Application of notoginsenoside Rg1 in preparation of cathepsin D inhibitor

Abstract

The invention discloses novel application of notoginsenoside Rg1, namely application of the notoginsenoside Rg1 in preparation of a cathepsin D (CTSD) inhibitor, the notoginsenoside Rg1 can inhibit CTSD enzyme activity so as to recover lysosome structure and function, relieve endoplasmic reticulum stress reaction and relieve alpha-syn aggregation and dopaminergic neuron apoptosis, has a remarkable protection effect on a Parkinson's disease model, and can be used for preparing a novel anti-inflammatory drug. The invention provides a new thought and a potential drug basis for targeted intervention of Parkinson's disease. The invention discloses a new mechanism that Rg1 plays a role in neuroprotection through a CTSD-lysosome-endoplasmic reticulum stress pathway.

Description

Technical Field

[0001] The present invention belongs to the field of drug therapy, and particularly relates to a method for treating Parkinson's disease by Ginsenoside Rg1 regulating lysosomal function and endoplasmic reticulum stress pathway through Cathepsin D. Background Art

[0002] Parkinson's disease (PD) is a common neurodegenerative disease of the central nervous system. Clinically, it is mainly manifested as symptoms such as bradykinesia, tremor, muscle rigidity, and postural instability. Its pathological features include the progressive loss of dopaminergic neurons in the substantia nigra pars compacta of the midbrain and the abnormal aggregation of α-synuclein (α-syn) in nerve cells. The current clinical treatment mainly focuses on dopamine replacement therapy (such as levodopa). Although it can relieve motor symptoms to a certain extent, it cannot prevent the continuous progression of the disease, nor can it effectively control non-motor symptoms. There are still significant treatment bottlenecks in clinical practice. Therefore, developing innovative targeted intervention strategies for the key pathogenic mechanisms of PD has become a research hotspot.

[0003] In recent years, studies have shown that the signal interaction pathway between lysosomes and endoplasmic reticulum stress plays an important role in the pathogenesis of PD. Lysosomal dysfunction can lead to the obstruction of the autophagy-lysosome pathway, the decrease in the degradation efficiency of α-syn, and then cause its abnormal aggregation and enhanced cytotoxicity. At the same time, it is accompanied by the aggravation of intracellular stress response, inducing mitochondrial damage and programmed cell death. Cathepsin D (CTSD) is an aspartic protease located in lysosomes, which participates in the degradation process of various substrate proteins and plays a key role in maintaining the normal function of lysosomes. However, in recent years, studies have found that in neurodegenerative diseases such as PD, the enzyme activity of CTSD abnormally increases, which may lead to enhanced lysosomal membrane permeability, leakage of lysosomal contents into the cytoplasm, triggering endoplasmic reticulum stress (ER stress) and cytotoxic reactions, and further aggravating neuronal damage and apoptosis.

[0004] Ginsenoside Rg1 is a natural triterpenoid saponin compound extracted from the traditional Chinese medicine Panax notoginseng. It has been proven to have a variety of biological activities, including antioxidant, anti-inflammatory, anti-apoptotic, and promoting nerve repair effects, and shows certain neuroprotective potential in various neurological disease models. However, the molecular target of Rg1 in the treatment of Parkinson's disease is not clear, and there is still a lack of systematic research on whether it regulates the lysosome-endoplasmic reticulum stress axis and whether it involves the regulation of CTSD enzyme activity and other mechanisms. Summary of the Invention

[0005] The present invention provides a new use of notoginsenoside Rg1, that is, its application in the preparation of a cathepsin D (CTSD) inhibitor. Notoginsenoside Rg1 can inhibit the enzymatic activity of CTSD, thereby restoring the structure and function of lysosomes, alleviating endoplasmic reticulum stress response, reducing α-syn aggregation and dopaminergic neuron apoptosis, and having a significant protective effect on the Parkinson's disease model, providing new ideas and potential drug basis for the targeted intervention of Parkinson's disease. The present invention reveals a new mechanism of Rg1 exerting neuroprotection through the "CTSD–lysosome–endoplasmic reticulum stress" pathway.

[0006] The component (or active ingredient) of the cathepsin D inhibitor of the present invention is notoginsenoside Rg1, and one or more pharmaceutically acceptable excipients can also be added, or it can be compounded with other active ingredients to exert an inhibitory effect; in addition to being made into tablets, the preparation can also be made into various pharmaceutically acceptable forms such as pills, powders, capsules, granules, oral liquids, and injections.

[0007] The inhibitor is used for the treatment of diseases related to the abnormal activation of cathepsin D, including neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease.

[0008] The alleviation of the endoplasmic reticulum stress response is verified by at least one of the following indicators:  (a) The decreased expression level of CHOP protein;  (b) The reduced level of GRP78 / BIP.

[0009] Compared with the prior art, the advantages of the present invention include: 1. The present invention for the first time reveals that Rg1 can restore lysosome function, reduce endoplasmic reticulum stress and neuronal damage by inhibiting the enzymatic activity of CTSD, providing a new pharmacological mechanism; the present invention applies Rg1 to the regulation of the lysosome-endoplasmic reticulum stress axis, and clarifies its key role in regulating lysosome homeostasis, reducing neurotoxicity and delaying the progression of Parkinson's disease; 2. The experimental models cover in vitro (SH-SY5Y cells) and in vivo (zebrafish) systems, and the verification is comprehensive; 3. The mechanism of action is clear, providing theoretical support for the development of CTSD-targeted neuroprotective agents. Description of the Drawings

[0010] Figure 1 Results of the inhibitory effect of notoginsenoside Rg1 on the enzymatic activity of CTSD. The data in the figure are expressed as mean ± standard deviation (n = 3), **p < 0.01, ****p < 0.0001; Figure 2 Schematic diagram of the results of QPCR detection of the transcriptional level of CTSD; Figure 3Schematic diagram of the results of QPCR detection of LAMP1 transcription level; Figure 4 Schematic diagram of the results of QPCR detection of SNCA transcription level; Figure 5 Schematic diagram of the results of Western blot detection of CTSD protein expression; Figure 6 Schematic diagram of the results of Western blot detection of LAMP1 protein expression; Figure 7 Schematic diagram of the results of Western blot detection of GRP78 protein expression; Figure 8 Schematic diagram of the results of Western blot detection of CHOP protein expression; Figure 9 Results of immunofluorescence detection of the distribution of CTSD and LAMP2 in cells; Figure 10 Fluorescence image of Lysotracker(Red); Figure 11 Fluorescence image of ER-Tracker(Green); Figure 12 Results of flow cytometry detection of cell apoptosis rate; Figure 13 Schematic of zebrafish movement trajectory; Figure 14 Quantification graph of zebrafish average speed; Figure 15 Quantification graph of zebrafish swimming distance; Figure 16 Schematic diagram of zebrafish brain section. Detailed implementation manners

[0011] The present invention will be further described below in conjunction with embodiments. However, the scope of the present invention is not limited to the following examples. Those skilled in the art can understand that various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention. The instruments, reagents, and materials involved in the following embodiments, unless otherwise specified, are all conventional instruments, reagents, and materials existing in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following examples, unless otherwise specified, are all conventional experimental methods and detection methods existing in the prior art.

[0012] Example 1: Effects of Notoginsenoside Rg1 on CTSD Enzyme Activity and Lysosome Function in SH-SY5Y Cells 1. SH-SY5Y cells were treated with MPTP (1 mmol / L) for 24 h to construct an in vitro model of Parkinson's disease; 2. The in vitro model of Parkinson's disease was intervened with notoginsenoside Rg1 at different concentrations (1, 10 μmol / L), and a blank group (SH-SY5Y cells without treatment) was set up simultaneously. 3. Detect the changes in the activity of CTSD enzyme in each experimental group in step 2. After rinsing the cells twice with PBS, carefully scrape the cells with a cell scraper. Centrifuge the culture medium at 3000 rpm for 10 min, discard the supernatant, and keep the cell pellet. Add 100 μL of the sample to be detected diluted appropriately into the coated reaction wells (at the same time, set up a blank well, a standard product well with serial dilutions, and if conditions permit, add a negative control well and a positive control well as quality control points). Seal the plate with a sealing film and incubate at 37°C for 1 - 2 h. Add 100 μL of the diluted biotinylated antibody working solution to each well; Incubation: Seal the plate with a sealing film and incubate at 37°C for 1 h. Add the enzyme conjugate: Add 100 μL of the diluted enzyme conjugate working solution to each well, and incubate: Seal the plate with a sealing film and incubate at 37°C in the dark for 30 min. Add the chromogenic substrate: Add 100 μL of the TMB substrate solution to each well, and react at 37°C in the dark for 10 - 30 min until an obvious color gradient appears in the standard product well with serial dilutions. Terminate the reaction: Add 100 μL of 2M sulfuric acid to each reaction well, and the color changes from blue to yellow. Within 10 min, on the microplate reader, at 450 nm, zero the instrument with the blank control well and then measure the OD value of each well. The results are shown in Figure 1 , and it can be seen from the figure that compared with the blank group, the activity of CTSD enzyme in the MPTP-induced model group was significantly increased, suggesting lysosomal enzyme hyperactivity in the Parkinson's model; after the intervention with Rg1, the activity of CTSD enzyme was significantly decreased, indicating that Rg1 can effectively inhibit the activity of abnormally activated CTSD enzyme, and the differences between groups were statistically significant.

[0013] 4. Detect the transcriptional levels of CTSD, LAMP1, and the SNCA gene encoding α-synuclein in each experimental group by QPCR. (1) RNA extraction: Wash the cells with 1×PBS, add 500 μL of TRIzol to lyse the cells, let stand on ice for 5 minutes, transfer to an ep tube, add 100 μL of chloroform, mix well, let stand for 5 minutes, centrifuge at 12000g at 4°C for 15 minutes, transfer the upper aqueous phase to a new tube, add an equal volume of isopropanol, invert 10 times up and down, let stand for 20 minutes, centrifuge at 12000g at 4°C for 20 minutes, discard the supernatant, add 75% ethanol to wash the precipitate, centrifuge at 7500g at 4°C for 10 minutes, dry for 10 minutes after centrifugation, dissolve in 10 - 20 μL of RNase-free water, and measure the absorbance value.

[0014] (2)Synthesize cDNA: Prepare a reaction system of 100 μL (5 μL of RNA, 20 μL of 5× Mix, 5 μL of Transcript, 70 μL of RNase-free water), place it in a PCR instrument, incubate at 50 °C for 5 minutes and then at 85 °C for 2 minutes; (3)Reverse transcription: In a QPCR plate, add samples to each well according to the following amounts: 5 μL of 2× Mix, 1 μL of cDNA, 0.4 μL + 0.4 μL of primers, and 3.2 μL of DEPC water. After adding the samples, seal the plate and place it in a QPCR instrument. The reaction conditions are as follows:

[0015] The primers used are as follows:

[0016] The results are shown in Figures 2 - 4 , and the results in the figure show that the transcriptional levels of CTSD, LAMP1, and SNCA in the MPTP-induced model group are all significantly increased, suggesting the presence of lysosomal stress and abnormal expression of α-synuclein in the model cells.

[0017] In the intervention group with notoginsenoside Rg1 (1 μM), the expression levels of CTSD and LAMP1 are significantly decreased compared with the model group, but the change in SNCA expression has no statistical significance; while in the intervention group with notoginsenoside Rg1 at a higher dose, the expression levels of CTSD, LAMP1, and SNCA are all significantly down-regulated, indicating that the regulation of notoginsenoside Rg1 on related targets is dose-dependent.

[0018] 5. Use Western blot to detect the expression levels of related proteins such as CTSD, LAMP1, GRP78, and CHOP, and combine immunofluorescence double staining to observe the localization and distribution of CTSD and LAMP2 in cells to evaluate the structural and functional status of lysosomes (1)Detect protein expression by Western blot: Collect cells, wash them 3 times with pre-cooled PBS, add RIPA lysis buffer (containing 1% protease inhibitor) and lyse on ice for 30 minutes, centrifuge (12,000 g, 4 °C, 15 min), take the supernatant to measure the protein concentration (BCA method), and adjust to equal amounts (20 - 50 μg / well). Select the gel concentration according to the protein molecular weight (e.g., use a 12% gel to separate CTSD and CHOP; use a 10% gel to separate LAMP1 and GRP78). After loading the samples, perform constant voltage electrophoresis (80 V stacking gel → 120 V separating gel). Transfer the protein to a PVDF membrane by wet transfer method, block it with 5% skim milk at room temperature for 1 hour, incubate with the primary antibody overnight at 4 °C, wash the membrane, and incubate with the secondary antibody at room temperature for 1 hour (1:5000); develop with ECL and analyze the gray value with Image Lab, and normalize with GAPDH and α-actin as internal references; The results are shown in Figures 5 - 8, The results in the figure show that in the MPTP-induced model group, the expression of the precursor form of CTSD (about 48 kDa) was significantly increased, while the expression of the mature form (about 14 kDa) was significantly decreased, suggesting that the processing and maturation of CTSD were blocked. After intervention with notoginsenoside Rg1, the expression of the precursor form of CTSD was down-regulated, and the expression of the mature form was up-regulated, indicating that notoginsenoside Rg1 helps to promote the maturation and activation process of CTSD.

[0019] At the same time, the expression levels of the lysosomal membrane marker protein LAMP1, the endoplasmic reticulum stress-related proteins GRP78 (Bip) and CHOP were all significantly up-regulated in the model group, suggesting obvious lysosomal dysfunction and endoplasmic reticulum stress in the model cells; while the treatment with notoginsenoside Rg1 could significantly reduce the expression levels of the above proteins, indicating its obvious effect in alleviating the disorder of the lysosome-endoplasmic reticulum stress axis. GAPDH and α-actin were used as internal references, and the bands were stable. The data of each group were statistically significant, supporting the mechanism that Rg1 exerts neuroprotective effects by regulating CTSD maturation and alleviating cellular stress.

[0020] (2) Immunofluorescence: The cell slides were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 10 min, washed 3 times with PBS, and blocked with 10% goat serum for 30 min; incubated with a mixture of primary antibodies (CTSD 1:200 + LAMP2 1:100) overnight at 4°C. Incubated with fluorescent secondary antibodies in the dark for 1 hour; stained the nuclei with DAPI for 5 min, sealed the slides with an anti-fluorescence quencher, and collected images with a confocal microscope; The results of immunofluorescence detection of the distribution of CTSD and LAMP2 in cells are shown in Figure 9 , The results show that in the MPTP-induced model group, the green fluorescence signal of CTSD was significantly enhanced and showed a diffuse distribution, suggesting an up-regulation of CTSD expression; the red fluorescence of LAMP2 was also significantly enhanced and aggregated in the region near the nucleus, indicating hyperactive lysosomal activity and structural disorder. After treatment with notoginsenoside Rg1, both fluorescence signals were significantly weakened, the expression of CTSD tended to be normal, and the distribution of LAMP2 became more uniform, suggesting that Rg1 can effectively inhibit the function of abnormally activated lysosomes and improve their spatial distribution state.

[0021] 6. Experiments using Lysotracker Red and ER-Tracker Green to label lysosomes and endoplasmic reticulum respectively Lysosome labeling (Lysotracker Red): Dilute Lysotracker Red with pre-warmed serum-free medium to a final concentration of 50 nM, aspirate the original medium, add the medium containing the probe, and incubate at 37°C in the dark for 30 - 60 minutes; Endoplasmic reticulum marker (ER-Tracker Green): Dilute ER-Tracker Green with pre-warmed medium to a final concentration of 0.5 μM; add it to the culture dish simultaneously with Lysotracker Red and incubate co-incubate for 30 minutes at 37°C in the dark.

[0022] The results are shown in Figure 10 、 11 , and the results showed that the Lysotracker red fluorescence signal was significantly enhanced in the MPTP-induced model group, suggesting an abnormal increase in the number or activity of lysosomes; at the same time, the ER-Tracker green fluorescence signal was also significantly enhanced, indicating an upregulation of endoplasmic reticulum stress levels.

[0023] In the Rg1 treatment group, both fluorescence signals were significantly weakened, approaching the level of the blank control group, indicating that Rg1 could effectively alleviate lysosomal dysfunction and endoplasmic reticulum stress. In addition, the cell morphological structure of the Rg1 group and the blank group was clear, and the endoplasmic reticulum reticular structure was well distributed, while the cell morphology of the model group was disordered and the fluorescence distribution was diffuse; the results showed that Rg1 could coordinately regulate the functions of lysosomes and endoplasmic reticulum and improve the cell stress state.

[0024] 7. Detect apoptosis in different experimental groups by Annexin V-FITC / PI double staining combined with flow cytometry Set up a blank control group, a model group (MPTP-induced Parkinson's disease cell model), and a notoginsenoside Rg1 intervention group (10 μM), with 3 replicates in each group; use human neuroblastoma SH-SY5Y cells (or primary midbrain dopamine neurons), and culture them routinely in MEM / F12 medium containing 20% fetal bovine serum (FBS) at 37°C and 5% CO2; when the cell density reaches 80%, add MPTP to the model group and incubate for 24 h. In the intervention group, add Rg1 for pretreatment for 2 h first, and then add MPTP and co-incubate for 24 h; the control group only adds an equal volume of medium; Detect cell apoptosis by Annexin V-FITC / PI double staining: Discard the medium, gently rinse twice with pre-cooled PBS, terminate after digestion with 0.25% trypsin (without EDTA), centrifuge at 100 rpm for 5 min to collect cells, resuspend with pre-cooled PBS, centrifuge again to remove the supernatant, and repeat 2 times. Add 500 μL of 1× Annexin V binding buffer (containing 2.5 mM CaCl2) and 5 μL of Annexin V-FITC (BD Biosciences, catalog number 556420) to each tube, and incubate at room temperature in the dark for 15 min; PI staining: Add 5 μL of propidium iodide (PI), and immediately incubate in the dark on ice for 5 min; detect using a flow cytometer within 1 h, with an excitation wavelength of 488 nm, and collect signals in the dual channels of FITC (530 / 30 nm) and PI (585 / 42 nm), and acquire 20,000 cell events for each sample. Analyze using FlowJo V10 software, with Annexin V + / PI - as early apoptotic cells, and AnnexinV + / PI + as late apoptotic / necrotic cells, and the total apoptosis rate = early apoptosis rate + late apoptosis rate.

[0025] The results are shown in Figure 12 . The results in the figure show that the proportions of early and late apoptotic cells in the MPTP-induced model group were significantly increased, suggesting that MPTP-induced cell damage can significantly promote cell apoptosis; after Rg1 intervention, the apoptosis rate was significantly decreased, indicating that Rg1 can effectively inhibit MPTP-induced apoptosis of neuron-like cells and has a good cell protection effect. This result is consistent with the inhibition of upstream CTSD activity, improvement of lysosome and endoplasmic reticulum functions, further supporting the anti-apoptotic mechanism of Rg1.

[0026] In summary, notoginsenoside Rg1 significantly inhibited the activity of CTSD enzyme, restored the expression of lysosome-related marker proteins, reduced the level of endoplasmic reticulum stress, effectively decreased the apoptosis rate, and improved lysosomal dysfunction. Notoginsenoside Rg1 has the effect of regulating the lysosome-endoplasmic reticulum pathway and protecting nerve cells in vitro.

[0027] Example 2: Effects of notoginsenoside Rg1 on the behavior and CTSD expression of zebrafish Parkinson's model Select 1-day-old wild-type AB strain zebrafish larvae. Under standard breeding conditions (water temperature 28.5°C, 14 h / 10 h light-dark cycle, density ≤ 20 tails / well), randomly divide the zebrafish into four groups (n = 20 / group) and raise them in 6-well plates (each well has a capacity of 4 mL). Blank control group: Normal culture without intervention; Model control group: Continuously expose to 50 μmol / L 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) solution for 5 days (change the drug solution regularly every day) to construct a Parkinson's disease model and only treat without drug administration; Rg1 (50 μmol / L) group: MPTP treatment + soak and administer notoginsenoside Rg1 (50 μmol / L) (24 h); Rg1 (100 μmol / L) group: MPTP treatment + soak and administer notoginsenoside Rg1 (100 μmol / L) (24 h). Use the ANY-maze behavior tracking system to track the spontaneous swimming trajectories of zebrafish and evaluate the motor behavior by mechanical stimulation response. At the same time, perform Nissl staining on the brain tissue to observe the nerve cell damage situation; The results are shown in Figures 13 - 16 , and the results indicate that the activity trajectories of zebrafish in the blank group are complete, with a longer movement distance and higher average speed; the movement trajectories of the MPTP-induced model group are significantly shortened, showing a decline in activity ability; after treatment with Rg1 (50 μmol / L and 100 μmol / L), the movement trajectories of zebrafish gradually recover, the activity range increases, and the behavioral ability improves. The quantitative analysis results also show that their movement distance and average speed are significantly increased; The brain tissue structure of the blank group is complete, and the neurons are densely distributed; obvious vacuolar-like damage appears in the brain of the MPTP-induced model group, and the number of neurons is significantly reduced. After Rg1 intervention, the brain tissue structure is improved, the vacuoles are reduced, and the number of neurons is somewhat restored, suggesting that Rg1 has a protective effect on the nervous system.

[0028] The above results further indicate that Rg1 can effectively improve the locomotor behavior of zebrafish and reduce neuronal damage, further verifying the in vivo basis of the neuroprotective effect of Rg1.

Claims

1. Use of notoginsenoside Rg1 in the preparation of a cathepsin D inhibitor.

2. The use according to claim 1, wherein: Notoginsenoside Rg1 inhibits the enzymatic activity of cathepsin D, regulates lysosome-endoplasmic reticulum interaction, reduces the level of endoplasmic reticulum stress, thereby reducing dopaminergic neuron apoptosis, and is used for preventing and / or treating Parkinson's disease.

3. The use according to claim 2, characterized in that: The reduction of the endoplasmic reticulum stress level is verified by at least one of the following indicators: (a) The expression level of CHOP protein decreases; (b) The level of GRP78 / BIP decreases.

Images