Application of rosmarinic acid in treatment of cervical spondylotic myelopathy

Abstract

The invention provides application of rosmarinic acid in treatment of cervical spondylotic myelopathy. Specifically, animal experiments and in-vitro cell experiments are carried out by constructing a rat cervical spondylotic myelopathy model, and it is verified that rosmarinic acid has a neuroprotective effect and can inhibit neuronal apoptosis under the conditions of ischemia and hypoxia. Experiments show that the rosmarinic acid can enhance mitochondrial autophagy by activating AMPK-TFEB and PINK1-PRKN signal pathways, so that neurons are protected from ischemia and hypoxia, and neuronal cell apoptosis is inhibited. Based on the invention, the rosmarinic acid or the derivative thereof can be used for preparing the medicine for treating cervical spondylotic myelopathy.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and in particular relates to the application of rosmarinic acid in treating cervical spondylotic myelopathy. BACKGROUND

[0002] Cervical spondylotic myelopathy, also known as degenerative cervical spondylotic myelopathy, is the most serious type of cervical spondylosis. With the development of the disease, patients can have decreased motor function, limb sensory dysfunction, and even paralysis. Existing studies have shown that the prevalence of cervical spondylotic myelopathy treated by surgery is about 1.6 cases per 100,000 people. However, the actual prevalence may be much higher. The high prevalence of cervical spondylotic myelopathy is an increasingly growing social problem that affects the quality of life of millions of middle-aged and elderly people.

[0003] So far, there are few ideal specific drugs that can be used to effectively treat or relieve cervical spondylotic myelopathy.

[0004] Therefore, there is an urgent need in the art to develop a new drug that can effectively treat cervical spondylotic myelopathy or effectively promote neuronal repair and promote recovery from cervical spondylotic myelopathy. SUMMARY

[0005] The present application provides a new drug that can effectively treat and / or relieve cervical spondylotic myelopathy.

[0006] In a first aspect of the present application, there is provided the use of an active ingredient, rosmarinic acid, or a rosmarinic acid derivative, or a pharmaceutically acceptable salt or ester thereof, for the preparation of a composition or a preparation,

[0007] The composition or preparation is used for one or more purposes selected from the group consisting of:

[0008] (a) preventing and / or treating cervical spondylotic myelopathy;

[0009] (b) activating neuronal cell AMPK-TFEB and PINK1-PRKN signaling pathways;

[0010] (c) enhancing mitochondrial autophagy;

[0011] (d) inhibiting neuronal apoptosis;

[0012] (e) promoting recovery of impaired motor function.

[0013] In another preferred embodiment, the composition or preparation is used to inhibit cell damage caused by glucose-oxygen deprivation.

[0014] In another preferred embodiment, the composition or preparation is used to inhibit cell damage caused by ischemia-hypoxia conditions.

[0015] In another preferred embodiment, the composition or formulation is used for enhancing mitochondrial autophagy.

[0016] In another preferred embodiment, the composition or formulation is used alone or in combination in the prevention and / or treatment of cervical spondylotic myelopathy.

[0017] In another preferred embodiment, the combination use comprises a combination use with other drugs for the prevention and / or treatment of cervical spondylotic myelopathy.

[0018] In another preferred embodiment, the composition is a pharmaceutical composition, a dietary supplement, a food composition, or a nutraceutical composition.

[0019] In another preferred embodiment, the composition is a pharmaceutical composition.

[0020] In another preferred embodiment, the formulation is a laboratory formulation.

[0021] In another preferred embodiment, the composition further comprises an additional drug for the treatment of cervical spondylotic myelopathy. In another preferred embodiment, the additional drug for the treatment of cervical spondylotic myelopathy is selected from the group consisting of non-steroidal anti-inflammatory drugs, dehydrating and detumescent drugs, nerve-nourishing drugs, steroid drugs, skeletal muscle relaxant drugs.

[0022] In another preferred embodiment, the pharmaceutical composition is a solid or liquid formulation.

[0023] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of oral formulations, injection formulations, enteric sustained-release formulations, lyophilized formulations.

[0024] In another preferred embodiment, the carrier of the injection formulation is selected from the group consisting of physiological saline, glucose, stabilizers, preservatives, suspending agents, emulsifying agents, or combinations thereof.

[0025] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of tablets, capsules, granules, powders, pastes, powders, injections, water preparations.

[0026] In another preferred embodiment, the dosage form of the pharmaceutical composition is an oral formulation, preferably a tablet, a capsule, or a granule.

[0027] In another preferred embodiment, the administration method of the pharmaceutical composition is selected from the group consisting of subcutaneous administration, intravenous administration, and anorectal administration.

[0028] In another preferred embodiment, the pharmaceutical composition comprises: (a) rosmarinic acid, or a rosmarinic acid derivative, or a pharmaceutically acceptable salt or ester thereof, as an active ingredient; and (b) a pharmaceutically acceptable carrier.

[0029] In another preferred embodiment, the composition or formulation is used for inhibiting neuronal apoptosis.

[0030] In a second aspect of the present application, there is provided a pharmaceutical composition comprising:

[0031] (a1) rosmarinic acid, or a rosmarinic acid derivative, or a pharmaceutically acceptable salt or ester thereof;

[0032] (a2) an optional other drug for preventing and / or treating cervical spondylosis of the spinal cord type; and

[0033] (b) a pharmaceutically acceptable carrier.

[0034] In another preferred embodiment, the component (a1) is present in an amount of 0.1 to 99.9 wt%, preferably 10 to 99.9 wt%, more preferably 70 to 99.9 wt%, based on the total weight of the pharmaceutical composition.

[0035] In another preferred embodiment, the other drug for treating cervical spondylosis of the spinal cord type is selected from the group consisting of non-steroidal anti-inflammatory drugs, dehydrating and detumescent drugs, nerve-nourishing drugs, steroid drugs, skeletal muscle relaxant drugs.

[0036] In another preferred embodiment, the pharmaceutical composition is a solid or liquid formulation.

[0037] In another preferred embodiment, the pharmaceutical composition is in a dosage form selected from the group consisting of oral formulations, injection formulations, enteric sustained-release formulations, lyophilized formulations.

[0038] In another preferred embodiment, the pharmaceutical composition is in an oral formulation.

[0039] In another preferred embodiment, the pharmaceutical composition is in an injection formulation.

[0040] In another preferred embodiment, the pharmaceutical composition is administered in a dose of 45 mg / kg to 55 mg / kg.

[0041] In another preferred embodiment, the pharmaceutical composition is administered in a dose of 50 mg / kg.

[0042] In another preferred embodiment, the carrier of the injection formulation is selected from the group consisting of physiological saline, glucose, stabilizers, preservatives, suspending agents, emulsifying agents, or combinations thereof.

[0043] In another preferred embodiment, the pharmaceutical composition is in a dosage form selected from the group consisting of tablets, capsules, granules, powders, pastes, powders, injections, water preparations.

[0044] In another preferred embodiment, the pharmaceutical composition is in an oral formulation, preferably in a tablet, capsule, or granule.

[0045] In another preferred embodiment, the pharmaceutical composition is administered by a mode selected from the group consisting of subcutaneous, intravenous, and anorectal.

[0046] In another preferred embodiment, the pharmaceutical composition is administered by a mode selected from the group consisting of subcutaneous, intravenous, and anorectal.

[0047] In another preferred embodiment, the pharmaceutical composition is administered by a mode selected from the group consisting of subcutaneous, intravenous, and anorectal.

[0048] In a third aspect of the present application, there is provided a method for inhibiting apoptosis of a neuronal cell in vitro under a sugar oxygen deprivation condition, comprising the steps of:

[0049] (a) culturing the neuronal cell in the presence of rosmarinic acid, thereby enhancing mitochondrial autophagy of the neuronal cell and inhibiting apoptosis of the neuronal cell.

[0050] In another preferred embodiment, the rosmarinic acid enhances mitochondrial autophagy of the neuronal cell by activating the AMPK-TFEB and PINK1-PRKN signaling pathways.

[0051] In another preferred embodiment, the neuronal cell is derived from a mammal.

[0052] In another preferred embodiment, the neuronal cell is derived from a mammal.

[0053] In another preferred embodiment, the mammal is a human or a non-human mammal.

[0054] It should be understood that, within the scope of the present application, each of the technical features of the present application described above and each of the technical features specifically described below (e.g., in the examples) can be combined with each other to form a new or preferred technical solution. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0055] Figure 1 A chemical structural formula of rosmarinic acid is shown.

[0056] Figure 2 A graph showing the behavior score of rats with cervical spondylotic myelopathy is shown.

[0057] Figure 3 A graph showing HE staining at multiple time points in the lesion site of rats with cervical spondylotic myelopathy is shown.

[0058] Figure 4 A graph showing immunofluorescence staining of apoptosis indicators at multiple time points in the lesion site of rats with cervical spondylotic myelopathy is shown.

[0059] Figure 5The immunofluorescence staining images of the path indicators at multiple time points in the lesion site of the rats with cervical spondylotic myelopathy are shown.

[0060] Figure 6 The CCK8, Western blot and immunofluorescence result images of PC12 cells after hypoxia (or sugar oxygen deprivation, ischemia and hypoxia) modeling are shown. DETAILED DESCRIPTION

[0061] The present inventors have made extensive and in-depth research and for the first time accidentally found that under hypoxia (or sugar oxygen deprivation, ischemia and hypoxia) conditions, rosmarinic acid can significantly enhance mitochondrial autophagy in neuron cells. Specifically, the present inventors have found that under hypoxia (or sugar oxygen deprivation, ischemia and hypoxia) conditions, rosmarinic acid can activate the AMPK-TFEB and PINK1-PRKN signaling pathways, enhance mitochondrial autophagy, and thus can effectively promote neuron repair and inhibit apoptosis of neuron cells. Therefore, it can be used for effectively treating CSM. On this basis, the present application is completed.

[0062] TERMS

[0063] In order that the disclosure can be more readily understood, certain terms are first defined. As used in this application, unless specifically stated otherwise, each of the following terms has the meaning given below. Additional definitions are set forth throughout the application.

[0064] The term "about" can refer to a value or a composition that is within an acceptable error range for the specific value or composition determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. For example, as used herein, the expression "about 100" includes all values between 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0065] As used herein, the term "containing" or "including" can be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0066] As used herein, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range is to be understood to include the entire range of values as well as any value within that range (e.g., one tenth and one hundredth of an integer) as appropriate.

[0067] As used herein, the term "and / or" relates to and encompasses any and all possible combinations of one or more of the associated listed items.

[0068] As used herein, "PC12 cells" refer to a cell line derived from a transplantable mouse pheochromocytoma that has a reversible neuronal phenotypic response to nerve growth factor (NGF).

[0069] As used herein, in the embodiments of the present application, M is molar concentration, i.e. mol / L; μM is micromole per liter.

[0070] Rosmarinic acid

[0071] As used herein, "Rosmarinic acid", "RA" are used interchangeably, is a natural polyphenol compound from many common herbs, which has been proved to play a role in neuron protection and activation of autophagy. Its chemical structural formula is shown in Figure 1 The rosmarinic acid used in the present application is commercially available or isolated from Rosmarinus officinalis of Labiatae according to the extraction method commonly used in the field of traditional Chinese medicine.

[0072] In the present application, rosmarinic acid can be administered in the form of its pharmaceutically acceptable salt or derivative. Representative pharmaceutically acceptable salts include salts formed by rosmarinic acid with alkali metals (such as Na, K) or alkaline earth metals (such as Ca, Mg, Fe) or ammonium ion, etc.

[0073] Representative derivatives of rosmarinic acid include derivatives formed by rosmarinic acid with acids and / or alcohols. The hydroxyl group of rosmarinic acid can form ester or ester bond derivatives with various groups, for example, one or more hydroxyl groups of rosmarinic acid can form esters with organic acids (such as acetic acid, aspartic acid, formic acid, citric acid, benzoic acid, malic acid, maleic acid, tartaric acid and lactic acid, etc.). In addition, the carboxyl group of rosmarinic acid can form ester or amide bond derivatives with various groups, for example, the carboxyl group of rosmarinic acid can form esters with C1-C6 lower alcohols (such as methanol, ethanol, propanol, butanol) or C6-C10 alkyl alcohols. In addition, the hydroxyl group of rosmarinic acid can be deprotonated, and a sugar group (such as monosaccharides, disaccharides composed of glucose, fructose and sucrose) can also be introduced.

[0074] In the present application, rosmarinic acid and its derivatives or its pharmaceutically acceptable salt or ester can be administered in the form of a pure substance (purified compound) or an extract.

[0075] Cervical spondylotic myelopathy

[0076] The pathological features of cervical spondylotic myelopathy are summarized as degeneration of intervertebral disc and its surrounding structures, leading to compression of the cervical spinal cord, ischemia and hypoxia of the spinal cord, and finally pathological changes such as neuron apoptosis, causing spinal cord dysfunction. The pathological mechanism of cervical spondylotic myelopathy is a dynamic process, involving different time-dependent processes such as inflammation, neuron apoptosis and scar formation.

[0077] Mitophagy

[0078] Mitophagy refers to the specific degradation of polarized or damaged mitochondria through the autophagy process, which maintains the homeostasis of mitochondria by removing damaged mitochondria. Under chronic spinal cord compression conditions, hypoxia induces oxidative stress, and the accumulation of reactive oxygen species (ROS) leads to mitochondrial depolarization and fragmentation, resulting in mitochondrial dysfunction, thereby inducing early enhancement of mitophagy.

[0079] In this state, mitophagy plays a neuroprotective role by accelerating the removal of fragmented and damaged mitochondria. However, sustained ischemia and hypoxia conditions lead to the massive production of ROS and mitochondrial damage, thereby impairing mitophagy. Then, the increase in mitochondrial membrane permeability promotes the cytosolic release of cytochrome C and activates caspase-3, subsequently inducing neuronal apoptosis.

[0080] The role of mitophagy in the progression of cervical spondylotic myelopathy has not been extensively studied.

[0081] AMPK-TFEB and PINK1-PRKN signaling pathways

[0082] PINK1 is a protein kinase synthesized in the cytoplasm and is hydrolyzed by related proteases under normal physiological conditions. When mitochondria are damaged or stressed, the hydrolysis of PINK1 is inhibited, anchoring it to the outer membrane of mitochondria, thereby playing a role in recruiting PRKN / Parkin to the outer membrane and phosphorylating it. Ubiquitinated PRKN can further ubiquitinate mitochondrial outer membrane substrate proteins. Ubiquitinated mitochondria can be recognized by SQSTM1 / p62 and combined with LC3, gradually forming autophagosomes, promoting the occurrence of mitophagy.

[0083] AMPK is known as the "energy regulator" of cells. When the body is in energy crisis such as ischemia, hypoxia, and oxidative stress, this enzyme can be activated to maintain the energy balance of the whole body by regulating metabolism. TFEB and TFE3 are downstream of AMPK and can regulate lysosome biogenesis and autophagy. AMPK can promote the nuclear translocation of TFEB to activate autophagy by directly dephosphorylating TFEB.

[0084] Studies have shown that the activation of the AMPK-TFEB signaling pathway can recruit PINK1 protein to mitochondria, inducing mitophagy, while the absence of TFEB prevents PINK1 protein from anchoring to mitochondria, leading to impaired mitophagy. Therefore, targeting the regulation of the AMPK-TFEB signaling pathway can promote mitophagy, remove fragmented mitochondria and their products, and improve related pathological changes such as inflammation and apoptosis.

[0085] Compositions and uses

[0086] The present application also provides a variety of different compositions containing the active ingredients of the present application, including, but not limited to, pharmaceutical compositions, dietary supplements, food compositions, nutraceutical compositions

[0087] As used herein, the term "active ingredient" in the context of a pharmaceutical composition refers to the rosmarinic acid, or rosmarinic acid derivative, or pharmaceutically acceptable salt or ester thereof, useful in the present application.

[0088] As used herein, the term "effective amount" or "effective dose" refers to an amount that is functional or active and acceptable to a human and / or animal.

[0089] As used herein, the term "pharmaceutically acceptable" refers to those substances that are appropriate for use with humans and / or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit / risk ratio, i.e., substances that are acceptable to the U.S. Food and Drug Administration as well as the European Medicines Agency. The term "pharmaceutically acceptable carrier" refers to a carrier for therapeutic administration including various excipients and diluents.

[0090] The pharmaceutical compositions of the present application comprise a safe and effective amount of the active ingredients of the present application and a pharmaceutically acceptable carrier. Such carriers include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof. Typically, the pharmaceutical preparation will be matched to the mode of administration, and the pharmaceutical compositions of the present application are in the form of injectables, oral preparations (tablets, capsules, oral solutions), transdermals, sustained release formulations. For example, they are prepared by conventional methods using, for example, physiological saline or aqueous solutions containing glucose and other adjuncts. The pharmaceutical compositions are preferably manufactured under aseptic conditions.

[0091] The effective amount of the active ingredients of the present application can vary depending on the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by one of ordinary skill in the art (e.g., through clinical trials) based on various factors. Such factors include, but are not limited to, the pharmacokinetic parameters of the active ingredients such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the weight of the patient, the patient's immune status, the route of administration, etc. Generally, satisfactory results are indicated to be obtained when the active ingredients of the present application are administered at a dosage of about 0.00001 mg to 50 mg / kg, preferably 0.0001 mg to 10 mg / kg, of animal body weight per day. For example, several divided doses can be administered daily, or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0092] The pharmaceutically acceptable carriers described in the present application include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptide substances, cellulose, nanogels, or combinations thereof. The selection of the carrier should be matched with the administration mode, which is well known to those skilled in the art.

[0093] The main advantages of the present application include:

[0094] (a) The present application verifies that rosmarinic acid can restore the hind limb motor dysfunction of rats after chronic compression of the cervical spinal cord, enhance the mitochondrial autophagy of neurons in the compressed part of the cervical spinal cord gray matter, and reduce neuronal apoptosis, and has good application prospects in inhibiting the occurrence and development of cervical spondylotic myelopathy.

[0095] (b) The present application verifies that rosmarinic acid can enhance the PINK1-PRKN-mediated mitochondrial autophagy regulated by AMPK-TFEB of neurons in a hypoxic environment, thereby inhibiting the apoptosis of neuronal cells and playing a therapeutic role.

[0096] The present application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. The experimental methods in the following examples are not specified, and the methods are usually carried out according to the conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0097] Example 1 animal model experiment

[0098] 1.1 Experimental method

[0099] 56 SD rats (8-9 weeks old, weighing 350±30g, purchased from Zhejiang Vintone Lihua Experimental Animal Co., Ltd.) were divided into 7 groups (8 rats in each group) according to the random number table method, namely: sham operation group, 3 days after operation group, 7 days after operation group, 28 days after operation group, 3 days after rosmarinic acid intervention group, 7 days after rosmarinic acid intervention group, and 28 days after rosmarinic acid intervention group.

[0100] The rats in the cervical spinal cord chronic compression operation model group and the rosmarinic acid intervention group were anesthetized with 30mg / kg sodium pentobarbital, and then the C4-C7 lamina was exposed by incision of the skin. Then, the yellow ligament between C5-C6 lamina was removed. Under a microscope, 1×1×1mm 3 Hydrogel. The hydrogel swells to 2×2×2mm in about 48-72 hours3 , a model of spinal cord compression.

[0101] The rats in the rosemary acid intervention group were injected with 50 mg / kg rosemary acid (structure shown) intraperitoneally at 1 day after the operation, and the rats in the chronic compression of the cervical spinal cord group were injected with the same amount of normal saline. The same operation was performed on the rats in the sham operation group, but no gel was implanted, and no drug injection treatment was performed. Figure 1

[0102] Behavioral tests were performed at three time points of 3, 7, and 28 days after the operation, including body weight measurement, BBB score evaluation of the hind limbs of the rats, incline board test, forelimb grip strength test, and rotarod fatigue test.

[0103] At three time points of 3, 7, and 28 days after the operation, the rats in the corresponding groups were anesthetized with 30 mg / kg sodium pentobarbital, perfused through the heart, and the cervical spinal cord was taken for pathological and immunofluorescence staining and Western Blot detection.

[0104] 1.2 Experimental results

[0105] The behavioral scores are shown in Table 1. The rats were evaluated for limb movement and balance ability using the BBB score scale, incline board test, forelimb grip strength test, and rotarod fatigue test. Each animal was tested three times for statistical analysis. Figure 2 The statistical results showed that the body weight of the rats in the chronic compression of the cervical spinal cord group decreased significantly compared with the sham operation group, reaching the lowest point at 7 days. From 7 to 28 days after the operation, the body weight of the rats in the chronic compression of the cervical spinal cord group gradually recovered. The body weight of the rats in the rosemary acid intervention group decreased more slowly than that in the chronic compression of the cervical spinal cord group, reaching the lowest point at 3 days after the operation, and then gradually recovering. The body weight was always higher than that in the chronic compression of the cervical spinal cord group, and the difference was statistically significant.

[0106] The behavioral results showed that the bilateral BBB scores and the maximum inclination angle of the incline board test of the rats in the chronic compression of the cervical spinal cord group decreased to the lowest point at 3 days after the operation, and then gradually recovered to 28 days after the operation, but were still lower than those in the sham operation group, and the difference was statistically significant. The behavioral scores of the rats in the rosemary acid intervention group were significantly higher than those in the chronic compression of the cervical spinal cord group at 3 days after the operation, and remained so until 28 days after the operation.

[0107] The rotarod fatigue test results showed that the rotarod time and distance of the rats in the chronic compression of the cervical spinal cord group were significantly lower than those in the sham operation group, indicating that the impaired motor function had not recovered. Although the above indicators of the rats in the rosemary acid intervention group had not recovered to the normal level, there was a significant difference compared with the rats in the chronic compression of the cervical spinal cord group.

[0108] HE staining of the cervical spinal cord of the rats at different days after spinal cord compression is shown in Figure 1.

[0109] Figure 3 ​​The results show that rosmarinic acid can improve the pathological damage of the spinal cord 3 to 28 days after modeling.

[0110] The results of immunofluorescence staining and the ratio of TUNEL, Bax and Bcl-2 positive neurons of the rat spinal cord at different days after spinal cord compression are shown in Figure 4 According to the relative expression and proportion of apoptosis-related protein positive neurons, it is proved that rosmarinic acid can inhibit neuronal apoptosis.

[0111] The results of the mitochondrial autophagy pathway indicators affected by the rat spinal cord compression are shown in Figure 5 As shown in the results, rosmarinic acid activates the PINK1-PRKN signaling pathway and enhances the level of mitochondrial autophagy in chronic cervical spinal cord compression model rats.

[0112] According to the results of Western Blot and immunofluorescence staining, immunofluorescence staining shows that the proportion of AMPK / TFEB / TFE3 positive cells in the rosmarinic acid intervention group is significantly higher than that in the model group, and the difference is statistically significant from the 7th day to the 28th day. Western Blot results show that rosmarinic acid can continuously activate the pathway molecules related to the AMPK-TFEB and PINK1-PRKN signaling pathways, and the effect is obvious on the 3rd day after the operation; the results of laser confocal microscope scanning show that from the 3rd day to the 28th day after the chronic cervical spinal cord compression operation, the mitochondria and PINK1 of the compressed neurons in the gray matter of the spinal cord of the rosmarinic acid intervention group rat are co-localized, and the co-localization rate of mitochondria and lysosome and LC3 is higher than that of the chronic cervical spinal cord compression group, and the difference is statistically significant, indicating that the level of mitochondrial autophagy is continuously enhanced after the administration of rosmarinic acid.

[0113] These results show that rosmarinic acid can continuously activate the AMPK-TFEB and PINK1-PRKN signaling pathways after injury, enhance mitochondrial autophagy, restore impaired motor function, and can effectively play a therapeutic role.

[0114] Example 2 cell experiment

[0115] 2.1 Experimental method

[0116] PC12 cells were taken and cultured in a carbon dioxide incubator at 37°C with complete culture medium.

[0117] Before the experiment intervention, 10 ng / ml of NGF (inducing directional differentiation of neuron cells) was added and cultured for 72-96 h, and after more than 80% of the cells showed neuron appearance reaction, the intervention was performed.

[0118] The cells were divided into: control group (i.e. ordinary culture medium group), model group (i.e. sugar and oxygen deprivation, simulating the ischemia and hypoxia environment of spinal cord neuron cells), blank intervention group (ordinary culture medium rosmarinic acid 10 μM intervention) group, and treatment group (rosmarinic acid 10 μM intervention after modeling) group.

[0119] The control group cells were not treated, the rosemary acid intervention group was pretreated with different concentrations of rosemary acid (10, 50 and 100 μM) for 6 h, and then the model group cells were added with hypoxic treatment (placed in a three-gas incubator environment for 30 min: 37℃, 94% N2, 5% CO2, 1% O2) DMEM sugar-free medium, and placed in a three-gas incubator at 37℃, 94% N2, 5% CO2, 1% O2 environment for different times.

[0120] Western Blot and cell immunofluorescence techniques were used to detect the apoptosis and mitochondrial autophagy-related indicators of PC12 cells.

[0121] 2.2 Experimental results

[0122] The experimental results are shown in Figure 6 Rosemary acid can enhance the AMPK-TFEB regulated PINK1-PRKN mediated mitochondrial autophagy of neurons under hypoxic (or glucose-oxygen deprivation, ischemia-hypoxia) environment, thereby inhibiting the apoptosis of neuronal cells, and thus playing a therapeutic role.

[0123] First, CCK8 experiment was performed to determine the safe concentration range of rosemary acid. There was no obvious cytotoxicity below 500 μM, and 10 μM, 50 μM and 100 μM were selected as the intervention concentrations.

[0124] The Western Blot results showed that compared with the control group, the apoptosis of neurons was significantly enhanced after modeling, as shown by the up-regulation of the expression of apoptosis proteins Bax and cleaved caspase-3 and the down-regulation of the expression of anti-apoptotic protein Bcl-2. After rosemary acid administration, the apoptosis level of cells was down-regulated in a dose-dependent manner, and the difference was statistically significant.

[0125] At the same time, the intervention of 100 μM rosemary acid did not increase the apoptosis of PC12 cells under normal culture conditions. The above results suggest that rosemary acid can inhibit the apoptosis of PC12 cells under hypoxic (or glucose-oxygen deprivation, ischemia-hypoxia) environment in a dose-dependent manner.

[0126] Similarly, Western Blot experiment was used to verify the effect of rosmarinic acid on mitochondrial autophagy and the influence of related signal pathways. The results showed that, compared with the control group, the expression of p-AMPK, TFEB and TFE3 in the model group was significantly up-regulated, indicating the activation of AMPK-TFEB signal pathway. At the same time, the expression of PINK1, p-PINK1 and the ratio of LC3II / I were also significantly up-regulated, and the expression of TOMM20 and SQSTM1 was significantly down-regulated, which had statistical significance, indicating that PINK1-PRKN mediated mitochondrial autophagy was enhanced, which was considered as a protective mechanism of neuronal cells. And rosmarinic acid intervention could further activate AMPK-TFEB signal pathway in a dose-dependent manner, enhance PINK1-PRKN mediated mitochondrial autophagy, and gradually up-regulate the expression level of corresponding proteins, which had statistical significance.

[0127] To observe the effect of rosmarinic acid on mitochondrial autophagy of primary neurons in the sugar-oxygen deprivation environment, the AMPK-TFEB signal pathway related indicators for regulating mitochondrial autophagy were detected by immunofluorescence staining.

[0128] The results showed that, consistent with the previous experimental results, the nuclear translocation of p-AMPK, TFEB and TFE3 was significantly enhanced after modeling, and the number of positive neurons was increased. Rosmarinic acid administration could further increase the nuclear translocation of p-AMPK, TFEB and TFE3 in neurons under sugar-oxygen deprivation conditions, and increase the number of positive neurons, and the results had significant. It is suggested that under hypoxic conditions, rosmarinic acid can inhibit neuronal apoptosis by activating the AMPK-TFEB signal pathway and enhancing PINK1-PRKN dependent mitochondrial autophagy.

[0129] All the documents mentioned in the present application are cited as references in the present application, just as each document is cited as a reference. In addition, it should be understood that those skilled in the art can make various modifications or modifications to the present application after reading the above teaching of the present application, and these equivalent forms also fall within the scope defined by the claims attached to the present application.

Claims

1. Use of an active ingredient or a preparation containing the active ingredient, characterized in that A composition or a preparation for preparing a composition or a preparation, the active ingredient being rosmarinic acid, or a rosmarinic acid derivative, or a pharmaceutically acceptable salt or ester thereof, The composition or the preparation is for one or more uses selected from the group consisting of: (a) preventing and / or treating cervical spondylotic myelopathy; (b) activating neuronal cell AMPK-TFEB and PINK1-PRKN signaling pathways; (c) enhancing mitochondrial autophagy; (d) inhibiting neuronal apoptosis; (e) promoting recovery of impaired motor function.

2. Use according to claim 1, characterized in that, The composition or the preparation is for inhibiting cell impairment caused by glucose-oxygen deprivation.

3. Use according to claim 1, characterized in that, The composition is a pharmaceutical composition, a dietary supplement, a food composition, or a health product composition.

4. Use according to claim 3, characterized in that, The composition is a pharmaceutical composition.

5. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises: (a1) rosmarinic acid, or a rosmarinic acid derivative, or a pharmaceutically acceptable salt or ester thereof; (a2) optionally, other drugs for preventing and / or treating cervical spondylotic myelopathy; and (b) a pharmaceutically acceptable carrier.

6. The pharmaceutical composition of claim 5, wherein The component (a1) accounts for 0.1-99.9 wt%, preferably 10-99.9 wt%, more preferably 70%-99.9 wt% of the total weight of the pharmaceutical composition.

7. The pharmaceutical composition according to claim 5, wherein The other drugs for preventing and / or treating cervical spondylotic myelopathy are selected from the group consisting of non-steroidal anti-inflammatory drugs, dehydrating and detumescent drugs, nerve-nourishing drugs, steroid drugs, and skeletal muscle relaxant drugs.

8. The pharmaceutical composition of claim 5, wherein The dosage form of the pharmaceutical composition is selected from the group consisting of oral preparations, injection preparations, enteric sustained-release preparations, and lyophilized preparations.

9. A method of inhibiting apoptosis in neuronal cells in vitro under conditions of glucose oxygen deprivation, characterized in that, The method comprises the step of: (a) culturing neuronal cells in the presence of rosmarinic acid, thereby enhancing mitochondrial autophagy of the neuronal cells and inhibiting apoptosis of the neuronal cells.

10. The method of claim 9, wherein, The rosmarinic acid enhances mitochondrial autophagy of the neuronal cells by activating AMPK-TFEB and PINK1-PRKN signaling pathways.

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