Application of crocetin in preparation of medicine for treating Parkinson's disease and verification method

Abstract

The invention discloses application of crocetin in preparation of a medicine for treating Parkinson's disease and a verification method. According to the application, the action mechanism that the traditional Chinese medicine monomer crocetin (CRO) relieves symptoms of the Parkinson's disease (PD) by improving mitochondrial dysfunction and autophagy pathways is proved for the first time, and the discovery can provide a new theoretical basis for research and development of treatment drugs related to the Parkinson's disease and has great scientific significance and clinical transformation value. On the basis, the application of the traditional Chinese medicine monomer crocetin (CRO) (even serving as a unique active component) to the medicine treatment of the Parkinson's disease is provided for the first time; the medicine can improve mitochondrial membrane potential, ATP generation, mitochondrial active oxygen generation, intracellular active oxygen generation and ubiquitin-dependent mitochondrial autophagy pathway, so that the symptom of the Parkinson's disease (PD) is relieved, and a novel and effective medicine treatment means is provided for the Parkinson's disease (PD).

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of crocin in preparation of a medicament for treating Parkinson's disease and a verification method. BACKGROUND

[0002] Parkinson's disease (PD) is a chronic and progressive neurodegenerative disease, and the main clinical manifestations are motor symptoms such as resting tremor, muscle rigidity, bradykinesia and postural instability. However, it also involves non-motor symptoms such as sensory impairment, sleep disorders, autonomic dysfunction and mental disorders. At present, drug therapy is the main treatment for Parkinson's disease in clinical practice, aiming to supplement dopamine or simulate the effect of dopamine. Commonly used drugs include levodopa, dopamine receptor agonists, monoamine oxidase B (MAO-B) inhibitors, catechol-O-methyltransferase (COMT) inhibitors and anticholinergic drugs. Levodopa is the precursor of dopamine, which can pass through the blood-brain barrier and be converted into dopamine in the brain, and is the "gold standard" for the treatment of Parkinson's disease. At present, the most commonly used levodopa supplement drug in clinical practice is madopa. COMT inhibitors are often used in combination with levodopa supplements, which can inhibit the activity of catechol-O-methyltransferase, reduce the degradation of levodopa, and improve its bioavailability and efficacy in the body. In addition, dopamine receptor agonists can directly stimulate dopamine receptors, simulating the effect of dopamine, thereby alleviating the series of effects caused by dopamine deficiency. MAO-B inhibitors can reduce the decomposition of dopamine by inhibiting the activity of monoamine oxidase B, thereby increasing the level of dopamine in the brain.

[0003] Deep brain stimulation (DBS) is a neuroregulation technique that uses electrodes implanted in specific nuclei of the brain to deliver high-frequency electrical pulses to regulate abnormal neural activity, often referred to as a "brain pacemaker". The core principle is to suppress overactive neurons or neural circuits through electrical stimulation, thereby improving disease symptoms. In the treatment of PD, DBS is mainly suitable for patients with advanced Parkinson's disease who have reduced drug efficacy (such as "on-off phenomenon") and motor complications. Clinical trials have shown that DBS can significantly improve tremor, muscle rigidity and bradykinesia, and reduce drug dosage by 30%-50%. However, DBS treatment also has some limitations and challenges. For example, since DBS requires craniotomy to implant electrodes and subcutaneous pulse generators, it may cause intracranial hemorrhage, infection or electrode displacement, and some patients may experience postoperative cognitive dysfunction, emotional disorders and other side effects.

[0004] Mitochondria, as the energy factory of cells, play a crucial role in cell metabolism, signal transduction, and maintenance of cellular homeostasis. As the organ with the highest energy demand in the human body, the brain consumes about 20% of the oxygen and 25% of the glucose. Brain neurons, especially dopaminergic neurons, mainly rely on oxidative phosphorylation to meet their energy needs, and mitochondria are the main site of ATP production by oxidative phosphorylation in eukaryotic cells. Therefore, high-quality mitochondria are essential for maintaining the survival and normal function of brain neurons. Mitochondrial dysfunction is an early event in the pathogenesis of PD, manifested as impaired mitochondrial autophagy, disrupted mitochondrial dynamics, bioenergetic defects, and inhibition of complex I of the electron transfer chain (ETC). These changes can lead to accumulation of damaged mitochondria, reduced ATP production, increased ROS load, and elevated calcium levels, ultimately resulting in the loss of dopaminergic neurons and accelerating the progression of PD.

[0005] Traditional Chinese medicines can regulate mitochondrial bioenergetic defects by restoring mitochondrial oxidative phosphorylation levels, increasing ATP production, and improving mitochondrial respiratory levels. For example, gastrodin can improve mitochondrial respiration and energy metabolism in rats, reducing mitochondrial dysfunction; ginsenoside Rb1 regulates mitochondrial energy metabolism through different signaling pathways to restore ATP production; and iron ore stone bamboo alkali also shows great potential in restoring ATP levels and improving mitochondrial respiration. Therefore, traditional Chinese medicines play an important role in improving mitochondrial bioenergetic defects and provide a new direction for the treatment of PD.

[0006] Saffron is the dried stigmas of the Crocus sativus L., a perennial herb of the Iridaceae family, and is one of the most expensive spices in the world. Saffron has a variety of chemical properties and contains over 150 volatile and non-volatile components, including carotenoids, a series of polyphenols and flavonoids, and terpenes. The specific properties of these compounds may vary depending on the geographical origin of saffron. In addition, saffron has been shown to have a wide range of biological activities, including antioxidant, anticancer, anti-inflammatory, anti-atherosclerotic, anti-diabetic, hypotensive, hypoglycemic, anti-hyperlipidemic, anti-degenerative, and anti-depressive properties. In terms of disease treatment, saffron also shows potential in treating cardiovascular diseases, mental illnesses, neurodegenerative diseases, atherosclerosis, learning and memory disorders, depression, diabetes, and cancer. Saffron contains four main bioactive compounds, including crocin, crocetin, picrocrocin, and saffranal. Among them, crocin and crocetin are carotenoids derived from zeaxanthin, which gives saffron its distinctive red color. Picrocrocin is also a type of carotenoid that gives saffron its unique taste. Saffranal is a terpene with an aldehyde functional group that is related to the characteristic aroma of saffron.

[0007] Studies have shown that both crocin and crocetin (CRO) have strong antioxidant effects, can effectively inhibit the formation of free radicals, reduce lipid peroxidation, and increase the activity of antioxidant enzymes such as glutathione and superoxide dismutase. In addition, crocin can also induce apoptosis. Crocinaldehyde has been proven to reduce the production of pro-inflammatory cytokines, reduce H2O2-induced oxidative stress, and exhibit the ability to promote or inhibit cell death. The positive role of saffron in reducing inflammation and oxidative stress makes saffron have the potential to treat neurological diseases. In addition, there is research evidence to support the advantages of saffron in alleviating age-related diseases, such as cardiovascular disease, eye disease, neurodegenerative disease, and type II diabetes. In terms of efficacy on the nervous system, saffron has the potential to resist neurocognitive disorders, can restore memory deficits to some extent, prevent the formation of neurofibrillary tangles, and has a regulatory effect on Alzheimer's disease. In addition, saffron can also increase the level of gamma-aminobutyric acid in the brain and inhibit excessive excitatory neurotransmission.

[0008] CRO can induce autophagy in N9 microglial cells and primary neuronal cells through the STK11 / LKB 1-mediated AMPK pathway, reduce the level of Aβ in the mouse brain and neuroinflammation, and improve the memory function of mice, thereby playing a neuroprotective role in Alzheimer's disease. In addition, CRO has also been verified to have beneficial effects in ischemic stroke disease: CRO can regulate the function of neural stem cells, promote the differentiation of neural stem cells to produce new neurons for the recovery of cerebral ischemia, and inhibit NOX2 and retain mitochondrial hexokinase-I to antagonize dependent cell death in ischemic stroke. Since CRO can pass through the blood-brain barrier by passive transcellular diffusion, it has the potential to have an important impact on the treatment of neurodegenerative diseases. This indicates that CRO has the potential to treat neurological diseases. In addition, CRO has a wide range of pharmacological effects such as antioxidant, anti-inflammatory, and anti-apoptotic, and oxidative stress, inflammation, and cell death are common to many neurological diseases, so CRO may also have beneficial effects on Parkinson's disease. SUMMARY

[0009] The purpose of the present application is to propose an application of crocetin in the preparation of a drug for treating Parkinson's disease and a verification method based on the establishment of an in vitro Parkinson's disease injury model and cutting-edge technologies such as electrophysiology, so as to alleviate the symptoms of Parkinson's disease, thereby helping to provide a new drug treatment for Parkinson's disease.

[0010] To solve the above technical problems, the present application realizes the following technical scheme:

[0011] The present application provides an application of a traditional Chinese medicine monomer crocetin in the preparation of a drug for treating Parkinson's disease.

[0012] The application provides application of a traditional Chinese medicine monomer crocetin as a unique active ingredient in preparation of a medicine for treating Parkinson's disease.

[0013] Further, the medicine utilizes the crocetin to regulate the function of mitochondria, and then improves mitochondrial dysfunction and autophagy, so that the treatment of Parkinson's disease is realized.

[0014] Further, the function of the mitochondria regulated by the crocetin comprises mitochondrial membrane potential, ATP production, mitochondrial reactive oxygen species production, intracellular reactive oxygen species production, and ubiquitin-dependent mitochondrial autophagy pathway.

[0015] The application further provides a method for verifying that the crocetin treats Parkinson's disease by regulating the function of mitochondria, which comprises the following verification contents: constructing a MPP + cell damage model in vitro by using MES23.5 cells to simulate the in-vivo Parkinson's disease damage environment, and evaluating the treatment effect of the crocetin on the Parkinson's disease model.

[0016] 1) detecting the influence of the crocetin on the membrane potassium current of the MPP + model MES23.5 cells;

[0017] 2) detecting the influence of the crocetin on the alpha-syn aggregation in the MPP + model MES23.5 cells;

[0018] 3) detecting the influence of the crocetin on the mitochondrial oxidative stress of the MPP + model MES23.5 cells;

[0019] 4) detecting the influence of the crocetin on the mitochondrial and intracellular reactive oxygen species production of the MPP + model MES23.5 cells.

[0020] 5) detecting the influence of the crocetin on the mitochondrial ATP production of the MPP + model MES23.5 cells;

[0021] 6) detecting the influence of the crocetin on the mitochondrial membrane potential of the MPP + model MES23.5 cells.

[0022] Further, the automatic patch clamp technology can be used to detect the influence of the crocetin on the MPP +Effect on the membrane potassium current of the molded MES23.5 cells. The whole-cell patch clamp technique is a method of contacting the cell membrane with a micro-bottle electrode (patch electrode or patch pipette), sealing it with an impedance of more than 1,000 ohms, and separating a small area of the cell membrane (patch) connected to the opening of the electrode tip from its surroundings in an electrical manner, on the basis of which a fixed point is monitored to record the ion current of the ion channel on the patch. It is the gold standard for detecting ion channels. Its core advantages lie in high throughput, automated operation and accurate data acquisition, which can improve efficiency, reduce drug consumption, save time and experimental costs through multi-well plate parallel operation.

[0023] Further, the expression of PD pathological marker a-syn in the molded MES23.5 cells can be detected by immunofluorescence staining method. + The expression of PD pathological marker a-syn in the molded MES23.5 cells can be detected by immunofluorescence staining method.

[0024] Further, the effect of curcumin on MPP + Effect on the mitochondrial oxidative stress of the molded MES23.5 cells. MitoSOX is a cationic fluorescent probe that can penetrate the mitochondrial membrane and accumulate in the mitochondrial matrix. In mitochondria, MitoSOX reacts with superoxide ions and is oxidized to a product with strong fluorescence. This oxidation process enables MitoSOX to emit bright red fluorescence. This red fluorescence can be observed by fluorescence microscopy, and the fluorescence intensity can reflect the level of oxidative stress in mitochondria.

[0025] Further, the effect of curcumin on MPP + Effect on the mitochondrial oxidative stress of the molded MES23.5 cells. MitoSOX is a cationic fluorescent probe that can penetrate the mitochondrial membrane and accumulate in the mitochondrial matrix. In mitochondria, MitoSOX reacts with superoxide ions and is oxidized to a product with strong fluorescence. This oxidation process enables MitoSOX to emit bright red fluorescence. This red fluorescence can be observed by fluorescence microscopy, and the fluorescence intensity can reflect the level of oxidative stress in mitochondria.

[0026] Further, the effect of curcumin on MPP +The influence of the modeling of the MES23.5 cell mitochondria ATP production. ATP is the most important energy molecule in the cell, and plays an important role in various physiological and pathological processes of the cell; generally, the ATP level of the cell will decrease in apoptosis, necrosis or in some toxic environment; the decrease of the ATP level indicates that the function of the mitochondria is impaired, and the decrease of the ATP level generally occurs simultaneously with the decrease of the membrane potential of the mitochondria in apoptosis; the detection principle of the kit used in the application is that: the luciferase of firefly catalyzes the luciferin to produce fluorescence, and ATP provides energy; when the luciferase of firefly and the luciferin are both excessive, the fluorescence production is proportional to the concentration content of ATP within a certain concentration range; thus, the ATP concentration in the solution can be detected with high sensitivity.

[0027] Further, the JC-1 fluorescence probe can be used to detect the effect of the saffloric acid on the MPP + The influence of the modeling of the MES23.5 cell mitochondria membrane potential. JC-1 is an ideal fluorescence probe widely used for detecting the mitochondrial membrane potential ΔΨm, and can rapidly and sensitively detect the membrane potential of cells, tissues or purified mitochondria; when the mitochondrial membrane potential is high, the JC-1 is aggregated in the matrix of the mitochondria, forms a polymer and produces red fluorescence; when the mitochondrial membrane potential is low, the JC-1 cannot be aggregated in the matrix of the mitochondria, but exists in the form of JC-1 monomer, and produces green fluorescence; therefore, the change of the mitochondrial membrane potential can be judged through the change of the fluorescence color. The relative proportion of the red and green fluorescence is generally used to measure the proportion of the mitochondrial depolarization; the decrease of the mitochondrial membrane potential is a sign event in the early stage of apoptosis; the change of the JC-1 from red fluorescence to green fluorescence can be used as a detection index in the early stage of apoptosis.

[0028] Further, the nerve repair can be evaluated through the dopamine neuron electrophysiology detection, specifically, 8-channel microfilament array electrodes are implanted into the dorsal striatum of the mouse, 7 days are recovered after the operation, then MPTP modeling and 50mg / kg CRO administration are carried out. The left implantation point coordinate of the electrode is AP: +1.5mm, ML: 1mm, DV: 3.5mm. The right side is symmetrically implanted. The 8-channel microfilament array electrodes collect and analyze data through the KEDB recording system. The 50Hz notch filter is used to weaken the power line interference. The local field potential signal is band-pass filtered in the frequency range of 0.5-250Hz, and the neuron discharge signal is band-pass filtered in the frequency range of 250Hz to 7.5kHz. The Spike sorting is carried out offline using the offline classifier, and then data analysis is carried out using NeuroExplorer and custom MATLAB scripts.

[0029] The application has the following beneficial effects:

[0030] The application first proves that the Chinese medicine monomer crocetin (CRO) can alleviate the symptoms of Parkinson's disease (PD) by improving the dysfunction of mitochondria and the autophagy pathway, which can provide a new theoretical basis for the research and development of drugs related to Parkinson's disease, and has great scientific significance and clinical transformation value. On this basis, the application first proposes that the Chinese medicine monomer crocetin (CRO) is used as the only active ingredient in the treatment of Parkinson's disease. The drug can alleviate the symptoms of Parkinson's disease (PD) by improving the mitochondrial membrane potential, ATP production, mitochondrial reactive oxygen species production, intracellular reactive oxygen species production and ubiquitin-dependent mitochondrial autophagy pathway, and provides a new and effective drug treatment for Parkinson's disease (PD).

[0031] The above description is only a summary of the technical scheme of the application. In order to more clearly understand the technical means of the application, and to implement the content of the description, the following will be described in detail with the preferred embodiments of the application and the accompanying drawings. The specific embodiments of the application are described in detail by the following examples and the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0032] The drawings described herein are used to provide further understanding of the application, and form a part of the application. The schematic embodiments of the application and their descriptions are used to explain the application, and do not constitute an improper limitation on the application. In the drawings:

[0033] Figure 1 A schematic diagram of the treatment system of the Chinese medicine monomer crocetin in the application;

[0034] Figure 2 Different substance concentration MPP in experimental example 1 of the application + Cell viability column chart after treating three candidate cell lines, wherein the left graph corresponds to PC12 cells, the middle graph corresponds to MES23.5 cells, and the right graph corresponds to SH-SY5Y cells;

[0035] Figure 3 Different candidate Chinese medicine monomers treated MPP in experimental example 2 of the application + Cell viability column chart after modeling PC12 cells, wherein A corresponds to chrysin, B corresponds to crocetin, C corresponds to ligustrazine, D corresponds to uncarine, E corresponds to gallic acid, F corresponds to paeoniflorin, G corresponds to gastrodin, and H corresponds to round yuzhi ketone;

[0036] Figure 4 Different candidate Chinese medicine monomers treated MPP in experimental example 3 of the application +Cell viability histogram after modeling SH-SY5Y cells, wherein A corresponds to apigenin, B corresponds to picrohhyrin, C corresponds to ligustrazine, D corresponds to uncarine, E corresponds to gallic acid, F corresponds to paeoniflorin, G corresponds to gastrodin, and H corresponds to nobiletin;

[0037] Figure 5 MPP treated by different candidate Chinese medicine monomers in experimental example 4 of the present application + Cell viability histogram after modeling MES23.5 cells, wherein A corresponds to apigenin, B corresponds to picrohhyrin, C corresponds to ligustrazine, D corresponds to uncarine, E corresponds to gallic acid, F corresponds to paeoniflorin, G corresponds to gastrodin, and H corresponds to nobiletin;

[0038] Figure 6 Working principle diagram of full-automatic patch clamp technology for detecting potassium current in experimental example 5 of the present application;

[0039] Figure 7 Membrane potassium current diagram (A) and I-V curve diagram (B) of MES23.5 cells treated by different concentrations of picrohhyrin for 7 days in experimental example 5 of the present application under voltage gradient of-80mV to +80mV;

[0040] Figure 8 Stable current density histogram of MES23.5 cells treated by different concentrations of picrohhyrin for 7 days in experimental example 6 of the present application;

[0041] Figure 9 Results diagram of α-syn expression in MES23.5 cells treated by different concentrations of picrohhyrin for 7 days in experimental example 7 of the present application, wherein A is fluorescence scanning diagram of confocal microscope, and B is α-syn expression level histogram of MES23.5 cells;

[0042] Figure 10 Results diagram of mitochondrial reactive oxygen species and ATP production of MES23.5 cells treated by different concentrations of picrohhyrin for 7 days in experimental example 8 of the present application, wherein A is fluorescence scanning diagram of confocal microscope, B is mitochondrial reactive oxygen species level histogram of MES23.5 cells, and C is ATP level histogram of MES23.5 cells;

[0043] Figure 11 Flow analysis results diagram of mitochondrial membrane potential of MES23.5 cells treated by different concentrations of picrohhyrin for 7 days in experimental example 9 of the present application;

[0044] Figure 12 Quantitative results diagram of mitochondrial membrane potential recovery of MES23.5 cells treated by different concentrations of picrohhyrin for 7 days in experimental example 9 of the present application;

[0045] Figure 13A flow cytometry analysis result diagram of the production of reactive oxygen species in MES23.5 cells after treatment with different concentrations of crocetin in the experimental example 10 of the present application;

[0046] Figure 14 A quantitative result diagram of the production of reactive oxygen species in MES23.5 cells after treatment with different concentrations of crocetin in the experimental example 10 of the present application;

[0047] Figure 15 A result diagram of the expression of LC3 in the mitochondria of MES23.5 cells after treatment with different concentrations of crocetin for 7 days in the experimental example 11 of the present application, wherein A is a fluorescence scanning diagram of a confocal microscope, and B is a columnar diagram of the expression level of LC3 in the mitochondria of MES23.5 cells;

[0048] Figure 16 A schematic diagram of PINK1 / Parkin-mediated mitochondrial autophagy in the experimental example 12 of the present application;

[0049] Figure 17 A result diagram of the expression of mitochondrial autophagy-related proteins in MES23.5 cells after treatment with different concentrations of crocetin for 7 days in the experimental example 12 of the present application, wherein A is a result diagram of the expression of PINK1 and Parkin in each group of MES23.5 cells, B is a quantitative result diagram of the expression of PINK1 in each group of MES23.5 cells, C is a quantitative result diagram of the expression of Parkin in each group of MES23.5 cells, D is a result diagram of the expression of Tim23 and TOM20 in each group of MES23.5 cells, E is a quantitative result diagram of the expression of Tim23 in each group of MES23.5 cells, and F is a quantitative result diagram of the expression of TOM20 in each group of MES23.5 cells;

[0050] Figure 18 A schematic diagram of the collection of brain electrical signals in the striatum of a mouse in the experimental example 14 of the present application;

[0051] Figure 19 A result diagram of a mouse motor behavior experiment in the experimental example 15 of the present application, wherein A is a result diagram of the open field movement trajectory of each group of mice, B is a columnar diagram of the total distance of the open field movement of each group of mice, C is a columnar diagram of the number of activities of each group of mice in the open field, D is a columnar diagram of the average speed of each group of mice in the open field, E is a columnar diagram of the number of times of climbing and standing of each group of mice in the open field, F is a columnar diagram of the rotarod fall latency of each group of mice, G is a columnar diagram of the rotation speed of the rotarod fall of each group of mice, H is a columnar diagram of the time of descending along the pole of each group of mice, and I is a columnar diagram of the time of crossing the balance beam of each group of mice;

[0052] Figure 20Figure for the results of Nissl staining and TH immunohistochemical staining of mouse brain sections treated with crocetin for 7 days in experimental example 16 of the present application, wherein Figure A is the results of TH immunohistochemical staining and Nissl staining of the striatal region of mice in each group after CRO treatment for 7 days, Figure B is the results of TH positive fiber number in the striatal region of mice in each group after CRO treatment for 7 days, Figure C is the results of TH positive cell number in the substantia nigra region of mice in each group after CRO treatment for 7 days, Figure D is the results of TH immunohistochemical staining and Nissl staining in the substantia nigra region of mice in each group after CRO treatment for 7 days, Figure E is the results of Nissl body number in the substantia nigra region of mice in each group after CRO treatment for 7 days, and Figure F is the results of Nissl body number in the striatal region of mice in each group after CRO treatment for 7 days;

[0053] Figure 21 Figure for the results of electrophysiological recording of mice treated with crocetin for 7 days in experimental example 17 of the present application, wherein Figure A is the butterfly diagram of dopamine neuron discharge of mice in each group after CRO treatment for 7 days, Figure B is the histogram of dopamine neuron discharge rate of mice in each group after CRO treatment for 7 days (bin = 5s), and Figure C is the neuron electrical signal diagram of β frequency band. DETAILED DESCRIPTION

[0054] The preferred embodiments of the present application will be described in detail with reference to the accompanying drawings, so that the purpose, characteristics and advantages of the application can be more clearly understood. It should be understood that the embodiments shown in the drawings are not a limitation on the scope of the present application, but are only intended to illustrate the essential spirit of the technical solutions of the present application. In addition, the technical features involved in the different embodiments of the present application described below can be combined with each other as long as they do not conflict with each other.

[0055] The present application provides the use of traditional Chinese medicine monomer crocetin (CRO) as the only active ingredient in the preparation of a drug for treating Parkinson's disease (PD).

[0056] Referring to Figure 1 As shown in the drawings, the drug can regulate the function of mitochondria using crocetin, including positively affecting mitochondrial membrane potential, ATP production, mitochondrial reactive oxygen species production, intracellular reactive oxygen species production, and ubiquitin-dependent mitochondrial autophagy pathway, thereby improving mitochondrial dysfunction and autophagy, and achieving the treatment of Parkinson's disease.

[0057] In order to verify that traditional Chinese medicine monomer crocetin (CRO) can treat Parkinson's disease (PD) by regulating mitochondrial function and autophagy, and to explain the mechanism of crocetin in treating Parkinson's disease by regulating mitochondria, the inventors of the present application have carried out the following experimental work based on in vitro Parkinson's disease injury models and electrophysiology and other frontier technologies.

[0058] The main steps for verifying that CRO can treat Parkinson's disease by regulating mitochondrial function and autophagy are as follows:

[0059] (1) Screening of Parkinson's disease model cell lines and traditional Chinese medicine monomers to determine the cell lines and traditional Chinese medicine monomers to be studied.

[0060] (2) Examining the effect of CRO on MPP + The scavenging effect of MES23.5 on α-synuclein in vitro cells and its influence on cell membrane hyperpolarization.

[0061] (3) To evaluate the regulatory effect and mechanism of CRO on mitochondrial dysfunction and mitochondrial autophagy in PD model cells, mainly to explore the effects on mitochondrial membrane potential, ATP production, mitochondrial reactive oxygen species production and intracellular reactive oxygen species production, as well as ubiquitin-dependent mitochondrial autophagy pathway.

[0062] (4) By evaluating the damage and repair of nerve and motor function in PD mouse models, we can further investigate the neuroprotective efficacy of CRO in vivo.

[0063] Experimental Example 1

[0064] PC12, SH-SY5Y, and MES23.5 cells were seeded into 96-well plates at a density of 1000 cells / well. Once the cells were in the logarithmic growth phase, different concentrations of MPP were added. + The optimal concentration for modeling is when cell viability decreases to approximately 50%. For PC12 cells, MPP was administered. + The concentration gradient was 0, 0.25, 0.5, 1, 2 mM; for SH-SY5Y cells, MPP was administered. + The concentration gradient was 0, 0.5, 1, 1.5, and 2 mM; for MES23.5 cells, MPP was administered. + The concentration gradients were 0, 0.05, 0.1, 0.5, 1, and 2 mM. The three cell lines were then respectively treated with the corresponding concentration gradients of MPP. + The cells were incubated for 24 hours, with three replicates for each concentration gradient. After incubation, 10 μL of CCK-8 solution was added to each well, and the cells were incubated for another hour in a cell culture incubator. Finally, the absorbance of each well was measured at 450 nm. Based on the different absorbance values, the optimal cell modeling concentration was screened, and the responsiveness of the three cell lines to MPP was determined. + The degree of sensitivity.

[0065] See Figure 2 As shown, Figure 2 Showing different concentrations of MPP + Bar charts showing cell viability for three cell lines: left bar chart corresponds to PC 12 cells, middle bar chart corresponds to MES23.5 cells, and right bar chart corresponds to SH-SY5Y cells. From... Figure 2It can be seen from the table that the cell viability is reduced to about 50% when the optimal modeling concentration of the modeling agent is reached. Therefore, 2 mM is the optimal modeling concentration of MPP + for the three cell lines. + The optimal modeling concentration. For PC12 cells, 2 mM MPP + reduces the cell viability to 44.79%; for MES23.5 cells, 2 mM MPP + reduces the cell viability to 49.61%; and for SH-SY5Y cells, 2 mM MPP + reduces the cell viability to 34.23%. This indicates that the three cell lines have similar sensitivity to MPP + . Therefore, 2 mM concentration of MPP + is used for modeling of the three cell lines.

[0066] Experimental Example 2

[0067] PC12 cells modeled with MPP + at different concentrations of each of the eight candidate Chinese medicine monomers were treated with apigenin, safflor yellow A, ligustrazine, uncarine, gallic acid, paeoniflorin, gastrodin, and round yuzhi ketone at concentrations of 0, 1, 10, 100, and 1000 μM, respectively, and the cell viability of each of the cells was then detected to observe the effect of each of the different concentrations of the Chinese medicine monomers on the cell viability of PC12 cells modeled with MPP + .

[0068] As shown in the table, Figure 3 the effect of the eight candidate Chinese medicine monomers on the cell viability of PC12 cells modeled with MPP + is shown, where A corresponds to apigenin, B corresponds to safflor yellow A, C corresponds to ligustrazine, D corresponds to uncarine, E corresponds to gallic acid, F corresponds to paeoniflorin, G corresponds to gastrodin, and H corresponds to round yuzhi ketone. As can be seen from the table, PC12 cells induced by MPP + are not sensitive to apigenin and safflor yellow A. Ligustrazine, uncarine, and paeoniflorin administration can reduce the cell viability of PC12 cells to some extent, 10 μM ligustrazine and uncarine can reduce the cell viability to 92.54% and 85.90% (compared with the MPP + group), respectively. Gallic acid at a concentration lower than 100 μM cannot restore the cell viability of PC12, and gallic acid at a concentration higher than 100 μM has a significant cytotoxic effect on PC12 cells. Gastrodin can increase the cell viability of PC12 cells to 1.03 times that of the model group at an optimal concentration of 1 μM, and the cytotoxic effect is significant as the concentration increases. Round yuzhi ketone at a concentration of 1 to 1000 μM promotes the cell viability of PC12 cells, and 100 μM is the optimal administration concentration, which can increase the cell viability to 1.07 times that of the model group. Therefore, MPP + is used for modeling of the three cell lines at a concentration of 2 mM.+ Induced PC12 cells were excluded from the candidate cell lines in the validation experiment.

[0069] Experimental Example 3

[0070] MPP + induced SH-SY5Y cells, and then the cell viability of each cell was detected to observe the effect of each Chinese medicine monomer at different concentrations on the MPP + induced SH-SY5Y cells.

[0071] Referring to Figure 4 the drawings, Figure 4 the effects of 8 candidate Chinese medicine monomers on the viability of MPP + induced SH-SY5Y cells are shown, where A corresponds to apigenin, B corresponds to crocetin, C corresponds to tetramethylpyrazine, D corresponds to uncarine, E corresponds to gallic acid, F corresponds to paeoniflorin, G corresponds to gastrodin, and H corresponds to morindone. As can be seen from Figure 4 , apigenin, uncarine, and morindone have no effect on the viability of MPP + induced SH-SY5Y cells, and 1000 μM apigenin even causes toxic damage. Gastrodin at a concentration lower than 100 μM also has no beneficial effect on the proliferation of MPP + induced SH-SY5Y cells, and only when the concentration reaches 1000 μM does it show a promoting effect, increasing the cell viability to 1.12 times that of the model group. Paeoniflorin has a similar effect to gastrodin, and when the concentration is greater than 10 μM, it shows a significant effect on cell proliferation, such as 100 μM, which increases the cell viability to 1.26 times that of the model group. CRO and tetramethylpyrazine also have a similar effect on cells: 1 μM and 10 μM have no effect on cell proliferation, 100 μM is the best concentration, which increases the cell viability to 1.11 times that of the model group, and high-concentration administration (1000 μM) has a significant toxic effect, which decreases the cell viability to 67.38% of the model group. For gallic acid, 1 μM administration is effective, which increases the cell viability to 1.07 times that of the model group. When the concentration is greater than 10 μM, it also shows a significant cell-killing effect. Therefore, MPP + induced SH-SY5Y cells are also excluded from the candidate cell lines in the validation experiment.

[0072] Experimental Example 4

[0073] MPP was treated with 0, 1, 10, 100, and 1000 μM concentrations of apigenin, crocin, tetramethylpyrazine, rhododendronine, gallic acid, paeoniflorin, gastrodin, and naringin, respectively. + MES23.5 cells were used to create a model, and the cell viability of each cell was then measured to observe the effects of different concentrations of various herbal monomers on MPP. + Effects on the viability of modeled MES23.5 cells.

[0074] See Figure 5 As shown, Figure 5 Eight candidate Chinese herbal monomers were demonstrated against MPP. + The effect of modeling on the viability of MES23.5 cells, where Figure A corresponds to salicylic acid, Figure B to crocin, Figure C to tetramethylpyrazine, Figure D to rhizoside, Figure E to gallic acid, Figure F to paeoniflorin, Figure G to gastrodin, and Figure H to naringin. Figure 5 As can be seen, concentrations of rhynchophylline below 100 μM had no effect on the cell state of MES23.5 cells, while concentrations above 100 μM began to exhibit cytotoxic effects. Naringin showed similar behavior to rhynchophylline in MES23.5 cells, showing no improvement in cell viability. Low concentrations of apigenin and tetramethylpyrazine were ineffective against MES23.5 cells, but showed harmful effects at concentrations greater than 10 μM: 100 μM apigenin reduced cell viability to 88.94% of the model group, while 1000 μM apigenin reduced it to 86.30%. The sensitivity of MES23.5 cells to gastrodin varied with the concentration: 1 μM and 100 μM gastrodin promoted cell viability, increasing it to 1.05 times that of the model group. 10 μM administration had no significant stimulating effect on cells, while 1000 μM administration had a cytotoxic effect. Compared to PC 12 cells, MES23.5 cells were more sensitive to gallic acid. Administration of 1 μM and 10 μM gallic acid significantly restored cell viability, increasing it to 1.12 and 1.06 times that of the model group, respectively. Gallic acid promoted cell proliferation, while administration of gallic acid at concentrations greater than 100 μM inhibited cell proliferation, impaired cell function, and reduced cell viability. MES23.5 cells also showed a response to paeoniflorin administration, with 1 μM paeoniflorin increasing cell proliferation to 1.05 times that of the model group (P<0.05). Notably, CRO administration had the greatest impact on MES23.5 cell viability, significantly enhancing it. Specifically, 10 μM CRO increased MES23.5 cell viability by nearly 2 times, while 1 μM and 100 μM CRO increased it by more than 1.5 times.

[0075] In Experiments 1-4 above, the sensitivity of three different cell lines (PC 12, SH-SY5Y, and MES23.5) to the PD modeling agent was first investigated using the CCK-8 assay, aiming to select the cell lines most responsive to MPP. + The most sensitive cell lines. Results from Experiment 1 showed that the three cell lines were resistant to MPP. + The sensitivities of the samples were similar, all showing a decrease in cell viability to nearly 50% at 2 mM administration, meeting the modeling criteria. Then, the CCK-8 assay was used to screen eight active monomers from traditional Chinese medicine (caprylic acid, crocin, ligustrazine, rhizomein, gallic acid, paeoniflorin, gastrodin, and naringin). These eight different monomers were then reacted with 2 mM MPP... + The modeled cells were co-incubated. Results from Examples 2-4 showed that SH-SY5Y cells had the lowest sensitivity to the eight candidate herbal monomers, while MES23.5 and PC12 cells showed similar sensitivities. The effects of the eight candidate herbal monomers varied across the three cell lines, but crocin (CRO) showed a significant effect on MES23.5 cells, increasing cell viability by nearly two times at 10 μM. The reasons for the poor efficacy of the other seven monomers when used alone are unclear and require further investigation.

[0076] In summary, Experiments 1-4 of this invention screened out the cell lines used in this invention using the CCK-8 assay. These two cell lines were MES23.5 cells and CRO cells, respectively, for subsequent research.

[0077] Experimental Example 5

[0078] See Figure 6 As shown, Figure 6 A schematic diagram illustrating the working principle of fully automated patch-clamp technology for detecting potassium current is shown. Figure 6 As can be seen, the fully automated patch-clamp technique uses microglass tube electrodes (patch electrodes or patch pipettes) to contact the cell membrane, sealing it with an impedance of gigahertz ohms or higher. This electrically isolates a small region of the cell membrane (the patch) connected to the electrode tip from its surroundings. By fixing a point on this membrane, the ion current of the ion channels on the patch is monitored and recorded. This method is the gold standard for ion channel detection. Its core advantages lie in high throughput, automated operation, and precise data acquisition. It can improve efficiency and reduce drug consumption through parallel operation using multi-well plates, saving time and experimental costs.

[0079] Potassium ion channels have been shown to be closely related to neuronal damage (PD). During PD, dopaminergic neurons are in a hyperpolarized state, leading to increased potassium ion efflux. Therefore, potassium currents are used to assess cellular damage and repair. To demonstrate that CRO restores MPP... +The ability of crocin to induce cell membrane electrophysiological disturbances was investigated in this experiment using fully automated patch-clamp technique to detect the effect of crocin on MPP. + The effect of the MES23.5 cell membrane potassium current on the model was investigated using the following method:

[0080] 1) MES23.5 cell culture, modeling, and CRO drug delivery: MES23.5 cells were seeded in 60°C culture dishes. When the MES23.5 cells reached 70% confluence, 2 mM MPP was added. + The drug solution was co-incubated with MES23.5 cells for 24 hours; after incubation, MPP was aspirated. + After washing MES23.5 cells three times with PBS, the cells were treated with CRO at concentrations of 0, 10, 100, and 1000 μM for 36 hours.

[0081] 2) Pretreatment of cells before loading: Aspirate the CRO drug solution, wash MES23.5 cells three times with PBS, add 0.2% trypsin to each dish for 1 minute digestion; after digestion, add cell culture medium to stop digestion; then centrifuge at 1000 rpm for 4 minutes; after centrifugation, aspirate the supernatant, add special extracellular buffer solution to mix MES23.5 cells by pipetting, and take the cell suspension;

[0082] 3) Cell loading: 15 μL of cell suspension is injected into the cell loading port of the fully automated patch clamp system. The system's fully automated pipetting device transfers the cell suspension to a multi-well plate or perfusion tank. The system's microscopic imaging device identifies the morphology and position of individual cells. The system's robotic arm controls the electrode to approach the target cell, applies a slight negative pressure to make the electrode tip contact the cell membrane, and monitors the impedance changes in real time through software.

[0083] 4) Cell sealing and membrane rupture: When the impedance reaches 1 GΩ or above, it is considered a successful sealing. At this time, the current recorded in the electrode only reflects the activity of a single channel in the sealing area.

[0084] 5) Data analysis and acquisition: The system automatically applies a gradient voltage from -80mV to +80mV, records the current signal and performs noise filtering, and exports the data using Patchmaster software.

[0085] See Figure 7 As shown, Figure 7 The study presents the potassium current of MES23.5 cell membranes and the intracellular velocity (IV) curves under a voltage gradient from -80mV to +80mV after 7 days of treatment with different concentrations of crocin. Figure A shows the potassium current of MES23.5 cell membranes after 7 days of treatment with different concentrations of CRO, and Figure B shows the IV curves under a voltage gradient from -80mV to +80mV after 7 days of treatment with different concentrations of CRO. Figure 7 As can be seen, CRO restored MPP.+ The disturbance of the cell membrane potassium current caused by the drug reduced the elevated potassium current. The 1000 mM drug concentration increased the potassium current due to the high concentration causing cytotoxicity.

[0086] Experimental Example 6

[0087] Referring to FIG. B of Figure 6 Since the current at 40 mV was moderate, the steady current density at 40 mV was used for the efficacy analysis in this experimental example. Referring to FIG. 6, the steady current density of the MES23.5 cells treated with different concentrations of crocin for 7 days was shown. Figure 8 Figure 8 The steady current density of the MES23.5 cells treated with different concentrations of crocin for 7 days was shown. As can be seen from Figure 8 , the steady current density of the normal group at 40 mV was 24.63 pA / pF, while the MPP + group was increased to 83.18 pA / pF, indicating the occurrence of cell damage. The 10 mM and 100 mM concentrations of CRO were able to reverse the K + current increase phenomenon caused by cell damage, and the steady current density was restored to 36.76 pA / pF and 29.67 pA / pF, respectively, close to the normal group level.

[0088] Experimental Example 7

[0089] In PD, the α-syn protein is misfolded to form insoluble aggregates - Lewy bodies, leading to the death of dopaminergic neurons. This aggregation phenomenon is not only a hallmark pathological change of PD, but also related to the severity of disease progression. Therefore, the expression of α-syn in the MPP + modeled MES23.5 cells was detected by immunofluorescence staining to analyze the efficacy of CRO on the PD cell model, which can partially know the recovery of the disease. The specific method is as follows:

[0090] 1) The MES23.5 cells were inoculated in a 60 mm x 15 mm cell culture dish and placed in a CO2 incubator for culture; when the MES23.5 cells were about 70% confluent, 2 mM MPP + was added and incubated with the MES23.5 cells for 24 hours; after the incubation, the MPP + solution was aspirated, washed with PBS three times, and then 0, 1, 10, 100, and 1000 mM concentrations of CRO were added, respectively, and incubated in a CO2 incubator for 36 hours;

[0091] ​2) Aspirate CRO drug solution, wash with PBS for 5 minutes each time for three times; after washing, remove PBS, add 5 mL of immunostaining fixation solution to each dish, and fix at room temperature for 15 minutes; after fixation, remove the fixation solution, and wash with PBS for 5 minutes each time for three times;

[0092] 3) Remove PBS, add 5 mL of immunofluorescence permeation solution to each dish, and permeate at room temperature for 30 minutes; after permeation, remove the immunofluorescence permeation solution, and wash with PBS for 5 minutes each time for three times;

[0093] 4) Remove PBS, add 5 mL of immunofluorescence blocking solution to each dish, and incubate at room temperature for 2 hours in the dark; after incubation, remove the immunofluorescence blocking solution, and wash with PBS for 5 minutes each time for three times;

[0094] 5) Remove PBS, dilute α-syn antibody with immunofluorescence primary antibody dilution solution at a dilution ratio of 1:100, add 5 mL of the diluted α-syn antibody to each dish, and incubate overnight in a 4°C refrigerator; after incubation, recover the α-syn primary antibody, and wash with immunofluorescence washing solution for 5 minutes each time for three times;

[0095] 6) Remove the immunofluorescence washing solution, dilute goat anti-mouse Alexa Fluor 488 with immunofluorescence secondary antibody dilution solution at a dilution ratio of 1:1000, add 5 mL of the diluted secondary antibody to each dish, and incubate at room temperature for 2 hours in the dark; after incubation, remove the antibody solution, and wash with immunofluorescence washing solution for 5 minutes each time for three times;

[0096] 7) Remove the immunofluorescence washing solution, add DAPI staining solution, and incubate at room temperature for 1 hour in the dark; after incubation, wash with PBS for 5 minutes each time for three times, and finally add an appropriate amount of PBS to keep the cells moist, and perform fluorescence scanning and photographing under a confocal microscope.

[0097] Referring to Figure 9 as shown in the drawings, Figure 9 the expression of α-syn in MES23.5 cells after treatment with different concentrations of crocetin for 7 days is shown, wherein A is a fluorescence scanning diagram under a confocal microscope, and B is a column chart of the expression level of α-syn in MES23.5 cells. As Figure 9 can be seen in the drawings, CRO administration at concentrations of 1, 10, 100, and 1000 μM can reduce the expression of α-syn to a certain extent. Among them, the administration concentration of 10 μM has the most significant effect, and the fluorescence intensity of α-syn is reduced to 1.94% (P<0.0001, compared with the MPP + group), close to the normal group level. This indicates that CRO can restore the increase in α-syn expression caused by MPP + , thereby being beneficial to the functional recovery of the PD cell model.

[0098] Experimental Example 8

[0099] To investigate the effects and intensity of CRO on mitochondrial dysfunction, a comprehensive and detailed analysis of several key indicators was conducted. Mitochondrial dysfunction is a complex pathological process that profoundly impacts overall cellular metabolism and energy status. When mitochondria malfunction, their internal electron transport chain may be disrupted, leading to electron leakage and inappropriate binding with oxygen, thereby increasing mitochondrial oxidative stress. This oxidative stress disrupts normal mitochondrial function, making the mitochondrial environment increasingly unstable. As mitochondrial oxidative stress intensifies, it further stimulates the production of intracellular ROS. These ROS are highly oxidizing and can attack various intracellular biomolecules. More seriously, mitochondrial dysfunction also leads to a reduction in ATP production. Mitochondria are the core site of ATP synthesis in cells, providing approximately 90% of the cell's energy through oxidative phosphorylation. They are the cell's direct energy source, driving various cellular life activities. When mitochondrial function is impaired, ATP production decreases significantly. This not only affects normal cellular metabolism and function but may also trigger a series of pathophysiological changes, posing a serious threat to the body's health. Therefore, this experimental example investigated the effect of florid acid on MPP. + Mitochondrial reactive oxygen species and ATP production in modeled MES23.5 cells.

[0100] First, the effect of crocin on MPP was detected using the MitoSOX staining method. + The effect of modeled MES23.5 cells on mitochondrial oxidative stress. MitoSOX is a cationic fluorescent probe capable of penetrating the mitochondrial membrane and accumulating in the mitochondrial matrix. Inside the mitochondria, MitoSOX reacts with superoxide anions and is oxidized to a strongly fluorescent product. This oxidation process causes MitoSOX to emit bright red fluorescence. This red fluorescence can be observed using fluorescence microscopy, and its intensity reflects the level of oxidative stress in the mitochondria; stronger red fluorescence indicates a higher mitochondrial ROS content. The specific method is as follows:

[0101] 1) Seed MES23.5 cells at a density of 50,000 cells / mL in 24-well plates. When the MES23.5 cells reached 70% confluence, add 2 mM MPP. + The drug solution was co-incubated with MES23.5 cells for 24 hours; after incubation, MPP was aspirated. + The cells were washed three times with PBS and then incubated with CRO at concentrations of 0, 1, 10, 100, and 1000 μM for 36 hours. After incubation, the CRO solution was discarded and the cells were washed three times with PBS.

[0102] 2) To 50 μg MitoSOX powder, add 13 μL DMSO to configure 5 mM MitoSOX mother liquor; then dilute the mother liquor to 4 μM MitoSOX working solution; after aspirating PBS, add 500 μL MitoSOX working solution to each well, and incubate in a 37°C incubator for 1 hour; after incubation, wash with PBS for 3 times, 5 minutes each time;

[0103] 3) Aspirate PBS solution, add 500 μL DAPI staining solution to each well, and incubate at room temperature in the dark for 45 minutes; after incubation, wash with PBS for 3 times;

[0104] 4) Add 400 μL PBS solution to each well, and observe under a confocal microscope.

[0105] Then use the ATP detection kit to detect the effect of crocetin on MPP + The ATP production of the mitochondria of the modelled MES23.5 cells. The detection principle of the kit used in this experiment is as follows: when firefly luciferase catalyzes luciferin to produce fluorescence, ATP provides energy. When firefly luciferase and luciferin are in excess, the fluorescence produced is proportional to the concentration of ATP within a certain concentration range. Thus, the ATP concentration in the solution can be detected with high sensitivity. The specific method is as follows:

[0106] 1) Seed MES23.5 cells in a 6-well plate and place in a CO2 incubator for culture; when the MES23.5 cells are about 70% confluent, add 2 mM MPP + drug solution and incubate with the MES23.5 cells for 24 hours; after incubation, aspirate the MPP + drug solution, wash with PBS for 3 times, and add 0, 1, 10, 100 and 1000 μM concentrations of CRO, respectively, and place in a CO2 incubator for incubation for 36 hours; after incubation, discard the CRO drug solution, and wash with PBS for 3 times;

[0107] 2) Dissolve the reagents to be used on ice, dilute the ATP standard solution with ATP detection lysis solution to concentrations of 0.01, 0.03, 0.1, 0.3, 1, 3 and 10 μM to draw an ATP standard curve;

[0108] 3) Aspirate PBS and lyse the MES23.5 cells on ice; add 200 μL lysis solution to each well, and use a pipette to perform repeated pipetting; after lysis, centrifuge the cells at 12000 g at 4°C for 5 minutes, and take the supernatant for subsequent determination;

[0109] 4) Prepare ATP detection working solution according to the proportion of 100 μL ATP detection working solution per sample or standard; melt the reagent to be used on ice bath, take a certain amount of ATP detection reagent, dilute the ATP detection reagent with ATP detection reagent dilution liquid according to the proportion of 1:9; the diluted ATP detection reagent is the ATP detection working solution used in subsequent experiments, which is temporarily stored on ice bath;

[0110] 5) Add 100 μL ATP detection working solution to the detection hole of the full black 96-hole plate; place at room temperature for 3-5 minutes to make the background ATP completely consumed; add 20 μL sample or standard to the detection hole,

[0111] Mix quickly with a gun, and then measure the relative light unit with a Luminometer.

[0112] Referring to Figure 10 , Figure 10 shows the mitochondrial reactive oxygen species and ATP production of MES23.5 cells after 7 days of treatment with different concentrations of crocetin, wherein A is the fluorescence scanning diagram of confocal microscope, B is the column chart of the mitochondrial reactive oxygen species level of MES23.5 cells, and C is the column chart of the ATP level of MES23.5 cells. From Figure 10 A and Figure 10 B, it can be seen that MPP + administration significantly increases the production of mitochondrial reactive oxygen species, which can increase the production of mitochondrial reactive oxygen species by nearly 2 times compared with the control group (P<0.0001). However, CRO administration, especially 10 and 100 μM administration, can reduce the production of reactive oxygen species by 1 / 2, close to the normal group (P<0.0001 compared with the MPP+ group). From Figure 10 C, it can be seen that the ATP production of the normal group is 14.47 μM, the MPP + administration significantly reduces the ATP content and damages the mitochondrial function, so that the ATP production is reduced to 8.30 μM. The 10 μM concentration of CRO can significantly restore the damage caused by MPP + , so that the ATP production is restored to 13.02 μM (P<0.0001 compared with the MPP + group).

[0113] Experimental Example 9

[0114] JC-1 is an ideal fluorescent probe widely used to detect mitochondrial membrane potential ΔΨm, which can quickly and sensitively detect the membrane potential of cells, tissues or purified mitochondria. When the mitochondrial membrane potential is high, JC-1 aggregates in the matrix of mitochondria to form polymers, producing red fluorescence; when the mitochondrial membrane potential is low, JC-1 cannot aggregate in the matrix of mitochondria, but exists in the form of JC-1 monomer, producing green fluorescence. Therefore, by the change of fluorescence color, the change of mitochondrial membrane potential can be judged. The relative proportion of red and green fluorescence is commonly used to measure the proportion of mitochondrial depolarization. The decrease of mitochondrial membrane potential is a hallmark event in the early stage of apoptosis. The change of JC-1 from red fluorescence to green fluorescence can be used as an indicator for detecting the early stage of apoptosis.

[0115] In this experimental example, the effect of crocetin on the mitochondrial membrane potential of MPP + The specific method is as follows:

[0116] 1) The MES23.5 cells were inoculated in a 6-well plate and placed in a CO2 incubator for culture; when the MES23.5 cells were about 70% confluent, 2mM MPP + The drug solution was incubated with the MES23.5 cells for 24 hours; after the incubation, the MPP + drug solution was aspirated, and the cells were washed with PBS three times, and 0, 1, 10, 100 and 1000 μM concentrations of CRO were added respectively, and the plate was placed in a CO2 incubator for incubation for 36 hours; after the incubation, the CRO drug solution was aspirated, and the cells were washed with PBS three times;

[0117] 2) Take appropriate amount of JC-1 (200x), dilute JC-1 according to the ratio of 8mL ultrapure water per 50μl JC-1 (200x); vortex vigorously, dissolve and mix well; then add 2mL JC-1 staining buffer (5x), mix well to prepare JC-1 staining working solution;

[0118] 3) The cells were digested with 0.2% trypsin for 1 minute, and then the digestion was terminated with complete culture medium; the cell suspension was centrifuged at 1000 rpm for 4 minutes, and the supernatant was discarded;

[0119] 4) Take 600,000 cells, resuspend in 0.5mL cell culture medium; then add 0.5mL JC-1 staining working solution, mix well, and incubate in a cell incubator for 20 minutes; during the incubation, prepare appropriate amount of JC-1 staining buffer (1x) according to the ratio of 4mL distilled water per 1mL JC-1 staining buffer (5x), and place it in ice bath;

[0120] 5) After incubation at 37°C, centrifuge at 600g for 4 minutes at 4°C to precipitate the cells; then discard the supernatant; add 1mL of JC-1 staining buffer (1×) to resuspend the cells, centrifuge at 600g for 4 minutes at 4°C to precipitate the cells, discard the supernatant; add another 1mL of JC-1 staining buffer (1×) to resuspend the cells, and repeat the above centrifugation steps.

[0121] 6) Finally, resuspend the sample in an appropriate amount of JC-1 staining buffer (1×) and analyze it using flow cytometry; refer to the FITC settings when detecting JC-1 monomers and refer to the propidium iodide settings when detecting JC-1 polymers.

[0122] See Figure 11 and Figure 12 As shown, Figure 11 The flow cytometry results of mitochondrial membrane potential in MES23.5 cells treated with different concentrations of crocin for 7 days are presented. Figure 12 This study quantitatively demonstrated the recovery of mitochondrial membrane potential in MES23.5 cells after 7 days of treatment with different concentrations of crocin. Figure 11 and Figure 12 As can be seen, the normal group had a higher mitochondrial membrane potential, with an average relative ratio of 27.95 for red and green fluorescence. Meanwhile, the MPP group... + It can damage mitochondrial membrane potential, thereby affecting normal mitochondrial function and reducing the relative ratio of red to green fluorescence to 5.18. After administration of 10 μM CRO, the mitochondrial membrane potential increased, recovering to 21.99, close to the level of the normal group (compared to MPP). + Compared with the previous group, P < 0.01).

[0123] Experimental Example 10

[0124] A decrease in mitochondrial membrane potential disrupts the integrity of the electron transport chain, leading to an abnormal increase in reactive oxygen species (ROS) and creating a positive feedback loop of "ROS-induced ROS release." This state can activate apoptosis pathways (such as cytochrome C release) and inhibit the activity of antioxidant enzymes, ultimately resulting in cellular energy metabolism exhaustion and ROS accumulation within the cell. Therefore, detecting intracellular ROS levels can also help assess the extent of mitochondrial damage.

[0125] This embodiment utilizes DCFH-DA to detect the effect of crocin on MPP. +Effect of modeling of mitochondria of MES23.5 cell line and intracellular reactive oxygen species production. DCFH-DA itself has no fluorescence and can freely penetrate the cell membrane. After entering the cell, intracellular trypsin will hydrolyze it into DCFH, which cannot penetrate the cell membrane and thus accumulates in the cell. When there is reactive oxygen species in the cell, DCFH will be oxidized to DCF with strong fluorescence. The intensity of fluorescence is proportional to the level of intracellular reactive oxygen species, which is usually detected at an excitation wavelength of 502 nm and an emission wavelength of 530 nm. The specific method is as follows:

[0126] 1) MES23.5 cells were inoculated in 60 mm x 15 mm cell culture dishes and placed in a CO2 incubator for culture; when the MES23.5 cells were about 70% confluent, 2 mM MPP was added + The drug solution was incubated with MES23.5 cells for 24 hours; after incubation, MPP + The drug solution was washed with PBS three times, and 0, 1, 10, 100, and 1000 μM concentrations of CRO were added, respectively, and incubated in a CO2 incubator for 36 hours;

[0127] 2) After washing the CRO-treated cells with PBS, the PBS was removed, 1 mL of 0.2% trypsin was added to each dish and digested for 1 minute, and then complete medium was added to terminate digestion; the suspended MES23.5 cells were collected in a 15 mL centrifuge tube and centrifuged at 1000 rpm for 4 minutes;

[0128] 3) Dilute DCFH-DA with PBS at a dilution ratio of 1:1000 in the dark to a final concentration of 5 mM / L; after cell collection, suspend the cells in the diluted DCFH-DA, adjust the cell density to 1 million / mL, and incubate in a 37°C cell incubator for 30 minutes; every 3 minutes, invert the centrifuge tube to mix well, so that the probe and the cells are in sufficient contact;

[0129] 4) Wash the cells with PBS three times to remove the DCFH-DA that has not entered the MES23.5 cells, and use a flow cytometer to detect the production of intracellular reactive oxygen species.

[0130] Referring to Figure 13 and Figure 14 , it is shown that Figure 13 the flow cytometry analysis results of the production of intracellular reactive oxygen species in MES23.5 cells after treatment with different concentrations of crocetin, Figure 14 show the quantification of the production of intracellular reactive oxygen species in MES23.5 cells after treatment with different concentrations of crocetin. From Figure 13 and Figure 14 , it can be seen that MPP +The administration of the drug caused a sharp increase in intracellular reactive oxygen species (ROS) levels, reaching 28.93 times that of the normal group, leading to cell death. Different concentrations of CRO could alleviate intracellular oxidative stress to some extent, with 10 μM and 100 μM concentrations showing the best effects, being 3 times and 2.01 times that of the normal group, respectively (P < 0.0001, compared with the control group).

[0131] Experimental Example 11

[0132] LC3 is a marker protein for autophagosome formation and a key executor of mitophagy. Mitotracker is a fluorescent mitochondrial probe primarily used for specific fluorescent staining of mitochondria in live cells. By analyzing the co-localization ratio of LC3 and Mitotracker, the expression status of LC3 in mitochondria can be determined.

[0133] See Figure 15 As shown, Figure 15 This study presents the expression of LC3 in the mitochondria of MES23.5 cells after 7 days of treatment with different concentrations of crocin. Figure A shows the fluorescence scan image obtained by confocal microscopy, and Figure B is a bar chart showing the LC3 expression level in the mitochondria of MES23.5 cells. Figure 15 The results show that LC3 expression was high (10.30%) in the normal group, and mitophagy was significant. Meanwhile, MPP... + This impairs mitophagy, thereby reducing LC3 expression in mitochondria to 4.15%. When CRO administered 10 μM, LC3 expression was significantly upregulated to 9.85% (P < 0.0001 compared to the model group), indicating partial recovery of mitophagy.

[0134] Experimental Example 12

[0135] See Figure 16 As shown, Figure 16 This diagram illustrates PINK1 / Parkin-mediated mitophagy. Mitophagy is a key mechanism for the selective clearance of damaged mitochondria by cells, and its dysfunction is closely related to the pathological progression of Parkinson's disease (PD). The most classic pathway in mitophagy is the ubiquitin-dependent mitophagy mediated by PINK1 / Parkin. In short, when mitochondria are damaged, PINK1 stably accumulates on the outer mitochondrial membrane, phosphorylates ubiquitin, and activates Parkin; Parkin then ubiquitinates damaged mitochondria, recruiting autophagosomes (such as LC3 protein) for clearance. Furthermore, impaired mitophagy leads to the accumulation of damaged mitochondria, thereby increasing the levels of the inner mitochondrial membrane protein TIM23 and the outer mitochondrial membrane protein TOM20.

[0136] See Figure 17 As shown, Figure 17This study presents the expression of mitophagy-related proteins in MES23.5 cells after 7 days of treatment with different concentrations of crocin. Figure A shows the expression of PINK1 and Parkin in each group of MES23.5 cells; Figure B shows the quantitative results of PINK1 expression in each group; Figure C shows the quantitative results of Parkin expression in each group; Figure D shows the expression of Tim23 and TOM20 in each group; Figure E shows the quantitative results of Tim23 expression in each group; and Figure F shows the quantitative results of TOM20 expression in each group. PINK1 / Parkin pathway-mediated ubiquitin-dependent mitophagy is the most classic mode of mitophagy. When mitophagy function is impaired, damaged mitochondria cannot be effectively cleared, leading to a continuous decrease in mitochondrial membrane potential and an increase in ROS levels. At this point, the cell may suppress PINK1 expression through a negative feedback mechanism to prevent continuous activation of mitophagy signals, thus avoiding excessive energy consumption or organelle degradation. Figure 17 As can be seen from this, MPP + It impairs mitophagy, thereby downregulating PINK1 expression to 32.81% in the normal group. However, 1000 μM crocin restored PINK1 expression to 80.31% of the normal group, protecting mitophagy function (P < 0.001, compared with MPP). + (Group comparison).

[0137] Experimental Example 13

[0138] This experiment used MPTP to model Parkinson's disease in mice. MPTP itself is non-toxic, but after entering the brain, it is converted into the toxic metabolite MPP by MAO-B in astrocytes. + MPP + It can selectively enter dopaminergic neurons via dopamine transporters, inhibiting mitochondrial complex I and leading to neuronal apoptosis. The experimental procedure for MPTP mice was as follows: after one week of acclimatization to the laboratory environment, mice were randomly divided into five groups: control group, MPTP group, CRO 10 mg / kg group, CRO 50 mg / kg group, and CRO 100 mg / kg group. Mice were intraperitoneally injected with 20 mg / kg MPTP twice daily for 7 days to establish a subacute PD model. After 5 days of model establishment, mice in the treatment groups were intraperitoneally injected with 10, 50, and 100 mg / kg CRO once daily for 7 consecutive days, while the MPTP group was treated with the same volume of saline. The control group received saline injections at the same frequency and volume as the other groups throughout the process. Behavioral experiments were conducted after three days of behavioral training.

[0139] Experimental Example 14

[0140] Referring to Figure 18 illustrated, Figure 18 The schematic diagram of collecting the brain electrical signals of the mouse in the striatum region. The 8-channel micro-wire array electrode was implanted into the dorsal striatum of the mouse, and the mouse was recovered for 7 days after the operation, and then the method of Example 13 was used for MPTP modeling and 50 mg / kg CRO administration. The left implantation point coordinates of the electrode were AP: +1.5 mm, ML: 1 mm, DV: 3.5 mm. The right side was symmetrically implanted. The 8-channel micro-wire array electrode collected and analyzed the data through the KEDB recording system. The 50 Hz notch filter was used to reduce the power line interference. The local field potential signal was band-pass filtered in the frequency range of 0.5-250 Hz, and the neuron discharge signal was band-pass filtered in the frequency range of 250 Hz to 7.5 kHz. Spike sorting was performed offline using an offline classifier, and then data analysis was performed using NeuroExplorer and custom MATLAB scripts.

[0141] Example 15

[0142] The behavior of the mouse can directly reflect the recovery of the mouse's neural function after CRO administration. Therefore, after the mouse was pre-trained for three days, the behavior detection was started. The open field experiment of the mouse is a classical behavior evaluation method, which is mainly used to analyze the spontaneous activity, exploration behavior and emotional state of the experimental animal in a new and unfamiliar environment. Referring to Figure 19 illustrated, Figure 19 The mouse motor behavior experiment results are shown, wherein A is the open field movement trajectory diagram of each group of mice, B is the open field movement total distance column chart of each group of mice, C is the open field activity frequency column chart of each group of mice, D is the open field activity average speed column chart of each group of mice, E is the open field wall climbing standing frequency column chart of each group of mice, F is the rotarod fall latency column chart of each group of mice, G is the rotarod fall rotation speed column chart of each group of mice, H is the time column chart of each group of mice along the pole climbing, and I is the time column chart of each group of mice crossing the balance beam.

[0143] From Figure 19 As can be seen from A, MPTP administration reduces the desire of the mouse to explore, and CRO administration can partially restore the mouse's interest in movement. From Figure 19 B- Figure 19EAs can be seen, MPTP can significantly reduce the total distance of mice and the average speed, respectively, to the normal group of 50.17% and 55.11%, while reducing the number of mice and wall climbing standing times, and 50mg / kg CRO can restore the total distance of mice to the normal group of 88.04%, the average speed to 21.27mm / s (normal group average speed of 22mm / s), and can increase the number of mice and wall climbing standing times. This means that the exploration behavior of mice is restored, and the spontaneous activity is increased. From Figure 19 Fand Figure 19 GAs can be seen, MPTP administration significantly shortens the rod latency of mice, and the corresponding rod rotation speed is low (compared with the normal group, P<0.0001), and CRO 50mg / kg and 100mg / kg administration can significantly improve the motor function of mice, prolong the rod time of mice, and enable the mice to move at a relatively high speed; Among them, 50mg / kg CRO can make the mouse rod time recover to 154.67s, and 100mg / kg CRO can make the mouse rod time recover to 157.67s. From Figure 19 Hand Figure 19 IAs can be seen in and, 100mg / kg CRO administration has the most significant effect on shortening the time of mice descending along the pole, and 50mg / kg and 100mg / kg CRO can significantly shorten the time of MPTP mice crossing the balance beam (compared with the MPTP group, P<0.001, P<0.0001, respectively).

[0144] Experimental Example 16

[0145] The brain slices of the mice in each group were subjected to Nissl staining and immunohistochemical staining in the substantia nigra and striatal regions to determine the repair effect of CRO on brain damage in mice. Referring to Figure 20 As shown, Figure 20 show the Nissl staining and TH immunohistochemical staining of mouse brain slices treated with crocin for 7 days, wherein A is a TH immunohistochemical staining and Nissl staining result graph of the striatal region of mice treated with CRO for 7 days, B is a TH positive fiber number result graph of the striatal region of mice treated with CRO for 7 days, C is a TH positive cell number result graph of the substantia nigra of mice treated with CRO for 7 days, D is a TH immunohistochemical staining and Nissl staining result graph of the substantia nigra of mice treated with CRO for 7 days, E is a Nissl body number result graph of the substantia nigra of mice treated with CRO for 7 days, and F is a Nissl body number result graph of the striatal region of mice treated with CRO for 7 days.

[0146] From Figure 20 A, Figure 20 B, Figure 20As can be seen from F, MPTP can cause the number of TH-positive fibers in the striatum region to decrease to 25.39% of the normal group, and the Nissl body shrinks, the structure is dissolved and broken, and the number is reduced, while the administration of CRO can significantly restore the decrease of the number of TH-positive fibers in the striatum region caused by MPTP, and up-regulate the number of Nissl-positive cells to close to the level of the normal group, and restore the morphology of the Nissl body. As can be seen from Figure 20 C、 Figure 20 D、 Figure 20 As can be seen from E, MPTP can also cause the number of TH-positive neurons in the substantia nigra region to decrease, causing the loss of dopaminergic neurons, which can be reversed by 50 mg / kg and 100 mg / kg CRO, wherein 50 mg / kg CRO can restore the number of TH-positive neurons to 97.40% of the normal group. At the same time, 50 mg / kg and 100 mg / kg CRO can significantly up-regulate the number of Nissl bodies (P<0.0001 compared with the MPTP group), improve the morphology of the Nissl body, and play a role in nerve repair.

[0147] Experimental Example 17

[0148] Referring to Figure 21 The, Figure 21 The treatment of crocetin on mice for 7 days is shown, wherein A is a butterfly diagram of the discharge of dopaminergic neurons of mice in each group after treatment with CRO for 7 days, B is a statistical diagram of the discharge rate of dopaminergic neurons of mice in each group after treatment with CRO for 7 days (bin=5s), and C is a diagram of the electrical signal of the β frequency band of neurons. The damage of dopaminergic neurons can cause the decrease of the discharge rate of neurons, and the treatment of CRO can restore the discharge function of neurons. Figure 21 A and Figure 21 As can be seen from A and B, the discharge rate of dopaminergic neurons of the normal group of mice is 53.86 counts / bin, while the discharge rate of dopaminergic neurons of the MPTP group of mice is significantly reduced to 38.22 counts / bin (P<0.05 compared with the control group), and 50 mg / kg CRO can increase the discharge rate of neurons to 50.95 counts / bin, and restore the discharge function of neurons. As can be seen from Figure 21 C, MPTP can increase the amplitude of the β frequency, which is consistent with the characteristics of the electrical signal of the β frequency band of neurons in the occurrence of PD, and the administration of CRO can restore the amplitude of the β frequency, thereby restoring the discharge function of neurons to a certain extent.

[0149] The above only describes the preferred embodiments of the present application and is not used to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of crocetin in the preparation of a drug for treating Parkinson's disease.

2. Use of crocetin as the only active ingredient in the preparation of a drug for treating Parkinson's disease.

3. Use according to claim 1 or 2, characterized in that: The drug utilizes the regulation of the function of mitochondria by the crocetin to improve mitochondrial dysfunction and autophagy, thereby achieving the treatment of Parkinson's disease.

4. Use according to claim 3, characterized in that: The function of mitochondria regulated by the crocetin includes mitochondrial membrane potential, ATP production, mitochondrial reactive oxygen species production, intracellular reactive oxygen species production, and ubiquitin-dependent mitochondrial autophagy pathway.

5. A method of validating crocetin for the treatment of Parkinson's disease by modulating mitochondrial function, characterized by, Using the full-automatic patch-clamp high-throughput technology to detect the effect of picroside on MPP + The effect of picroside on the membrane potassium current of the model MES23.5 cells, comprising the following steps: Step 1) MES23.5 cell culture, modeling and CRO administration: MES23.5 cells were inoculated in culture dishes, and when the culture was completed, MPP was added + The drug solution was incubated with MES23.5 cells; after incubation, the MPP was removed + The drug solution was used to wash the MES23.5 cells with PBS, and finally different concentrations of CRO were added to treat the MES23.5 cells; Step 2) cell pre-treatment before loading: discard CRO reagent, wash MES23.5 cells with PBS, then add trypsin to each dish for digestion; after digestion, add cell culture medium to terminate digestion; then centrifuge and discard the supernatant, and finally add extracellular buffer to mix the MES23.5 cells, and take the cell suspension; Step 3) cell loading: the electrode of the automatic patch clamp system applies slight negative pressure to the target cells in the cell suspension to make the electrode tip contact the cell membrane, and real-time monitoring of impedance changes; Step 4) cell sealing and membrane rupture: when the impedance reaches a set value or more, it is determined that the sealing is successful, at which time the current recorded in the electrode only reflects the activity of a single channel in the sealed area; Step 5) data analysis and collection: the system automatically applies a gradient voltage in a set range, records the current signal and performs noise filtering, and finally exports the data.

6. The method of verifying crocetin for treating Parkinson's disease by modulating mitochondrial function according to claim 5, characterized in that: The specific method of MES23.5 cell culture, modeling and CRO administration in step 1 is as follows: MES23.5 cells are inoculated in 60 culture dishes, and when the cells are fused to 70%, 2mM MPP is added + The drug solution is incubated with MES23.5 cells for 24 hours; after incubation, the MPP is removed by suction + The drug solution, after washing the MES23.5 cells with PBS three times, 0, 10, 100, 1000 μM concentration of CRO is added to treat the MES23.5 cells for 36 hours.

7. The method of verifying crocetin for treating Parkinson's disease by modulating mitochondrial function according to claim 5, wherein, In step 2, the specific method for cell pre-treatment before loading is as follows: discard CRO reagent, wash MES23.5 cells with PBS three times, add 0.2% trypsin to each dish for 1 minute of digestion; after digestion, add cell culture medium to terminate digestion; then centrifuge at 1000 rpm for 4 minutes; after centrifugation, discard the supernatant, add extracellular buffer to mix the MES23.5 cells, and take the cell suspension.

8. The method of verifying crocetin for treating Parkinson's disease by modulating mitochondrial function according to claim 5, wherein, In step 3, the specific method for cell loading is as follows: 15 μL of cell suspension is punched into the cell loading port of the automatic patch clamp system, the automatic pipetting device of the system transfers the cell suspension to a multi-well plate or a perfusion chamber, and the microscopic imaging device of the system recognizes the morphology and position of a single cell; the mechanical arm of the system controls the electrode to approach the target cell, applies slight negative pressure to make the electrode tip contact the cell membrane, and real-time monitors the impedance changes through software.

9. The method of verifying crocetin for treating Parkinson's disease by modulating mitochondrial function according to claim 5, wherein, In step 4, the specific method for cell sealing and membrane rupture is as follows: when the impedance reaches 1 GΩ or more, it is determined that the sealing is successful, at which time the current recorded in the electrode only reflects the activity of a single channel in the sealed area.

10. The method of verifying crocetin for treating Parkinson's disease by modulating mitochondrial function according to claim 5, wherein, In step 5, the specific method for data analysis and collection is as follows: the system automatically applies a gradient voltage of -80 mV to +80 mV, records the current signal and performs noise filtering, and uses Patchmaster software to export the data.

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