Diterpene derivative ST-4 and application of sodium salt ST-4Na thereof

Abstract

The invention relates to application of a diterpene derivative ST-4 and a sodium salt ST-4Na thereof, the compound is ST-4 or the sodium salt ST-4Na thereof, activation of NLRP3 in microglial cells can be inhibited, neuroinflammation activated and mediated by the microglial cells is further inhibited, loss of dopaminergic neurons induced by MPTP can be reversed, and the effect of inhibiting neuroinflammation is achieved. The apoptosis of dopaminergic neurons is reduced, so that the effect of treating Parkinson's disease is achieved. The treatment effect on the Parkinson's disease is equivalent to or even better than that of an existing medicine L-Dopa, other neuronal cells cannot be damaged, and the L-Dopa can be applied to preparation of the medicine for treating the neurodegenerative disease, especially the medicine for treating the Parkinson's disease.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and more specifically to the use of a diterpene derivative ST-4 and its sodium salt ST-4Na in the preparation of drugs for treating neurodegenerative diseases. Background Art

[0002] Neurodegenerative diseases are a group of diseases characterized by the gradual degeneration of the structure and function of the nervous system. These diseases mainly affect neurons in the brain and spinal cord, leading to a gradual loss of cognitive and physical abilities. Common neurodegenerative diseases include Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), etc. Studies have shown that the NLRP3 inflammasome plays a key role in specific neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis.

[0003] The NLR family protein 3 (NLRP3) inflammasome is a large multi-protein complex composed of NLRP3, the adaptor protein ASC, and the effector protein caspase-1. The assembly of this complex complex leads to the activation of caspase-1, which promotes the proteolytic processing of pro-IL-1β and pro-IL-18 into mature and biologically active IL-1β and IL-18. In the central nervous system, the activation of the NLRP3 inflammasome can lead to the release of inflammatory mediators, including interleukin-1β (IL-1β) and interleukin-18 (IL-18). These inflammatory factors can promote neuronal damage and microglia activation, thereby initiating and exacerbating neuroinflammation. Among them, the activation of microglia leads to the strong secretion of cytokines, chemokines, reactive oxygen species, and other neurotoxic mediators, which directly leads to neuroinflammation in the brain. Neuroinflammation and neurodegenerative diseases can form a positive feedback loop, that is, neuroinflammation can promote the occurrence and development of neurodegenerative diseases, and neurodegenerative diseases will further aggravate the inflammatory response. For example, in Parkinson's disease patients, the levels of inflammatory biomarkers IL-1β, IL-18 and caspase-1 are significantly increased.

[0004] Parkinson's disease (PD) is characterized by motor symptoms such as rigidity, tremor, and bradykinesia, accompanied by a range of disabling non-motor symptoms. Its characteristic neuropathology includes the loss of dopamine-producing neurons in the midbrain and the formation of intraneuronal protein aggregates, such as the massive loss of TH neurons in the substantia nigra, which leads to a decrease in dopamine levels, causing movement disorders and other symptoms.

[0005] At present, the treatment strategies for Parkinson's disease are mainly aimed at increasing the level of dopamine in the brain or simulating the effects of dopamine. Among them, Levodopa is one of the most effective drugs for the treatment of Parkinson's disease. Levodopa is a precursor of dopamine, which can cross the blood-brain barrier and be converted into dopamine in the brain, thereby replenishing the dopamine reduced by Parkinson's disease. However, long-term use of Levodopa can cause serious side effects, mainly manifested in massive damage and death of non-dopaminergic neuronal cells. Given the important role of NLRP3 in neurodegenerative diseases, studying more small molecule drugs that target and inhibit NLRP3 activation is of great significance for delaying the progression of neurodegenerative diseases. Summary of the invention

[0006] In view of the above defects or improvement needs of the prior art, the present invention provides an application of a diterpene derivative ST-4 and its sodium salt ST-4Na, the purpose of which is to discover that compound ST-4 and its sodium salt ST-4Na can inhibit NLRP3-mediated neuroinflammation and reverse the loss of dopaminergic neurons, thereby improving the symptoms of Parkinson's disease, thereby solving the technical problems of the lack of existing effective drugs for the treatment of Parkinson's disease and large side effects.

[0007] To achieve the above object, according to one aspect of the present invention, there is provided a use of a diterpene small molecule compound in the preparation of a drug for treating neurodegenerative diseases, wherein the compound is ST-4 or its sodium salt ST-4Na;

[0008] The structural formula of compound ST-4 is as follows:

[0009]

[0010] The structural formula of its sodium salt ST-4Na is as follows:

[0011]

[0012] Preferably, the compound is used in the preparation of a drug for treating Parkinson's disease.

[0013] Preferably, in the application, the dosage of the compound ST-4 or ST-4Na in the drug for treating Parkinson's disease is 1.5 to 6.0 mg / kg.

[0014] Preferably, the compound ST-4 or ST-4Na is used for preparing a drug for resisting neuroinflammation caused by neurodegenerative diseases.

[0015] Preferably, the compound ST-4 or ST-4Na is used for preparing a drug for resisting neuroinflammation caused by Parkinson's disease.

[0016] Preferably, in the application, the dosage of compound ST-4 or ST-4Na in the anti-neuroinflammatory drug is 2 to 4 μM, or 1.5 to 6.0 mg / kg.

[0017] According to another aspect of the present invention, a drug for treating Parkinson's disease is provided, wherein the drug comprises compound ST-4 or its sodium salt ST-4Na, and pharmaceutically acceptable excipients.

[0018] Preferably, the compound in the drug is ST-4Na, and the dosage is 1.5 to 3.0 mg / kg.

[0019] Preferably, the compound in the drug is ST-4, and the dosage is 3.0 to 6.0 mg / kg.

[0020] Preferably, the drug for treating Parkinson's disease includes oral preparations and injection preparations. In general, the above technical solutions conceived by the present invention can achieve the following beneficial effects compared with the prior art:

[0021] The present invention uses Rubescensine A as a precursor compound, synthesizes and purifies to obtain compound ST-4, and prepares its sodium salt ST-4Na. Through microglial neuroinflammation model experiments, it is found that ST-4 can inhibit the activation of NLRP3 inflammasomes in microglial cells, thereby inhibiting neuroinflammation mediated by microglial activation. Through animal model experiments, it is found that both ST-4 and ST-4Na can significantly improve the symptoms of Parkinson's mice, mainly by reversing the loss of dopaminergic neurons, reducing the apoptosis of dopaminergic neurons, and thus playing an effect of treating Parkinson's disease. Its therapeutic effect is equivalent to that of the existing drug L-Dopa, or even better, and will not cause damage to other neuronal cells. It can be used to prepare drugs for treating neurodegenerative diseases, especially for preparing drugs for treating Parkinson's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is ST-2 positive ion mass spectrometer;

[0023] Figure 2 It is ST-2 negative ion mass spectrometer;

[0024] Figure 3 It is ST-3 positive ion mass spectrometer;

[0025] Figure 4 It is ST-3 negative ion mass spectrometer;

[0026] Figure 5 It is ST-4 positive ion mass spectrometer;

[0027] Figure 6 It is ST-4 negative ion mass spectrometer;

[0028] Figure 7 It is 1H NMR (600MHz) spectrum of ST-4in DMSO-d6;

[0029] Figure 8 is the 13C NMR (150MHz) spectrum of ST-4 in DMSO-d6;

[0030] Fig. 9 It is the HMBC (600MHz) spectrum of ST-4in DMSO-d6;

[0031] Fig.10 It is HSQC(600MHz)spectrum of ST-4in DMSO-d6;

[0032] Fig.11 It is NOESY(600MHz)spectrum of ST-4in DMSO-d6;

[0033] Fig.12 is NOESY(600MHz)spectrum of ST-4Na in DMSO-d6;

[0034] Fig.13 It is 13CNMR(600MHz)spectrum of ST-4Na in Acetone-d6;

[0035] Fig.14 ST-4 inhibits LPS-induced activation of NLRP3 inflammasome in BV2 cells and its effect on apoptosis of SH-SY5Y neurons;

[0036] Fig.15 It is ST-4 that alleviates the behavioral deficits in the MPTP-induced Parkinson's mouse model;

[0037] Fig.16 It is that ST-4 restores the loss of dopamine-positive cells in the MPTP-induced Parkinson's mouse model;

[0038] Fig.17 ST-4 alleviates the loss of TH-positive cells in the substantia nigra and striatum of PD mice;

[0039] Fig.18 It is a mouse behavioral experiment. A: Rotarod test; B: Climbing pole test; C: Hanging test; D: Y maze test;

[0040] Fig.19is the effect of ST-4 and ST-4Na on dopaminergic neurons in the SNpc region of the mouse midbrain (scale bars in A and B on the left = 125 μm, scale bars in A and B on the right = 20 μm);

[0041] Fig. 20 It is ST-4 that inhibits the proliferation of microglia in the substantia nigra of PD mice;

[0042] Fig.21 It is that ST-4 inhibits the transformation of substantia nigra microglia in PD mice to the M1 type;

[0043] Fig. 22 It is that ST-4 inhibits the NLRP3 inflammasome pathway in the substantia nigra of the brain of PD mice;

[0044] Fig.23 is the effect of ST-4 and ST-4Na on the expression level of inflammatory factors in the SNpc region of the mouse midbrain;

[0045] Fig.24 is the volcano plot of differentially expressed genes in the substantia nigra of PD mice after ST-4 treatment;

[0046] Fig.25 It is the KEGG pathway of the top 25 differentially expressed genes enriched in the substantia nigra of the midbrain of mice after ST-4 administration;

[0047] Fig.26 It is a key differentially expressed gene in the substantia nigra of PD mice after ST-4 administration. DETAILED DESCRIPTION

[0048] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation mode, structure, characteristics and effects of the present invention are described in detail below in combination with the accompanying drawings and preferred embodiments.

[0049] Oridonin is a biologically active diterpenoid isolated from the genus Oridonium of the Lamiaceae family. It has been shown to have specific and covalent inhibitory effects on the NLRP3 inflammasome. Recent studies have shown that Oridonin is a promising candidate molecule for the treatment of human neurodegenerative diseases. In the early stage, this research team used Oridonin as a precursor compound to transform and synthesize a new compound ST-4. In order to improve the water solubility of ST-4, ST-4 was further used as a lead compound and the sodium salt form of ST-4 was synthesized by reacting it with sodium ions under alkaline conditions.

[0050] However, the spatial structure of drug molecules has a significant impact on their biological activity, metabolic pathways and toxicity. For example, the R-isomer of thalidomide has a strong sedative effect, while the S-isomer is a strong teratogen. The chemical structures of these two enantiomers are similar, but their performance in vivo is completely different. Therefore, although the relationship between chemical structure and efficacy provides a certain basis for prediction, the determination of actual efficacy still depends on experimental data.

[0051] MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) is an organic compound belonging to the tetrahydropyridine class. It has attracted attention because it can pass through the blood-brain barrier and transform into the neurotoxin MPP+. MPTP can selectively damage dopaminergic neurons in the substantia nigra and striatum system in the brains of humans and primates, leading to a decrease in the levels of dopamine and its metabolites, thereby causing typical Parkinson's disease-like symptoms. It is often used to induce Parkinson's disease models.

[0052] The present invention studies the effect of compound ST-4 on inflammasome NLRP3-mediated neuroinflammation based on cell experiments. The results show that compound ST-4 can inhibit LPS-induced activation of NLRP3 in microglia and reduce neuroinflammation mediated by microglia activation. Further, the effect of ST-4 on LPS-induced neuronal inflammation and apoptosis was studied. The results show that ST-4 can inhibit LPS-induced dopaminergic neuron inflammation and apoptosis, which is beneficial to alleviate the progression of Parkinson's disease.

[0053] In addition, based on the animal model MPTP, the effects of ST-4 and its sodium salt ST-4Na on the motor conditions of Parkinson's model mice were studied. The results showed that ST-4 and its sodium salt ST-4Na can significantly improve the movement disorders of Parkinson's mice, have good effects in treating Parkinson's disease, and there is no significant difference in the therapeutic effects of the two. They can be used to prepare drugs for the treatment of Parkinson's disease.

[0054] Based on this, the present invention provides a use of a diterpene small molecule compound ST-4 or ST-4Na in the preparation of a drug for treating neurodegenerative diseases;

[0055] The compound ST-4 has the following structural formula:

[0056]

[0057] The compound ST-4Na has the following structural formula:

[0058]

[0059] Preferably, the compound ST-4 or ST-4Na is used for preparing a drug for treating Parkinson's disease.

[0060] In some embodiments, the dosage of the compound ST-4 or ST-4Na in the drug for treating Parkinson's disease is 1.5-6.0 mg / kg (converted to human dosage based on a mouse dosage to human dosage ratio of 10:1).

[0061] The compound in the drug for treating Parkinson's disease is ST-4, and its dosage is preferably 3.0 to 6.0 mg / kg.

[0062] The compound in the drug for treating Parkinson's disease is ST-4Na, and its dosage is preferably 1.5 to 3.0 mg / kg.

[0063] In some embodiments, the compound ST-4 or ST-4Na is used to prepare a drug for treating neuroinflammation.

[0064] The dosage of the compound ST-4 or ST-4Na in the drug for treating neuroinflammation is 1 μM or more, preferably 2 to 4 μM, or 1.5 to 6.0 mg / kg.

[0065] The compound ST-4 or ST-4Na is preferably used in the preparation of a drug for preventing neuroinflammation caused by neurodegenerative diseases, and more preferably used in the preparation of a drug for preventing neuroinflammation caused by Parkinson's disease.

[0066] In addition, the present invention also provides a drug for treating Parkinson's disease, which comprises compound ST-4 or ST-4Na, and pharmaceutically acceptable excipients.

[0067] The compound in the drug is ST-4Na, and its dosage is preferably 1.5 to 3.0 mg / kg;

[0068] The compound in the drug is ST-4, and its dosage is preferably 3.0 to 6.0 mg / kg;

[0069] The medicine includes oral preparations and injection preparations.

[0070] The following are examples

[0071] Example 1 Preparation of compound ST-4 and its sodium salt ST-4Na

[0072] 1.1 Preparation of compound ST-4

[0073] (1) Weigh 20 g of stevioside (ST-1), dissolve it in 1 L of pure water at room temperature, add 30 g of sodium periodate and stir to dissolve, and stir at room temperature for 18 h.

[0074] (2) The reaction solution was filtered to obtain a white solid. The white solid was added back into 1 L of pure water, stirred and 15 g of potassium hydroxide was slowly added. After the solid was completely dissolved, the mixture was heated under reflux at 95° C. for 1.5 hours and cooled naturally.

[0075] (3) After the reaction solution was cooled to room temperature, the pH was adjusted to 5.0 with glacial acetic acid, extracted three times with ethyl acetate, and the organic phase was concentrated. The organic phase was washed three times with saturated brine, anhydrous sodium sulfate was added to dry the organic phase for 24 hours, and then the solvent was recovered under reduced pressure.

[0076] (4) The residual crude product was dissolved by heating methanol at 50°C. After the solution was clarified, it was filtered while hot. The filtrate was crystallized at room temperature. White needle-like crystals were filtered out and dried at 60°C for 24 hours to obtain the purified product ST-2.

[0077] (5) Weigh 2 g of the product ST-2 and dissolve it in 200 mL of a mixed solvent of 1,4-dioxane and pure water in a ratio of 1:1. Stir until steviol is completely dissolved, then add 2 mL of tert-butyl peroxide, and then add 2 g of selenium dioxide. Heat the mixture in a water bath at 60°C for 5 h.

[0078] (6) TLC was used to detect the progress of the reaction. After all the reactants had reacted, 200 mL of pure water was added to terminate the reaction. The reaction solution was extracted three times with dichloromethane, the organic phases were combined and washed three times with saturated brine, and the organic phases were dried over anhydrous sodium sulfate for 24 h and then the solvent was recovered under reduced pressure to obtain a white solid crude product.

[0079] (7) Methanol was heated to 50°C for dissolution, and the solution was filtered while hot after being clarified. The filtrate was crystallized at room temperature, and white needle-shaped crystals were obtained by filtration. The purified product ST-3 was obtained after drying at 60°C for 24 hours.

[0080] (8) Weigh 1 g of the product ST-3 and dissolve it in 70 mL of pyridine. Take another 56 mL of pyridine and slowly add it to 7 mL of aqueous solution containing 0.9 g of chromium trioxide to prepare a pyridine-chromium trioxide reagent. Slowly add the pyridine-chromium trioxide reagent to the pyridine solution containing 15-hydroxysteviol and stir overnight at room temperature.

[0081] (9) TLC was used to detect the progress of the reaction. After the reaction was completed, 300 mL of pure water was added to terminate the reaction. The reaction solution was extracted three times with ethyl acetate, and the organic phases were combined and washed three times with saturated brine. The organic phase was dried over anhydrous sodium sulfate for 24 hours and then the solvent was recovered under reduced pressure to obtain a white solid crude product. After the crude product was dissolved in methanol, it was separated by a silica gel chromatography column to obtain a preliminary purified product. It was further dissolved in methanol at 50°C, and the solution was filtered while hot after it was clarified. The filtrate was crystallized at room temperature, and white needle-shaped crystals were filtered. After drying at 60°C for 24 hours, the purified product ST-4 was obtained.

[0082] NMR analysis was performed using mass spectrometry (MS), 1H-NMR (600MHz), 13C-NMR (150MHZ), HMBC, HSQC and NOESY, and the test solvent was deuterated DMSO. The spectral data were compared with relevant literature to characterize the structure of each compound, such as Figures 1 to 11 As shown. Figure 1 and Figure 2 This is the positive and negative ion mass spectra of compound ST-2. Figure 3 and Figure 4 This is the positive and negative ion mass spectra of compound ST-3. Figure 5 and Figure 6 It is the positive and negative ion mass spectra of compound ST-4; Figure 7-Figure 11 The analysis results of compound ST-4 were 1H-NMR (600 MHz), 13C-NMR (150 MHZ), HMBC, HSQC and NOESY.

[0083] 1.2 Preparation of compound sodium salt ST-4Na

[0084] (1) Weigh 0.9308 g ST-4, dissolve it in 40 mL anhydrous ethanol, add 0.1091 g NaOH, heat under reflux and stir until the solution turns yellow and clear, and the reaction is complete;

[0085] (2) Filter, collect the filtrate, recover part of the solvent under reduced pressure, fully crystallize, and dry to obtain 0.89 g of slightly yellow crystals, namely ST-4Na. The structural identification results are as follows: Fig.12 and Fig.13 shown.

[0086] Example 2 ST-4 inhibits LPS-induced NLRP3 inflammasome activation in microglia

[0087] BV-2 cells, also known as BV2 cells, are a microglial cell line derived from mice. In neuroscience research, BV-2 cells are often used as a tool to study microglial biology and neuroinflammation models because they have the characteristics of primary microglia. In this example, bacterial lipopolysaccharide (LPS) was used to induce the activation of the BV-2 cell line to construct a microglial neuroinflammation model, and then study the therapeutic effect of drugs on neuroinflammation-related diseases. The experimental mouse microglia cells (Mouse microglia cells, BV2) were cultured in the CO2 cell culture incubator of our research group at Huazhong Agricultural University, and the mode was set to 37°C, 5% CO2, and 100% humidity. The specific experimental process is as follows:

[0088] BV-2 cells were inoculated in a 6-well plate with a coverslip for 24 hours. When the cell density reached about 60-80%, the plasmid (pEGFP-N1-NLRP3 or pmCherry-N1-caspase-1) was transfected into the BV-2 cells using ExFect2000 transfection reagent (Vazyme Biotech Co., Ltd., Nanjing, Jiangsu, China) according to the manufacturer's instructions. After 18-24 hours of transfection, they were divided into a normal control group, an LPS-induced group, and an ST-4 drug group (1 μM, 2 μM, and 4 μM), and the treatment of each group of cells was as follows:

[0089] Normal control group (Control): cells BV-2 were transfected without bacterial lipopolysaccharide (LPS) induction and ST-4 treatment; LPS induced group: cells BV-2 were transfected with LPS (200 ng / mL) for 3 hours and stimulated with ATP (2.5 μM) for 12 hours; ST-4 drug group: cells BV-2 were transfected with ST-4 (1 μM, 2 μM and 4 μM) for 3 hours, then treated with LPS (200 ng / mL) for 3 hours and stimulated with ATP (2.5 μM) for 12 hours.

[0090] After the cells in the above groups were treated, the cells were collected for immunofluorescence and WB analysis. After the cells in each group were treated, the cells were fixed with 4% PFA and stained with DAPI solution. Representative images were captured and merged by fluorescence microscopy. Scale bar: 75 μm. The results are shown in Fig.14 As shown in (a), the cells with NLRP3 signal or ASC signal were quantitatively analyzed, and the results are shown in Fig.14 As shown in b and c, b is the percentage of cells with NLRP3 signals to cells with DAPI signals, and c is the percentage of cells with ASC signals to cells with DAPI signals.

[0091] The cells in each group were collected for cell lysis, and then the cell lysates of each group were collected. Western blotting was used to detect the protein levels of NLRP3, ASC, pro-caspase-1, caspase-1, pro-IL-1β, IL-1β and iNOS in the cells of each group. The results are shown in Fig.14 As shown in middle d.

[0092] Depend on Fig.14The experimental results show that the NLRP3 inflammasome was induced by quantifying the fluorescence intensity of NLRP3 and caspase-1 in BV-2 cells transiently transfected with pEGFP-N1-NLRP3 and pmCherry-N1-caspase-1. Compared with the normal control group, these BV-2 cells showed a very significant increase in the fluorescence intensity of green fluorescent protein (GFP) and red fluorescent protein (RFP) after LPS treatment (P < 0.001), indicating that the NLRP3 inflammasome in BV-2 cells was activated after LPS induction treatment, and the neuroinflammation model was successfully established.

[0093] Compared with the LPS-induced group, ST-4 treatment could significantly reduce the fluorescence intensity of GFP and RFP (P < 0.001), and showed a certain dose-dependent effect ( Fig.14 In addition, ST-4 dose-dependently inhibited the expression of NLRP3 and ASC proteins, as well as the expression of caspase-1 and the mature form of IL-1β ( Fig.14 These results suggest that ST-4 treatment can inhibit LPS-induced NLRP3 inflammasome activation in BV-2 cells, thereby inhibiting neuroinflammation mediated by microglia activation.

[0094] ST-4Na is the sodium salt of ST-4, which is more water soluble and easier to apply.

[0095] Example 3ST-4 inhibits apoptosis of neuronal cells

[0096] SH-SY5Y cells are a human neuroblastoma cell line that can differentiate into neuron-like cells and have become an ideal model for studying neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. This example uses SH-SY5Y cells to study the pathology of neurodegenerative diseases and evaluates the effect of ST-4 on LPS-induced SH-SY5Y cell apoptosis. The human neuroblastoma cells (Human neuroblastoma SH-SY5Y, SH-SY5Y) used in the experiment were cultured in the CO2 cell culture incubator of our research group at Huazhong Agricultural University, and the mode was set to 37°C, 5% CO2, and 100% humidity. The specific experiment is as follows:

[0097] Aspirate the cell culture medium into a suitable centrifuge tube, wash the adherent cells once with PBS, and add an appropriate amount of trypsin cell digestion solution to treat the cells. Incubate at room temperature until the adherent cells can be blown off by gently blowing, and then aspirate the trypsin cell digestion solution. Avoid excessive digestion with trypsin. Add the original cell culture medium, gently blow the cells off, transfer them to a centrifuge tube, centrifuge at 1000r / min for 5min, discard the supernatant, collect the cells, gently resuspend the cells with PBS and count them.

[0098] Take 50,000-100,000 resuspended cells, centrifuge at 1000r / min for 5 minutes, discard the supernatant, add 195μL Annexin V-FITC binding solution and gently resuspend the cells. Add 5μL Annexin V-FITC and mix gently. Add 10μL propidium iodide staining solution and mix gently. Incubate at room temperature in the dark for 10-20 minutes, then place in an ice bath. Aluminum foil can be used to protect from light. Resuspend the cells 2-3 times during the incubation process to improve the staining effect. Immediately load the flow cytometer for detection. Annexin V-FITC is green fluorescence and propidium iodide (PI) is red fluorescence.

[0099] Among them, normal control group: SY-SH5Y cells were not induced by LPS and treated with ST-4; LPS induced group: SY-SH5Y cells were incubated with conditioned medium exposed to LPS+ATP for 12 hours; ST-4 drug group: SY-SH5Y cells were first pretreated with ST-4 (1μM, 2μM and 4μM) for 3 hours, and then exposed to conditioned medium of LPS+ATP and incubated for 12 hours.

[0100] After the treatment, the SY-SH5Y cells in each group were detected by flow cytometry using a cell apoptosis detection kit (C1005, Beyotime). Specifically, the cells were collected and centrifuged (2000 rpm, 5 min). The cell pellet was resuspended in 500 μL of 1×Annexin V solution containing 5 μL FITC and 10 μL PI reagent. After incubation in the dark for 15 minutes, the flow cytometer (Guava easyCyte TM 8 The apoptosis rate of cells was analyzed by Millipore. Data acquisition and analysis were performed by FlowJo software. Fig.14 As shown in ej.

[0101] in Fig.14 The middle ei is the representative dot plot of cell apoptosis after double staining of Annexin V and PI corresponding to the normal control group, LPS group, ST-4 group (1μM), ST-4 group (2μM), and ST-4 group (4μM); and the cell apoptosis rate of each group was statistically analyzed. The results are shown in Fig.14 As shown in Figure 9.

[0102] Depend on Fig.14 From the cell apoptosis rate statistics in Figure 1, we can see that compared with the LPS-induced group, the SY-SH5Y cell apoptosis rate in the ST-4 drug group was significantly reduced, especially when treated with ST-4 at a concentration of more than 2 μM, the SY-SH5Y cell apoptosis rate was extremely significantly reduced (P < 0.001).

[0103] Example 4 ST-4 improves behavioral disorders in MPTP-induced PD mice

[0104] MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) is a neurotoxin that is often used in the laboratory to induce symptoms of Parkinson's disease. In the study of Parkinson's disease, the MPTP model is one of the widely used animal models. This example uses the MPTP-induced Parkinson's mouse model to study the behavioral effects of ST-4 on MPTP-induced PD mice. The experimental mice were C57BL / 6 male mice (6-8 weeks old, 18-22g) and SD rats (7 weeks old, 200-250g), which were purchased from Liaoning Changsheng Bio-Science Technology Co., Ltd. The animal experiment protocol was approved by the Animal Ethics Committee of Huazhong Agricultural University (Animal Experiment Ethics Approval Number: HZAUMO-2019-014) and was carried out in accordance with the specifications, the same below. The experimental process is as follows Fig.15 As shown in A, the details are as follows:

[0105] After the experimental animals were sent to the laboratory, they were first raised in the animal room (to ensure ventilation, sufficient food and water, and regular alternation of light and temperature and humidity) for 7 days. Six cages were randomly selected, and ordinary mice and rats were fed for one week to maintain feed adaptability, and new feed, bedding and drinking water were replaced every three days. When the average body weight reached about 22g, MPTP modeling was performed. The MPTP suspension was injected intraperitoneally every day for 7 consecutive days at a dose of 30 mg / kg. ST-4 (8 mg / kg / d) was injected intraperitoneally for 7 consecutive days before MPTP administration, and then performed together with MPTP modeling. Behavioral testing began on the second day after completing 14 days of ST-4 treatment, and the mice were euthanized after the behavioral test (on the 20th day), and the mouse brain tissue was taken for immunohistochemical analysis and protein blot analysis. The experimental methods of behavioral testing are as follows:

[0106] 1-1 Rotarod test

[0107] To evaluate the motor coordination of mice, a rotating test was performed. During the first 3 days of modeling, all mice were trained on the rotating body until no falls were observed within 300 seconds. During the experiment, mice ran on a rod that accelerated from 5 rpm to 40 rpm over a period of 90 seconds. The maximum time each mouse could stay on the rod was recorded, and the test results of each group are shown in Figure 2. Fig.15 As shown in B.

[0108] 1-2 pole test

[0109] Before starting modeling, mice were trained three times, and the time it took for the mice to successfully descend from the top of a pole (55 cm high and 1 cm in diameter) was recorded. The results of the three trials were averaged, with an interval of more than 30 minutes between trials. The test results of each group are shown in Fig.15 As shown in C.

[0110] 1-3 Suspension Test

[0111] The suspension test was used to assess the muscle strength of mice. The mice were suspended by their front paws on a horizontal line (30 cm above the ground, 0.1 cm in diameter); the time it took for the front paws to release and slide off was recorded. The average time for each mouse to perform the test three times was calculated, and the test results for each group are shown in Figure 2. Fig.15 As shown in D.

[0112] Depend on Figure 15-17 The experimental results show that compared with the control group, the mice in the Parkinson's disease group were significantly prolonged in the rotating rod test after ST-4 intervention; the time for the mice in the Parkinson's disease group to return to the ground was significantly shortened in the extreme point test; and the time for the mice in the Parkinson's disease group to maintain their position was prolonged in the hanging test. It can be seen that the motor behavior ability of the mice in the Parkinson's disease group was significantly improved after ST-4 intervention, indicating that ST-4 can significantly improve the motor function of Parkinson's disease mice, and can significantly improve the symptoms of slow movement (bradykinesia), muscle stiffness, difficulty in maintaining posture and balance in Parkinson's disease mice, and can play a role in the treatment of Parkinson's disease.

[0113] The mechanism of action was further investigated, and the experimental methods of immunohistochemistry analysis and western blot analysis were as follows:

[0114] The paraffin-embedded sections were subjected to immunohistochemical staining and WB was used to detect the TH protein level in the midbrain striatum. After obtaining the brain slices by paraffin sectioning, the paraffin sections were first dewaxed and dehydrated. The sections were treated with 5% bovine serum albumin and sealed at room temperature for 30 minutes. Then, the diluted primary antibody rabbit anti-TH (A12756, ABclonal) was added dropwise to each section and incubated in a wet box at 4°C overnight. After washing away the excess primary antibody, the diluted HRP-labeled secondary antibody was added to the brain sections. For Nissl staining, stain with Nissl solution (Beyotime Biotechnology) at room temperature for 10 minutes, then dehydrate with 95% ethanol and make it transparent with xylene, and observe and photograph under a bright field microscope; WB detected the TH protein level in the midbrain striatum, and the results of immunohistochemical analysis and western blot analysis are shown in the following figure. Fig.16 and Fig.17 shown.

[0115] Fig.16 The experimental results showed that compared with the normal control group, the dopaminergic neurons in the striatum of the MPTP group were severely damaged, but ST-4 administration largely reversed the damage to dopaminergic neurons, thereby playing its role in treating Parkinson's disease. Nissl staining further confirmed the neuroprotective effect of ST-4. The specific experimental results are as follows Fig.17These results indicate that ST-4 can significantly reduce the loss of TH neurons in the substantia nigra pars compacta of Parkinson's disease mice, thereby improving MPTP-induced degeneration of dopaminergic neurons in the midbrain of mice.

[0116] Example 5 Effects of different concentrations of ST-4 and ST-4Na on the improvement of behavioral disorders in MPTP-induced PD mice

[0117] L-DOPA (levodopa) is a white or off-white crystalline powder with a chemical formula of C9H 11 NO4 is a dopamine precursor formed by tyrosine, which can cross the blood-brain barrier and be converted into dopamine in the brain, thereby exerting a therapeutic effect. It is currently one of the most effective drugs for treating Parkinson's disease. In order to further study the therapeutic effect of ST-4 and ST-4Na on Parkinson's disease, this example uses L-DOPA as a positive control to study the therapeutic effect of different concentrations of ST-4 and ST-4Na on Parkinson's disease, as follows:

[0118] 6-7 week old C57BL / 6 male mice were randomly selected and divided into cages. Ordinary mice and rats were fed with feed adaptability for one week, and new feed, bedding and drinking water were replaced every three days. When the average body weight reached about 22g, MPTP modeling and drug administration experiments were performed. The mice were grouped and randomly divided into MPTP modeling group, ST-4 group (MPTP+ST-4 low, medium and high doses), ST-4Na group (MPTP+ST-4Na low, medium and high doses), L-Dopa group (MPTP+L-Dopa), and blank control group. Each group had one cage, and each cage had 6 mice. MPTP was intraperitoneally injected at 25mg / kg / d for six consecutive days. Low-dose group of drugs (ST-4 or ST-4Na): 15mg / kg; medium-dose group: 30mg / kg; high-dose group: 60mg / kg. The volume of each gavage was 100 μL / mouse (solvent was 90% water + 5% ethanol + 5% Tween-80). The control group and model group mice were gavaged with equal volume of solution once a day for 14 consecutive days. Behavioral tests were performed 14 days after administration, and then samples were collected for subsequent experiments.

[0119] Behavioral testing was started on the second day after the 14-day drug treatment, and mice were euthanized after the behavioral testing (day 20), and samples were collected for subsequent experiments. The experimental methods of behavioral testing are as follows:

[0120] 1-1 Rotarod test: Same as in Example 2, the test results are as follows Fig.18 As shown in A.

[0121] 1-2 Pole test: Same as Example 2, the test results are as follows Fig.18 As shown in B.

[0122] 1-3 Suspension test: Same as Example 2, the test results are as follows Fig.18 As shown in C.

[0123] 1-4Y maze behavioral alternation test

[0124] The Y-maze apparatus is made of three opaque plastic arms (labeled A, B, and C), with an angle of 120° between the arms; the mouse is gently placed in the center of the maze and allowed to explore freely for 10 minutes. The criterion for the mouse to enter each arm is that all four limbs enter. The mouse enters three different arms (such as ABC, ACB, BCA, etc.) in sequence, which is considered a correct alternation. The alternation score is calculated by dividing the total number of alternations by the total number of choices minus 2 and multiplying it by 100, as shown in the following equation: Correct alternation rate (%) = (number of correct alternations) / (total number of arm entries - 2) × 100; the test results are as follows Fig.18 As shown in D.

[0125] Depend on Fig.18 The results showed that compared with the normal control group, the time that the mice in the MPTP group stayed on the rotating rod and the hanging time in the suspension experiment were greatly reduced, the crawling time of the mice in the pole experiment was significantly prolonged, and the correct alternation rate of the mice in the Y-maze experiment was significantly decreased, indicating that MPTP induction did reduce the motor coordination ability of the mice, indicating that the injection of MPTP can successfully induce Parkinson's disease mice, that is, the MPTP model was successfully constructed.

[0126] Fig.18 The results in Figure A showed that compared with the MPTP model group, administration of ST-4 or ST-4Na at a concentration of 15-60 mg / kg could significantly prolong the time that Parkinson's mice stayed on the rotating rod (P < 0.05), among which the dose of 30 mg / kg showed a very significant difference (P < 0.001), and there was no significant difference between the two groups given ST-4 or ST-4N.

[0127] Fig.18 The results in Figure B showed that compared with the MPTP model group, administration of ST-4Na at a concentration of 15-60 mg / kg could significantly reduce the crawling time of Parkinson's disease mice in the extreme point experiment (P < 0.05), and the effect was better than the drug L-Dopa. Among them, the effect of ST-4Na at a concentration of 30 mg / kg was better, and the effect of ST-4 at a concentration of 15-60 mg / kg was equivalent to that of the drug L-Dopa.

[0128] Fig.18The results in Figure 3 showed that compared with the MPTP model group, oral gavage of ST-4 or ST-4Na at a concentration of 15-60 mg / kg could increase the hanging time of Parkinson's disease mice in the suspension experiment, and there was no significant difference between the two groups given ST-4 or ST-4Na, especially when given a dose of 30 mg / kg ST-4 or ST-4Na, the hanging time of mice was longer than that of mice in the normal control group.

[0129] Fig.18 The results in Figure D showed that compared with the MPTP model group, ST-4 or ST-4Na at concentrations of 15 to 60 mg / kg could increase the correct alternation rate of Parkinson's disease mice in the Y-maze experimental model.

[0130] In summary, both ST-4 and ST-4Na showed good therapeutic effects on Parkinson's disease mice, among which the therapeutic effect of ST-4Na was better overall.

[0131] The experimental method of brain tissue immunohistochemical analysis is the same as that of Example 4. Fig.19 shown.

[0132] The results of immunohistochemical analysis showed that the expression of tyrosine hydroxylase in the substantia nigra pars compacta increased after treatment with ST-4 and ST-4Na, indicating that ST-4 and ST-4Na can restore the dopaminergic neuron loss caused by MPTP.

[0133] Example 6 ST-4 inhibits MPTP-induced NLRP3 inflammasome activation in the substantia nigra pars compacta of mice midbrain Neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD) and Huntington's disease (HD), are complexly associated with neuroinflammation. This example further studies the effect of ST-4 on neuroinflammation caused by neurodegenerative diseases. The specific experimental grouping and experimental methods are the same as those in Example 5.

[0134] Experimental methods: Immunofluorescence staining was performed on paraffin-embedded brain tissue sections; paraffin sections were first dewaxed and hydrated. Sections were treated with 5% bovine serum albumin and sealed at room temperature for 30 minutes. Then, diluted primary antibodies were added to each section and incubated in a wet box at 4°C overnight. After washing away excess primary antibodies, diluted fluorescent secondary antibodies were added to brain sections and incubated in the dark at 20 or 37°C for 1 hour. 4′, 6-diamino-2-phenylindole (DAPI) staining was then performed for 5 to 10 minutes to mark cell nuclei. Sections were observed using the Olympus automatic scanning system SV120. The following primary antibodies were used: rabbit anti-IBA1 (NO.019-19741, Wako), rabbit anti-iNOS (18985-1-AP, Proteintech), and rabbit anti-GFAP (A19058, ABclonal). WB was used to detect the level of inflammasome protein in the midbrain; Elisa was used to detect the level of inflammatory factors in the midbrain.

[0135] The experimental results are as follows Fig. 20 As shown, its Fig. 20 A is a representative double immunofluorescence staining image of iNOS (red) and IBA1 (green) in the substantia nigra of the mouse midbrain; B is the IBA1 + Statistical results of cells; C is the iNOS in each group + Statistical results of cells.

[0136] IBA1 is a specific protein that marks microglia (immune cells in the brain). Under normal and pathological conditions, IBA1 is a marker of microglial activation. In many neurodegenerative diseases, the activation of IBA1-positive microglia is a common phenomenon, and the activation of microglia leads to the strong secretion of cytokines, chemokines, reactive oxygen species and other neurotoxic mediators, which directly leads to neuroinflammation in the brain.

[0137] iNOS is an enzyme that is induced in response to inflammation, infection or cellular stress. It is not normally expressed in neurons, but can be induced under certain pathological conditions. In neurodegenerative diseases, iNOS expression increases in neurons, indicating that these neurons are responding to inflammation or injury.

[0138] Depend on Fig. 20 The results of immunofluorescence staining showed that compared with the control group, the number of microglia in the substantia nigra of the MPTP model group increased significantly. At the same time, compared with the normal control group mice, the expression levels of iNOS, NLRP3, ASC, mature IL-1β and caspase-1 in the substantia nigra of the MPTP model group mice were significantly increased, indicating that the NLRP3 inflammasome was activated in the brain of the model group mice, accompanied by neuroinflammation.

[0139] Compared with the MPTP model group, the number of co-localized iNOS-positive neurons and IBA1-positive neurons in the ST-4 drug treatment group was significantly reduced ( Fig.21 ,22), and the expression levels of iNOS, NLRP3, ASC, mature IL-1β and caspase-1 in the substantia nigra of the model mice were significantly reduced ( Fig. 22 ). These results indicate that ST-4 can promote the transformation of microglia from M1 phenotype to M2 phenotype in the brain of MPTP-induced model mice, inhibit the activation of NLRP3 inflammasome, and thus reduce neuroinflammation and neuronal damage in the brain caused by neurodegenerative diseases.

[0140] Example 7 Effects of different concentrations of ST-4 and ST-4Na on inhibition of MPTP-induced NLRP3 inflammasome activation in the substantia nigra of mice midbrain

[0141] The experimental mice were randomly divided into MPTP modeling group, ST-4 group (MPTP + ST-4 low, medium and high doses), ST-4Na group (MPTP + ST-4Na low, medium and high doses), L-Dopa group (MPTP + L-Dopa), and blank control group. Each group had one cage, and each cage had 6 mice. MPTP was intraperitoneally injected at 25 mg / kg / d for six consecutive days.

[0142] The low-dose group: 15 mg / kg; the medium-dose group: 30 mg / kg; the high-dose group: 60 mg / kg, each intragastric administration volume 100 μL / mouse (solvent is 90% water + 5% ethanol + 5% Tween-80), the control group and the model group mice were given equal volume of solution intragastric administration once a day for 14 consecutive days. Behavioral tests were performed 14 days after administration, and then samples were taken for subsequent experiments.

[0143] By detecting inflammatory factors (IL-18, IL-1β) in the substantia nigra region of the mouse midbrain, the results are as follows Fig.21 shown.

[0144] Depend on Fig.21 It can be seen that compared with the normal control group, the levels of inflammatory factors IL-18 and IL-1β in the substantia nigra region of the midbrain of mice in the MPTP model group were significantly increased; after treatment with ST-4 or ST-4Na at a concentration of 15 to 60 mg / kg, the levels of inflammatory factors IL-1β and IL-18 in the substantia nigra region of the midbrain of the model mice were significantly decreased, and there was no significant difference in the therapeutic effect between the ST-4 group and the ST-4Na group, and the preferred concentration was 30 mg / kg; these results indicate that ST-4 and ST-4Na can reduce neuroinflammation in the brain of Parkinson's mice and can be used to prepare drugs for neuroinflammation caused by neurodegenerative diseases.

[0145] Fig.21The results of the experiment of ST-4 and ST-4Na inhibiting the activation of NLRP3 inflammasome in MPTP-induced mouse SN. Among them, GFAP labeled astrocytes in green, Iba-1 labeled microglia in red, and DAPI labeled cell nuclei in blue. Fig.21 Immunofluorescence staining of GFAP and Iba1, scale bar = 20 μm.

[0146] like Fig.23 As shown in the figure, compared with the normal control group, the number of microglia (red) and astrocytes (green) in the MPTP model group increased significantly in the substantia nigra region. Compared with the model group, the number of activated microglia and astrocytes in the substantia nigra of mice in the ST-4 and ST-4Na drug treatment groups was significantly reduced. These results indicate that ST-4 and ST-4Na can reduce the activation of microglia and astrocytes in the substantia nigra region, thereby inhibiting the activation of the NLRP3 / IL-1β signaling pathway in the substantia nigra induced by MPTP.

[0147] Example 8 Transcriptomic changes in Parkinson's disease mice after ST-4 treatment

[0148] The experimental modeling grouping and drug treatment method are the same as in Example 5;

[0149] Experimental methods: RNA isolation, cDNA library construction and sequencing were performed by Meiji Biotechnology (Shanghai, China). Data analysis was performed using i-Sanger (https: / / www.i-sanger.com / ). Data were also analyzed using Kyoto Gene, Genome Encyclopedia Database, Protein Database, Swiss-Prot and Pfam database tools.

[0150] The experimental results are as follows Figure 24-26 As shown, the transcriptomic changes of Parkinson's disease mice after ST-4 treatment Fig.24 The volcano plot shows the differentially expressed genes between the ST-4 treatment group and the Parkinson's disease model group, where significantly upregulated genes are dyed red, significantly downregulated genes are dyed green, and genes that are not significantly changed are dyed blue; Fig.25 The top 25 KEGG pathways of genes significantly changed in the substantia nigra of PD mice after ST-4 treatment; Fig.26 Comparison of the number of changes in genes related to Parkinson's disease and immune inflammatory response in mice in the ST-4 treatment group and the Parkinson's disease model group.

[0151] The transcriptomic analysis results of the above mice showed that there were 4460 differentially expressed genes between the ST-4 drug treatment group and the Parkinson's disease model group, of which 2123 genes were upregulated and 2337 genes were downregulated ( Fig.24). Further functional analysis of the differentially expressed genes revealed that the genes with significantly changed expression levels caused by ST-4 were mainly related to "inflammatory response" and "Parkinson's disease", mainly involving ribosome-related pathways (Ribosome, ko03010), oxidative phosphorylation pathways (Oxidative phosphorylation, ko00190) and Parkinson's disease-related pathways (Parkinson disease, ko05012). In neuroscience research, tyrosine hydroxylase is often used as a marker of dopaminergic neurons to study Parkinson's disease and other neurodegenerative diseases. Among them, the decrease in tyrosine hydroxylase activity is related to the pathological mechanism of Parkinson's disease.

[0152] Depend on Fig.26 It can be seen that after administration of ST-4, the expression of tyrosine hydroxylase (TH) in the compact tissue of the substantia nigra in the midbrain was significantly upregulated, indicating that ST-4 can restore the loss of dopaminergic neurons and inhibit the death of dopaminergic neurons, thereby playing its role in treating Parkinson's disease.

[0153] The experimental results of the above examples demonstrate that ST-4 and ST-4Na have significant therapeutic effects on neuroinflammation and neurodegenerative diseases such as Parkinson's disease mediated by microglia activation, and have drug development potential.

[0154] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. Use of a diterpene small molecule compound in the preparation of a drug for treating neurodegenerative diseases, characterized in that: The compound is ST-4 or its sodium salt ST-4Na; The structural formula of compound ST-4 is as follows: The structural formula of its sodium salt ST-4Na is as follows:

2. The use according to claim 1, characterized in that The compound is used for preparing medicine for treating Parkinson's disease.

3. The use according to claim 2, characterized in that The dosage of the compound ST-4 or ST-4Na in the drug for treating Parkinson's disease is 1.5 to 6.0 mg / kg.

4. The use according to claim 1, characterized in that The compound ST-4 or ST-4Na is used for preparing drugs for resisting neuroinflammation caused by neurodegenerative diseases.

5. The use according to claim 4, characterized in that The compound ST-4 or ST-4Na is used for preparing drugs for resisting neuroinflammation caused by Parkinson's disease.

6. The use according to claim 5, characterized in that The dosage of compound ST-4 or ST-4Na in the anti-neuroinflammatory drug is 2-4 μM, or 1.5-6.0 mg / kg.

7. A drug for treating Parkinson's disease, characterized in that: The drug comprises compound ST-4 or its sodium salt ST-4Na, and pharmaceutically acceptable auxiliary materials.

8. The drug according to claim 7, characterized in that The compound is ST-4Na, and its dosage is 1.5 to 3.0 mg / kg.

9. The drug according to claim 7, characterized in that The compound is ST-4, and the dosage thereof is 3.0 to 6.0 mg / kg.

10. The drug for treating Parkinson's disease according to any one of claims 7 to 9, characterized in that: The medicine is an oral preparation or an injection preparation.

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