Application of ethanolamine in the preparation of products for preventing, alleviating and / or treating diseases related to neuroinflammation

Abstract

The present invention discloses the use of ethanolamine in the preparation of products for preventing, alleviating and / or treating diseases related to neuroinflammation, and relates to the field of medicine. The neuroinflammation is caused by excessive activation of microglia, and the diseases related to neuroinflammation include ophthalmic diseases and central nervous system diseases. The present invention evaluated the inhibitory effect of ethanolamine on microglia activation through a series of experiments, and the results showed that ethanolamine can significantly reduce the activation of microglia and the expression of inflammatory factors by inhibiting the NF-κB pathway, thereby reducing the level of inflammation. Therefore, ethanolamine is used for pharmaceuticals or health products, etc., and products suitable for preventing, alleviating and treating diseases related to neuroinflammation can be obtained.

Description

Technical Field

[0001] The present invention belongs to the field of medicine and relates to a new use of ethanolamine, and specifically relates to the use of ethanolamine in preparing products for preventing, alleviating and / or treating diseases related to neuroinflammation. Background Art

[0002] Dysregulated inflammatory responses can lead to tissue and organ damage, thus promoting the development of diseases. The inflammatory response of the nervous system, or "neuroinflammation", has its own unique characteristics and is present in neurodegenerative diseases of the central nervous system, including multiple sclerosis (MS), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), etc., as well as ophthalmic diseases such as optic nerve damage, diabetic retinopathy (DR) and glaucoma. Among the related diseases, although the causes of onset vary, such as stroke, hypoxia, genetic factors, infection, neurotoxins, aggregation of abnormal proteins, trauma or immune mediation, etc., these diseases are characterized by progressive neuronal dysfunction and have a common chronic immune response process.

[0003] Neurodegenerative diseases mediated by inflammatory response are mainly caused by the activation of microglia, which in turn leads to excessive release of inflammatory factors such as NO, ROS, TNF-α, IL-1β, etc. Microglia are one of the main types of glial cells in the central nervous system (CNS). They play the role of macrophages and participate in the immune response of the CNS. In the resting state, microglia are branched and constantly monitor various pathological changes in the microenvironment around them. Under the stimulation of various damaging factors, microglia are immediately activated, change their morphology and migrate to the lesion area, playing a dual role of protecting and damaging tissues: on the one hand, microglia promote nerve cell survival and tissue regeneration by phagocytizing foreign components and secreting neurotrophic factors, while on the other hand, excessive activation of microglia may also induce neuronal apoptosis by secreting cytotoxic substances such as interleukin (IL)-1 and tumor necrosis factor (TNF)-α, thereby aggravating tissue damage.

[0004] Taking diabetic retinopathy as an example, the most common treatments include various anti-VEGF injections (such as ranibizumab, conbercept, etc.), hormones (such as sustained-release dexamethasone injection, triamcinolone acetonide, fluocinolone acetonide, etc.), laser photocoagulation and vitrectomy, but there are still some patients in clinical practice who cannot achieve the ideal expected results of the above treatment measures.

[0005] Studies have found that inhibiting neuroinflammatory responses can reduce neuronal lesions in damaged brain areas or delay the progression of neurodegenerative diseases. Therefore, finding and studying inhibitors of neuroinflammatory responses in the brain is of great significance for the development of new drugs for the treatment of a variety of acute and chronic neurodegenerative diseases. Summary of the invention

[0006] In view of the problems existing in the background technology, the present invention provides a new use of ethanolamine, namely, the use of ethanolamine in the preparation of products for preventing, alleviating and / or treating neuroinflammation-related diseases, thereby providing new drugs for clinical improvement or treatment of neuroinflammation-related diseases.

[0007] In order to achieve the above object, the present invention first provides the use of ethanolamine in the preparation of products for preventing, alleviating and / or treating diseases related to neuroinflammation.

[0008] Another aspect of the present invention provides the use of ethanolamine in preparing a product for inhibiting neuroinflammation.

[0009] Preferably, the neuroinflammation is neuroinflammation caused by excessive activation of microglia.

[0010] Preferably, the product comprises medicine and / or health care product.

[0011] Preferably, the drug further comprises a pharmaceutically acceptable carrier or excipient.

[0012] Furthermore, the carrier or auxiliary material includes any one or more of a diluent, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, a lubricant, and an excipient.

[0013] Furthermore, the dosage form of the drug includes any one or a combination of any two or more of oil, granules, tablets, powders, capsules, pills, powders, oral liquids, sols, sprays, and atomizers.

[0014] Preferably, the neuroinflammation-related diseases include ophthalmic diseases and central nervous system diseases.

[0015] Furthermore, the ophthalmic diseases include but are not limited to optic nerve damage, diabetic retinopathy, and glaucoma; the central nervous system diseases include but are not limited to multiple sclerosis, Huntington's disease, and Parkinson's disease.

[0016] The ethanolamine is used to inhibit the excessive activation of microglia and reduce the expression of inflammatory factors.

[0017] Compared with the prior art, the beneficial effects of the present invention are:

[0018] The present invention evaluates the inhibitory effect of ethanolamine on microglia activation through a series of experiments, and the results show that ethanolamine can significantly reduce microglia activation and the expression of inflammatory factors by inhibiting the NF-κB pathway, thereby reducing the level of retinal inflammation. Therefore, using ethanolamine for pharmaceutical or health care products can obtain products suitable for preventing, alleviating and treating neuroinflammatory diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The effect of ethanolamine treatment on the expression of inflammatory factors in BV2 induced by hypoxia; wherein:

[0020] A is the comparison of protein expression of inflammatory factors IL-1β and CD11b in cells of the control group and ethanolamine treatment group;

[0021] B is the comparison of the mRNA levels of inflammatory factor IL-1β in the control group and ethanolamine-treated group.

[0022] Figure 2 The immunofluorescence staining results of NFκB distribution in the control group and ethanolamine-treated group cells;

[0023] Control was the normal culture control group; Hypoxia was the hypoxic culture control group; Hypoxia+Ethanolamine was the hypoxic culture+ethanolamine treatment group.

[0024] Figure 3 These are the results of immunofluorescence staining of microglial activation levels in the retina of rats in the control group and ethanolamine-treated group. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0026] Ethanolamine is the second most abundant head group of phospholipids and the main structural component of biological membranes. Phosphatidyl-ethanolamine (PE) exists in all living cells and accounts for 25% of all phospholipids. The present invention provides a new use of Ethanolamine, namely, the use of Ethanolamine in preparing products for preventing, alleviating and / or treating diseases related to neuroinflammation, and the use of Ethanolamine in preparing products for inhibiting neuroinflammation.

[0027] Preferably, the neuroinflammation described in the present invention is neuroinflammation caused by excessive activation of microglia; the neuroinflammation-related diseases include ophthalmic diseases and central nervous system diseases, and further, the ophthalmic diseases include but are not limited to optic nerve damage, diabetic retinopathy, and glaucoma; the central nervous system diseases include but are not limited to multiple sclerosis, Huntington's disease, and Parkinson's disease.

[0028] Preferably, the product comprises medicine and / or health care product.

[0029] The Ethanolamine of the present invention can be used alone when preparing drugs for inhibiting neuroinflammation or drugs for preventing, alleviating and / or treating diseases related to neuroinflammation; it can also be used in the form of a pharmaceutical composition. In addition to containing a therapeutically effective amount of Ethanolamine, the drug also includes a pharmaceutically acceptable carrier or excipient, and the carrier or excipient includes any one or more of a diluent, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, a lubricant, and an excipient. Further, the dosage form of the drug includes oil, granules, tablets, powders, capsules, pills, powders, oral liquids, sols, sprays, atomizers, etc. When used, a safe and effective amount of ethanolamine is applied to a mammal (such as a human). Of course, the specific dose should also consider factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians. The exact effective amount for a subject depends on the subject's body shape and health status, the nature and extent of the disease, and the therapeutic agent and / or combination of therapeutic agents selected for administration. For a given condition, the effective amount can be determined by routine experiments, and clinicians can judge it.

[0030] The active ingredient ethanolamine in the product of the present invention is used to inhibit the excessive activation of microglia and reduce the expression of inflammatory factors.

[0031] The present invention evaluates the inhibitory effect of ethanolamine on microglia activation through cell and animal experiments, and the results show that ethanolamine can significantly reduce microglia activation and the expression of inflammatory factors by inhibiting the NF-κB pathway, thereby reducing the level of retinal inflammation. The therapeutic effect of ethanolamine on diabetic retinopathy is proved, and it can be used to prepare drugs, health products and other products for treating diabetic retinopathy.

[0032] The experimental process and experimental results of the present invention are described in detail below.

[0033] Example 1 Ethanolamine inhibits hypoxia-induced inflammatory response of microglia

[0034] 1. Experimental Methods

[0035] This experiment used hypoxia-induced BV2 cells as an in vitro model to verify the effect of ethanolamine on microglia by detecting the expression of inflammatory factors. The specific experimental process is as follows:

[0036] BV2 cells were seeded in 6-well plates and cultured in high-glucose (containing 4.5 g / L glucose) DMEM culture medium (catalog number 10-013-CV; Corning) containing 10% FBS and penicillin / streptomycin double antibodies. When the cells grew to about 70%, different concentrations of ethanolamine (catalog number: S6210; SELLCK) were added under hypoxic conditions and continued to be cultured. At the same time, a group of cells cultured under conventional conditions without ethanolamine was retained as a control. After 24 hours, the cells were collected and RNA was extracted, and the expression of related inflammatory factors was detected by qRT-PCR and Western blot.

[0037] 2. Experimental results

[0038] The results are as follows Figure 1 As shown in the figure: Compared with the control group, the protein expression of inflammatory factors IL-1β and CD11b in microglia BV2 cells increased significantly under hypoxic conditions, but after ethanolamine treatment, the protein expression of IL-1β and CD11b could be reduced, and the reduction effect became more significant with the increase of concentration ( Figure 1 In addition, the PCR test results also showed that ethanolamine treatment could significantly reduce the mRNA level of inflammatory factor IL-1β ( Figure 1 B). This indicates that ethanolamine can effectively reduce the activation of microglia and the expression of inflammatory factors, thereby inhibiting neuroinflammation caused by excessive microglia activation, thereby achieving the effect of preventing, alleviating and treating neuroinflammation-related diseases.

[0039] Example 2 Ethanolamine inhibits hypoxia-induced NF-κB nuclear translocation in microglia

[0040] 1. Experimental Methods

[0041] NF-κB plays a key role in DR inflammatory response. Hypoxic stimulation of BV2 cells promotes inflammatory response through activation of the NF-κB pathway. To further characterize the molecular mechanism of Ethanolamine inhibiting inflammatory response, we detected the translocation of NF-κBp65, which is a marker of NF-κB activation. The specific experimental process is as follows:

[0042] After hypoxia induction, the culture medium was discarded and the cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature and permeabilized with 0.1% Triton X-100 for 10 minutes at room temperature. The cells were blocked with 5% goat serum for 1 hour at room temperature and then incubated with rabbit anti-NF-κBp65 antibody at 4°C overnight. After washing with PBS three times, they were incubated with Alexa 488-conjugated anti-rabbit secondary IgG (1:500; Thermo Fisher Scientific, A-11008) for 1 hour at room temperature. After washing with PBS three times, they were counterstained with DAPI (Sigma-Aldrich) for 5 minutes. After completion, the coverslips were visualized and photographed under a confocal fluorescence microscope (LSM 510, Carl Zeiss, Gottingen Germany).

[0043] 2. Experimental results

[0044] The results are as follows Figure 2 As shown, the immunofluorescence images showed that in the normal control group, NF-κBp65 was mainly distributed in the cytoplasm, while in the hypoxia-induced group (Hypoxia group), NF-κBp65 was significantly translocated from the cytoplasm to the nucleus, indicating the transcriptional activity of inflammatory factors; and Ethanolamine treatment can greatly reduce the hypoxia-induced NF-κBp65 nuclear translocation from the cytoplasm. The above results show that Ethanolamine can reduce the expression of inflammatory factors in microglia by inhibiting the activation of the NF-κB pathway induced by hypoxia or hypoxia.

[0045] Example 3 Ethanolamine reduces retinal microglial activation in diabetic rats

[0046] 1. Experimental Methods

[0047] All animal experiments were performed in accordance with the ARVO statement for the use of animals in ophthalmic and vision research and adhered to the guidelines required by the Ethics Committee of Shanghai General Hospital, School of Medicine, Shanghai Jiao Tong University, China.

[0048] (1) Diabetic rat modeling

[0049] Male Sprague Dawley rats weighing 120-160g were randomly assigned to a diabetic group and a control group. Rats in the diabetic group were intraperitoneally injected with streptozotocin (STZ, solvent: citrate buffer, 60mg / kg body weight) after fasting for 24 hours; rats in the control group were injected with the same volume of citrate buffer. Animals in the STZ model group were confirmed to be diabetic when their blood glucose exceeded 13.9mmol / L (250mg / dL) for 3 consecutive days.

[0050] (2) Ethanolamine administration

[0051] After the model was successfully established, rats in the diabetic group were repeatedly gavaged with ethanolamine (60 mg / kg body weight) every day (DM+Etha group for short), and rats in the diabetic control group were repeatedly gavaged with an equal volume of ddH2O every day (DM group for short), and untreated age-matched normal rats were used as controls (Con group for short). Rats were killed 4 weeks after the onset of diabetes, and samples were taken for relevant tests.

[0052] (3) Immunofluorescence staining to observe microglial activation levels

[0053] Eyes were fixed with 4% paraformaldehyde for 1 h at 4°C, and the anterior segment including the cornea, iris, and lens was removed. After dehydration with a sucrose gradient, the eye cups were embedded in embedding medium (Sakura Finetek, Tokyo, Japan) and stored at −80°C until sectioning. For sectioning, serial sections (15 μm thick) were made on a cryostat (Leica, Wetzlar, Germany). After cryosectioning at room temperature, the sections were blocked and permeabilized with 1% BSA and 0.1% Triton X-100 in PBS for 1 h at room temperature. The sections were incubated with rabbit anti-IBA-1 antibody (1:500, catalog number 019-19741; WAKO, Osaka, Japan) overnight at 4°C. After washing three times with PBS (10 min each time), the sections were incubated with donkey anti-rabbit IgG H&L (Alexa Fluor 555, 1:500; catalog number ab150074; Abcam, USA) at room temperature for 1 hour; then washed three times with PBS, 10 minutes each time, and the sections were counterstained with DAPI for 5 minutes and mounted and observed under a confocal fluorescence microscope (LSM 510, Carl Zeiss, Gottingen Germany).

[0054] 2. Experimental results

[0055] The results are as follows Figure 3 As shown in the results, compared with the normal control group, the microglia in the retina of diabetic rats were activated and their number increased, while intraperitoneal injection of Ethanolamine could greatly reduce the activation level of microglia. The above results show that Ethanolamine can inhibit the activation of microglia, proving the preventive, alleviating and therapeutic effects of Ethanolamine on diabetic retinopathy.

[0056] In summary, ethanolamine can effectively reduce the activation level of retinal microglia and the expression of inflammatory factors. It can be used to prepare products that inhibit neuroinflammation, and can also be used to prepare drugs and health products for preventing, alleviating and / or treating neuroinflammation-related diseases.

[0057] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. Use of ethanolamine in the preparation of a product for preventing and / or treating neuroinflammatory diseases, characterized in that: The neuroinflammation is neuroinflammation caused by excessive activation of microglia, the neuroinflammation-related disease is an ophthalmic disease, the ophthalmic disease is diabetic retinopathy or glaucoma, and the treatment is early treatment.

2. The use according to claim 1, characterized in that The products described include pharmaceuticals.

3. The use according to claim 2, characterized in that The medicine also includes a pharmaceutically acceptable carrier or excipient.

4. The use according to claim 3, characterized in that The carrier or auxiliary material includes any one or more of a diluent, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, and a lubricant.

5. The use according to claim 3, characterized in that The dosage form of the drug includes any one of oil, granules, tablets, powders, capsules, pills, powders, oral liquids, and sols, or a combination of any two or more thereof.

6. The use according to any one of claims 1 to 5, characterized in that The ethanolamine is used to inhibit the excessive activation of microglia and reduce the expression of inflammatory factors.

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