Application of PDCD4 gene in preparation of cerebral hemorrhage treatment medicine as target gene

Abstract

The invention relates to the technical field of biological medicines, in particular to application of a PDCD4 gene as a target gene to preparation of a medicine for treating cerebral hemorrhage. The PDCD4 gene is used as a target gene to prepare a medicine for treating cerebral hemorrhage. The shRNA for inhibiting PDCD4 is transferred into the microglial cells, so that the intercellular function of the microglial cells in an inflammatory environment is promoted, the anti-inflammatory effect of the microglial cells is enhanced, and damage repair after inflammatory reaction is promoted. The method is helpful for inhibiting or slowing down sustainable development of neuroinflammation after cerebral hemorrhage, and has important significance for improving prognosis of cerebral hemorrhage patients.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of the PDCD4 gene in preparing a drug for treating intracerebral hemorrhage as a target gene. Background Art

[0002] Intracerebral hemorrhage (ICH) is a severe disease caused by the rupture of cerebral blood vessels and the entry of blood into the brain parenchyma. In the Global Burden of Disease Study 2019, ICH accounted for 27.9% of all strokes. Although its incidence is relatively low compared to ischemic stroke, the mortality and disability rates remain high. Currently, in clinical practice, in addition to conventional treatment methods such as controlling blood pressure, correcting coagulation function, and surgical operations, there is still a lack of high-performance prognostic scales or biomarkers, as well as effective drug treatment means. Recent studies have found that the treatment of neuroinflammation after intracerebral hemorrhage may become a new breakthrough.

[0003] After intracerebral hemorrhage, due to blood-brain barrier damage, hematoma expansion, and mass effect, cell death will occur within a short period. This acute-phase cell death will lead to a rapid inflammatory response, which helps to limit tissue damage. However, these inflammatory responses need to be well controlled and terminated to avoid secondary injury and provide a suitable environment for tissue repair or regeneration.

[0004] Microglia are a type of phagocytic cell naturally present in the central nervous system (CNS) and play an important role in regulating waste clearance and inflammatory responses. Efferocytosis specifically refers to the process by which phagocytic cells phagocytose and digest dying cells. (Nat Rev Immunol. 2020) First, dying cells release "find me" signals to attract and / or activate phagocytic cells. Then, phagocytic cells distinguish apoptotic cells from healthy living cells through specific phagocytic receptors, and phagocytic receptors (such as TYRO3, AXL, MERTK, etc.) recognize the "eat me" signals on dying cells. Next, phagocytic cells undergo extensive cytoskeletal rearrangement to internalize corpses of generally the same size (for example, epithelial cells phagocytose neighboring cells). The last step is to process the ingested cargo and trigger specific phagocytic cell responses, mainly the secretion of anti-inflammatory mediators to help inhibit local immune responses.

[0005] Programmed cell death 4 (PDCD4) was first discovered and cloned in apoptosis experiments and has since received extensive attention and research due to its tumor transformation inhibitory effect. The human PDCD4 gene is located on chromosome 10q25.2.2, with a full length of 26.9 kb, containing 13 exons. The full length of cDNA is approximately 3.5 kb, and the coding region is approximately 1.4 kb. The PDCD4 protein contains nearly 500 amino acids, with a nuclear localization signal region at the N-terminus and a nuclear export signal region at the C-terminus, allowing it to freely enter and exit the nucleus. The PDCD4 protein contains several phosphorylation sites and can be phosphorylated and modified by various protein kinases such as PKB, p70S6K, and ERK. The PDCD4 protein interacts with the translation initiation factor eIF4A through the MA3 domain, competitively inhibiting the binding of eIF4A to eIF4G, thereby interfering with the helicase activity of eIF4A and inhibiting eukaryotic protein translation.

[0006] In addition to being a novel tumor suppressor, PDCD4 also plays important roles in apoptosis and autophagy, immunity and inflammation, and its function in central nervous system inflammation has also begun to attract attention. Studies have shown that PDCD4 deficiency alleviates neurodegeneration in a PD mouse model; conditional knockout of PDCD4 in microglia protects mice from LPS-induced microglial overactivation and depressive-like behaviors. In addition, PDCD4 promotes microglial activation and neuronal apoptosis through the MAPK-NFκB positive feedback loop. However, whether PDCD4 is involved in microglial efferocytosis remains an unknown area.

[0007] References: Doran AC, Yurdagul A Jr, Tabas I. Efferocytosis in health and disease. Nat Rev Immunol. 2020 Apr;20(4):254-267. doi: 10.1038 / s41577-019-0240-6. Epub 2019 Dec 10. PMID: 31822793; PMCID: PMC7667664. Summary of the Invention

[0008] To solve the above problems, the present invention provides the application of the PDCD4 gene in the preparation of a drug for treating cerebral hemorrhage as a target gene. The present invention reveals that the PDCD4 gene is a target gene that inhibits the efferocytosis function of microglia. By transferring shRNA that inhibits the PDCD4 gene into microglia, the expression of the microglial efferocytosis-related molecule MERTK is promoted, the secretion of pro-inflammatory factors by microglia is inhibited, and the repair of damage after inflammation is promoted.

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] The present invention provides the use of the PDCD4 gene in the preparation of a drug for treating cerebral hemorrhage as a target gene.

[0011] Preferably, the phagocytic function of microglia is promoted by inhibiting the expression of the PDCD4 gene to treat cerebral hemorrhage.

[0012] Preferably, the cerebral hemorrhage includes neuroinflammation after cerebral hemorrhage.

[0013] Preferably, the secretion of pro-inflammatory factors by microglia is inhibited by inhibiting the expression of the PDCD4 gene, and the repair of damage after the inflammatory response is promoted.

[0014] The present invention also provides an shRNA for treating cerebral hemorrhage based on the PDCD4 gene, and the nucleotide sequence of the shRNA is shown in any one of SEQ ID No.1-3.

[0015] The present invention also provides the use of the shRNA described in the above technical solution in the preparation of a drug for treating cerebral hemorrhage.

[0016] The present invention also provides the use of the shRNA described in the above technical solution in the preparation of a reagent for promoting the phagocytic function of microglia at the cellular level.

[0017] The present invention also provides an expression vector for treating cerebral hemorrhage based on the PDCD4 gene, and the expression vector contains the shRNA described in the above technical solution.

[0018] Preferably, the expression vector is a viral vector.

[0019] The present invention also provides an inhibitor of PDCD4 gene expression, and the inhibitor of PDCD4 gene expression contains the shRNA described in the above technical solution or the expression vector described in the above technical solution.

[0020] Advantages of the present invention:

[0021] (1) The present invention reveals that the PDCD4 gene (ID: 18569) is a target gene that inhibits the phagocytic function of microglia. Therefore, by inhibiting the expression of the PDCD4 gene, the present invention promotes the phagocytic function of microglia and inhibits the pro-inflammatory response of microglia.

[0022] (2) The present invention provides a method for constructing a PDCD4 knockdown cell line, namely viral infection.

[0023] (3) The present invention provides 3 small interfering RNA sequences for inhibiting the expression of PDCD4, as shown in SEQ ID No.1-3.

[0024] The efficiency of infecting microglia with the shRNA sequence shown in SEQ ID No.1 exceeds 90%, that is, a method for more controllable knockdown of PDCD4 and influence on ICH is provided.

[0025] (4) In the present invention, by transferring shRNA that inhibits PDCD4 into microglia, the efferocytosis of microglia in an inflammatory environment is promoted, the anti-inflammatory effect of microglia is enhanced, and the damage repair after the inflammatory response is promoted.

[0026] (5) The preparation process of the present invention is simple, the expression is stable, the preparation system is easy to control the quality, and it has good clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments.

[0028] Figure 1 Results of the expression levels of the target protein PDCD4 and the microglia activation marker IBA1 protein (A) and statistical results (B, C) after intracerebral hemorrhage in mice;

[0029] Figure 2 Results of the protein expression levels of microglial efferocytosis molecules (TYRO3, AXL, MERTK) (A) and statistical results (B, C, D) after intracerebral hemorrhage in mice;

[0030] Figure 3 Colocalization result diagram of the target protein PDCD4 and microglia after intracerebral hemorrhage in mice;

[0031] Figure 4 Results of the expression levels of the target protein PDCD4 and the efferocytosis protein (A) and statistical results (B, C, D, E) in the in vitro hemoglobin (HB)-stimulated microglia activation model;

[0032] Figure 5 Result diagram of the protein expression level for screening PDCD4 knockdown sequences;

[0033] Figure 6 Infection efficiency diagram of PDCD4 knockdown virus infecting BV2 microglia cell line;

[0034] Figure 7 Results of the protein expression levels of PDCD4, efferocytosis marker (MERTK), and pro-inflammatory factor IL-6 in microglia (A) and statistical results (B, C, D) after PDCD4 knockdown intervention and HB intervention of the target protein;

[0035] Figure 8Fluorescence images showing the effects of PDCD4 knockdown intervention and HB intervention on the efferocytosis function of microglia. Detailed implementation manners

[0036] The present invention provides the use of the PDCD4 gene as a target gene for preparing a drug for treating cerebral hemorrhage. Preferably, the present invention promotes the efferocytosis function of microglia to treat cerebral hemorrhage by inhibiting the expression of the PDCD4 gene. In the present invention, the cerebral hemorrhage preferably includes neuroinflammation after cerebral hemorrhage. Preferably, the present invention inhibits the secretion of pro-inflammatory factors by microglia and promotes the repair of damage after the inflammatory response by inhibiting the expression of the PDCD4 gene.

[0037] The present invention also provides an shRNA for treating cerebral hemorrhage based on the PDCD4 gene, and the nucleotide sequence of the shRNA is shown as any one of SEQ ID No.1 to 3.

[0038] SEQ ID No.1:

[0039] GAGGCTATGAGAGAATTTA;

[0040] SEQ ID No.2:

[0041] GGGCCAGTTTATTGCTAGA;

[0042] SEQ ID No.3:

[0043] GGACGGTGATGAGCACAAA.

[0044] The present invention also provides the use of the shRNA described in the above technical solution in the preparation of a drug for treating cerebral hemorrhage.

[0045] The present invention also provides the use of the shRNA described in the above technical solution in the preparation of a reagent for promoting the efferocytosis function of microglia at the cellular level.

[0046] The present invention also provides an expression vector for treating cerebral hemorrhage based on the PDCD4 gene, and the expression vector contains the shRNA described in the above technical solution. In the present invention, the expression vector is a viral vector.

[0047] The present invention also provides an inhibitor for PDCD4 gene expression, and the inhibitor for PDCD4 gene expression contains the shRNA described in the above technical solution or the expression vector described in the above technical solution.

[0048] In the present invention, the "PDCD4 gene", whose full name is Programmed cell death 4, is a gene encoding the PDCD4 protein in humans. The PDCD4 protein refers to a protein having two MA3 domains and an unstructured RNA-binding domain located at the N-terminus. The "small interfering RNA" refers to a small interfering RNA capable of inhibiting the expression of the PDCD4 gene. The small interfering RNAs show different degrees of interfering with the expression of the PDCD4 gene, but as long as they show inhibition of the expression of the PDCD4 gene, it is acceptable. PEI is Polyethylenimine, a cationic polymer widely used for gene transfection, that is, delivering exogenous DNA or RNA into cells.

[0049] As shown in Table 1, the present invention provides 3 small interfering RNAs for interfering with the expression of the PDCD4 gene, including small interfering RNA1 (shRNA1), small interfering RNA2 (shRNA2), and small interfering RNA3 (shRNA3). The base sequence of small interfering RNA1 is as shown in SEQ ID No.1, the base sequence of small interfering RNA2 is as shown in SEQ ID No.2, and the base sequence of small interfering RNA3 is as shown in SEQ ID No.3.

[0050] Among them, the above three small interfering RNAs can all inhibit the expression of the PDCD4 gene.

[0051] Table 1 Base sequence information of the small interfering RNAs involved in the present invention

[0052] Small interfering RNA SEQ ID No. Base sequence (5' to 3') shRNA1 1 GAGGCTATGAGAGAATTTA3 shRNA2 2 GGGCCAGTTTATTGCTAGA3 shRNA3 3 GGACGGTGATGAGCACAAA3

[0053] To further illustrate the present invention, the present invention will be described in detail below in conjunction with examples, but they should not be construed as limiting the protection scope of the present invention.

[0054] Example 1

[0055] Expression of the PDCD4 gene after cerebral hemorrhage

[0056] A collagenase-induced intracerebral hemorrhage model in mice was established. Briefly, after a one-week adaptation period, the housed mice were randomly divided into 6 groups, namely the Sham group, and the intracerebral hemorrhage 12h, 1d, 3d, 5d, and 7d groups. The mice were anesthetized with 3% isoflurane until they lost response to the tail clip test. Then, a nasal cone blowing 1.5% isoflurane was installed on the stereotaxic frame to maintain the anesthetic state. The skull was exposed by separating the muscle and periosteum, and a hole with a diameter of 1 mm was drilled in the skull (0.2 mm in front of the bregma and 2 mm laterally), and collagenase was injected into the right striatum (3.5 mm under the skull). 1 μl of 0.05 U type VI-S collagenase was dissolved in TESCA buffer and injected at a rate of 2 μl / min. After the injection, the needle was slowly removed in two steps over 10 minutes, with a 5-minute interval. During the operation, the rectal temperature was maintained at 37.0 ± 0.5 °C using a temperature-controlled heating pad. Sham-operated animals received the same anesthesia and surgery without striatal injection.

[0057] The expression of the PDCD4 gene in the mouse brain tissue was determined by immunoblotting. The results of the expression levels of the target protein PDCD4 and the microglial marker IBA1 are as Figure 1 shown. It can be seen that compared with the normal cerebral cortex, the protein expression level of PDCD4 increased after injury, and the protein expression level of PDCD4 reached its peak 7 days after injury; the expression level of IBA1 increased after injury, and with the extension of time, the activation level of microglia reached its peak on the 7th day. In addition, the protein expression levels of the efferocytosis markers (TYRO3, AXL, MERTK) in the mouse cerebral cortex were detected by immunoblotting, and the results are as Figure 2 shown. After ICH, TYRO3 and AXL increased after injury and reached their peaks on the 5th day, and their expression levels decreased over time; MERTK reached its peak on the 3rd day after injury and then gradually decreased. To further observe the localization of PDCD4 in different cell types after ICH, the present invention used double immunofluorescence staining with cell-specific markers to identify cell types. Frozen sections of the mouse brain tissue in the sham-operated group and 5 days after ICH were fluorescently stained with an antibody against PDCD4. As Figure 3 the data results showed that compared with the sham-operated group, the positive signals of PDCD4 and IBA1 (a microglial activation marker) increased after ICH; in addition, the positive signal of PDCD4 co-localized with the positive signal of IBA1, indicating that the PDCD4 gene may be a potential molecule involved in microglial activation after ICH.

[0058] Example 2

[0059] Expression of the PDCD4 gene in microglia

[0060] (1) Cell culture medium and culture conditions:

[0061] Complete medium formula for BV2 microglial cell line: 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).

[0062] (2) In vitro neuroinflammatory inducer: Use 300 μM hemoglobin (HB) to stimulate and induce the activation of BV2 microglial cell line. The stimulation time is determined according to the expression level of the target protein PDCD4. Western blotting is used to verify the efficiency of hemoglobin stimulation and conduct cell function experiments. The results of protein expression levels are as Figure 4 shown. It can be seen that stimulation with 300 μM HB for 24 h can significantly induce an increase in the expression of PDCD4 and proteins related to efferocytosis in BV2 microglial cells.

[0063] Example 3

[0064] Construction and identification of PDCD4 small interfering fragments

[0065] Small interfering RNAs can be roughly divided into siRNA (small interfering RNA) and shRNA (short hairpin RNA). siRNA is single-stranded RNA interference that blocks protein expression. Through external expression, a decrease in gene expression is shown, that is, this is an exogenous expression experiment. While shRNA is a kind of virus that integrates into the genome for gene interference, so it can achieve a long-term gene suppression effect. That is to say, the interference effect of shRNA lasts longer, and the effective number of product uses is more than that of siRNA. There are usually two ways for shRNA to inhibit gene expression: by plasmid transfection or virus infection. The present invention provides the infection method of the shRNA expression vector.

[0066] Establishment of knockdown cell line:

[0067] The PDCD4 knockdown lentivirus was purchased from Weinanjian Biotechnology Co., Ltd. Infection procedure: Add 10 μl of PDCD4 knockdown lentivirus to microglial cells at a density of 5×10 6 cells, and its MOI (multiplicity of infection) is 60. Three days after infection, GFP (green fluorescent protein)-positive cells were screened for stable transduction of PDCD4 knockdown BV2 cells by adding 1 μg / ml of Puromycin to the medium. Proteins were extracted for subsequent experiments.

[0068] The results of protein expression levels are as Figure 5As shown, it can be seen that compared with the control group, the expression levels of PDCD4 protein in the experimental groups transfected with small interfering RNA1 (shRNAA), small interfering RNA2 (shRNA B), and small interfering RNA3 (shRNA C) all decreased. From the relative expression levels, it is clear that the experimental group transfected with small interfering RNA1 had the greatest interference on PDCD4 protein expression and the best effect. Therefore, in the subsequent examples, shRNA1 was applied to interfere with the expression of the PDCD4 gene, and the sequence of shRNA is as follows: 5’GAGGCTATGAGAGAATTTA3’.

[0069] Among them, Figure 5 shA shown above refers to shRNA1, shB refers to shRNA2, and shC refers to shRNA3.

[0070] Mouse microglial cell line BV2 was infected with lentivirus carrying PDCD4 knockdown sequence. The infection efficiency of microglial cells was as Figure 6 shown. The knockdown sequence carried GFP green fluorescence, and the infected cells showed green fluorescence. From the results in the figure, the infection efficiency was >90%. Uninfected cells were killed by puromycin to obtain a stable PDCD4 knockdown stable transfected cell line.

[0071] Example 4

[0072] Cytological experiment for detecting the promotion of efferocytosis by PDCD4 small interfering fragment

[0073] According to the experimental protocol in Example 3, shRNA interference experiments were carried out, that is, shRNA1 was applied to inhibit the expression of PDCD4, named shPDCD4. Among them, the experimental groups included NC group (without stimulation), shPDCD4 group (without stimulation), NC group (stimulated with HB300uM), and shPDCD4 group (stimulated with HB 300uM).

[0074] 1. Western blot was used to detect the protein expression levels of the efferocytosis marker MERTK and the pro-inflammatory factor IL-6.

[0075] (1) Extraction of total protein: Extraction of cell protein: After the cells were treated to the appropriate time point, the culture medium was discarded, and the cells were washed twice with pre-cooled PBS to wash away the residual culture medium. The cell protein was lysed with TBS + 1% TritonX-100 containing phosphatase and protease inhibitors, and the mixture was placed on a rotating table at 4°C for 45 min. Then, centrifugation was performed (12000 r / min, 20 min), and the supernatant was aspirated and transferred to a prepared centrifuge tube for standby.

[0076] (2) Protein concentration determination: There are 8 standard wells, and 2 replicate wells are prepared for each sample to reduce experimental error. The concentration of the target sample is determined according to the BCA kit operating instructions. 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.

[0077] (3) Protein electrophoresis: Mix the prepared sample with 5×SDS-PAGE protein loading buffer in a 4:1 volume ratio and denature the protein in a 100°C water bath. Place the pre-prepared 10% SDS-PAGE gel into the electrophoresis apparatus. After adding the electrophoresis buffer, add the protein sample into the gel wells and add the protein standard to mark the location of the molecular bands. Run the electrophoresis at a constant voltage of 80V until the separation gel is formed. Once the standard bands appear, run the electrophoresis at a constant voltage of 120V until the protein standard bands are completely separated.

[0078] (4) Membrane transfer: Under a constant current of 300 mA, the protein bands on the gel were transferred in parallel to a PVDF membrane soaked in methanol, and then the PVDF membrane was blocked with 5% skim milk prepared in 1×TBST for 2 h.

[0079] (5) Antibody incubation: After blocking, the PVDF membrane strips corresponding to the target protein were cut out and the antibodies PDCD4 (CST / 9535S), IBA1 (FUJIFILM Wako / 019-29741), MERTK (Abcam / ab197643), IL-6 (CST / D5W4V), GAPDH (Proteintech / 60004-10-Ig), and α-Tubμlin (Proteintech / 66031-1-Ig) were prepared with primary antibody diluent. The concentration of the MERTK primary antibody was 1:500, and the concentration of the other primary antibodies was 1:1000. The antibodies were fully covered with the target strips and incubated at 4°C overnight. After overnight incubation with the primary antibody, the target strips were washed three times with 1×TBST for 5 minutes each time. After sufficient washing, the strips were incubated with the corresponding secondary antibody prepared in 1×TBST at room temperature for 2 hours. The strips were washed again with 1×TBST for three times for 5 minutes each time.

[0080] (6) Development and Analysis: Place the washed PVDF membrane on the development platform of a Bio-Rad gel imager and add an appropriate amount of ECL developer to cover the PVDF membrane for development. Finally, use Image J software to analyze the grayscale value of the protein blot.

[0081] The results are as follows Figure 7As shown, compared with the NC group (without stimulation), the protein expression of IL-6 (a pro-inflammatory factor) in the NC group (treated with HB) was significantly up-regulated, indicating that HB can significantly induce the inflammatory response of microglia. Compared with the NC group (treated with HB), the protein expression of MERTK (a microglial efferocytosis molecule) in the shPDCD4 group (treated with HB) was significantly up-regulated, and the protein expression of IL-6 was significantly decreased, indicating that the small interfering fragment of PDCD4 promotes microglial efferocytosis and inhibits the pro-inflammatory response of microglia at the cellular level.

[0082] 2. Detection of microglial efferocytosis level by cellular immunofluorescence.

[0083] The microglia transfected with the small interfering RNA of PDCD4 were seeded at 1×10 5 cells / well on coverslips in 24-well plates coated with polylysine. At the same time, latex beads with red fluorescence were added. After stimulating the cells with 300 μM HB for 24 h, the cells were washed 3 times with PBS for 5 min each time; fixed with methanol at 4 °C for 15 min, then the residual methanol was washed with pre-cooled PBS, and phalloidin was added and incubated at room temperature in the dark for 20 min; washed 3 times with PBS for 5 min each time, and finally sealed with a mounting medium (glycerol:PBS = 1:1), and observed with an inverted fluorescence microscope.

[0084] The results are as Figure 8 shown. Compared with the NC group (treated with HB), the number of latex beads phagocytosed by microglia in the shPDCD4 group (treated with HB) was significantly increased, indicating enhanced microglial efferocytosis function.

[0085] This experiment proves that the small interfering fragment of PDCD4 promotes the efferocytosis function of microglia at the cellular level.

[0086] In summary, the small interfering RNA provided in the embodiments of the present invention that inhibits the expression of the PDCD4 gene can inhibit the pro-inflammatory response of microglia and promote the efferocytosis function of microglia by inhibiting the expression of the PDCD4 gene with small interfering RNA, and has good application prospects for the clinical development of targeted drugs for the treatment of neuroinflammation after cerebral hemorrhage.

[0087] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments according to this embodiment without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of the PDCD4 gene in the preparation of a medicament for treating cerebral hemorrhage as a target gene.

2. The application according to claim 1, characterized in that, Promote the efferocytosis function of microglia to treat cerebral hemorrhage by inhibiting the expression of the PDCD4 gene.

3. The application according to claim 1, characterized in that, The cerebral hemorrhage includes neuroinflammation after cerebral hemorrhage.

4. The application according to claim 3, characterized in that, Inhibit the secretion of pro-inflammatory factors by microglia by inhibiting the expression of the PDCD4 gene, and promote the repair of damage after the inflammatory response.

5. A shRNA for treating cerebral hemorrhage based on the PDCD4 gene, characterized in that, The nucleotide sequence of the shRNA is shown as any one of SEQ ID No.1-3.

6. Use of the shRNA according to claim 5 in the preparation of a medicament for treating cerebral hemorrhage.

7. Use of the shRNA according to claim 5 in the preparation of a reagent for promoting the efferocytosis function of microglia at the cellular level.

8. An expression vector for treating cerebral hemorrhage based on the PDCD4 gene, the expression vector containing the shRNA according to claim 5.

9. The expression vector according to claim 8, characterized in that, The expression vector is a viral vector.

10. An inhibitor of PDCD4 gene expression, characterized in that, The PDCD4 gene expression inhibitor contains the shRNA according to claim 5 or the expression vector according to any one of claims 8-9.

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