Application of capsaicin in preparation of medicine for improving autophagy dysfunction related diseases

Abstract

The invention relates to the technical field of medicines, in particular to application of capsaicin to preparation of a medicine for improving autophagy dysfunction related diseases. The capsaicin is trans-8-methyl-N-vanillyl-6-nonenamide, and the molecular formula of the capsaicin is C18H27NO3; the structural formula of the capsaicin is as shown in the specification; the autophagy dysfunction related diseases comprise Alzheimer's disease. After the capsaicin is used as a food additive to feed the mouse, the expression of the PPAR gene is promoted, the signal channel is enhanced, and the autophagy function is improved. The capsaicin is used as an additive, the purpose of improving the autophagy function is achieved by changing the ATP6V0E1 promoter region PPARA and enhancing the formation of autophagy lysosome, and the degradation of the organism on pathological sample precipitates, lipids and the like is enhanced, so that the symptoms of the neurodegenerative disease are improved.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to an application of capsaicin in preparing a medicine for improving diseases related to autophagy dysfunction. Background Art

[0002] Autophagy is one of the important physiological processes for the human body to clear foreign bodies. Autophagy disorders can cause related diseases, among which autophagy disorders causing Alzheimer's disease (AD)-related pathology is one of the hot spots. Restoring or even enhancing autophagy function to clear human Aβ-like deposits and pathological tau proteins is an important direction for treating related diseases.

[0003] However, there are currently few drugs that can treat AD by improving autophagy function and the effects are poor. In addition, some of them are still only in animal experiments, and the specific mechanism of improving AD pathology has not been clearly explored.

[0004] Based on the above reasons, the present invention explores the application of capsaicin in the preparation of drugs for improving diseases related to autophagy dysfunction. Summary of the invention

[0005] The purpose of the present invention is to address the problems existing in the background technology and to propose an application of capsaicin in the preparation of a drug for improving diseases related to autophagy dysfunction.

[0006] The technical solution of the present invention is the use of capsaicin in preparing a drug for improving autophagy dysfunction-related diseases, characterized in that the capsaicin is trans-8-methyl-N-vanillyl-6-nonenamide, with a molecular formula of C 18 H 27 NO3; The autophagy dysfunction-related diseases include Alzheimer's disease.

[0007] Preferably, when capsaicin is fed to mice as a food additive, it promotes the expression of PPAR genes, enhances the signal pathway and improves autophagy function.

[0008] Further preferably, capsaicin as an additive enhances the formation of autophagic lysosomes by changing PPARA in the promoter region of ATP6V0E1.

[0009] Preferably, the capsaicin intake is 20-40 mg / Kg per day.

[0010] More preferably, the capsaicin intake is 30 mg / Kg per day.

[0011] Application of capsaicin as an additive in improving spatial memory impairment in mice.

[0012] A medicine for improving diseases related to autophagy dysfunction, comprising 300-1000 mg of capsaicin.

[0013] The drug is in the form of tablets, pills or capsules.

[0014] Compared with the prior art, the present invention has the following beneficial technical effects:

[0015] The present invention studies autophagy dysfunction and explores capsaicin C through experiments. 18 H 27 The application of NO3 in improving autophagy dysfunction, specifically based on mouse experiments, found that feeding mice with capsaicin as a food additive can promote the expression of PPAR genes, enhance signaling pathways and improve autophagy function; specifically, capsaicin as an additive can improve autophagy function by changing PPARA in the ATP6V0E1 promoter region, enhancing the formation of autophagic lysosomes, and enhancing the body's degradation of pathological deposits, lipids, etc., thereby improving the symptoms of neurodegenerative diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This indicates that capsaicin in the embodiments of the present invention can activate the PPAR signaling pathway in 3xTg-AD mice;

[0017] Figure 2 This indicates that capsaicin in the present invention can increase the lysosomal function mediated by atp6v0e1 in 3xTg-AD mice;

[0018] Figure 3 It shows that capsaicin in the embodiments of the present invention improves AD-related pathology by enhancing autophagy and lysosome;

[0019] Figure 4 This indicates that capsaicin in the embodiments of the present invention can reduce the inflammatory activity of glial cells and the accumulation of glial lipids;

[0020] Figure 5 This indicates that capsaicin in the examples of the present invention can improve the spatial memory impairment of 3xTg-AD mice. DETAILED DESCRIPTION

[0021] Example 1

[0022] This example illustrates that the experimental group of mice were treated with capsaicin.

[0023] First, the temperature in the breeding area was controlled at 22-26°C, the relative humidity was controlled at 50-60%, sufficient food and water were guaranteed, and the light and dark were alternated for 12 hours. It is worth noting that all experiments performed with these mice were in accordance with ethical considerations and animal welfare. All mice were given a normal diet.

[0024] Triple transgenic AD male mice (3xTg-AD) were fed a diet containing capsaicin starting from 4 months of age, with a daily capsaicin intake of 30 mg / Kg. Animal experiments were performed when they reached old age at 8 months, and they were killed at 8 months of age, and their brain tissues were taken for experiments.

[0025] Example 2

[0026] This example shows that capsaicin can improve autophagy function by promoting the expression of PPAR genes to enhance related signaling pathways.

[0027] The specific process is as follows:

[0028] Bodipy staining and reversal group analysis of hippocampal slices from mice fed capsaicin

[0029] Materials and reagents required for Bodipy staining:

[0030] 35 mm cell culture dishes / well, flow cytometer tubes with cell filter caps (Corning, catalog number: 352235, 15 ml conical tubes, 35 um filters, FACS tubes, glass slides, oleic acid (OA), bovine serum albumin, hydroxyethyl cellulose, sodium chloride, phosphate buffered saline (PBS), trypsin-EDTA (0.25%), phenol red, 4,4-difluoro-1,3,5,7,8-pentamethyl-4-boron-3a,4a-diaza-s-phendomethacin, dimethyl sulfoxide (DMSO), 4% paraformaldehyde (PFA), Dulbecco's modified Eagle's medium with high glucose and L-glutamine.

[0031] Specific steps of Bodipy staining:

[0032] 1. Grow cells under culture conditions relevant to your study. A 35 mm dish / well is sufficient for the number of cells required for this assay.

[0033] Incubation of cells with 30 μM oleic acid overnight served as a positive control for increased neutral lipid content.

[0034] Prepare 2 μM BODIPY staining solution in PBS.

[0035] Rinse cells quickly with 3 ml PBS to remove media / serum.

[0036] Incubate in BODIPY staining solution in the dark for 15 min at 37 °C. Include an unstained flow cytometry control.

[0037] Rinse cells quickly with 3 ml PBS to remove the staining solution.

[0038] The experimental results are as follows Figure 1As shown, it can be determined that the PPAR-rich expression region is produced under the action of capsaicin.

[0039] (A, B) Immunofluorescence staining analysis of Bodipy (fat droplet fluorescent dye) in the DG region of the hippocampus of 3xTg-AD mice and C57 mice treated with or without capsaicin (A) and the number of Bodipy+ particles in the field of view (B). Comparison showed that the aggregation of lipid droplets was significantly reduced under the administration of capsaicin.

[0040] (C) is the differentially expressed genes, and the Z value of gene abundance is represented by a red and blue scale, with red and blue representing increased and decreased protein expression, respectively. Differentially expressed genes (DEGs) in the hippocampus of 3xTg-AD+Cap mice compared with 3xTg-AD mice (p<0.05, fold change>1.5), indicating that capsaicin can significantly reverse gene expression in the hippocampus of 3xTg-AD mice.

[0041] (D and E) KEGG enrichment analysis of decreased DEGs (D) and increased DEGs (E). Downregulated differentially expressed genes (deg) were significantly enriched in neuroactive ligand-receptor interaction, cell adhesion molecules, Delta-Notch signaling pathway, ErbB signaling pathway, nuclear receptors, etc. (Figure D). Upregulated differentially expressed genes (deg) were significantly enriched in adipose cytokine signaling pathway and PPAR signaling pathway, etc. (E).

[0042] (F and G) are the heat maps of DEGs involved in the PPAR signaling pathway (F) and the PPI network module (G). The analysis found that the PPAR signaling pathway mainly includes Acadl, Acadm, Acox2, Acsl3, Angptl4, Cpt1a, Cpt2, Hmgcs2, Plin4, Sorbs1, etc., which constitute a closely related network module.

[0043] (H) Real-time fluorescence quantitative PCR (qRT-PCR) confirmed specific target genes in the heat map, genes related to PPAR (n = 4 / group). The qPCR data were consistent with transcriptomics, and the genes related to the PPAR signaling pathway in the hippocampus of 3xTg-AD mice were significantly reduced, which was significantly reversed after the administration of capsaicin.

[0044] (I) TRRUST analysis showed that Ppara may be a key transcription factor for the action of capsaicin, which is consistent with the report that Ppara is a key regulator of the PPAR signaling pathway ( Figure 1 )

[0045] (J, K) Western blot (WB) validation experiments to verify the expression of PPARA in the hippocampus of C57 mice, 3xTg-AD mice, and 3xTg-AD mice. The images showed that PPARA was significantly decreased in 3xTg-AD and significantly increased after capsaicin administration.

[0046] Example 3

[0047] This example illustrates that capsaicin affects autophagy function through ATP6V0E1.

[0048] The specific process is as follows:

[0049] Chromatin immunoprecipitation (ChIP) was performed on the hippocampal cells of the experimental group of mice

[0050] Specific steps of ChIP experiment:

[0051] Formaldehyde cross-linking and ultrasonic disruption of cells (first day)

[0052] One dish of cells (10 cm dish) was taken out, and 243 μl of 37% formaldehyde was added to make the final concentration of formaldehyde 1% (a total of 9 ml of culture medium).

[0053] Incubate at 37°C for 10 min.

[0054] Terminate cross-linking: Add glycine to a final concentration of 0.125 M. Add 450 μl of 2.5 M glycine to the dish. Mix well and leave at room temperature for 5 minutes.

[0055] The culture medium was aspirated and the cells were washed twice with ice-cold PBS.

[0056] Collect the cells with a cell scraper in a 15 ml centrifuge tube (PBS is 5 ml, 3 ml and 3 ml respectively). Collect the cells at 2000 rpm for 5 min after precooling.

[0057] Pour off the supernatant. Add SDS Lysis Buffer according to the cell volume. Make the final cell concentration be 2×106 cells per 200ul.

[0058] Ultrasonic fragmentation: VCX750, 25% power, 4.5s impact, 9s rest, 14 times in total.

[0059] Cleaning and antibody incubation (first day)

[0060] 1. After ultrasonic disruption, centrifuge at 10000g for 10 min at 4°C to remove insoluble matter;

[0061] 2. Keep 300ul for experiment and store the rest at -80℃;

[0062] 3. In the 300ul, 100ul was added with antibody as the experimental group; 100ul was not added with antibody as the control group; 100ul was added with 4ul5MNaCI (final concentration of NaCI was 0.2M), treated at 65℃ for 3h to dissolve cross-linking, and then run electrophoresis to detect the effect of ultrasonic disruption;

[0063] 4. Add 900ul ChlP Dilution Buffer and 20ul 50×PIC to 100ul of ultrasonic disruption product. Then add 60ul ProteinAAgarose / SalmonSpermDNA. Mix by inversion at 4℃ for 1h.

[0064] 5. After 1 hour, let it stand at 4°C for 10 minutes to precipitate and centrifuge at 700 rpm for 1 minute.

[0065] 6. Take the supernatant. Keep 20ul each as input. Add 1ul antibody to one tube and no antibody to the other tube. Invert at 4℃ overnight.

[0066] Test the effect of ultrasonic fragmentation (first day)

[0067] 1. Take 100ul of the product after ultrasonic disruption, add 4ul of 5M NaCl, and treat at 65℃ for 2h to decrosslink.

[0068] 2. Separate half of the sample and extract with phenol / chloroform. Detect the effect of ultrasound by electrophoresis.

[0069] Precipitation and washing of immune complexes (second day)

[0070] 1. After overnight incubation, add 60ul ProteinAAgarose / SalmonSperm DNA to each tube. Invert at 4℃ for 2h

[0071] 2. After standing at 4°C for 10 min, centrifuge at 700 rpm for 1 min and remove the supernatant.

[0072] The precipitated complex was washed with the following solutions in sequence. Washing steps: add solution, invert at 4°C for 10 min, let stand at 4°C for 10 min to precipitate, centrifuge at 700 rpm for 1 min, and remove the supernatant.

[0073] After cleaning, wash off.

[0074] Decrosslinking: Add 20ul 5M NaCl to each tube (final NaCl concentration is 0.2M).

[0075] Mix well and incubate at 65°C to decrosslink overnight.

[0076] Recovery of DNA samples (Day 3)

[0077] 1. After the cross-linking is resolved, add 1ul RNaseA (MBI) to each tube and incubate at 37℃ for 1h.

[0078] 2. Add 10ul 0.5M EDTA, 20ul 1M Tris.HCl (PH6.5), and 2ul 10mg / ml proteinase K to each tube and treat at 45℃ for 2h.

[0079] 3. Recovery of DNA fragments - Omega gel recovery kit. The final sample was dissolved in 100ul ddH2O.

[0080] PCR analysis (day 3)

[0081] The results are as follows Figure 2 As shown, the experiment found that capsaicin had a significant effect on PPARA in the promoter region of ATP6V0E1.

[0082] (Figure AB) is a transcriptome analysis, (A) Volcano plot of lysosome-related genes in 3xTg-AD+Cap and 3xTg-AD mice: the x-axis is the ratio of the 3xTg-AD+Cap to 3xTg-AD groups, and the y-axis is the p-value after -log10 transformation. Red indicates increased expression, and blue indicates decreased expression. The red box indicates ATP6V0E1, which is involved in the regulation of lysosomal activity. (B) Lysosome-related gene protein expression profile; the dotted line connects the average abundance of proteins in different animal groups.

[0083] (Figure AB) shows that lysosome-related genes ATP6V0E1, Dmxl1, Ctso, Gaa, and Ap4b1 are dysregulated in 3xTg-AD mice, which can be reversed by capsaicin administration. Among them, ATP6V0E1 controls lysosomal acidification, Dmxl1 can induce V-ATPase assembly, and Ctso is a hydrolase that controls protein degradation.

[0084] (C) The mRNA levels of lysosomal-related genes in the volcanic area were verified by qRT-PCR analysis (n=4 / group), which was consistent with the transcriptome data, showing that ATP6V0E1, Dmxl1, and Ctso were significantly decreased in 3xTg-AD mice compared with WT mice, which could be reversed by capsaicin treatment.

[0085] (Figure DF) shows the relative expression of ATP6V0E1 mRNA in GSE5281 brain (D), GSE63060 peripheral blood (E), and GSE63061 peripheral blood (F) of the AD, MCI, and control groups. GEO data analysis showed that the V0 complex of the cytosolic (H+)-ATPase ATP6V0E1 was significantly reduced in AD brain tissue (Figure D, GSE5281) and peripheral blood of MCI and AD patients (Figure EF, GSE63060, GSE63061). In summary, the significant dysregulation of lysosomal acidification-related genes, especially the reduction of ATP6V0E1, which is directly involved in hydrogen ion transport, leads to AD autophagy lysosomal dysfunction, and capsaicin may improve this process by upregulating ATP6V0E1.

[0086] (Figure G) is a chromatin immunoprecipitation (Chip) experiment, the results of which showed that the enrichment of PPARA in the ATP6V0E1 promoter region of 3xTg-AD mice was significantly reduced, while capsaicin could significantly promote the enrichment of PPARA in the ATP6V0E1 promoter region.

[0087] In this embodiment, if Figure 3-4 As shown, ATP6V0E1 is a subunit of V-ATPse, and V-ATPse is an important component for the normal function of lysosomes. Capsaicin enhances the function of lysosomes through the PPAR-V-ATPse-ATP6V0E1 pathway, thereby enhancing the formation of autophagic lysosomes to achieve the purpose of improving autophagic function, enhancing the body's degradation of pathological deposits, lipids, etc., and reducing the inflammatory activity of glial cells, thereby improving the symptoms of neurodegenerative diseases.

[0088] Improve AD-related pathologies such as Figure 3 As shown:

[0089] (Figure A) Immunofluorescence analysis, double-labeling immunofluorescence staining of Aβ-4g8 and LAMP1 in the DG region of the hippocampus of 3xTg-AD mice and C57 mice treated with or without capsaicin, (Figure B) Aβ-4g8+ visual field plaque % area, (Figure C) Aβ-4g8+ visual field plaque area, (Figure D) LAMP1+ visual field plaque % area, (Figure E) The number of Aβ-4g8+LAMP1+ cells in the field of view.

[0090] Image (A) shows that after capsaicin administration, the size and percentage of 4G8+ staining area of ​​Aβ pathological plaques in the 3xTg-AD group increased significantly (Figure B); the percentage of 4G8+ staining area of ​​Aβ pathological plaques in the 3xTg-AD group decreased significantly (Figure C); the lysosomal biomarker LAMP1+ was significantly increased in 3xTg-AD mice, but significantly decreased after capsaicin administration (Figure D); the co-localization of 4G8+ and LAMP1+ in 3xTg-AD mice was significantly reduced, while the co-localization of 4G8+ and LAMP1+ was significantly increased after capsaicin administration (Figure E)

[0091] (Figure F) Western blotting (WB) detected the changes of Aβ pathology and Tau pathology in the DG region of 3xTg-AD mice with or without capsaicin and C57 mice (n=4 / group). Data are expressed as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001. The results showed that pS262, pS214, pS396 and pS404 were significantly increased in 3xTg-AD, but decreased after the administration of capsaicin (Figure FG).

[0092] Eliminate inflammatory activity of nerve cells such as Figure 4 As shown:

[0093] (Figure AD) shows the immunofluorescence double staining analysis of Iba1 and Bodipy in the hippocampal DG region of 3xTg-AD mice and C57 mice treated with or without capsaicin (A), the number of Bodipy+ particles in the field of view (Figure B), the number of Iba1+ cells in the field of view (Figure C), and the number of Bodipy+Iba1+ cells in the field of view (Figure D). (Figures E and F) show the changes in the relative protein levels of GFAP and Iba1 (n=4 / group). Data are expressed as mean ± SEM. ***P<0.05, P<0.01, ***P<0.001.

[0094] (Figure A) shows that the glial cells of 3xPTg-AD mice were significantly activated, and (Figure C) shows that the activation of glial cells was significantly inhibited after the administration of capsaicin. Correspondingly, (Figure Ad) found that the lipid accumulation of microglia was also significantly reduced after the administration of capsaicin. (Figures E and F) GFA and Iba1 protein levels were detected. The GFAP and Iba1 protein levels in the 3xTg-AD group decreased, and the GFAP and Iba1 protein levels in the 0.01% capsaicin group increased. Therefore, lipid deposition in glial cells can promote glial cell activation, and capsaicin can inhibit glial cell inflammatory activation by reducing lipid droplets in glial cells.

[0095] Example 4

[0096] Water maze test and novel object test were performed on three groups of mice: 3xTg-AD mice fed with capsaicin, normal mice and 3xTg-AD mice not fed with medicine.

[0097] The specific steps are as follows:

[0098] a. Water maze experiment:

[0099] The experiment lasted for 7 days, with 4 training sessions every day at a fixed time period. The platform was fixed in one quadrant, and the mice were placed in different quadrants during the 4 training sessions. The system recorded the time it took the rats to find the platform and the swimming path.

[0100] During the first three days, the experiment was stopped when the mice found the platform or failed to find the platform within 60 seconds. The experimenter guided the mice to the platform, memorized the platform for 30 seconds, took them out, let them rest and dry, and then conducted the next round of experiments.

[0101] On the 4th to 6th day, training was carried out in the same manner as the first three days, but without guiding the rats to the platform and training for 30 seconds.

[0102] On the 7th day, the platform was removed, and the number of times the mouse crossed the original platform position within 60 seconds was recorded to observe the spatial positioning ability of the mouse.

[0103] b. New object recognition experiment:

[0104] The experiment lasted for two days. On the first day, two objects A and B were placed at the left and right ends of one side wall (objects A and B were the same object, but with different codes). The rats were placed in the arena with their backs facing the two objects. The video recording equipment was turned on immediately after they were placed in the arena, and the experimenter left the test room immediately. The mice explored the two objects, but only the total time the mice explored object B within 10 minutes was recorded (the time started when the front paws were placed on the object, the nose sniffed the object, the nose licked the object, etc., all of which were considered as exploring the object, and the time was stopped when the rats left the object).

[0105] On the second day, object B was replaced with object C (object C had the same shape as objects A and B, but a different color). As in recording the time the mice spent exploring the objects on the first day, only the total time the mice spent exploring the new object C within 10 min was recorded.

[0106] The results are as follows Figure 5 As shown:

[0107] Figure 5 (A) Experimental procedure for in vivo studies. Figure 5(BE) Morris water maze (MWM) was used to detect the effect of capsaicin treatment on spatial learning and memory of 3xTg-AD mice fed with 0.01% capsaicin. The latency to escape to the hidden platform during the training phase (B), the latency to escape to the hidden platform (C), the path to the hidden platform on the 7th day (D), and the count of the original position of crossing the hidden platform (E). The MWM experiment showed that the time for 3xTg-AD mice to find the platform during training was significantly higher than that of WT mice, while capsaicin treatment shortened the time to find the platform (Figure B). This shows that capsaicin can significantly improve the learning ability of AD mice. During the exploration period, the time for 3xTg-AD mice to pass the platform for the first time was significantly higher than that of WT mice, while capsaicin treatment shortened the time to pass the platform for the first time (Figure CD), indicating that capsaicin can significantly improve the memory ability of AD mice.

[0108] Figure (FH) shows the use of the novel object recognition test (NOR) to evaluate the learning and memory ability of mice treated with 0.0% capsaicin. Changes in exploration time from day 1 to day 2 (F), changes in exploration time for new objects from day 1 to day 2 (G), and changes in the recognition index for new objects (H) in the three groups. NOR showed that the time spent by 3xTg-AD mice exploring new objects and entering the new object area was significantly less than that of WT mice, while capsaicin significantly increased the time spent by 3xTg-AD mice exploring new objects and entering the new object area (Figure FG). In addition, the recognition index of 3xTg-AD for new and old objects was significantly lower than that of WT mice, and capsaicin administration significantly increased this index (Figure H). These results indicate that capsaicin can significantly improve the memory ability of AD mice.

[0109] In two behavioral experiments, it was found that the learning and memory abilities of 3xTg-AD mice were significantly lower than those of the normal group, while the 3xTg-AD mice given capsaicin had higher abilities than the 3xTg-AD mice, indicating that capsaicin (C 18 H 27 NO3) can improve AD symptoms behaviorally.

[0110] The embodiments of the present invention are described in detail above with reference to the accompanying drawings, but the present invention is not limited thereto, and various changes can be made within the knowledge scope of technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. The use of capsaicin in preparing a drug for improving diseases related to autophagy dysfunction, characterized in that: The capsaicin is trans-8-methyl-N-vanillyl-6-nonenamide, with a molecular formula of C 18 H 27 NO3.

2. The use of capsaicin according to claim 1 for preparing a drug for improving diseases related to autophagy dysfunction, characterized in that: The autophagy dysfunction-related diseases include Alzheimer's disease, Parkinson's disease, and Huntington's disease.

3. The application of capsaicin as an additive to improve the autophagy function of mice, characterized in that: When capsaicin is fed to mice as a food additive, it promotes the expression of PPAR genes, enhances the signaling pathway and improves autophagy function.

4. The use of capsaicin as an additive to improve the autophagic function of mice according to claim 3, characterized in that: Capsaicin as an additive enhances the formation of autophagolysosomes by altering PPARA in the promoter region of ATP6V0E1.

5. The use of capsaicin as an additive to improve the autophagic function of mice according to claim 3, characterized in that: The daily intake of capsaicin is 20-40 mg / Kg.

6. The use of capsaicin as an additive to improve the autophagic function of mice according to claim 5, characterized in that: The daily intake of capsaicin is 30 mg / Kg.

7. Application of capsaicin as an additive to improve spatial memory impairment in mice.

8. A drug for improving diseases related to autophagy dysfunction, characterized in that: The medicine contains 300-1000 mg of capsaicin.

9. The drug for improving diseases related to autophagy dysfunction according to claim 8, characterized in that: The drug is in the form of tablets, pills or capsules.

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