CN111632058A - Application of verbascoside in preparing medicament for preventing and treating neurodegenerative diseases - Google Patents
Application of verbascoside in preparing medicament for preventing and treating neurodegenerative diseases Download PDFInfo
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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
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to application of verbascoside in preparation of medicines for preventing and treating neurodegenerative diseases.
Background
Neurodegenerative diseases are diseases which are mainly characterized by abnormal behavior and death of individuals caused by neuronal degeneration or apoptosis, and the incidence of the neurodegenerative diseases is increased rapidly with the gradual global aging. However, the diagnosis of the diseases is difficult, and no effective treatment measures are available at present. Intracellular or extracellular accumulation of misfolded proteins is a feature of most neurodegenerative diseases, such as the accumulation of amyloid β (Α β) and tau au in Alzheimer Disease (AD), and the accumulation of α -Synuclein (α -Syn) in Parkinson Disease (PD). Autophagy dysfunction is an important marker for 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 to lysosomes and finally degraded by them. Autophagy is a conserved important pathway in eukaryotes for the recycling of intracellular material. In recent years, a particular function of autophagy has been found in many tissues and organs. Neuronal autophagy disorders and abnormal aggregation of proteins and misfolding are major pathological changes in neurodegenerative diseases. The role of autophagy in the treatment of neurodegenerative diseases is becoming increasingly important. Autophagy modulation has been shown to be closely related to amyloid clearance in alzheimer's disease, lewy bodies in parkinson's disease and huntingtin clearance in huntington's disease.
Mitochondria are the indispensable energy generator for tissue homeostasis, and are also the channel for apoptosis and necrotic cell death, and their core function is such that the quality and quantity of mitochondria need to be strictly controlled. It is important to maintain a healthy population of mitochondria, which requires tight control of proteolysis and mitophagy. Mitophagy (Mitophagy) is the targeted phagocytosis and destruction of mitochondria by autophagy devices and is generally considered to be the primary mechanism of quality control of mitochondria. Mitophagy is a special form of autophagy that regulates the turnover of damaged and dysfunctional mitochondria through lysosomal degradation. Mitochondrial dysfunction is a key factor in causing 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 enhancing mitochondrial autophagy are helpful in delaying or treating neurodegenerative diseases such as parkinson.
Many natural drugs have been reported to play a positive protective role in neurodegenerative diseases by inducing autophagy. Neurodegenerative diseases belong to refractory diseases, prognosis is poor, western medicine has no specific medicine for the diseases so far, traditional Chinese medicine has unique theoretical understanding on the diseases, has good curative effect on delaying and preventing senile dementia, and becomes a new field for developing new medicines for treating senile dementia in recent years.
Verbascoside is one of the important active ingredients of many Chinese herbal medicines. Acteoside has multiple biological activities, such as anticancer, antivirus, antioxidant, yang supporting, antiaging, memory improving, blood lipid reducing, and antiinflammatory. In view of the above, the invention provides a new application of verbascoside in preparing medicines for preventing and treating neurodegenerative diseases.
Disclosure of Invention
The invention aims to provide a new application of verbascoside in preparing a medicine or a health-care product for preventing and treating neurodegenerative diseases, and particularly relates to an application of verbascoside in preparing a novel medicine for preventing and treating neurodegenerative diseases.
In order to realize the purpose, the adopted technical scheme is as follows:
application of verbascoside in preparing medicine for preventing and treating neurodegenerative diseases is provided.
Further, the acteoside, ChineseAlias: acteoside, acteoside，English name (Acteoside or Verbascoside), molecular weight: 624.59 formula C29H36O15CAS number: 61276-17-3, the structural formula is as follows:
further, application of verbascoside in preparing mitochondria protective agent is provided.
Furthermore, the verbascoside can protect the mitochondria of nerve cells from being damaged, reduce the active oxygen of the nerve cells and inhibit the nerve cells from apoptosis.
Further, use of verbascoside in the preparation of an autophagy-inducing agent for protecting nerve cells by inducing mitophagy in the cells.
Furthermore, the verbascoside is non-toxic and easily soluble in water, can obviously induce autophagy of cells in vivo and in vitro, and reduces the accumulation of alpha-Syn in the cells.
Further, the verbascoside is applied to the preparation of Parkinson medicines, but is not limited to the prevention and treatment of Parkinson diseases.
Still further, the anti-Parkinson's drug is a mitophagy agonist drug.
Compared with the prior art, the invention has the beneficial effects that:
the invention utilizes a plurality of advanced biochemical and cell experimental techniques to confirm that the verbascoside has the nerve cell protection function, and further proves that the verbascoside plays the nerve protection function by inducing autophagy. The application of the verbascoside in preparing novel, low-toxicity and high-efficiency autophagy inducer clinically and treating diseases benefiting from autophagy induction, in particular to the application of the verbascoside in preparing novel medicaments or health care products for safely treating and preventing neurodegenerative diseases. Meanwhile, the molecular and cytopharmacological research of the protective effect of verbascoside on nerve cell injury provides theoretical support for the clinical application of verbascoside in the future.
Drawings
FIG. 1 shows that verbascoside has an injury repairing effect on rotenone-stimulated PC-12 cells;
FIG. 2 shows that verbascoside induces autophagy in NRK, PC-12 and SH-SY5Y cells;
FIG. 3 shows that verbascoside induces autophagy in fat body cells of Drosophila trilliota larvae;
FIG. 4 shows that verbascoside has anti-apoptotic effect on rotenone-stimulated PC-12 cells;
FIG. 5 shows that verbascoside inhibits the rotenone-stimulated production of reactive oxygen species in PC-12 cells and protects mitochondria;
FIG. 6 is a graph of the effect of verbascoside on rotenone-stimulated mitochondrial membrane potential in PC-12 cells;
FIG. 7 shows that verbascoside decreases alpha-synuclein accumulation after 24h of action on GFP-alpha-Syn-GN-link-alpha-Syn GC (alpha-Syn) -stably expressed HEK cells;
FIG. 8 shows that verbascoside induces mitophagy;
FIG. 9 is a graph showing the effect of verbascoside in protecting nerve cells.
Detailed Description
In order to further illustrate the application of verbascoside in the preparation of drugs for preventing and treating neurodegenerative diseases, and to achieve the intended purpose of the invention, the following detailed description of the specific implementation, structure, characteristics and efficacy of verbascoside provided by the present invention is provided with reference to the preferred embodiments. In the following description, different "one embodiment" or "an embodiment" refers to not necessarily the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
The following will be described in detail with reference to specific examples for the use of verbascoside of the present invention in the preparation of a medicament for the prevention and treatment of neurodegenerative diseases:
the verbascoside is taken from cistanche and radix rehmanniae, and according to the research reports at home and abroad, the cistanche and the radix rehmanniae have the pharmacological effects of resisting aging, enhancing the learning and memory capacity, protecting nerves, improving sexual function, regulating immunity, resisting fatigue, resisting ischemia, protecting liver and the like. Acteoside has low toxicity, good water solubility, and easy absorption.
The invention takes nerve cell injury model cells human neuroblastoma cell SH-SY5Y, rat adrenal medulla chromaffin tumor PC-12 cells and exogenous alpha-synuclein stable expression human embryonic kidney HEK-293 cells as experimental cell models, uses verbascoside to carry out drug intervention, detects the injury repair function of verbascoside on nerve cell induced autophagy, mitochondrial integrity protection, active oxygen generation reduction, mitochondrial membrane potential improvement and alpha-Syn accumulation final anti-apoptosis, and further researches reveal that the verbascoside has the improvement function on nerve cell injury caused by neurodegenerative diseases through autophagy induction.
The invention researches whether verbascoside can induce autophagy of cells to protect nerve cells by using cytobiology and molecular biochemistry research means, and the specific operation is as follows:
1. the protection effect of verbascoside on the PC-12 cell model damaged by rotenone is detected by applying a morphological observation method, an MTT colorimetric method and a crystal violet staining method respectively.
2. In established LC3-GFP stably expressed NRK cells, the function of verbascoside for inducing autophagy of cells is observed by using a laser confocal microscope.
3. The expression level of LC3 protein was examined by immunoblotting technique after treating PC-12 and SH-SY5Y cells with verbascoside.
4. Detecting the influence of verbascoside on autophagy of fat bodies of fruit fly third-instar larvae: the drosophila trilorks (after 96 hours of oviposition of drosophila) were transferred to a culture broth containing verbascoside, yeast powder (1.5%) and sucrose (10%) or starvation culture broth with sucrose (10%) and DMSO equivalent to the experimental group, respectively, and incubated for a designated time, after which the liposomes of the trilorks were stained with a lysosome Red fluorescent probe (LysoTracker Red) and observed.
5. Detecting the influence of verbascoside on PC-12 apoptosis induced by rotenone stimulation by flow cytometry, and detecting the expression levels of apoptosis-related proteins Bax and Caspase-3 by immunoblotting technology. Drug intervention was performed simultaneously with autophagy inhibitor 3-MA, and whether the anti-apoptotic effect of verbascoside on nerve cells was exerted by inducing autophagy was analyzed.
6. Active oxygen, mitochondrial integrity and mitochondrial membrane potential changes after the acteoside acts on PC-12 cells induced by rotenone stimulation are detected by using a Reactive Oxygen Species (ROS) detection reagent (DCFH-DA), a mitochondrial red fluorescent probe (MitoTracker Red) and a mitochondrial membrane potential detection reagent (JC-1) respectively. Drug intervention was performed simultaneously with 3-MA to analyze whether the effect of verbascoside on neuronal reactive oxygen species and mitochondria was exerted by inducing autophagy.
7. The influence of verbascoside on the accumulation of exogenous alpha-synuclein in HEK293 cells is detected by using a laser confocal microscope and a protein immunoblotting technology, and the influence of the verbascoside on the alpha-synuclein is analyzed by using 3-MA and an autophagy inducer Rapamycin (Rapamycin) during drug intervention and by using autophagy induction.
8. After the acteoside acts on NRK cells stably expressed by TOM20-GFP for 4h, the cells are stained by a lysosome Red fluorescent probe Lysotracker Red, and the function of the acteoside on the NRK cells for inducing mitochondrion autophagy is observed by laser confocal observation. In addition, after 24h of action of verbascoside (50. mu.M) on SH-SY5Y cells, mitochondria and cytoplasm were extracted and changes in mitochondrial expression levels of p62, LC3 and PINK1 proteins were examined, respectively.
The protective effect of the nerve cells is beneficial to preventing and treating neurodegenerative diseases such as Parkinson and the like. Mitochondria are the "motor center" of cells, and impaired mitochondrial function may lead to a decrease in cellular energy levels, as mitochondrial membrane permeability changes, a decrease in membrane potential, and leakage of electrons promotes the formation of Reactive Oxygen Species (ROS), thereby destroying proteins, membrane lipids, and nucleic acids, ultimately leading to cell death. Mitochondria play an important role in neurons, and maintenance of mitochondria directly affects development, function, and survival of neurons. Maintaining a healthy population of mitochondria is critical to neuronal health, and thus there are many mitochondrial quality control pathways such as misfolded protein degradation, fission, and fusion, and mitochondrial autophagy.
The verbascoside can obviously improve the expression level of LC3 protein in nerve cells by up-regulating autophagy level, and is favorable for preparing novel autophagy inducers and treating diseases which can benefit from autophagy inducement, in particular neurodegenerative diseases.
The method comprises the following specific steps:
example 1: acteoside for protecting nerve cells
1. Cell culture and drug formulation
Rat adrenal pheochromocytoma cell PC-12 cell line (purchased from cell bank of Chinese academy of sciences) cultured in DMEM medium containing 10% fetal bovine serum and 5% horse serum, and 1% penicillin/streptomycin at 37 deg.C and 5% CO2Cultured in an incubator. Respectively taking the powders of verbascoside and rotenone standard substance, dissolving with DMSO, and finally preparing into mother liquor of 0.05Mol/L and 0.01 Mol/L.
2. Experiment grouping
The PC-12 cells were divided into 9 groups, a blank group, a verbascoside (6 different doses), a model (rotenone) group and a model drug intervention group, each group was set with 5 replicate wells and incubated for the indicated time.
3. Morphological observation of cells
Morphological changes of each group of cells were observed using an inverted microscope.
4. MTT assay
1) MTT solution (0.5g/L) was added at 200. mu.L/well and incubation was continued at 37 ℃ for 4 h.
2) The supernatant was discarded, DMSO was added at 150. mu.L/well, and shaken on a constant temperature shaker for 10 min.
3) And detecting the absorbance value of each hole at 490nm of the microplate reader.
5. Cell crystal violet staining
1) The supernatant was discarded, washed 1 time with PBS, and cells were fixed with 4% paraformaldehyde for 10 min.
2) The supernatant was discarded, washed with PBS 2-3 times, added with 0.05% crystal violet and incubated at room temperature for 10 min.
3) The supernatant was discarded, washed 2-3 times with PBS, naturally dried at room temperature, and the cell pellet was counted.
6. Statistical treatment
All data results were expressed as mean ± standard deviation, and one-way anova was performed on each set of data using GraphPad Prism 5 software, and significance was analyzed by Tukey test for comparison between groups, with p <0.05 being the significance level and p <0.01 being the extreme significance level.
7. Results of the experiment
FIG. 1 shows that verbascoside has an injury repairing effect on rotenone-stimulated PC-12 cells. (A) The morphological change of cells is observed after the verbascoside acts on PC-12 cells within the concentration range of 50-200 mu M for 24h, the verbascoside is found to have no toxic effect on the cells within the concentration range of 200 mu M, and 50 mu M of the verbascoside has certain injury repairing effect on the PC-12 cells treated by 0.2 mu M of rotenone. (B) After 50 μ M verbascoside was exposed to 0.2 μ M rotenone-treated PC-12 cells for 24, 48 and 72h, respectively, MTT colorimetry was used to detect cell activity, and found that verbascoside had damage-repairing effects on PC-12 cells, and that the repairing effects were more pronounced with increasing exposure time (p <0.05, > p <0.01, > p <0.001, single-factor ANOVA). (C) After 50 mu M verbascoside acts on 0.2 mu M rotenone-treated PC-12 cells for 96h, crystal violet is used for detecting the color development intensity of living cells, and the result further proves that the verbascoside has obvious damage repair effect on the rotenone-treated PC-12 cells.
As shown in figure 1, compared with blank group, verbascoside has no toxicity to PC-12 cells in the concentration range of 0-200 μ M, and obvious damage repair phenomenon can be observed when 50 μ M verbascoside acts on 0.2 μ M rotenone-stimulated PC-12 cells for 24 h; MTT experimental results show that 50 mu M verbascoside has obvious damage repair effect after 24, 48 and 72 hours of action on 0.2 mu M rotenone stimulated PC-12 cells, and the effect is more obvious when the action time is longer; the results of crystal violet staining experiments further prove that 50 mu M verbascoside has protective effect on PC-12 cells stimulated by 0.2 mu M rotenone, and is safe and nontoxic.
Example 2: verbascoside-induced autophagy of cells
1. Cell culture procedure as in example 1
2. Experiment grouping
The stably expressed NRK cells, PC-12 and SH-SY5Y cells of GFP-LC3 were divided into 5 groups, a blank group, a rapamycin (100nM concentration) group and a verbascoside (3 different doses) group, 3-5 replicates were set for each group and incubated for the indicated time.
3. Confocal laser microscopy
Cells were cultured using glass-bottomed dishes and drug intervention, and sites of fluorescent GFP-LC3 were observed and counted in cells using confocal laser microscopy.
4. Detection of LC3 protein expression by immunoblotting experiment
Detecting the expression of LC 3I and II proteins by immunoblotting, analyzing and adopting Protein bands by a Protein Simple chemiluminescence gel analysis system, analyzing and obtaining the gray value (IOD) of each band, and reflecting the expression level of the target Protein according to the ratio of the gray value of the target band to the gray value of the internal reference band.
5. Statistical procedure as in example 1
6. Results of the experiment
FIG. 2 shows that verbascoside effects on NRK cells induce autophagy. (A) The NRK cells stably expressing GFP-LC3 were subjected to intervention with verbascoside at concentrations of 25, 50 and 100. mu.M and rapamycin at 100nM for 12h, and the autophagy-lysosome development was observed using confocal laser microscopy. (B) Autophagy-lysosome counts were counted and statistically analyzed, (. p <0.05,. p <0.01,. p <0.001, one-way anova). (C) Effect of verbascoside on LC3 protein expression levels 24h after PC-12 and SH-SY5Y cells were affected.
As shown in figure 2, after the acteoside dose groups act on NRK cells stably expressed by GFP-LC3 for 12 hours, obvious GFP-LC3 aggregation points can be observed, and the statistical analysis result of the number of the GFP-LC3 aggregation points shows that the acteoside dose groups have significant difference compared with the blank group; the verbascoside has the effect of obviously improving the expression level of LC3 II protein after 24 hours of action on PC-12 and SH-SY5Y cells.
Example 3: activity of verbascoside for activating fat body tissue lysosome of drosophila melanogaster third-instar larvae
1. Raising fruit flies
The drosophila was fed with the corn medium specific for drosophila at 25 ℃ and humidity of 75%.
2. Obtaining fruit fly third instar larva and grouping
After the drosophila lay eggs for 96 hours, the third-instar larvae are respectively transferred to a blank group (containing 1.5 percent of yeast powder, 20 percent of sucrose and the same amount of DMSO) culture solution, an experimental group (containing different dosages of verbascoside, 1.5 percent of yeast powder and 20 percent of sucrose) culture solution and a hunger group (containing 20 percent of sucrose and the same amount of DMSO) culture solution, and are incubated for a designated time, so that 3-5 third-instar larvae are transferred for each group.
3. Detection of fat body lysosome activity of drosophila melanogaster third-instar larvae
Adipose tissue was isolated using a stereomicroscope and stained with LysoTracker Red and Hoechst33342, lysosomes were visualized by confocal laser microscopy and analyzed by counting.
4. Statistical procedure as in example 1
5. Results of the experiment
FIG. 3 shows that verbascoside induces autophagy in fat body cells of Drosophila trilliota larvae. (A) After the drosophila lay eggs for 96 hours, collecting third instar larvae, incubating in drosophila larvae culture solution containing 100 mu M and 500 mu M verbascoside for 6 hours, starving the third instar larvae for 4 hours by using starvation culture medium as a positive control group, separating and staining adipose body tissues under a body scope, and observing by using a laser confocal microscope with a scale bar of 25 mu M. (B) The fluorescence intensity of each group was analyzed statistically, and the data from three independent replicates were analyzed (p <0.05, p <0.01, p <0.001, single factor analysis of variance).
The results are shown in figure 3, 100 μ M and 500 μ M verbascoside can activate the lysosome activity of fat body tissues of drosophila trilling larvae, and have very significant difference compared with the control group; the fluorescence intensity of lysosomes generated by 500 mu M verbascoside group is stronger than that generated by 100 mu M verbascoside group, but the difference of the quantity of lysosome fluorescence sites of the two dose groups is not obvious, and the fluorescence intensity and the quantity of the lysosome fluorescence sites generated by the starvation group are both high.
Example 4: verbascoside against apoptosis
1. Cell culture and drug preparation procedures were the same as in example 1
2. Experiment grouping
PC-12 and SH-SY5Y cells were divided into 5 groups, a blank group, a verbascoside (50. mu.M) group, a model (0.2. mu.M rotenone) group, a model drug intervention group and a model drug intervention combination 3-MA (5mM) group, with 3-5 replicates set for each group and incubated for the indicated time.
3. Detecting apoptosis
The cells were treated with 7-ADD apoptosis assay kit from BD, and the incidence of apoptosis in each group of cells was determined using a flow cytometer from BD.
4. Immunoblotting assay for detecting Bax and Caspase-3 protein expression procedure as in example 2
5. Statistical procedure as in example 1
6. Results of the experiment
FIG. 4 shows that verbascoside has anti-apoptotic effect on rotenone-stimulated PC-12 cells. (A) Flow cytometry experiment results show that the effect of verbascoside (50 mu M) on apoptosis of rotenone (0.2 mu M) treated PC-12 cells after 24h, and the anti-apoptosis function of the verbascoside after the combined intervention of the verbascoside and an autophagy inhibitor 3-MA (5mM) on the cells is obviously inhibited. (B) The incidence of apoptosis was analyzed statistically for each group and data from three independent replicates were analyzed (. p <0.05,. p <0.01,. p <0.001, single factor anova). (C) Effect of verbascoside (50. mu.M) on Bax and Caspase-3 protein expression levels 24h after PC-12 and SH-SY5Y cells, and 3-MA (5mM) blocks the effect of verbascoside on apoptosis-related protein expression levels.
The results are shown in figure 4, compared with the blank control group, 50 mu M verbascoside has no rapid apoptosis after interfering PC-12 cells for 24h, 0.2 mu M rotenone has obvious rapid apoptosis after interfering PC-12 cells for 24h, verbascoside is added into the model group to treat the cells for 24h, the apoptosis incidence rate is reduced, and the anti-apoptosis effect of the verbascoside is obviously prevented after additionally adding 3-MA with 5mM concentration into the drug interference group, and the statistical results show that the verbascoside plays the anti-apoptosis effect by inducing autophagy. The result of the immunoblotting experiment shows that 50 mu M verbascoside acts on PC-12 and SH-SY5Y cells for 24h, then the expression level of apoptosis-related proteins Bax and cleared-caspase-3 is rapidly advanced, and 3-MA inhibits the anti-apoptosis function of the cells.
Example 5: acteoside has active oxygen resisting and mitochondrion protecting effects
1. Cell culture and drug preparation procedures were the same as in example 1
2. Experiment grouping
PC-12 cells were divided into 6 groups, blank, acteoside (50. mu.M), model (0.2. mu.M rotenone), model drug intervention combined 3-MA (5mM), 3-MA, and 3-5 replicates for each group and incubated for the indicated time.
3. Observation of active oxygen and mitochondria
The glass-bottom culture dish is used for cell culture and fluorescence observation, and is stained by an active oxygen detection probe DCFH-DA, a mitochondrial activity detection probe MitoTracker-Red and Hoechst33342, and active oxygen and mitochondria are observed by a laser confocal microscope.
4. Results of the experiment
FIG. 5 shows the effect of verbascoside on rotenone-stimulated PC-12 cell reactive oxygen species and mitochondria, which was stained with DCFH-DA and MitoTracker Red, respectively, and observed, showing that verbascoside (50. mu.M) has the function of reducing reactive oxygen species and maintaining mitochondrial integrity in rotenone (0.2. mu.M) -treated PC-12 cells, and that 3-MA (5mM) blocks the normal functioning of verbascoside.
The results are shown in FIG. 5, compared with the blank control group, after 50 μ M verbascoside intervenes in PC-12 cells for 24h, no fast-entry active oxygen is generated, and mitochondria are kept in a stable state; after 0.2 mu M rotenone acts on PC-12 cells for 24h, the active oxygen content is obviously improved, and mitochondria are fragmented and accumulated on the cells and the periphery; after the verbascoside is added into the model group to treat the cells for 24 hours, the incidence rate of active oxygen release is reduced, and mitochondrial damage is repaired; after the drug intervention group is additionally added with 3-MA with the concentration of 5mM, the active oxygen resisting and mitochondrion protecting effects of verbascoside are obviously blocked, and the effect of the 3-MA with the concentration of 5mM on the active oxygen and mitochondrion of PC-12 cells after being independently used for 24h is not obvious. The results indicate that verbascoside exerts its anti-reactive oxygen and mitochondrial protection functions by inducing autophagy.
Example 6: acteoside protects mitochondrial membrane integrity and maintains mitochondrial membrane potential
1. Cell culture and drug preparation procedures were the same as in example 1
2. Experimental grouping procedure as in example 5
3. Detecting mitochondrial membrane potential
The cell culture and fluorescence observation are carried out by using a glass-bottom culture dish, and then the cell culture and fluorescence observation are stained by using a mitochondrial membrane potential detection probe JC-1 and Hoechst33342, and the higher the red fluorescence intensity is compared with the blue fluorescence intensity ratio, the higher the mitochondrial membrane potential is, the normal mitochondria are indicated.
4. Statistical procedure as in example 1
5. Results of the experiment
FIG. 6 shows the effect of verbascoside on rotenone-stimulated PC-12 cell mitochondrial membrane potential, (A) cells were stained with JC-1 and observed, showing that verbascoside (50. mu.M) has the function of increasing mitochondrial membrane potential in rotenone (0.2. mu.M) -treated PC-12 cells and that 3-MA (5mM) blocks the normal function of verbascoside. (B) The fluorescence intensity of each group was analyzed statistically and data from three independent replicates were analyzed (. p <0.05,. p <0.01,. p <0.001, single factor analysis of variance).
The results are shown in FIG. 6, compared with the blank control group, the effect of 50 μ M verbascoside on PC-12 cells after 24h intervention is not obvious, and mitochondria are kept in a stable state; after 0.2 mu M rotenone acts on PC-12 cells for 24h, the mitochondrial membrane potential is obviously reduced; the mitochondrial membrane potential is obviously improved after the model group is added with verbascoside to treat cells for 24 hours; the function of verbascoside for improving mitochondrial membrane potential is obviously blocked after the drug intervention group is additionally added with 3-MA with the concentration of 5mM, and the influence of the 3-MA with the concentration of 5mM on the mitochondrial membrane potential of PC-12 cells after being independently used for 24 hours is not obvious. The results indicate that verbascoside exerts its function of maintaining mitochondrial membrane potential by inducing autophagy.
Example 7: verbascoside reduces alpha-synuclein accumulation
1. Cell culture and drug preparation procedures were the same as in example 1
2. Experiment grouping
GFP-a-Syn-GN-link-a-Syn GC (. alpha. -Syn) -stably expressing HEK cells were divided into 4 groups, a blank group, a verbascoside (50. mu.M) group, a rapamycin (100nM) group, a verbascoside-3-MA (5mM) group, and 3-5 replicates for each group and incubated for the indicated time.
3. Observation of alpha-Syn and lysosomal Activity
Cell culture and fluorescence observation were performed using glass-bottom petri dishes, and stained with LysoTracker Red and Hoechst33342, and observed with a confocal laser microscope, the more Red fluorescent sites indicate more active autophagy, and the more green fluorescent sites and the stronger the sites indicate more alpha-Syn stacking.
4. Immunoblotting experiment for detecting alpha-Syn and LC3 protein expression steps as in example 2
5. Statistical procedure as in example 1
6. Results of the experiment
FIG. 7 shows that verbascoside decreases alpha-synuclein accumulation 24h after acting on alpha-Syn stably expressed HEK cells. (A) The effect of alpha-synuclein is observed after the verbascoside (50 mu M) acts on HEK cells stably expressed by alpha-Syn for 24 hours, the proportion is 10 mu M, the verbascoside has the function of reducing alpha-synuclein accumulation, the function of reducing the alpha-synuclein accumulation by the verbascoside is more obvious compared with the autophagy inducer rapamycin (100nM), and the function of reducing the alpha-synuclein accumulation by the verbascoside is obviously inhibited after the verbascoside is combined with 3-MA (5mM) to intervene the cells. (B) Effect of verbascoside (50. mu.M) on alpha-Syn-stably expressed HEK cells after 24h of alpha-Syn and LC3 protein expression levels, 3-MA (5mM) blocks the normal function of verbascoside.
The results are shown in FIG. 7, compared with the blank control group, after 50 mu M verbascoside intervenes HEK cells stably expressed by alpha-Syn for 24h, the accumulation and expression quantity of the alpha-Syn are obviously reduced, and the expression level of LC3 II is improved; after the 100nM rapamycin intervenes in alpha-Syn stably expressed HEK cells for 24h, the effect on alpha-Syn accumulation and expression quantity is not obvious, but the expression level of LC3 II is improved; after the cells are intervened by combination of 50 mu M verbascoside and 3-MA with the concentration of 5mM for 24 hours, the accumulation and expression quantity of alpha-Syn are obviously improved, and the LC3 II/I ratio is reduced. The results indicate that verbascoside reduces alpha-Syn stacking and expression levels by inducing autophagy.
Example 8: mullein glucoside induced mitochondrion autophagy
1. Cell culture and drug preparation procedures were the same as in example 1
2. Experiment grouping
The stably expressed NRK cells of GFP-TOM20 were divided into 2 groups, a blank group and a verbascoside (50. mu.M) group, and 3-5 replicates were set for each group and incubated for the indicated time.
3. Observation of TOM20-GFP, localization of lysosomes
Cell culture and fluorescence observation were performed using glass-bottomed culture dishes, and LysoTracker Red staining was performed on NRK cells stably expressing TOM 20-GFP. The observation of a laser confocal microscope shows that the more yellow sites co-located by the green fluorescence sites and the red fluorescence sites indicate that the mitophagy is more active.
4. For different treated cells cultured in 10cm cell culture dishes, the procedure for preparing the drug was the same as in example 1, and mitochondria were extracted using the solibao (beijing) mitochondria extraction kit, respectively.
5. Immunoblotting experiments to examine the expression of cytoplasmic and mitochondrial protein fractions p62, PINK1 and LC3 proteins
6. Results of the experiment
FIG. 8 shows that verbascoside induces mitophagy. (A) After acteoside (50 μ M) had been allowed to act on NRK cells stably expressing TOM20-GFP for 4h, the cells were stained with LysoTracker Red and observed at a scale of 10 μ M, showing that acteoside (50 μ M) had a mitophagia-inducing function on NRK cells. (B) After acteoside (50 mu M) acts on SH-SY5Y cells for 24h, mitochondria and cytoplasm are extracted, and the change of the expression level of p62 and LC3 proteins is detected respectively, and the result shows that after the acteoside treatment, the enrichment of mitochondria p62, PINK1 and LC3 prompts the increase of the level of mitophagy, and prompts the acteoside treatment to exert the damage repair effect on SH-SY5Y cells treated by rotenone (0.2 mu M) by inducing mitophagy.
The results are shown in fig. 8-a, compared with the blank control group, after intervention of NRK cells stably expressing TOM20-GFP by 50 μ M verbascoside for 4h, the co-localization probability of lysosomes with green fluorescence TOM20-GFP and red fluorescence sites localized on mitochondria is obviously improved, and the results show that the verbascoside obviously induces mitophagy. As shown in FIG. 8-B, 50 μ M verbascoside reacted with untreated SH-SY5Y cells and 0.2 μ M rotenone-treated SH-SY5Y cells to significantly increase the expression levels of the mitochondria-localized p62, PINK1 and LC3 proteins, as compared to the blank control group. The results indicate that verbascoside exerts its function of protecting nerve cells by inducing mitophagy.
The protective effect of the nerve cells is helpful for delaying or treating neurodegenerative diseases such as Parkinson and the like (as shown in figure 9). Mitochondria are the "motor center" of cells, and impaired mitochondrial function may lead to a decrease in cellular energy levels, as mitochondrial membrane permeability changes, a decrease in membrane potential, and leakage of electrons promotes the formation of Reactive Oxygen Species (ROS), thereby destroying proteins, membrane lipids, and nucleic acids, ultimately leading to cell death. Mitochondria play an important role in neurons, and maintenance of mitochondria directly affects development, function, and survival of neurons. Maintaining a healthy population of mitochondria is critical to neuronal health, and thus there are many mitochondrial quality control pathways such as misfolded protein degradation, fission, and fusion, and mitochondrial autophagy.
The verbascoside can obviously improve the expression level of LC3 protein in nerve cells by up-regulating autophagy level, and is favorable for preparing novel autophagy inducers and treating diseases which can benefit from autophagy inducement, in particular neurodegenerative diseases.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and any simple modification, equivalent change and modification made to the above embodiments according to the technical spirit of the present invention are within the scope of the technical solution of the present invention.
Claims (8)
1. Application of verbascoside in preparing medicine for preventing and treating neurodegenerative diseases is provided.
2. The use according to claim 1, characterized in that said verbascoside, the alternative name of Chinese: acteoside, molecular weight: 624.59 formula C29H36O15CAS number: 61276-17-3, the structural formula is as follows:
3. application of verbascoside in preparing mitochondria protective agent is provided.
4. The use of claim 3, wherein verbascoside protects neuronal mitochondrial damage, reduces neuronal reactive oxygen species, and inhibits neuronal apoptosis.
5. The application of verbascoside in preparing autophagy inducer is characterized in that the verbascoside protects nerve cells by inducing mitophagy of cells.
6. The use of claim 5, wherein verbascoside induces autophagy and reduces the intracellular accumulation of α -Syn.
7. The use according to claim 1, characterized in that the verbascoside is used for preparing Parkinson's medicine.
8. The use of claim 7, wherein the anti-Parkinson's drug is a mitophagy agonist drug.
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