Application of angiotensin converting enzyme 2 as drug target for treating traumatic brain injury

Abstract

The invention discloses an application of angiotensin converting enzyme 2 (ACE2) as a drug target for treating traumatic brain injury, and researches find that ACE2 protein abundance is reduced around a mouse injury area, and a TBI mouse has cognitive impairment which can be relieved by activating ACE2. Transcriptomics finds that the Casp-1 and Gsdmd genes are up-regulated by the knockout of the ACE2, a pyroptosis pathway of the cells is activated, and the process can be reversed by AVE0991. ACE2 seems to regulate pyroptosis to influence the progress of TBI by regulating the balance of PTGIS and TXA2. These findings provide creative evidence for the complex effect of ACE2 in post-traumatic neurological function recovery and regulation. The discovery provides a promising new target for promoting traumatic brain injury treatment intervention.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and particularly relates to the application of angiotensin converting enzyme 2 as a drug target for treating traumatic brain injury. Background Art

[0002] Traumatic brain injury (TBI) is a major public health problem that increases the risk of chronic traumatic encephalopathy, dementia, and various neurodegenerative diseases. The severity of TBI can vary greatly due to the progression of secondary molecular injury responses such as oxidative stress, apoptosis, and neuroinflammation. Therefore, timely therapeutic interventions during the acute phase of injury are critical to reduce tissue damage and improve outcomes.

[0003] Angiotensin-converting enzyme 2 (ACE2) is an enzyme involved in the conversion of angiotensin II to angiotensin (1-7) and has been shown to exhibit neuroprotective effects. However, little research has been conducted on the role of ACE2 in the progression of traumatic brain injury.

[0004] The renin-angiotensin system (RAS) is well known for its role in regulating blood pressure and fluid balance. However, recent studies have shown that it is involved in active gene expression and signaling pathways within the central nervous system. Clinical studies have shown that the renin-angiotensin system is significantly upregulated in patients with TBI. Angiotensin (Ang) II is considered to be the main bioactive molecule of the RAS. After brain trauma, increased levels of Ang II can enter the brain through a weakened blood-brain barrier and activate microglia to cause inflammation. Although related to ACE, ACE2 plays a different role in regulating the renin-angiotensin system and has specific functions and substrates. ACE2 mediates the enzymatic conversion of Ang II to Ang (1-7) and interacts with the G protein-coupled receptor MasR, producing biological effects opposite to those of Ang II. Multiple studies have shown that the ACE2 / Ang II / MasR pathway can effectively attenuate the overactivation of the neuroimmune system, thereby reducing neurological damage and brain inflammation caused by intracerebral hemorrhage and ischemia-reperfusion. Although the role of ACE2 in neuroinflammation is well established, comprehensive studies on its expression and regulation remain scarce.

[0005] Arachidonic acid (ARA) is an important lipid component in animal cells and a key precursor for the synthesis of bioactive lipid mediators, including prostaglandins, leukotrienes, and epoxyeicosatrienoic acid. In the field of neurological diseases, ARA and its metabolites are closely related to neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Thromboxane A2 (TXA2) is a prostaglandin derived from arachidonic acid through the enzymatic activity of cyclooxygenase-1 (COX-1), COX-2, or TXA2 synthase (TXAS). Research reports indicate that inhibition of the TXAS / TXA2 / TP signaling pathway can protect the kidney from ischemia / reperfusion (I / R) injury by exerting anti-inflammatory and anti-pyroptotic effects. There are currently no literature reports on the relationship between TXA2 and cell pyroptosis in TBI, and no public literature has been retrieved for its neuroprotective effect after brain trauma. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present invention provides the use of angiotensin converting enzyme 2 as a drug target for treating traumatic brain injury.

[0007] The present invention aims to explore the potential neuroprotective effect of ACE2 after brain trauma and its effect on the progression of TBI by regulating arachidonic acid metabolism-related metabolites and metabolic enzymes to regulate the cell pyroptosis pathway.

[0008] The present invention provides the following technical solutions:

[0009] In one aspect, the present invention provides the use of angiotensin converting enzyme 2 as a drug target for treating traumatic brain injury.

[0010] In another aspect, the present invention provides the use of angiotensin (1-7) receptor agonist or thromboxane A2 (TXA2) inhibitor in the preparation of a drug for treating traumatic brain injury.

[0011] Preferably, the angiotensin (1-7) receptor agonist is used to optimize motor coordination and balance ability caused by traumatic brain injury.

[0012] Preferably, the angiotensin (1-7) receptor agonist is used to upregulate the expression levels of angiotensin converting enzyme 2 and MasR to improve cell pyroptosis.

[0013] Preferably, the thromboxane A2 inhibitor is used to alleviate cognitive dysfunction caused by traumatic brain injury.

[0014] Preferably, thromboxane A2 inhibitors alleviate movement disorders and balance deficits caused by traumatic brain injury, and reduce inflammation and pyroptosis in the damaged brain.

[0015] A therapeutic drug for traumatic brain injury, the active ingredient of which is an angiotensin (1-7) receptor agonist or a TXA2 inhibitor.

[0016] Furthermore, the angiotensin (1-7) receptor agonist is Telmisartan.

[0017] Furthermore, the thromboxane A2 (TXA2) inhibitor is Ozagrel and a pharmaceutically acceptable salt thereof, and the acid used for salt formation is selected from hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and citric acid.

[0018] The therapeutic agent also includes one or more pharmaceutically acceptable carriers or excipients.

[0019] The administration route can be oral, parenteral or topical administration, preferably oral and injection. Oral pharmaceutical preparations include capsules and tablets.

[0020] Traumatic brain injury (TBI) is a complex pathological condition with unclear underlying mechanisms; this study found that downregulation of ACE2 aggravated neurological dysfunction in TBI mice, manifested as significant behavioral impairment and memory deficits. The use of angiotensin (1-7) receptor agonist AVE0991 can effectively prevent brain damage and behavioral deficits caused by TBI. After TBI, microglia, as the core of neuroimmune response, are significantly activated when ACE2 is significantly reduced. The present invention observed that TBI-induced microglial activation is associated with downregulation of ACE2 expression, which further confirms the results of previous studies. Studies have shown that the release of pyroptotic factors such as Caspase-1 and GSDMD plays a key role in the pathological progression of TBI. Studies have shown that the ACE2 / Ang(1-7) / Mas axis can negatively regulate pyroptosis, thereby protecting mice from LPS-induced acute lung injury. In SARS-CoV-2 studies, ACE2 is considered to be a key factor in inhibiting the release of pyroptotic bodies and the formation of cell membrane pores. This study further confirmed this viewpoint, as ACE2 deficiency aggravated cell pyroptosis in TBI mice, while treatment with angiotensin (1-7) receptor agonist AVE0991 could alleviate this phenomenon. In addition, supplementation of Ang (1-7) to microglia also alleviated LPS-induced pyroptosis.

[0021] The ACE2 / Ang(1-7) / MasR axis can effectively inhibit neuroimmune activation and reduce neurological damage and inflammation caused by cerebral hemorrhage and ischemia-reperfusion injury. This study showed that ACE2 is naturally expressed in mouse brain tissue, and its level decreases significantly near the injured area. This suggests that targeting the ACE2 pathway may improve the treatment prognosis of head injury. After TBI, reduced ACE2 expression may lead to the accumulation of AngII, a key inflammatory mediator. AngII further activates AT1R, forming a feedback loop that enhances the effect of AngII. The reduction in ACE2 function may directly lead to neurological deficits in TBI mice and is closely related to behavioral and memory disorders.

[0022] Arachidonic acid (ARA) and its metabolites, as bioactive lipids, play an important role in inflammation. The prostaglandin family derived from ARA regulates the body's homeostasis and inflammatory response. Studies have shown that ARA levels in rat serum increased 24 hours after TBI, suggesting that it may be a biomarker of acute TBI metabolism. This study found that ACE2 deficiency exacerbated arachidonic acid metabolic disorders after TBI and changed metabolite levels and enzyme activities. Non-targeted metabolomics analysis showed that TXA2 metabolite TXB2 was significantly increased, while PTGIS, the key enzyme responsible for metabolizing PGI2, was significantly decreased in proteomics. Multi-omics studies consistently point to the arachidonic acid metabolic pathway. Studies have suggested that PGI2 analogs may alleviate stroke-induced neurological damage, while impaired PGI2 signaling can aggravate hippocampal cell damage in TBI. Ang(1-7) can promote the release of PGI2, suggesting that ACE2 deficiency hinders PTGIS function, thereby reducing PGI2 synthesis and leading to TXA2 accumulation. The lack of TXA2 synthase and receptor can reduce ischemia-reperfusion-induced cell pyroptosis. This finding further supports the hypothesis of the present invention that arachidonic acid metabolism disorders and the imbalance of TXA2 and PGI2 play a key role in ACE2 deficiency-induced cell pyroptosis. In addition, the metabolomics data for arachidonic acid vividly demonstrate the severity of metabolic disorders.

[0023] PGI2 is produced by cyclooxygenase and PTGIS and has cardiovascular protective and anti-inflammatory properties. ACE2 deficiency reduces PTGIS function, resulting in reduced PGI2 synthesis. Overexpression of PTGIS in BV2 cells or use of the TXA2 inhibitor Ozagrel in animals can prevent TXA2 accumulation and reduce cellular inflammation caused by ACE2 deficiency. In contrast, the TXA2 agonist U46619 disrupts the PGI2 / TXA2 balance and aggravates LPS-induced microglial inflammation. PTGIS helps stabilize arachidonic acid metabolism, but cell experiments may not fully reflect the complex physiological conditions in vivo, resulting in differences in in vivo and in vitro research results.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The present invention provides an application of angiotensin-converting enzyme 2 as a drug target for treating traumatic brain injury. The present invention has found that the abundance of ACE2 protein is downregulated around the injured area of ​​mice, and TBI mice have cognitive impairment that can be alleviated by activating ACE2.

[0026] (2) The present invention found through transcriptomics that the knockout of ACE2 upregulated the Casp-1 and Gsdmd genes and activated the cell pyroptosis pathway, and this process could be reversed by Telmisartan (AVE0991). It is worth noting that the loss of ACE2 disrupted the thromboxane A2 (TXA2)-mediated arachidonic acid metabolism process and hindered the expression of prostaglandin I2 synthase (PTGIS). Overexpression of PTGIS can effectively alleviate cell pyroptosis and arachidonic acid metabolism disorders. The results of U46619 and Ozgrel treatment showed that regulating TXA2 metabolism can affect the pyroptosis process. ACE2 seems to regulate cell pyroptosis by regulating the balance of PTGIS and TXA2, affecting the progression of TBI. These findings provide groundbreaking evidence for the complex role of ACE2 in regulating post-traumatic neurological recovery. This discovery provides a promising new target for advancing therapeutic interventions for traumatic brain injury.

[0027] (3) This study illustrates the neuroprotective effect of ACE2 in TBI, which mainly affects the balance of arachidonic acid metabolism by regulating the key metabolic enzyme PTGIS, thereby regulating the inflammation and pyroptosis process. This regulatory mechanism is closely related to the behavioral disorders and neural damage caused by TBI. In-depth research on ACE2-related signaling pathways and pyroptosis mechanisms in the future is expected to provide new targets and intervention strategies for the treatment of TBI. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0029] Figure 1.Renin-angiotensin system (RAS) damage caused by traumatic brain injury (TBI) affects the motor ability and cognitive function of mice. Among them, A is the change in the expression level of ACE2 24 hours after brain injury. B is the cellular localization of ACE2 protein in the brain determined by immunofluorescence staining. C is the fluorescence intensity analysis showing that the expression of ACE2 in microglia after brain injury is significantly different. DG is the verification of the dysregulation of the RAS system after TBI by immunoblotting and ELISA. HJ is the co-expression analysis of ACE2 and IBA1 and the effect of TBI on ACE2 expression in microglia. K is a schematic diagram of the behavioral experiment of mice after brain injury. L is a representative trajectory diagram showing the path of mice in spatial exploration and directional navigation tests. M is that AVE0991 treatment reduces the time to find the underwater platform. N is the shortened distance covered in the exploration of the platform quadrant. OR is that AVE0991 improves the motor coordination and balance ability of brain-injured mice, which is manifested in the shortened completion time and improved motor scores in the horizontal ladder and balance beam tests. *p<0.05, **p<0.01, ****p<0.0001 indicate the comparison between Con-TBI group and control group, #p<0.05, ##p<0.01, ###p<0.001 indicate the comparison between Con-TBI group and Con-TBI-AVE group.

[0030] Figure 2 .The effect of ACE2 deficiency on cell pyroptosis in TBI and the intervention effects of ACE2 agonists AVE0991 and Ang(1-7). A is a heat map of differential gene expression levels. B is Reactome pathway enrichment analysis highlighting the pyroptosis pathway. CE is Western blot verification of the activation of pyroptosis markers Gsdmd and Caspase-1, indicating that ACE2 deficiency exacerbated cell pyroptosis. FH is Sholl analysis to evaluate the activation of microglia after brain injury. I is the colocalization of microglial marker IBA1 and pyroptosis factors. J is Western blot verification that AVE0991 alleviates cell pyroptosis and inflammation caused by ACE2 deficiency. K is AVE0991 reducing the fluorescence intensity of pyroptosis factors. L is the use of qRT-PCR to verify pyroptosis and inflammation-related genes. M is transmission electron microscopy showing that Ang(1-7) significantly alleviates LPS-induced cell pyroptosis. N is Western blot and O is corresponding analysis to further confirm this conclusion. Data are expressed as the mean ± SEM of three independent experiments. *p<0.05, **p<0.01 indicates the comparison between Con-TBI group and ACE2-TBI group, ##p<0.01 indicates the comparison between LPS group and LPS+Ang(1-7) group.

[0031] Figure 3.Comprehensive analysis of metabolic and proteomic changes caused by ACE2 deficiency in TBI. A is a dot plot of the permutation test results of the untargeted metabolomics OPLS-DA model. B is a scatter plot of the PCA model scores, red represents the ACE2-TBI group, and blue represents the Con-TBI group. C is a volcano plot of differential metabolite screening, red represents up-regulated metabolites, and blue represents down-regulated metabolites. D is a differential abundance score plot, comparing the Con-TBI group with the ACE2-TBI group. A score of 1 indicates an increase in the expression of the differential metabolite pathway, and the line length represents the absolute value of the DAScore. E is a Z-score analysis of 20 significantly differential metabolites based on VIP scores. F is a bubble plot of metabolite pathway enrichment.

[0032] Figure 4 .Effect of ACE2 deficiency on PTGIS expression and arachidonic acid metabolism in the process of TBI. A is a heat map of differential protein levels, with colors indicating expression levels, from red (high expression) to blue (low expression). B is a KEGG enrichment chord diagram showing the enrichment of proteins in the KEGG pathway. C is immunofluorescence co-expression of PTGIS and pyroptosis markers in microglia. D is immunofluorescence colocalization of ACE2 and PTGIS in microglia. EF is ELISA verification of the dysregulated levels of two arachidonic acid metabolites, TXB2 and 6-keto-PGF1a. G is a metabolome clustering heat map for arachidonic acid and H is a quantitative result analysis. Red arrows indicate increased metabolic levels, and green arrows indicate decreased metabolic levels. Data are expressed as the mean ± SEM of three independent experiments. **p<0.01, ***p<0.001 for comparison between Con-TBI group and control group, ##p<0.01, ###p<0.001 for comparison between ACE2-TBI group and Con-TBI group.

[0033] Figure 5.The effect of PTGIS supplementation on cell pyroptosis and arachidonic acid metabolism. A is a schematic diagram of the construction of the PTGIS lentiviral overexpression vector. B is a Western blot to verify the effect of PTGIS overexpression on LPS-induced cell pyroptosis. C is a scanning electron microscopy observation of the effect of PTGIS on cell pyroptosis morphology. D is the immunofluorescence staining detection of GSDMD and Caspase-1 in PTGIS-transfected microglia. E is the verification of pyroptosis markers and inflammatory factor gene expression. F is the metabolomics verification of arachidonic acid-targeted metabolites at the cellular level. G is the use of nucleic acid dyes to assess membrane damage in cell morphology. Data are expressed as the mean ± SEM of three independent experiments. *p<0.05, **p<0.01, ****p<0.0001 indicate comparison with the LPS group; #p<0.05, ##p<0.01, ###p<0.001 indicate comparison between the LPS group and the LPS+PTGIS group.

[0034] Figure 6 .TXA2 exacerbates the process of cell pyroptosis. A is the effect of thromboxane A2 agonist U46619 on cell pyroptosis detected by nucleic acid dye. B is a representative image showing the effect of U46619 on the immunofluorescence expression of pyroptosis markers. C is a Western blot to verify the effect of U46619 on the expression level of pyroptosis-related proteins. DE is the effect of U46619 on the levels of TXB2 and 6-keto-PGF1a. F is that U46619 enhances the fluorescence intensity of pyroptosis and inflammatory factors after TBI. Data are expressed as the mean ± SEM of three independent experiments. ****p<0.0001 indicates comparison with the U46619 group; *p<0.05, **p<0.01.

[0035] Figure 7 .Ozagrel alleviates behavioral deficits after TBI. A is a schematic diagram of the mouse behavioral experiment. B is a representative trajectory diagram of mice to study the effect of Ozagrel on learning and cognition after TBI. C is that Ozagrel reduces the moving distance of the platform quadrant. D is that Ozagrel has little effect on the time of finding the underwater platform. EH is that AVE0991 improves the motor coordination and balance ability of brain-injured mice, which is manifested by shortened completion time and increased motor scores in horizontal ladder and balance beam tests. I and J are that Ozagrel increases the correct walking behavior and score of mice within 60 seconds. Data are expressed as the mean ± SEM of three independent experiments. **p<0.01, ***p<0.001 represent the comparison between the Con-TBI group and the control group; #p<0.05, ##p<0.01 represent the comparison between the TBI+AO group and the Con-TBI group.

[0036] Figure 8 . Schematic diagram summarizing the findings of the research on the mechanism of the present invention.

[0037] Fig. 9 .The effect of TBI on ACE2 expression and its improvement on the behavioral performance of TBI mice. Among them, A and D show that the ACE2 expression level reaches the lowest value within 24 hours after TBI. B is a schematic diagram of the construction of ACE2 knockout mice using CRISPR / Cas9 technology. C, E and F are immunoblotting and PCR verification of the successful knockout of the ACE2 gene in mice. G is a schematic diagram of behavioral experiments after TBI. H is a representative trajectory diagram of mice in spatial exploration and directional navigation tests. I and J show that AVE0991 treatment reduces the time to find the underwater platform and shortens the moving distance in the platform quadrant. KM shows that AVE0991 improves the motor coordination and balance ability of TBI mice, which is manifested by shortened completion time of horizontal ladder and balance beam tests and increased motor scores.

[0038] Fig.10 .The effect of ACE2 deficiency on the expression of inflammatory factors after TBI and the improvement of AVE0991 on pyroptosis and inflammation. A is the principal component analysis (PCA) of transcriptome data. B is a volcano plot showing 385 upregulated genes and 74 downregulated genes. C is PCR verification of the expression of inflammation and pyroptosis-related factors. DI is Western blot analysis showing increased levels of IL-1β and IL-18 in ACE2 knockout TBI mice. JN is immunoblotting verification that AVE0991 alleviates pyroptosis and RAS disorder caused by ACE2 deficiency. O is that AVE0991 treatment reduces the fluorescence intensity of GSDMD and Caspase-1. N is nucleic acid dye evaluation confirming the protective effect of Ang (1-7) on cell pyroptosis.

[0039] Fig.11 .ACE2 gene deletion leads to lipid metabolism disorders and inhibits PTGIS expression. A is a cluster diagram of differential lipid metabolites. B is a KEGG classification analysis of differential metabolites. C is a volcano map showing upregulated (red) and downregulated (blue) proteins. D is a schematic diagram of GO enrichment analysis of differential proteins. E and F show that ACE2 gene deletion after TBI inhibits the expression of PTGIS. G is the immunohistochemical localization of PTGIS in the TBI lesion of mice.

[0040] Fig.12 .PTGIS alleviates LPS-induced cell pyroptosis, and Ang(1-7) improves arachidonic acid metabolism disorder. Figure 5 Western blot analysis. E: PTGIS alleviates the morphological changes of LPS-induced cell pyroptosis. F: Cluster heat map of arachidonic acid metabolome. G: Analysis of the effects of Ang(1-7) on ARA, 6-keto-PGF1a, PGE2, and TXB2 metabolism. DETAILED DESCRIPTION

[0041] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0042] AVE0991, generic name: Telmisartan, purchased from: MedChemExpress, specification: 10 mg. Its structural formula is:

[0043]

[0044] Telmisartan is a potent angiotensin II type 1 receptor antagonist that can inhibit its activity. Telmisartan tablets are currently used to treat hypertension.

[0045] Ozagrel: Ozagrel hydrochloride hydrate; purchased from aladdin; 1g; its structural formula is:

[0046]

[0047] U46619: 9,11-dideoxy-9A,11A-methylene epoxy prostaglandin F2A solution; purchased from GLPBIO; 5 mg; its structural formula is:

[0048]

[0049] Embodiment 1,

[0050] 1.1 Primary cell culture

[0051] The experiment used newborn C57BL / 6J mice at 24 hours of age. The bilateral cerebral cortex was isolated and mechanically sheared under sterile conditions. Subsequently, the cells were separated by mechanical shearing and layering pipette, filtered and centrifuged, and resuspended in complete DMEM / F12 medium containing 10% fetal bovine serum and inoculated in poly-D-lysine coated culture flasks. The mixed glial cells were cultured in an incubator at 37°C and 5% CO2 until they reached optimal growth state after about 7 days of culture.

[0052] 1.2 Experimental animals

[0053] Gender was not used as a biological variable. Male C57BL / 6J mice (weight 20-25 g, 6-8 weeks old) were used in this study and were provided by Jinan Pengyue Experimental Animal Breeding Co., Ltd. ACE2 knockout (ACE2-KO) mice were bred by SPF (Beijing) Biotechnology Co., Ltd. on a C57BL / 6J background. The mice were kept in an environment with a constant temperature (23°C ± 2°C) and a 12-h light / dark cycle, with free access to food and water. Before the experiment, the mice were acclimated to the environment for 7 days. Subsequently, they were randomly assigned to the surgical (TBI) group, the sham operation (Con) group, or the drug treatment (AVE0991, Ozagrel, U46619) group.

[0054] 1.3 TBI model

[0055] Mice were anesthetized by intraperitoneal injection of 100 mg / kg of sodium pentobarbital, and the body temperature was maintained at 37±0.5°C using a heating blanket. Subsequently, the mice were fixed on a stereotaxic instrument, and a 1.5 cm longitudinal incision was made in the middle of the head to expose the parietal bone. A 3 mm diameter craniotomy was performed on the right parietal bone, and the impact device was used to vertically impact the trauma area at a speed of 6.0±0.2 m / s, a contact time of 50 ms, and an impact depth of 1.4 mm. After surgery, the incision was sutured and the mice were returned to the cage after recovery from anesthesia. Con-TBI group and ACE2-TBI group were obtained after TBI modeling of normal mice (Con) and ACE2-KO mice.

[0056] 1.4 Drug treatment

[0057] To activate the ACE2 / Ang(1–7) / MasR axis, mice were intraperitoneally injected with AVE0991 (3 mg / kg) for 2 weeks. To modulate TXA2 signaling, selective TXA2 inhibitor Ozagrel (5 mg / kg) and TXA2 agonist U46619 (30 mg / kg) were administered daily for 7 days before TBI until the animals were euthanized or completed the Morris water maze test. The mortality rate after TBI surgery is approximately 20%. The AVE0991-treated groups were designated as the Con-TBI-AVE group and the ACE2-TBI-AVE group. Ozagrel and U46619 treatments resulted in the TBI+AO group and the TBI+U46619 group, respectively.

[0058] 1.5 Lentiviral transfection

[0059] Healthy BV2 microglia were seeded in 24-well plates at a concentration of 1×105 cells per well. The plates were incubated at 37°C and 5% CO 2Incubate overnight under the same conditions. After the virus was gradually thawed on ice, the primary infection was performed at an MOI (multiplicity of infection) of 20 (virus titer 1×108TU / mL). 24 hours after infection, a secondary infection was performed with fresh virus to improve the infection efficiency. 48 hours after infection, the GFP expression efficiency was observed by fluorescence microscopy. Subsequently, fresh culture medium was replaced, and the optimal concentration of puromycin was added to select stably transfected cell lines for subsequent experiments.

[0060] 1.6 Western blot

[0061] Total protein was extracted from mouse tissues and BV2 cells using lysis buffer. The concentration of protein lysates was quantified using BCA protein assay reagent (Boster, Wuhan, China). Denatured proteins were separated by electrophoresis and transferred to nitrocellulose membranes. After blocking in skim milk for 1 h, the membranes were incubated with antibodies overnight at 4 °C. The secondary antibodies were then incubated at room temperature for 1 h, and the membranes were placed in a dual-color infrared laser scanning imaging system for band detection.

[0062] ImageJ and GraphPad Prism 8 were used for quantitative analysis of the scan results.

[0063] The main antibodies included: ACE2 (Abcam, ab108252, 1:1000), MasR (Proteintech, 20080-1-AP, 1:2000), IL-1β (Abcam, ab9722, 1:1000), PTGIS (Affinity, DF4745, 1:1000), GSDMD (Santa Cruz Biotechnology, SC-393581, 1:1000), Caspase1 (Santa Cruz Biotechnology, SC-56036, 1:1000), IL-18 (Proteintech, 10663-1-AP, 1:2000) and β-actin (Proteintech, 20536-1-AP, 1:5000). Protein expression levels were normalized to β-actin. Data were analyzed using ImageJ and GraphPad Prism 8 software.

[0064] 1.7 Enzyme-linked immunosorbent assay (ELISA)

[0065] Ang II (CSB-E04495m), Ang (1-7) (CSB-E13763m), TXB2 (CSB-E08048m) and 6-keto-PGF1a (CSB-E09422m) were detected using ELISA kits provided by CUSABIO, and the operations were performed strictly in accordance with the manufacturer's instructions.

[0066] 1.8 Immunohistochemistry and immunofluorescence staining

[0067] Experimental mice were perfused with PBS and fixed with 4% paraformaldehyde (PFA). The brains were quickly removed, fixed in 4% PFA overnight, dehydrated with gradient sucrose solution, and frozen sections were made at a thickness of 10 μm. The sections were treated with 0.25% TritonX-100 for 25 minutes, blocked with 2% bovine serum albumin (BSA) for 90 minutes, and incubated with specific primary antibodies at 4°C overnight. The primary antibodies included: ACE2 (Proteintech, ab108252, 1:100), IL-1β (Santa Cruz Biotechnology, SC-12742, 1:100), PTGIS (Affinity, DF4745, 1:100), GSDMD (Santa Cruz Biotechnology, SC-393581, 1:100), Caspase-1 (Santa Cruz Biotechnology, SC-56036, 1:100), IL-18 (Proteintech, 10663-1-AP, 1:2000), MAP2 (Cell Signaling Technology, 8707S, 1:200), GFAP (Santa Cruz Biotechnology, SC-33673, 1:100) and Iba1 (Abcam, ab178846, 1:200). The next day, the secondary antibodies were added and incubated at room temperature for 1 hour in the dark, and the cell nuclei were stained with DAPI for 15 minutes. Images were acquired by an Olympus confocal microscope and processed using uniform parameters.

[0068] 1.9 PCR and quantitative real-time PCR

[0069] Total RNA from tissues and cells was extracted using Trizol reagent (Invitrogen, Shanghai), and RNA concentration was determined using NanoDrop2000 (Thermo Scientific). Total RNA was reverse transcribed into cDNA using SuperScript III reverse transcriptase (Invitrogen, Shanghai). Quantitative real-time PCR was performed on a CFX96 real-time PCR detection system (Bio-Rad). β-actin was used as an internal reference, and the experiment was repeated three times, using three samples each time. Primers were ordered from Origene, and the specific sequences are shown in Table 1.

[0070] Table 1 RT-PCR primer sequences

[0071]

[0072]

[0073] 1.10 Transmission Electron Microscopy (TEM)

[0074] The cells obtained by centrifugation were first fixed in an electron microscope fixative for 2-4 hours and then embedded in a 1% agarose solution. They were fixed in 0.1M phosphate buffer (pH 7.4) with 1% osmic acid for 2 hours in the dark, and then dehydrated, infiltrated and embedded in alcohol and acetone solutions. After the inlay was polymerized at 60°C for 48 hours, ultrathin sections with a thickness of 60-80nm were made using an ultrathin slicer. The copper grid was stained with 2% uranyl acetate for 8 minutes, rinsed three times with 70% alcohol, and then stained with 2.6% lead citrate for 8 minutes and naturally air-dried overnight in a CO2-free environment. Finally, it was observed under a transmission electron microscope.

[0075] 1.11 Behavioral Analysis

[0076] Behavioral experiment process Figure 7 As shown in A, on the second day after TBI induction, the neurological deficits of mice were evaluated using the balance beam test, Morris water maze test, rotarod test, and foot slip test. There were 7 mice in each group, and all evaluations were performed by two independent experimenters who were blind to the experimental conditions.

[0077] 1.11.1 Balance beam experiment

[0078] Balance beam walking training was performed three days before TBI modeling. The assessment was performed on the second day after TBI induction, and the time required for mice to cross the balance beam and the gait score (score range 0 to 6) were recorded. The experiment was performed three times a day, with an interval of 15-20 minutes, for five days.

[0079] 1.11.2 Foot slip test

[0080] The paw placement ability of mice on the grid was assessed by the foot slip test (FFT). A 3-day training session was conducted before surgery, with each session lasting 5 minutes at least twice a day. On the third day after TBI induction, mice were placed on a horizontal metal grid, and the total number of steps and the number of left forelimb slips within 2 minutes were recorded.

[0081] 1.11.3 Morris water maze test

[0082] The spatial learning and memory abilities of mice after TBI were evaluated through a series of training. Five days of training were conducted before TBI modeling, four times a day. The time it took for mice to find and stand on the hidden platform after entering the water (escape latency) and the total swimming distance were recorded.

[0083] 1.11.4 Mouse Rotarod Test

[0084] The rotarod test was used to assess the balance ability and sensorimotor coordination of mice. The test was performed on a five-channel rotating apparatus with an initial speed of 4 rpm, gradually accelerated to 40 rpm, and lasted for 300 seconds. Two days of training were performed before surgery, with a 30-minute interval between each session. During the test, the average falling time of the mice was recorded.

[0085] 1.12 Statistical analysis

[0086] SPSS 22.0 (IBM Corp., New York) was used for data analysis. Data were expressed as mean ± standard deviation (SD). Statistical analysis included unpaired Student's t test and one-way analysis of variance (ANOVA). GraphPad Prism 8.0 (GraphPad Software, USA) was used for statistical charting. The significance level was set at p < 0.05.

[0087] Embodiment 2,

[0088] 2.1 Traumatic brain injury (TBI) disrupts the renin-angiotensin system (RAS) and inhibits the motor and learning abilities of mice

[0089] After TBI, the level of ACE2 in the injured cortical area decreased significantly ( Figure 1 A), and the expression level was lowest 24 hours after injury ( Fig. 9 A, Fig. 9 D). The results of ACE2 protein localization in the brain showed that ACE2 was expressed in MAP2, IBA1 and GFAP positive cells ( Figure 1 B). It is noteworthy that the fluorescence intensity of ACE2 in microglia was significantly affected ( Figure 1 C), the co-expression analysis of ACE2 and IBA1 further explains this conclusion ( Figure 1HJ). In addition, TBI disrupts the normal functioning of the renin-angiotensin system (RAS). After TBI, MasR protein ( Figure 1 A) and Ang(1-7) levels were inhibited, while AngII level was significantly increased ( Figure 1 DG). As ACE2 inhibition may be a major factor in the occurrence of this event, the present invention conducted additional studies to investigate its effects on traumatic brain injury. In order to confirm the in vivo neuroprotective effect of ACE2, the present invention used behavioral experiments to verify ( Figure 1 K). AVE0991 significantly alleviated TBI-induced cognitive decline, as evidenced by a reduction in escape latency and distance in the learning curve ( Figure 1 LN). In addition, AVE0991 can improve motor coordination and balance caused by TBI. The use of AVE0991 shortened the time it took for mice to complete the test in the horizontal ladder and balance beam experiments, and significantly improved the scores of TBI mice ( Figure 1 OR).

[0090] CRISPR / Cas9 successfully constructed the mouse ACE2-KO model ( Fig. 9 B), the present invention uses PCR and WB to verify the model ( Fig. 9 C, 9E, 9F). The behavioral experiment of ACE2 gene knockout mice once again verified that TBI caused a serious decline in the learning and memory ability and motor ability of mice ( Fig. 9 HM). However, the knockout of ACE2 gene limits the improvement ability of AVE0991 ( Fig. 9 HM).

[0091] 2.2 Loss of ACE2 is involved in the activation of pyroptosis

[0092] To explore the effect of ACE2 deficiency on TBI progression, the present invention performed transcriptome sequencing. PCA analysis verified the integrity of the samples ( Fig.10 A total of 385 up-regulated genes and 74 down-regulated genes were identified ( Fig.10 B). Reactome enrichment analysis of differentially expressed genes (https: / / reactome.org / ?ref=blog.opentargets.org) showed that the pyroptosis pathway was significantly enriched ( Figure 2 B), and found that pyroptosis-related factors Gsdmd, Casp1, II18rap and Illr2 were significantly changed ( Figure 2 A). qRT-PCR confirmed that the mRNA level of pyroptosis factors was significantly upregulated ( Fig.10C). Western blot analysis showed that ACE2 deficiency exacerbated the activation of pyroptosis key factors Gsdmd and Casp1 ( Figure 2 CE). Immunofluorescence double staining further confirmed this result ( Figure 2 I). In addition, the activation levels of mature forms of the proinflammatory cytokines IL-1β and IL-18, which are associated with pyroptosis, were significantly increased in the absence of ACE2 ( Fig.10 DI). At the same time, Sholl analysis was used to evaluate the morphological changes of microglia around the injury after TBI, and the results showed that their state changed from resting to activated ( Figure 2 FH).

[0093] To further clarify the role of ACE2 in cell pyroptosis, the present invention administered AVE0991 to mice. AVE0991 upregulated the expression levels of ACE2 and MasR, thereby improving cell pyroptosis ( Fig.10 JN), which is also reflected in the attenuation of pyroptosis-related proteins GSDMD and Caspase-1 immunofluorescence ( Fig.10 O). It is noteworthy that AVE0991 has shown good therapeutic effects in animal models ( Figure 2 JL). In addition, the present invention uses LPS to induce cell pyroptosis model, and supplements with Ang (1-7) as an adjuvant for ACE2. Transmission electron microscopy (TEM) observations showed that cells in the LPS-treated group underwent severe disintegration, manifested by extensive membrane rupture, cytoplasmic outflow, nuclear disintegration, and chromatin condensation ( Figure 2 M). In contrast, Ang(1-7) significantly alleviated LPS-induced cell pyroptosis ( Figure 2 N). The role of Ang(1-7) in alleviating the severity of cell pyroptosis was further confirmed by nucleic acid dye detection ( Fig.10 P).

[0094] 2.3 ACE2 downregulation exacerbates arachidonic acid metabolism disorders

[0095] In order to further explore the relationship between ACE2 downregulation or loss and cell pyroptosis, the present invention uses a combination of omics technology and bioinformatics analysis to reveal the underlying molecular mechanism. First, through non-targeted metabolomics analysis, it was found that the protein composition within the group was highly correlated, while the differences between the groups were significant, which provided a basis for subsequent differential protein analysis ( Figure 3 A, 3B). 134 differential metabolites were identified and screened, and displayed intuitively in the form of a volcano plot ( Figure 3 C). It is worth noting that TXB2 (a metabolite of TXA2) was significantly increased in the ACE2-TBI group. Through KEGG pathway analysis, the present invention found that TXB2 was significantly enriched in the arachidonic acid metabolic pathway, suggesting that it may be involved in regulating cell pyroptosis ( Figure 3 D). This pathway is classified as a lipid metabolism pathway ( Figure 3 D) and was further verified by lipid metabolomics analysis ( Fig.11 A, 11B), and at the same time, differential lipid metabolites were detected multiple times in the arachidonic acid metabolic pathway ( Figure 3 EF). Subsequently, 43 differentially expressed proteins were identified by TMT quantitative proteomics analysis ( Fig.11 C), some of which are involved in metabolic processes such as lipid metabolism ( Fig.11 D). It is worth noting that PTGIS, as a key metabolic enzyme in arachidonic acid metabolism, is significantly downregulated under ACE2 deficiency conditions ( Figure 4 A), and this result was confirmed by Western blotting and immunohistochemistry experiments ( Fig.11 EG). Further KEGG pathway analysis showed that PTGIS was significantly enriched in the arachidonic acid metabolic pathway ( Figure 4 B) These results indicate that ACE2 deficiency leads to the disorder of arachidonic acid metabolites and metabolic enzyme expression, revealing the complex interaction mechanism between ACE2 and cell pyroptosis.

[0096] 2.4 ACE2 deficiency exacerbates pyroptosis by regulating arachidonic acid metabolism

[0097] In view of the correlation between ACE2 deficiency and PTGIS downregulation, the present invention further explored the potential role of PTGIS in brain injury. Immunolocalization experiments were performed in the damaged cerebral cortex tissue of mice with traumatic brain injury (TBI). The results showed that PTGIS (pink) was co-expressed with the microglial marker protein IBA1 (green), suggesting that microglial activation after TBI is accompanied by a decrease in PTGIS expression levels ( Figure 4 D). In addition, in the ACE2-TBI group, the level of pyroptosis was significantly increased, while the expression level of PTGIS was significantly decreased ( Figure 4 C). ELISA results showed that compared with normal mice, the arachidonic acid metabolite TXB2 in TBI mice was significantly increased, and ACE2-TBI mice showed a stronger expression level ( Figure 4 E). In contrast, low PTGIS expression led to a significant decrease in the level of 6-keto-PGF1α, a metabolite of PGI2 ( Figure 4 F). The hypothesis of the present invention was further verified by the analysis of arachidonic acid targeted metabolomics, metabolic clustering diagram and correlation analysis ( Figure 4 G). In the metabolic pathway diagram, red arrows indicate increased metabolic levels, and green arrows indicate decreased metabolic levels. The loss of ACE2 significantly disrupts the balance between TXA2 and PGI2, thereby exacerbating the pyroptosis mechanism ( Figure 4H). These results suggest that ACE2 plays an important role in TBI-related pyroptosis by regulating arachidonic acid metabolism.

[0098] 2.5PTGIS plays a key role in pyroptosis regulation

[0099] Based on the above research results, it is speculated that supplementation with PTGIS may alleviate arachidonic acid metabolic disorders and inhibit the process of pyroptosis. In in vitro studies, LPS was used to induce cell pyroptosis, and the effect of microglial PTGIS overexpression vector on pyroptosis was evaluated ( Figure 5 A). LPS-induced cell pyroptosis significantly increased the expression of pyroptosis-related factors such as GSDMD and IL-18 ( Fig.12 AD). PTGIS can inhibit the release of pyroptosis-related inflammatory mediators and restore cell deformation caused by pyroptosis ( Fig.12 E), and reduced the mature levels of GSDMD and Caspase-1 ( Figure 5 B). Scanning electron microscopy results further confirmed the inhibitory effect of PTGIS on the pyroptosis process and its effect in delaying cell disintegration ( Figure 5 C). This conclusion was further supported by immunofluorescence and transcriptional level analysis ( Figure 5 D, 5E).

[0100] Targeted metabolite analysis showed that there were differences between different cell physiological functions and tissue types, and the effects of LPS and PTGIS intervention on arachidonic acid metabolism were not completely consistent with the results of animal studies. The present invention first made a cluster heat map of arachidonic acid metabolome ( Fig.12 F), statistical analysis results showed that LPS treatment significantly increased the expression levels of ARA and PGE2, but the effect on TXB2 did not reach statistical significance ( Figure 5 F). This result may be affected by sample size and individual differences. Although LPS had a weak inhibitory effect on 6-keto-PGF1a, PTGIS intervention restored its level to normal. PTGIS intervention promoted arachidonic acid metabolism, reduced cellular expression levels, and restored PGE2 to normal. Using nucleic acid dyes to assess cell morphology, it was found that LPS significantly increased cell membrane permeability, causing YO-PRO-1 to enter the cell nucleus and cause fluorescence enhancement ( Figure 5 G). At the same time, EthD-2 penetrates the damaged cell membrane and binds to more nucleic acids ( Figure 5 G). In addition, PTGIS significantly inhibited the LPS-induced cell pyroptosis process. At the same time, the present invention found that Ang (1-7) had similar regulatory results ( Fig.12 G).

[0101] 2.6TXA2 exacerbates pyroptosis

[0102] TXA2 is functionally antagonistic to PGI2. The TXA2 agonist U46619 directly exacerbates the process of cell pyroptosis, leading to increased cell membrane permeability and upregulation of pyroptosis-related protein expression levels ( Figure 6 AC, F). This process is accompanied by disturbances in TXB2 and 6-keto-PGF1a levels ( Figure 6 D, 6E). Ozagrel acts as a TXA2 synthesis inhibitor by blocking the production of TXA2. In vivo studies, the role of TXA2 in the progression of TBI was further verified by administering Ozagrel to mice after TBI. The application of Ozagrel significantly alleviated the cognitive dysfunction caused by traumatic brain injury, as shown by a shortened escape latency in the learning curve and improved performance in the probe test ( Figure 7 BD). In addition, inhibition of TXA2 alleviated TBI-induced movement disorders and balance deficits ( Figure 7 EJ). These results suggest that targeting the TXA2 signaling pathway can reduce inflammation and pyroptosis in the injured brain, thus providing a potential intervention strategy for the treatment of TBI.

[0103] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of angiotensin-converting enzyme 2 as a drug target for the treatment of traumatic brain injury.

2. Use of angiotensin (1-7) receptor agonists or thromboxane A2 inhibitors in the preparation of drugs for treating traumatic brain injury.

3. The use according to claim 2, characterized in that: Angiotensin (1-7) receptor agonists are used to improve motor coordination and balance after traumatic brain injury.

4. The use according to claim 2, characterized in that: The angiotensin (1-7) receptor agonist is used to up-regulate the expression levels of angiotensin converting enzyme 2 and MasR, and improve cell pyroptosis.

5. The use according to claim 2, characterized in that: Thromboxane A2 inhibitors are used to alleviate cognitive impairment caused by traumatic brain injury.

6. The use according to claim 2, characterized in that: Thromboxane A2 inhibitors alleviate movement disorders and balance deficits caused by traumatic brain injury and reduce inflammation and pyroptosis in the damaged brain.

7. A therapeutic drug for traumatic brain injury, characterized in that: Its active ingredients are angiotensin (1-7) receptor agonists or thromboxane A2 inhibitors.

8. The therapeutic drug for traumatic brain injury according to claim 7, characterized in that: The angiotensin (1-7) receptor agonist is Telmisartan.

9. The therapeutic drug for traumatic brain injury according to claim 7, characterized in that: The thromboxane A2 inhibitor is Ozagrel and its pharmaceutically acceptable salts, and the acid used for salt formation is selected from hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and citric acid.

10. The therapeutic drug for traumatic brain injury according to claim 7, characterized in that: The therapeutic agent also includes one or more pharmaceutically acceptable carriers or excipients.

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