Application of HIF-1alpha inhibitor in preparation of product for inducing transformation of astrocytes from pro-inflammatory type to anti-inflammatory type

Abstract

The invention belongs to the technical field of biological medicines, and relates to application of an HIF-1alpha inhibitor in preparation of a product for inducing transformation of astrocytes from a pro-inflammatory type to an anti-inflammatory type. The invention reveals that activation of an HIF-1 signal channel is an important mechanism for driving neuroinflammatory response and pro-inflammatory A1 type astrocyte formation, and inhibition of HIF-1 activation can effectively promote generation of neuroprotective A2 type astrocytes. By applying the HIF-1alpha small-molecule inhibitor KC7F2 to astrocytes, HIF-1alpha activation is blocked, and the astrocytes are promoted to be converted from a proinflammatory A1 phenotype to an anti-inflammatory A2 phenotype, so that inflammatory response is inhibited, neurotoxicity is reduced, and a neuron microenvironment is improved. The invention provides a novel intervention strategy based on HIF-1alpha signal regulation and control, and a novel theoretical basis and a novel potential drug treatment scheme are provided for treatment of nervous system injury and related neurodegenerative diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of HIF-1α inhibitors in the preparation of products that induce the transformation of astrocytes from pro-inflammatory to anti-inflammatory types. Background Technology

[0002] Neurological diseases are a class of major illnesses characterized by the progressive loss of neuronal function and eventual death. These include Alzheimer's disease (AD), Parkinson's disease (PD), stroke, and amyotrophic lateral sclerosis (ALS). Elucidating the molecular pathological mechanisms of neurological diseases is crucial for developing effective clinical interventions and treatments.

[0003] In recent years, numerous studies have confirmed that neuroinflammation plays a crucial role in the occurrence and progression of various neurological diseases. Chronic, persistent inflammatory responses are a major contributing factor to neuronal dysfunction and death. The primary driver of neuroinflammation is glial cell dysfunction. In the central nervous system, glial cells mainly include astrocytes and microglia. Functionally normal glial cells play a vital role in maintaining neuronal homeostasis, synaptic support, and metabolic balance. However, when stimulated by external damaging signals or inflammation, these cells become activated and undergo phenotypic polarization, typically exhibiting neuroprotective and neurotoxic phenotypes.

[0004] Under inflammatory conditions, microglia activation can induce resting astrocytes to transform into a neurotoxic phenotype, the so-called A1 phenotype astrocytes. A1 astrocytes lose many of the functions of normal astrocytes but upregulate the expression of genes harmful to synapse formation and secrete a series of cytotoxic molecules, leading to rapid neuronal and oligodendrocyte death. Studies have found that in inflammatory environments, the main factor inducing neuronal death is not pro-inflammatory microglia (M1 type), but rather the overactivation of A1 astrocytes. Conversely, another type of astrocyte with neuroprotective functions, the A2 type, also exists at the site of injury. These cells promote synaptic repair, growth, and neuronal survival by upregulating various neuroprotective factors. Therefore, regulating astrocyte polarization from the neurotoxic A1 type to the neuroprotective A2 type is considered a potential therapeutic direction for neurological diseases.

[0005] Currently, the molecular mechanisms regulating astrocyte phenotypic transition remain incompletely understood, potentially involving the interaction of multiple signaling pathways, such as nuclear factor kappa-B (NF-κB), mitogen-activated protein kinase (MAPK), Janus kinase / signal transducer and activator of transcription 3 (JAK / STAT3), and phosphatidylinositol-3-kinase / protein kinase B (PI3K / AKT). Previous studies have demonstrated that hypoxia-inducible factor 1 (HIF-1) participates in inducing M1 polarization in macrophages and microglia, exerting its effects through mechanisms such as enhanced secretion of pro-inflammatory factors and glycolytic metabolism. However, the role of the HIF-1 signaling pathway in astrocyte polarization has not been fully investigated and reported.

[0006] Therefore, exploring the regulatory mechanism of the HIF-1 pathway in astrocyte polarization and developing corresponding regulatory methods are of great scientific significance and potential application value for elucidating the pathogenesis of neuroinflammation and its role in nervous system diseases. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide the application of HIF-1α inhibitors in the preparation of products that induce the transformation of astrocytes from pro-inflammatory to anti-inflammatory types.

[0008] To achieve this objective, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides the application of HIF-1α inhibitors in the preparation of products that induce the transformation of astrocytes from pro-inflammatory to anti-inflammatory types.

[0010] To address the lack of clarity regarding the mechanisms of reactive polarization in astrocytes and the scarcity of regulatory targets in existing technologies, this invention reveals the crucial role of the HIF-1 pathway in regulating the reactive phenotype of astrocytes. It also demonstrates that inhibiting this pathway with specific small-molecule inhibitors can significantly reduce neuroinflammation and neurotoxicity, promoting the formation of a neuroprotective phenotype. Furthermore, this invention reveals that under hypoxic conditions, activation of the HIF-1 pathway can induce astrocytes to transform into the neurotoxic A1 phenotype; while under normoxic conditions, inflammatory stimulation can also upregulate HIF-1α and induce the formation of A1 phenotype astrocytes.

[0011] By using KC7F2, a specific small molecule inhibitor of HIF-1α, the overactivation of the HIF-1 pathway can be effectively inhibited, the expression of A1 phenotype-related genes can be reduced, and the upregulation of neuroprotective A2 phenotype genes can be promoted, thereby alleviating neuroinflammation and reducing neuronal damage. This invention reveals the core regulatory role of the HIF-1 pathway in astrocyte polarization and provides a novel intervention strategy targeting HIF-1 signaling, which has significant theoretical implications and potential clinical translational value.

[0012] Preferably, the product includes a drug or reagent.

[0013] The reagents described in this invention are for laboratory research purposes only and are not intended for human treatment; they are solely for scientific experimental research.

[0014] Preferably, the HIF-1α inhibitor is KC7F2.

[0015] Preferably, the astrocytes are derived from mammals.

[0016] Preferably, the astrocytes are primary rat astrocytes.

[0017] Preferably, activation of the HIF-1 pathway induces astrocytes to transform into pro-inflammatory astrocytes.

[0018] Preferably, the pro-inflammatory astrocytes are obtained by cobalt chloride induction.

[0019] Preferably, the induction concentration is 100-200 μM, and the time is 12-48 h.

[0020] Concentrations can be selected from 100 μM, 120 μM, 150 μM, 180 μM, 200 μM, etc., and time can be selected from 12 h, 15 h, 18 h, 20 h, 25 h, 30 h, 35 h, 40 h, 45 h, 48 h, etc. Other specific point values ​​within the above range can be selected, which will not be elaborated here.

[0021] In a second aspect, the present invention provides a method for inducing the transformation of astrocytes from pro-inflammatory to anti-inflammatory types for non-disease treatment purposes, the method comprising: inducing pro-inflammatory astrocytes using an HIF-1α inhibitor.

[0022] Preferably, the induction concentration of the HIF-1α inhibitor is 5-40 μM, and the induction time is 12-48 h.

[0023] Concentrations can be selected from 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, etc., and time can be selected from 12 h, 15 h, 18 h, 20 h, 25 h, 30 h, 35 h, 40 h, 45 h, 48 h, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0024] HIF-1α inhibitors can simultaneously reduce HIF-1α expression levels in both the nucleus and cytoplasm. KC7F2 treatment inhibits C3 protein degradation and blocks the formation of the A1 phenotype. KC7F2 treatment also increases the expression of A2 phenotype-related genes, inducing cell polarization towards the A2 phenotype.

[0025] Thirdly, the present invention provides the application of HIF-1α inhibitors in the preparation of activation inhibitors of C3 protein.

[0026] Fourthly, this invention provides the application of HIF-1α inhibitors in the preparation of neuroprotective gene expression promoters.

[0027] Preferably, the neuroprotective genes include any one or a combination of at least two of Tgm1, Sphk1, Ptx3, Tm4sf1, B3galt5, Ptgs2, S100a10, Clcf1, Emp1, or CD14.

[0028] According to the research results of this invention, HIF-1α inhibitors can inhibit C3 protein activation and promote the expression of neuroprotective genes. Therefore, HIF-1α inhibitors can be used as in vitro formulations for basic scientific research.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] This invention elucidates the core mechanism of action of the HIF-1 pathway in astrocyte phenotypic polarization. By inhibiting HIF-1α signaling through pharmacological methods, it facilitates the transformation of astrocytes from a neurotoxic A1 phenotype to a neuroprotective A2 phenotype. This method significantly reduces neuroinflammatory responses, inhibits neurotoxic factors secreted by A1 phenotype astrocytes, thereby reducing neuronal death. Simultaneously, it promotes the expression of neuroprotective genes, improves the neural microenvironment, and enhances neural repair capabilities. This invention provides a novel therapeutic approach and drug development direction for neuroinflammatory treatment targeting the HIF-1 pathway. Attached Figure Description

[0031] Figure 1 Immunofluorescence identification image of purified rat astrocytes.

[0032] Figure 2 This image shows Western blot analysis of HIF-1α protein expression in astrocytes 12 h and 24 h after induction by inflammatory factors. Figure A shows representative protein bands, and Figure B shows the quantitative analysis of protein expression.

[0033] Figure 3 Immunofluorescence images of HIF-1α protein localization in astrocytes 12 h and 24 h after ITC (IL-1α, TNF-α, C1q) induction. Image A shows a representative immunofluorescence image of HIF-1α, while images B and C are statistical graphs of HIF-1α fluorescence intensity in the cell nucleus and cytoplasm, respectively.

[0034] Figure 4 Figures show the qPCR detection of gene expression in astrocytes after 24 h of induction with ITC and CoCl2, respectively. Figure A shows HIF-1α expression; Figures B-D show the expression of A1-type marker genes C3, Gbp2, and Serping1; Figures E-G show the expression of pro-inflammatory factors TNF-α, IL-6, and IL-1β.

[0035] Figure 5 Immunofluorescence images showing the localization of HIF-1α protein in astrocytes under different treatment conditions. Image A shows a representative immunofluorescence image of HIF-1α, while images B and C are statistical graphs of its fluorescence intensity in the cell nucleus and cytoplasm, respectively.

[0036] Figure 6 This image shows Western blot images of protein expression in astrocytes under different treatment conditions. Image A shows representative protein bands for C3 and C3α; Images B and C are statistical graphs of C3 and C3α expression quantification, respectively.

[0037] Figure 7 The graph shows the changes in the expression of A2 phenotype-related genes in astrocytes under different treatment conditions. Detailed Implementation

[0038] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0039] Example 1

[0040] In vitro astrocyte A1 phenotype induction and HIF-1 pathway analysis

[0041] 1. Rat astrocyte culture

[0042] Newborn rats, 24 hours old, were placed on ice and anesthetized with hypothermia for 5 minutes. After disinfection with 75% alcohol, they were transferred to a clean bench for further processing. The rats were euthanized by decapitation. The skin, subcutaneous tissue, and skull were cut along the midline of the brain from back to front to the brow. The skin was then pulled to the sides with forceps to expose the skull. The skull was bluntly dissected to expose the brain tissue. The brain was then removed with curved forceps and placed in pre-cooled PBS. The meninges and blood vessels were carefully dissected, and the brain was cut into small pieces of approximately 1 mm using ophthalmic scissors. Papain was added, and the cells were digested at 37 °C for 30 minutes (with a 10-minute interval between pipetting). Digestion was then stopped by adding 4 volumes of PBS or basal culture medium. The cells were centrifuged at 1200 rpm for 3 minutes, the supernatant was discarded, and high-glucose DMEM complete culture medium was added and mixed thoroughly. The cell suspension was filtered through 100 μm and 40 μm sieves, respectively, and then transferred to cell culture flasks for culture. The medium was changed every other day. After the cells reached confluence, they were incubated at 37°C with shaking at 200 rpm for 1 hour to remove contaminating cells and continue culturing. P1 or P2 cells were used for subsequent experiments. Immunofluorescence was used to detect whether microglia had been removed. Astrocytes were labeled with GFAP, microglia were labeled with Iba1, and DAPI was used to label the cell nuclei.

[0043] The results are as follows Figure 1 As shown, there were almost no Iba1-positive cells, demonstrating that high-purity astrocytes were obtained and can be used for subsequent experiments.

[0044] 2. A1 phenotype astrocyte induction

[0045] Once the cell confluence reached 80%, the culture medium was changed to serum-free medium for 48 h of induction, followed by induction for 24 h with a combination of ITC factors (3 ng / mL IL-1α, 30 ng / mL TNF-α, 400 ng / mL C1q).

[0046] 3. HIF-1 pathway analysis

[0047] Under normoxic conditions, HIF-1α in the cytoplasm is rapidly degraded within minutes and cannot cross into the nucleus to form the HIF-1 complex. The activation of the HIF-1 pathway can be determined by detecting the expression and localization of HIF-1α. Immunofluorescence and Western blotting were performed on cells at different time points (12 h and 24 h) after ITC induction.

[0048] Western blot analysis of HIF-1α protein expression in astrocytes is shown below. Figure 2As shown in Figure A, HIF-1α protein expression increases with prolonged ITC induction time. Figure B shows the ratio of HIF-1α to the internal reference protein β-actin, indicating that HIF-1α expression is upregulated over time with significant differences. Figure B also shows that compared with the uninduced group, HIF-1α expression increased by approximately 1.3 times after 12 h of induction and by approximately 1.8 times after 24 h of induction.

[0049] Immunofluorescence assay results for HIF-1α protein localization are as follows: Figure 3 As shown in Figure A, the distribution of HIF-1α in cells indicates that the nuclear HIF-1α content significantly increases with prolonged ITC induction time. Figure B statistically analyzes the nuclear fluorescence intensity, and the results are statistically significant. Figure C statistically analyzes the cytoplasmic fluorescence intensity, and the results show no statistical difference. These results indicate that the A1 phenotype induces protein stabilization of HIF-1α, leading to its accumulation under normoxic conditions. The increased HIF-1α is manifested in a higher nuclear distribution, suggesting the formation of the HIF-1 complex and activation of the HIF-1 pathway.

[0050] 4. Compare the differences in gene expression in astrocytes treated with ITC and CoCl2.

[0051] To further investigate the correlation between the A1 phenotype and the HIF-1 pathway in astrocytes, HIF-1 pathway activation was induced by simulating a hypoxic environment with 100 μM CoCl2 treatment. The differences in gene expression between CoCl2-treated and ITC-treated astrocytes were compared. After treatment with serum-free medium for 48 h, one group of cells was treated with 100 μM CoCl2 for 24 h, another group was treated with ITC combination factor for 24 h, and the control group was treated with the same medium without any drugs for 24 h. Cells were then harvested, lysed, and total RNA was extracted for RT-qPCR.

[0052] qPCR test results as follows Figure 4 As shown in Figure A, both CoCl2 and ITC treatments upregulated HIF-1α expression. Figures B, C, and D show that both CoCl2 and ITC treatments upregulated A1 phenotype-related genes (C3, Gbp2, Serping1). Figures E, F, and G show that both CoCl2 and ITC treatments upregulated pro-inflammatory factors (TNF-α, IL-6, IL-1β), but the ITC treatment group showed significantly higher upregulation than the CoCl2 treatment group. These results indicate that CoCl2 and ITC treatments share the same gene expression trend, further demonstrating the correlation between the A1 phenotype and the HIF-1 pathway in astrocytes.

[0053] Example 2

[0054] KC7F2 induces the transformation of astrocytes from phenotype A1 to A2.

[0055] KC7F2 is a commercially available HIF-1α inhibitor that inhibits HIF-1α protein synthesis. Based on the above experimental results demonstrating the correlation between the HIF-1 pathway and the A1 phenotype, it is reasonable to hypothesize that inhibiting HIF-1α protein levels can alleviate the A1 phenotype in astrocytes. The experimental design is as follows:

[0056] 1. KC7F2 inhibits HIF-1 activation.

[0057] After astrocytes were induced with ITC combination factor for 24 h, they were treated with 10 μM KC7F2 (KCF) for 24 h. The control group was treated with the same amount of DMSO for 24 h. Cells were then harvested to detect HIF-1α expression.

[0058] Immunofluorescence staining of cells yielded the following results: Figure 5 As shown, 24 h after ITC treatment was stopped, the nuclear HIF-1α content returned to the blank control level, while the cytoplasmic HIF-1α content was still higher than that of the blank control group. In contrast, the nuclear and cytoplasmic HIF-1α content of the experimental group (KCF treatment group) was significantly reduced, indicating that KCF can inhibit the protein level of HIF-1α.

[0059] 2. KC7F2 inhibits the expression of A1 phenotype-related genes.

[0060] Complement C3 is produced by reactive astrocytes. Upregulation of complement C3 expression, especially the increased expression of C3 degradation products, is considered a classic marker of the A1 phenotype (the addition of C1q induces the activation of C3 convertase, thereby degrading C3 into α and β chains and initiating downstream signal transduction). Therefore, after determining that KCF can inhibit the protein level of HIF-1α (inhibiting the activation of the HIF-1 pathway), we further verified whether KCF can inhibit the A1 phenotype.

[0061] Total protein was extracted from the cells after drug treatment, and Western blotting experiments were performed. Results are as follows: Figure 6 As shown, compared with the control group, the expression of C3 was increased after treatment with the ITC combination factors, but the expression of the α chain of C3 degradation products was significantly reduced after KCF treatment, indicating that KCF blocked the activation of the C3-C3α signaling pathway.

[0062] 3. KC7F2 upregulates genes related to the A2 phenotype.

[0063] The above results confirm that KCF can suppress the A1 phenotype in astrocytes, further suggesting whether it can induce the A2 phenotype. Therefore, qPCR analysis was performed on cells treated for 48 h to detect changes in the expression of a series of genes related to the A2 phenotype.

[0064] The results are as follows Figure 7 As shown, compared with ITC treatment alone, A2-related genes in cells treated with ITC+KCF were generally upregulated, demonstrating that KCF can induce A2 phenotypic polarization.

[0065] The above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. Application of HIF-1α inhibitors in the preparation of products that induce the transformation of astrocytes from pro-inflammatory to anti-inflammatory types.

2. The application according to claim 1, characterized in that, The HIF-1α inhibitor is KC7F2.

3. The application according to claim 1, characterized in that, The astrocytes mentioned are derived from mammals.

4. The application according to claim 1, characterized in that, The astrocytes mentioned are primary rat astrocytes.

5. The application according to claim 1, characterized in that, Activation of the HIF-1 pathway induces astrocytes to transform into pro-inflammatory astrocytes; The pro-inflammatory astrocytes were obtained by induction with cobalt chloride at a concentration of 100-200 μM for 12-48 h.

6. A method for inducing the transformation of astrocytes from pro-inflammatory to anti-inflammatory types for non-disease treatment purposes, characterized in that, The method includes inducing pro-inflammatory astrocytes using an HIF-1α inhibitor.

7. The method for inducing astrocytes to transition from a pro-inflammatory to an anti-inflammatory phenotype according to claim 6, characterized in that, The induction concentration of the HIF-1α inhibitor is 5-40 μM, and the induction time is 12-48 h.

8. Application of HIF-1α inhibitors in the preparation of activation inhibitors of C3 protein.

9. Application of HIF-1α inhibitors in the preparation of neuroprotective gene expression promoters.

10. The application according to claim 9, characterized in that, The neuroprotective genes include any one or a combination of at least two of the following: Tgm1, Sphk1, Ptx3, Tm4sf1, B3galt5, Ptgs2, S100a10, Clcf1, Emp1, or CD14.

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