Application of PDCD4 expression inhibitor in preparation of traumatic craniocerebral injury repair medicine

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of a PDCD4 expression inhibitor in preparation of a traumatic craniocerebral injury repair medicine. The research finds that the PDCD4 gene is positioned in a cell nucleus of an astrocyte, the PDCD4 is transferred to cytoplasm after traumatic craniocerebral injury, and an inhibition effect is generated on autophagy. Based on the research result, the invention provides the sh-PDCD4 lentivirus which is relatively good in lentivirus infection effect and can stably knock down the expression level of PDCD4, and the sh-PDCD4 lentivirus remarkably promotes the autophagy of astrocytes and has a relatively good application prospect on targeted drugs for clinically developing and treating traumatic craniocerebral injury.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of a PDCD4 expression inhibitor in the preparation of a drug for repairing traumatic craniocerebral injury. Background Art

[0002] Traumatic brain injury (TBI) refers to a serious injury caused by external force acting on the head, resulting in damage to the brain tissue structure and function. Common causes include traffic accidents, falls from heights, violent blows, sports accidents, etc. According to the severity of the injury, it can be divided into mild, moderate and severe. Its pathophysiological process is relatively complex, including primary injury and secondary injury. Primary injury occurs at the moment of injury, and external force directly causes contusion, laceration, hemorrhage, etc. of brain tissue. Secondary injury is based on primary injury, and due to a series of pathological and physiological changes, such as cerebral edema, increased intracranial pressure, cerebral ischemia and hypoxia, inflammatory response, oxidative stress, etc., further aggravate brain damage. Traumatic brain injury not only has a serious impact on the patient's physical health, but may also lead to psychological and social dysfunction, bringing a heavy burden to patients and their families.

[0003] Astrocytes are a very important type of glial cell in the nervous system. First, they play a key role in providing nutritional support. Like a hard-working "logistics force", they provide the neurons in the central nervous system with the energy substances and nutrients they need. Secondly, they play an indispensable role in maintaining the stability of the extracellular environment. They can regulate the ion concentration in the extracellular fluid, such as potassium ions and calcium ions, to ensure that neurons can normally generate and transmit nerve impulses. Furthermore, astrocytes are essential for the metabolism of neurotransmitters. They can take up and degrade some neurotransmitters, such as glutamate, to prevent excessive accumulation of neurotransmitters in the synaptic cleft, thereby ensuring the accuracy and effectiveness of nerve signal transmission. In addition, astrocytes are also actively involved in the formation and maintenance of the blood-brain barrier. The blood-brain barrier is like a "protective shield" for the central nervous system. Astrocytes strictly control the entry and exit of substances into the central nervous system through interaction with vascular endothelial cells, protecting the brain from the invasion of harmful substances.

[0004] Autophagy is an intracellular degradation and recycling process that is essential for cell survival, homeostasis, and stress adaptation. In astrocytes, autophagy plays an important role. Under normal physiological conditions, autophagy helps maintain the normal function and metabolic balance of astrocytes. It can remove damaged organelles and misfolded proteins to provide cells with necessary substances and energy. For example, when astrocytes face nutrient deficiency or energy pressure, autophagy is activated to break down intracellular substances to provide energy and nutrition to help cells overcome difficulties. Under pathological conditions, the autophagy process of astrocytes will also change accordingly. For example, in neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, astrocyte autophagy may be abnormal. Excessive or insufficient autophagy may affect the support and protection of astrocytes for neurons. In addition, some studies have shown that external stimuli, such as oxidative stress and inflammatory factors, may affect the autophagy level of astrocytes and thus affect their function. In conclusion, autophagy in astrocytes is a complex and finely regulated process, and its normal function is essential for maintaining the health of the central nervous system. In-depth research on astrocyte autophagy is expected to provide new targets and strategies for the treatment of related neurological diseases.

[0005] The human programmed cell death 4 (PDCD 4) gene was discovered in 1999. It is located on chromosome 3q21.3, has a total length of 28212bp, and encodes a protein containing 496 amino acid residues. As a tumor suppressor, PDCD4 also plays an important role in the central nervous system. First, PDCD4 is involved in the regulation of apoptosis in the central nervous system. During normal development, it helps to remove excess or damaged nerve cells to shape and optimize the structure and function of the nervous system. Secondly, PDCD4 is closely related to neuroinflammatory response. When the central nervous system is infected, injured or otherwise stimulated to cause inflammation, the expression and function of PDCD4 may change, thereby affecting the process and outcome of inflammation. Furthermore, PDCD4 plays a role in the occurrence of tumors in the central nervous system. In brain tumors, the expression level of PDCD4 often changes. Highly expressed PDCD4 may inhibit the growth and spread of tumor cells, while reduced or absent expression may promote tumor progression. In addition, PDCD4 may also be involved in the development of neurodegenerative diseases. In diseases such as Parkinson's disease and Alzheimer's disease, PDCD4 may affect the survival and function of neurons by affecting processes such as cellular stress response, protein homeostasis, and autophagy. In short, PDCD4 plays an important role in the normal development, homeostasis maintenance, and disease occurrence of the central nervous system. In-depth research on it will help provide new ideas and methods for the treatment of central nervous system-related diseases. However, the regulatory mechanism of PDCD4 in traumatic brain injury and whether it has therapeutic value are still unknown. Summary of the invention

[0006] In order to solve the above problems, the present invention discloses a new discovery of PDCD4 in astrocytes after TBI, that is, PDCD4 inhibits autophagy by entering the cytoplasm and exerts an anti-inflammatory effect in astrocytes.

[0007] In a first aspect, the present invention provides the use of a PDCD4 expression inhibitor in the preparation of a drug for repairing traumatic brain injury, wherein the repair of traumatic brain injury is to promote astrocyte autophagy.

[0008] Preferably, the PDCD4 expression inhibitor comprises at least one of the following:

[0009] i) compounds that specifically inhibit PDCD4 expression;

[0010] ii) interfering molecules that specifically interfere with PDCD4 expression;

[0011] iii) A PDCD4 gene knockdown reagent, which is a gene editing reagent that specifically knocks down PDCD4.

[0012] Preferably, the PDCD4 gene knockdown agent comprises a viral vector, wherein the viral vector comprises a sequence of a shRNA fragment, wherein the shRNA fragment comprises a sequence as shown in SEQ NO ID: 1, SEQ NO ID: 2 or SEQ NO ID: 3.

[0013] Furthermore, the traumatic brain injury repair drug promotes activation and inflammatory response in astrocytes and autophagy in astrocyte cytoplasm by knocking down the expression of PDCD4.

[0014] Preferably, the dosage form of the drug is any pharmaceutically therapeutically acceptable dosage form.

[0015] Preferably, the dosage of the drug is any therapeutically acceptable dosage.

[0016] The second aspect of the present invention provides a method for constructing a cell line with stable knockdown of PDCD4, comprising the following steps:

[0017] i). Design multiple specific shRNA interference sequences targeting the PDCD4 gene;

[0018] ii). inserting the shRNA interference sequence into the shRNA lentiviral vector to construct a recombinant plasmid;

[0019] iii). Transforming competent cells and screening positive clones to obtain the cell line with stable knockdown of PDCD4.

[0020] Preferably, the shRNA interference sequence is a sequence such as SEQ NO ID: 1, SEQ NO ID: 2 or SEQ NO ID: 3.

[0021] Preferably, the cell line is C8-D1A cells.

[0022] The third aspect of the present invention provides a cell line with stable knockdown of PDCD4, wherein the cell line is obtained by the above method.

[0023] The beneficial effects of the present invention are:

[0024] 1. The lentiviral infection effect is good and the PDCD4 expression level is stable;

[0025] 2. Inhibition of inflammation: The present invention found that PDCD4 plays an anti-inflammatory role in astrocytes.

[0026] 3. Inhibition of autophagy: PDCD4 significantly inhibited autophagy in the in vitro astrocyte TBI model;

[0027] 4. Broad application prospects: The present invention provides a new type of simple intervention method, which is expected to play an important role in the treatment of brain injury diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1. The experimental results of knocking down PDCD4 by infecting cells with lentivirus, where A is the GFP observation after 24 hours, and B is the experimental result of Western blot after extracting protein after 48 hours. The results show that the knockdown effect of target A is the most significant.

[0029] Figure 2 Western Blot was used to detect the protein expression levels of cell activation markers (GFAP) and inflammatory markers (IL-1β). A shows that sh-PDCD4 plays a role in promoting cell activation and inflammatory response in C8-D1A cells, and B shows that sh-PDCD4 significantly promotes autophagy.

[0030] Figure 3 shows the related experiments of PDCD4 cytoplasmic translocation after LPS stimulation. A shows that in C8-D1A cells, PDCD4 is mainly expressed in the nucleus, and the expression level of PDCD4 in the cytoplasm increases with LPS stimulation. B shows that after LPS stimulation, the expression level of PDCD4 in the cytoplasm increases, and there is cytoplasmic translocation.

[0031] Figure 4 This is a cell immunofluorescence experiment. The results showed that before LPS stimulation, PDCD4 and LC3 were co-localized in the cell nucleus. After LPS stimulation, PDCD4 was translocated to the cytoplasm, and the co-localization with LC3 in the cytoplasm increased. DETAILED DESCRIPTION

[0032] The experimental methods in the following examples of the present invention, for which specific conditions are not specified, are generally carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0033] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0034] The terms "including" and "having" and any variations thereof of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or equipment.

[0035] In the present invention, the term "multiple" refers to two or more than two. "And / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can represent the following three situations: A exists alone, A and B exist at the same time, and B exists alone. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0036] In the present invention, an astrocyte TBI model was constructed in vitro, and lipopolysaccharide (1 μg / ml) was used to induce autophagy in astrocyte cell lines.

[0037] Astrocytes were infected with lentivirus to knock down the expression level of PDCD4, which was then identified by Western blotting.

[0038] Cell immunofluorescence and immunoblotting were used to analyze the effect of PDCD4 on autophagy in astrocyte cell lines.

[0039] The following is a description with reference to specific embodiments.

[0040] Example 1. Construction of PDCD4 interference virus vector.

[0041] The present invention provides three GFP-labeled specific shRNA lentiviral vectors, namely shRNA-A (shA), shRNAB (shB), and shRNA-C (shC), and the base sequences are as follows:

[0042] ShA: 5'GAGGCTATGAGAGAATTTA3' (SEQ NO ID: 1);

[0043] ShB: 5'GGGCCAGTTTATTGCTAGA3' (SEQ NO ID: 2);

[0044] ShC: 5'GGACGGTGATGAGCACAAA3' (SEQ NO ID: 3).

[0045] The following is the method for constructing PDCD4 interference virus vector:

[0046] 1. Design of shRNA interference sequence

[0047] According to the target gene sequence of the target gene, multiple RNA interference target sequences were designed using the RNA interference sequence design principles provided on the public website.

[0048] 2. Construction of RNAi Lentivirus Recombinant Plasmid

[0049] The oligo single-stranded DNA was annealed into double-stranded DNA, and the double-stranded shRNA oligo was respectively inserted into the shRNA lentiviral vector to construct the shRNA lentiviral recombinant plasmid, which was then transformed into the competent cell DH5.

[0050] 1) Annealing of shRNA

[0051] Dilute the primers with sterile TE buffer to a final concentration of 100 μMol. Pipette 10 μL of the upstream and downstream primers, mix them, pipette evenly, and place them in a PCR tube for annealing. The procedure is as follows:

[0052] 95℃30s

[0053] 72℃2min

[0054] 37℃2min

[0055] 25℃2min

[0056] After completion, place on ice for a few minutes and connect directly or store in a freezer at -20°C.

[0057] 2) Enzyme digestion and recovery of shRNA vector

[0058] The enzyme digestion system is as follows:

[0059] shRNA vector 5μg

[0060] 10*Buffer 5μL

[0061] BamHI 2 μL

[0062] EcoRI 2 μL

[0063] Make up to 50 μL with ddH2O

[0064] Digest at 37℃ for about 30min. During this period, 0.8% agarose gel can be prepared, and nucleic acid electrophoresis can be performed after the digestion is completed. After the electrophoresis is completed, cut the gel strip containing the target fragment. Use a balance to weigh the total weight and subtract the weight of the empty tube to calculate the weight of the gel. Calculate the volume of the gel based on 100mg being approximately 100μL, and add 3 times the volume of QG buffer to place in a 50℃ water bath to melt the gel completely. Shake the EP tube appropriately during this period to accelerate the dissolution of the gel. After the gel is completely melted, add isopropanol equal to the volume of the gel and mix well. Transfer all the above liquids to the filter column and centrifuge at 13000rpm for 30s. Discard the liquid in the tube, add 750μL of PE buffer to the column, and centrifuge for 1min. Discard the liquid in the tube and centrifuge for another 1min. Change to a new 1.5mL EP tube, add 50μL of EB buffer to the column, centrifuge for 1min, and the recovered vector is in the centrifuge tube.

[0065] 3) Ligation of shRNA vector and primers

[0066] The connection system is as follows:

[0067] Recover vector 3μL (50ng)

[0068] Oligo primer 1μL (0.5μM)

[0069] T4 DNA ligase buffer 1.5μL

[0070] T4 DNA ligase 1 μL

[0071] Make up to 15 μL with ddH2O

[0072] Incubate in a 25°C water bath for 30 min.

[0073] 4) Conversion

[0074] Place the competent cells on ice and wait for them to thaw naturally. Add all the ligation products to the competent cells, place them on ice for 20 minutes, and then heat shock them in a 42℃ water bath for 90 seconds. Then quickly place them on ice for 2-3 minutes. Add 1000μL of LB culture medium without antibiotics and culture them at 37℃, 150rpm and shake for 45 minutes. Centrifuge at 3000rpm for 2 minutes, discard about 850μL of supernatant, blow and disperse the bacterial liquid at the bottom of the tube, add it to the culture dish containing the corresponding resistance, spread it evenly with a sterilized applicator, and invert it in a 37℃ constant temperature incubator for overnight culture.

[0075] 5) Preparation of recombinant plasmid

[0076] Pick several single colonies, perform small-scale shaking culture, and conduct PCR identification.

[0077] 6) Sequencing to identify positive clones

[0078] Example 2: Establishment of knockdown cell lines.

[0079] Step 1: Cultivation of astrocyte cell line C8-D1A

[0080] 1. Cell culture conditions: complete culture medium DMEM (4.5 g / L D-Glucose) + 10% fetal bovine serum + 1% penicillin-streptomycin (100 mg / ml). Culture environment: 37°C constant temperature incubator (containing 5% CO2).

[0081] 2. Cell inducer: 1 μg / ml lipopolysaccharide (LPS) induces astrocyte activation.

[0082] Step 2: Infect cells with lentivirus to knock down PDCD4

[0083] Cell infection: GFP-labeled specific shRNA lentiviral vector (sh-PDCD4) was used to knock down PDCD4 in C8-D1A cells at three different targets. C8-D1A cells were plated in five large dishes, namely complete control group (NC), empty virus group (Con), ShA, ShB, and ShC. An appropriate amount of lentivirus (MOI = 40) and an equal amount of PBS were added to the culture dish, gently mixed, and placed in the incubator. The culture medium was replaced after 12 hours. This lentiviral vector carries the pac gene, and subsequent cell culture is screened with puromycin (beyotime / ST551) to stabilize the expression of the cell line.

[0084] Step 3: Detect knockdown efficiency

[0085] GFP expression was observed 24 hours after cell infection. Figure 1A ), and protein was extracted after 48 hours. The knockdown efficiency of three different targets was analyzed by Western blot. The results showed that the knockdown efficiency of target A and target B was significant, and both could achieve a stable knockdown effect of PDCD4 gene ( Figure 1B ). The target Sh-A was selected as a prerequisite for the subsequent knockout of PDCD4.

[0086] Example 3: Cytological experiment on PDCD4 knockout virus promoting inflammatory response and autophagy in astrocytes.

[0087] In order to further study the effect of PDCD4 knockout on astrocyte behavior, the NC group was a control group without virus infection, and the ShA group was an experimental group infected with the virus. In order to study the changes in astrocytes after lipopolysaccharide (LPS)-induced activation, PBS treatment was the control group, and LPS (1 μg / ml) treatment was the experimental group. Four experimental groups were set up, divided into NC group (PBS treatment), NC group (LPS treatment), ShA group (PBS treatment) and ShA group (LPS treatment). Among them, astrocytes can be stimulated by 1ug / ml LPS to simulate the inflammatory response after TBI.

[0088] Western Blot was used to detect the protein expression levels of cell activation markers (GFAP) and inflammatory markers (IL-1β).

[0089] (1) Cell protein extraction: discard the culture medium, wash twice with pre-cooled PBS to remove the residual culture medium. Lyse the cell protein with TBS (ph=7.2) + 1% TritonX-100 with phosphatase and protease inhibitors, place on a 4°C rotating table to react for 45 minutes, then centrifuge (15000r / min, 20min), and transfer the supernatant to the prepared centrifuge tube for later use.

[0090] (2) Protein concentration determination: There are 8 standard sample wells. The concentration of the target sample is determined according to the operating instructions of the BCA kit. The absorbance value at a wavelength of 562 nm is measured using a full-wavelength microplate reader. A standard concentration curve is drawn to calculate the final concentration of the protein sample.

[0091] (3) Protein electrophoresis: Mix the reserved sample with 5x SDS-PAGE protein loading buffer in a volume ratio of 4:1, and denature the protein in a 100°C water bath. Place the pre-prepared 7.5% SDS-PAGE gel in the electrophoresis device, add electrophoresis buffer (Tris 3.02g, glycine 18.8g, SDS1g, ddH2O to 1000ml), add 10μg of protein into the gel well, and add protein standards to mark the location of the molecular bands. Perform electrophoresis at a constant voltage of 80V to the separation gel. After the standard bands appear, perform electrophoresis at a constant voltage of 120V until the protein standard bands are completely separated.

[0092] (4) Transfer: Prepare transfer solution (Tris 3.03 g, glycine 14.4 g, methanol 200 ml, ddH2O to 1000 ml), and transfer the protein bands on the gel in parallel to the PVDF membrane soaked in methanol under a constant current of 300 mA. Then, block the PVDF membrane with 5% skim milk prepared in 1×TBST (Tris 2.42 g, NaCl 8 g, Tween20 0.5 ml, ddH2O to 1000 ml) for 2 h.

[0093] (5) Antibody incubation: After blocking, cut out the PVDF membrane strips corresponding to the target protein, and use the primary antibody diluent to prepare the antibodies PDCD4 (CST / #9535T), GFAP (CST / #3670), IL-1β (Abcam / ab9722), α-Tubulin (proteintech / 66031-1-Ig), p-mTOR (proteintech / 67778-1-Ig), LC3 (CST / #83506), and P62 (CST / #23214). The concentration of the primary antibody is 1:1000. The antibody fully covers the target strips and incubate at 4°C overnight. After overnight incubation with the primary antibody, wash the target strips three times with 1×TBST, each time for 5 minutes. After sufficient washing, incubate with the corresponding secondary antibody prepared in 1×TBST at room temperature for 2 hours, and wash the strips again with 1×TBST three times, each time for 5 minutes.

[0094] (6) Development and analysis: Place the washed PVDF membrane on the development platform of the Bio-Rad gel imager, add an appropriate amount of ECL developer to cover the PVDF membrane for development.

[0095] The results showed that sh-PDCD4 played a role in promoting cell activation and inflammatory response in C8-D1A cells ( Figure 2 A). In addition, sh-PDCD4 also significantly promoted autophagy ( Figure 2 B). It can be seen that PDCD4 is located in the nucleus of astrocytes, and autophagy occurs in the cytoplasm. In order to explore how PDCD4 in the nucleus affects autophagy in the cytoplasm, the present invention conducted the following research.

[0096] Example 4: PDCD4 was translocated to plasma after LPS stimulation.

[0097] Cell immunofluorescence: An appropriate amount of C8-D1A cells were plated on a 6-well plate cell slide. After stimulating the cells with 1ug / ml LPS for 24h, the culture medium was discarded, and the cells were gently washed 3 times with pre-cooled PBS, and then fixed with 4% paraformaldehyde for 15min at room temperature. The fixative was removed, and the cells were gently washed 3 times with PBS. The cells were permeabilized with 0.2% Triton X-100 at room temperature for 5min. The permeabilization solution was removed, and the cells were gently washed 3 times with PBS. The cells were blocked with blocking solution (10% BSA) at room temperature for 60min. The blocking solution was removed, and the primary antibodies PDCD4 (CST / #9535T) and LC3 (CST / #83506) were diluted 1:200 and incubated at 4°C overnight. After the primary antibody incubation, the primary antibody was removed, and the cells were gently washed 3 times with PBS. The fluorescent secondary antibody (Thermo Fisher Scientific, 1:1000) was added and incubated at room temperature for 2h in the dark. After the secondary antibody incubation, remove the secondary antibody, wash lightly 3 times with PBS, dilute DAPI (1:1000) with PBS, and incubate at room temperature in the dark for 10 minutes. After washing lightly with PBS 3 times, use filter paper to absorb the liquid on the slide, drop a drop of anti-fluorescence quenching sealing liquid on the slide, cover the cell slide with cells, avoid bubbles, make the cells contact the sealing liquid, and observe the fluorescence under a fluorescence microscope.

[0098] The results showed that in C8-D1A cells, PDCD4 was mainly expressed in the nucleus, and the expression level of PDCD4 in the cytoplasm increased with LPS stimulation ( Figure 3A ).

[0099] Nuclear and cytoplasmic separation test: The nuclear and cytoplasmic proteins were extracted using the Biyuntian nuclear and cytoplasmic protein extraction kit according to the instructions. The expression level of PDCD4 in the nucleus and cytoplasm before and after LPS stimulation was analyzed by Western Blot. Figure 3B ).

[0100] The results showed that after LPS stimulation, the expression level of PDCD4 in the cytoplasm increased, and there was cytoplasmic translocation. PDCD4 located in the nucleus of astrocytes was transferred to the cytoplasm after LPS stimulation. Because autophagy occurs in the cytoplasm, and LC3, an important marker of autophagy, is located in the cytoplasm, in order to explore how PDCD4 transferred from the nucleus to the cytoplasm affects autophagy, the present invention conducted the following research.

[0101] Example 5: After LPS stimulation, the co-localization of PDCD4 and LC3 in the cytoplasm increased.

[0102] C8-D1A cells before and after LPS stimulation were prepared for cell immunofluorescence experiments. PDCD4 and LC3 were selected as primary antibodies to observe the localization of the two molecules in the cells.

[0103] The results showed that before LPS stimulation, PDCD4 and LC3 were co-localized in the nucleus. After LPS stimulation, PDCD4 was translocated to the cytoplasm and its co-localization with LC3 in the cytoplasm increased ( Figure 4 ). So far, the present invention has found that after LPS stimulation, PDCD4 localized in the nucleus is transferred to the cytoplasm, and co-localizes with LC3 in the cytoplasm, and has an inhibitory effect on autophagy. From the results of Example 3, it can be seen that after sh-PDCD4 lentivirus knocks out PDCD4, the autophagy level in the cytoplasm is not inhibited by PDCD4, and the autophagy level increases, that is, sh-PDCD4 significantly promotes the principle process of autophagy.

[0104] In summary, the sh-PDCD4 lentivirus provided in this experiment can promote activation and inflammatory response in astrocytes, and has a significant promoting effect on autophagy. Taking into account the localization of PDCD4 in the nucleus, the present invention has performed cell immunofluorescence to find that PDCD4 is displaced after LPS stimulation, and is confirmed by nuclear cytoplasmic separation protein and immunoblotting (Western Blot). The present invention further explores the mechanism of action of PDCD4 on autophagy. Through cell immunofluorescence experiments, it is found that PDCD4 is displaced after LPS stimulation, and co-localization with LC3 increases, achieving an inhibitory effect. The sh-PDCD4 lentivirus in this experiment can stably regulate autophagy, and has a good application prospect for the clinical development of targeted drugs for the treatment of traumatic brain injury.

[0105] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For ordinary technicians in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. The use of a PDCD4 expression inhibitor in the preparation of a drug for repairing traumatic brain injury, characterized in that: The traumatic brain injury repair is to promote astrocyte autophagy.

2. The use according to claim 1, characterized in that: The PDCD4 expression inhibitor includes at least one of the following: i) compounds that specifically inhibit PDCD4 expression; ii) interfering molecules that specifically interfere with PDCD4 expression; iii) A PDCD4 gene knockdown reagent, which is a gene editing reagent that specifically knocks down PDCD4.

3. The use according to claim 2, characterized in that: The PDCD4 gene knockdown reagent includes a viral vector, wherein the viral vector includes a sequence of an shRNA fragment, wherein the sequence of the shRNA fragment is one or more of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 in the sequence listing.

4. The use according to claim 1, characterized in that: The traumatic brain injury repair drug inhibits or knocks down the expression of PDCD4, thereby promoting activation and inflammatory response in astrocytes and promoting autophagy in astrocyte cytoplasm.

5. The use according to claim 4, characterized in that The dosage form of the drug is any pharmaceutically therapeutically acceptable dosage form.

6. A pharmaceutical composition for treating traumatic brain injury, characterized in that: The active ingredient of the pharmaceutical composition includes a PDCD4 expression inhibitor.

7. A method for constructing a cell line with stable knockdown of PDCD4, characterized in that: The following steps are involved: i). Designing specific shRNA interference sequence targeting PDCD4 gene; ii). inserting the shRNA interference sequence into the shRNA lentiviral vector to construct a recombinant plasmid; iii). Transforming competent cells and screening positive clones to obtain the cell line with stable knockdown of PDCD4.

8. The method according to claim 7, characterized in that The shRNA interference sequence is shown in SEQ NO ID: 1, SEQ NO ID: 2 or SEQ NO ID:

3.

9. The method according to claim 7, characterized in that: The cell line is C8-D1A cells.

10. A cell line with stable knockdown of PDCD4, characterized in that: Obtained by the method according to any one of claims 7 to 9.

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