WO2002017931A1 - Use of tripterygium wilfordii hook.f's extracts for preparation of medicaments for preventing and treating nervous system disorde rs - Google Patents

Abstract

This invention relates to the use of one or more extracts of Tripterygium Wilfordii Hook.f for the preparation of medicaments for preventing and treating nervous system disorders, said Tripterygium Hook. f's extracts selected from: Triptolide, tripchloride, tripdiolide, triptriolide, 16-hydroxytriptolide, triptolidemol, triptolidenol, tripterine and wilfortrine. These tripterygium wilfordii' extracts have immunosuppressive activity, as well as have significant nutritional effect of dopaminergic (DA) neuron. It can stimulate the growth of mesenphalic nerve cells, and stimulate elongation of process of primary cultured cortical nerve cells, they can antagonized the injury effect of exogenous toxin, endogenous toxin and excitatory nerve toxin on the nerve cells, and have remarkable protection on cell survival.

Description

 Use of tripterygium plant extract in prevention and treatment of nervous system diseases

 The present invention relates to the use of tripterygium plant extracts in the preparation of a medicament for the prevention and treatment of neurological diseases. The neurological diseases include Alzheimer's disease, Parkinson's disease, and Huntington's neurodegenerative disease. The invention also relates to a medicament for the prevention and treatment of neurological diseases. technical background

 Senile neurodegenerative diseases are a type of neurological diseases that appear with age and are characterized by progressive necrosis of certain neurons in a specific region of the brain parenchyma, with learning and memory disorders or motor, behavioral, and psychological disorders as the main manifestations. Among them, Alzheimer's disease (AD) and Parkinson's disease (PD) are the most common, with a high disability rate and the greatest impact on the health of the elderly.

 The PD is a common extrapyramidal degenerative disease of the nervous system that occurs frequently in the middle and old age, and patients often experience symptoms such as tremor, muscle stiffness, and bradykinesia. The main pathological change of PD is dopamine (DA) in the mesencephalic and substantia nigra striatum pathways, which can cause neuronal degeneration and necrosis, leading to a significant reduction in striatum DA content. Therefore, supplementation with DA precursor L-DOPA (L-DOPA) can alleviate the symptoms of PD. However, L-DOPA itself cannot delay the further necrosis of dopaminergic neurons and has other side effects. Therefore, people have been trying to find a drug that can delay the degeneration and necrosis of DA neurons and have nutritional protective effects on DA neurons.

 Neurotrophic factors (NTFs) are a class of specific peptides or proteins that maintain and promote the normal survival, growth and differentiation of nerve cells and promote their regeneration in the case of nerve damage. Including nerve growth factor (NCF), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), neurotrophic Factor-3 (neuro'trophic-3, NT-3) and so on. Among them, GDNF can be more specific-nutritionally and protect DA neurons, and can promote the regeneration of DA neurons, which has become a hot spot in the current prevention and treatment of PD.

 However, NGF, GDNF, BDNF, etc. are macromolecular proteins that cannot penetrate the blood-brain barrier and should not be administered peripherally. There are two ways to solve this difficulty: 1. The foreign GDNF gene is introduced into the brain by vectors such as engineered cells and adenovirus, so that it can express and secrete GDNF protein for a long time, and play a role in nutrition, protection and repair; 2. Look for small molecules that have neurotrophic effects or promote the expression of endogenous neurotrophic factors.

Alzheimer's disease, also known as senile dementia, is a primary cerebral degenerative disease that occurs in middle-aged and elderly people. The main clinical manifestations are cognitive and learning and memory dysfunction, and even emotional or personality defects. The main pathological changes of AD are senile plaques (also known as amyloid plaques), nerve fiber tangles, and selective cholinergic neurons and synaptic loss. Pathological anatomy showed that neurons in the neocortex, hippocampus, Menet basal nucleus, and blue nucleus of AD patients had a large number of neurons, especially the reduction of acetylcholinergic nerves in the cortex and hippocampus.

 Modern research suggests that acetylcholine (ACh) is a neurotransmitter that promotes learning and memory, and M-cholinergic synapses are the basis of memory. Degeneration of cholinergic neurons is considered to be an important pathological factor causing dementia. Therefore, drugs that can prevent cholinergic neurodegeneration are the first choice for future AD treatment. NGF has been shown to be effective in preventing degeneration and death of basal forebrain cholinergic neurons in animals that mimic AD disease. However, due to the large molecular weight of NGF, it is difficult to reach the brain by oral or injection. Some people tried to repeatedly inject NGF into the brain with lateral ventricle intubation, and received positive results. In order to solve the problems caused by repeated intracerebral administration, in recent years, researchers have been trying to find alternative routes for NGF administration. These pathways include: 1. Transplantation of genetically engineered NGF-producing cells in the brain; 2. Drugs that promote NGF biosynthesis in the brain.

 Recent studies have found that the immunosuppressants cyclosporin A (CsA), FK506, and Rapamycin (RAPA) also have neurotrophic effects. In vitro experiments by Snyder et al. Showed that very small amounts (ng-grade) of immunosuppressants FK506 and RAPA can cooperate with neurotrophic factor (NGF) to promote the axonal growth effect of PC12 cells, enabling NGF to induce an effective concentration of optimal axonal growth effect 1ng / ml。 Reduced by 20-50 times, half the optimal effect dose was also reduced to 0.1 ng / ml. On rat dorsal root ganglion cells cultured in vitro, FK506 alone can produce significant neurotrophic effects. It is believed that the mechanism is still based on NGF produced by glial cells such as Schwann cells in ganglia.

 In vivo experiments have also shown that CsA can slow the degeneration of DA neurons induced by 6-hydroxydopamine (6-HDA) in the mouse brain. Similarly, FK506 and CsA can also protect against DA depletion in MP57-induced C57 / BLACK Parkinson's disease mice. Both CsA and FK506 can significantly increase the content of DA and D0PAC in the striatum, which is of significant significance. Of the two, FK506 is particularly important. CsA 'also protects against lack of brain tissue' after repeated blood perfusion injury.

At present, the mechanism of neurotrophic effects of immunosuppressants is that CsA, FK506, and RAPA act on their respective intracellular receptors. CsA corresponds to Cyclophilin (Cyp), and FK506 and RAPA correspond to FK506 binding proteins. (FK506 binding protein, FKBP). The immunosuppressant specifically forms a complex with FKBP and Cyp, and then binds to phospholipase 2B (Calcineurin, CaN), which inhibits the activity of this enzyme to catalyze protein dephosphorylation. Further research found that CaN affects the phosphorylation of two enzymes in the brain: 1. Nitric oxide synthase (N0S), the phosphorylated form of N0S will inhibit its catalytic activity, and immunosuppressants can increase NOS by The degree of phosphorylation inhibits the generation of NO to block the neurotoxic effects of excessive glutamate; 2. Growth-associated protein (GAP-43). GAP-43 is involved in the growth of neurons and its phosphorylated form can enhance this growth-promoting effect. Axon elongation can occur in PC12 cells under NGF stimulation. Very low concentration (nmol level) of FK506 can increase the sensitivity of cells to NGF, induce axonal growth effect of PC12 cells, and increase the effect of NGF by 100 times. Therefore, drugs that selectively inhibit certain substrates of CaN, such as inhibitors of GAP-43 dephosphorylation, may have significant potential therapeutic value in the treatment of neurodegenerative diseases.

 Tripterygium is a plant belonging to the genus Celastraceae (Tripterygium Wilfordii Hook, f). Tripterygium also includes T. Hypoglacum (Levi) Hutch and T. Regelli Sprague et Tak. , Have medicinal value. Tripterygium wilfordii contains alkaloids, diterpenes, triterpenes, and sesquiterpenes. Among them, diterpenes are the main active ingredients, and triterpenes and alkaloids are also active. Currently known monomeric constituents of tripterygium include triptolide, triptolide, triptolide, tripdiolide, and tripteride compounds such as triptolide and tripterylide Triterpenes such as tripterine are shown in Figure 1.

The extract of Tripterygium wilfordii has various pharmacological activities such as immunosuppressive, anti-inflammatory, anti-tumor and anti-fertility, especially the immunosuppressive effect is its main pharmacological activity. 06mg / kg。 The strongest immunosuppressive activity of the monomer component extracted from tripterygium wilfordii is triptolide, and its immunosuppressed ED ₅₀ ¾ 0. 06mg / kg. Tripterygium decoction, triptolide "total glycosides" (TII) or triptolide and triptolide have significantly inhibited the effects of concanavallin A (ConA) -induced mouse spleen cell secretion of IL-2 effect.

 In addition, triptolide chlorolactone, one of the main active ingredients of tripterygium wilfordii, has a dose-dependent two-way regulation effect on mouse spleen NK cell activity, indicating that tripterygium wilfordii is not only immunosuppressive.

 Previously, the total extract of Tripterygium wilfordii was used clinically to treat rheumatoid arthritis and nephropathy such as chronic glomerulonephritis, nephrotic syndrome, and allergic purpura nephritis. In addition, it has certain effects on connective tissue diseases such as systemic lupus erythematosus, polymyositis, dermatitis, and some skin diseases. However, the total extract of tripterygium wilfordii or its single body component has been used as a neuron protective agent for the prevention and treatment of senile neurodegenerative diseases, and there have been no reports so far. Summary of invention

 An object of the present invention is to provide use of a tripterygium plant extract in the manufacture of a medicament for preventing and treating a nervous system disease.

 Another object of the present invention is to provide a medicament for preventing and treating a nervous system disease.

Therefore, the present invention provides a kind selected from triptolide, triptolide, triptolide, Triptolide triol, 16-hydroxy triptolide, triptolide, triptolide, and one or more of triptolide and triptolide are used in the preparation Application in medicine for preventing and treating neurological diseases.

 The neurological disease may be Alzheimer's disease, Parkinson's disease, Huntington's neurodegenerative disease and spinal cord injury, or lateral spinal cord sclerosis disease.

 The present invention also provides a medicament for the prevention and treatment of neurological diseases, which contains a drug selected from the group consisting of triptolide, triptolide, triptolide glycol, triptolide triol, 16-hydroxy triptolide Extracts of triptolide, triptolide, triptolide, and one or more of triptolide and triptolide. ·

 The above-mentioned medicine of the present invention may further contain a neurotrophic factor, so as to achieve a better therapeutic effect. The neurotrophic factor may be a nerve growth factor, a glial-derived neurotrophic factor, a brain-derived neurotrophic factor, and / or a ciliary neurotrophic factor.

In order to find immunosuppressive agents with neuroprotective and nutritional effects from natural medicines, a variety of single-agent and compound natural medicines with immunosuppressive activity currently used in clinical evaluation were evaluated. The monomer component in the serotonin has significant immunosuppressive activity under both retired and in vivo experimental conditions. Further in vivo and in vitro studies have shown that as the main active ingredient in tripterygium wilfordii extract, triptolide lactone (hereinafter referred to as 968) has a significant nutritional effect on cultured DA energy neurons. Compared with cyclosporin as a control group, 968 was cultured in DMEM containing 10% fetal bovine serum for 4 days at a lower concentration (0.01 ng / ml) and a higher concentration (0.1 ng / ml). surviving DA neurons than the control group were 87% and 29% lower than the additional 10- ¹³ mol / L to 968 can promote the growth of nerve cells, neurite length compared with the control group the 113% Gao. The neurotrophic effect of 968 does not seem to be confined to DA energy neurons. At a very low concentration, 968 can promote the elongation of the primary cultured cortical neurons. This axonal growth-promoting effect of 968 is of great significance for the reconstruction of synapses between nerve cells in diseases such as PD, AD and spinal cord injury.

Another important feature of triptolide is its ability to antagonize the damage to nerve cells caused by some neurotoxins. Environmental toxins, endogenous toxins, and excitatory neurotoxins are one of the important mechanisms for the pathogenesis of neurodegenerative diseases such as PD and AD. It was found in the experiment that: (1), ΙρΜ triptolide and triptolide (T _1Q ) can antagonize the toxic effect of DA neurotoxin MPP + on PC12 and have a significant protective effect on cell survival; (2) ) 968 can resist the damage of primary cortical neurons by large doses of excitatory glutamic acid, which is conducive to maintaining the integrity of nerve cell morphology and reducing apoptosis induced by glutamic acid; (3), in whole animals It was found in the experiment that 968 can effectively prevent DATP neuronal damage induced by DATP neurotoxin MPTP in the mouse brain. Brief description of the drawings

 The invention is further described below with reference to the drawings and specific experiments.

 Figure 1 shows the chemical structure of the main active monomer in the extract of Tripterygium wilfordii. Among them, (1) is triptolide, (2) is triptolide, (3) is triptolide triol, (4) is triptolide, (5) is triptolide diol, (6) is 16-hydroxytriptolide, and (7) is triptolide.

 Figure 2 shows the effect of different concentrations of 968 on the survival of dopaminergic neurons in the brain of primary cultured rats. Counted dopaminergic neurons are TH immunohistochemically positive cells. Each point of data is the average count of three parallel experiments.

 Figure 3 shows the effect of different concentrations of 968 on the survival percentage of rat dopaminergic neurons in the primary culture.

 Figure 4 shows the effects of pretreatment with different doses of 968 on DA content in striatum of C57BL / 6J mice damaged by MPTP.

 Figure 5 shows the effect of pretreatment with different doses of 968 on DA metabolic rate (DOPAC + HVA / DA) in the striatum of C57BL I 6J mice damaged by MPTP. Experiment 1: In vitro Study of Triptolide on Protecting Dopaminergic Neurons

SD pregnant rats with a gestational age of 14 to 17 days were decapitated and sacrificed. The embryonic rat midbrain was cultured in a 24-well cell culture plate for primary culture of DA brain neurons. The next day, serum medium or serum-free medium was changed and different concentrations of 968 (10, 1, 0.1, 0.01, 0.001 ng / ml) were added to identify the survival status of DA neurons by immunohistochemistry. . The experiment found that after adding different concentrations of 968 on the fourth day of culture in serum medium, the number of cells in the 0.001, 0.01, 0.1 ng / ml group was 187%, 148%, 129%, NEWMAN—KEULS single factor analysis of variance is significant (Table 1, Figure 2, Figure '3). Means ± standard errors listed in the table, and the control group-significant differences are marked with *, * indicates p <0. 05. * * means P <0. 01. The results indicate that 968 has a clear nutritional effect on primary dopaminergic neurons cultured in vitro in serum-containing medium. Table 1 Protective effects of different concentrations of 968 on fetal rat midbrain dopaminergic neurons (TH positive neurons).

另外， 在原代的无血清培养基中没有发现 968具有明确的 DA能神经元的营 作 用实验组与对照组的神经元数目没有显著性差别。 这与 CsA和 FK506在无血清培养基 中末发现有营养作用是一致的。 实验二 雷公藤氯内酯醇保护多巴胺能神经元的在体研究。

In addition, no significant difference was found in the number of neurons between the experimental group and the control group in the primary serum-free medium. This is consistent with the fact that CsA and FK506 have no nutritional effect in serum-free medium. Experimental study of triptolide lactone protection of dopaminergic neurons in vivo.

 MPTP is a toxin that specifically damages DA neurons. Peripheral injection can specifically penetrate the blood-brain barrier and specifically destroy nigrostriatal dopaminergic neurons. C57BL I 6J mice, ip MFTP 30mg I kg body weight per day, for three consecutive days, can reduce the DA content in the striatum by more than 80%.

In 968 experiments on the protective effects of dopaminergic meridians in the brain of C57 / BLACK Parkinson model mice, we divided into 8 groups: (1) N. S + NS group, as blank control; (2) N. S + MPTP, as a negative control; (3)-(7) groups are gesdo-g / kg body weight-io g / k _g body weight) experimental group; (8) CsA (10mg / kg body weight) + MPTP, As a positive control group.

 Inject 968 or saline once a day before MPTP damage; after that, inject 968 or normal saline every morning, and then inject MPTP or normal saline for three days in the afternoon; continue to inject 968 or normal saline once a day until the day before the animal is sacrificed, and inject lld . The HPLC method was used to detect the contents of DA and its metabolites HVA (vanillin) and D0PAC (dihydroxyphenylacetic acid) in the striatum of mice, and the ratio of DA, (HVA + DOPAC) / DA Calculations and statistical analysis were performed.

We found that 968 in the 0.1 ng / ml concentration group significantly increased the DA content in the striatum of mice (Figure 4); meanwhile, the ratio of (HVA + DOPAC) I DA was the lowest in all groups (Figure 5) ). HVA and DOPAC are the final metabolites of DA in the brain. (HVA + DOPAC) / DA ratio increases, indicating that the metabolic rate of DA is increased, and the production of metabolites is increased. It is more common when DA neurons are incompletely damaged. 968 can reduce the ratio of (HVA + DOPAC) / DA, indicating that it can slow the DA metabolism renewal rate in DA neurons in MPTP-injured mice, thereby effectively protecting the survival of neurons. It has a certain protection for the damage of DA neurons in the midbrain of Parkinson mice caused by MPTP. 护 效应。 Protective effect.

从图 4可以看出， 0.01—0. l g/ kg的 968能够维持 DA的正常代谢。 Effect of 968 pretreatment on dopamine content in striatum of C57BL I 6J mice caused by PTP injury (± SE)

It can be seen from Figure 4 that 0.01-0. Lg / kg of 968 can maintain the normal metabolism of DA.

 Through ex vivo cell experiments and in vivo animal experiments, we found that 968 does have a certain neurotrophic effect. According to the difference of the role of 968 in serum-free medium and serum-free medium, it is speculated that the mechanism may be to increase the susceptibility of neurons to serum nerve growth factor (NGF) and so on, thereby exerting neurotrophic effects. Effect of Experiment 3 968 on the synaptic length of mesencephalic neurons in primary cultured fetal rats.

SD pregnant rats with fetal fetuses for 16 days Take the fetal rat midbrain for primary neuron culture. The midbrain tissue is chopped, digested, and blown out to make it a single cell. It is planted at a density of 3 × 10 ⁵ cells / well in 24 wells. In a culture plate. First add 10% fetal bovine serum medium with DMEM, incubate at 37 ° C, 5% ∞ _{2 for} 24 hours, then change to DMEM / F-12 (1: 1) medium, add! ^ Additives continued to be cultured (mainly to inhibit glial cell proliferation), other conditions remained unchanged. While adding 10- ¹⁵ - 10- ol / L 968 process, 4 holes per dose group. After 72 hours, observe under an inverted phase-contrast microscope (200 X), and acquire images with an image processing system, collect 4 fields of view per well, and measure the axon length of nerve cells with the image processing system. Axons are defined as the longest for each cell The longest 8 cells of the uranium process were taken from each view. The data from each dose group are combined, averaged, and the standard deviation is calculated, as listed in the table below (Table 3). AN0VA data analysis, and by Durmet multiple comparison test, the control group with a significance difference marked with *, * p <0.05, ** p <0.01 o The results showed 10- ¹⁵ - 10- ¾ol / L968 could significantly promote The axonal length of mesencephalic nerve cells grew by 213% to 150% longer than the control group, and the difference was significant. This shows that 968 can promote the axon growth of nerve cells, and its effective concentration range is wide, which increases the choice of future clinical dosage and the safety of medication.

Continued Table 3

实验四 968对原代培养的胎鼠中脑神经细胞存活数目的影响

Effect of Experiment 4 968 on the Survival Number of Nerve Cells in Primary Cultured Fetal Rats

The method of primary culture of fetal rat midbrain was the same as that in Example 3, except that the medium was changed to DMEM / F-12 + 1% [^ medium, and then treated with 968 for 7 days, and the culture solution was aspirated on the 8th day, and used Rinse in PBS. Add 0.01% pyridine orange staining, observe under a fluorescence microscope (100X), the nucleus shows green fluorescence, take three fields per well, and count with a manual counter. AN0VA data were analyzed, and by Dunnet multiple comparison test, was found 10_ ¹² -10- "mol / L 968 treated group was significantly higher number of viable cells, contribute to the survival of nerve cells 968 described in Table 4: treatment with different concentrations 968 Effect on Fetal Rat Midbrain Primary Cultured Neuronal Cell Survival (± SF)

实验五 968可以拮抗多巴胺能神经毒素 MPP+对 PC12细胞的毒性。

Experiment five 968 can antagonize the toxicity of dopaminergic neurotoxin MPP + to PC12 cells.

PC12 cells were cultured with RPMI 1640 + 10% newborn bovine serum, and then uniformly at a density of 1.5 × 10 ⁵ Plant in a 96-well plate. After the cells adhere well, add 968 or ^. After pretreatment for 2 hours, PCI2 cells were injured by adding dopaminergic neurotoxin MPP + 60 μιηο1 / L to the experimental group. We have demonstrated in other experiments that the median lethal concentration (LC ₅ ) of MPP + injured PC12 cells is 60 μπο1 / L. After 72 hours, the medium was aspirated, and 0.5 mg / ml of thiazole blue (MTT) 100 μl was added to each well, and incubated at 37 ° C for 4 hours. At this time, the original yellow-green TT was catalyzed by the succinase enzyme in living cells. Blue particles were formed below, and 100 μl of co-solvent (isopropanol: Triton X-100: water = 5: 1: 4) was added to each well, and the blue particles were completely dissolved by shaking at 37 ° C overnight. An ELISA plate reader detects the absorbance of each well at a wavelength of 490 nm. Under the same conditions, the absorbance is directly proportional to the number of cells in each well. The ratio of the absorbance of the experimental group (including the control group and the 968 and. Treatment groups) to the MPP + uninjured group is the percentage of cell survival. Table 5 lists the mean ± standard deviation of the cell viability of 8 wells per group. * Indicates that after a fine OVA analysis, followed by Dunnet multiple comparison test, the difference with the control group is significant, * P <0.05, * P <0.01. The results showed ^{that: 1CT 9 - l (T 7} mol / L and 968 have an antagonistic 7. Processing of MPP + toxicity in PC12 cells, so that cell viability was improved by about 30%.

Antagonistic effects of 968 and T _1Q on MP12 + PC12 cytotoxicity under 10% serum conditions ± SE)

上述实验是在 RPMI 1640+ 10%新生牛血清条件下的结果， 为了排除血清在其中 起的作用， 我们降低了血清的 *度， 由 10%降为 1 %， 其它条件木变， 观察了 968和 T,。对 PC12细胞存活的影响。 数据列于表 6。 968和 T。在 10%血清条件对 ΜΡΡ+的 PC12细胞毒性的拮抗作用（5 [土 SE)

The above experiment is the result under the condition of RPMI 1640 + 10% newborn bovine serum. In order to exclude the role of serum in it, we reduced the serum degree of serum from 10% to 1%. The other conditions were woody. Observed 968 And T ,. Effect on PC12 cell survival. The data are listed in Table 6. 968 and T. Antagonistic effect of MP12 + on PC12 cytotoxicity in 10% serum conditions (5 [土 SE)

 The above results indicate that at low concentrations of serum (1%), 968 and 1 were obtained. Still has the effect of antagonizing MPP + toxicity. Experiment 6 Inhibitory effect of 968 on glutamate-induced apoptosis of fetal rat cerebral cortex neurons

 Materials and Method

 1. Plate: 2 to 3 days before inoculation, plate with 12. 5 g / ml poly-L-lysine, 6-well plate, 500 μί / well, 24-well plate 400 μί / well, and air dry.

 2. Inoculation culture:

Isolate the cerebral cortex of 16-18d fetal rats under sterile conditions in pre-chilled DMEM / F-12 medium, carefully remove the pia mater and blood vessels, and transfer the brain tissue to another ice-containing DMEM / F-12 ( In a small beaker containing a volume fraction of 10% fetal bovine serum and 10% horse serum), gently blow 20-30 times with a pointed polished glass tube until all brain tissue is dispersed into a cell suspension and passed through a 200 mesh nylon sieve Filter to remove undigested tissue pieces, and use DMEM / F-12 medium for the filtrate (10% fetal bovine serum, 10% horse serum, 100kU. L- ¹ streptomycin, pH 7. 2-7. 4), Taiwan After Pan-Ilan staining, 500 cells were counted under an inverted phase-contrast microscope, and the stained cells were dead cells, and the density was adjusted to 1 × 10 ⁹ cells. 0.5 cell suspension was inoculated into 12.5 μ _§ 1 ml overnight cultured 6-well culture plate, cultured in 37 ° C, 5% C0 ₂ incubator, after 24 hours, the cells were adhered to the wall to change the solution once to remove Dead cells were changed every 3 to 4 days thereafter, and 5-fluorouracil (final concentration 10 μm Ι.Γ) was added on day 3 for 24 hours to inhibit non-neuronal proliferation. Continue to culture until the 5th day and start the experiment.

 Experimental results:

1. 968 has a significant nutritional effect on primary cortical neurons cultured in vitro in a medium with serum. Use different concentrations of 968 (1 (T ¹³ — 10_ ⁷ mol / L) in serum (10% fetal bovine serum). + 10% horse serum) DMEM / F1-12 incubated primary cortical neurons cultured in vitro (5d, Wistar suckling rat 14-16d), solidified after 24h The number of cells, the length of the protrusions and the area of the cell body were determined and analyzed by microscopic images. The results show that the number of cells

No significant changes in 24h, whereas the area of cell bodies and neurite length in ^10-13--10-8 each concentration were significantly increased. Table 7: Effect of 968 on the growth status of fetal rat cerebral cortex neurons in the presence of serum (± SE)

选取在含血清的培养基中神经营养作用最显著的三个浓度 10_】², 10—"， 10"¹⁰ mol / L的 968孵育不含血清的 DMEM 7 F— 12 (亦不含 ^添加剂）中的原代皮层神经元 (5b， Wistar乳鼠 14一 16d)， 24h后固定同时对细胞数目、 突起长度和胞体面积进行显微 图象分析。 发现细胞数目无明显变化， 而胞体面积显著减小， 突起长度也有一定的缩 短。 说明 968在无血清的培养基中对体外培养的原代皮层神经元无营养作用。 从机理 上推测 968是通过增强血清中 NGF的营养作用发挥功能的。

Select the three concentrations with the most significant neurotrophic effect in serum-containing medium 10_] ² , 10— ", 10" ¹⁰ mol / L of 968 to incubate serum-free DMEM 7 F-12 (also without ^ additives) The primary cortical neurons (5b, Wistar 14-14d) in the medium were fixed after 24 hours while microscopic image analysis was performed on the number of cells, protrusion length and cell body area. It was found that there was no significant change in the number of cells, the area of the cell body was significantly reduced, and the length of the protrusions was also shortened to some extent. This indicates that 968 has no nutritional effect on primary cortical neurons cultured in vitro in serum-free medium. It is speculated that 968 functions by enhancing the nutritional effect of NGF in serum.

 2, 968 can obviously resist Glu-induced primary cortical neuronal injury

First incubate 968 ( ^10-12 — 10 mol / L) with primary cortical cells (5d) cultured in vitro in DMEM / F-12 + 10% fetal bovine serum for 24 h, and add .100ng / ml NGF as Positive control. Then add 250 μπιο / L of Giu for 30 min, immediately fix for microscopic image analysis. The excitatory neurotoxicity of Glu can shorten or delete cell synapses, while 968 can significantly protect the cell body area and process length of cortical neurons caused by Glu. Table 8 Effect of 968 on glutamate-induced damage to fetal rat cortical neurons (鼠 土 SE)

大量文献报道， 细胞凋亡是 PD、 AD等神经退行性疾病中大量神经元死亡的主要 形式， 而大剂量 Glu处理可以诱导神经细胞的凋亡， 我们在原代培养的胎鼠大脑皮层 神经细胞中加入 Glu 25(^mol / L孵育 30rain诱导细胞凋亡， 收集细胞， 进行碘化丙 啶 (PI)荧光染色， 用流式细胞仪检测、 分析凋亡细胞的百分率。 发现 Glu在给定浓度 和作用时间下可以使 38. 9%的神经细胞发生凋亡，而 l(T^1Qmol I L 968处理组只有 22. 5 %的细胞发生凋亡， 与 100ng / ml的 NGF的作用相当（21. 9%)。 表 9: 不同浓度 968处理对谷氨酸诱导原代培养的胎鼠皮层神经细胞凋亡的影响 ¾±SE)

A large number of literatures have reported that apoptosis is the main form of death of a large number of neurons in neurodegenerative diseases such as PD and AD, and high-dose Glu treatment can induce apoptosis of neural cells. Glu 25 (^ mol / L was added and incubated for 30rain to induce apoptosis. Cells were collected and subjected to propidium iodide (PI) fluorescence staining. The percentage of apoptotic cells was detected and analyzed by flow cytometry. Glu was found at a given concentration and Under the action time, 38.9% of the nerve cells can undergo apoptosis, while only 22.5% of the cells in the l (T ^1Q mol IL 968 treatment group undergo apoptosis, which is equivalent to the effect of 100ng / ml NGF (21.9 %). Table 9: Effects of different concentrations of 968 treatment on glutamate-induced apoptosis of fetal rat cortical neurons in primary culture ¾ ± SE)

 NGF 968 concentration (log mol / L) Normal group Control group

 (lOOng / ml) -12 -11 -10 apoptotic cell percentage 16. 13 + 38. 91 ± 21. 94 ± 25.2 ± 29. 91 + 22. 45 ± fraction (%) 1. 44 1. 92 '1. 01 ** 3. 25 ** 1. 89 ** 1. 85 **

Claims

Rights request 1. Selected from triptolide, triptolide, triptolide glycol, triptolide triol, 16-hydroxy triptolide alcohol, triptolide, triptolide and triptolide And the application of one or more of tripterygium wilfordii plant extracts in the preparation of a medicament for preventing and treating neurological diseases.  2. The use according to claim 1, wherein the neurological disease is Alzheimer's disease, Parkinson's disease, Huntington's neurodegenerative disease and spinal cord injury, and / or lateral spinal cord sclerosis disease.  3. A medicament for the prevention and treatment of nervous system diseases, which contains a drug selected from triptolide, triptolide, triptolide, diol, triptolide, and 16-hydroxy triptolide Esterol, rapide, triptolide and triptolide and one or more of triptolide extracts of Tripterygium plants.  4. The medicament according to claim 3 ', further comprising a neurotrophic factor.  The medicament according to claim 4, characterized in that the neurotrophic factor is a nerve growth factor, a glial-derived neurotrophic factor, a brain-derived neurotrophic factor and / or a ciliary neurotrophic factor.  6. The medicament according to any one of claims 3 to 5, wherein the neurological disease is Alzheimer's disease, Parkinson's disease, Huntington's neurodegenerative disease and spinal cord injury, and / or Spinal cord sclerosis.