KR101922903B1 - Method for screening natural product-derived antineurodegenerative ingredients - Google Patents

Abstract

The present invention relates to a method for evaluating the survival rate of hippocampal neurons, comprising the steps of: (a) evaluating the survival rate of mouse-derived hippocampal neurons for a natural material, and determining the concentration of the natural material not affecting the viability of the hippocampal neurons; (TDP-43) present in the cytoplasm of the hippocampal neuron, and screening the natural material; and (c) using the antigen-antibody reaction to assess the aggregation rate of the transcriptional response DNA-binding protein 43 , And evaluating the amount of TDP-43 expression in the cytoplasm and nucleus of hippocampal neurons for the screened natural material.

Description

TECHNICAL FIELD The present invention relates to a method for screening natural antineurodegenerative ingredients,

The present invention relates to a method for screening an anti-neural degenerate natural material, and more particularly, to a method for screening a natural neural material, which comprises (a) evaluating the survival rate of a mouse-derived hippocampal neuron for a natural material, (B) evaluating the aggregation rate of the transcriptional response DNA-binding protein 43 (TDP-43) present in the cytoplasm of the hippocampal neuron to the determined concentration of the natural material, , And (c) evaluating the amount of TDP-43 expression in the cytoplasm and nucleus of hippocampal neurons for the screened natural material using an antigen-antibody reaction. Screening method.

There are several hypotheses about the causes of neuronal loss or death in patients with neurodegenerative diseases. However, no definite cause or treatment method has been proposed yet.

(TDP-43) protein accumulation in the cytoplasm of ubiquitinated transactivated DNA-binding protein 43 (DDP-43) in patients with atrophic lateral sclerosis and frontotemporal dementia as a cause of another neurodegenerative disease Is presented.

Normal TDP-43 is located in the nucleus but is present in the cytoplasm in pathological conditions. The abnormal location of the TDP-43 protein in the neuron leads to the loss of normal function of the neuron, which leads to neuronal dysfunction and death, resulting in neurodegenerative disease.

The TDP-43 protein mutation induces apoptosis and motor neuron deficiency in the zebrafish embryo, and excessive expression of TDP-43 induces neuronal death and motor nerve damage, Terminal fragments exhibit intracellular toxicity and are involved in the death of nerve cells (Human molecular genetics, 19, 671-683, 2010; Molecular Therapy 17, 607-613, 2009; Proceedings of the National Academy of Sciences. 106, 7607-7612, 2009, etc.).

These studies can be suggested as a basis for the hypothesis that denatured TDP-43 protein induces neurodegenerative diseases. Therefore, exploring effective functional components by using TDP-43 protein, a biomarker of new neurodegenerative diseases, can be a basic research for developing materials capable of preventing and treating neurodegenerative diseases .

The TDP-43 protein is a 414 amino acid DNA / RNA binding protein with a molecular weight of 43 kDa. The TDP-43 protein was first identified as a protein that binds to TAR DNA of human immunodeficiency virus type 1 (HIV-1) and inhibits gene transcription (J. Virol, 69, 3584-96, 1995).

Since then, the TDP-43 protein has been implicated in intracellular physiological activities such as transcriptional regulation of genes, selective splicing, and mRNA delivery (Int J Clin Exp Pathol, 4, 147-55, 2011; Journal of Biological Chemistry, 276, 3633-36343, 2001).

In 2006, we found that TDP-43 is the major protein of the ubiquitin-positive inclusion body in the frontal dementia patients with dichotomous frontal lobe dementia and ALS patients, and the relationship between this protein and other neurodegenerative diseases has been studied (Biochemical and biophysical research communications, 351, 602-611, 2006; Annals of neurology, 61, 435-445, 2007; Neuropathology, 29, 566-73, 2009).

In normal cells, TDP-43 is mainly a protein expressed in the nucleus. However, when the cell is placed in a stressed environment, the TDP-43 protein is cleaved by the activated caspase, and TDP-43 C- (TDP-43) is degraded and removed, and aggregation and accumulation in the cytoplasm results in loss of function or death of the nerve cell (Science, 314, 130-133, 2006; Experimental neurology, 237, 90-95, 2012, etc.).

Thus, the neuronal cells of neurodegenerative patients exhibit the properties of cytoplasmic inclusion, poorly soluble or insolubility, hyperphosphorylated, ubiquitinated, and cleaved into small fragments of TDP-43 protein (Nature Reviews Neurology, 6, 211-220, 2010, Neurosignals, 16, 41-51, 2008).

Korean Patent Laid-Open No. 10-2009-0046576 discloses a method for preparing a pharmaceutical composition comprising the steps of (a) depositing an amyloid protein on a solid substrate to which an amyloid protein is bound, (b) And (c) measuring the activity of the cultured neuron and screening the candidate drug candidate for promoting or maintaining the activity with a therapeutic agent for degenerative neurological disease, comprising the steps of: Is disclosed.

Korean Patent Laid-Open No. 10-2015-0015226 discloses a method for producing a TDP-43 degenerative disorder including TDP-43 mismatch mutation that abnormally locates in the cytoplasm and nerve cell branches, induces abnormal loss of normal TDP-43, A marker for diagnosing neurological diseases is disclosed.

However, the Korean Patent Laid-Open No. 10-2015-0015226 is the same as the present invention in that it is a screening method of an anti-neuron-degenerate material. However, the prior art screened the physiologically active substance which inhibits deposition of amyloid protein, Since the invention screened for a physiologically active substance which inhibits aggregation of TDP-43 protein, both inventions are completely different from each other in screening.

Korean Patent Laid-Open No. 10-2015-0015226 discloses a marker for diagnosing degenerative neurological diseases including a TDP-43 mismatch mutation which causes abnormal structural change of nerve cells. The present invention and the configuration are completely different.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made to solve the above problems occurring in the prior art, and it is an object of the present invention to provide a method for inhibiting accumulation of TDP-43 on a natural material effective for neurodegenerative diseases by inducing aggregation of TDP- To provide a method for effectively screening for TDP-43 accumulation-related cognitive or memory-enhancing targeting material.

(A) evaluating the survival rate of a mouse-derived hippocampal neuron for a natural material and determining the concentration of the natural material not affecting the survival rate of the hippocampal neuron, (b) evaluating the aggregation rate of the transcriptional response-DNA-binding protein 43 (TDP-43) present in the cytoplasm of the hippocampal neuron to the determined concentration of the natural material, screening the natural material and c) evaluating the expression level of TDP-43 in the cytoplasm and nucleus of hippocampal neuron cells against the screened natural material using an antigen-antibody reaction.

According to the screening method of the neurodegenerative natural material of the present invention, it is possible to rapidly select the natural material having the effect of inhibiting TDP-43 protein aggregation in the cytoplasm of hippocampal neurons in vitro.

FIG. 1 shows cell viability of hippocampal neuron HT-22 cell line according to natural material.
FIG. 2 shows fluorescence image evaluation results of the TDP-43 aggregation inhibitory effect of gastronidin.
FIG. 3 shows fluorescence image evaluation results of TDP-43 aggregation inhibitory effect of thymidine.
Figure 4 shows fluorescence image evaluation results of quercetin against TDP-43 aggregation inhibitory effect.
FIG. 5 shows the results of the TDP 43 accumulation inhibitory antigen-antibody reaction assay for the natural material screened by the fluorescent image evaluation.

The present invention relates to a method for evaluating the survival rate of hippocampal neurons, comprising the steps of: (a) evaluating the survival rate of mouse-derived hippocampal neurons for a natural material, and determining the concentration of the natural material not affecting the viability of the hippocampal neurons; (TDP-43) present in the cytoplasm of the hippocampal neuron, and screening the natural material; and (c) using the antigen-antibody reaction to assess the aggregation rate of the transcriptional response DNA-binding protein 43 , And evaluating the amount of TDP-43 expression in the cytoplasm and nucleus of hippocampal neurons for the screened natural material.

In the method for screening the anti-neuronigenic natural material of the present invention, the natural material may be at least one selected from the group consisting of gastrodine, sizandrine, and quercetin.

Examples of typical natural materials with anti-neurodegenerative effects include sizzindrin, gastrodine, quercetin, and schizandrin are known as Schisandra As a functional ingredient contained in chinensis (Turcz.) Baill, inhibition of apoptosis of cerebral cortical cells induced by glutamate (Molecules, 18, 354-372, 2012 etc.), by β-amyloid Induced immunosuppressive effect (Phytotherapy Research, 25, 435-443, 2011), anti-inflammatory effect (International immunopharmacology, 17, 415-426, 2013) , 1136-1140, 2006) have been reported to prevent neurodegenerative diseases including dementia.

In addition, gastrodin is a major component contained in Gastrodia elata, which inhibits glutamate-induced apoptosis (Cellular and molecular neurobiology, 34, 591-602, 2014), by β-amyloid Induced neurotoxicity (Brain research, 1482, 13-21, 2012), protection of oxidative stress induced by MPP + (Neurochemistry international, 75, 79-88, 2014), inhibition of LPS induced inflammation (PloS one, 6, e21891, 2011) have been proven.

Quercetin, on the other hand, is a type of flavonoid that is contained in many fruit or plant leaves. It is known as an antioxidant (Pharmacological research, 51, 117-123, 2005), anticancer research (26, 1177-1181, 2006; Apoptosis, 12, 411-421, 2007) and anti-inflammatory effects (Atherosclerosis, 218, 44-52, 2011).

In addition, inhibition of cytotoxicity induced by? -Amyloid 1-42 (The Journal of nutritional biochemistry, 20, 269-275, 2009), inhibition of cell death induced by hydrogen peroxide (Neuroscience letters, 504, 223-227, 2011), it is known that 6-OHDA-induced neurotoxicity mitigation effects by inhibiting the formation of NO and iNOS (International Journal of Molecular Medicine, 27, 195-203, 2011) .

In the screening method for the anti-neural degenerate natural material of the present invention, the mouse-derived hippocampal neuron may be HT-22 cells.

In the method for screening the anti-neuronigenic natural material of the present invention, the natural material is isozinc or quercetin, and the concentration is 0.1 to 10 μM, preferably 1 to 10 μM, more preferably 5 to 10 μM, most preferably May be 5 [mu] M.

In the method for screening the anti-neural degenerate natural material of the present invention, the natural material may be gastron, and the concentration may be 0.1-1 μM, preferably 0.5-1 μM, and most preferably 1 μM.

In the screening method of the anti-neural degenerate natural material of the present invention, the coagulation ratio can be calculated by the following equation (1).

[Formula 1]

Coagulation ratio (%) of TDP-43 = (number of TDP-43 protein aggregated cells / total number of cells present in cytoplasm) × 100

In the screening method for the anti-neuronigenic natural material of the present invention, the screening in the step (b) may be performed by selecting a natural material having an aggregation ratio of TDP-43 of 50% or more as compared to the aggregation rate of the control group .

In the screening method for the anti-neural degenerate natural material of the present invention, the control group may be a proteasome inhibitor, preferably MG-132.

The present invention also relates to a natural material screen screened by the above method.

In the present invention, the material may be at least one selected from the group consisting of gastrodine, sizandrine, and quercetin.

Hereinafter, the present invention will be described in detail with reference to Examples and Test Examples. However, these are for the purpose of illustrating the present invention in more detail, and the scope of the present invention is not limited thereto.

<Examples>

1. Experimental material

(One). material

Gastrodin was purchased from Biopurity (Biopurity phytochemicals Ltd. China), and schizandrin was purchased from Korea Traditional Medical Industry (Gyeongsan, S. Korea).

Quercetin and MG-132 were purchased from Sigma-Aldrich (Saint Louis, Mo.).

(2). Cell line

HT-22 cells were harvested from mouse-derived hippocampal neurons using the Salk institute (La Jolla, CA, USA).

2. Experimental Method

(One). Cell culture and differentiation

The nerve cells used in the experiments were hippocampal cell line (HT-22) derived from mouse and were used in Salk institute (USA). Were cultured in Dulbecco's modified Eagle's medium (DMEM) at 37 ° C and 5% CO ₂ with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.

The cells were incubated at 3-day intervals. The cells were washed with phosphate-buffered saline (PBS) at 80% confluent on the bottom of the plate, allowed to stand with 0.05% trypsin-EDTA solution, Followed by subculture.

HT-22 cells were cultured in NeuroBasal medium for 24 hours after replacing with differentiation induction medium supplemented with 2 mM glutamine, 1 × N ₂ , 100 μM 6,2'-O-Dibutyryladenosine 3 ', 5'-cyclic monophosphate sodium salt Differentiation was induced.

(2). Cell survival rate measurement of natural material

Cells were plated at 5 × 10 ⁴ cells / well in a 96-well plate, and cultured at 37 ° C. for 24 hours at 5% CO ₂ . The cells were treated with each sample in a concentration-dependent manner, and then cultured for a period of time.

After removing both the medium and the treated samples, 100 μl of PBS and 10 μl of CCK-8 were treated, followed by reaction at 37 ° C in 5% CO ₂ for 2 hours. The reaction was stopped with 10 μl 1 N HCl and the absorbance at 450 nm was measured.

The inhibitory effects of cell - growth inhibition were studied by calculating the ratios of the untreated control and the treated groups.

(3). Immunostaining fluorescent image evaluation

Cells were inoculated on a coverslip coated with 10 [mu] g / ml poly-L-ornithine and 10 [mu] g / ml laminin. After incubation at 37 ° C and 5% CO ₂ for 24 hours, differentiation was induced as described above, followed by culturing for 24 hours.

Gastrodine, and the other two functional components were treated with MG-132 (proteasome inhibitor) 1 μl for 12 hours, followed by culturing for 2 hours. After incubation, the cells were fixed with 4% paraformaldehyde.

Then, the cells were washed with PBS, and then blocked for 2 hours using a blocking solution (0.4% BSA, 0.2% Triton X-100 and 10% chicken serum, PBS). The primary antibody (TDP-43, EdU) of the protein to be identified was dissolved in the blocking solution and treated at 4 ° C for 12 hours or longer.

After washing with PBS, 2 secondary antibody ^{(Alexa Flour ® 488-conjuated chicken} anti-mouse IgG or ^{Alexa Flour ® 594 conjugated chicken anti-} rabbit IgG) with 4 ', 6-dia mi dino-2-phenyl-indole, Dirac Tate (DAPI, nuclear staining) for 2 hours at room temperature.

After washing with PBS, Fluoromount ^TM Cover slips were attached to microscope slides using an aqueous mounting medium and imaged via fluorescence microscopy.

The ratio of cells exhibiting TDP-43 protein aggregation in the cytoplasm of all cells was calculated by the following formula 1 to compare the TDP-43 aggregation inhibitory effect of the functional components.

[Formula 1]

Coagulation ratio (%) of TDP-43 = (number of TDP-43 protein aggregated cells / total number of cells present in cytoplasm) × 100

(4). TDP -43 protein antigen-antibody reaction validation evaluation

Cells were inoculated on a 10-cm plate and cultured at 37 ° C, 5% CO ₂ for 24 hours. And gastrodine were treated respectively. After 12 hours, 1 μM of MG-132 (proteasome inhibitor) was treated and cultured for 2 hours.

After the culture, all of the cells were collected, and the nucleus and cytoplasmic proteins of the cells were separated and extracted with NE-PER ^® nuclear and cytoplasmic extraction reagents. Then, 10 μg of protein was dissolved in 10% sodium dodecyl sulfate polyacrylamide gel And electrophoresis was carried out at a voltage of 100 V for 1 hour and 40 minutes.

The separated proteins were transferred to a nitrocellulose membrane at 100 V for 1 hour. Then, the nitrocellulose membrane was blocked with a PBST solution containing 5% skim milk, and then reacted with the primary antibody (TDP-43, Lamin B1, GAPDH) at 4 ° C for 12 hours or longer.

After washing with PBS, the cells were reacted with a secondary antibody (goat anti-rabbit IgG-HRP) for 2 hours at room temperature and washed with PBS. The film was developed in a dark room using a chemiluminescence detection kit and an X-ray film.

The expression level of TDP-43 protein in the cytoplasm and nucleus was compared among the treatment groups to analyze the inhibitory effect of the functional component on TDP-43 protein aggregation.

(5). Statistical processing

Statistical analysis was performed using SAS 9.0 (SAS Institute Inc., SAS Campus Drive, Cary, North Carolina 27513, USA) and Duncan's multiple range test (p <0.05).

3. Results and Discussion

(One). Assessment of hippocampal neuron survival rate of natural material

In order to examine the effect of the natural materials used in the present invention on the survival of hippocampal neurons, mouse-derived HT-22 cells were treated with 0 to 10 μM of gastrodine, And the cell viability was measured. The results are shown in Fig.

As shown in FIG. 1, in the case of sizadrine and quercetin, the cell growth was promoted to a level of 130 to 150% at a concentration range of 0.1 to 10 μM, and the highest cell growth rate was observed at a concentration of 5 μM.

On the other hand, in the case of gastrodine, the cell growth was accelerated in the concentration range of 0.1 to 1 μM, but the cell survival rate was decreased by more than 60% at the concentration of 5 μM or more.

From these results, we evaluated the inhibition of TDP-43 protein accumulation by determining the highest concentration of natural material that does not affect the viability of hippocampal neurons.

(2). TDP -43 Cell Immunostaining Fluorescence image evaluation

The TDP-43 protein is mainly located in the nucleus of normal neurons, but when treated with a proteasome inhibitor such as MG-132, the TDP-43 protein accumulates in the cytoplasm and forms aggregates.

Therefore, we determined the concentration of MG-132, which can induce TDP-43 aggregation inhibition in hippocampal neurons (HT-22) cytoplasm by assessing TDP-43 aggregation inhibition.

As a result, when treated with 1 μM of MG-132, it was confirmed that the TDP-43 protein in the nucleus disappeared from the cytoplasm (inducing TDP-43 aggregation of 30% or more).

As shown in FIG. 2, the accumulation of TDP-43 protein in 1 μM of gastrointin was slightly decreased, but the activity of MG There was no significant difference between the control group treated with -132 (MG-132 induced cells and TDP-43 aggregation-induced inhibitory effect = 23.5%) (p <0.05).

In FIG. 2, A shows the TDP-43 immunoreactive image of HT-22 cells, wherein the treatment concentrations of MG-132 and gastronin were 1 μM, respectively. In Fig. 2, B represents the coagulation ratio of TDP-43.

From these results, it was concluded that Gastrodine natural material, which has anti-neurodegenerative effect, has little or no inhibitory effect on TDP-43 protein accumulation in cytoplasm of hippocampal neurons.

In the case of sizandrine and quercetin, the inhibitory effect of TDP-43 protein accumulation on the hippocampal neuronal cell was evaluated at a concentration of 5 μM, which is less toxic to hippocampal neurons and has the highest cell survival rate.

As shown in FIG. 3, the TDP-43 protein accumulation was 15.6 ± 3.1% in case of iszanidin, and the decrease was about 50% (p <0.05) as compared with the MG- This result suggests that the possibility of rapid screening of TDP-43 protein target antineutrophiligenic natural material among various materials by using in vitro fluorescence image evaluation method is suggested.

In FIG. 3, A shows the TDP-43 immunoreactivity image of HT-22 cells, wherein the treatment concentration of MG-132 was 1 μM and the treatment concentration of thiazolidine was 5 μM. 3, B shows the coagulation rate of TDP-43.

As shown in FIG. 4, the accumulation of TDP-43 protein in quercetin was 21.5 ± 2.8%, which was about 33% lower than that of the MG-132-treated control, (P <0.05). However, there was no significant difference from the control group treated with MG-132 alone (p <0.05).

In FIG. 4, A shows the TDP-43 immunoreactivity image of HT-22 cells, wherein the treatment concentration of MG-132 was 1 μM and the treatment concentration of thiazolidine was 5 μM. In Fig. 4, B represents the coagulation ratio of TDP-43.

(3). TDP -43 protein antigen-antibody reaction validation evaluation

Antibody-antibody reaction assays were performed to examine the cytoplasmic and nuclear intracellular TDP-43 protein expression levels of hippocampal neurons for sizadrine screened by in vitro fluorescence image assay.

As shown in FIG. 5, the expression level of TDP-43 protein in the cytoplasm was increased by about 41.0 ± 15.5% by treatment with MG-132, but the amount of protein expression in cytoplasm when treated with sizzlin was not treated with MG-132 control.

This means that it is effective as a method for rapidly screening a natural material having an inhibitory effect on TDP-43 protein accumulation by an in vitro fluorescent image evaluation method.

In FIG. 5, A is the result of immunoblotting of TDP-43 using lysates from HT-22 cells, wherein the treatment concentration of MG-132 was 1 μM and the treatment concentration of thiazolidine was 1 μM And 5 [mu] M.

In FIG. 5, B represents the amount of TDP-43 protein expression in the cytoplasm, and C represents the amount of TDP-43 protein expression in the nucleus.

4. Conclusion

(One). Concentrations of natural materials (gastrodine, iszandrine, quercetin) for evaluation of survival rate of hippocampal neurons (HT-22) in this outbreak were performed in the concentration range of 0.1 ~ 10 μM, and the representative neurodegeneration The natural materials that are effective for the disease promoted the growth of hippocampal neurons by 130 ~ 150%.

Thus, the inhibitory effect of TDP-43 protein aggregation was evaluated in the concentration range of the natural material to which hippocampal neurons had been grown (1 μM of gastrodine, 5 μM of thiazolidine, and 5 μM of quercetin). In order to evaluate the effect of inhibiting TDP-43 protein aggregation, it is preferable to determine the concentration of natural materials in the range of hippocampal neuron growth. However, in the toxic natural materials, the hippocampal neuron exhibits a survival rate of at least 80% desirable.

(2). In addition, the optimal concentration and time of MG-132 inducing agent for inducing TDP-43 protein aggregation into the cytoplasm of hippocampal neurons (HT-22) were 1 μM and 2 hours, respectively, It is preferable that the TDP-43 protein is induced by 30% or more.

(3). In the in vitro fluorescence image evaluation method of the present invention, in which natural material materials having hippocampal neuron cell (HT-22) inhibitory effect on TDP-43 protein aggregation in cytoplasm can be rapidly selected, 50-300 immunofluorescently stained cells It is preferable to select materials having an average TDP-43 aggregation inhibiting effect of 50% or more compared to the MG-132 induction control. In addition, it is preferable to perform the TDP-43 protein antigen-antibody reaction confirmation step in the materials selected in the in vitro fluorescence image evaluation method.

Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims. It will be understood that the present invention can be changed.

Since the screening method of the neurodegenerative natural material of the present invention can provide a method for rapidly selecting natural material having the effect of suppressing TDP-43 protein aggregation in the cytoplasm of hippocampal neurons in vitro, It can be usefully applied to the field.

Claims (10)

(a). Determining the concentration of the natural product material that does not affect the viability of the hippocampal neuron by evaluating the survival rate of the mouse derived hippocampal neuron with respect to the natural product material, wherein the natural product material is gastrodine, sizandrine, or quercetin , The concentration of thymindrine or quercetin is 0.1 to 10 μM, and the concentration of gastronine is 0.1 to 1 μM;
(b). Evaluating the aggregation rate of the transcriptional response DNA-binding protein 43 (TDP-43) present in the cytoplasm of the hippocampal neuron to the determined concentration of the natural material, and screening the natural material; And
(c). And evaluating the amount of TDP-43 expression in the cytoplasm and nucleus of hippocampal neuron cells against the screened natural material using an antigen-antibody reaction. delete The method according to claim 1, wherein the mouse-derived hippocampal neuron is HT-22 cells. delete delete 2. The method for screening anti-neuronigenic natural material according to claim 1, wherein the coagulation ratio is calculated by the following formula 1:
[Formula 1]
Coagulation ratio (%) of TDP-43 = (number of TDP-43 protein aggregated cells / total number of cells present in cytoplasm) × 100 The method according to claim 1, wherein the screening of step (b) comprises selecting a natural material having a coagulation ratio of TDP-43 of 50% or more as compared to the coagulation ratio of the control group Screening method. 8. The method according to claim 7, wherein the control group is treated with a proteasome inhibitor MG-132. delete delete

Images