Application of oxalisin A in the preparation of anti-Parkinson's disease drugs

Abstract

The invention discloses Buddlejasaponin IVb (BJP-IVb) for preparing anti-Parkinson's disease (Parkinson , The application of the saponin A in PD (PD) drugs. The saponin A has a significant improvement effect on the motor function damage and dopaminergic neuron loss of PD mice induced by MPTP; + The induced PD cell neurotoxicity has a significant improvement effect. Among them, the blood-breaking saponin A inhibits the iron deposition of dopaminergic neurons mediated by IRP2, thereby inhibiting the ferroptosis of dopaminergic neurons, and finally exerts an anti-PD effect.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and particularly relates to the application of oxalicylic acid saponin A in the preparation of anti-Parkinson's disease drugs. Technical Background

[0002] Parkinson's disease (PD) is a neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra compacta of the midbrain, and with progressive bradykinesia, resting tremor, muscle rigidity and other movement disorders as clinical symptoms. Clinical therapeutic drugs can be divided into dopamine mimetics represented by levodopa and central cholinergic receptor blockers represented by trihexyphenidyl according to their mechanism of action. However, the above drugs can only temporarily control the clinical symptoms of PD patients such as movement disorders, and cannot inhibit the loss of dopaminergic neurons, thus failing to fundamentally prevent the progression of the disease.

[0003] Buddlejasaponin IVb (BJP-Ⅳb) is the main active ingredient of the traditional Chinese medicine Buddlejasaponin, and its structure is as follows. At present, there are no related research reports on the application of Buddlejasaponin A in the preparation of anti-PD drugs. The present invention is the first to discover that Buddlejasaponin A has anti-PD uses. The experimental results show that Buddlejasaponin A can inhibit IRP2-mediated dopaminergic neuron iron deposition, thereby inhibiting dopaminergic neuron ferroptosis and dopaminergic neuron loss, and exerts an anti-PD effect, revealing that it has the prospect of being developed into an anti-PD drug.

[0004]

[0005] Chemical structure of Buddlejasaponin IVb (BJP-Ⅳb) Summary of the invention

[0006] The purpose of the present invention is to disclose a new application of saponin A for the preparation of anti-PD drugs.

[0007] The experimental results of the present invention show that: saponin A can inhibit the iron deposition of dopaminergic neurons mediated by IRP2, thereby inhibiting the ferroptosis of dopaminergic neurons and inhibiting the loss of dopaminergic neurons.

[0008] The experimental results of the present invention prove that Duanxueliu saponin A has an anti-PD effect and can be used in the preparation of anti-PD drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 The experimental results of the improvement of the behavior of MPTP-induced PD mice by saponin A;

[0010] Figure 2The experimental results of the effect of saponin A on Nissl bodies and TH-positive cells in the substantia nigra of MPTP-induced PD mice;

[0011] Figure 3 Effect of saponin A on MPP + The induced PD cell morphology affects the experimental results;

[0012] Figure 4 Effect of saponin A on MPP + The survival rate of induced PD cells affects the experimental results;

[0013] Figure 5 Effect of saponin A on MPP + The induced lactate dehydrogenase release rate of PD cells affects the experimental results;

[0014] Figure 6 Effect of saponin A on MPP + The induced lipid ROS in PD cells affected the experimental results;

[0015] Figure 7 Effect of saponin A on MPP + The induced MDA content of PD cells affects the experimental results;

[0016] Figure 8 The experimental results of the effect of saponin A on the MDA content in the substantia nigra pars compacta of MPTP-induced PD mice;

[0017] Fig. 9 Effect of saponin A on MPP + The induced 4-HNE content of PD cells affects the experimental results;

[0018] Fig.10 The experimental results of the effect of saponin A on the 4-HNE content in the substantia nigra pars compacta of MPTP-induced PD mice;

[0019] Fig.11 Effect of saponin A on MPP + Induced effects on cell membrane and mitochondrial status of PD cells;

[0020] Fig.12 Effect of saponin A on MPP + The induced iron deposition in PD cells affects the experimental results;

[0021] Fig.13 The experimental results of the effect of saponin A on iron deposition in the substantia nigra pars compacta of PD mice induced by MPTP;

[0022] Fig.14 Effect of saponin A on MPP +The induced PD cell IRP2, FTH, TFR, and FPN protein expression levels affect the experimental results;

[0023] Fig.15 The experimental results of the effect of saponin A on the expression levels of IRP2, FTH, TFR and FPN proteins in the substantia nigra compacta of MPTP-induced PD mice;

[0024] Fig.16 The results of IRP2 protein expression in cells after transfection with IRP2 overexpression plasmid;

[0025] Fig.17 Effect of saponin A on MPP after overexpression of IRP2 protein + The survival rate of induced PD cells affects the experimental results;

[0026] Fig.18 Effect of saponin A on MPP after overexpression of IRP2 protein + The induced LDH release rate of PD cells affects the experimental results;

[0027] Fig.19 Effect of saponin A on MPP after overexpression of IRP2 protein + The effects of induced lipid ROS levels in PD cells;

[0028] Fig. 20 Effect of saponin A on MPP after overexpression of IRP2 protein + The induced MDA content of PD cells affects the experimental results;

[0029] Fig.21 Effect of saponin A on MPP after overexpression of IRP2 protein + Effects of induced PD cell membrane and mitochondrial status DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with specific embodiments. The features and advantages of the present invention will become clearer as the description proceeds, but the exemplary embodiments are only used to illustrate the present invention and do not constitute any limitation on the scope of the present invention.

[0031] Example 1

[0032] Effects of saponin A on movement disorders and dopaminergic neuron loss induced by MPTP in PD mice.

[0033] 1. Experimental Animal Grouping

[0034] Fifty C57BL / 6 male mice, weighing 20-25 g, were randomly divided into normal group (con group), model group (MPTP group), positive drug group (L-DOPA group), low-dose group (L-BJP-Ⅳb group), and high-dose group (H-BJP-Ⅳb group).

[0035] 2. Behavioral training

[0036] In order to study whether saponin A can protect animals from MPTP-induced motor impairment, the PD animal model was tested using the rotarod test, the suspension test, and the pole climbing test. Three days before modeling, all mice were trained with rotarods 3 times a day, each time at a speed of 25r / min for 5 minutes; pole climbing training 3 times a day; and suspension training 3 times a day. The motor ability of all mice was kept at the same level.

[0037] 3. Modeling and drug administration

[0038] High dose, low dose and positive drug were administered by gavage 3 days in advance until the 14th day, and MPTP was continuously administered at 25 mg / kg.d -1 Intraperitoneal injection for 7 days. Behavioral tests were performed after the administration. After the behavioral tests were completed, the mice were anesthetized with 4% chloral hydrate, fixed on the surgical board in supine position, and the abdominal cavity was cut open horizontally below the sternum to completely expose the entire chest cavity. PBS and 4% paraformaldehyde were perfused through the left ventricle of the mouse until the liver turned white. The entire brain tissue was completely removed and soaked in 4% paraformaldehyde for 24 hours. The fixed brain tissue was taken and dehydrated in 30% sucrose solution until the brain tissue completely sank to the bottom. The dehydrated brain tissue was embedded in OCT embedding agent, and then the location of the substantia nigra and striatum was selected using a freezing slicer to slice at a thickness of 30μm per slice, and stored in antifreeze at -20°C.

[0039] 4. Nissl staining

[0040] Take frozen sections of the mouse brain from the substantia nigra, fix them in 4% paraformaldehyde for 30 minutes, wash them three times with PBS, 5 minutes each time, and then fully immerse the washed brain sections in Nissl dye and incubate them at 40°C for 10 minutes. After staining, wash them in PBS three times, dehydrate them three times with 95% ethanol, and then transparentize them three times with xylene, seal them with neutral resin, and observe the distribution of Nissl bodies in the substantia nigra under a microscope.

[0041] 5. TH staining

[0042] Take frozen sections of the substantia nigra, wash them in PBS three times, 5 minutes each time, and then shake them in 1% H2O2 at room temperature for 10 minutes, and then wash them in PBS three times, 5 minutes each time. Incubate the brain slices in PBS containing 0.3% Triton-100 for 15 minutes at room temperature, then transfer the brain slices to the blocking solution for 1 hour, incubate them in anti-TH at 4°C overnight, wash the brain slices in PBS three times, 5 minutes each time, incubate them in secondary antibodies at room temperature for 1 hour, and continue to wash them with PBS three times. Incubate in SABC solution at room temperature for 1 hour, wash in PBS three times, 5 minutes each time, color with DAB for 2 minutes, dehydrate in gradients, air dry, and soak in xylene twice in turn, and seal the slices. Observe the number of TH in the substantia nigra under a microscope.

[0043] 6. Experimental results

[0044] The results of the mouse behavioral experiment were as follows Figure 1 As shown. In the pole climbing test, the time that the MPTP group spent on the straight pole was significantly longer than that of the normal group (P < 0.01). After the drug was administered, the time that the mice spent on the pole was shortened. For the hanging test and the rotating rod test, the time that the MPTP group mice spent hanging on the rod and staying on the fatigue rotating rod instrument was significantly shortened (P < 0.05). After the administration of blood-breaking saponin A, the time that the animals spent on the instrument was prolonged. The results of the mouse behavioral experiment showed that the MPTP modeling had an impaired effect on the motor function of mice, and blood-breaking saponin A could improve the damage to the motor function.

[0045] The results of Nissl staining and TH (tyrosine hydroxylase) staining of frozen sections of the substantia nigra in the mouse brain are as follows Figure 2 The expression of Nissl bodies and TH in the MPTP group was decreased (P<0.01), and the number of Nissl bodies and TH positive cells increased after pre-administration of saponin A to cut off blood flow (P<0.01).

[0046] The results of animal experiments have shown that MPTP can cause damage to the motor function of mice, and reduce the number of Nissl bodies and TH-positive cells in the substantia nigra of mice. Blood flow-destroying saponin A can reduce the effects of MPTP.

[0047] Example 2

[0048] Effect of saponin A on MPP + Induced neurotoxicity in PD cells.

[0049] 1. Cell culture

[0050] SH-SY5Y cells were quickly thawed and inoculated in 1640 culture medium containing 15% fetal bovine serum and 1% double antibody. The cells were gently blown to disperse evenly. When the cells grew to 90% confluence, modeling or passaging was performed.

[0051] 2. Cell experiment grouping and drug administration

[0052] The experimental groups were normal group, model group (MPP + For the different concentrations of saponin A, cells were cultured for 24 h until the cells adhered to the wall, and then saponin A at the corresponding concentration was added. After 2 h, 4 μM MPP was added to the model group and the different concentrations of saponin A. + , and continue culturing for 24h.

[0053] 4. Cell morphology observation

[0054] The cells were seeded in 6-well plates (1.0 × 10 4 / well), set up a normal group, a model group and a drug-treated group (50 μM), cultured the cells according to the experimental modeling method, and took pictures under an inverted microscope.

[0055] 5. Cell viability detection

[0056] The cells were seeded in 96-well plates (1.0 × 10 5 / well), treat cells with different concentrations of drugs for 24 hours according to the experimental modeling method, add 10 μL CCK-8 solution to each well, continue incubation in the cell culture incubator for 2 hours, measure the absorbance at 450 nm, and record the experimental results. Cell viability = (absorbance value of experimental group - absorbance value of blank group) / (absorbance value of control group - absorbance value of blank group) × 100%.

[0057] 6. LDH lactate dehydrogenase assay

[0058] The cells were seeded in 96-well plates (1.0 × 10 5 / well), treat cells according to the experimental grouping and modeling method, and set up a maximum enzyme activity control group. 1 hour before the end of the experiment, add LDH release agent to the maximum enzyme activity control group and repeatedly blow the cells and continue incubation. After the experiment, take the plate and centrifuge it at 1000rpm for 5 minutes, take 50μL of the supernatant, transfer it to another 96-well plate, and add 50μL of basal culture medium and 50μLDH detection working solution in accordance with the instructions. Keep it away from light for 30 minutes at room temperature, measure the absorbance at 490nm, and record the experimental results. LDH release rate = (sample absorbance value - background blank control well absorbance value) / (maximum enzyme activity absorbance value - background blank control well absorbance value) × 100%.

[0059] 7. Experimental results

[0060] The results of cell morphology experiments are as follows Figure 3 MPP+ After the cells were damaged, the morphology of the cells changed compared to the normal group. The cells were round and shrunken, and the protrusions were broken. After the cells were protected by saponin A in advance, the cell state was improved, and the cells were irregular or triangular.

[0061] The results of the cell survival experiment are as follows Figure 4 The results of the lactate dehydrogenase release rate experiment are shown in Figure 5 As shown. In MPP + After modeling and drug administration, the cell survival rate was significantly decreased and the LDH release rate was increased (P < 0.01). After pre-treatment with saponin A, the cell survival rate was increased and the LDH release rate was decreased (P < 0.01).

[0062] Cell experiment results show that saponin A has an effect on MPP + It has a protective effect on SH-SY5Y cell damage induced by TNF-α.

[0063] Example 3

[0064] Effect of saponin A on MPTP / MPP + Effects of induced ferroptosis in in vitro and in vivo PD models.

[0065] 1. Lipid peroxidation detection

[0066] The cells were modeled according to the cell culture method and modeling method of Example 2. After the modeling was completed, C11BODIPY582 / 591 at a concentration of 50 μM was added to each well of the 6-well plate and incubated for 1 hour. The cells were washed twice with PBS, and after trypsin digestion, the cells were collected and operated on a flow cytometer. The 505-550 nm signal was measured in the F1 channel, and the signal greater than 580 nm was detected in the F2 channel. The collected signals were made into a histogram, and the proportion of lipid peroxidation was calculated based on the fluorescence signal of the histogram.

[0067] 2. MDA content detection

[0068] The cells were modeled according to the cell culture method and modeling method of Example 2. After the modeling was completed, PBS was added to the 6-well plate for washing twice. After adding the cell lysis solution containing phosphatase inhibitors and PMSF, the cells were fully scraped into an EP tube using a cell scraper, and the supernatant was obtained by centrifugation. The MDA detection working solution was added to the supernatant according to the instructions of the kit, and the solution was boiled in a boiling water bath for 15 minutes. After cooling, the absorbance was detected at 530-540 nm. The experiment was repeated three times, and the data were collected for calculation.

[0069] Extract the protein from the substantia nigra of the animal, homogenize it and then perform the test according to the above steps.

[0070] 3. 4-HNE content detection

[0071] The cells were modeled according to the cell culture method and modeling method of Example 2. After the modeling was completed, the cells were washed with PBS, digested with trypsin, and centrifuged to collect the cells, and 200 μL PBS was added for resuspending. After ultrasonic disruption of the cells, the supernatant was taken by centrifugation. 50 μL of the test solution was added to each well of the ELISA plate, and then 50 μL of the biotinylated antibody working solution was added and coated. Incubate at 37 degrees for 45 minutes, dry the liquid in the plate, wash the plate three times, add 100 μL of enzyme-bound substrate to each well, incubate at 37 ° C for 30 minutes, wash the plate 5 times, add 90 μL of substrate solution to each well, incubate at 37 ° C for 15 minutes, add 50 μL of stop solution, and immediately detect the absorbance at 450 nm. Repeat the experiment three times. Calculate the 4-HNE content in the sample according to the standard curve.

[0072] Extract the substantia nigra of the animal brain tissue, wash it, add the corresponding volume of PBS into it, grind it thoroughly, centrifuge it, take the supernatant and repeat the above experimental steps.

[0073] 4. Transmission electron microscopy

[0074] The cells were modeled according to the cell culture method and modeling method of Example 2. After the modeling was completed, they were washed twice with PBS, and the cells were scraped into an EP tube with a cell scraper, centrifuged, and 2.5% glutaraldehyde fixative was slowly added. After overnight at 4°C, they were fixed, dehydrated, infiltrated, embedded, sliced, stained, and photographed under a transmission electron microscope to observe the internal state of the cells.

[0075] 5. Experimental results

[0076] The results of lipid peroxidation test in cell experiments are as follows Figure 6 The results of MDA content test are shown in Figure 7 As shown in the figure, the 4-HNE content test results are as follows Fig. 9 Compared with the normal group, in MPP + After stimulation of the cells, the levels of lipid ROS, MDA and 4-HNE in SH-SY5Y cells increased significantly (P<0.01), while the levels of lipid ROS, MDA and 4-HNE in the cells of the drug-treated group decreased.

[0077] The results of MDA content test in animal tissues are as follows Figure 8 As shown in the figure, the 4-HNE content test results are as follows Fig.10 After MPTP induced the Parkinson's mouse model, compared with the normal group, the MDA and 4-HNE contents in the substantia nigra of the model group mice were significantly increased (P<0.01), and compared with the model group, the MDA and 4-HNE contents in the substantia nigra of the mice in the drug administration group were decreased (P<0.01).

[0078] Transmission electron microscopy results of the cell model are as follows Fig.11As shown in the figure, compared with the normal group, the mitochondrial diameter in the model group was smaller, the membrane density was increased, the mitochondrial cristae were fused, and the cell membrane was damaged. When saponin A was added, the state of mitochondrial cristae in the cells was improved, the swelling was reduced, and the degree of cell membrane damage was alleviated.

[0079] Example 4

[0080] Effect of saponin A on MPTP / MPP + Effects of iron deposition in induced PD models in vitro and in vivo.

[0081] 1. Experimental methods

[0082] The cells were modeled according to the cell culture method and modeling method of Example 2. After the modeling was completed, the cells were washed 3 times with PBS, and 4% paraformaldehyde was added to fix the cells for 15 minutes. The prepared Prussian blue working solution was added to the cells, and the cells were incubated at 37°C for 30 minutes. The cells were rinsed twice under tap water, and then counterstained with Prussian blue counterstaining solution for 30 seconds. After rinsing with tap water, the cells were observed under a microscope.

[0083] Take frozen sections of the substantia nigra, add Prussian blue working solution, immerse for 20 minutes at room temperature, rinse with distilled water twice, 2 minutes each time, then use nuclear fast red staining solution to counterstain the cell nucleus for 5 minutes, rinse with distilled water for 30 seconds, dry, observe under a microscope and take pictures.

[0084] 2. Experimental results

[0085] The results of cell experiment were as follows Fig.12 MPP + After modeling, the blue color in the cells increased, indicating that iron deposition occurred in the cells. Compared with the model group, the iron deposition in the cells of the drug-treated group was improved.

[0086] Animal tissue test results Fig.13 Compared with the normal group, iron deposition occurred in the substantia nigra in the MPTP group, and the iron deposition symptoms in the drug-treated group were improved.

[0087] Example 5

[0088] Effect of saponin A on MPTP / MPP + Effects of iron transport disturbances induced in vitro and in vivo PD models.

[0089] 1. Experimental methods

[0090] The cells were modeled according to the cell culture and modeling method of Example 2, and the cell proteins were extracted. The cell protein concentration was detected by the BCA method, 5× Loading Buffer was added, boiled, and stored at -80°C. After protein loading, electrophoresis, transfer, and blocking, anti-Fpn, anti-FTH, anti-TFR, anti-actin, and anti-IRP2 were added overnight at 4°C, and then the membrane was washed 6 times with TBST, each time for 5 minutes. Under room temperature, the secondary antibody was incubated for 2 hours, and the TBST membrane washing step was repeated, and the development was exposed.

[0091] The protein from the substantia nigra of the animal was extracted and the concentration was detected, and the above experimental steps were repeated to detect the expression of related proteins.

[0092] 2. Experimental results

[0093] Western Blot results of cell experiments are as follows Fig.14 The results of Western Blot of animal tissues are shown in Fig.15 In the model group, the relative expression levels of TFR (P<0.01) and FTH increased (P<0.01 or P<0.05), the relative expression level of Fpn decreased (P<0.01 or P<0.05), and the relative expression level of IRP2 increased (P<0.01 or P<0.05), suggesting that Duanxueliusaponin A may regulate the iron transport process by regulating IRP2 protein.

[0094] Example 6

[0095] Experimental results show that saponin A inhibits ferroptosis of PD neurons by inhibiting IRP2-mediated iron deposition.

[0096] 1. Determination of overexpressed IRP2 protein

[0097] The cells were cultured at 1×10 6 / well were inoculated on the cell culture plate, and after 12 hours of adherent growth, when the cells grew to 70-80% full, new culture medium was replaced in each well, and the transfection reagent Lipo8000 containing the overexpression plasmid and the basic culture medium without serum and antibiotics were mixed according to the instructions and added to the cells. The cells were cultured for 24 hours, and the cell proteins were extracted according to the above steps, and the expression of cell IRP2 protein was detected by Western Blot.

[0098] 2. Detection of cell survival rate after overexpression of IRP2 protein

[0099] The cells were cultured at 1×10 5 The cells were seeded at a density of 100 μg / well on a 96-well plate and the normal group, model group, drug-treated group, empty vector + MPP group, and ++BJP-IVb, and overexpression of IRP2 protein + MPP + After 12 h of adherence culture, the cells in the 5 groups of the +BJP-IVb group and the empty vector + drug-treated group, as well as the overexpression of IRP2 protein + MPP + The corresponding plasmids were transfected in the groups, and the cells were cultured for 24 hours. The corresponding groups were stimulated with drugs, and the cell viability was detected according to the experimental method of Example 2.

[0100] 3. Detection of LDH release rate after overexpression of IRP2 protein

[0101] According to the above experimental steps, the cells were grouped and transfected with corresponding plasmids. After transfection, the cells were modeled and the cell LDH release was detected according to the experimental method of Example 2.

[0102] 4. Lipid peroxidation detection

[0103] The cells were cultured at 1×10 6 / well were seeded on a 6-well plate, the cells were grouped and transfected with corresponding plasmids according to the above experimental steps, and after the transfection was completed, the lipid peroxidation between the groups was detected using a flow cytometer according to the method of Example 3.

[0104] 5. Detection of MDA content after overexpression of IRP2 protein

[0105] The cells were cultured at 1×10 6 / well were seeded on a 6-well plate, the cells were grouped and transfected with overexpression plasmids according to the above experimental steps, after transfection, drugs were added to the corresponding groups, after modeling was completed, cell proteins were extracted, and the MDA content in the cells was detected according to Example 3.

[0106] 6. Transmission electron microscopy photography after overexpression of IRP2 protein

[0107] The cells were cultured at 1×10 6 / well were seeded on a 6-well plate, the cells were grouped and transfected with overexpression plasmids according to the above experimental steps, after transfection, drugs were added to the corresponding groups, and after modeling was completed, the samples were processed and photographed according to the transmission electron microscopy sample processing steps in Example 3.

[0108] 7. Experimental results

[0109] The results of IRP2 protein determination in cells are as follows Fig.16 After plasmid transfection, the relative protein expression of IRP2 in the plasmid transfection group increased (P<0.05), indicating that the plasmid transfection process was successful.

[0110] Effect of saponin A on MPP after overexpression of IRP2 protein + The results of the stimulated cell survival rate assay were as follows Fig.17 As shown, the effect of saponin A on MPP after overexpression of IRP2 protein + The results of the stimulated cell LDH release rate determination are as follows Fig.18 The results of the lipid peroxidation assay of MPP+ stimulated cells after overexpression of IRP2 protein by saponin A are shown in Fig.19 As shown, saponin A has an effect on MPP after overexpression of IRP2 protein. + The results of MDA content determination in stimulated cells are as follows Fig. 20 As shown, transmission electron microscopy images of the effect of saponin A on MPP after overexpression of IRP2 protein + After stimulation, the cell membrane and mitochondrial status Fig.21 Compared with empty vector + MPP + +BJP-IVb, IRP2 overexpression group +MPP + In the +BJP-IVb group, the cell survival rate decreased (P<0.01), the LDH content increased significantly (P<0.01), the lipid peroxidation level increased significantly (P<0.01), and the MDA content increased significantly (P<0.01). Transmission electron microscopy showed that mitochondria were wrinkled, the inner cristae were thickened, and the mitochondrial membrane density increased.

[0111] The above description is only a preferred embodiment of the present invention. It should be pointed out that, for ordinary technicians in this technical field, the content of the present invention can be improved and modified without departing from the technical principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. Application of saponin A in the preparation of anti-Parkinson's disease drugs.

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