CN111632058A - The application of verbascoside in the preparation of drugs for preventing and treating neurodegenerative diseases - Google Patents

Abstract

The invention relates to an application of verbascoside in preparing a medicament for preventing and treating neurodegenerative diseases. Firstly, the verbascoside is found to have a repairing effect on rotenone-induced nerve cell injury; researches show that verbascoside can obviously reduce active oxygen of nerve cells, protect the integrity of mitochondria of the nerve cells, induce autophagy of the cells, reduce the accumulation of alpha-Synuclein (alpha-Syn) related to Parkinson's disease and resist apoptosis. The experimental result shows that the verbascoside plays the damage repair function on nerve cells by inducing autophagy of cells and has the activity of resisting neurodegenerative diseases. The invention provides the application prospect of the verbascoside which has exact novel medicine for preventing and treating neurodegenerative diseases.

Description

The application of verbascoside in the preparation of drugs for preventing and treating neurodegenerative diseases

technical field

The invention belongs to the field of biomedicine, in particular to the application of verbascoside in the preparation of medicines for preventing and treating neurodegenerative diseases.

Background technique

Neurodegenerative diseases are a class of diseases characterized by neuronal degeneration or apoptosis leading to abnormal behavior and even death. As the world gradually ages, the incidence of neurodegenerative diseases increases rapidly. Diagnosis of these diseases is difficult, and there is currently no effective treatment. Intracellular or extracellular accumulation of misfolded proteins is characteristic of most neurodegenerative diseases, such as the accumulation of β-amyloid (Aβ) and tau in Alzheimer's disease (AD) , α-Synuclein (α-Synuclein, α-Syn) accumulation in Parkinson's disease (Parkinson disease, PD). Autophagy dysfunction is an important hallmark of several neurodegenerative diseases in which misfolded proteins or dysfunctional mitochondria are found in pathological sections. During autophagy, protein aggregates or damaged organelles are engulfed by vesicles called autophagosomes and then fused with lysosomes for final degradation. Autophagy is an important pathway for the recycling of intracellular substances conserved in eukaryotes. In recent years, special functions of autophagy have been discovered in many tissues and organs. Neuronal autophagy disorders and abnormal aggregation of proteins and misfolding are the main pathological changes in neurodegenerative diseases. The role of autophagy in the treatment of neurodegenerative diseases has received increasing attention. Studies have shown that autophagy regulation is closely related to the clearance of amyloid in Alzheimer's disease, Lewy bodies in Parkinson's disease, and huntingtin in Huntington's disease.

Mitochondria are essential energy generators for tissue homeostasis and channels for programmed apoptosis and necrotic cell death, and their core functions require strict control of mitochondrial quality and quantity. The importance of maintaining a healthy mitochondrial population requires tight control of proteolysis and mitophagy. Mitophagy is the targeted phagocytosis and destruction of mitochondria by the autophagic apparatus, and is generally considered to be the main mechanism of mitochondrial quality control. Mitophagy is a specialized form of autophagy that regulates the turnover of damaged and dysfunctional mitochondria through lysosomal degradation. Mitochondrial dysfunction is a key factor in aging and a range of aging-related diseases, including neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease, and other pharmacological approaches to enhance mitophagy could help delay or treat Parkinson's disease and other neurodegenerative diseases.

Many natural medicines have been reported to exert positive protective effects in neurodegenerative diseases by inducing autophagy. Neurodegenerative diseases are refractory diseases with poor prognosis. So far, there is no specific medicine for this disease in Western medicine. Traditional Chinese medicine has a unique theoretical understanding of this disease, and has a good effect in delaying and preventing senile dementia. In recent years, it has become an emerging field of research and development of new drugs for the treatment of senile dementia.

Verbasin is one of the important active components of various Chinese herbal medicines. Verbasin has a variety of biological activities, such as anti-cancer, anti-virus, anti-oxidation, aphrodisiac, anti-aging, memory enhancement, hypolipidemic, anti-inflammatory and other purposes. In view of this, the present invention proposes a new application of verbascoside-application in the preparation of medicaments for preventing and treating neurodegenerative diseases.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide new uses of verbascoside in the preparation of drugs or health care products for preventing and treating neurodegenerative diseases, particularly the application of verbascoside in preparing new drugs for preventing and treating neurodegenerative diseases.

In order to achieve the above purpose, the adopted technical scheme is:

Application of verbascoside in the preparation of medicaments for preventing and treating neurodegenerative diseases.

Further, described Verbascoside, Chinese alias: ergosteroside, acteoside, cimicifloside _, English name (Acteoside or Verbascoside), molecular weight: 624.59, molecular formula C ₂₉ H ₃₆ O ₁₅ , CAS number: 61276 -17-3, the structural formula is as follows:

Further, the application of verbascoside in the preparation of mitochondrial protective agent.

Still further, the verbascoside can protect nerve cell mitochondria from damage, reduce nerve cell reactive oxygen species, and inhibit nerve cell apoptosis.

Further, the application of verbascoside in the preparation of an autophagy-inducing agent, which protects nerve cells by inducing mitochondrial autophagy.

Still further, the verbascoside is non-toxic and easily soluble in water, can obviously induce autophagy in vitro and in vivo, and reduce the accumulation of α-Syn in cells.

Further, the application of the verbascoside in the preparation of Parkinson's medicine is not limited to the prevention and treatment of Parkinson's disease.

Still further, the anti-Parkinsonian drug is a mitophagy agonist drug.

Compared with the prior art, the beneficial effects of the present invention are:

The present invention uses a variety of advanced biochemical and cellular experimental techniques to confirm that verbascoside has neuroprotective effects, and further proves that verbascoside exerts its neuroprotective function by inducing autophagy. Clinical preparation of novel, low-toxicity and high-efficiency autophagy-inducing agents and new uses in the treatment of diseases that can benefit from autophagy induction, especially the preparation of new drugs for safe treatment and prevention of neurodegenerative diseases or Application in health products. At the same time, the molecular and cellular pharmacological studies on the protective effect of verbascoside on nerve cell injury will provide theoretical support for its clinical application in the future.

Description of drawings

Figure 1 shows that verbasin has a repairing effect on PC-12 cells stimulated by rotenone;

Figure 2 shows the autophagy of NRK, PC-12 and SH-SY5Y cells induced by verbascoside;

Fig. 3 shows that verbascoside induces autophagy in fat body cells of Drosophila third instar larvae;

Figure 4 shows that verbasin has an anti-apoptotic effect on PC-12 cells stimulated by rotenone;

Figure 5 shows that Verbasin inhibits the production of reactive oxygen species in PC-12 cells stimulated by rotenone and has a protective effect on mitochondria;

Figure 6 shows the effect of verbasin on the mitochondrial membrane potential of PC-12 cells stimulated by rotenone;

Figure 7 shows that the accumulation of α-synuclein was reduced after 24 hours of action of verbascoside on HEK cells stably expressing GFP-α-Syn-GN-link-α-Syn GC (α-Syn);

Fig. 8 shows mitophagy induced by verbascoside;

Figure 9 is a diagram showing the mode of action of verbasin in protecting nerve cells.

Detailed ways

In order to further illustrate the application of the verbascoside of the present invention in the preparation of drugs for the prevention and treatment of neurodegenerative diseases, and to achieve the intended purpose of the invention, the following combined with the preferred embodiments, the preparation of the verbascoside proposed by the present invention for the prevention and treatment of neurodegenerative diseases. The application in medicine, its specific embodiment, structure, characteristics and efficacy are described in detail as follows. In the following description, different "an embodiment" or "embodiments" do not necessarily refer to the same embodiment. Furthermore, the particular features, structures or characteristics in one or more embodiments may be combined in any suitable form.

Below in conjunction with specific embodiment, the application of verbascoside of the present invention in the preparation of medicine for preventing and treating neurodegenerative diseases will be further introduced in detail:

The verbascoside of the present invention is obtained from Cistanche deserticola and Rehmannia glutinosa. According to research reports at home and abroad, Cistanche deserticola and Rehmannia glutinosa have anti-aging properties, enhance learning and memory ability, neuroprotection, improve sexual function, immune regulation, anti-fatigue, and anti-ischemia. and liver protection and other pharmacological effects. Verbasin has the characteristics of low toxicity, easy water solubility, and easy absorption by tissues.

The invention uses nerve cell injury model cells human neuroblastoma cell SH-SY5Y, rat adrenal medulla pheochromoma PC-12 cell and exogenous α-synuclein stably expressing human embryonic kidney HEK-293 cell as In an experimental cell model, the drug intervened with Verbasin to detect its effects on nerve cells to induce autophagy, protect mitochondrial integrity, reduce reactive oxygen species production, increase mitochondrial membrane potential, reduce α-Syn accumulation and ultimately resist apoptosis. Further research revealed the ameliorating effect of verbascoside on nerve cell damage caused by neurodegenerative diseases by inducing autophagy.

The present invention will use cell biology and molecular biochemical research methods to study whether verbascoside can induce autophagy to protect nerve cells, and the specific operations are as follows:

1. Morphological observation method, MTT colorimetric method and crystal violet staining method were used to detect the protective effect of verbascoside on rotenone-injured PC-12 cell model.

2. In the established NRK cells stably expressing LC3-GFP, the autophagy-inducing function of verbascoside was observed by laser confocal microscope.

3. Detect the expression level of LC3 protein in PC-12 and SH-SY5Y cells treated with verbasin by western blotting technique.

4. To test the effect of verbascoside on autophagy in the fat body of Drosophila third instar larvae: Drosophila third instar larvae (96 hours after Drosophila lay eggs) were transferred to a solution containing verbascoside, yeast powder (1.5%) and sucrose (10 %) in the culture medium or in the starvation medium with sucrose (10%) and the same amount of DMSO as the experimental group, and incubate for the specified time, then take the fat body of the third instar larvae with lysosome red fluorescent probe (LysoTracker Red) to stain and visualize lysosomes.

5. The effect of verbascoside on rotenone-induced apoptosis of PC-12 cells was detected by flow cytometry, and the expression levels of apoptosis-related proteins Bax and Caspase-3 were detected by western blotting. The autophagy inhibitor 3-MA was used in combination with drug intervention to analyze whether the anti-apoptotic effect of verbascoside on nerve cells was exerted by inducing autophagy.

6. Reactive oxygen species (ROS) detection reagent (DCFH-DA), mitochondrial red fluorescent probe (MitoTrackerRed) and mitochondrial membrane potential detection reagent (JC-1) were used to detect the effect of verbascoside on PC-12 cells induced by rotenone stimulation Changes in reactive oxygen species, mitochondrial integrity, and mitochondrial membrane potential. Drug intervention was used in combination with 3-MA to analyze whether the effects of verbascoside on reactive oxygen species and mitochondria in neurons were induced by autophagy.

7. The effect of Verbasin on the accumulation of exogenous α-synuclein in HEK293 cells was detected by laser confocal microscopy and Western blotting. ), to analyze whether the effect of verbascoside on α-synuclein is exerted by inducing autophagy.

8. NRK cells stably expressing TOM20-GFP were treated with verbasin for 4 hours, and the cells were stained with Lysotracker Red, a lysosomal red fluorescent probe, and the function of inducing mitophagy on NRK cells was observed by laser confocal. . In addition, after the action of Verbasin (50μM) on SH-SY5Y cells for 24h, mitochondria and cytoplasm were extracted to detect the changes of p62, LC3 and PINK1 protein expression levels in mitochondria, respectively.

The protective effect of the nerve cells described in the present invention is helpful for the prevention and treatment of neurodegenerative diseases such as Parkinson's disease. Mitochondria are the "power centers" of cells. Impaired mitochondrial function may lead to a decrease in cellular energy levels. At the same time, mitochondrial membrane permeability changes, membrane potential decreases, and electron leakage promotes the formation of reactive oxygen species (ROS), thereby damaging proteins and membrane lipids. and nucleic acids, ultimately causing cell death. Mitochondria play an important role in neurons, and mitochondrial maintenance directly affects neuronal development, function, and survival. Maintaining a healthy mitochondrial population is critical to neuronal health, so there are many mitochondrial quality control pathways such as misfolded protein degradation, fission and fusion, and mitophagy.

Verbasin can significantly increase the expression level of LC3 protein in nerve cells by up-regulating the level of autophagy, which is beneficial for the preparation of novel autophagy inducers and for the treatment of diseases that can benefit from autophagy induction, especially neurodegenerative diseases.

details as follows:

Example 1: Verbasin protects nerve cells

1. Cell culture and drug preparation

Rat adrenal pheochromocytoma cell PC-12 cell line (purchased from the Chinese Academy of Sciences Cell Bank) was cultured in DMEM medium containing 10% fetal bovine serum and 5% horse serum, and 1% penicillin/streptomycin at 37 °C in a 5% CO ₂ incubator. The powders of the standard products of verbascoside and rotenone were taken respectively, dissolved in DMSO, and finally prepared into mother solutions of 0.05Mol/L and 0.01Mol/L.

2. Experimental grouping

The PC-12 cells were divided into 9 groups, namely blank group, verbascoside (6 different doses) group, model (rotenone) group and model drug intervention group, 5 replicate wells were set for each group and incubated for the specified time.

3. Cell morphology observation

Use an inverted microscope to observe the morphological changes of each group of cells.

4. MTT detection

1) Add 200 μL/well of MTT solution (0.5 g/L), and continue to incubate at 37°C for 4 h.

2) Discard the supernatant, add 150 μL/well of DMSO, and shake on a constant temperature shaker for 10 min.

3) Detect the absorbance value of each well at 490nm with a microplate reader.

5. Cellular crystal violet staining

1) Discard the supernatant, wash with PBS once, and add 4% paraformaldehyde to fix the cells for 10 min.

2) Discard the supernatant, wash 2-3 times with PBS, add 0.05% crystal violet and incubate at room temperature for 10 min.

3) Discard the supernatant, wash 2-3 times with PBS, dry naturally at room temperature, and count the cell mass.

6. Statistical processing

All the obtained data results are expressed as mean ± standard deviation, using GraphPad Prism 5 software to perform one-way ANOVA, and Tukey test for comparison between groups to analyze significance, p<0.05 is the significance level, p <0.01 is the very significant level.

7. Experimental results

Figure 1 shows that verbascoside has a repairing effect on PC-12 cells stimulated by rotenone. (A) The morphological changes of PC-12 cells were observed after 24h of verbasin in the concentration range of 50-200 μM, and it was found that verbasin had no toxic effect on the cells within the concentration of 200 μM, and 50 μM verbasin had no toxic effect on PC-12 cells treated with 0.2 μM rotenone. -12 cells have a certain damage repair effect. (B) After 50 μM verbasin was treated with 0.2 μM rotenone-treated PC-12 cells for 24, 48 and 72 h, the cell viability was detected by MTT colorimetry. It was more significant with prolonged action time, (*p<0.05, **p<0.01, ***p<0.001, one-way ANOVA). (C) After 50 μM verbasin was treated with 0.2 μM rotenone-treated PC-12 cells for 96 h, crystal violet was used to detect the color intensity of living cells. The results further proved that verbascoside has obvious damage repair effect on rotenone-treated PC-12 cells. .

As shown in Figure 1, compared with the blank group, verbascoside was not toxic to PC-12 cells in the concentration range of 0-200 μM, and 50 μM verbascoside treated PC-12 cells stimulated with 0.2 μM rotenone for 24 h, and obvious damage repair could be observed. The results of MTT experiment showed that 50μM verbasin had obvious damage repair effect on PC-12 cells stimulated by 0.2μM rotenone after 24, 48 and 72 hours, and the longer the effect time, the more obvious the effect; the results of crystal violet staining experiment were more obvious. It was further proved that 50 μM verbasin had a protective effect on PC-12 cells stimulated by 0.2 μM rotenone, and was safe and non-toxic.

Example 2: Verbasin induces autophagy

1. Cell culture steps are the same as in Example 1

2. Experimental grouping

The NRK cells, PC-12 and SH-SY5Y cells stably expressing GFP-LC3 were divided into 5 groups: blank group, rapamycin (100nM concentration) group and verbasin (3 different doses) groups, respectively. Groups are set up in 3-5 replicates and incubated for the indicated times.

3. Laser confocal microscope observation

The cells were cultured in glass-bottom dishes and drug intervention was used, and the intracellular fluorescent sites of GFP-LC3 were observed and counted by laser confocal microscope.

4. Western blot assay to detect LC3 protein expression

Western blot was used to detect the expression of LC3Ⅰ and Ⅱ proteins. Protein Simple chemiluminescence gel analysis system was used to analyze and map the protein bands. The gray value (IOD) of each band was analyzed and obtained. The expression level of the target protein was determined according to the target strip. It is reflected by the ratio of the gray value of the band to the gray value of the internal reference band.

5. Statistical processing steps are the same as in Example 1

6. Experimental results

Figure 2 shows the effect of verbascoside on NRK cells to induce autophagy. (A) NRK cells stably expressing GFP-LC3 were intervened with 25, 50 and 100 μM concentrations of verbascoside and 100 nM rapamycin for 12 h, and the occurrence of autophagy-lysosomes was observed by confocal laser microscopy. (B) The number of autophagy-lysosomes was counted and analyzed statistically, (*p<0.05, **p<0.01, ***p<0.001, one-way ANOVA). (C) The effect of verbasin on the expression level of LC3 protein in PC-12 and SH-SY5Y cells for 24 h.

As shown in Figure 2, obvious GFP-LC3 aggregation points could be observed after 12 h of action on NRK cells stably expressing GFP-LC3 in each dose group of verbascoside. Statistical analysis of the number of GFP-LC3 aggregation points showed that each There were significant differences between the dose group and the blank group. Verbasin significantly increased the expression level of LC3Ⅱ protein after 24 hours of action on PC-12 and SH-SY5Y cells.

Example 3: Verbasin activates lysosomal activity in the fat body tissue of Drosophila third instar larvae

1. Feeding fruit flies

Drosophila were reared in Drosophila-specific corn medium at 25°C and 75% humidity.

2. Obtain Drosophila third instar larvae and group them

96 hours after Drosophila lay eggs, the third instar larvae were transferred to the blank group (containing 1.5% yeast powder, 20% sucrose and the same amount of DMSO) culture medium, the experimental group (containing different doses of verbascoside, 1.5% yeast powder and 20 % sucrose) and starvation groups (containing 20% sucrose and an equal amount of DMSO) and incubated for the indicated times, transferring 3-5 third instar larvae for each group.

3. Detection of fat body lysosome activity in Drosophila third instar larvae

The fat body tissue was isolated using a stereomicroscope and stained with LysoTracker Red and Hoechst33342, and the lysosomes were observed and counted under a laser confocal microscope.

4. Statistical processing steps are the same as in Example 1

5. Experimental results

Figure 3 shows the induction of autophagy in fat body cells of Drosophila third instar larvae by verbascoside. (A) After 96 hours of Drosophila egg laying, the third instar larvae were collected and incubated in Drosophila larvae medium containing 100 μM and 500 μM verbascoside for 6 h, respectively. The third instar larvae were starved with starvation medium for 4 h as a positive control group. The fat body tissue was isolated and stained under the stereo microscope, and observed with a laser confocal microscope, the scale bar was 25 μm. (B) Statistical analysis was performed on the fluorescence intensity of each group, and statistical analysis was performed on the data of three independent replicate experiments, (*p<0.05, **p<0.01, ***p<0.001, one-way ANOVA).

The results are shown in Figure 3. Both 100μM and 500μM Verbasin can activate the lysosome activity of Drosophila third instar larvae, and there is a very significant difference compared with the control group; Compared with the 100 μM verbascoside group, the fluorescence intensity of lysosomes was stronger, but the number of lysosomal fluorescence sites was not significantly different between the two dose groups, and the fluorescence intensity and number of lysosomal sites in the starvation group were higher.

Example 4: Verbasin anti-apoptosis

1. Cell culture and drug configuration steps are the same as in Example 1

2. Experimental grouping

The PC-12 and SH-SY5Y cells were divided into 5 groups: blank group, verbascoside (50 μM) group, model (0.2 μM rotenone) group, model drug intervention group and model drug intervention combined with 3-MA (5mM) group, respectively. Set up 3-5 replicates for each group and incubate for the indicated times.

3. Detection of apoptosis

The cells were treated with BD's 7-ADD apoptosis detection kit, and the apoptosis rate of each group of cells was detected by BD's flow cytometer.

4. The steps of detecting Bax and Caspase-3 protein expression by immunoblotting experiment are the same as those in Example 2

5. Statistical processing steps are the same as in Example 1

6. Experimental results

Figure 4 shows that verbasin has an anti-apoptotic effect on PC-12 cells stimulated by rotenone. (A) The results of flow cytometry experiments showed that the effect of verbascoside (50 μM) on the apoptosis of PC-12 cells treated with rotenone (0.2 μM) for 24 h, combined with the autophagy inhibitor 3-MA (5 mM) The anti-apoptotic function of verbascoside was significantly inhibited after intervening cells. (B) Statistical analysis was performed on the occurrence rate of apoptosis in each group, and the data of three independent repeated experiments were analyzed, (*p<0.05, **p<0.01, ***p<0.001, one-way ANOVA). (C) The effect of verbascoside (50 μM) on the expression levels of Bax and Caspase-3 proteins in PC-12 and SH-SY5Y cells for 24 h, and the effect of 3-MA (5 mM) blocking the effect of verbascoside on the expression levels of apoptosis-related proteins .

The results are shown in Figure 4. Compared with the blank control group, PC-12 cells did not undergo rapid apoptosis after 24 hours of intervention with 50 μM verbascoside, while 0.2 μM rotenone significantly accelerated the apoptosis of PC-12 cells after 24 hours of treatment. In the model group, adding verbascoside to the cells for 24 hours reduced the incidence of apoptosis, and adding 5 mM 3-MA to the drug intervention group significantly prevented the anti-apoptotic effect of verbascoside. and exert its anti-apoptotic effect. The results of western blotting showed that 50μM verbasin treated PC-12 and SH-SY5Y cells for 24 hours, and the expression levels of apoptosis-related proteins Bax and Cleaved-caspase-3 were rapidly advanced in PC-12 and SH-SY5Y cells, and 3-MA inhibited their anti-apoptotic function.

Example 5: Verbasin against reactive oxygen species and protection of mitochondria

1. Cell culture and drug configuration steps are the same as in Example 1

2. Experimental grouping

The PC-12 cells were divided into 6 groups, respectively blank group, verbascoside (50μM) group, model (0.2μM rotenone) group, model drug intervention group, model drug intervention combined with 3-MA (5mM) group, 3-MA group , and set 3-5 replicates for each group and incubate for the indicated times.

3. Observe reactive oxygen species and mitochondria

Glass-bottom culture dishes were used for cell culture and fluorescence observation, and stained with reactive oxygen species detection probe DCFH-DA, mitochondrial activity detection probe MitoTracker-Red and Hoechst 33342, and laser confocal microscope was used to observe reactive oxygen species and mitochondria.

4. Experimental results

Figure 5 shows the effect of verbascoside on the reactive oxygen species and mitochondria of PC-12 cells stimulated by rotenone. The cells were stained and observed with DCFH-DA and MitoTracker Red, respectively. The PC-12 cells have the functions of reducing reactive oxygen species and maintaining mitochondrial integrity, and 3-MA (5mM) blocks the normal function of verbascoside.

The results are shown in Figure 5. Compared with the blank control group, 50 μM verbasin did not generate fast-moving reactive oxygen species and the mitochondria remained stable after 24 hours of intervention on PC-12 cells; The content of reactive oxygen species and mitochondria were fragmented and accumulated in the cells and surrounding areas; the model group was treated with verbascoside to reduce the incidence of reactive oxygen species release and repair mitochondrial damage after 24 hours of treatment; the drug intervention group added 5mM 3-MA significantly Blocking the anti-ROS and mitochondrial protective effects of verbascoside, the effect of 5mM 3-MA on PC-12 cells alone for 24h was not obvious. The results indicated that verbasin exerted its anti-ROS and mitochondrial protection functions by inducing autophagy.

Example 6: Verbasin protects mitochondrial membrane integrity and maintains mitochondrial membrane potential

1. Cell culture and drug configuration steps are the same as in Example 1

2. The experimental grouping steps are the same as those in Example 5

3. Detection of mitochondrial membrane potential

Cell culture and fluorescence observation glass-bottomed dishes were stained with the mitochondrial membrane potential detection probe JC-1 and Hoechst 33342, and observed by laser confocal microscope. The higher ratio of red fluorescence intensity to blue fluorescence intensity indicates mitochondrial membrane potential Higher, mitochondria are normal.

4. Statistical processing steps are the same as in Example 1

5. Experimental results

Figure 6 shows the effect of verbascoside on the mitochondrial membrane potential of rotenone-stimulated PC-12 cells. (A) JC-1 was used to stain and observe the cells. -12 cells have the function of increasing mitochondrial membrane potential, and 3-MA (5mM) blocks the normal function of verbascoside. (B) Statistical analysis of the fluorescence intensity of each group, data from three independent replicate experiments, (*p<0.05, **p<0.01, ***p<0.001, one-way ANOVA).

The results are shown in Figure 6. Compared with the blank control group, the effect of 50 μM verbascoside on PC-12 cells was not obvious after 24 h of intervention, and the mitochondria remained stable; ;In the model group, the mitochondrial membrane potential was significantly increased after 24 hours of treatment with verbascoside; the addition of 5mM 3-MA to the drug intervention group significantly blocked the function of verbascoside to increase the mitochondrial membrane potential. The effect of mitochondrial membrane potential in -12 cells was not obvious after 24h of single treatment. The results suggest that verbasin exerts its function of maintaining mitochondrial membrane potential by inducing autophagy.

Example 7: Verbasin reduces the accumulation of alpha-synuclein

1. Cell culture and drug configuration steps are the same as in Example 1

2. Experimental grouping

HEK cells stably expressing GFP-a-Syn-GN-link-a-Syn GC (α-Syn) were divided into 4 groups: blank group, verbascoside (50μM) group, rapamycin (100nM) group, Verbasin combined with 3-MA (5 mM) groups and set 3-5 replicates for each group and incubated for the indicated times.

3. Observation of α-Syn and lysosomal activity

Cell culture and fluorescence observation in glass-bottom culture dishes, and stained with LysoTracker Red and Hoechst33342, and observed by laser confocal microscope, the more red fluorescence sites indicate the more active autophagy, the more and stronger green fluorescence sites indicate α -Syn piles up more.

4. Western blotting experiments to detect the expression of α-Syn and LC3 proteins are the same as in Example 2

5. Statistical processing steps are the same as in Example 1

6. Experimental results

Figure 7 shows that Verbasin reduces the accumulation of α-synuclein after 24 hours of action on HEK cells stably expressing α-Syn. (A) The effect of verbascoside (50 μM) on α-synuclein stably expressed in HEK cells for 24 h was observed, and the scale bar was 10 μm. The inducer rapamycin (100nM) has a more obvious function of reducing the accumulation of α-synuclein than Verbasin, and the function of Verbasin in reducing the accumulation of α-synuclein is obvious after intervening cells with 3-MA (5mM) suppressed. (B) The effect of verbascoside (50 μM) on the expression levels of α-Syn and LC3 proteins in HEK cells stably expressing α-Syn for 24 h, and 3-MA (5 mM) blocked the normal function of verbascoside.

The results are shown in Figure 7. Compared with the blank control group, 50 μM Verbasicose significantly reduced the accumulation and expression of α-Syn and increased the expression level of LC3II after 24 hours of intervention on HEK cells stably expressing α-Syn; 100 nM rapamycin After 24 hours of intervention on HEK cells with stable expression of α-Syn, the effect on the accumulation and expression of α-Syn was not obvious, but the expression level of LC3Ⅱ was increased. The accumulation and expression of α-Syn were significantly increased, and the ratio of LC3Ⅱ/Ⅰ was decreased. The results showed that verbascoside reduced the accumulation and expression levels of α-Syn by inducing autophagy.

Example 8: Verbasin induces mitophagy

1. Cell culture and drug configuration steps are the same as in Example 1

2. Experimental grouping

The NRK cells stably expressing GFP-TOM20 were divided into 2 groups, the blank group and the verbascoside (50 μM) group, respectively, and 3-5 replicates were set for each group and incubated for the indicated times.

3. Observe the localization of TOM20-GFP and lysosome

Glass bottom dishes were used for cell culture and fluorescence observation, and LysoTracker Red staining was performed on NRK cells stably expressing TOM20-GFP. Confocal laser observation showed that the more yellow sites co-localized with green fluorescent sites and red fluorescent sites, the more active mitophagy was.

4. For cells cultured in a 10cm cell culture dish with different treatments, the drug preparation steps were the same as those in Example 1, and mitochondria were extracted separately using Solebao (Beijing) mitochondrial extraction kit.

5. Western blot experiment to detect cytoplasmic protein components and mitochondrial protein components p62, PINK1 and LC3 protein expression steps are the same as Example 2

6. Experimental results

Figure 8 shows the induction of mitophagy by verbasin. (A) NRK cells stably expressing TOM20-GFP were treated with verbascoside (50 μM) for 4 h. LysoTracker Red was used to stain and observe the cells. The scale bar was 10 μm. The results showed that verbascoside (50 μM) could induce mitophagy in NRK cells. function. (B) The mitochondria and cytoplasm were extracted from SH-SY5Y cells for 24 h after the action of verbascoside (50 μM), and the changes of p62 and LC3 protein expression levels were detected, respectively. The level of mitophagy was increased, suggesting that it exerted its repairing effect on SH-SY5Y cells treated with rotenone (0.2 μM) by inducing mitophagy.

The results are shown in Fig. 8-A. Compared with the blank control group, 50 μM verbasin significantly increased the concentration of green fluorescent TOM 20-GFP and red fluorescent sites localized in mitochondria after 4h intervention on NRK cells stably expressing TOM 20-GFP. Probability of colocalization of lysosomes, the results showed that verbasin significantly induced mitophagy. The results are shown in Figure 8-B, compared with the blank control group, 50 μM verbasin on untreated SH-SY5Y cells and 0.2 μM rotenone-treated SH-SY5Y cells significantly increased the mitochondrial localization of p62, PINK1 and SH-SY5Y cells. Expression levels of LC3 protein. The results showed that verbasin exerted its function of protecting nerve cells by inducing mitophagy.

The protective effect of the nerve cells of the present invention is helpful for delaying or treating neurodegenerative diseases such as Parkinson's disease (as shown in FIG. 9 ). Mitochondria are the "power centers" of cells. Impaired mitochondrial function may lead to a decrease in cellular energy levels. At the same time, mitochondrial membrane permeability changes, membrane potential decreases, and electron leakage promotes the formation of reactive oxygen species (ROS), thereby damaging proteins and membrane lipids. and nucleic acids, ultimately causing cell death. Mitochondria play an important role in neurons, and mitochondrial maintenance directly affects neuronal development, function, and survival. Maintaining a healthy mitochondrial population is critical to neuronal health, so there are many mitochondrial quality control pathways such as misfolded protein degradation, fission and fusion, and mitophagy.

Verbasin can significantly increase the expression level of LC3 protein in nerve cells by up-regulating the level of autophagy, which is beneficial for the preparation of novel autophagy inducers and for the treatment of diseases that can benefit from autophagy induction, especially neurodegenerative diseases.

The above descriptions are only preferred embodiments of the embodiments of the present invention, and are not intended to limit the embodiments of the present invention in any form. Any simple modifications, equivalent changes, and Modifications still fall within the scope of the technical solutions of the embodiments of the present invention.

Claims (8)

1. The application of verbascoside in the preparation of medicaments for preventing and treating neurodegenerative diseases. 2. application according to claim 1, is characterized in that, described verbascoside, Chinese alias: ergosteroside, acetoside, phylloside, molecular weight: 624.59, molecular formula C ₂₉ H ₃₆ O ₁₅ , CAS number: 61276-17-3, the structural formula is as follows:

3. The application of verbascoside in the preparation of mitochondrial protective agent. 4 . The application according to claim 3 , wherein the verbascoside can protect nerve cell mitochondrial damage, reduce nerve cell reactive oxygen species, and inhibit nerve cell apoptosis. 5 . 5. The application of verbascoside in the preparation of an autophagy-inducing agent, wherein the method protects nerve cells by inducing mitochondrial autophagy. 6 . The application according to claim 5 , wherein the verbascoside induces autophagy and reduces the accumulation of α-Syn in cells. 7 . 7. application according to claim 1, is characterized in that, the application of described verbascoside in the preparation of Parkinson's medicine. The application according to claim 7, wherein the anti-Parkinsonian drug is a mitophagy agonist drug.

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