阅读说明：本技术 胡黄连素二聚体类似物衍生物在制备防治帕金森病的药物或保健品中的应用 (Application of kutkin dimer analogue derivative in preparation of medicine or health-care product for preventing and treating Parkinson's disease ) 是由 蒋杰 李沙 王高芳 冯国帅 藕洋 高瑞涛 *** 于 2019-03-21 设计创作，主要内容包括：本发明公开了胡黄连素二聚体类似物衍生物在制备防治帕金森病的药物或保健品中的应用。该胡黄连素二聚体类似物衍生物的结构式如式I所示。本发明中发现胡黄连素二聚体类似物衍生物或其药学上可接受的盐在帕金森病体外细胞模型上,显示出比母体化合物胡黄连素更优的药效活性,且其可以明显改善帕金森病模型小鼠的行为学,增加黑质致密部多巴胺能神经元数量,提高纹状体内多巴胺和3,4-二羟基苯乙酸水平。因此,可将胡黄连素二聚体类似物衍生物或其药学上可接受的盐制成相关药物或保健品,用于防治帕金森病。<Image he="471" wi="592" file="DDA0002002018540000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>(The invention discloses an application of a kutkin dimer analogue derivative in preparing a medicine or a health-care product for preventing and treating Parkinson's disease. The structural formula of the kutkin dimer analogue derivative is shown as a formula I. The invention discovers that the kutkin dimer analogue derivative or the pharmaceutically acceptable salt thereof shows better pharmacodynamic activity than that of a parent compound kutkin in vitro cell model of the Parkinson disease, can obviously improve the behaviours of mice of a Parkinson disease model, increase the number of dopaminergic neurons at the substantia nigra pars compacta and improve the number of striate dopaminergic neurons on the parkinsonism cell modelLevels of dopamine and 3, 4-dihydroxyphenylacetic acid in the body. Therefore, the kutkin dimer analogue derivative or the pharmaceutically acceptable salt thereof can be prepared into related medicines or health products for preventing and treating the Parkinson's disease.)

1. The application of the kutkin dimer analogue derivative or the pharmaceutically acceptable salt thereof in preparing the medicine or the health care product for preventing and treating the Parkinson disease is characterized in that the structural formula of the kutkin dimer analogue derivative is shown as a formula I:

wherein R is₁、R₂、R₃The same or different, are respectively selected from: hydrogen, substituted or unsubstituted, heteroatom-containing or heteroatom-free, straight-chain, branched or cyclic hydrocarbon carbon chains of up to 10 carbon atoms, substituted or unsubstituted monocyclic aryl, heteroaryl, alkoxycarbonylalkyl, lipoyl, substituted or unsubstituted cysteinyl, nitrate, acyl, sulfonate.

2. The use of the kutkin dimer analog derivative or the pharmaceutically acceptable salt thereof according to claim 1 for the preparation of a medicament or health product for preventing and treating parkinson's disease, wherein: the kutkin dimer analogue derivative is at least one of JJA-D0 and JJA-D1-JJA-D40; wherein the structural formulas of JJA-D0 and JJA-D1-JJA-D40 are shown as follows:

3. the use of the kutkin dimer analog derivative or the pharmaceutically acceptable salt thereof according to claim 1 for the preparation of a medicament or health product for preventing and treating parkinson's disease, wherein:

the medicine or health care product is a medicine or health care product for activating PI3K/Akt and Nrf2/HO-1 signal channels in neuron cells, inhibiting MAPKs and NF-kB signal channels in the neuron cells, inhibiting the activation of NADPH oxidase in the neuron cells and/or inhibiting the apoptosis of the neuron cells.

4. The use of the kutkin dimer analog derivative or the pharmaceutically acceptable salt thereof according to claim 3 for the preparation of a medicament or health product for preventing and treating Parkinson's disease, wherein:

the inhibition of the activation of NADPH oxidase in neuronal cells inhibits the activation of NADPH oxidase in neuronal cells by down-regulating the protein expression of the NADPH oxidase gp91-phox and p47-phox subunits.

5. The use of the kutkin dimer analog derivative or the pharmaceutically acceptable salt thereof according to claim 3 for the preparation of a medicament or health product for preventing and treating Parkinson's disease, wherein:

the inhibition of the apoptosis of the neuron cells inhibits the apoptosis of the cells by down-regulating the expression of apoptotic protein Cleaved caspase-3 and increasing the ratio of Bcl-2/Bax.

6. The use of the kutkin dimer analog derivative or the pharmaceutically acceptable salt thereof according to claim 1 for the preparation of a medicament or health product for preventing and treating parkinson's disease, wherein:

the medicine or health care product is a medicine or health care product for reducing the ROS level in neuron cells, reducing the level of a lipid peroxidation product malondialdehyde MDA in the neuron cells, improving the relative activity of superoxide dismutase SOD in the neuron cells, reversing the reduction of mitochondrial membrane potential in the neuron cells, reducing the expression of inflammatory factor TNF-alpha in the neuron cells, reducing the expression of alpha-synuclein protein in the neuron cells, increasing the number of dopaminergic neuron elements in substantia nigra, improving the dopamine level in striatum, and/or improving the 3, 4-dihydroxy phenylacetic acid level in the striatum, and improving the behaviourology of Parkinson's disease.

7. The use of the kutkin dimer analog derivative or the pharmaceutically acceptable salt thereof according to any one of claims 1 to 6 in the preparation of a medicament or health product for preventing and treating Parkinson's disease, wherein the kutkin dimer analog derivative or the pharmaceutically acceptable salt thereof is characterized in that:

the medicine or health care product can also contain one or at least two pharmaceutically acceptable carriers;

the carrier is a sustained release agent, an excipient, a filler, an adhesive, a wetting agent, a disintegrating agent, an absorption enhancer, an adsorption carrier, an absorbent, a surfactant or a lubricant.

8. The use of the kutkin dimer analog derivative or the pharmaceutically acceptable salt thereof according to claim 7 for the preparation of a medicament or health product for preventing and treating parkinson's disease, wherein:

the medicine or health product is further prepared into injection, tablet, pill, granule or capsule.

Technical Field

The invention belongs to the technical field of medicines, and relates to an application of a kutkin dimer analogue derivative in preparation of a medicine or a health-care product for preventing and treating Parkinson's disease, in particular to an application of the kutkin dimer analogue derivative or a pharmaceutically acceptable salt thereof in preparation of the medicine or the health-care product for preventing and treating Parkinson's disease.

Background

Parkinson's Disease (PD), also known as parkinsonism tremor, is a common nervous system degenerative disease, is common in the elderly, has an average onset age of about 60 years, is less common in young Parkinson's disease with onset under 40 years, and has a prevalence rate of PD of about 1.7% in people over 65 years in China.

Parkinson's disease is a disease which is difficult to cure clinically, and has great influence on physical and mental health and social and economic pressure of patients. The most prominent pathological feature of parkinson's disease is degenerative death of Dopaminergic (DA) neurons of the midbrain substantia nigra, accompanied by the formation of lewy bodies with alpha-synuclein as the major component. The massive loss of dopaminergic neurons in the substantia nigra pars compacta of the midbrain can cause the significant reduction of the striatum DA level and the relative increase of the acetylcholine level, break the balance between the two and further cause the clinical symptoms of PD. To date, the pathogenesis of PD is still not well defined, and oxidative stress, inflammatory response, genetic factors, aging and the like may be involved in the occurrence and development of PD, wherein the research on the pathogenesis of PD from the level of oxidative stress and inflammatory response is still a current research hotspot. Mitochondria are known to provide large amounts of ATP to the body as a site for energy production by cells. When mitochondria in neuronal cells are damaged, they are not supplied with sufficient energy to cause degenerative death of the neuronal cells. Meanwhile, mitochondrial respiratory chain dysfunction can also lead to the generation of a large amount of Reactive Oxygen Species (ROS) and the explosion of oxidative stress. The large amount of ROS in cells can cause cells to produce large amounts of neurotoxic substances, such as inflammatory factors TNF-alpha, interleukins, apoptotic proteins, and the like, which all contribute to the degeneration and death of neuronal cells. In PD patients, α -synuclein levels within dopaminergic neurons are significantly elevated, and α -synuclein is an important component of the Lewy body. The abnormal aggregation of alpha-synuclein can promote the inflammation and oxidative stress of neurons, and the inflammation and oxidative stress can further cause the abnormal aggregation of the protein, and the protein is difficult to be degraded by cells, so that the condition is worsened.

At present, the most main treatment means for treating the Parkinson's disease is drug treatment, and clinical drugs mainly comprise the following categories: dopamine replacement therapy, dopamine receptor agonists, anticholinergic drugs, monoamine oxidase B inhibitors, catechol-oxygen-methyltransferase inhibitors, and the like. Dopamine substitutes for drugs, supplements the deficiency of dopamine in the brain, which represents that the drugs are levodopa. Dopamine receptor agonists, which stimulate the postsynaptic membrane dopamine receptors to exert their effect, represent the drug bromocriptine. Monoamine oxidase B (MAO-B) inhibitors, inhibit MAO-B in the nigrostriatal, prevent degradation of dopamine, increase the concentration of dopamine in the brain, which represents the drug selegiline. A catechol-o-methyltransferase inhibitor (COMTI), which inhibits levodopa metabolism in the periphery and increases dopamine levels in the brain, which is an indication of the drug entacapone. Anticholinergic drugs, which maintain the balance of dopamine and acetylcholine in the brain of patients, represent the drug diphenhydramine. The medicines mainly improve the symptoms of the Parkinson disease by changing the levels of dopamine and acetylcholine in the brain, are not directly aiming at the conditions of oxidative stress and inflammatory reaction in the brain, are difficult to cure the disease, and simultaneously have side effects after being taken for a long time. Therefore, the development of specific drugs for treating the Parkinson's disease is always a focus of attention of domestic and international large pharmaceutical enterprises and scientific research institutions.

Rhizoma picrorhizae (Apocynin, chemical name is 3-methoxy-4-hydroxy-acetophenone, molecular formula is C)₉H₁₀O₃) Is an active ingredient separated from the root of the traditional Chinese medicine picrorhiza rhizome, and is widely present in various plants. The picrorhizin can selectively inhibit the release of ROS (reactive oxygen species) in human neutrophils, has been used for research of anti-inflammatory and antioxidant medicines for many years, but has not ideal activity. Since various diseases seriously threatening human health such as diabetes, asthma, acute lung injury, arthritis, ischemic injury, tumor, Parkinson's disease, senile dementia and the like are found to be related to oxidative stress and inflammation, and NADPH oxidase activation is one of main pathways for generating ROS in human bodies, NADPH oxidase inhibition is regarded as a drug research target. Picrorhizin, a classical NADPH oxidase inhibitor, has been of interest as an anti-inflammatory, anti-oxidant drug in the treatment of these conditions, although its activity is less than ideal. Currently, few studies are conducted on the structural modification of picrorhizin to improve the activity of picrorhizin. The patent of derivatives of kutkin and their preparation and use (ZL200610037302.1) and the patent of derivatives of kutkin and their preparation and use (ZL201010185981.3) were previously disclosed by researchers in the field aiming at structural modification of kutkin. The two patents mainly disclose the derivatives of the kutkin monomer and the application of the compounds in preparing the medicines and health care products for preventing and treating immune system diseases, NADPH oxidase related diseases, and antioxidation and anti-inflammation. Researchers in the field also disclose the application of picrorhizin nitrone in preparing medicines for preventing and treating asthma (patent application No. 201510955782.9).

Picrorhizin is used in the therapeutic study of many diseases involving oxidative stress injury and inflammatory response, such as parkinson's disease, senile dementia, acute lung injury, ischemic cerebral stroke, etc. Flavonoids can exert some protective effect on neuronal cells by inhibiting NADPH oxidase-induced ROS production, inflammatory responses and apoptosis ((1) Sharma N, Kapor M, Nehru B. Apocynin, NADPH oxidase inhibitor precursors in lipid polysaccharides induced-synthesis and amides functions in rates of polymeric roll of biochemical and inorganic assays [ J ]. Behav brain Res.2016,296:177-190.(2) Stefanska J, Pawlickk R. Apocynin: molecular assays [ J ]. initiators in fluidic 2008, 106507.doi: 10.1155/2008/106507.). Flavonoids can play a role in scavenging oxygen free radicals by blocking the subunits of NADPH oxidase to down-regulate the expression of p47-phox and gp91-phox subunits of NADPH oxidase, thereby specifically inhibiting NADPH oxidase activity (Kanegae MP, Condino-NetoA, Pedroza LA, et al. Diaphynin versacin as a precursor of NADPH oxidase and cytokine production by lipid membrane of NADPH oxidase and cytokine production [ J ]. Biochem Biophys Res Commun.2010,393(3): 551-554.). Under normal physiological conditions, ROS have the effect of conducting cellular signals and killing externally invading bacteria. In pathological conditions, excess ROS exceeds the responsiveness of cells, causing an inflammatory response in cells, resulting in damaged mitochondria, activation of apoptotic pathways, production of caspases and Bax-series apoptotic proteins, and finally in apoptosis of neuronal cells (Ramalingam M, kimsj. reactive oxidative/nitrogen species and the functional synergistic oxidative diseases [ J ]. J Neural trans (Vienna) 2012,119(8): 891-.

The key to the pharmacological activity of kutkin was found to be its conversion to dimers in vivo, and researchers in the field have designed and synthesized kutkin dimer analogs JJA-D0, kutkin (Apocynin), kutkin dimer (Apocynin dimer) and kutkin dimer analogs JJA-D0 with the following structures.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide the application of the kutkin dimer analogue derivative or the pharmaceutically acceptable salt thereof in preparing the medicine or the health care product for treating the Parkinson disease. The invention discloses antioxidant, anti-inflammatory and anti-apoptosis activities and related action mechanisms of the derivatives on a Parkinson disease cell model, discloses therapeutic action of a kutkin dimer analogue on a Parkinson disease model mouse, and proves that the kutkin dimer analogue JJA-D0 and the derivatives thereof have remarkable action of preventing and treating the Parkinson disease, mainly achieve the protection of nerve cells through the antioxidant, anti-inflammatory and anti-apoptosis actions, increase the number of dopaminergic neurons and the dopamine level, and improve the behaviouristics of the Parkinson disease model mouse.

The purpose of the invention is realized by the following technical scheme: an application of a kutkin dimer analogue derivative or a pharmaceutically acceptable salt thereof in preparing a medicine or a health-care product for preventing and treating Parkinson's disease, wherein the structural formula of the kutkin dimer analogue derivative is shown as a formula I:

wherein R is₁、R₂、R₃The same or different, are respectively selected from: hydrogen, substituted or unsubstituted, heteroatom-containing or heteroatom-free, straight, branched or cyclic hydrocarbyl carbon chains of up to 10 carbon atoms (preferably 1 to 8, more preferably 1 to 4 carbon atoms), substituted or unsubstituted monocyclic aryl, heteroaryl, alkoxycarbonylalkyl, lipoyl, substituted or unsubstituted cysteinyl, nitrate, acyl, sulfonate.

The kutkin dimer analogue derivative is preferably at least one of JJA-D0 and JJA-D1-JJA-D40; wherein the structural formulas of JJA-D0 and JJA-D1-JJA-D40 are shown as follows:

the kutkin dimer analogue derivative or the pharmaceutically acceptable salt thereof is applied to the preparation of medicaments or health care products for preventing and treating the Parkinson's disease, wherein the medicaments or health care products are medicaments or health care products for activating PI3K/Akt and Nrf2/HO-1 signal channels in neuron cells, inhibiting MAPKs and NF-kB signal channels in the neuron cells, inhibiting the activation of NADPH oxidase in the neuron cells and/or inhibiting the apoptosis of the neuron cells.

The inhibition of the activation of NADPH oxidase in neuronal cells can inhibit the activation of NADPH oxidase in neuronal cells by down-regulating the protein expression of the NADPH oxidase gp91-phox and p47-phox subunits.

The inhibition of the apoptosis of the neuron cell can inhibit the apoptosis of the cell by down-regulating the expression of apoptosis protein clear caspase-3 and increasing the ratio of Bcl-2/Bax.

The medicine or the health-care product is used for reducing the ROS level in neuron cells, reducing the level of lipid peroxidation product Malondialdehyde (MDA) in the neuron cells, improving the relative activity of superoxide dismutase (SOD) in the neuron cells, reversing the reduction of mitochondrial membrane potential in the neuron cells, reducing the expression of inflammatory factor TNF-alpha in the neuron cells, reducing the expression of alpha-synuclein protein in the neuron cells, increasing the number of dopaminergic neurons in substantia nigra, improving the dopamine level in striatum, and/or improving the level of 3, 4-dihydroxy phenylacetic acid (DOPAC) in the striatum, and improving the behaviouristics of Parkinson disease.

The medicine or health care product can also contain one or at least two pharmaceutically acceptable carriers; the kutkin dimer analogue derivative or pharmaceutically acceptable salt thereof is used as an active ingredient, and a pharmaceutically acceptable carrier is added to prepare a medicament or a health-care product.

The carrier is preferably a sustained-release agent, an excipient, a filler, an adhesive, a wetting agent, a disintegrating agent, an absorption enhancer, an adsorption carrier, an absorbent, a surfactant or a lubricant, and the like.

The medicine or health care product can be further prepared into various forms such as injection, tablet, pill, granule or capsule, and the medicine or health care product with various dosage forms can be prepared according to the conventional method in the pharmaceutical field.

The technical scheme of the invention is as follows:

1. with 1-methyl-4-phenyl-pyridinium ion (MPP)⁺) Inducing human neuroblastoma cell strain SH-SY5Y cells, and establishing an in vitro Parkinson disease cell model; 1-methyl-4-phenyl-1, 2,3, 6-tetrahydropyridine (MPTP) is adopted to induce male C57BL/6 mice to establish an in vivo Parkinson disease model.

2. Investigating the relation of kutkin dimer analogue JJA-D0 and its derivatives on MPP⁺The effect of the survival rate of treated SH-SY5Y cells, intracellular ROS levels and mitochondrial membrane potential, the pharmacodynamic activity of the above compounds on in vitro Parkinson's disease cell models was initially evaluated. Compared with a model group, the kutkin dimer analogue JJA-D0 and the derivatives thereof can improve the survival rate of cells of a dosing group, reduce the intracellular ROS level and reverse the reduction of the mitochondrial membrane potential in the cells, and the activity of most derivatives is better than that of the parent compound kutkin.

3. The compound JJA-D10 is used as a molecular probe, the pharmacological action mechanism of the kutkin dimer analogue derivative for preventing and treating the Parkinson disease is discussed from the aspects of mechanism, target point, signal path and the like, and the specifically investigated indexes and signal path comprise: intracellular lipid peroxide Malondialdehyde (MDA), superoxide dismutase (SOD) activity, inflammatory factor TNF-alpha, apoptosis protein caspase-3 and Bcl-2/Bax levels; intracellular levels of NADPH oxidase subunit gp91-phox and p47-phox proteins; PI3K/Akt signal path, Nrf2/HO-1 signal path, MAPKs signal path and NF-kappa B signal path. The research result shows that JJA-D10 can reduce the level of intracellular lipid peroxide MDA, improve the relative activity of intracellular superoxide dismutase SOD, and reduce the protein expression of NADPH oxidase gp91-phox and p47-phox subunits, thereby reducing the level of intracellular oxidative stress; can reduce the expression of apoptosis protein clear caspase-3 and increase the ratio of Bcl-2/Bax to inhibit apoptosis; and also reduces the expression of the inflammatory factor TNF-alpha. JJA-D10 achieves the effects of antioxidation, anti-inflammation and anti-apoptosis by activating PI3K/Akt and Nrf2/HO-1 signal pathways, inhibiting MAPKs and NF-kB signal pathways and inhibiting the activation of NADPH oxidase, thereby protecting neuronal cells and realizing the prevention and treatment of Parkinson's disease.

4. The treatment effect of the kutkin dimer analogue JJA-D0 on a Parkinson disease model mouse is examined. The results show that JJA-D0 can obviously improve the model mouse ethology, increase the number of dopaminergic neurons in substantia nigra pars compacta and increase the levels of dopamine and 3, 4-dihydroxyphenylacetic acid (DOPAC) in striatum.

Compared with the prior art, the invention has the following advantages and effects:

1. based on the fact that the existing Parkinson disease treatment medicine which is clinically applied realizes the alleviation of symptoms mainly by adjusting the levels of dopamine and acetylcholine, and can not cure diseases aiming at the pathogeny, the invention utilizes the kutkin dimer analogue derivative in a Parkinson disease cell model (adopting MPP)⁺SH-SY5Y cell processing to cause an in vitro Parkinson disease cell model) and screening to obtain a compound for preventing and treating Parkinson disease better from the aspects of oxidation resistance, inflammation resistance and apoptosis resistance aiming at a pathogenesis generated by oxidative stress and inflammation. The kutkin dimer analogue derivative or the pharmaceutically acceptable salt thereof can protect dopaminergic neuron cells from being oxidized and inflammatory damaged and reduce apoptosis by the antioxidant, anti-inflammatory and anti-apoptosis functions aiming at the pathogenesis of high oxidative stress level and inflammatory reaction of the Parkinson disease, thereby realizing the effective prevention and treatment of diseases.

2. The kutkin dimer analogue derivative or the pharmaceutically acceptable salt thereof shows better pharmacodynamic activity than the parent compound kutkin on a Parkinson disease in-vitro cell model. The antioxidant and anti-inflammatory effects of the medicine are obviously superior to that of the picrorhizin, and the medicine has an anti-apoptosis effect.

3. The invention takes the kutkin dimer analogue derivative JJA-D10 as a molecular probe, and finds that the compound protects neuronal cells by dual action mechanisms of antioxidation and anti-inflammation and prevents and treats Parkinson's disease mainly by activating PI3K/Akt and Nrf2/HO-1 signal pathways, inhibiting MAPKs and NF-kB signal pathways and inhibiting the activation of NADPH oxidase.

4. The invention adopts MPTP to induce C57BL/6 mice to establish an in-vivo Parkinson disease animal model, and inspects the treatment effect of the kutkin dimer analogue on the Parkinson disease model mice. The results show that the kutkin dimer analogue JJA-D0 shows obvious Parkinson disease treatment effect on a Parkinson disease model mouse, can obviously improve the model mouse ethology, increase the number of dopaminergic neurons in substantia nigra pars compacta, and increase the levels of dopamine and 3, 4-dihydroxy phenylacetic acid (DOPAC) in striatum.

Drawings

FIG. 1 is a graph showing the results of the toxic effects of kutkin and JJA-D0 on SH-SY5Y cells (n;. P < 0.01; P < 0.001; drug addition versus blank control); wherein, the graph A shows the toxic effect of the picrorhizin on SH-SY5Y cells; panel B is the toxic effect of JJA-D0 on SH-SY5Y cells.

FIG. 2 is an MPP⁺MPP with different concentrations when SH-SY5Y cells are induced to establish PD model⁺Graph of the effect on SH-SY5Y cells (n;. 3;. P)<0.001, additive group compared to blank control group).

FIG. 3 is a graph of picrorhizine versus MPP at various concentrations⁺Effect profile of induced PD model cells (n; # 3; # P;)<0.001 model group compared to blank control group; p<0.01, the additive group compared to the model group).

FIG. 4 is a graph of picrorhizin and picrorhizin dimer analog derivatives versus MPP⁺Effect profile of induced PD model cells (n; # 3; # P;)<0.001, model group compared to blank control group; p<0.05，**P<0.01，***P<0.001, the drug addition group compared with the model group); wherein, the graph A shows that Apocynin, JJA-D0-JJA-D10 are opposite to MPP⁺Protection of induced PD model cells; FIG. B shows JJA-D11-JJA-D22 pairs of MPP⁺Protection of induced PD model cells; FIG. C shows JJA-D23-JJA-D34 pairs of MPP⁺Protection of induced PD model cells.

FIG. 5 shows the pairs of MPP of kutkin and kutkin dimer analog derivatives⁺Effect of induced ROS scavenging ability of SH-SY5Y in cells (n ═ 3; ### P)<0.001, model group compared to blank control group; p<0.05，**P<0.01，***P<0.001, the drug addition group compared with the model group); wherein, the graph A shows that Apocynin, JJA-D0-JJA-D10 are opposite to MPP⁺Induced clearance of intracellular ROS of SH-SY 5Y; FIG. B shows JJA-D11-JJA-D22 pairs of MPP⁺Induced clearance of intracellular ROS of SH-SY 5Y; FIG. C shows JJA-D23-JJA-D34 pairs of MPP⁺Induced clearance of intracellular ROS of SH-SY 5Y.

FIG. 6 shows the pairs of MPP of kutkin and kutkin dimer analog derivatives⁺Graph of the Effect of induced intracellular mitochondrial membrane potential in SH-SY5Y cells (n 3; ## P)<0.001, model group compared to blank control group; p<0.05,**P<0.01，***P<0.001, the drug addition group compared with the model group); wherein, the graph A shows that Apocynin, JJA-D0-JJA-D10 are opposite to MPP⁺The induced reversal of mitochondrial membrane potential drop in SH-SY5Y cells; FIG. B shows JJA-D11-JJA-D22 pairs of MPP⁺The induced reversal of mitochondrial membrane potential drop in SH-SY5Y cells; FIG. C shows JJA-D23-JJA-D34 pairs of MPP⁺Induced reversal of mitochondrial membrane potential drop in SH-SY5Y cells.

FIG. 7 is JJA-D10 vs. MPP⁺Effect of induced SH-SY5Y Total SOD Activity in cells and MDA levels (n; ### P)<0.001, model group compared to blank control group; p<0.05，**P<0.01，***P<0.001, the drug addition group compared with the model group); wherein, the diagram A is JJA-D10 pairs of MPP⁺Effect of induced total SOD activity in SH-SY5Y cells; FIG. B shows JJA-D10 pairs of MPPs⁺Effect of induced MDA levels in SH-SY5Y cells.

FIG. 8 is JJA-D10 vs. MPP⁺Effect of induced SH-SY5Y intracellular TNF- α, cleared caspase-3 and Bax, Bcl-2 protein expression levels (n ═ 3; ### P)<0.001, model group compared to blank control group; p<0.05，**P<0.01，***P<0.001, the drug addition group compared with the model group); wherein, the graphs A and B are JJA-D10 pairs of MPP⁺Induced TNF- α expression levels in SH-SY5Y cells(ii) an effect; graphs C and D are JJA-D10 vs MPP⁺The effect of the level of Cleavedcaspase-3 expression in induced SH-SY5Y cells; graphs E and F are JJA-D10 vs MPP⁺Influence of induced expression level of Bax, Bcl-2 proteins in SH-SY5Y cells.

FIG. 9 is JJA-D10 vs. MPP⁺Graph of the effect of the levels of expression of gp91-phox and P47-phox proteins in induced SH-SY5Y cells (n.gtoreq.3; ## P; #<0.001, model group compared to blank control group; p<0.05，**P<0.01，***P<0.001, the drug addition group compared with the model group); wherein, the graphs A and B are JJA-D10 pairs of MPP⁺The effect of induced gp91-phox protein expression levels in SH-SY5Y cells; graphs C and D are JJA-D10 vs MPP⁺Influence of the induced expression level of the p47-phox protein in SH-SY5Y cells.

FIG. 10 is JJA-D10 vs. MPP⁺Graph showing the influence of the expression levels of PI3K-Akt signal pathway P-PI3K and P-Akt protein in the induced SH-SY5Y cell (n is more than or equal to 3; #### P)<0.001, model group compared to blank control group; p<0.001, the drug addition group compared with the model group); wherein, the graphs A and B are JJA-D10 pairs of MPP⁺The influence of the induced expression level of PI3K-Akt signal pathway p-PI3K protein in SH-SY5Y cells; graphs C and D are JJA-D10 vs MPP⁺Influence of the induced expression level of the P-Akt protein in the PI3K-Akt signaling pathway in SH-SY5Y cells.

FIG. 11 is JJA-D10 vs. MPP⁺Induced SH-SY5Y intracellular Nrf2/HO-1 signal path Nrf2 and HO-1 protein expression level influence graph (n is more than or equal to 3; # P; ## P<0.01, model group compared to blank control group; p<0.05，***P<0.001, the drug addition group compared with the model group); wherein, the graphs A and B are JJA-D10 pairs of MPP⁺Influence of induced expression level of Nrf2/HO-1 signal channel Nrf2 protein in SH-SY5Y cells; graphs C and D are JJA-D10 vs MPP⁺Influence of induced SH-SY5Y intracellular Nrf2/HO-1 signal pathway HO-1 protein expression level.

FIG. 12 is JJA-D10 vs. MPP⁺Induced MAPKs signaling pathway p-ERK in SH-SY5Y cells_1/2Influence patterns of protein expression levels of P-JNK and P-P38 (n.gtoreq.3; ## P)<0.001, model group compared to blank control group; p<0.05，**P<0.01, the drug-adding group is compared with the model group); wherein, the graphs A and B are JJA-D10 pairs of MPP⁺Induced MAPKs signaling pathway p-ERK in SH-SY5Y cells_1/2The effect of protein expression level; graphs C and D are JJA-D10 vs MPP⁺The influence of the induced expression level of MAPKs signaling pathway p-JNK protein in SH-SY5Y cells; graphs E and F are JJA-D10 vs MPP⁺Induced effects of MAPKS signaling pathway and P-P38 protein expression levels in SH-SY5Y cells.

FIG. 13 is JJA-D10 vs. MPP⁺Graph of the effects of the levels of expression of the NF-. kappa.B signaling pathways P-IKK, P-Ikappa B and P-P65 proteins in induced SH-SY5Y cells (n; #######P;)<0.001, model group compared to blank control group; p<0.05，**P<0.01，***P<0.001, the drug addition group compared with the model group); wherein, the graphs A and B are JJA-D10 pairs of MPP⁺Influence of induced expression level of NF-kB signal pathway p-IKK protein in SH-SY5Y cells; graphs C and D are JJA-D10 vs MPP⁺Influence of the induced expression level of the NF-kB signal pathway p-IkB protein in SH-SY5Y cells; graphs E and F are JJA-D10 vs MPP⁺Influence of the induced expression level of the NF-kB signal pathway P-P65 protein in SH-SY5Y cells.

FIG. 14 is a graph showing the effect of JJA-D10 on the expression level of α -synuclein in SH-SY5Y cells (SH-SY5Y (WT α -syn)) transfected with wild-type α -synuclein; wherein, the picture A is SH-SY5Y (WT alpha-syn) cells highly expressing alpha-synuclein observed under a fluorescence microscope (picture I: 10X; picture II: 20X); and the graphs B and C show the relative expression quantity of alpha-synuclein (n is 3; ## # P <0.001, SH-SY5Y (WT alpha-syn) cell model group is compared with the common SH-SY5Y cell control group;. P <0.05, P <0.01, and the drug addition group is compared with the model group).

FIG. 15 is a graph of the effect of Catwalk automatic gait analyser detecting JJA-D0 on MPTP induced Parkinson disease model mouse behaviours (mean + -SD, n 18; # p <0.05, # p <0.01, # p <0.001, compared to normal control;. p <0.05, # p <0.01, compared to model group); wherein, the graph A shows the influence of JJA-D0 on the MPTP-induced distance between forelimb and hind limb of a Parkinson disease model mouse; FIG. B is a graph of the effect of JJA-D0 on the step circumferences of the forelimbs and hindlimbs of MPTP-induced Parkinson's disease model mice; FIG. C is a graph of JJA-D0 effects on MPTP-induced phase of forelimb and hindlimb support in mice models of Parkinson's disease; FIG. D is a graph showing the effect of JJA-D0 on the swing velocity of the forelimb and hindlimb of MPTP-induced Parkinson's disease model mice; FIG. E is a graph of the effects of JJA-D0 on MPTP-induced walking cycles of forelimbs and hindlimbs in a model mouse of Parkinson's disease.

FIG. 16 is a graph of the effect of the Open field assay JJA-D0 on MPTP-induced Parkinson's disease model mouse behaviours (mean + -SD, n 18; # p <0.05, # p <0.01, compared to normal control; # p <0.05, # p <0.01, compared to model group); wherein, the graph A shows the influence of JJA-D0 on the total movement path of MPTP-induced Parkinson disease model mice; FIG. B is a graph of the effect of JJA-D0 on total activity time in MPTP-induced Parkinson's disease model mice; FIG. C is a graph of the effect of JJA-D0 on the residence time in the central region of MPTP-induced Parkinson's disease model mice; FIG. D is a graph representing the effect of JJA-D0 on the trajectory of MPTP-induced Parkinson's disease model mice in the Open field test.

FIG. 17 is a graph of the effect of JJA-D0 on the number of dopaminergic neurons staining positive for TH-staining of substantia nigra in MPTP-induced Parkinson's disease model mice (means + -SD, n; #########p <0.001, compared to the normal control group;. p <0.05,. p <0.01,. p <0.001, compared to the model group); wherein, Panel A is a count chart of TH-staining positive dopaminergic neurons in each experimental group; panel B is a graph of the number of TH-staining positive dopaminergic neurons in each experimental group as a percentage of the number of control groups; panel C is a representative image (20X) of micrographs of various groups of brain tissue sections after TH-staining.

Figure 18 is a graph of the therapeutic effect of the kutkin dimer analog derivatives of the present invention on parkinson's disease.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. The reagents, methods and apparatus employed in the following examples are conventional in the art, unless otherwise indicated. The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer.

SH-SY5Y cells (purchased from ATCC, Manassas, Va., USA) in the following examples were cultured in DMEM/F12 medium with the addition of FBS at a final concentration of 10% (v/v), penicillin 100U/mL and streptomycin 100. mu.g/mL; that is, the fresh medium (solution) referred to in the examples was DMEM/F12 medium containing FBS, penicillin and streptomycin, and the medium was DMEM/F12 medium.

The derivatives of the kutkin dimer analogue are JJA-D0 and JJA-D1-JJA-D34, and can be prepared by the method described in Chinese patent application (with the application number of 201710347578.8, named as the derivatives of kutkin dimer analogue JJA-D0 or pharmaceutically acceptable salts, preparation methods and application thereof).