferristatin Ⅱ are significantly alleviated, and can effectively improve the anxiety of anxiety model animals.

[0013] The present invention verifies through animal experiments that the injection of a virus that interferes with the expression of transferrin receptor 1 in cells in the ventral hippocampus region can effectively improve the anxiety behavior of anxiety model animals, and has a certain effect of improving anxiety.

[0014] As a preferred embodiment of the application of the present invention, the interference with the ventral hippocampal region cells is interference with the ventral hippocampal region neuronal cells. The present invention specifically knocks down TfR1 in the neuronal cells, astrocytes and microglia in the ventral hippocampal region of the animal brain and finds that the decrease in the TfR1 expression level of the neuronal cells can significantly reduce the anxiety behavior of the animal, indicating that interference with the TfR1 expression of the neuronal cells in the ventral hippocampal region can effectively improve anxiety.

[0015] As a preferred embodiment of the application of the present invention, the virus that interferes with the expression of transferrin receptor 1 in cells in the ventral hippocampus contains transferrin receptor 1 shRNA targeting the ventral hippocampus, and the nucleotide sequence of the transferrin receptor 1 shRNA targeting the ventral hippocampus is shown in SEQ ID NO. 1. The virus of the present invention can reduce the expression of TfR1 in the ventral hippocampus through RNA interference technology to achieve the effect of preventing and / or treating anxiety.

[0016] As a preferred embodiment of the application of the present invention, the virus that interferes with the expression of transferrin receptor 1 in cells in the ventral hippocampus region is a recombinant adeno-associated virus type 9 containing transferrin receptor 1 shRNA targeting the ventral hippocampus region.

[0017] As a preferred embodiment of the application of the present invention, the recombinant adeno-associated virus type 9 containing transferrin receptor 1 shRNA targeting the ventral hippocampal region also contains a DIO element.

[0018] As a preferred embodiment of the application of the present invention, the recombinant adeno-associated virus type 9 containing transferrin receptor 1 shRNA targeting the ventral hippocampal region also contains a specific promoter.

[0019] As a preferred embodiment of the application of the present invention, the specific promoter includes at least one of Syn promoter, mecp2 promoter, c-fos promoter, CaMKIIα promoter, TUBA1A promoter and VGAT promoter.

[0020] The recombinant adeno-associated virus type 9 containing transferrin receptor 1 shRNA targeting the ventral hippocampal region described in the present invention can use a variety of expression strategies to achieve the reduction of TfR1 expression levels, such as by adding a DIO element in combination with Cre recombinase to reduce the TfR1 expression level of the target cells, or by using a specific promoter (such as Syn, etc.) to enable the recombinant adeno-associated virus type 9 to be transfected into the target cells and then initiate the expression of shRNA to reduce the expression level of TfR1.

[0021] As a preferred embodiment of the application of the present invention, the dosage form of the drug includes at least one of tablets, solutions, suspensions, emulsions, microcapsules, microspheres, injections, liposomes and aerosols.

[0022] As a preferred embodiment of the application of the present invention, when the ventral hippocampal transferrin receptor 1 inhibitor is Ferristatin II, the content of the ventral hippocampal transferrin receptor 1 inhibitor in the drug for preventing and / or treating anxiety, the drug for inhibiting the iron transfer capacity of ventral hippocampal cells, or the drug for preventing and / or treating iron metabolism disorders in the ventral hippocampus is 0.1-0.5 μg / μL.

[0023] As a preferred embodiment of the application of the present invention, when the ventral hippocampal region transferrin receptor 1 inhibitor is a virus that interferes with the expression of transferrin receptor 1 in ventral hippocampal region cells, the content of the ventral hippocampal region transferrin receptor 1 inhibitor in the drug for preventing and / or treating anxiety, the drug for inhibiting the iron transfer capacity of ventral hippocampal region cells, or the drug for preventing and / or treating ventral hippocampal region iron metabolism disorder is (1-2.5)×10 12 VG / mL.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The present invention proposes for the first time a strategy of intervening transferrin receptor 1 for use in the preparation of drugs for the treatment or prevention of anxiety disorders. Experiments have shown that the expression level of TfR1 in the ventral hippocampus of animals with anxiety disorders related to social isolation stress is significantly increased, and iron metabolism disorders occur in nerve cells. Ferristatin Ⅱ (NSC306711) can significantly improve iron deposition in the ventral hippocampus of anxious animals and improve the anxiety symptoms of animals. Ferristatin Ⅱ (NSC306711) does not affect the cognitive, memory, and action abilities of animals. It has no obvious toxic and side effects within an appropriate dose, is easy to prepare, extract, and preserve, and has high stability. Local injection of a virus that reduces the expression of TfR1 in the ventral hippocampus of the brain can effectively reduce the expression of TfR1 in the ventral hippocampus, improve the abnormal metabolism of neuronal cells in the ventral hippocampus of socially isolated animals, and prevent or improve anxiety disorders caused by social isolation stress in a targeted manner.

[0026] 2. This invention is the first to deeply explain the mechanism of anxiety disorder caused by social isolation from the perspective of TfR1 overexpression, propose an intervention plan for the key links in this mechanism, and prove the effectiveness and huge application potential of the intervention plan, laying the foundation for the further development of drugs for anxiety disorders caused by social isolation stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The statistical results of the open field test of mice in different treatment groups in Experimental Example 1 of the present invention are as follows;

[0028] Figure 2The statistical results of the elevated plus maze test of mice in different treatment groups in Experimental Example 1 of the present invention;

[0029] Figure 3 The statistical results of the black-and-white box experiment of mice in different treatment groups in Experimental Example 1 of the present invention;

[0030] Figure 4 The statistical results of iron concentration in the ventral hippocampus of the brain of mice in different treatment groups in Experimental Example 1 of the present invention;

[0031] Figure 5 The iron staining results of the ventral hippocampus region of the brain of mice in different treatment groups in Experimental Example 1 of the present invention;

[0032] Figure 6 The Fe in the ventral hippocampus region of the brain of mice in different treatment groups in Experimental Example 1 of the present invention 2+ , 4-HNE, DMT1, FPN and TfR1 content and expression level difference fold statistical results;

[0033] Figure 7 is a fitting curve of the TfR1 protein expression level and iron concentration in the ventral hippocampus region of the brain of anxiety model mice in Experimental Example 1 of the present invention;

[0034] Figure 8 The statistical results of iron concentration in the ventral hippocampus of the brain, open field test and elevated plus maze test of mice in different treatment groups in Experimental Example 2 of the present invention;

[0035] Fig. 9 The results of fluorescence microscopy detection after injection of recombinant adeno-associated virus type 9 into the ventral hippocampus of the brains of mice in different treatment groups in Experimental Example 2 of the present invention, as well as the statistical results of the open field test and the elevated plus maze test;

[0036] Fig.10 The results of fluorescence microscopy detection after injection of recombinant adeno-associated virus type 9 into the ventral hippocampus of the brains of mice in different treatment groups in Experimental Example 2 of the present invention, as well as the statistical results of the open field test and the elevated plus maze test;

[0037] Fig.11 The results of fluorescence microscopy detection after injection of recombinant adeno-associated virus type 9 into the ventral hippocampus of the brains of mice in different treatment groups in Experimental Example 2 of the present invention, as well as the statistical results of the open field test and the elevated plus maze test;

[0038] In the above figures, the "*" mark indicates that there are significant differences between the two groups, where "*" indicates p < 0.05, "**" indicates p < 0.01, and "***" indicates p < 0.001. DETAILED DESCRIPTION

[0039] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0040] Unless otherwise specified, the reagents and consumables used in the following experimental examples can be obtained through commercial channels.

[0041] The SPF-grade C57BL / 6J mice used in the following experimental examples were purchased from the Experimental Animal Center of Southern Medical University and bred at South China University of Technology. The mice were housed in a standard animal room with a temperature of 23±2°C and a humidity of 50±10%, a 12h / 12h (lighting on at 8:00), and free access to food. This experiment was approved by the Animal Ethics Committee of South China University of Technology and was carried out in strict accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

[0042] In the following experimental examples, CaMK2α-Cre mice (strain number: 005359), Aldh1l1-Cre mice (strain number: 023748), and Cx3Cr1-Cre mice (strain number: 025524) were all from the Jackson Laboratory and bred at South China University of Technology. Mice were housed in a standard animal room at a temperature of 23±2°C and a humidity of 50±10%, with a 12h / 12h (light on at 8:00) circadian rhythm, and free access to water and food.

[0043] In the following experimental examples, the kits used include a Prussian blue staining kit (purchased from Beijing Solebow Technology Co., Ltd., item number G1428); an enzyme-linked immunosorbent assay kit for 4-HNE purchased from Biomatik, item number EKF58313; and a Fe 2+ The kit was purchased from DOJINDO with the catalog number I291. If there are no detailed operation steps in the experimental examples, the steps in the kit manual were followed.

[0044] The primers for RT-qPCR of DMT1, FPN and TfR1 are:

[0045] DMT1, forward: 5-ACAAATATGGCTTGCGGAAGCTGG-3, reverse: 5-ACATGTTGTGCGGCATGATCACAG-3;

[0046] FPN, forward: 5-TTGTTGTTGTGGCAGGAGAA-3, reverse: 5-AGCTGGTCAA TCCTTCTAATGG-3;

[0047] Tfr, forward: 5-CTTTCCTTGCATATTCTGG-3, reverse: 5-CCAAATAAGGATA GTCTGCATCC-3.

[0048] The antibody used for immunoblotting experiment of TfR1 is anti-TfR1 (1:1000, ab214039, purchased from Abcam), and the internal reference is anti-GAPDH (1:2000, catalog number ab8245, purchased from Abcam). Immunoblotting experiment is a routine technique in this field, and reference can be made to "Molecular Biology Experiments" (2021, Zhejiang University Press), "Biochemistry Experiments" (2020, Science Press), etc.

[0049] Ferristatin II (NSC306711) was purchased from MedChemExpress with the product number HY-D0256A.

[0050] In the following experimental examples, the recombinant adeno-associated virus type 9 was commissioned to Brain Case (Shenzhen, China) to prepare; the recombinant adeno-associated virus type 9 for TfR1 is AAV9-CAG-DIO-TfR1-shRNA-GFP, and the nucleotide sequence of shRNA is shown in SEQID NO.1. AAV9-CAG-DIO-TfR1-shRNA-GFP carries the DIO element, which is an element that cooperates with the Cre recombinase to control the expression of the target gene (TfR1-shRNA). If there is no Cre recombinase, the target gene (TfR1-shRNA) will not be expressed. This DIO strategy relies on the virus to react with the above-mentioned cell-specific transgenic mice to produce a cell-specific infection effect (specifically manifested as a decrease in TfR1 expression). Mice that do not carry Cre recombinase will not be specifically infected, and their TfR1 expression will not be affected. The titers of the recombinant adeno-associated virus type 9 mentioned above are all 2.50×10 12 VG / mL, VG / mL is the number of gene copies contained in 1 mL of virus liquid (VG, vector genomes).

[0051] Specific steps of the elevated plus maze experiment: On the morning of the experiment, an elevated plus maze (closed wall length 65cm, open arm length 65cm) was used. Before the experiment, the mice to be tested were gently soothed. Each mouse was gently placed in the center of the device and allowed to move freely for 5 minutes. The mouse's movement trajectory was recorded by a camera above the elevated plus maze. After each mouse completed an elevated plus maze experiment, the device was cleaned and disinfected with 75% (v / v) ethanol, and the next mouse was placed after removing the odor. When the mouse enters the elevated plus maze device, due to the fear of the mouse's surrounding uncovered and high-altitude environment, the mouse will instinctively prefer to be in the closed wall, while normal mice have a strong characteristic of exploring novelty, and usually explore the open arm area a certain number of times and a certain proportion of time. The exploration novelty characteristics of anxiety model mice are greatly weakened, and the fear of open and high-altitude environments is enhanced, so they will obviously prefer to be in a closed wall environment. In this experiment, Ethovision software was used to record and analyze the mouse's movement trajectory preference.

[0052] Specific steps of the open field experiment: The equipment needed in the open field experiment is composed of a rectangular box of 40cm×40cm×40cm (length×width×height), with a rectangular frame at the bottom as the central area. Gently soothe the mice to be tested before the experiment. At the beginning of each experiment, place the mice in the central rectangular frame and let them move freely in the open field. The sensing equipment will record and save the path they walk in the open field. After each mouse completes an open field experiment, clean and disinfect the device with 75% (v / v) ethanol to remove odors. When the mouse enters the open field device, due to the fear of rodents for the uncovered open environment, the mouse will instinctively prefer to move along the four walls of the device, while normal mice have a strong characteristic of exploring novelty, and usually have a certain proportion of time to explore the central open area. The exploration novelty characteristics of anxiety model mice are greatly weakened, and the fear of the open environment is enhanced, so they will obviously dislike being in the central open area. The anxiety state of the animal can be reflected by capturing and recording the trajectory of the mouse's movements through the camera lens and analyzing and calculating the duration of the mouse's stay in the central area. The experimental results were recorded and analyzed using Ethovision software.

[0053] Specific steps of the black and white box experiment: The black and white box device is a rectangular box of 45cm×27cm×27cm (length×width×height). The long side is vertically divided into two parts. The white box part is an open bright area without a roof. A 30W fluorescent lamp is set 40cm above the bright box and 40cm from the bottom of the box. The black box part is a closed dark area with no light source. There is a passage between the two areas that can accommodate a mouse. Before the experiment, gently comfort the mouse to be tested. During the experiment, the mouse is gently placed in the middle of the white box with its back to the black box. The detection time is 5 minutes. The camera above records the length of time the mouse stays in the white box and the black box. The experimental results are recorded and analyzed using Ethovision software. After each mouse completes an open field test, the device is cleaned and disinfected with 75% (v / v) ethanol, and the next mouse is placed after removing the odor. Due to the nature of rodents to avoid light, mice instinctively prefer to be in a dark environment. Normal mice have a strong ability to explore novelty, and usually spend a certain proportion of time exploring the white box area. However, the exploration of novelty in anxiety model mice is greatly weakened, and the fear of bright environments is enhanced, so they will obviously not like to be in the white box. The anxiety of mice can be evaluated by analyzing the time the animals stay in the black box and the white box. The longer the time in the black box, the more serious the anxiety of the mice.

[0054] Experimental Example 1

[0055] In order to verify that anxiety disorder is associated with iron content in the ventral hippocampus of the brain, an anxiety disorder model mouse was constructed and the iron content in the ventral hippocampus was detected. The specific scheme is as follows:

[0056] 1 Construction of anxiety disorder model mice

[0057] 1.1 Healthy 8-week-old male C57BL / 6J mice were divided into a social isolation group and a control (group living) group. The social isolation group had a single mouse in a cage for 28 days, and the control group had four mice in a cage for 28 days. Both groups of mice had free access to water and food.

[0058] 1.2 The mice in the control group and the mice in the social isolation group were subjected to three anxiety behavior tests, including the elevated plus maze test, the open field test, and the black-and-white box test. The experimental data of each group of mice were recorded. All three experimental devices were kept free of special odors. The results are shown in Figure 1-3 .

[0059] like Figure 1 As shown in the open field test, the time that mice in the social isolation group stayed in the central area of ​​the open field was significantly reduced; Figure 2 As shown, compared with the control group, the mice in the social isolation group spent significantly less time in the open arm in the elevated plus maze test, and the number of times they entered the open arm was also reduced; Figure 3As shown in the figure, in the black and white box experiment, the number of times the mice in the social isolation group entered the light box decreased, and the latency of entering the bright box increased, that is, the time they stayed in the light box decreased. The above results show that social isolation stress can cause anxiety-like behavior in mice, indicating that the anxiety model mice were successfully established.

[0060] 2 Determination of iron content in the ventral hippocampus of anxiety disorder model mice

[0061] 2.1 The mice in the social isolation group and the control group obtained in step 1.1, which were socially isolated or group-housed for 28 days, were anesthetized by intraperitoneal injection of 0.3wt% sodium pentobarbital solution at a dose of 0.1mg / 10g. After anesthesia, the mice were fixed on a foam board, and the heart and liver were exposed. The mice were first perfused by heart with 50mL of normal saline, and then fixed by heart perfusion with 50mL of 4% formaldehyde solution. Then the brain was removed by decapitation, and the ventral hippocampal region slices with a thickness of 1000μm were separated and taken out. The isolated brain tissue to be tested was weighed, and then pre-cooled PBS (0.1M phosphate, 1% v / v HCl) was added for homogenization. The homogenate was centrifuged at 4℃, 5000rpm for 10min, and the supernatant was taken for detection using a serum metal element detection kit. The sample was burned in an atomic absorption spectrometer, and the sample to be tested was atomized by high-temperature combustion of acetylene. The generated atoms were in a free radical state, which absorbed the characteristic spectrum of the element to be tested emitted by the hollow cathode lamp to complete the detection. By simultaneously measuring the standard in the kit, a standard curve is obtained to complete the measurement process. The results are shown in Figure 4 .

[0062] 2.2 Obtain the ventral hippocampal tissue of the brain according to the method of step 2.1, cut it into a thickness of about 3-5mm, and quickly place the cut brain tissue in a 4% paraformaldehyde solution at 4℃ for 1-2d. After routine dehydration, transparency and paraffin embedding, cut the tissue into 6μm slices, and the slices are routinely dewaxed and rehydrated. Take the Prussian blue staining kit out of the refrigerator and rewarm it to room temperature (25-30℃) for 20min, and fill the wet box with water and place it in a 37℃ constant temperature box for preheating for 20min. Then prepare the Perls staining working solution in a 1:1 ratio, add it to the slices until the tissue is completely covered, and place it in a wet box at 37℃ for incubation for 20min. After taking out the slices, rinse them gently with distilled water three times, 10s each time. Prepare the incubation working solution in a 1:9 ratio, add it to the slices until the tissue is completely covered, and place it in a wet box at 37℃ for incubation for 10-20min. Then take out the slices and gently soak them three times with 1×PBS, 60s each time. Prepare the enhanced working solution in a ratio of 19:1, add it to the slice until the tissue is completely covered, and incubate it in a humidified box at 37°C for 10-20 minutes. Take out the slice, gently rinse it three times with 1×PBS, 5 seconds each time, add the counterstain solution for 3-5 minutes, and then rinse it with distilled water for 10 minutes. Dehydrate with gradient ethanol, make it transparent with xylene, and seal the slice with neutral gum. Observe and analyze it under a microscope. The results are shown in Figure 5 .

[0063] like Figure 4 As shown in Figure 2, compared with the control group, the iron content in the ventral hippocampus of mice in the social isolation group was significantly increased, indicating that anxiety is associated with changes in iron content in the ventral hippocampus. Figure 5 As shown, the ventral hippocampus of socially isolated mice had darker iron staining, suggesting that anxiety disorders may be associated with increased iron content in the ventral hippocampus.

[0064] 3. Detection of specific changes in iron metabolism in the ventral hippocampus of anxiety disorder model mice

[0065] 3.1 Enzyme-linked immunosorbent assay kit was used to quantitatively detect the amount of divalent iron (Fe) in the ventral hippocampus of the two groups of mice (blank control group and social isolation stress group). 2+ ), 4-hydroxyazelaate (4-HNE) content, and the expression levels of divalent metal ion transporter protein 1 (DMT1), ferroportin (FPN), and transferrin receptor 1 (TfR1) were quantitatively detected by RT-qPCR technology, and correlation analysis was performed. The results are shown in Figure 6 .

[0066] like Figure 6 As shown, compared with the control group, social isolation led to a specific increase in TfR1 expression, while Fe 2+, 4-HNE, DMT1 and FPN did not change significantly, proving that social isolation led to an increase in the ability of cells to transfer iron into, while the ability to transfer iron out did not change significantly. It also proved that social isolation caused iron metabolism disorders but did not cause cell iron death.

[0067] 4. Verify the correlation between TfR1 protein expression and iron content in the ventral hippocampus of anxiety model mice

[0068] During the construction of the anxiety disorder model mice, the data of iron content (iron concentration) and TfR1 protein expression (measured by immunoblotting) in the ventral hippocampus of the brain were collected at the same time. The correlation between the iron concentration and the TfR1 protein expression in the ventral hippocampus of the anxiety disorder model mice was analyzed by linear regression. The results are shown in Figure 7 .

[0069] like Figure 7 As shown in Figure 2, the iron content in the ventral hippocampus of the brain was significantly positively correlated with the expression of TfR1 in the ventral hippocampus. 2 =0.7475, P = 0.0027, indicating that the increased iron content in the ventral hippocampus of anxiety model mice was associated with the increased expression of TfR1 in the ventral hippocampus.

[0070] Experimental Example 2

[0071] In order to further verify the effect of iron disorder in the ventral hippocampus of anxiety model mice, the iron inhibitor Ferristatin II was used to intervene in the ventral hippocampus of mice by in situ cannula administration, and TfR1 was knocked down in the ventral hippocampus cells. The specific scheme is as follows:

[0072] 1. Iron inhibitor intervention trial

[0073] 1.1 SPF adult male C57BL / 6J mice (22±2g, 8 weeks old) were divided into NSC306711 group and control group. After intraperitoneal injection of 0.3wt% sodium pentobarbital solution 0.1mL / 10g, the mouse brain was fixed on a stereotaxic instrument, the skull was leveled, the skin on the top of the skull was cut, the skull was exposed, and the tip of the lambdoid suture on the skull surface was used as a surface landmark to locate the bilateral ventral hippocampal region (coordinates: AP: -3.28mm; ML: ±3.25mm; DV: -4.50mm). A cranial drill was used to drill holes, and 2-3 small holes were drilled near the hole position for screwing in the skull pins as the dental cement fulcrum. Then the dura mater above the target brain area was slightly punctured with a needle to expose the skull, the skull pin was screwed into the hole for about three turns, and then the prepared catheter was slowly implanted into the skull through the clamp, and the screw and catheter were wrapped and fixed with dental cement. After waiting for the dental cement to solidify for 15 minutes, the clamp was removed and the catheter cap was slowly inserted, tightened, and the skin was sutured. The mice were allowed to recover for two weeks and were fed normally during this period.

[0074] 1.2 After two weeks, when no abnormal health conditions were observed, the two groups of mice were respectively put into group housing and social isolation housing, and each group of mice was administered FerristatinⅡ(NSC306711) (0.5μL, 0.1μg / μL) and artificial cerebrospinal fluid (1μL) into the bilateral ventral hippocampus daily for 28 days. The specific operation was as follows: the drug was extracted and injected with a micropipette. After the injection, the mice were left to stand for 10 minutes to allow the drug to diffuse fully, then the injection inner tube was slowly pulled out, the catheter cap was reinserted, and tightened.

[0075] 1.3 After all the injection days, the mice were subjected to anxiety behavior tests (elevated plus maze test and open field test). The results are shown in Figure 8 .

[0076] like Figure 8 As shown, compared with the control group mice, the socially isolated NSC306711 group mice spent significantly more time in the central area of ​​the open field; the time spent in the open arm of the elevated plus maze increased and the number of times they entered the open arm increased. The above results further prove that FerristatinⅡ (NSC306711) as a TfR1 degradation drug has a therapeutic effect on anxiety caused by social isolation stress, and also prove the effectiveness and feasibility of the strategy of intervening transferrin receptor 1 in the preparation of drugs for the prevention and / or treatment of anxiety.

[0077] 2 Intervention experiment on TfR1 expression in ventral hippocampal cells

[0078] 2.1 The three types of mice, CaMK2α-Cre mice, Aldh1l1-Cre mice, and Cx3Cr1-Cre mice, were set up as interference groups and negative control groups. The mice in the interference group were stereotactically injected with recombinant adeno-associated virus type 9 (AAV9) that lowly expressed transferrin receptor 1 (TfR1) in the bilateral ventral hippocampus, and the expression of transferrin receptor 1 (TfR1) was knocked down in neurons (using CaMK2α-Cre mice), astrocytes (using Aldh1l1-Cre mice), and microglia (using Cx3Cr1-Cremice) in the ventral hippocampus using a DIO-dependent cell-specific strategy. A preliminary experiment was conducted in the early stage to prove the effectiveness of this intervention strategy, and the health status was observed after injection. The mice in the interference group and the control group were divided into a normal feeding group and a social isolation group. The mice in the social isolation group were subjected to social isolation feeding. The mice in the normal feeding group were kept in a cage of four mice. After 4 weeks of normal feeding, anxiety behavior tests (elevated plus maze test and open field test) were performed. The experimental results were recorded and analyzed, and the accuracy of the virus injection position was confirmed by fluorescence microscopy (because the recombinant adeno-associated virus type 9 carries a green fluorescent protein marker, when it knocks down the target gene, it will cause the tissue to emit green fluorescence, thereby judging whether the target gene is knocked down in the expected ventral hippocampal area). The results are shown in Figure 9-11 .

[0079] like Figure 9-11 As shown, the anxiety symptoms of mice in the social isolation group after specific low expression of neuronal cells were significantly reduced, the time they stayed in the open arm in the elevated plus maze test was significantly increased, and the number of times they entered the open arm was significantly increased; in the open field test, the time that mice in the social isolation group after specific neuronal cells stayed in the central area of ​​the open field was significantly increased; while no significant reduction in the anxiety symptoms of mice in the social isolation group with specific low expression of astrocytes and microglia was observed, indicating that in anxiety disorder caused by social isolation stress, TfR1-mediated iron deposition occurs specifically in neuronal cells in the ventral hippocampus, and has no obvious relationship with astrocytes and microglia.

[0080] In summary, the above experiments have proved that social isolation stress leads to increased expression levels of TfR1 in the ventral hippocampus of animals' brains, causing iron metabolism disorders in the neuronal cells in this area, which in turn leads to the occurrence of anxiety. This experiment also proves that the use of the TfR1 inhibitor FerristatinⅡ and recombinant adeno-associated virus that interferes with TfR1 expression has a therapeutic and preventive effect on anxiety caused by social isolation stress, and can be used in the preparation of drugs for the prevention and / or treatment of anxiety disorders.

[0081] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. Use of a transferrin receptor 1 inhibitor in the ventral hippocampal region in the preparation of a drug for preventing and / or treating anxiety disorders.

2. Application of transferrin receptor 1 inhibitor in ventral hippocampus region in the preparation of drugs that inhibit the iron transfer ability of cells in ventral hippocampus region.

3. Use of transferrin receptor 1 inhibitors in the ventral hippocampus region in the preparation of drugs for preventing and / or treating iron metabolism disorders in the ventral hippocampus region.

4. The use according to any one of claims 1 to 3, characterized in that: The ventral hippocampal region transferrin receptor 1 inhibitor includes Ferristatin II and / or a virus that interferes with the expression of transferrin receptor 1 in cells in the ventral hippocampal region.

5. The use according to claim 4, characterized in that The interfering with the ventral hippocampal region cells is interfering with the ventral hippocampal region neuronal cells.

6. The use according to claim 4, characterized in that The virus interfering with the expression of transferrin receptor 1 in cells of the ventral hippocampus region contains transferrin receptor 1 shRNA targeting the ventral hippocampus region, and the nucleotide sequence of the transferrin receptor 1 shRNA targeting the ventral hippocampus region is shown in SEQ ID NO.

1.

7. The use according to claim 6, characterized in that The virus interfering with the expression of transferrin receptor 1 in cells in the ventral hippocampus region is a recombinant adeno-associated virus type 9 containing transferrin receptor 1 shRNA targeting the ventral hippocampus region.

8. The use according to any one of claims 1 to 3, characterized in that: The dosage form of the drug includes at least one of tablets, solutions, suspensions, emulsions, microcapsules, microspheres, injections, liposomes and aerosols.

9. The use according to any one of claims 1 to 3, characterized in that: When the ventral hippocampal transferrin receptor 1 inhibitor is Ferristatin II, the content of the ventral hippocampal transferrin receptor 1 inhibitor in the drug for preventing and / or treating anxiety, the drug for inhibiting the iron transfer capacity of cells in the ventral hippocampal region, or the drug for preventing and / or treating iron metabolism disorders in the ventral hippocampal region is 0.1-0.5 μg / μL.

10. The use according to any one of claims 1 to 3, characterized in that: When the ventral hippocampal region transferrin receptor 1 inhibitor is a virus that interferes with the expression of ferritin receptor 1 in ventral hippocampal region cells, the content of the ventral hippocampal region transferrin receptor 1 inhibitor in the drug for preventing and / or treating anxiety, the drug for inhibiting the iron transfer capacity of ventral hippocampal brain region cells, or the drug for preventing and / or treating ventral hippocampal region iron metabolism disorder is (1-2.5)×10 12 VG / mL.

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