A method to study the mechanism of microglia activation in depression

Abstract

The invention discloses a method for studying the activation mechanism of microglia in depression, comprising the following steps: S1: constructing a mouse inflammatory depression model; S2: experimentally evaluating the depressive-like behavior of the constructed model, and retaining the model with depressive behavior; S3: collecting hippocampal tissue samples of the retained model; S4: detecting and evaluating the state of microglia; S5: injecting adeno-associated viruses that target microglia and knock down USP22 into the hippocampus of mice; S6: repeating steps S1-S4 two weeks after the targeted injection; S7: constructing a BV2 microglial inflammatory cell model; S8: detecting and evaluating the state of microglia; S9: comparing the states of microglia in steps S4, S6 and S8 to determine the activation mechanism of microglia in depression; the invention is more accurate in studying the activation mechanism of microglia in depression.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to a method for studying the activation mechanism of microglia in depression. Background Art

[0002] Depression is an affective mental disorder or mood disorder, which is mainly manifested by significant and persistent low mood. In severe cases, it may lead to self-harm and suicidal behavior.

[0003] Currently, the treatment of depression generally relies on antidepressants, but existing antidepressants have problems such as slow onset of effect and serious side effects, and some patients with depression are insensitive to antidepressants.

[0004] Studies have shown that microglia-mediated central inflammatory response is an important factor in the occurrence and development of depression. Inhibiting microglial activation can effectively reduce the level of inflammation in the brain of patients with depression and improve the depressive-like behavior of patients with depression.

[0005] In view of this, a research method for the activation mechanism of microglia in depression was designed to study the activation mechanism of microglia in depression and lay a theoretical foundation for the benign development of antidepressant drugs. Summary of the invention

[0006] To solve the problems raised in the above background technology, the present invention provides a method for studying the activation mechanism of microglia in depression, which has the characteristics of being relatively accurate in studying the activation mechanism of microglia in depression and being able to lay a theoretical foundation for the benign development of antidepressant drugs.

[0007] To achieve the above object, the present invention provides the following technical solution: a method for studying the activation mechanism of microglia in depression, characterized in that it comprises the following steps:

[0008] S1: Construction of mouse inflammatory depression model;

[0009] S2: Evaluate the depressive-like behavior of the model constructed in step S1 through the sugar water preference test, tail suspension test and forced swimming test, and retain the model that exhibits depressive behavior;

[0010] S3: Collect hippocampal tissue samples from the model with depressive behavior retained in step S2;

[0011] S4: performing real-time fluorescence quantitative PCR detection, immunofluorescence detection and protein immunoblotting detection on the hippocampal tissue samples of the model with depressive behavior collected in step S3 to evaluate the status of microglia in the hippocampal tissue samples of the model;

[0012] S5: Adeno-associated virus was injected into the hippocampus of mice to knock down USP22 in microglia;

[0013] S6: Two weeks after the localization injection, repeat steps S1-S4;

[0014] S7: Construction of BV2 microglial inflammatory cell model;

[0015] S8: Performing protein immunoblotting detection on the BV2 microglial inflammatory cell model constructed in step S7 to evaluate the status of microglia in the BV2 microglial inflammatory cell model;

[0016] S9: Compare the status of microglia in steps S4, S6, and S8 to determine the mechanism of microglia activation in depression.

[0017] Furthermore, in step S1, the specific steps of constructing the mouse inflammatory depression model include:

[0018] S11: dissolving lipopolysaccharide in physiological saline to form a 0.1 mg / mL lipopolysaccharide solution;

[0019] S12: Inject lipopolysaccharide solution into the abdominal cavity of adult male inbred mice at a dose of 1 mg / kg / d for five consecutive days to establish a mouse inflammatory depression model.

[0020] Furthermore, in step S3, the specific steps of retaining the hippocampal tissue sample of the model with depressive behavior are:

[0021] S31: Anesthetize the mouse, open the mouse chest, and perform cardiac perfusion. Physiological saline is infused from the left ventricle, flows through the systemic circulation, and flows out from the right ventricle. When the liver turns white, stop the perfusion.

[0022] S32: Use surgical scissors to open the mouse skull and take out the mouse brain. Among them, one part of the mouse brain is used to remove the mouse hippocampus region according to the brain atlas for real-time fluorescence quantitative PCR and protein immunoblotting detection, and the other part of the mouse brain is preserved in 4% paraformaldehyde, and then precipitated and dehydrated in 30% sucrose solution for immunofluorescence detection.

[0023] Furthermore, in step S5, the injection coordinates for positioning the mouse hippocampus are 2.06 mm caudal to the anterior fontanelle, 1.5 mm to the left and right of the sagittal midline, and 2 mm deep from the skull surface. The adeno-associated virus targeting microglia to knock down USP22 is AAVMG1.2-USP22-RNAi from GeneCare Biotechnology, with a viral titer of 109TU / mL, and the RNAi sequence of USP22 contained in the vector is 5'-GUGGACAACUGGAAGCAAA-3'.

[0024] Furthermore, in step S7, the specific steps of constructing the BV2 microglial inflammatory cell model include:

[0025] BV2 microglia were treated with 100 ng / mL lipopolysaccharide for 24 h to induce BV2 microglial inflammation.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention constructs a mouse inflammatory depression model, evaluates the depressive-like behavior of the model based on a saccharide preference experiment, a tail suspension experiment and a forced swimming experiment, retains the model with the depressive-like behavior, collects hippocampal tissue samples of the model with the depressive-like behavior, evaluates the state of microglia based on real-time fluorescence quantitative PCR detection, immunofluorescence detection and protein immunoblotting detection, then injects adeno-associated viruses that target microglia to knock down USP22, repeats the above steps two weeks later, constructs a BV2 microglial inflammatory cell model at the same time, evaluates the state of microglia based on protein immunoblotting detection, and then studies the activation mechanism of microglia in depression based on the comparison of the state of microglia. This method is more accurate in studying the activation mechanism of microglia in depression, and can lay a theoretical foundation for the benign development of antidepressant drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 These are the pictures of the depressive-like behavior, sugar water preference, tail suspension test and forced swimming test of the mice of the present invention;

[0029] Figure 2 This is a graph showing the levels of inflammatory factors in the hippocampus of mice of the present invention;

[0030] Figure 3 This is a schematic diagram of the data of IBA1, a microglial cell marker in the hippocampus of mice of the present invention;

[0031] Figure 4 A is a graph showing the expression changes of USP22 in the hippocampus of the inflammatory depression model mice of the present invention, B and C are graphs showing the expression changes of USP22 in the BV2 cells of the present invention, and the effects of knocking down USP22 expression using USP22 siRNA on the inflammatory indicator iNOS of BV2 cells;

[0032] Figure 5 It is a schematic diagram of the structure of the adeno-associated virus vector of the present invention;

[0033] Figure 6 A is a diagram of the experimental process of the present invention, B is a diagram of the specific effect of the adeno-associated virus of the present invention targeting microglia, and C, D and E are diagrams of the detection of depression-like behaviors of mice of the present invention;

[0034] Figure 7A is a diagram showing the activation state of mouse microglia after knocking down microglia USP22 in the present invention, and B and C are diagrams showing the levels of inflammatory factors in the hippocampus of mice after knocking down microglia USP22 in the present invention. DETAILED DESCRIPTION

[0035] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] See also Figure 1-7 The present invention provides the following technical solution: a method for studying the activation mechanism of microglia in depression, comprising the following steps:

[0037] S1: Construction of mouse inflammatory depression model;

[0038] The specific steps of constructing the mouse inflammatory depression model include:

[0039] S11: dissolving lipopolysaccharide in physiological saline to form a 0.1 mg / mL lipopolysaccharide solution;

[0040] S12: Inject lipopolysaccharide solution into the abdominal cavity of adult male inbred mice at a dose of 1 mg / kg / d for five consecutive days to establish a mouse inflammatory depression model;

[0041] S2: Evaluate the depressive-like behavior of the model constructed in step S1 through the sugar water preference test, tail suspension test and forced swimming test, and retain the model that exhibits depressive behavior;

[0042] Before the experiment, a control group needs to be constructed. Based on the control evaluation of the two groups, the depressive-like behavior of the model constructed in step S1 is evaluated. The specific steps for constructing the control group include:

[0043] Normal saline was injected into the peritoneal cavity of adult male inbred mice at a dose of 1 mg / kg / d for five consecutive days;

[0044] (1) Sugar water preference experiment:

[0045] Three days before the experiment, mice were given a bottle of normal water and a bottle of 1% sucrose solution for acclimatization;

[0046] During the experiment, the position of the water bottle was changed every 6 hours, and the consumption of 1% sucrose solution by mice was detected after 24 hours. The consumption was calculated by weighing the bottle at the beginning and end of the experimental period, which can calculate the sucrose preference (%) of mice, that is, the percentage of sucrose solution consumption in the total drinking amount;

[0047] (2) Tail suspension test:

[0048] Use tape to stick the mouse's tail 1 cm from the tip, and hang the mouse upside down on the beam. After acclimation for 2 minutes, count the mouse's immobility time within the next 4 minutes. Except for the limb or body movement caused by breathing, no other movement is considered as immobility;

[0049] (3) Forced swimming test:

[0050] During the experiment, 20 cm of water was poured into a transparent cylindrical container at room temperature, and the mice were gently placed in the water. After acclimating for 2 minutes, the immobility time of the mice within the next 4 minutes was counted. Except for the necessary floating of the mice to keep their heads above the water, no other movement was considered as immobility.

[0051] Sugar water preference, tail suspension test and forced swimming test were used to evaluate the depressive-like behavior of mice, as shown in the attached manual. Figure 1 As shown in Figure A, the results of the sugar water preference experiment are as shown in the instruction manual. Figure 1 As shown in Figure B, the results of the tail suspension test are as shown in the instruction manual. Figure 1 As shown in Figure C, the results of the forced swimming test are as shown in the instruction manual. Figure 1 As shown in Figure D, it can be seen that the preference for sugar water in LPS-injected mice was significantly reduced, the immobility time in the tail suspension test was increased, and the immobility time in the forced swimming test was increased, which indicates that LPS-injected mice showed depressive behavior;

[0052] S3: Collect hippocampal tissue samples from the model with depressive behavior retained in step S2;

[0053] The specific steps for preserving hippocampal tissue samples from models showing depressive behavior are:

[0054] S31: Anesthetize the mouse, open the mouse chest, and perform cardiac perfusion. Physiological saline is infused from the left ventricle, flows through the systemic circulation, and flows out from the right ventricle. When the liver turns white, stop the perfusion.

[0055] S32: using surgical scissors to open the mouse skull and remove the mouse brain, wherein a part of the mouse brain was taken out of the mouse hippocampus according to the brain atlas for real-time fluorescence quantitative PCR and protein immunoblotting detection, and another part of the mouse brain was preserved in 4% paraformaldehyde, and then precipitated and dehydrated in 30% sucrose solution for immunofluorescence detection;

[0056] S4: performing real-time fluorescence quantitative PCR detection, immunofluorescence detection and protein immunoblotting detection on the hippocampal tissue samples of the model with depressive behavior collected in step S3 to evaluate the status of microglia in the hippocampal tissue samples of the model;

[0057] Real-time fluorescence quantitative PCR detection:

[0058] RNA from mouse hippocampal tissue samples was extracted by the guanidine isothiocyanate-phenol method, and then the reverse transcription process was completed using a reverse transcription kit, and then the RT-PCR kit was used to detect the expression of IL-1β mRNA and IL-6 mRNA, where:

[0059] IL-1β Primer Information:

[0060] Upstream: 5'-GAAATGCCACCTTTTGACAGTG-3'

[0061] Downstream: 5'-TGGATGCTCTCATCAGGACAG-3'

[0062] IL-6 Primer Information:

[0063] Upstream: 5'-TAGTCCTTCCTACCCCAATTTCC-3'

[0064] Downstream: 5′-TTGGTCCTTAGCCACTCCTTC-3′;

[0065] The test results of IL-1β mRNA and IL-6 mRNA are as shown in the instruction manual. Figure 2 As shown in Figures A and B, it can be seen that both IL-1β mRNA and IL-6 mRNA increased significantly, indicating that microglia in LPS mice were activated;

[0066] Immunofluorescence detection:

[0067] Cryosections of mouse hippocampal tissue samples were prepared and incubated overnight in antibody IBA1 solution, followed by Alexa Fluor TM The fluorescent secondary antibodies labeled with 488 and 594 were incubated for 1 h, and the slides were mounted with nuclear stain DAPI solution;

[0068] The results are as shown in the instruction manual Figure 3 As shown in Figure A, it can be seen that the microglial marker IBA1 in the hippocampus of LPS mice increased, indicating that the microglia of LPS mice were activated;

[0069] Western Blot Detection:

[0070] The mouse hippocampus tissue was lysed using RAPI lysis buffer, 1 / 4 volume of protein buffer was added, and the protein sample was obtained by denaturation in a 100°C water bath.

[0071] Use polyacrylamide gel method to separate equal amounts of protein samples and identify different protein expressions;

[0072] Antibody information is as follows: iNOS, USP22 antibody, GAPDH antibody;

[0073] The expression of inflammation-related protein iNOS and deubiquitinase USP22 is as shown in the instructions. Figure 4 As shown in Figure A;

[0074] It can be seen that the expression of the inflammation-related protein iNOS increased in the hippocampal tissue samples of LPS mice, and the expression of USP22 also increased;

[0075] S5: Adeno-associated virus was injected into the hippocampus of mice to knock down USP22 in microglia;

[0076] Mouse hippocampus brain localization injection:

[0077] The mice were anesthetized by injecting 1% sodium pentobarbital into the peritoneal cavity;

[0078] The mouse was fixed on a stereotaxic apparatus;

[0079] The microinjection syringe was used to inject 1.5 μL of adeno-associated virus targeting microglia to knock down USP22 into the mouse hippocampus at an injection speed of 0.3 μL / min. The injection coordinates of the mouse hippocampus were 2.06 mm caudal to the anterior bregma, 1.5 mm to the left and right of the sagittal midline, and 2 mm deep from the skull surface. The adeno-associated virus targeting microglia to knock down USP22 was AAVMG1.2-USP22-RNAi from GeneCare Biotechnology, with a virus titer of 109 TU / mL. The structure of the adeno-associated virus vector was as shown in the instructions. Figure 5 As shown, the RNAi sequence of USP22 contained in the vector is 5′-GUGGACAACUGGAAGCAAA-3′;

[0080] S6: Two weeks after the localization injection, repeat steps S1-S4;

[0081] Two weeks later, the mouse inflammatory depression model was established according to the above method, and the mice were subjected to sugar water preference test, tail suspension test and forced swimming test. At the same time, the mouse hippocampal tissue samples were collected, and real-time fluorescence quantitative PCR detection, immunofluorescence detection and protein immunoblotting detection were performed to evaluate the status of microglia in the model hippocampal tissue samples;

[0082] Sugar water preference, tail suspension test and forced swimming test were used to evaluate the depressive-like behavior of mice, as shown in the attached manual. Figure 6 As shown in Figure A, the overlap between the adeno-associated virus GFP infection signal and the microglial marker IBA1 in the mouse hippocampal tissue sample is shown in the attached manual. Figure 6 As shown in Figure B, the results of the sugar water preference experiment are as shown in the instruction manual. Figure 6 As shown in Figure C, the tail suspension test results are as shown in the instruction manual. Figure 6 As shown in Figure D, the results of the forced swimming test are as shown in the instruction manual. Figure 6 As shown in Figure E, it can be seen that the adeno-associated virus GFP infection signal and the microglial marker IBA1 in the mouse hippocampal tissue sample are highly overlapped, indicating that the adeno-associated virus has a high efficiency in targeting microglia. Knocking down the expression of microglial USP22 can increase the mouse's preference for sugar water and reduce the immobility time in the tail suspension test and the forced swimming test, indicating that inhibiting microglial USP22 can alleviate the depressive behavior of mice;

[0083] Knockdown of USP22 expression in microglia and IBA1 expression in the hippocampus of LPS mice are shown in the instructions. Figure 7 As shown in Figure A, the inflammatory factors IL-1β mRNA and IL-6 mRNA in the hippocampus of LPS mice were Figure 7 As shown in Figures D and C, it can be seen that knocking down microglial USP22 expression can inhibit microglial activation, and at the same time inhibit the inflammatory factors IL-1β mRNA and IL-6 mRNA, which indicates that inhibiting microglial USP22 expression can reduce microglial activation and inflammation levels in the hippocampus of depressed mice;

[0084] S7: Construction of BV2 microglial inflammatory cell model;

[0085] The specific steps of constructing the BV2 microglial inflammatory cell model include:

[0086] BV2 microglia were treated with 100 ng / mL lipopolysaccharide for 24 h to induce inflammation in BV2 microglia;

[0087] S8: Performing protein immunoblotting detection on the BV2 microglial inflammatory cell model constructed in step S7 to evaluate the status of microglia in the BV2 microglial inflammatory cell model;

[0088] Before the test, a control group needs to be constructed. Based on the control evaluation of the two groups, the state of microglia in the BV2 microglial inflammatory cell model in step S8 is evaluated. The specific steps for constructing the control group include:

[0089] BV2 microglia were not treated;

[0090] Western Blot Detection:

[0091] BV2 microglial inflammatory cells and the control group were lysed using RAPI lysis buffer, 1 / 4 volume of protein buffer was added, and the cells were denatured in a 100°C water bath to obtain protein samples;

[0092] Use polyacrylamide gel method to separate equal amounts of protein samples and identify different protein expressions;

[0093] Antibody information is as follows: iNOS, USP22 antibody, GAPDH antibody;

[0094] The expression of inflammation-related protein iNOS and deubiquitinase USP22 is as shown in the instructions. Figure 4 As shown in Figure B;

[0095] It can be seen that the expression of inflammation-related protein iNOS increased, indicating that the validation model was successful, while USP22 expression increased, indicating that lipopolysaccharide also promoted USP22 expression;

[0096] Small interfering RNA transfection:

[0097] Empty siRNA was configured to a concentration of 20 μM and mixed with an equal volume of transfection reagent GP-transfect-Mate in serum-free culture medium for 20 min, then added to BV2 microglia and cultured in a culture plate for 24 h. Cell samples were collected and subjected to protein immunoblotting experiments, which served as control group 1.

[0098] Empty siRNA was configured to a concentration of 20 μM and mixed with an equal volume of transfection reagent GP-transfect-Mate in serum-free medium for 20 min, then added to BV2 microglia and cultured in a culture plate for 24 h. BV2 microglia were treated with 100 ng / mL lipopolysaccharide for 24 h, and cell samples were collected for protein immunoblotting experiments, which served as the second control group;

[0099] USP22 siRNA was configured at a concentration of 20 μM and mixed with an equal volume of transfection reagent GP-transfect-Mate in serum-free medium for 20 min, then added to BV2 microglia and cultured in a culture plate for 24 h. BV2 microglia were treated with 100 ng / mL lipopolysaccharide for 24 h, and cell samples were collected for protein immunoblotting experiments, wherein the USP22 siRNA sequence was as follows: 5′-GUGGACAACUGGAAGCAAATT-3′;

[0100] After using USP22 siRNA to inhibit the expression of USP22 in BV2 microglia, the expression of inflammatory-related protein iNOS was as shown in the attached manual. Figure 4 As shown in Figure C;

[0101] It can be seen that the inhibition of USP22 expression in BV2 cells using USP22 siRNA can significantly reduce the expression of iNOS induced by lipopolysaccharide, which indicates that knocking down USP22 can inhibit the inflammation of BV2 microglia in vitro;

[0102] S9: Compare the status of microglia in steps S4, S6, and S8 to determine the mechanism of microglia activation in depression;

[0103] Inhibiting microglial USP22 expression can reduce microglial activation and inflammation levels, that is, the activation mechanism of microglia in depression is affected by USP22 expression.

[0104] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Use of an adeno-associated virus vector targeting microglia to knock down USP22 in the preparation of an antidepressant drug, characterized in that: The adeno-associated virus vector for targeting microglia to knock down USP22 is AAVMG1.2-USP22-RNAi from GeneCare Biotechnology, with a virus titer of 109TU / mL, and the RNAi sequence of USP22 contained in the vector is 5'-GUGGACAACUGGAAGCAAA-3'.

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