Application of GPNMB in preparation of medicine for treating neonatal hypoxic ischemic brain injury

Abstract

The invention belongs to the technical field of biological medicines, and discloses application of GPNMB in preparation of a medicine for treating hypoxic ischemic brain injury of newborns. The invention develops a new application field. The medicine taking the GPNMB as the raw material can be used for inhibiting neuron death caused by hypoxia and ischemia in the development period of the newborn, and the GPNMB can also be used as an accelerant for activating an AKT signal channel.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and relates to medical application of glycoprotein non-metastatic melanoma protein B (GPNMB), in particular to application of GPNMB in a treatment drug for developmental period hypoxic-ischemic brain injury; the application proves that GPNMB plays a neuroprotective role in hypoxic-ischemic brain injury of a newborn mouse. BACKGROUND

[0002] Hypoxic-ischemic encephalopathy (HIE) in newborns is mainly caused by asphyxia or hypoxia during the perinatal period. About 1-6 cases occur per 1000 live births, and 25%-30% of the survivors have long-term sequelae, including cerebral palsy, epilepsy, and mental retardation, and other long-term neurological dysfunctions. At present, the treatment of full-term children in clinical practice is mainly hypothermia therapy, and early treatment can significantly improve the prognosis and reduce acute death; however, the optimal treatment window is 6 hours after HIE, and 30%-70% of children still have disabilities or even die after treatment. Therefore, it is very important to explore the pathogenesis of HIE and find new treatment strategies.

[0003] GPNMB is an endogenous protein and also a type 1 transmembrane glycoprotein. The extracellular fragment can be cleaved by metalloproteinase ADAM10 and play a biological function by binding to various receptors such as Na+ / K+-ATPase (NKA), CD44, heparin, and syndecan-4. Studies have shown that GPNMB plays an important role in regulating inflammation, autophagy, oxidative stress, cell proliferation, cell differentiation, nerve regeneration, and immune suppression. However, the role of GPNMB in neuronal death in developmental period hypoxic-ischemic brain injury is not clear, and whether GPNMB-targeted interventions can improve developmental period hypoxic-ischemic brain injury has not been reported. SUMMARY

[0004] The purpose of the application is to explore and prove the new use of GPNMB in drug preparation in order to provide new drug treatment measures for the treatment of developmental period hypoxic-ischemic brain injury in view of the current technical situation that there is a lack of effective treatment drugs for neonatal hypoxic-ischemic brain injury.

[0005] The application provides application of GPNMB in preparation of a drug for treating neonatal hypoxic-ischemic brain injury.

[0006] In one implementation manner, the GPNMB is used for preparing a drug for inhibiting neuronal death caused by hypoxia-ischemia in the developmental period of a newborn.

[0007] In an implementation manner, the GPNMB is used as an activator of the AKT signal channel.

[0008] In an implementation manner, the dosage form of the medicine is an injection.

[0009] Firstly, the application constructs a new mouse HIBD model, screens the differentially expressed gene GPNMB through transcriptome sequencing, transfects the si-RNA to knock down the expression of GPNMB in the HT22 cell line, and prepares a lentivirus, and carries out in vivo experiments by injecting the sh-GPNMB lentivirus and the GPNMB recombinant protein into the lateral ventricle of the HIBD mouse. Secondly, the application constructs a neuron oxygen-glucose deprivation (OGD) model, and adds the GPNMB recombinant protein and the AKT inhibitor, and explores the mechanism of the protective effect of GPNMB on neurons through cell in vitro experiments. The results show that after the HIBD of the new mouse, the expression of GPNMB is up-regulated, the area of cerebral infarction is significantly reduced, the memory and learning ability of the HIBD mouse is improved, and the growth and development of the HIBD mouse is improved. On the contrary, the memory and learning ability of the HIBD mouse injected with shRNA-GPNMB into the lateral ventricle is reduced, and the growth and development is poor, so it can be known that GPNMB plays a protective role in the ischemia and hypoxia brain damage of the new mouse. The results of the cell in vitro experiment show that the GPNMB recombinant protein can significantly improve the survival rate of the neuron cell after OGD; reduce the apoptosis of the neuron cell, and activate the AKT signal pathway; when the AKT inhibitor (LY294002) is added, the death rate of the neuron cell is significantly increased, so it can be known that GPNMB plays a protective role on the OGD neuron by activating the AKT signal pathway. Therefore, the GPNMB recombinant protein can be used as an effective component for treating the hypoxic-ischemic brain damage in the development period, and a potential medicine is prepared.

[0010] Compared with the prior art, the application has the following beneficial effects: 1) The application provides the application of GPNMB in the preparation of a medicine for treating hypoxic-ischemic brain damage of a newborn, and opens up a new application field; 2) The medicine using GPNMB as a raw material can be used for inhibiting the neuron death caused by hypoxia and ischemia in the development period of a newborn; 3) The GPNMB in the medicine provided by the application can be used as an activator of the AKT signal channel; 4) The application provides a new research direction for the treatment medicine of the hypoxic-ischemic brain damage in the development period by injecting the GPNMB recombinant protein into the lateral ventricle. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 is a HIBD model molding diagram; Figure 2is the TTC staining observation of brain tissue infarction test results 1 day after HIBD; it is shown that the brain section of the sham group mice is dyed red, and no white infarction is found; the right half of the brain tissue of the HIBD group mice is obviously white, and the HIBD model is successfully established; Figure 3 is the mouse brain tissue pathological test result after HIBD 24 h; HIBD 24 h HE staining shows that the cerebral cortex and hippocampal structure are relatively loose, the number of cells is reduced, the arrangement is disorderly, and the interstitial space is widened; Figure 4 is the Morris water maze experiment result after HIBD; compared with the sham group, the escape time of the HIBD group is prolonged, and the number of crossing the platform is reduced; Figure 5 is the mouse laser speckle cerebral blood flow test result after HIBD 24 h; the cerebral blood flow ratio of the HIBD group is reduced compared with the sham group; Figure 6 is the clustering analysis diagram of brain tissue differentially expressed genes statistics grouping; compared with the sham group, 1050 genes in the HIBD brain tissue are differentially expressed, of which 982 are up-regulated and 68 are down-regulated; Figure 7 is the result of GO enrichment analysis of differential genes; Figure 8 is the RT-PCR verification diagram; 10 genes with high differential gene expression and most obvious differential expression are selected for PCR verification, and the results show that GPNMB is one of the most obvious up-regulated genes (p<0.001); Figure 9 is the KEGG enrichment function bubble chart; combined with bioinformatics analysis, it is shown that GPNMB plays a role in apoptosis and production of inflammatory factors, suggesting that GPNMB is involved in biological processes such as apoptosis inhibition and cytokine production; Figure 10 is the test result of GPNMB expression in mouse brain tissue after HIBD modeling; compared with the sham group, the expression of GPNMB in mouse brain tissue after HIBD is up-regulated, which has statistical significance (p<0.001); Figure 11 is the expression of GPNMB in neurons, microglial cells and astrocytes after HIBD detected by immunofluorescence (IF); GPNMB is co-stained with neuron marker NEUN (red), astrocyte ALDH1L1 (green) and microglial cell marker IBAI (red), and the results show that GPNMB is expressed in these cells, but mainly expressed in neuron cells after HIBD; Figure 12RT-PCR detection of GPNMB expression in NC group and 24 h after OGD in neurons and HT22 cells; compared with the NC group, GPNMB expression in neurons and HT22 cells in the OGD group was up-regulated, and the difference between groups was statistically significant (p<0.001); Figure 13 Calcein-AM / PI staining results and CCK8 calculation results after the establishment of the neuron OGD cell model; A is the Calcein-AM / PI staining result, B is the PI positive rate, and the results show that the number of dead neurons increases after OGD (PI positive rate 55%), and the difference is statistically significant compared with the normoxic group (PI positive rate 21%) (p<0.001) Figure 13 A, B); C is the cell viability calculation result, and the results show that the neuron cell viability in the OGD group (59%) is significantly lower than that in the normoxic group (85.5%), and the difference is statistically significant (p<0.001); these results show that the neuron OGD model is successfully established; NC: normoxic control group; OGD: oxygen-glucose deprivation group; scale bar: 100 ; Figure 14 siRNA inhibition efficiency verification results; in the HT22 mouse hippocampal neuron cell line, si-RNA transfection was used to knock down the expression of GPNMB. RT-PCR showed that siRNA-1, siRNA-2, and siRNA-3 (small interfering Si-RNA of GPNMB) could significantly down-regulate GPNMB in HT22, and the difference was statistically significant (p<0.01), among which siRNA-2 had the highest knockdown efficiency and was used to prepare lentivirus subsequently; Figure 15 GPNMB mRNA expression in mouse brain tissue after intracerebroventricular injection of shRNA-GPNMB and negative control shRNA-Control; Figure 16 GPNMB protein expression in mouse brain tissue after intracerebroventricular injection of shRNA-GPNMB and negative control shRNA-Control; in mice, shRNA-GPNMB lentivirus was injected into the lateral ventricle, and shRNA-Control was used as a control, and the knockdown efficiency was detected by PCR after 7 days; RT-PCR results showed that intracerebroventricular injection of shRNA-GPNMB lentivirus could reduce GPNMB in the brain tissue of the sham operation group (p<0.001); Figure 17The Morris water maze test was used to observe the learning and spatial memory abilities of mice in the sham group, HIBD group, HIBD+shRNA-Control group, and HIBD+shRNA-GPNMB group; sham: sham operation group; HIBD: ischemia-hypoxia group; compared with the HIBD+shRNA-Control group, the escape latency of the HIBD+shRNA-GPNMB group was prolonged (p<0.05). Figure 17 A); Space exploration experiments showed that compared to the HIBD+shRNA-Control group, the HIBD+shRNA-GPNMB group had fewer platform crossings (p<0.05). Figure 17 B); sham: sham surgery group; HIBD: ischemia-hypoxia group; HIBD+shRNA-Control: ischemia-hypoxia + shRNA-Control lateral ventricle injection group; HIBD+shRNA-GPNMB: ischemia-hypoxia + shRNA-GPNMB lateral ventricle injection group; Figure 18 The study used cranial magnetic resonance imaging to observe the long-term brain tissue morphology and structure of mice. Compared with the HIBD+shRNA-Control group, the HIBD+shRNA-GPNMB group showed an increased volume of necrotic cysts in the right hemisphere of the mice. HIBD: ischemia-hypoxia group; HIBD+shRNA-GPNMB: hypoxia-ischemia + shRNA-GPNMB lateral ventricle injection group; HIBD+shRNA-Control: hypoxia-ischemia + shRNA-Control lateral ventricle injection group. Figure 19 The results are pathological examinations of liver and kidney function after lentivirus injection; no significant pathological changes were observed in the liver and kidney tissues of the sham group, HIBD group, HIBD+shRNA-Control group, and HIBD+shRNA-GPNMB group. Figure 20 The results are the serum urea (UREA), serum creatinine (CREA), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels measured after lentiviral injection to assess liver and kidney function. There were no significant differences in ALT, AST, UREA, and CREA levels among the sham group, HIBD group, HIBD+shRNA-Control group, and HIBD+shRNA-GPNMB group. Sham: sham surgery group; HIBD: ischemia-hypoxia group; HIBD+shRNA-Control: ischemia-hypoxia + shRNA-Control lateral ventricle injection group; HIBD+shRNA-GPNMB: ischemia-hypoxia + shRNA-GPNMB lateral ventricle injection group. Figure 21CCK test results obtained at different GPNMB administration concentrations; the cell survival rate of neurons 24 h after OGD was the lowest, and the GPNMB concentration of 50 ng / ml was able to improve the survival rate of neurons after OGD, and the recombinant GPNMB protein (50 ng / ml) was determined as the optimal concentration, and the treatment time was 24 h; cell viability: cell survival rate; OGD / R6h: neurons after 6 h of reoxygenation after hypoxia; OGD / R12h: neurons after 12 h of reoxygenation after hypoxia; OGD / R24h: neurons after 24 h of reoxygenation after hypoxia; Figure 22 Calcein-AM / PI staining results of different experimental groups; the Calcein-AM / PI experiment results show that compared with the OGD group, the addition of the GPNMB recombinant protein reduces the death of neurons after OGD (p<0.001); NC: normoxic control group; OGD: oxygen-glucose deprivation group; OGD+GPNMB: oxygen-glucose deprivation+GPNMB recombinant protein group; Figure 23 BCL-2, BAX, and BCL-2 / BAX expression of different experimental groups; the Western Blotting results show that compared with the OGD+PBS group, the addition of the GPNMB recombinant protein increases the expression of the anti-apoptotic protein BCL-2, reduces the expression of the pro-apoptotic protein BAX, and increases the BCL-2 / BAX ratio, indicating that GPNMB can reduce the apoptosis of neurons after OGD; NC: normoxic control group; OGD: oxygen-glucose deprivation group; OGD+GPNMB: oxygen-glucose deprivation+GPNMB recombinant protein group; Figure 24 AKT detection results of different experimental groups; the Western Blotting results show that after the addition of the GPNMB recombinant protein, the P-AKT / AKT ratio increases, and the AKT signaling pathway is activated. NC: normoxic control group; OGD: oxygen-glucose deprivation group; OGD+GPNMB: oxygen-glucose deprivation+GPNMB recombinant protein group; Figure 25 Calcein-AM / PI staining results and CCK test results of different experimental groups after the addition of an AKT inhibitor; the Calcein-AM / PI and CCK8 results show that compared with the OGD+GPNMB group, the addition of the LY294002 (10 ) group increases the death rate of neurons (p<0.001) (Fig. A, B) and reduces the cell viability (p<0.001) (Fig. C); Figure 26Figure 6 is TTC staining to observe the cerebral infarction area after intracerebroventricular injection of GPNMB recombinant protein; compared with the HI+PBS group, the cerebral infarction area of the HI+GPNMB group was reduced (p<0.001), and the extracellular fragment of GPNMB can reduce the cerebral infarction volume and improve the ischemia and hypoxia brain injury. HIBD+PBS group: hypoxia-ischemia+intracerebroventricular injection group; HIBD+GPNMB group: ischemia and hypoxia+intracerebroventricular injection of GPNMB recombinant protein group; Figure 27 Figure 7 is transmission electron microscopy (TEM) detection of apoptosis after intracerebroventricular injection of GPNMB recombinant protein; compared with the HIBD+PBS group, the HIBD+GPNMB group has no karyopyknosis, no nuclear membrane folding, and compact cell body; sham: sham operation group; HIBD: ischemia and hypoxia group; HIBD+PBS group: hypoxia-ischemia+intracerebroventricular injection of PBS group; HIBD+GPNMB group: ischemia and hypoxia+intracerebroventricular injection of GPNMB recombinant protein group; scale bar: 2 ; Figure 28 Figure 8 is the growth and development of mice after intracerebroventricular injection of GPNMB to improve HIBD; the body weight of HIBD mice was measured every day within one week after intracerebroventricular injection of GPNMB recombinant protein, and the body weight of the HIBD+GPNMB group increased (p<0.05), indicating that intracerebroventricular injection of GPNMB recombinant protein can improve the growth and development of mice and increase the body weight of mice; sham: sham operation group; HIBD: ischemia and hypoxia group; HIBD+PBS group: hypoxia-ischemia+intracerebroventricular injection of PBS group; HIBD+GPNMB group: ischemia and hypoxia+intracerebroventricular injection of GPNMB recombinant protein group; Figure 29 Figure 9 is the brain atrophy amount test result; the brain atrophy amount of the HIBD+GPNMB group was reduced (p<0.05), and intracerebroventricular injection of GPNMB recombinant protein can reduce the brain atrophy amount of mice; sham: sham operation group; HIBD: ischemia and hypoxia group; HIBD+PBS group: hypoxia-ischemia+intracerebroventricular injection of PBS group; HIBD+GPNMB group: ischemia and hypoxia+intracerebroventricular injection of GPNMB recombinant protein group; Brain weight ration: brain atrophy ratio; R / L (right brain / left brain). DETAILED DESCRIPTION

[0012] (I) Experimental materials and reagents 1. Experimental animals SPF level 7-day-old C57BL / 6JGpt mice were provided by Chengdu Dasuo Company, and the gender was not limited, which were used to construct an animal model of hypoxia-ischemia brain injury. 1-day-old mice were used for extraction of primary neuron cells and construction of oxygen-glucose deprivation (OGD) model.

[0013] 2. Main reagents The main reagents used in this example are shown in Table 1.

[0014]

[0015]

[0016] The cell culture reagents are shown in Table 2.

[0017] Table 2 Cell culture reagents

[0018] (B) Experimental methods and analysis of experimental results This example uses SPSS19.0 to process experimental data. Normality test is performed on continuous variables, and mean ± standard deviation (mean ± SD) is used to represent data, t test is used for comparison between groups, and least significant difference (least significant difference, LSD) method is used for comparison between groups; p <0.05, p <0.01, p <0.001 are considered to be statistically different. ImageJ software is used to calculate the TTC infarction area and the gray value of the immunoblotting band. Excel is used to calculate the relative expression of the target protein and RNA (target protein and RNA internal reference). Column chart is drawn using GraphPad Prism 7.0.

[0019] 1. Construction of neuron cell OGD model

[0020] 1.1 Extraction and culture of mouse cortical neurons 1.1.1 Prepare neuron culture medium: add 50 mL neuron base medium, 1 mL B-27 serum, 0.5 mL glutamine, 25 double antibodies, and 0.66 mL glucose, mix well; 1.1.2 Culture plate coating: use PDL to coat 6-well plates, and then place them in a biological safety cabinet for air drying; 1.1.3 Brain removal: surgical instruments are sterilized in advance in a high-pressure sterilization pot; 4 mL of digestion solution is placed in a 6 cm x 6 cm culture dish, and pre-cooled PBS (pre-cooled in a 4°C refrigerator) is poured into two 10 cm x 10 cm culture dishes (10 mL each) placed in an ice box; take 10 P1 day newborn mice, anesthetize them under low temperature, and then put them in a 75% alcohol beaker for disinfection, decapitate them, take out the whole brain, and put it into a pre-cooled PBS culture dish; 1.1.4 Separation: use surgical scissors to remove the cerebral cortex on a clean bench, put it into a culture dish containing PBS, peel off the vascular membrane, and put it into a culture dish containing digestion solution; 1.1.5 Digestion: The cerebral cortex was broken into small pieces, and 0.5 mL of papain and 100 mL of... Digest with DNA enzymes for 30 minutes; 1.1.6 Termination of digestion and filtration: Remove the culture dish containing the cerebral cortex from the incubator and add 4.5 mL of neutralization solution (composed of 50 mL of neuronal basal culture medium and 25 mL of...). Digestion was terminated by mixing the two antibodies, and the tissue was aspirated 10 times with a pipette. The tissue was then tilted and allowed to settle, allowing the brain tissue mass to precipitate. The supernatant digestive fluid was aspirated, and then... (The sentence is incomplete and requires more context to translate accurately.) Cells were filtered through a sieve into 50 mL centrifuge tubes, centrifuged at 1500 rpm for 10 min, and the cell filtrate was collected. 1.1.7 Inoculation: Discard the supernatant, add the neuronal culture medium preheated to 37℃, and gently mix by pipetting; add 2 mL of neuronal culture medium containing neuronal cells to each six-well plate, mix by figure-eight method, and place in an incubator; 1.1.8 Culture: After culturing for 24 h, observe the cells under a microscope. During this period, supplement the neuronal culture medium as needed according to the cell condition. For cell treatment, place the cells in a 37℃, 5% CO2 incubator and change the medium once at half volume on the 3rd day.

[0021] 1.2 Culture of hippocampal neurons in HT22 mice

[0022] 1.2.1 Cell resuscitation: HT22 mouse hippocampal neurons frozen at -80℃ were rehydrated in cell culture medium preheated at 37℃ (45 mL DMEM high glucose + 5 mL 10% FBS + 500 mg / L). Resuscitation overnight in 100x P / S (dual antibody) solution; 1.2.2 Cell passage: On the second day, observe cell growth and cell density under a microscope. When the cell density reaches 80%-90%, it is ready for passage. Discard the culture medium in the 10 cm culture dish, wash twice with PBS, add 1 mL of trypsin (0.025%) and mix well. Use a 1 mL pipette to remove the trypsin, then place the culture dish in a 37℃ incubator for about 1 min. Observe the cell digestion under a microscope. If most of the cells become round and detach, quickly return the dish to the operating table, add 2 mL of cell culture medium to stop digestion and mix well. Aspirate the cell culture medium to remove the cells from the dish, gently mix, and then aspirate and seed them into 4 separate culture dishes. Add cell culture medium to 10 mL. 1.2.3 Cell Plate Separation: On day 3, cells were digested with 0.025% trypsin (trypsin level covering the bottom of the culture dish). 2 mL of cell culture medium was added to each dish and mixed thoroughly. All cell culture was collected into 15 mL centrifuge tubes for cell counting. The tubes were then centrifuged at 1500 rpm for 10 min. Finally, the desired cell number was determined (2-3 × 10⁶ cells per well in a 6-well plate). 6 For a 24-well plate, inoculate 5-6 × 10⁶ cells per well. 5 The cells were then separated into plates for further culture and processing. 1.2.4 Cell cryopreservation: After neutralizing trypsin with cell culture medium, the cells were centrifuged at 1500 rpm for 10 min and then resuspended in cryopreservation solution (purchased from Sewell) and frozen into 3 tubes. Each tube contained 1 mL of cryopreservation solution and was placed at -80℃.

[0023] 1.3 siRNA transfection of hippocampal neurons in HT22 mice

[0024] 1.3.1 Cell resuscitation and passage (same as steps 1.2.1 and 1.2.2 above). 1.3.2 In a 6-well plate, use 2×10 6 Seed cells and transfected them when the cells are in good growth condition and the plate-forming rate reaches 70-80%. 1.3.3 To 125 Add 5% of serum-free DMEM culture medium lip3000 transfection reagent was prepared as liquid one and allowed to stand for 5 minutes; 125 Add 5% of serum-free DMEM culture medium 20 Prepare liquid 2 with mol si-RNA. Mix liquid 1 and liquid 2 thoroughly to prepare the transfection solution. Add 260 μmol si-RNA to each 6-well plate. Transfection solution, and replenish with 1.75 mL of high-glucose DMEM medium; 1.3.4 24 h after transfection, observe the cell growth status, change the cell culture medium, and perform subsequent operations.

[0025] 1.4 Establishment of a neuronal oxygen deprivation (OGD) model Primary neurons were subjected to OGD treatment on day 5: pre-hypoxic DMEM (1×) medium was used for overnight pre-hypoxia. The neuronal medium was discarded and washed with PBS. 2 mL of pre-hypoxic medium was added to each well and the cells were placed in a 37°C incubator for 1.5 h of hypoxia. The pre-hypoxic DMEM (1×) medium was then aspirated, washed with PBS, and normal neuronal medium was added for continued culture.

[0026] 1.5 CCK-8 test CCK-8 test was used to detect the survival and damage of mouse neurons after OGD: 50 μL of CCK-8 solution was added to 100 μL of cell culture medium in each well of the 24-well plate after the neurons in each group were inoculated into the 24-well plate and subjected to OGD for 24 hours. Then the plate was incubated in a carbon dioxide incubator (37°C, 5% CO2) for 2-6 hours. The OD value at 450 nm was measured using an enzyme-labeled instrument, and the data was recorded for cell viability calculation.

[0027] The grouping of neuron cells is as follows: NC group: normal culture group of neuron cells under normoxic conditions; OGD group: neuron glucose-oxygen deprivation treatment group; OGD+GPNMB group: neuron glucose-oxygen deprivation treatment+GPNMB group; OGD+PBS group: neuron glucose-oxygen deprivation treatment+PBS group (as a GPNMB control group); OGD+LY294002+GPNMB group: neuron glucose-oxygen deprivation treatment+AKT inhibitor+GPNMB group.

[0028] Cell viability calculation: cell viability (%) = [A-B] / [C-B] x 100.

[0029] A: absorbance of the well with cells, CCK-8 solution and drug; B: absorbance of the well with CCK-8 solution, cell culture medium and no cells; C: absorbance of the well with cells, CCK-8 solution and no drug.

[0030] The test results are shown in Figure 21 , Figure 25 .

[0031] The related test steps after the HIBD model of newborn mice are the same.

[0032] 1.6 Calcein-AM / PI staining 1.6.1 After OGD for 24 hours, the neuron culture medium in the well was removed, and the cells were washed with PBS three times; 1.6.2 Preparation of Calcein AM / PI staining solution: 1 μL of Calcein (calcein) and 1 μL of PI were added to 1 mL of cell culture medium and mixed well, and stored in the dark; 1.6.3 Add 200 μL of Calcein and PI-containing medium to each well, and stain in the cell culture box in the dark for 10 minutes. 1.6.4 Aspirate the staining solution and wash once with PBS. Add the neuron culture medium and observe the survival and growth of the neurons under an inverted microscope.

[0033] The test results are shown in Figure 13 .

[0034] The relevant test steps after the HIBD model of the newborn mice are the same.

[0035] 2 The HIBD model of the newborn mice 2.1 Establishment of the HIBD model of the newborn mice

[0036] Here, first, the model is grouped as follows: Sham group: Only the right common carotid artery is isolated.

[0037] HIBD group: The right common carotid artery is isolated, electrocoagulated, and subjected to hypoxic treatment. HIBD+shRNA-GPNMB group: After the HIBD model is successfully established for 1 hour, 2 μL of shRNA-GPNMB (provided by Shanghai Oubio Biomedical Technology Co., Ltd.) with a virus titer of TU / μL is injected through the lateral ventricle.

[0038] HIBD+shRNA-control group: After the HIBD model is successfully established for 30 minutes, 2 μL of shRNA-Control negative control (provided by Shanghai Oubio Biomedical Technology Co., Ltd.) with a virus titer of TU / μL is injected through the lateral ventricle.

[0039] HIBD+GPNMB group: After the HIBD model is successfully established for 1 hour, 2 μL of GPNMB recombinant protein (50 ng / μL) is injected through the lateral ventricle.

[0040] HIBD+PBS group: After the HIBD model is successfully established for 1 hour, 2 μL of PBS solution is injected through the lateral ventricle.

[0041] Surgical instruments: scalp needle, suture soaked in 75% alcohol for one hour in advance, P7 SPF level and well-grown C57 mice (Chengdu Dasuo).

[0042] As Figure 1As shown, HIBD group mice were anesthetized in an isoflurane-containing anesthesia chamber, then removed and fixed in a supine position on a 37°C heating pad. Surgical procedures were performed while the mice continued to inhale the anesthetic. The neck skin was wiped with alcohol using a cotton swab. A longitudinal incision of approximately 0.5 cm was made slightly to the right of the midline of the neck with surgical scissors to separate and expose the right common carotid artery, which was then severed using an electrocautery scalpel. The incision was sutured and disinfected with an alcohol swab. The oxygen concentration in the hypoxia chamber was set to 10% (the gas valve was opened to supply N2 to the chamber, and the oxygen concentration was allowed to drop to 10%). C57 P7 mice with their left common carotid artery ligated were placed in the hypoxia chamber and subjected to 35°C hypoxia for 70 minutes. In the sham group mice, only the right common carotid artery was separated; it was neither ligated nor severed, and no hypoxia treatment was administered.

[0043] 2.2 RNA-Seq of brain tissue from the HIBD group 2.2.1 Brain tissue was extracted from mice in the sham group and HIBD group (n=3), and DNA was extracted from the samples. The DNA was then digested with DNase to interfere with the DNA. 2.2.2 Enrichment of eukaryotic mRNA; 2.2.3 Breaking mRNA → One-stranded cDNA → Double-stranded cDNA → Purifying double-stranded cDNA; 2.2.4 After the cDNA is tested and the results are satisfactory, sequencing is performed. 2.2.5 Sequencing results analysis.

[0044] Test results are as follows Figure 6 As shown.

[0045] 2.3 Intraventricular injection of shRNA-GPNMB lentivirus and recombinant GPNMB protein Thirty minutes after establishing the HIBD model, mice were fixed in a supine position and anesthetized with isoflurane until they became unresponsive to stimulation. The skin on the top of the head was disinfected, and a longitudinal incision was made along the midline to locate the anterior fontanelle. The drug was drawn into a microsyringe, and referring to a stereotactic map of the newborn mouse brain, the injection site in the lateral ventricle was determined to be 2 mm posterior to the anterior fontanelle and approximately 1.5 mm lateral to the sagittal suture. The needle was inserted perpendicularly at this point to a depth of 2 mm for 5 minutes, followed by a 2-minute pause before slowly withdrawing the needle and observing for any bleeding or leakage. The skin was then sutured and disinfected. After injection, the mice were returned to their cages to continue feeding with their mothers, and their condition was monitored for mortality, weight, and growth. The shRNA-GPNMB lentivirus titer was 2.23 × 10⁻⁶. 6 TU / μL, injection dose is 2μL; GPNMB recombinant protein concentration is 50ng / μL, injection dose is 2μL.

[0046] 2.4 Preparation of paraffin-embedded brain tissue specimens and HE staining 2.4.1 Prepare two 20 mL syringes in advance, draw 4% PFA (polyformaldehyde) and 4°C refrigerator pre-cooled normal saline solution respectively, and connect the scalp needle; 2.4.2 After the mouse is anesthetized in ice, it is fixed on the foam board in supine position. After no response to stimulation, the skin and subcutaneous muscle layer are cut transversely under the xiphoid process with ophthalmic forceps and scissors, taking care not to damage the liver to cause bleeding. The chest cavity is opened with scissors to expose the heart. The scalp needle is pierced into the left ventricle from the apex of the heart. When the normal saline is injected, the heart is filled. A small notch is cut in the right atrial appendage; 2.4.3 Slowly and evenly inject the normal saline solution until the lungs and liver turn white, and no obvious bloody substances are observed in the normal saline solution; 2.4.4 Slowly and evenly inject 4% PFA solution again until the mouse limbs are rigid. Then, the brain tissue is quickly removed and placed in the PFA solution for fixation for 48 h (4°C); 2.4.5 Trim the brain tissue: according to the stereotactic map of the newborn mouse brain, select the brain tissue at the coronal position before the fontanel and about 2 mm before the optic chiasm, and about 6 mm back in the sagittal position. Rinse with running water and place in a paraffin embedding box; 2.4.6 Dehydrate the brain tissue in gradient alcohol (70%, 85%, 95% alcohol, 15 min for each concentration), and then dehydrate in 100% ethanol for 3 times, 5 min each time; 2.4.7 Soak the brain tissue in 60°C wax to complete embedding, and fix the embedded brain tissue on a paraffin sectioning machine to start continuous sectioning. The thickness is 5 µm. The section should contain the cortex and hippocampal structure of the brain tissue. Then, place the section on a non-dropping glass slide, and finally place it in a 60°C incubator for 6 h; 2.4.8 De-paraffinization of brain tissue paraffin sections: de-paraffinization with xylene (30 min) to remove embedded paraffin, and then dehydrate with gradient alcohol (as above); 2.4.9 After staining with hematoxylin for 5 min, rinse with running water for 5 sec; differentiate with 1% hydrochloric acid alcohol for 30 sec, and rinse with running water for 5 sec; stain with eosin solution for 1 min, and rinse with running water for 2 min; 2.4.10 Dehydration and transparency: dehydrate gradually in gradient alcohol (70%, 85%, 95%, 100%) for 5 min; transparentize with xylene for 2 times, 10 min each time; add neutral balsam for mounting, and examine under a microscope.

[0047] 2.5 TTC staining The brain mold and the vessel containing PBS solution were pre-cooled on ice. After the mice were anesthetized, the brain tissue was quickly removed and placed in the pre-cooled PBS solution. The brain tissue was placed in an ice water bath for 30 min. After the brain tissue was slightly frozen, it was removed and placed in the brain mold. The brain mold was placed in an ice water bath and sliced (about 2 mm thick). The brain tissue slice was placed in a well plate containing TTC solution and stained in the dark.

[0048] The test results are shown in Table 2. Figure 2 、 Figure 26 The test results are shown in Table 2.

[0049] 2.6 Immunofluorescence staining of brain tissue 2.6.1 Paraffin section deparaffinization: the paraffin section was placed in a 70°C oven for 60 min; 2.6.2 The paraffin section was immersed in a dye vat containing xylene for 30 min, and then immersed in gradient alcohol (as above) and ddH2O (double deionized water) for 5 min; 2.6.3 Antigen repair: the section was placed in a section box filled with citric acid: a pot was filled with water and placed on an electromagnetic oven to pre-heat to boiling. The section immersed in citric acid was placed in the boiling water, and heating was continued for 30 min. Then, the section was placed at room temperature and allowed to cool naturally; 2.6.4 The section was washed with PBS three times, each for 5 min (5 min x 3); 2.6.5 Draw a water-blocking circle: the section tissue was wiped dry with a handkerchief, and a water-blocking pen was used to draw a circle around the tissue; 2.6.6 0.3% TritonX-100, 4°C membrane penetration treatment for 15-30 min; the section was washed with PBS three times, each for 5 min; 2.6.7 Blocking solution (BSA 2.5 g added to TBST 50 mL) was added to the water-blocking circle, and the section was blocked in a refrigerator at 4°C for 1 hour; 2.6.8 The blocking solution was gently shaken off, and the primary antibody was added dropwise. The section was placed in a wet box and incubated in a refrigerator at 4°C overnight; 2.6.9 The primary antibody was recovered, and the section was washed with PBS three times, each for 5 min; 2.6.10 The corresponding secondary antibody was added dropwise to the tissue in the water-blocking circle, and the section was incubated at room temperature in the dark for 1 hour; 2.6.11 The secondary antibody was gently shaken off, and DAPI staining solution was added dropwise. The section was incubated at room temperature for 5 min; 2.6.12 The section was washed with PBS, and anti-fluorescence quencher was added dropwise. The section was mounted and observed under a fluorescence microscope.

[0050] The test results are shown in Table 2. Figure 11

[0051] 2.7 Mouse head MRI examination ​Each group of experimental C57 mice (sham group, HIBD group, HIBD+shRNA-Control group, HIBD+shRNA-GPNMB group) after inhalation of isoflurane anesthesia, take the prone position fixed, and maintain the mouse anesthesia, using small animal nuclear magnetic resonance imaging (MRI) for mouse head nuclear magnetic T2 sequence scanning, to understand the change of mouse brain tissue morphological structure. Test results are shown in Figure 18 .

[0052] TEM experiment C57 mice after modeling 24h, each group (sham group, HIBD group, HIBD+shRNA-Control group, HIBD+shRNA-GPNMB group) cerebral cortex (grain size) was collected into electron microscope fixing fluid. First, use 3% glutaraldehyde-4% paraformaldehyde, 1% osmium tetroxide to fix the sample, then dehydrate with acetone step by step, then use Epon812 for embedding, after embedding, prepare ultrathin sections, thickness is about 50nm; spread, to the copper net. Stained with uranyl acetate, lead citrate at room temperature, observed under transmission electron microscope and took pictures.

[0053] Test results are shown in Figure 27 .

[0054] 2.9 Morris water maze experiment Each experimental group of C57 mice 10 (sham group, HIBD group, HIBD+shRNA-Control group, HIBD+shRNA-GPNMB group), P 28-P 36 days to carry out Morris water maze experiment. The first 5 days of positioning navigation experiment, 6 days of space exploration experiment.

[0055] 2.9.1 Experimental setup Water is injected into a cylindrical water tank with a diameter of 120 cm and a height of 50 cm to make the water depth reach 32 cm, and the water maze heating device is turned on to make the water temperature reach a constant temperature of 25℃; Mark the I, II, III, IV quadrant around the pool. The platform is placed in the first quadrant, and white dye is poured into the water until the pool is completely dyed white and the platform disappears.

[0056] 2.9.2 Positioning navigation experiment Each group of mice (sham group, HIBD group, HIBD+shRNA-Control group, HIBD+shRNA-GPNMB group) was trained for 5 days. The mice were placed in the water close to the pool wall, and were placed in the water from the four quadrants in different order every day, and the position of the mouse was kept unchanged every time. The escape latency was the time from the mouse entering the water to finding the platform position, and the search time was 90 seconds. If the platform was not found within 90 seconds, the mouse was guided to find the platform and stay on the platform for 10 seconds. The average time of reaching the platform in the four quadrants was the escape latency.

[0057] 2.9.3 Spatial exploration experiment The spatial exploration experiment was performed on the 6th day. The existing experimental conditions were kept unchanged, the platform was removed, a virtual platform was set, and each group of mice was placed in the water from the III quadrant. The number of times the mice crossed the platform in 60 seconds was recorded.

[0058] The test results are shown in Figure 4 , Figure 17 .

[0059] 2.10 Western blot experiment 2.10.1 Gel preparation: 10% or 12% separation gel was added to the gel plate with a pipette, anhydrous ethanol was added, and the gel plate was placed at room temperature for 30 minutes. After the anhydrous ethanol was poured out, it was absorbed with a handkerchief. 5% concentrated gel was added on top of the separation gel, a comb was inserted, and it was left to stand for 30 minutes. 2.10.2 Sample loading: the protein sample was taken out and placed on ice. The metal bath was boiled (100°C, 5 minutes). The prepared gel plate was loaded. 2.10.3 Electrophoresis: 1x electrophoresis buffer was added to the electrophoresis tank, and the separation gel and the concentrated gel were connected. The bromophenol blue was run to the lower edge of the separation gel, and the electrophoresis was stopped. 2.10.4 Membrane transfer: 1x transfer solution was prepared, and PVDF membrane (length: 8 cm, width: 7 cm) was cut in advance. The PVDF membrane was soaked in formaldehyde (10 seconds) and then immersed in the transfer solution. The glass plate was removed, and the gel was cut according to the protein band control marker and placed on filter paper. The membrane transfer was performed according to the "sandwich" model, the current was constant current 250 mA, and the time was (molecular weight + 10) min. 2.10.5 Blocking: the PVDF membrane was taken out from the electric tank and placed in 5% milk blocking solution (5 g skim milk added to 100 mL TBST) with the front side facing down. It was incubated at room temperature for 1-2 hours (shaking bed about 75 rpm / min). 2.10.6 First antibody hybridization: dilute the antibody to be tested with blocking solution (BSA 2.5 g added to TBST 50 mL), Bcl2 Bax 1:1000, GPNMB 1:2000, Actin 1:5000, add the diluted first antibody to the antibody incubation box, shake at room temperature overnight at 4°C; 2.10.7 Washing: add 1x TBST solution to the antibody incubation box where the PVDF membrane is placed, wash thoroughly on a shaker, 5 min x 6 times; 2.10.8 Second antibody hybridization: dilute the corresponding second antibody 1:5000, shake gently at room temperature for about 1 h; add the diluted second antibody to the antibody incubation box, shake at room temperature overnight at 4°C; 2.10.9 Washing and color development: wash the membrane with TBST, mix A and B liquids in the exposure solution at a ratio of 1:1, add to the PVDF membrane, and expose.

[0060] The test results are shown in Figure 10 , Figure 16 , Figure 23 , Figure 24 .

[0061] 2.11 Real-time PCR Real-time fluorescent quantitative PCR 2.11.1 Extraction of brain tissue RNA sample ① Cut open the mouse skull, place the mouse brain tissue in a culture dish, separate the tissue with surgical forceps in the culture dish, and place it in a clean 1.5 mL enzyme-free EP tube and on ice; ② Add 1 mL Trizol to the EP tube containing the brain tissue, grind to complete lysis in a tissue homogenizer, add 0.2 mL chloroform to each 1 mL Trizol, shake for 15 sec, and place at room temperature for 3-5 min, centrifuge at 4°C and 12000 rcf for 15 min in a high-speed refrigerated centrifuge; ③ After centrifugation, the upper aqueous phase is removed with a pipette and placed in another EP tube. Add 0.5 mL isopropanol to the 1.5 mL EP tube, and after placing at room temperature for 10 min, centrifuge in a high-speed refrigerated centrifuge (same centrifugation conditions as above); ④ After centrifugation, the supernatant is aspirated, and the white substance at the bottom of the EP tube is RNA. Add 1 mL of 75% alcohol and gently shake the EP tube; ⑤ Centrifuge in a high-speed refrigerated centrifuge at 4°C and 12000 rcf for 10 min. The supernatant after centrifugation is directly aspirated with a pipette and dried at room temperature for 5-10 min; ⑥ 40 Dissolve the RNA precipitate in DEPC water, measure the RNA concentration with a spectrophotometer, and store at -80°C.

[0062] 2.11.2 Brain tissue RNA sample extraction ① Remove genomic DNA: Put gDNA Eraser Buffer 2 (5x) 1 μL, gDNA Eraser 1 μL, and RNA sample into a 200 μL EP tube, add DEPC water to 10 μL, 42°C, reaction for 2 min, remove genomic DNA; (5x), gDNA Eraser 1 μL, and RNA sample into a 200 EP tube, add DEPC water to 10 μL, 42°C, reaction for 2 min, remove genomic DNA; ② Reverse transcription to synthesize cDNA: Put PrimeScript RT Enzyme Mix 1 (1 μL), RT Primer Mix (1 μL), 5x PrimeScript Buffer 2 (4 μL), DEPC water (4 μL) into the above 10 system after removing genomic DNA, 37°C, reaction for 15 min, 85°C, reaction for 5 s, to obtain cDNA; ③ Dilution: Add 180 μL DEPC water to dilute the above cDNA; ④ Mixture preparation: Upstream primer 0.3 μL, downstream primer 0.3 μL, qPCR SYBR Green Master Mix 5 Preparation 5.6 μL system mixture, shake and mix well; ⑤ Loading and centrifugation: Put 4.4 μL cDNA sample and 5.6 μL above mixture into each well of a 96-well plate, centrifuge for about 1-2 min, and shake down the sample; ⑥ Sample reaction: Perform reaction according to Table 3; ⑦ Ct value recording: Record Ct value after reaction, and calculate results; ⑧ Result calculation: The calculation formula of the relative expression amount of the target gene is: ΔΔCt =ΔCt 实验组 -ΔCt 对照组 .

[0063] Table 3 PCR translation conditions and process settings

[0064] Table 4 PCR primer design

[0065] 3 Experimental result analysis

[0066] (i) Newborn mouse HIBD model and neuron OGD model were successfully constructed After the HIBD model of newborn mice was established, pathological staining and neurobehavioral tests were performed to evaluate brain injury. As shown in FIG. 1A, TTC staining results showed that there were white infarction lesions in the right brain of HIBD mice. As shown in FIG. 1B, HE staining showed that the arrangement structure of the cerebral cortex and hippocampal tissue of HIBD mice was disordered, and the number of cells was reduced. As shown in FIG. 1C, laser speckle results showed that the cerebral blood flow of the injured side of HIBD mice was significantly reduced. As shown in FIG. 1D, Morris water maze results showed that the number of times of crossing the platform of HIBD mice was reduced, and the escape time was prolonged (P<0.05). These results suggest that the HIBD model of newborn mice is successfully established. Figure 2 Figure 3 Figure 5 Figure 4 p <0.05). These results suggest that the HIBD model of newborn mice is successfully established.

[0067] After OGD treatment of primary cultured neurons, CCK8 and Calcein-AM / PI double staining were performed to evaluate cell injury (results shown in FIG. 2). As shown in FIG. 2A, the cell viability of the OGD group (59%) was significantly lower than that of the normoxic group (85.5%), and the difference was statistically significant (P<0.001). As shown in FIG. 2B, the Calcein-AM / PI staining results showed that the number of neuron death increased after OGD (PI positive rate 55%), and compared with the normoxic group (PI positive rate 21%), the difference was statistically significant (P<0.001). These results indicate that the OGD model of neurons is successfully established. Figure 13 Figure 13 p Figure 13 p <0.001). These results indicate that the OGD model of neurons is successfully established.

[0068] (ii) Transcriptional sequencing of brain tissue of HIBD mice and verification of differential genes

[0069] As shown in FIG. 3A, the RNA-seq results of mouse brain tissue showed that there were 1050 differential genes in the HIBD group compared with the sham group, of which 982 genes were up-regulated and 68 genes were down-regulated. As shown in FIG. 3B, bioinformatics analysis showed that the differential genes played a role in biological processes such as apoptosis, TNF signaling pathway, chemokine signaling pathway, and Toll-like receptor signaling pathway. Figure 6 Figure 7 As shown in FIG. 4, RT-PCR detection was performed on the genes with high expression and high differential fold, and the results were consistent with the sequencing data. GPNMB was one of the most up-regulated genes (P<0.001).

[0070] As shown in FIG. 4, RT-PCR detection was performed on the genes with high expression and high differential fold, and the results were consistent with the sequencing data. GPNMB was one of the most up-regulated genes (P<0.001). Figure 8 p

[0071] As shown in FIG. 4, RT-PCR detection was performed on the genes with high expression and high differential fold, and the results were consistent with the sequencing data. GPNMB was one of the most up-regulated genes (P<0.001). Figure 9 ​​​​​​​​​​​As shown, bioinformatics analysis indicates that GPNMB is involved in biological processes such as apoptosis inhibition and cytokine production, suggesting that GPNMB may play an important role in the inhibition of apoptosis in HIBD neurons.

[0072] (iii) Distribution of GPNMB in brain tissue and expression changes in hypoxic-ischemic brain injury after HIBD like Figure 11 As shown in the IF results, GPNMB was localized in both neurons and microglia after HIBD, but mainly in neurons.

[0073] like Figure 10 As shown, Western blotting results indicated that GPNMB expression increased after HIBD. p <0.05); Figure 12 This indicates that GPNMB expression increased in neurons and HT22 cells after OGD ( p <0.001).

[0074] (iv) Knockdown of GPNMB in newly regenerated mice with hypoxic-ischemic brain injury HIBD mice were injected intracerebroventricularly with shRNA-GPNMB and a negative control shRNA-Control. Seven days later, GPNMB expression was detected by RT-PCR and Western blotting. Figure 15 , Figure 16 As shown. RT-PCR and Western blotting results showed that, compared with the shRNA-Control group (100%), the expression level of GPNMB mRNA in the brain tissue of mice in the shRNA-GPNMB group decreased by 85% ( p <0.001, protein levels decreased by 30% ( p The value <0.05 indicates that shRNA-GPNMB can effectively inhibit the expression of GPNMB in brain tissue.

[0075] like Figure 19 , Figure 20 As shown, H&E staining results of liver and kidney tissues showed that the structure and cell morphology of mouse liver and kidney tissues were normal, with no significant differences among groups. The levels of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (UREA), and creatinine (CREA) in each group were all within the normal range, suggesting that intraventricular injection of shRNA virus had no adverse effects on liver and kidney function in mice.

[0076] like Figure 17 As shown, the Morris water maze test revealed that, compared to the control group, HIBD mice with knocked-down GPNMB had a longer average escape latency and a significantly reduced number of platform crossings. p<0.05), suggesting that inhibition of GPNMB aggravates the neurobehavioral impairment of HIBD mice.

[0077] As shown in Figure 18 , the mouse head magnetic resonance examination (T2 sequence) showed that the volume of necrotic lesions in the injured side of the brain increased, indicating that knockdown of GPNMB aggravated the HIBD injury in mice. These results suggest that knockdown of GPNMB aggravates hypoxic-ischemic brain injury in neonatal mice.

[0078] (v) GPNMB recombinant protein can improve the survival rate of neuron cells

[0079] After OGD of primary cultured neuron cells, different concentrations of GPNMB recombinant protein (25 ng / ml, 50 ng / ml, 100 ng / ml) were given, and the CCK test and Calcein-AM / PI staining test results are shown in Figure 21 , Figure 22 .

[0080] As shown in Figure 21 , the CCK8 results showed that the cell survival rates of the 25 ng / ml, 50 ng / ml, and 100 ng / ml GPNMB groups were 78%, 81%, and 75%, respectively, and the differences were statistically significant compared with the PBS group (59%) (all P<0.001). p

[0081] As shown in Figure 22 , the Calcein-AM / PI staining results showed that when the dose of GPNMB recombinant protein was 50 ng / ml, the neuron death was significantly reduced, and the PI positive rate of neurons was 48% (P<0.001), which was statistically significant compared with the OGD group. p

[0082] As shown in Figure 23 , the WB results showed that after adding GPNMB, the expression of BCL-2 increased and the expression of BAX decreased. The above experiments comprehensively showed that GPNMB recombinant protein has a protective effect on OGD neuron cells.

[0083] As shown in Figure 24 , the WB results showed that after adding GPNMB recombinant protein, the ratio of P-AKT / AKT increased, and the AKT signaling pathway was activated.

[0084] As shown in Figure 25 , after adding AKT inhibitor LY294002 (10 ), the CCK8 results showed that the cell survival rates were 12%, and the differences were statistically significant compared with the OGD+GPNMB group (59%) (all P<0.001). p ​​All <0.01). Calcein-AM / PI staining results showed a significant increase in neuronal death, with a neuronal PI positivity rate of 57% compared to the OGD+GPNMB group ( p <0.01), which is statistically significant.

[0085] (vi) Recombinant GPNMB protein can alleviate hypoxic-ischemic brain injury in newborn mice.

[0086] like Figure 26 As shown, after administration of recombinant GPNMB protein, the cerebral infarction volume in mice in the HIBD+GPNMB group was significantly smaller compared to the HIBD+PBS group. p <0.05).

[0087] like Figure 27 As shown, after administration of recombinant GPNMB protein, mice in the HIBD+GPNMB group did not exhibit condensation, their nuclear membranes did not fold, and their cell bodies were compact, compared to the HIBD+PBS group.

[0088] like Figure 28 As shown, the weight statistics indicate that the HIBD group had better weight development compared to the HIBD+PBS group. p <0.05).

[0089] like Figure 29 As shown, the brain atrophy results indicate that brain atrophy is reduced in the HIBD+GPNMB group ( p <0.05).

[0090] These results indicate that recombinant GPNMB protein plays a neuroprotective role in hypoxic-ischemic brain injury in newborn mice.

Claims

1. Application of GPNMB in the preparation of drugs for treating hypoxic-ischemic brain injury in newborns.

2. The application according to claim 1, characterized in that, The GPNMB is used to prepare drugs that inhibit neuronal death caused by hypoxia and ischemia during neonatal development.

3. The application according to claim 1, characterized in that, The GPNMB is used as a promoter to activate the AKT signal channel.

4. The application according to any one of claims 1 to 3, characterized in that, The drug is in the form of an injection.

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