Application of forsythiaside in preparing medicine for treating Parkinson's disease

Abstract

The present invention discloses the application of forsythiaside in the preparation of a drug for treating Parkinson's disease, and relates to the field of biomedicine technology. The present invention uses the model organism Caenorhabditis elegans as a carrier to study the therapeutic effect of forsythiaside on the neurodegenerative disease Parkinson's disease. On the one hand, the effect of forsythiaside on the lifespan, motor ability and α-Syn protein accumulation of the Caenorhabditis elegans model NL5901 nematode of Parkinson's disease was studied. On the other hand, the effect of forsythiaside on the expression of DA neurons and food perception behavior of BZ555 nematodes was studied. The results show that forsythiaside can enhance the activity of antioxidant enzymes in nematodes, thereby inhibiting and removing excess reactive oxygen free radicals in nematodes, and reducing the abnormal aggregation of α-Syn protein, thereby protecting nerve cells from damage by reactive oxygen free radicals and treating Parkinson's disease.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to application of forsythiaside in preparing medicine for treating Parkinson's disease. Background Art

[0002] To date, the number of Parkinson's patients worldwide has exceeded 6 million. Clinically, when patients show obvious motor symptoms, 50% of dopaminergic neurons (also known as DA neurons) have been lost, and existing drugs cannot save the lost dopaminergic neurons. In addition, the main drug currently used to treat Parkinson's disease is levodopa, and after taking levodopa for about 2 years, Parkinson's patients will have no response or a reduced response to the original dose of levodopa. Therefore, there is a great need to find a drug that can prevent the loss of dopaminergic neurons and can effectively treat Parkinson's disease in the long term.

[0003] Phillygenin is a monomeric compound of lignans. Most of it is in a free state in nature and mainly comes from Forsythia leaves and Osmanthus fragrans. Forsythia is also known as forsythia aglycone. As the main active ingredient in lignans and the fingerprint ingredient in Forsythia fustris, it has been found to have a variety of pharmacological activities in recent years. The pharmacological activities of forsythia include its good antioxidant activity, anti-inflammatory activity, anti-hyperlipidemic activity and anti-tumor activity, but there are no reports on its research on the anti-neurodegenerative disease Parkinson's disease. Summary of the invention

[0004] The purpose of the present invention is to provide the use of forsythiaside in the preparation of drugs for treating Parkinson's disease, so as to solve the problems existing in the above-mentioned prior art. The present invention has found that forsythiaside has the effect of treating Parkinson's disease.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides application of forsythiaside in preparing medicine for treating Parkinson's disease.

[0007] The present invention also provides a medicine for treating Parkinson's disease, wherein the active ingredient of the medicine comprises forsythiaside.

[0008] Furthermore, the medicine also includes pharmaceutically acceptable excipients.

[0009] Furthermore, the dosage form of the drug is granules, tablets, capsules, pills or injections.

[0010] The present invention discloses the following technical effects:

[0011] The present invention uses the model organism Caenorhabditis elegans as a carrier to study the therapeutic effect of forsythiaside on the neurodegenerative disease Parkinson's disease. On the one hand, the effect of forsythiaside on the lifespan, motor ability and α-Syn protein accumulation of the Caenorhabditis elegans model NL5901 of Parkinson's disease was studied, and on the other hand, the effect of forsythiaside on the expression of DA neurons and food perception behavior of BZ555 nematodes was studied. The results show that forsythiaside can enhance the activity of antioxidant enzymes in nematodes, thereby inhibiting and removing excess reactive oxygen free radicals in nematodes, and reducing the abnormal aggregation of α-Syn protein, thereby protecting nerve cells from damage by reactive oxygen free radicals and treating Parkinson's disease.

[0012] The present invention is the first to discover the pharmacological effect of forsythiaside in treating Parkinson's disease. Compared with other drugs for treating Parkinson's disease, forsythiaside is a natural product that is easy to extract and separate, and has a significant therapeutic effect at a relatively low concentration. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0014] Figure 1 The effect of forsythiaside on the life span of NL5901 nematodes;

[0015] Figure 2 The effect of forsythiaside on the motility of NL5901 nematodes;

[0016] Figure 3 The effect of forsythiaside on NL5901 nematode synaptic nucleoprotein α-syn; Figure A shows the results of fluorescence microscopy observation, and Figure B shows the statistical results of GFP fluorescence intensity; the scale bar in A is 50μm;

[0017] Figure 4 The effect of forsythiaside on the expression of DA neurons in BZ555 nematodes; Figure A shows the results of fluorescence microscopy observation, and Figure B shows the statistical results of GFP fluorescence intensity; the scale bar in A is 50 μm;

[0018] Figure 5 The effect of forsythiaside on the food perception behavior of BZ555 nematodes;

[0019] Figure 6 The effect of forsythiaside on the lifespan of nematodes under oxidative stress;

[0020] Figure 7The effect of forsythiaside on the endogenous ROS content in nematodes;

[0021] Figure 8 This is the effect of forsythiaside on the levels of antioxidant enzymes SOD (A) and GSH (B) in nematodes. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] definition

[0028] The term "forsythiaside" used in the present invention is a monomeric compound of lignans, abbreviated as PHI, with a molecular formula of C 21 H 24 O6, molecular weight is 372.41, structural formula is as follows:

[0029]

[0030] In the following examples, NL5901 nematode refers to Caenorhabditis elegans NL5901, purchased from Caenorhabditis Genetics Center (CGC); BZ555 nematode refers to transgenic Caenorhabditis elegans strain BZ555, purchased from Caenorhabditis Genetics Center (CGC); E. coli OP50 was kindly donated by Professor Ma Enbo of the Institute of Applied Biology, Shanxi University.

[0031] Example 1 Forsythiaside can prolong the lifespan of NL5901 nematode, a Parkinson's disease model

[0032] S1. After forsythiaside was dissolved in DMSO solution, different concentrations of forsythiaside solution (0, 10, 20, 40 μg / mL) were mixed with E. coli OP50 suspension (OD 600 0.2-0.3) and mixed evenly at a volume ratio of 1:1, and 100 μL of the mixture was applied to the surface of the plate with nematode NGM medium. Then, 30 NL5901 nematodes synchronized to the L4 stage were selected and added to the above-applied plates, and then placed in a constant temperature incubator at 20°C for culture, which was recorded as day 0 of the lifespan experiment.

[0033] S2. Transfer NL5901 nematodes to a new NGM medium containing forsythiaside and E. coli OP50 every other day (the concentration of forsythiaside in the medium and the coating method are the same as step S1), and count the number of nematodes that survive, die, and escape (escaped nematodes need to be removed from the statistical data) until all nematodes die. The basis for judging the death of nematodes is that when the nematodes cannot move and do not swallow, they are judged to be dead if there is no reaction when touched with a metal platinum wire. Escaped nematodes refer to nematodes that drill into the agar of the NGM culture medium or crawl onto the wall of the culture dish and die. The experiment was repeated 3 times independently.

[0034] The data of the nematode lifespan experiment were analyzed using the Kaplan-Meier survival function in SPSS22.0 (demo version, Armonk, NY) software, and the log-rank test was used for significance analysis. The experimental results were plotted using OriginLab plotting software. P<0.05 was considered to be statistically significant between the two groups of data. (P<0.05* indicates a significant difference between the two groups, and P<0.01** indicates an extremely significant difference between the two groups).

[0035] Experimental results:

[0036] α-syn in NL5901 nematodes is the pathogenic factor of Parkinson's disease. The toxins produced by it can affect the lifespan of nematodes. Therefore, to study the effect of forsythiaside on Parkinson's disease, we must first study the effect of forsythiaside on the lifespan of NL5901 nematodes. The experimental results showed that compared with the control group, 20μg / mL and 40μg / mL forsythiaside significantly prolonged the average lifespan of nematodes, with the extension rates of 23% (P<0.01) and 17% (P<0.05), respectively. Figure 1 As shown in Table 1. From the data, it can be seen that the lower the concentration of forsythiaside, the higher the extension rate of the life span of NL5901 nematodes, which has the advantage of low concentration and significant effect.

[0037] Table 1 Effects of forsythiaside on the lifespan of Parkinson's disease model nematodes

[0038]

[0039] Example 2 Forsythiaside can delay the weakening of NL5901 nematode movement ability

[0040] After forsythiaside was dissolved in DMSO solution, different concentrations of forsythiaside solution (0, 10, 20, 40 μg / mL) were mixed with E. coli OP50 bacterial suspension (OD 600 0.2~0.3) and mixed evenly at a volume ratio of 1:1, and 100μL of the mixture was applied to the surface of the plate with nematode NGM culture medium. Then 30 NL5901 nematodes synchronized to the L4 stage were selected and added to the above-coated plates, and then placed in a constant temperature incubator at 20°C for culture, which was recorded as the 0th day of the experiment. The movement of the nematodes was observed and recorded on the 5th, 10th, and 15th days. In this experiment, the movement of nematodes was divided into three levels: Level A means that the nematodes can move freely without any touch, Level B means that the nematodes need to be stimulated by the outside world (touched by platinum wire) before the body can move freely, and Level C means that the nematodes cannot move freely after being touched with platinum wire and can only swallow. The experiment was repeated 3 times independently.

[0041] Experimental results:

[0042] The harmful α-Syn aggregation in NL5901 nematodes can cause DA neuron toxicity, leading to movement disorders in nematodes and producing certain side effects on the movement ability of nematodes. Therefore, the present invention studies the effect of forsythiaside on the movement ability of NL5901 nematodes. The results show that compared with the control, the movement ability of nematodes treated with forsythiaside on the 10th day was improved by 12%, 27%, and 21% for grade A, and on the 15th day, the movement ability of grade B was improved by 16%, 16%, and 11% (such as Figure 2), proving that forsythiaside delayed the weakening of motility of Caenorhabditis elegans NL5901.

[0043] Example 3 Forsythiaside reduces the aggregation of α-Syn in NL5901 nematodes

[0044] After forsythiaside was dissolved in DMSO solution, different concentrations of forsythiaside solution (0, 10, 20, 40 μg / mL) were mixed with E. coli OP50 bacterial suspension (OD 600 0.2~0.3) and mixed evenly at a volume ratio of 1:1, and 100 μL of the mixture was applied to the surface of the plate with nematode NGM culture medium. Then, 20 NL5901 nematodes synchronized to the L4 stage were selected and added to the above-applied plate, and placed in a constant temperature incubator at 20°C for 2 days. The aggregation of nematode green fluorescent protein was observed with a deltavision deconvolution fluorescence microscope and statistically analyzed.

[0045] Experimental results:

[0046] α-Syn with YFP fluorescence signal is expressed in the body wall muscle cells of NL5901 nematodes. α-Syn is a disordered protein involved in the pathogenesis of PD. Therefore, the present invention studies the effect of forsythiaside on the aggregation of α-Syn in NL5901 nematodes. The results show that forsythiaside reduces the aggregation of α-Syn. Figure 3 As shown in AB.

[0047] Example 4 Forsythiaside enhances the expression of DA neurons in BZ555 nematodes

[0048] After forsythiaside was dissolved in DMSO solution, different concentrations of forsythiaside solution (0, 10, 20, 40 μg / mL) were mixed with E. coli OP50 bacterial suspension (OD 600 After mixing evenly at a volume ratio of 1:1, 100 μL of the mixture was applied to the surface of the plate containing the nematode NGM culture medium. + The solution was shaken every 10 minutes. After 1 hour, the cells were washed three times by centrifugation with M9 buffer and transferred to the NGM medium coated with different concentrations of forsythiaside, 30 per plate, and cultured at 20°C for 2 days. Then, BZ555 nematodes synchronized to the L4 stage were taken and cultured without MPP. + The cells were treated with the solution and directly transferred to the surface of NGM medium containing only E. coli OP50. They were cultured at 20°C for 2 days, and the aggregation of green fluorescent protein in the pharynx of the nematodes was observed using a delta vision deconvolution fluorescence microscope.

[0049] Experimental results:

[0050] The present invention uses the transgenic mutant nematode BZ555 expressing GFP in DA neurons to evaluate the effect of forsythiaside on the activity of DA neurons in nematodes. Figure 4 As shown in Figure AB, compared with the control, nematodes treated with 2 mM MPP + After 1 h of incubation, GFP fluorescence decreased in the cell bodies and dendrites from the nerve ring to the nose tip. + When the nematode was treated with MPP, the GFP fluorescence intensity of DA neurons in the nematode increased significantly. + The activity of DA neurons in BZ555 nematodes can be reduced, and this reduction was significantly attenuated by forsythiaside treatment, proving that forsythiaside can enhance the expression of DA neurons in nematodes.

[0051] Example 5 Forsythiaside enhances the food perception behavior of BZ555 nematodes

[0052] After forsythiaside was dissolved in DMSO solution, different concentrations of forsythiaside solution (0, 10, 20, 40 μg / mL) were mixed with E. coli OP50 bacterial suspension (OD 600 After mixing evenly at a volume ratio of 1:1, 100 μL of the mixture was applied to the surface of the plate containing the nematode NGM culture medium. + The solution was shaken every 10 minutes. After 1 hour, the cells were washed three times by centrifugation with M9 buffer and transferred to the NGM medium coated with different concentrations of forsythiaside, 30 per plate, and cultured at 20°C for 2 days. Then, BZ555 nematodes synchronized to the L4 stage were taken and cultured without MPP. + Solution treatment, directly transferred to the surface of clean NGM culture medium, and cultured at 20℃ for 2 days. After 2 days of culture, each group of nematodes was transferred to NGM culture medium with food and without food respectively. After being placed at room temperature for 5 minutes, the movement of nematodes within 30 seconds was observed and recorded under a stereo microscope, and the reduction rate of nematode food perception was calculated according to the formula:

[0053] Reduction rate = (number of movements of nematodes under sterile conditions - number of movements of nematodes under sterile conditions) / number of movements of nematodes under sterile conditions × 100%.

[0054] Experimental results:

[0055] The rate of movement of C. elegans varies with the presence or absence of food and the feeding state of the worm. For example, well-fed wild-type worms move more slowly in the presence of bacteria than when there is no bacterial food source on a petri dish. This foraging behavior relies on the function of dopaminergic neurons to sense food, and when food is present, the animal's movement slows down. This slowing down in response to plentiful food is called basal slowing. Defects in dopaminergic function result in higher movement in the presence of food, as evidenced by a lower basal response. MPP + The treated nematodes showed a phenotype of DA neuron degeneration, which directly led to abnormal food perception behavior in the nematodes. The research results of the present invention show that exposure to MPP + elegans exposed to MPP + Compared with the control group, the food perception behavior parameters of nematodes decreased by 35% (P<0.01). + After treatment, the nematodes were treated with 20 μg / mL of forsythiaside. + Compared with the control nematodes in the experiment, the parameters of food perception behavior increased by 16.3% (P<0.05). Figure 5 This indicates that forsythiaside can enhance the food sensing behavior of BZ555 nematodes.

[0056] Example 6 Forsythiaside can increase the lifespan of NL5901 nematodes under oxidative stress

[0057] The NL5901 nematodes were synchronized to the L4 stage and treated with 0 μg / mL, 10 μg / mL, 20 μg / mL and 40 μg / mL forsythiaside for 2 days (the method of action is the same as in Example 1). The nematodes were then transferred to NGM medium containing 240 μM juglone, with 30 nematodes per group. The survival status of the nematodes was observed and recorded every hour (the escaped nematodes needed to be removed from the statistical data) until all the nematodes died. The judgment principle of nematode death and escape was the same as in Example 1.

[0058] Experimental results:

[0059] Many research results show that oxidative stress is a key driver of the complex degenerative cascade of dopaminergic neurodegeneration in PD. Therefore, the present invention examines the effect of forsythiaside on the ability of nematodes to resist oxidative stress. The present invention uses juglone to stimulate acute oxidative stress in nematodes. The results show that 20 μg / mL and 40 μg / mL forsythiaside can prolong the average survival time of nematodes under juglone stimulation conditions compared with the control group, with extension rates of 39% and 34%, respectively (e.g. Figure 6 As shown in Table 2). This indicates that forsythiaside can reduce the damage of oxidative stress to nematodes.

[0060] Table 2 Effect of forsythiaside on the survival time of NL5901 under oxidative stress

[0061]

[0062] Example 7 Forsythiaside can reduce the content of ROS in nematodes

[0063] The NL5901 nematodes were synchronized to the L4 stage, and the nematodes were treated with 0 μg / mL, 20 μg / mL and 40 μg / mL forsythiaside for 2 days (the method of action was the same as in Example 1), and then the nematodes were washed by centrifugation with M9 buffer for 2-3 times, and the lysate was added to be lysed with a homogenizer, and the supernatant was collected by high-speed centrifugation. 40 μL of the supernatant was added to a 96-well plate (protected from light), and 100 μL of H2DCF-DA probe was added, incubated at 20°C, read once every 20 minutes, read for 140 minutes, the excitation light was 485 nm, the emission light was 528 nm, and the remaining supernatant was measured for protein concentration by BCA method.

[0064] Experimental results:

[0065] Excessive ROS produced after cellular redox imbalance can lead to the death of dopaminergic neurons. Therefore, the present invention studies the effect of forsythiaside on the ROS content in nematodes. Figure 7 As shown, the ROS content in the nematodes treated with 20 μg / mL and 40 μg / mL forsythiaside significantly decreased compared with the control group (P<0.05).

[0066] Example 8 Forsythiaside can enhance the activity of antioxidant enzymes in nematodes

[0067] The NL5901 nematodes were synchronized to the L4 stage, and the nematodes were treated with 0 μg / mL, 20 μg / mL and 40 μg / mL forsythiaside for 4 days (the treatment method was the same as in Example 1). The nematodes were washed by centrifugation with M9 buffer, homogenized in a homogenizer, and the supernatant was collected by high-speed centrifugation. The SOD and GSH contents in the nematodes were detected using a kit.

[0068] Experimental results:

[0069] The antioxidant enzyme proteins SOD and GSH are mainly used to remove reactive oxygen free radicals in nematodes, and play an anti-Parkinson's disease role from a molecular perspective. Figure 8 As shown in Figure AB, the content of antioxidant enzymes SOD and GSH in the nematodes was significantly increased after treatment with 20μg / mL and 40μg / mL forsythiaside compared with the control group (P<0.01).

[0070] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of forsythiaside in the preparation of drugs for treating Parkinson's disease.

Images