Application of antimicrobial peptide MU1

Abstract

The present invention provides an application of antimicrobial peptide MU1, especially an application in preparing a drug for treating hypoxic-ischemic encephalopathy and a drug for protecting neurons, and belongs to the field of biomedicine. The present invention verifies through experiments that antimicrobial peptide MU1 has the effects of alleviating the symptoms of hypoxic-ischemic encephalopathy and protecting neurons, and can be used to prepare a drug for treating hypoxic-ischemic brain injury, especially neonatal hypoxic-ischemic encephalopathy. The antimicrobial peptide MU1 has good therapeutic effect and high safety, filling the gap in the prior art that neonatal hypoxic-ischemic encephalopathy has no drug to treat it, and has good application prospects.

Description

Technical Field

[0001] The invention relates to an application of an antimicrobial peptide MU1, in particular to an application of the antimicrobial peptide MU1 in preparing a drug for hypoxic-ischemic encephalopathy and a drug for protecting neurons after hypoxia-ischemia, and belongs to the field of biomedicine. Background Art

[0002] Hypoxic-ischemic brain damage (HIBD) is a common brain injury caused by cerebral hypoxia or ischemia due to various factors, and is mainly manifested by changes in consciousness and muscle tone. Neonatal hypoxic-ischemic encephalopathy (HIE) is a brain injury caused by cerebral hypoxia or ischemia due to various perinatal factors. It is one of the main causes of neonatal death and long-term neurobehavioral abnormalities caused by brain damage worldwide. It can be divided into mild, moderate and severe according to the changes in the disease. Mild and moderate symptoms are excitement or dullness, and muscle tone is normal or reduced. Severe cases may have coma, loose muscle tone, frequent convulsions, etc., and are often accompanied by serious sequelae such as cerebral palsy, epilepsy, learning difficulties, etc. It is a serious complication of patients after asphyxia, with a high mortality rate and disability rate. The pathophysiological pathogenesis of HIE involves the interaction between complex neuronal excitotoxicity, neuroinflammation and oxidative stress, and its pathogenesis and pathophysiological manifestations are different from those of adult stroke. Neonates are at higher risk of neuronal excitotoxicity than adults and are more susceptible to epileptic seizures; in addition, the antioxidant capacity of the neonatal brain is underdeveloped, and the permeability of the blood-brain barrier increases more significantly after injury than in adults.

[0003] At present, HIE still lacks effective clinical treatment. Brain protection measures for HIE have always been a hot topic and difficulty of international concern. At present, the most effective intervention measure is hypothermia therapy, but the efficacy is limited, and there are great differences in the specific treatment plans of hypothermia therapy in the laboratory and clinic. The cooling time and degree of cooling are different in different cases, which has caused troubles in the design of clinical treatment plans. Based on the complex pathogenesis of HIE and the uniqueness of the dosage and method of neonatal medication, the current drug intervention plan is still immature. For example, studies have shown that the effect of erythropoietin combined with hypothermia therapy in rat HIE models varies greatly, showing mild injury relief or no obvious rescue effect, while in the study of newborn macaques, this regimen can reduce the incidence of cerebral palsy sequelae. Although neurotrophic factors have repair functions, pharmacological clinical results suggest that neurotrophic factor drugs have large molecular weight and short half-life, so they need to be administered in large doses, but there is still a problem of low efficacy, and large-dose systemic administration also brings large drug side effects. In addition, studies have shown that the rescue effect of antioxidants may be related to gender. In addition, large doses of antioxidants can over-activate glutamate receptors and produce neurotoxicity. The metabolism of nanoformulation encapsulation materials in newborns is still lacking. In addition, the characteristics and expression levels of receptors and enzymes in the brains of newborns are different from those of adults, and the development of organs for drug metabolism, the liver and kidneys, is also very different from that of adults. Therefore, the treatment plans for adult diseases cannot be fully applied to newborns.

[0004] Host defense peptides (HDPs) are small molecules for potential drug development, but their research in the central nervous system is very limited. Innate immune molecules cathelicidins are host defense peptides in vertebrates and are members of the family. Only one cathelicidin is found in humans and mice, LL-37 in humans and CRAMP in mice. Cathelicidins are mainly expressed in a variety of immune cells, such as neutrophils, mast cells, macrophages, etc., and are also expressed in some non-immune cells, such as epithelial cells and keratinocytes. The latest studies have shown that they are also expressed in pancreatic islet cells, neurons and glial cells. Most host defense peptide cathelicidins have a broad spectrum of antimicrobial activity, including Gram-positive and Gram-negative bacteria, fungi, viruses and parasites. In addition, host defense peptide cathelicidins also have diverse immunomodulatory functions on immune cells and non-immune cells. More importantly, host defense peptide cathelicidins have great advantages in the field of drug development, mainly manifested in: 1) broad-spectrum and efficient antibacterial activity; 2) not easy to cause microbial resistance; 3) strong immunomodulatory activity; 4) small molecular weight, simple structure, low toxicity and immunogenicity; 5) large molecular modification space. Current studies have confirmed that endogenous cathelicidins are involved in the pathogenesis of a variety of autoimmune and autoinflammatory diseases. Studies have shown that CRAMP (mouse cathelicidins) can reduce the activation of microglia cells (MGs) after binding to Aβ protein, so that the protein cannot form a protein structure unique to Alzheimer's disease; knockout of the CRAMP gene leads to post-meningitis immune cell activation, increased release of inflammatory mediators, and increased mortality. Although the mechanism of action of endogenous cathelicidins in the body has received widespread attention, it is not widely used as an exogenous drug to treat neurological diseases. Most exogenous cathelicidins are used to prepare disinfectants for their antibacterial effects. Laboratory studies have found that they have a certain degree of effect in epidermal repair and tumor treatment. However, the therapeutic application of exogenous cathelicidins for neurological diseases is still little known. A recently published study suggests that exogenous cathelicidins can alleviate the oxidative stress response of the nervous system in Parkinson's disease, but there are still no reports on the use of exogenous cathelicidins in the treatment of HIE.

[0005] Exogenous antimicrobial peptides acting on nervous system diseases face common problems of exogenous drugs targeting the nervous system: blood-brain barrier penetration, toxicity and safety, whether drug administration will aggravate inflammatory response, etc. Most exogenous antimicrobial peptides have a small molecular weight and carry a positive charge, which may react with the blood-brain barrier cell membrane. Crossing the blood-brain barrier requires reducing the interaction with the blood-brain barrier and drug polarity. Exogenous drugs must also have plasma stability before entering the blood-brain barrier to ensure that they will not be degraded before entering the body and reaching the central nervous system. In addition, the occurrence of neurological diseases may be accompanied by damage to the blood-brain barrier. When administering drugs, it is also necessary to consider whether the effects of exogenous drugs will aggravate the damage to the blood-brain barrier caused by neuroinflammation.

[0006] The MU1 antimicrobial peptide is extracted and purified from the skin secretions of a Yunnan frog, Rana fasciatus. It is a polypeptide composed of 24 amino acid residues (ARGKKECKDDRCRLLMKRGSFSYV), and its sequence is shown in SEQ ID NO: 1. It belongs to the cathelicidin family of antimicrobial peptides and is a host defense peptide. Existing studies have shown that MU1 still has no cytotoxicity and hemolytic activity on mammalian cells at high concentrations, and is safe, stable, and highly effective. After searching, there is no literature report on the role of the antimicrobial peptide MU1 in hypoxic-ischemic encephalopathy and neuronal protection. Summary of the invention

[0007] In view of the above technical problems, the present invention intends to provide an application of an antimicrobial peptide, in particular, an application of the antimicrobial peptide MU1 in the preparation of drugs for hypoxic-ischemic encephalopathy and drugs for neuron protection.

[0008] The invention discloses an application of the protective antimicrobial peptide MU1 in preparing medicines for hypoxic-ischemic encephalopathy.

[0009] The present invention also protects the use of the antimicrobial peptide MU1 in preparing a drug for protecting neurons after hypoxia-ischemia.

[0010] Furthermore, the hypoxic-ischemic encephalopathy includes hypoxic-ischemic brain damage.

[0011] Furthermore, the hypoxic-ischemic encephalopathy is neonatal hypoxic-ischemic encephalopathy.

[0012] Furthermore, the dosage form of the drug includes injection and capsule.

[0013] The present invention proves the change of endogenous host defense peptide expression in the acute time window after HIE through the expression spectrum of host defense peptides in neurons and microglia in the brain of suckling mice, suggesting that it may play a certain role in resisting exogenous damage. Based on this basis, the inventor exogenously supplemented MU1, an antimicrobial peptide extracted from the skin of Yunnan frogs, to mice after HIE to study whether it plays an effect in brain protection and how it plays a role. In vivo experiments in the embodiments of the present invention prove that the antimicrobial peptide MU1 reduces the incidence of liquefaction injury and the liquefaction area of ​​lesions in neonatal hypoxic-ischemic encephalopathy. In vitro experiments have shown that primary neurons can slow down cell apoptosis caused by oxygen-glucose deprivation (OGD) treatment after MU1 is given, indicating that the antimicrobial peptide MU1 can protect neurons during hypoxia and ischemia, and play a role in brain protection by reducing neuronal apoptosis after hypoxia and ischemia. After intraperitoneal injection of MU1 in HIE mice, the incidence of liquefaction injury and the liquefaction area of ​​lesions in neonatal hypoxic-ischemic encephalopathy were reduced, verifying the brain protection effect of exogenous host defense peptide MU1 on HIE. In further mechanism exploration, it was found that after human microglia HMC3 and mouse primary neurons MU1 were given in vitro, the apoptosis of primary neurons under ischemic and hypoxic conditions was reduced, while the phagocytic function of microglia under hypoxic and ischemic conditions did not change after HMC3 was given to MU1. This shows that MU1 does not affect the phagocytic function of microglia, and can maintain the balance of the brain environment to a certain extent, and play a role in brain protection by reducing neuronal apoptosis after hypoxia and ischemia. The experiment also proved that MU1 prevented microglia from excessively transforming to a proinflammatory phenotype after OGD through the expression of important neurotrophic factors and homeostasis-maintaining factor TGFβ1 expressed by microglia, combined with its morphological changes. This shows that MU1 can protect neurons during hypoxia and ischemia, reduce neuronal apoptosis after hypoxia and ischemia, and maintain the homeostasis of microglia, maintain phagocytic function, and is conducive to the repair of the brain microenvironment after hypoxia and ischemia. This shows that MU1 plays a brain protective role by reducing neuronal apoptosis and restoring microglial homeostasis.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention is the first to discover changes in the expression of endogenous host defense peptides in the acute time window after HIE in suckling mice, prove that exogenous supplementation of the antimicrobial peptide MU1 can play a brain protective effect on HIE damage, and prove the use of the antimicrobial peptide MU1 in the preparation of drugs for neonatal hypoxic-ischemic encephalopathy. The antimicrobial peptide MU1 protects neurons during hypoxia and ischemia, reduces neuronal apoptosis after hypoxia and ischemia, and can maintain microglial homeostasis and has no effect on the phagocytosis of microglial cells. This makes the application of MU1 for brain protection safe, stable and efficient, and has potential application prospects in HIE. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The expression profile of endogenous host defense peptide CRAMP in neurons in the central area of ​​cortical infarction at different time windows after HIE in neonatal rats. Figure 1 A-B are the expression location and expression level statistical analysis of the cortical infarct center area in the sham group (Sham) and the severe HIE (HI-S) group 1 day after the model; Figure 1 C~D are the expression location and expression level statistical analysis of the cortical infarct center area at 3 days after the model in the sham operation group (Sham) and the severe injury HIE (HI-S) group;

[0017] Figure 2 The expression profile of endogenous host defense peptide CRAMP on microglia in the cortical infarct core area at different time windows after HIE in neonatal rats. Figure 2 A-B are the expression location and expression level statistical analysis of the cortical infarct center area in the sham group (Sham) and the severe HIE (HI-S) group 1 day after the model; Figure 2 C~D are the expression location and expression level statistical analysis of the cortical infarct center area at 3 days after the model in the sham operation group (Sham) and the severe injury HIE (HI-S) group;

[0018] Figure 3 The results of the MU1 intervention in HIE mice showed the incidence of severe brain tissue damage and the gross observation of liquefaction lesions. Figure 3 A is a statistical chart showing the percentage of mild injury (HI-M) and severe injury (HI-S) in HIE control mice (HIBD + NS) and HIE mice (HIBD + MU1) treated with normal saline. Figure 3 B is the gross results of liquefaction lesions in HIE control mice (HIBD + NS) and HIE mice (HIBD + MU1) with mild injury (HI-M) and severe injury (HI-S) given normal saline.

[0019] Figure 4 This is the result of MU1 intervening in glucose-oxygen deprivation primary neuron apoptosis. Figure 4 (AI) are the TUNEL and DAPI staining results of primary neurons in different treatment groups: control group, hypoxia-ischemia group (OGD group), hypoxia-ischemia+MU1 peptide group (OGD+MU1 group); Figure 4 J is the statistical result of neuronal apoptosis rate.

[0020] Figure 5 This is the result of MU1 intervening in the phagocytic ability of glucose-oxygen deprived HMC3 cells. Figure 5(AC) are the flow cytometry results of HMC3 phagocytic ability in different treatment control group (Control group), ischemia-hypoxia group (OGD group), ischemia-hypoxia + MU1 peptide group (OGD + MU1 group); Figure 5 D is the statistical result of phagocytic ability.

[0021] Figure 6 Figure 6A-C represent the morphological changes of the microglial cell line HMC3 and the changes in TGFβ1 protein expression after OGD and MU1 administration; 6A-C represent the DAPI and Iba1 expression and morphological images of HMC3 cells in the control group (Control group), ischemia-hypoxia group (OGD group), ischemia-hypoxia + MU1 peptide group (OGD + MU1 group), respectively. Figure 6 D represents the results of western blot analysis of TGFβ1 protein expression in the control group (Control group), ischemia-hypoxia group (OGD group), and ischemia-hypoxia + MU1 peptide group (OGD + MU1 group). Figure 6 E represents the statistical analysis of TGFβ1 protein expression in the control group (Control group), ischemia-hypoxia group (OGD group), and ischemia-hypoxia + MU1 peptide group (OGD + MU1 group). DETAILED DESCRIPTION

[0022] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention. It should be noted that if specific techniques or conditions are not specified in the embodiments, the experimental results can be obtained according to the description of relevant literature in the field or product instructions. The reagents, instruments, cells and animal models used are all conventional products that can be purchased on the market if the manufacturer is not specified.

[0023] Experimental animals: C57BL / 6J mice, animal license number SCXK (Yunnan) 2015-0002, were provided by the Animal Care and Use Committee of Kunming Medical University. Pregnant mice were purchased from Kunming Medical University and kept in a sterile animal room with independent ventilation cages. Their offspring were fed with their mothers until 10 days of age (Day 10, 3.0-6.0 g) to establish the hypoxic-ischemic brain injury model.

[0024] Equipment: 5-0 surgical sutures, surgical scissors, ophthalmic forceps, surgical forceps, needle holders, syringes, hypoxia chamber (BillupsRothenburg, HF151), fluorescence microscope, an improved constant temperature hypoxia model experimental device (patent number: ZL201320101770.6), a simple small experimental animal operating box (patent number: ZL201520214776.3)

[0025] Reagents: isoflurane for inhalation (Maruishi Pharmaceutical Co., Ltd., H20150020, Japan), anhydrous ether, sugar-free culture medium (Solebol, 90113, China), PBS, penicillin-streptomycin double antibody, Neurobasal plus Medium, B27 plus supplement, glutamine, papain, TUNEL kit, DAPI mounting medium, FBS, Gibco 0.25% trypsin, environmentally friendly tissue dewaxing solution, anhydrous ethanol, donkey serum, MEM culture medium, FITC-Dextran40, high-efficiency tissue / cell lysis buffer (Solebol), TGF beta 1 polyclonal antibody (Proteintech), TBST, SDS-Page running buffer, Transfer buffer, methanol, skim milk powder, GAPDH antibody, Goat anti-Rabbit IgG H&LHRP antibody, Goat anti-Mouse IgG H&L HRP antibody, 5X reduced protein loading buffer, dual-color pre-stained protein marker (10-250kd), Iba1 antibody

[0026] Example 1 Expression of endogenous CRAMP on neurons in the central area of ​​cortical infarction in HIE mice

[0027] Experimental methods:

[0028] 1) C57BL / 6J pregnant mice were purchased from Kunming Medical University and housed in a sterile animal room with independent ventilation cages. Their offspring were fed next to their mothers until they were 10 days old (Day 10, 3.0-6.0 g) to establish a hypoxic-ischemic brain injury model.

[0029] 2) Establishment of HIE model: Newborn mice were anesthetized by inhalation of sevoflurane (30 μL / mouse), and fixed in supine position on the operating table in a small experimental animal operating box. The left common carotid artery was found in a sterile environment, and the proximal and distal ends were ligated and then cut in the middle. The mice were allowed to rest for 1.5 h after surgery. Then, the mice in the model group were placed in a hypoxia model experimental device, and soda lime and an oxygen meter were placed in the square groove at the bottom of the device. The condition in the device was 8% oxygen-balanced nitrogen, and the mice were exposed to an ambient temperature of 34-34.5°C for 45 min.

[0030] 3) Mice were deeply anesthetized with 10% chloral hydrate (0.3 mL / 100 g), and saline was perfused from the apex of the heart. Brain tissue was obtained after perfusion with 4% paraformaldehyde solution, fixed with 4% paraformaldehyde for 24 h, dehydrated with gradient alcohol, immersed in wax, and embedded into wax blocks. The specific steps are as follows: tissue washing with running water (2 hours) → 70% alcohol (3 hours) → 80% alcohol (overnight) → 90% alcohol (1 hour) → 95% alcohol (1 hour) → absolute alcohol I (30 minutes) → absolute alcohol II (30 hours) → xylene (25 minutes) → paraffin I (1 hour) → paraffin II (1 hour) → paraffin III (1 hour) → embedded into wax blocks with an embedding machine. Brain tissue was sliced ​​for immunofluorescence staining, and the slice thickness was 7 μm.

[0031] 4) The specific staining steps are as follows: environmentally friendly dewaxing transparent liquid is placed in a constant temperature box and preheated at 45°C in advance, dewaxing transparent liquid I (15 minutes) → dewaxing transparent liquid II (15 minutes) → dewaxing transparent liquid III (15 minutes) → anhydrous alcohol I (3 minutes) → anhydrous alcohol II (3 minutes) → 95% alcohol (3 minutes) → 85% alcohol (3 minutes) → 75% alcohol (3 minutes) → distilled water (3 minutes) → antigen repair (citric acid tissue antigen buffer: pure water = 1:100 high temperature and high pressure cooking for 90 seconds), PBST wash brain slices 3 times, 5% donkey serum blocked incubation at room temperature for 2 hours, prepare primary antibody, incubate primary antibody at 4°C overnight, PBS please wash brain slices 3 times, secondary antibody incubation at room temperature for 2 hours

[0032] Results: The inventors found that CRAMP was expressed in neurons of sham-operated rats. The expression of CRAMP in neurons showed an upward trend at the 1-day and 3-day expression time window after HIE. Interestingly, the expression level on the 3rd day after HIBD was significantly different from that in the sham-operated group. On the 1st day after HIBD, the expression level of CRAMP in neurons showed an upward trend compared with that in normal rats, but there was no significant difference. Figure 1 As shown in A and C, CRAMP was expressed in the cytoplasm of all Ns in the central area of ​​cortical infarction. The statistical results of the mean fluorescence intensity of CRAMP co-expressed with NeuN showed that 1 day after HIE, the mean fluorescence intensity of CRAMP co-stained with NeuN and DAPI in the HI-S group was higher than that in the sham group, but there was no statistical significance (Supplementary Figure 2). Figure 1 B) (F = 0.1513, p = 0.6416, HI-S vs. Sham; p = 0.5111, n = 3-5 / group); The statistical results of the number of NeuN-positive cells showed that the number of NeuN-positive cells in the severe HIE (HI-S) group was significantly reduced compared with the sham operation group (Sham group), which was statistically significant. Figure 1As shown in C, 3 days after HIBD (F = 18.63), in the severe HIE (HI-S) group, the average fluorescence intensity of CRAMP co-stained with NeuN and DAPI was significantly increased compared with that in the sham group (***p = 0.0006<0.001, HI-S vs. Sham). The statistical results of the number of NeuN-positive cells showed that the number of NeuN-positive cells in the severe HIE (HI-S) group was significantly reduced compared with that in the sham group (Supplementary Figure 5). Figure 1 D).

[0033] The above experiments show that the number of neurons in the central area of ​​cortical infarction in severely injured mice decreased 1d and 3d after HIE, and endogenous CRAMP was expressed on surviving neurons, and increased significantly 3d after HIE. The changes in the expression of endogenous host defense peptides in the acute time window after HIE suggest that endogenous host defense peptides may play a certain role in resisting exogenous damage.

[0034] Example 2 Colocalization of endogenous CRAMP expression on microglia in the central area of ​​cortical infarction in HIE mice

[0035] The experimental method is as follows:

[0036] 1) C57BL / 6J pregnant mice were purchased from Kunming Medical University and housed in a sterile animal room with independent ventilation cages. Their offspring were fed next to their mothers until they were 10 days old (Day 10, 3.0-6.0 g) to establish a hypoxic-ischemic brain injury model.

[0037] 2) Establishment of HIE model: Newborn mice were anesthetized by inhalation of sevoflurane (30 μL / mouse), and fixed in supine position on the operating table in a small experimental animal operating box. The left common carotid artery was found in a sterile environment, and the proximal and distal ends were ligated and then cut in the middle. The mice were allowed to rest for 1.5 h after surgery. Then, the mice in the model group were placed in a hypoxia model experimental device, and soda lime and an oxygen meter were placed in the square groove at the bottom of the device. The condition in the device was 8% oxygen-balanced nitrogen, and the mice were exposed to an ambient temperature of 34-34.5°C for 45 min.

[0038] 3) Mice were deeply anesthetized with 10% chloral hydrate (0.3 ml / 100 g), and saline was perfused from the apex of the heart. Brain tissue was obtained after perfusion with 4% paraformaldehyde solution, fixed with 4% paraformaldehyde for 24 hours, dehydrated with gradient alcohol, immersed in wax, and embedded into wax blocks. The specific steps are as follows: tissue washing with running water (2 hours) → 70% alcohol (3 hours) → 80% alcohol (overnight) → 90% alcohol (1 hour) → 95% alcohol (1 hour) → absolute alcohol I (30 minutes) → absolute alcohol II (30 hours) → xylene (25 minutes) → paraffin I (1 hour) → paraffin II (1 hour) → paraffin III (1 hour) → embedded into wax blocks with an embedding machine. Brain tissue was sliced ​​for immunofluorescence staining, and the slice thickness was 7 μm.

[0039] 4) The specific staining steps are as follows: environmentally friendly dewaxing transparent liquid is placed in a constant temperature box and preheated at 45°C, dewaxing transparent liquid I (15 minutes) → dewaxing transparent liquid II (15 minutes) → dewaxing transparent liquid III (15 minutes) → anhydrous alcohol I (3 minutes) → anhydrous alcohol II (3 minutes) → 95% alcohol (3 minutes) → 85% alcohol (3 minutes) → 75% alcohol (3 minutes) → distilled water (3 minutes) → antigen repair (citric acid tissue antigen buffer: pure water = 1:100 high temperature and high pressure cooking for 90 seconds), wash the brain slices 3 times with PBST, block and incubate with 5% donkey serum at room temperature for 2 hours, prepare the primary antibody, incubate the primary antibody at 4°C overnight, wash the brain slices 3 times with PBS, and incubate the secondary antibody at room temperature for 2 hours.

[0040] As attached Figure 2 A, In the central area of ​​cortical infarction, 1 day after HIBD, the statistical results of the percentage of Iba1, CRAMP, and DAPI co-localized cells in all Iba1-positive cells showed an upward trend in the number of Iba1 cells co-stained with CRAMP compared with the Sham group, with no statistical significance (F = 2.317, p = 0.0581, HI-S vs. Sham; p = 0.7084, n = 3-5 / group) (Appendix Figure 2 B). The statistical results of the average fluorescence intensity of CRAMP co-stained with Iba1 and DAPI showed that the HI-S group showed a significant increase compared with the Sham group, which was statistically significant (F=3.638, *p = 0.0456<0.05, HI-Svs.Sham; p = 0.1758, n = 3-5 / group) (Appendix Figure 2 B); The statistical results of the number of Iba1-positive cells showed that the number of Iba1-positive cells in the HI-S group increased significantly compared with the Sham group, which was statistically significant (F = 5.070, ***p = 0.0006 < 0.001, HI-S vs. Sham; p = 0.1695, n = 3-5 / group) (Appendix Figure 2B). As attached Figure 2 C, 3 days after HIBD, in the statistical results of the percentage of Iba1, CRAMP, and DAPI co-localized cells in the total number of Iba1-positive cells in the region, the number of Iba1 cells co-stained with CRAMP in the HI-S group increased compared with the Sham group, but there was no statistical significance (F = 0.6805, p = 0.4074, HI-S vs. Sham; p = 0.4942, n = 3-5 / group) (Appendix Figure 2 D). In the statistical results of the average fluorescence intensity of CRAMP co-stained with Iba1 and DAPI, the HI-S group was significantly higher than the Sham group (F = 37.07, **p = 0.0035<0.01, HI-S vs. Sham; p = 0.78717, n = 3-5 / group) (Appendix Figure 2 D). The statistical results of the number of Iba1-positive cells showed that the number of Iba1-positive cells in the HI-S group increased significantly compared with the Sham group, which was statistically significant (F = 5.070, ***p = 0.0002<0.001, HI-S vs. Sham; **p = 0.0061<0.01, n=3-5 / group) (Appendix Figure 2 D). In addition, CRAMP was found to be translocated to the nucleus of microglia.

[0041] The above results indicate that endogenous CRAMP expression was co-localized on microglia in the central area of ​​cortical infarction in severely injured mice 1d and 3d after HIE, and the expression was significantly increased 3d after HIE.

[0042] In summary, the experiments demonstrated the expression profile of host defense peptides in neurons and microglia in the brain of neonatal mice, and confirmed the changes in the expression of endogenous host defense peptides in the acute time window after HIE, suggesting that they may play a certain role in resisting exogenous damage.

[0043] Example 3 Effects of antimicrobial peptide MU1 on brain damage after HIE in mice

[0044] When it was found that neurons and microglia in the brains of suckling mice expressed CRAMP and that the expression increased in the acute time window after HIE, the inventors believed that host defense peptides played an important role in the brain injury process after HIE, but the specific function was still unclear. This increase in host defense peptides after injury may also be the body's role in resisting exogenous damage. After screening, the inventors first gave HIE mice exogenous host defense peptide MU1 from a holistic perspective, trying to use exogenous host defense peptide supplementation to verify the function of host defense peptides after HIE.

[0045] The specific method is as follows:

[0046] 1) C57BL / 6J pregnant mice were purchased from Kunming Medical University and housed in a sterile animal room with independent ventilation cages. Their offspring were fed next to their mothers until they were 10 days old (Day 10, 3.0-6.0 g) to establish a hypoxic-ischemic brain injury model.

[0047] 2) Establishment of HIE model: Newborn mice were anesthetized by inhalation of sevoflurane (30 μL / mouse), and fixed in supine position on the operating table in a small experimental animal operating box. The left common carotid artery was found in a sterile environment, and the proximal and distal ends were ligated and then cut in the middle. The mice were allowed to rest for 1.5 h after surgery. Then, the mice in the model group were placed in a hypoxia model experimental device, and soda lime and an oxygen meter were placed in the square groove at the bottom of the device. The condition in the device was 8% oxygen-balanced nitrogen, and the mice were exposed to an ambient temperature of 34-34.5°C for 45 min.

[0048] 3) The model group mice were divided into HIE+NS group and HIE+MU1 group. The antimicrobial peptide MU1 was dissolved in normal saline (NS) to a concentration of 2 mg / mL. The HIE+MU1 group was intraperitoneally injected with host defense peptide MU1 (10 mg / kg) 2 h (Day 0), 1 day (Day 1), and 2 days (Day 2) after HIE; the mice in the HIE+NS group were given an equal volume of normal saline NS. The brain damage of the mice was observed and recorded on the third day (Day 3) after HIE.

[0049] 4) After HIE mice were randomly given NS or MU1, the degree of brain tissue damage was counted after scalp incision and anesthesia perfusion 3 days after HIE. 64 mice were given NS (43 in HI-M group and 21 in HI-S group), and 29 mice were given MU1 (26 in HI-M group and 3 in HI-S group). Figure 3 As shown in A. HI-S indicates severe injury, which means that there are macroscopically visible injury lesions (liquefaction) in the mouse brain, and microscopic pathology shows severe neuronal damage and microglial activation; HI-M indicates mild injury, which means that there are no macroscopically visible injury lesions in the mouse brain, and microscopic pathology shows mild neuronal damage and microglial activation.

[0050] 5) Mice 3 days after HIE were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.3 mL / 100 g), fixed in the supine position, the chest cavity was cut open to expose the heart, and the brain tissue was removed after perfusing 4% paraformaldehyde solution through the heart, postfixed in 4% paraformaldehyde solution for 24 h, and photographed. Figure 3 As shown in B.

[0051] The results showed that after intraperitoneal injection of MU1 into HIE mice, the area of ​​severe liquefaction lesions in the brain tissue of the mice was reduced (see attached Figure 3 A), and the severe injury rate of mice injected with MU1 and mice not injected with MU1 was statistically analyzed after modeling. It was found that the incidence of severe injury in mice injected with MU1 was reduced (such as the attached Figure 3 B) The gross observation showed that the scope of brain tissue liquefaction injury in the severe injury (HI-S) group given MU1 was reduced to a certain extent compared with the solvent control group, indicating that MU1 has a brain protective effect.

[0052] Example 4 Effect of antimicrobial peptide MU1 on apoptosis of primary neurons

[0053] In vivo experiments have shown that exogenous supplementation of the host defense peptide MU1 can significantly improve the degree of HIE brain damage, but the mechanism is unclear. Neuronal loss is significant after HIE. This implementation case explores the role of MU1 in directly protecting neurons. The oxygen-glucose deprivation model (Oxygen-Glucose Deprivation, OGD), also known as the in vitro ischemia model, is a model that simulates ischemia / hypoxia stimulation on cells. Cells will produce necrosis, apoptosis, autophagy and other phenomena. In particular, the central nervous system is very sensitive to ischemia and hypoxia. The in vitro primary neuron oxygen-glucose deprivation model can simulate the pathophysiological progression of brain ischemia in vivo and study its damage mechanism. The experimental steps of this example are as follows:

[0054] 1) Primary neuron culture: Place a 14mm cell slide on a 24-well plate, add 500μL of diluted poly-lysine (100μg / mL) to each well, and incubate at 37°C overnight. Wash with sterile water 2-3 times the next day, dry at 37°C, and set aside. Use pregnant mice at embryonic stage 16.5-18.5 days, kill by cervical dislocation, soak in alcohol for a few seconds, take out the fetus on ice, put it in dissection fluid, quickly separate the fetal brain tissue, and transfer it to new dissection fluid. Under a stereoscopic microscope, peel off the meninges, separate the hippocampus and cortex, and transfer them to an EP tube filled with dissection fluid. Use sterile scissors to cut the cortex into pieces (to 1mm 3 size), add papain, digest at 37℃ for 10 minutes. Add DNase, digest at 37℃ for 8 minutes. Use a Pasteur pipette to blow 10 times and continue digestion for 5 minutes. Add appropriate amount of seed plate solution (DMEM-high glucose medium + 10% FBS + 1% double antibody), blow 10 times with a Pasteur pipette, let it stand for a few minutes, count, and calculate 2*10 4 Cells / well were seeded into 24-well plates for culture. After 4-6 hours, the medium was changed to neuronal culture medium (Neurobasal plus Medium + 2% B27plus supplement + 1% glutamine + 1% double antibody). The medium was changed every 2-3 days.

[0055] 2) Glucose-oxygen deprivation of primary neurons: The cells were randomly divided into a control group, an oxygen-glucose deprivation group (OGD), and an oxygen-glucose deprivation plus MU1 group (OGD+MU1-100μg / mL). Host defense peptide MU1 was added to the neuronal culture medium 12 h in advance to pretreat the cells. After 12 h, the culture medium of the OGD and OGD+MU1 groups was replaced with a sugar-free culture medium and placed in an oxygen-deficient chamber filled with 5% CO2+95% N2 gas. The oxygen-deficient chamber was placed in an incubator for 3 h of hypoxia, and the control group was not treated in any way. After the end of hypoxia, the cells in the control group, OGD, and OGD+MU1 groups were subjected to TUNEL staining to detect the apoptosis rate of the three groups of cells after oxygen-glucose deprivation.

[0056] Results from Figure 4 As shown in the figure, the antimicrobial peptide MU1 reduced the death rate of neurons after glucose-oxygen deprivation. After glucose-oxygen deprivation, the neuronal apoptosis rate in the OGD group reached 22.24%, while the neuronal apoptosis rate in the host defense peptide MU1 treatment group was 10.14%. This shows that the antimicrobial peptide MU1 can play a protective role in primary neurons in the in vitro neuronal hypoxia model.

[0057] After glucose and oxygen deprivation of neurons, neuronal cell viability decreases, leading to functional disorders. Neuronal cells treated with MU1 showed better cell viability than the control group in the glucose and oxygen deprivation experiment, and there was a certain dose dependence, indicating that MU1 can protect neurons and significantly reduce the apoptosis rate of primary neurons after glucose and oxygen deprivation.

[0058] Example 5 Effect of MU1 on the phagocytic function of human microglial cells HMC3 after OGD

[0059] In vivo experiments have shown that exogenous supplementation of the host defense peptide MU1 can significantly improve the degree of brain damage in HIE, and can also reduce the apoptosis of neurons after OGD in vitro, indicating that MU1 has the brain protection function of HIE, but the mechanism is unclear. Therefore, this implementation case further explores the target and mechanism of MU1. Microglia are the main immune function cells in the brain and the main cells that cause neuroinflammation after HIE. They have multiple functions such as phagocytosis, migration, pro-inflammatory, and anti-inflammatory. Among them, the phagocytosis of microglia is one of the important functions of microglia-mediated brain damage and brain protection mechanisms during HIE. Moderate phagocytosis of microglia is of great significance for maintaining the balance of the microenvironment under pathological conditions. In this example, MU1 was given to human microglia HMC3 to detect changes in its phagocytic function after OGD.

[0060] The specific operations are as follows:

[0061] 1) HMC3 cell culture and MU1 treatment: HMC3 cells were cultured at 4 x 10 5The cells were seeded at a density of 1:1 in a six-well plate, and MU1 was added 12 hours before the experiment to a final concentration of 100 μg / mL.

[0062] 2) Establishment of cell OGD model: The culture medium of cells in the OGD group and the MU1 group was discarded, PBS was washed once, and the sugar-free and serum-free culture medium was replaced. The cells were placed in a hypoxic chamber filled with a 5% CO2+95% N2 mixed gas and a 5% oxygen concentration and placed in an incubator for 2 hours of hypoxia.

[0063] 3) Phagocytic function: Add FITC-dextran reagent (final concentration 1 mg / mL) to each well of a six-well plate, mix the liquid in the well, and incubate at room temperature in the dark for 30 min.

[0064] 4) Prepare single-cell suspension: discard the culture medium, wash 1-2 times with PBS, digest the cells with 0.25% trypsin for 30 seconds, collect the cell fluid after digestion, centrifuge at 300g for 3 minutes, discard the supernatant, resuspend in PBS and place on ice for testing.

[0065] 5) Prepare single-stained tubes: add one drop of positive and negative magnetic beads to each tube, add 80uL PBS to each tube, add antibody, mix well, incubate at 4℃ for 30min, centrifuge at 1000g for 10min, discard the supernatant, resuspend in PBS and place on ice for testing.

[0066] 6) Resuspend the cells in the blank tube with PBS solution and place on ice for testing.

[0067] The results showed that in the resting state, the phagocytic function of microglia was low, with about 22.1% of the cells performing phagocytic function (see Appendix Figure 5 A), while the number of phagocytic microglia increased significantly after OGD, and the number of cells with phagocytic function reached 88.6% (Appendix Figure 5 B), after MU1 cells were given OGD, the number of cells that performed phagocytic function accounted for 85.7% (Appendix Figure 5 C), the statistical results showed that the number of phagocytic cells in the ischemia-hypoxia group (OGD group) was significantly higher than that in the control group (**p = 0.0058) (Appendix Figure 5 D), the number of phagocytic cells after MU1 administration was significantly higher than that in the control group (**p = 0.0076), but there was no difference with the ischemia-hypoxia group (OGD group) (Appendix Figure 5 D), indicating that the phagocytic ability of microglia after OGD is greatly improved, and MU1 does not affect the phagocytic function of microglia after OGD. In the brain, the phagocytic function of microglia can remove necrotic neurons and debris within a certain range and participate in synaptic pruning, which is one of the key mechanisms of nervous system development and injury repair. Therefore, the present invention believes that MU1 does not change the phagocytic function of microglia after OGD, and can maintain the balance of the brain environment to a certain extent.

[0068] Example 6 MU1 plays a brain protective role by restoring homeostasis by upregulating TGF-β1 responsive expression in MGs after OGD

[0069] Microglia are highly heterogeneous cells that can polarize under pathological conditions, become overactivated, and play a damaging role. TGFβ1 is a very important neuroprotective factor expressed by microglia. Recent studies have shown that it has the function of maintaining microglial homeostasis. CreER Tgfb1 fl / fl iKO treated at P3-P5) produces a similar activated microglial phenotype, with reduced branching morphology and significant loss of homeostatic microglial markers throughout the brain. This suggests that microglia-derived TGFβ1 ligands are also required to maintain homeostasis in the early postnatal mouse brain, and microglia in MGS-Tgfb1 iKO mice are activated (Bedolla, Alicia et al. “Adult microglial TGFβ1 is required for microgliahomeostasis via an autocrine mechanism to maintain cognitive function inmice.” Nature communications vol. 15,1 5306. 21 Jun. 2024, doi:10.1038 / s41467-024-49596-0), indicating that TGFβ1 plays an important role in maintaining microglial homeostasis. The inventors first confirmed that the HMC3 cell line expressed TGFβ1 in vitro, and further studied the changes of this molecule after OGD and administration of MU1.

[0070] The specific operations are as follows:

[0071] 1) Cell culture: Complete culture medium: MEM + 10% FBS + 1% penicillin / streptomycin was used to culture HMC3 cells at 37°C and 5% carbon dioxide. MU1 (100 μg / mL) was added to the experimental group and cultured for 12 h before the experiment.

[0072] 2) Immunofluorescence morphological staining: cells were seeded in a 24-well plate with a slide, the culture medium was discarded, the cells were washed 2-3 times with PBS, incubated with 4% paraformaldehyde for 20 min, washed 2-3 times with PBS, incubated with 5% donkey serum for 40 min, incubated with primary antibody (Iba1 antibody) at 4°C overnight, washed 3 times with PBS, incubated with secondary antibody (donkey anti rabbit AF488) at room temperature for 2 h, washed 3 times with PBS, and sealed with anti-fluorescence quenching sealing agent containing DAPI and observed under a microscope.

[0073] 3) Protein sample preparation: Discard the culture medium of HMC3 cells, wash 1-2 times with PBS, add high-efficiency tissue / cell lysis buffer, lyse the cells on ice for 30 min, scrape the cells with a cell scraper, collect the lysate, centrifuge at 12000g for 15 min at 4°C, transfer the supernatant to a new tube, determine the protein concentration, add 5X protein loading buffer to prepare the sample, and heat in a 100°C metal bath for 10 min.

[0074] 4) Protein gel electrophoresis: Use a pipette to add samples and markers to the wells of a 4-20% SDS-Page gel in sequence, add running buffer to the electrophoresis tank, run the gel at 80V for 30 min, then at 120V for 60 min, until the bromophenol blue front indicator reaches the bottom of the electrophoresis tank.

[0075] 5) Transfer: Soak the PVDF membrane in methanol for 5 minutes, then in the transfer solution for 1 minute. Assemble the transfer plate in the order of sponge, filter paper, gel, PVDF membrane, filter paper, sponge, and transfer at 250 mA in the transfer solution for 60 minutes.

[0076] 6) Wash the membrane 3 times with TBST, 10 min each time, add the prepared primary antibody solution, incubate at 4°C overnight, wash the membrane 3 times with TBST, 10 min each time, incubate with secondary antibody at room temperature for 1 hour, wash the membrane 3 times with TBST, 10 min each time, and develop

[0077] The results showed that most of the microglia in the control group showed protrusions, and the protrusions of microglia were slender ( Figure 6 A yellow arrow); after OGD, it can be seen that the processes of microglia become shorter and the cell bodies become rounder, and the number of round microglia increases ( Figure 6 B); After pretreatment with MU1, it can be seen that the number of round microglia has decreased significantly, and the processes of microglia have elongated ( Figure 6 C). The experimental results confirmed that the HMC3 cell line in vitro expressed TGFβ1, which has the function of maintaining homeostasis. The acute microglial hypoxia model was used in vitro, and it was found that microglial cells were significantly activated and the cell bodies became round ( Figure 6B), at this time, the cells need to take adequate defensive responses to compensate for the serious imbalance of microglial homeostasis caused by hypoxia. The experimental results of the present invention confirmed that the expression of TGFβ1 increased after OGD (**p = 0.0014) ( Figure 6 D, E), which is a manifestation of severe dysfunction of microglia. The high expression of TGFβ1 helps microglia to restore homeostasis. After MU1 administration, the number of protruding microglia representing steady-state microglia increased compared with the simple ischemia-hypoxia group (OGD group). Figure 6 C), at this time, the change of TGFβ1 expression was not as drastic as that in the ischemia-hypoxia group (OGD group) (lower than that in the OGD group, but still higher than that in the control group) (*p = 0.0133) ( Figure 6 D, E). This indicates that MU1 can alleviate the excessive activation of microglia caused by OGD, thereby maintaining their homeostasis and playing a role in repairing the brain microenvironment after OGD.

[0078] Endogenous host defense peptides are co-expressed on neurons and microglia, and their expression increases in the acute time window after HIE injury, proving that host defense peptides may be important players in regulating the pathophysiological progression of HIE. Severely injured HIE model mice will cause liquefaction necrosis of brain tissue and affect neurological function. In vivo experiments have shown that after administering MU1 peptide to HIE model mice, the range of liquefaction in severely injured mice is reduced, and the probability of severe injury after HIE is reduced, indicating that the antimicrobial peptide MU1 reduces the incidence of liquefaction injury and the area of ​​lesion liquefaction in neonatal hypoxic-ischemic encephalopathy, confirming the protective effect of exogenous antimicrobial peptide MU1 on the brain after HIE. Further in vitro experiments proved that the administration of antimicrobial peptide MU1 to primary neurons could slow down the cell apoptosis caused by OGD treatment. The phagocytic function of microglia HMC3 was not changed after administration of MU1, but the increased expression of reactive TGFβ1 caused by OGD was restored, and the morphology of microglia also changed to steady state, indicating that MU1 can protect neurons during hypoxia-ischemia, reduce neuronal apoptosis after hypoxia-ischemia, maintain the homeostasis of microglia, and phagocytic function, which is beneficial to the repair of brain microenvironment after hypoxia-ischemia. This shows that MU1 exerts its brain protective effect by reducing neuronal apoptosis and restoring microglial homeostasis.

Claims

1. Use of antimicrobial peptide MU1 in the preparation of a drug for hypoxic-ischemic encephalopathy, characterized in that: The amino acid sequence of the antimicrobial peptide MU1 is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that: The hypoxic-ischemic encephalopathy includes hypoxic-ischemic brain damage.

3. The use according to claim 1, wherein the hypoxic-ischemic encephalopathy includes neonatal hypoxic-ischemic encephalopathy.

4. The use according to any one of claims 1 to 3, characterized in that The dosage form of the drug includes injection and capsule.

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