KR102623664B1 - Composition and treatment method for diabetic Alzheimer's disease using urolitin A - Google Patents

Abstract

The present invention relates to a composition and treatment method for diabetic Alzheimer's disease using urolithin A, and specifically, to reduce the generation of mitochondrial reactive oxygen species induced by high blood sugar or high glucose conditions, or to inhibit mitochondrial calcium accumulation; It relates to a composition that inhibits the production of amyloid beta and neuronal degeneration.

Description

Composition and treatment method for diabetic Alzheimer's disease using urolitin A {Composition and treatment method for diabetic Alzheimer's disease using urolitin A}

The present invention relates to a composition and treatment method for diabetic Alzheimer's disease using urolithin A, and specifically, to reduce the generation of mitochondrial reactive oxygen species induced by high blood sugar or high glucose conditions, or to inhibit mitochondrial calcium accumulation; It relates to a composition that inhibits the production of amyloid beta and neuronal degeneration.

Hyperglycemia in diabetes mellitus (DM) patients is a major pathogenic factor contributing to amyloidogenesis-induced neuropathological changes, such as neuronal damage, neurodegeneration, and cognitive impairment. Previous studies have shown that high glucose conditions increase amyloid precursor protein (APP) accumulation and amyloid beta (Aβ) plaque formation in neurons. Increased expression of the APP processing enzyme β-secretase 1 (BACE1) by high glucose is important for Aβ production in neurons, and inactivation of BACE1 causes Aβ-based Alzheimer's disease (AD)-like pathologies such as synaptic and memory deficits. alleviate. Hyperglycemia-induced diabetes complications, including cognitive impairment, are mainly related to oxidative stress. Mitochondrial reactive oxygen species (mtROS) play a key role in the pathogenesis of AD by promoting mitochondrial dysfunction, RNA and DNA damage, lipid peroxidation, and Aβ oxidation. Since mtROS is involved in amyloid production and neuronal apoptosis, inhibiting mtROS accumulation under hyperglycemic conditions can be considered a strategy to prevent AD-related diseases in DM patients.

Hyperglycemia-induced dysregulation of mitochondrial calcium homeostasis is a risk factor for mtROS accumulation, which leads to diabetic neuropathy. The mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) is an organelle between mitochondria and the ER that controls cellular metabolism and calcium transport from the ER to the mitochondria. Aβ-peptide-enhanced MAM-dependent calcium transport is mediated by voltage-dependent anion selective channel protein 1 (VDAC1)-inositol 1,4,5-tris phosphate receptor (IP3R) cross-link formation in primary hippocampal neurons implicated in AD. . Regulators of MAM that regulate IP3R-VDAC1 interaction, such as GRP75, Bax, Bcl2, and TGM2, are being studied.

Meanwhile, 3,8-Dihydroxy-urolithin (urolithin A) is a metabolite of ellagitannin produced in intestinal microorganisms. The bioavailability of urolithin extracted from ellagitannin and ellagic acid varies depending on the overall composition of individual intestinal microorganisms, and previous studies on the activity of urolithin showed that urolithin A was more active than its precursor, ellagitannin, and other urolithins. It was studied that it is a high metabolite (J Ethnopharmacol. 2014;155:801-9.). However, the specific effects of urolithin A on MAM formation and mitochondrial calcium influx from the ER, which regulates mtROS production in neurons under high glucose conditions, have not been studied.

Korean Patent Publication No. 10-2018-0037052 Korean Patent Publication No. 10-2015-0036224

One object of the present invention is to provide a pharmaceutical composition for preventing or treating Alzheimer's disease, containing urolithin A as an active ingredient.

Another object of the present invention is to provide a health functional food composition for preventing or improving Alzheimer's disease, containing urolithin A as an active ingredient.

Another object of the present invention is to treat a subject with a candidate substance or urolithin A; The present invention provides a method for screening candidates for the prevention or treatment of Alzheimer's disease, which includes comparing the degree of mitochondrial reactive oxygen species, LDH production, TGM2 expression, or mitochondrial calcium accumulation in the subject.

One aspect of the present invention for achieving the above object provides a pharmaceutical composition for preventing or treating Alzheimer's disease, comprising urolithin A as an active ingredient.

Another aspect of the present invention provides a health functional food composition for preventing or improving Alzheimer's disease, comprising urolithin A as an active ingredient.

Another aspect of the present invention includes treating a subject with a candidate substance or urolithin A; Provided is a method for screening candidates for the prevention or treatment of Alzheimer's disease, comprising comparing the degree of mitochondrial reactive oxygen species, LDH production, TGM2 expression, or mitochondrial calcium accumulation of the subject.

In the present invention, it was found that urolithin A inhibits the mitochondrial-endoplasmic reticulum junction by inhibiting TGM2 expression, thereby blocking the influx of endoplasmic reticulum calcium into the mitochondria. Through this, urolitin A, an intestinal microbial metabolite, is used to treat diabetic Alzheimer's disease. It can be used as a preventive and therapeutic agent.

Figures 1 to 7 relate to the effects of urolithin on mitochondrial calcium and ROS in neurons under high glucose conditions.
Figures 8-18 relate to the effect of urolithin A on amyloid production in neurons under high glucose conditions.
Figures 19-29 relate to the effect of urolithin A on MAM regulating mitochondrial calcium under high glucose conditions.
Figures 30-34 relate to the role of TGM2 in high glucose-induced MAM formation and amyloidogenesis.
Figures 35-42 relate to the effect of urolitin A on suppressing TGM2 expression through AIP-AhR transcription complex formation.
Figures 43-48 relate to the protective effects of urolithin A against Aβ-induced mitochondrial calcium influx, mtROS accumulation and neuronal cell death.
Figures 49-54 relate to the role of TGM2 in Aβ-induced mitochondrial calcium overload, mtROS accumulation, tau phosphorylation and neuronal cell death.

This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Additionally, the scope of the present invention cannot be considered limited by the specific description described below.

One aspect of the present invention provides a pharmaceutical composition for preventing or treating Alzheimer's disease, comprising urolithin A as an active ingredient.

Another aspect of the present invention provides a health functional food composition for preventing or improving Alzheimer's disease, comprising urolithin A as an active ingredient.

Another aspect of the present invention includes treating a subject with a candidate substance or urolithin A; Provided is a method for screening candidates for the prevention or treatment of Alzheimer's disease, comprising comparing the degree of mitochondrial reactive oxygen species, LDH production, TGM2 expression, or mitochondrial calcium accumulation of the subject.

In the present invention, the Alzheimer's disease may be diabetic Alzheimer's disease.

In the present invention, the composition reduces the generation of mitochondrial reactive oxygen species induced by high blood sugar or high glucose conditions, the composition inhibits mitochondrial calcium accumulation induced by high blood sugar or high glucose conditions, or the production of amyloid beta. and suppressing neuronal degeneration, lowering the production of LDH induced by high blood sugar or high glucose conditions, suppressing TGM2 expression, and/or promoting the formation of the AIP-AhR complex.

In the present invention, the term “prevention” refers to all actions that suppress or delay the onset of a disease by administering the composition according to the present invention. In the present invention, the term “treatment” refers to any action that improves or beneficially changes the symptoms of the disease by administering the composition according to the present invention. The term “improvement” of the present invention refers to any action in which the disease is improved or beneficially changed by administration of the composition of the present invention.

As used in the present invention, the term "administration" means introducing the pharmaceutical composition of the present invention into an individual by any appropriate method, and the route of administration of the composition of the present invention is oral or parenteral as long as it can reach the target tissue. It can be administered through various routes.

The term 'individual' used in the present invention refers to all animals, including humans, that have already developed or may develop Alzheimer's disease, and that the disease can be effectively prevented and treated by administering the composition of the present invention to the subject.

Another aspect of the present invention is to treat cells under high glucose conditions with urolithin A in vitro to lower the production of cellular LDH, reduce the generation of mitochondrial reactive oxygen species, inhibit mitochondrial calcium accumulation, or , it provides a cell treatment method that reduces the expression level of amyloid beta, inhibits TGM2 expression, and/or promotes the formation of the AIP-AhR complex.

Another aspect of the present invention is a method of treating a model mouse, comprising treating a diabetic Alzheimer's disease model mouse with urolithin A,

Urolithin A is treated or administered to model mice under hyperglycemic conditions to lower the production of LDH in mice, reduce the generation of mitochondrial reactive oxygen species, inhibit mitochondrial calcium accumulation, or reduce the expression level of amyloid beta. , inhibiting TGM2 expression, and/or promoting the formation of the AIP-AhR complex.

The composition of the present invention is administered in a pharmaceutically effective amount. The term 'pharmaceutically effective amount' used in the present invention refers to an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level refers to the type and severity of the individual, age, It can be determined based on factors including gender, type of viral infectious disease, activity of the drug, sensitivity to the drug, administration time, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And it can be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and can be easily determined by a person skilled in the art.

In the present invention, the composition may further include appropriate carriers, excipients, and diluents commonly used in manufacturing.

The composition can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injectable solutions according to conventional methods, respectively. Possible carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, not determined. Vaginal cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate and mineral oil.

When formulating the composition, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations are made by mixing the above compound with at least one excipient, such as cotton, starch, calcium carbonate, sucrose or lactose, gelatin, etc. It is prepared. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral administration of the pharmaceutical composition include suspensions, oral solutions, emulsions, syrups, etc. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients, such as wetting agents, Sweeteners, flavoring agents, preservatives, etc. may be included. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, Withepsol, Macrogol, Tween 61, cacao, laurin, glycerol gelatin, etc. can be used.

In the present invention, the preferred dosage of the composition varies depending on the patient's condition and weight, degree of disease, drug form, administration route and period, but can be appropriately selected by a person skilled in the art. However, for a desirable effect, the composition is preferably administered at 0.01 mg/kg to 10 g/kg per day, preferably at 1 mg/kg to 1 g/kg. Administration may be administered once a day, or may be administered in several divided doses.

The composition can be administered to mammals such as rats, mice, livestock, and humans through various routes. All methods of administration may be conventional methods, for example, oral, rectal or intravenous administration.

The term “health functional food” used in the present invention refers to food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and pills using raw materials or ingredients with functional properties useful to the human body. Here, functionality means controlling nutrients for the structure and function of the human body or obtaining useful effects for health purposes such as physiological effects. The health functional food of the present invention can be manufactured by a method commonly used in the art, and can be manufactured by adding raw materials and ingredients commonly added in the art. In addition, unlike general drugs, it is made from food, so it has the advantage of not having any side effects that may occur when taking the drug for a long time, and it can be highly portable.

When using the composition of the present invention in a health functional food, the composition can be added as is or used together with other health functional foods or health functional food ingredients, and can be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). Generally, when manufacturing food, the composition of the present invention is added in an amount of 1 to 10% by weight, specifically 5 to 10% by weight, of the raw material composition. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be used even below the above range.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1: Experimental Materials and Methods

1-1. experiment material

Neuroblastoma cell line SH-SY5Y was purchased from the Korean Cell Line Bank. The SK-N-MC neuroblastoma cell line stably expressing the Swedish mutation (K595N/M596L) of amyloid precursor protein (APPSwe) was provided by Dr. Ki-Won Park (Yonsei University) and was supplied with FBS, urolithin A, urolithin B, and urolithin. Materials such as ritin C, urolithin D, ellagic acid, D-glucose, hydrogen peroxide, carbonyl cyanide m-chlorophenylhydrazone, Ru360, and antimycin A were purchased and prepared.

1-2. cell culture

SH-SY5Y cells were cultured in high-glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with 1% penicillin-streptomycin solution (Gibco, Grand Island, NY, USA) and 10% FBS (Hyclone, Logan). . APPSwe SK-N-MC cells were cultured in high-glucose DMEM containing 1% penicillin-streptomycin solution and 10% FBS. Cells were grown in 60 mm dishes, 100 mm culture dishes, or 96-well plates in an incubator (37 °C, 5% CO ₂ and 95% air). When cells reached 80% confluency, the culture medium was replaced with serum-free low-glucose DMEM for 24 h for starvation. After incubation, cells were incubated in serum-free low-glucose DMEM supplemented with the indicated agents for the indicated treatment times. To create high glucose conditions, cells were treated with 25mM D-glucose.

1-3. Neural differentiation from iPSCs

iPSCs were obtained from Kangstem Biotech (Seoul, Korea). iPSCs were cultured on recombinant human vitronectin (rhVTN, A14700, Thermo Fisher) coated plates. PSC neural induction medium (A1647801, Thermo Fisher) was used for neural stem cell induction. After neuronal differentiation from iPSCs, neural stem cells (NSCs) were spread on laminin-(23017, Thermo Fisher) and poly-L-ornithine (Sigma, P3655) coated dishes. For neural differentiation, NSCs were cultured in Neurobasal medium (21103, Thermo Fisher) with serum-free supplements B27 (17504, Thermo Fisher) and GlutaMax (35050, Thermo Fisher) for 10 days. To enhance neural differentiation, dibutyryl-cAMP (D0627, Sigma) was added to the neural differentiation medium between days 7 and 10.

1-4. LDH cytotoxicity assay

Cell concentration was optimized according to the protocol provided in the LDH release assay kit (EZ-LDH, DoGenBio, Seoul, Korea, DG-LDH500). SH-SY5Y cells were seeded in 96-well plates at a density of 1 Х 10 ⁴ cells/well. When cells reached 90% confluency, the medium was replaced with serum-free, low-glucose DMEM. After centrifugation of the plate at 600Хg, the supernatant was collected and incubated with the LDH assay mixture for 30 minutes at room temperature (RT). LDH emission levels were analyzed by measuring optical density at 450 nm using an Epoch 2 spectrophotometer (BioTek, VT, USA).

1-5. Water-soluble tetrazolium salt (WST-1) cell viability assay

The viability of SH-SY5Y cells was measured by WST-1 cell viability assay (EZ-Cytox; Daeil Labservice, Seoul, Korea, #EZ-1000). After culturing the cells in a 96-well plate until 80% confluency, the growth medium was replaced with serum-free, low-glucose DMEM. Urolithin A (100 nM) or hydrogen peroxide (100 μM) was added to cells for 30 min and then treated with D-glucose for 72 h. Cells were incubated in 10 μl of EZ-Cytox reagent in 100 μl of DMEM for 30 min at 37 °C. Absorbance at 450 nm was measured with an Epoch 2 spectrophotometer.

1-6. mtROS and calcium measurements and mitochondrial permeability transition pore analysis

Intracellular ROS, mtROS and mitochondrial calcium levels were measured using MitoSOX Red (Thermo Fisher, M36008) and rhod-2 (Thermo Fisher, R1244). The level of mPTP opening was analyzed using Calcein AM (Thermo Fisher, C1430) to determine cell death by measuring mPTP opening. Cytosolic calcium was quenched by adding CoCl ₂ (400 μM). Each experiment was performed using a Cytoflex flow cytometer (Beckman Coulter, FL, USA), and results were obtained by comparing the proportion of cells with high fluorescence intensity.

1-7. Experimental design of animal studies

The animal research protocol was approved by the Seoul National University Committee (SNU-190801-4-1). Nine-week-old male CrljOri:CD1 (ICR) mice were obtained from Orient-Bio. Each mouse was weighed and housed for 3 days under controlled germ-free conditions at 22°C, 70% relative humidity, 12 h light:dark cycle and provided with a normal diet and water ad libitum. STZ was dissolved in 0.05 M citrate buffer, and mice were intraperitoneally injected with STZ (75 mg/kg/day) or citrate buffer (0.05 M) in a total volume of 200 μl for 3 days. All mice were provided with 10% sucrose water for 7 days to reduce the likelihood of low-glucose shock in STZ-injected mice. Blood was collected from the tail and blood glucose levels were measured 7 days after STZ injection using a glucometer (Accu-Chek, Roche Diagnostics, Indianapolis, IN). Urolithin A was dissolved in 0.5mM NaOH in PBS. Daily intraperitoneal injections of urolithin A (2.5 mg/kg/day) or 0.5 mM NaOH in a volume of 200 μl were administered for 8 weeks. Mice were monitored twice daily during all experiments. After 8 weeks of urolithin A or vehicle treatment, each mouse was weighed and the blood glucose level of tail blood was measured.

1-8. Y-maze voluntary alternation test

STZ-induced diabetic mice and normal mice were injected with urolithin A (2.5 mg/kg/day) or vehicle for 8 weeks. Mice were housed in the test room for 3 h before the Y-maze voluntary alternation test to reduce the influence of external environmental stimuli or unintentional stress on behavior. First, the mouse was placed in the Y-maze apparatus. Next, mice were given the opportunity to explore the Y-maze for 8 min, and their movements were recorded using a video camera.

1-9. Transglutaminase 2 activity measurement

Transglutaminase (TGM) 2 activity assay kit is applied to the production of hydroxamate product from deamidation activity. Hydroxamate finally formed a purple complex with the stop solution, and the optical absorbance was measured at 525 nm with a microplate reader. TGM2 activity was normalized by total protein in cell lysate samples.

1-10. siRNA transfection

SH-SY5Y cells were incubated with 25 nM of the indicated siRNA and transfection reagent TurboFect (Thermo Fisher, R0531) for 24 h without antibiotics. The medium was changed to serum-free low glucose DMEM. The siRNA efficacy was confirmed to be over 70% by real-time qPCR for TGM2, and NT siRNA was used as a control.

1-11. Real-time quantitative PCR

Cells were treated with high glucose or vehicle for 24 h. Cells were then washed twice with PBS and lysed with buffer RL containing 50X dithiothreitol solution. Total RNA was extracted using an RNA extraction kit (Takara, Japan, 9767) according to the manufacturer's instructions. Reverse transcription PCR was performed with 1 μg of total RNA using the Maxime RT premix kit (iNtRON, Sungnam, Korea, 25081). cDNA was amplified using the Maxime PCR PreMix Kit (iNtRON, 25165) and MyGenie 96 (Bioneer, Daejeon, South Korea). Relative mRNA expression levels of target genes were analyzed using a Rotor-Gene 6000 device (Corbett Research, Cambridge, UK) with TB Green Premix Ex Taq (TaKaRa, RR420A). PCR products were identified and melting curves were analyzed to verify the specificity, efficiency and fidelity of PCR primers for real-time quantitative PCR. The relative mRNA expression levels of VDAC1, MCU1, MICU1, MICU2, MCUR1, MCUB, BAX, BCL2L1, BCL2, GRP75, TGM2, PTGES3, AIP and HSP90AA1 were analyzed by delta-delta Ct method. 18s rRNA was used as a reference gene for data normalization.

1-12. Western blotting and subcellular fractionation

Protein concentration was determined by the bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA, 23225). Sample proteins were resolved by SDS-PAGE gel electrophoresis and transferred to PVDF membranes. Membranes were incubated with primary antibodies overnight at 4 °C. Specific bands were visualized by ChemiDoc XRS + System (Bio-Rad, Richmond, CA, USA). Subcellular fractionation was performed to separate cytoplasmic and nuclear proteins. Cells were cultured in 100 mm dishes and treated with the indicated reagents. The EzSubcell subcellular fractionation/extraction kit (Atto, Tokyo, Japan, WSE-7421) was used for preparation of cytoplasmic and nuclear fraction samples. Cytoplasmic and nuclear samples for Western blot analysis were prepared according to the manufacturer's instructions. α-tubulin and lamin A/C were used as cytoplasmic and nuclear protein markers, respectively.

1-13. ELISA for Aβ secretion

For quantification of Aβ(142) in cell culture medium, Aβ 42 Human ELISA (Thermo Fisher, KHB3544) was performed according to the supplier's protocol. SH-SY5Y cells were cultured to grow to 80% confluency, supernatants were collected and ELISA was performed. Mouse brain tissue samples from the prefrontal cortex and hippocampus were collected and lysed with RIPA buffer (ATTO, Tokyo, Japan). Aβ 42 mouse ELISA (Thermo Fisher, KMB3441) was performed according to the supplier's instructions.

1-14. immunocytochemistry

For immunocytochemistry, SH-SY5Y cells were fixed with 4% paraformaldehyde for 10 min and then incubated in 0.5% Tween-20 for 10 min. Cells were incubated with primary antibodies in PBS containing 0.1% Tween-20 (PBST; 1:100 dilution) for 2 h and washed three times with PBS. Cells were incubated with Alexa Fluor 488 or 555-conjugated secondary antibodies in PBST (1:100 dilution) for 1 h. Immunofluorescently stained samples were visualized by a super-resolution radial fluctuation (SRRF) imaging system (Andor Technology, Belfast, UK). The relative fluorescence intensity of AhR/DAPI was quantified with ImageJ software. To analyze ER-mitochondrial contacts, cells were treated with chemicals or transfected with TGM2 siRNA and cultured for 24 h. After washing the cells three times with PBS, cells were incubated with MitoTracker green (200 nM) and ER-Tracker (200 nM) for 20 min at 37 °C in serum-free medium, and nuclei were stained with Hoechst 33342 (Thermo Fisher). .

1-15. in situ PLA

VDAC1/IP3R1 interaction was detected in situ using Duolink II secondary antibodies and detection kits (Sigma-Aldrich, DUO92001, DUO92005 and DUO92008) according to the supplier's protocol. Cells were fixed and PLA probe anti-VDAC1 and anti-IP3R1 antibodies were applied followed by the addition of secondary antibodies. When antibodies are in close proximity (<40 nm), they become linked to each other. The signal in the closed circle (red) was amplified by supplementing the polymerization and amplification solution and visualized by SRRF microscopy. DAPI was used to counterstain nuclei.

1-16. Co-immunoprecipitation

SH-SY5Y cells transfected with NT or TGM2 siRNA were treated with vehicle or D-glucose (25mM) for 24 h and then incubated in co-immunoprecipitation lysis buffer (20mM Tris-HCl pH) containing protease inhibitor cocktail for 30 min on ice. 8.0, 137mM NaCl, 1% Nonidet P-40, and 2mM EDTA). The concentration of protein in each lysate was determined by BCA quantitative analysis (Thermo Fisher, 23225). VDAC1 or rabbit IgG antibodies were immobilized with protein G magnetic beads (Sure Beads, Bio-Rad, CA, USA, 161-4021). The immobilized magnetic beads were incubated with cell lysates for 6 h at 4 °C. Washed beads were eluted with 20mM glycine buffer (pH 2.0) for 5 min and neutralized with 1M phosphate buffer and Laemli sample buffer. The protein sample was then heated at 100 °C for 5 min.

1-17. statistical analysis

All quantitative data were expressed as mean ± standard error of the mean. Data were analyzed using SigmaPlot 12 software, and sample sizes for animal studies were determined by SigmaPlot 12 software. Comparison between the two experimental groups was performed using the two-tailed Student's t test. The means of multiple experimental groups were compared using one-way analysis of variance, followed by the Student-Newman-Keuls test for multiple comparisons. A p value of <0.05 was considered statistically significant.

Example 2: Experimental results

2-1. Effect of urolithin A on high glucose-induced mitochondrial calcium influx and mtROS accumulation.

First, we investigated the effects of ellagic acid and urolithin on cell viability of SH-SY5Y cells under hyperglycemic conditions. Pretreatment with urolitin A, urolitin B, urolitin C, and urolitin D significantly reduced the level of high glucose-induced LDH release (Figure 1). In the hyperglycemia group, the LDH release level was lowest in the urolithin A pretreatment compared to other pretreatments. In the WST-1 cell viability assay, it was confirmed that the survival rate of SHSY5Y cells pretreated with urolithin A with high glucose was higher than that of SH-SY5Y cells pretreated with vehicle with high glucose. MitoSOX staining analysis showed that urolithin A pretreatment reduced high glucose-induced mtROS levels (Figure 2). However, the effects of ellagic acid and other urolithins were not found to be significant. Additionally, high glucose conditions for 24 to 48 h increased the rhod-2-positive cell population in SH-SY5Y, indicating that high glucose induces mitochondrial calcium influx (Figure 3). We found that urolithin A pretreatment inhibited high glucose-stimulated mitochondrial calcium accumulation but not mitochondrial permeability transition pore (mPTP) opening (Figures 4, 5). High glucose-induced intracellular and mtROS levels were suppressed by pretreatment with the MCU (mitochondrial calcium uniporter) inhibitor Ru360 (Figures 6, 7). These results indicate that the effect of urolithin A on reducing mitochondrial calcium accumulation is important for maintaining mtROS homeostasis in SH-SY5Y cells under high glucose.

2-2. Effects of urolithin A on high glucose-induced amyloidogenesis and neurodegeneration in neurons and streptozotocin (STZ)-induced diabetic mice.

To determine the effect of urolithin on amyloid production in neuronal cells under high glucose, we measured the levels of secreted Aβ(1-42) in SH-SY5Y and iPSC-derived neural differentiated cells (iPSC-ND). We found that high concentrations of glucose increased APP and BACE1 expression in a time-dependent manner. In addition, we confirmed that Aβ (1-42) concentration levels were significantly reduced in SH-SY5Y cells cultured in medium containing urolithin A and urolithin B but not ellagic acid, urolithin C, or urolithin D ( Figure 8). Since the inhibitory effect of urolithin A on Aβ secretion was the strongest compared to other substrates, we tested the effect of urolithin A on Aβ concentration in iPSC-ND cells under high glucose. In iPSCND conditioned medium, the Aβ(1-42) concentration of urolithin A pretreatment with high glucose group was lower than vehicle pretreatment with high glucose group (Figure 9). Urolithin A pretreatment inhibited high glucose-induced APP and BACE1 expression in both SH-SY5Y and iPSC-ND cells (Figure 10). High glucose-induced APP and BACE1 expression levels were suppressed by pretreatment with Ru360 or the mtROS-specific scavenger Mito-TEMPO (Figures 11, 12). These results suggest that urolithin A-inhibited Aβ production is mediated by inhibition of mitochondrial calcium and mtROS accumulation. Additionally, the effects of urolithin A on STZ-induced cognitive impairment, amyloidogenesis, Tau phosphorylation, and Aβ deposition in the prefrontal cortex and hippocampus of mouse brain tissue were determined. Urolithin A had no effect on body weight gain in control or STZ mice (Figure 13). Blood glucose levels in urolithin A-injected control mice were significantly lower than vehicle-injected control mice, but this difference between vehicle-injected STZ and urolithin A-injected STZ mice was not statistically significant (Figure 14). The Y-maze voluntary alternation test was performed to assess cognitive function. We observed that STZ infusion reduced the rate of spontaneous alternation, which was reversed by urolithin A infusion (Figure 15). In prefrontal cortex and hippocampus tissue, urolithin A injection suppressed APP, BACE1, p-Tau (S262 and S396) and Aβ levels (1-42) in STZ-induced diabetic mice (Figures 16-18). Therefore, the in vitro and in vivo experimental results suggest that urolithin A treatment can inhibit amyloid production and neuronal degeneration in the DM model.

2-3. Role of transglutaminase type 2 (TGM2) Effects of urolithin A on reducing mitochondrial-ER contacts, mitochondrial calcium influx, mtROS accumulation, and amyloidogenesis under high glucose.

mRNA expression of proteins that regulate mitochondrial calcium influx, such as VDAC1, MCU1, mitochondrial calcium uptake 1 (MICU1), mitochondrial calcium uptake 2 (MICU2), MCU regulator 1 (MCUR1) and MCU-dominant negative beta subunit (MCUB) The effect of hyperglycemia on was investigated. We found that the mRNA expression levels of VDAC1 and MCU1 were upregulated in SH-SY5Y under high glucose conditions (Figure 19). However, urolithin A pretreatment had no effect on high glucose-induced VDAC1 and MCU1 mRNA and protein expression (Figures 20, 21). Additionally, Ru360 pretreatment did not alter high glucose-stimulated VDAC1 and MCU1 protein expression. We further investigated the effects of high glucose and urolithin A on the interaction between ER and mitochondria. As shown in Figure 22, high glucose increased co-localization of MitoTracker-positive and ER-Tracker-positive fluorescence in SH-SY5Y cells, which was reversed by urolithin A pretreatment. In coimmunoprecipitation and in situ proximity ligation assay (PLA) experiments, high glucose increased the direct interaction between IP3R1, IP3R3, and VDAC1, which was inhibited by urolithin A pretreatment (Figures 23, 24, 25). Additionally, the expression of mitochondria-ER contact regulatory adapter proteins such as Bcl-2-associated Changes in mRNA expression were investigated. However, high glucose only stimulated TGM2 mRNA expression, which was reversed by urolithin A pretreatment (Figure 26). TGM2 induction by high glucose was inhibited by MitoTEMPO pretreatment. We confirmed the inhibitory effect of urolithin A pretreatment on high glucose-induced TGM2 expression in both SH-SY5Y and iPSC-ND cells (Figure 27). In addition to urolithin A inhibited TGM2 expression, it also reduced high glucose-induced TGM2 activity in SH-SY5Y cells (Figure 28). In mouse brain samples, urolithin A injection significantly reduced STZ-induced TGM2 expression in both prefrontal cortex and hippocampal tissue (Figure 29). Taken together, urolithin A inhibited TGM2 expression, which is important for inhibiting IP3R1-VDAC1 interaction in neurons exposed to a high glucose environment. To determine the role of TGM2 in mitochondria-ER contacts and amyloidogenesis under high glucose, we examined the effect of TGM2 silencing in SH-SY5Y cells. We observed that TGM2 silencing inhibited high glucose-induced colocalization of MitoTracker-positive and ER-Tracker-positive fluorescence in SH-SY5Y cells (Figure 30). In co-immunoprecipitation and PLA experiments, TGM2 silencing downregulated high glucose-induced IP3R1-VDAC1 interaction (Figs 31, 32). High glucose-induced mitochondrial calcium and mtROS levels in SH-SY5Y cells were suppressed by TGM2 silencing (Figure 33). Additionally, TGM2 silencing prevented high glucose-induced Aβ secretion as well as APP and BACE1 expression (Figure 34). Based on these findings, urolithin-denormalized high-glucose-induced TGM2 expression disrupts the IP3R1-VDAC1 interaction, inhibiting mitochondrial calcium influx and mtROS accumulation under high glucose.

2-4. Role of urolithin A-mediated AIP-AhR complex promotion in high-glucose-induced TGM2 expression.

We sought to determine the mechanism of how urolithin A suppresses high glucose-induced TGM2 expression. It was confirmed that the protein expression level of aryl hydrocarbon receptor (AhR) increased at 24 and 48 hours of high glucose treatment, but that of AhR-interacting protein (AIP) did not increase (FIG. 35). Afterwards, we confirmed the effect of urolitin A on the mRNA expression of AhR-regulated adapter proteins such as prostaglandin E synthase 3 (PTGES3), AIP, and heat shock protein 90 α family class A member 1 (HSP90AA1) in SH-SY5Y high-glucose cells. did. However, high glucose and urolithin A did not affect the mRNA or protein expression of AIP in SHSY5Y cells. High glucose increased nuclear AhR expression levels, which was suppressed by urolithin A pretreatment (Figures 36, 37). High glucose-induced TGM2 mRNA and protein expression levels were suppressed by pretreatment with the AhR inhibitor CH-223191 (Figures 38, 39). Additionally, high glucose significantly inhibited the interaction between AhR and AIP, which was restored by urolithin A pretreatment (Figures 40, 41, 42). These findings indicate that urolithin A inhibits high glucose-activated AhR signaling by promoting the formation of the AIP-AhR complex to suppress TGM2 expression.

2-5. Protective effects of urolithin A and TGM2 silencing on Aβ -stimulated mitochondrial calcium influx, mtROS accumulation, Tau phosphorylation, and neuronal apoptosis.

Next, we investigated the effects of urolithin A on Aβ-stimulated mitochondrial calcium influx, mtROS accumulation and neuronal cell death. Aβ treatment significantly increased the level of LDH release in SH-SY5Y cells at 48 and 72 h (Figure 43). In SH-SY5Y and iPSC-ND cells, urolithin A pretreatment inhibited Aβ increased mitochondrial calcium levels (Figures 44, 45). It was confirmed that urolithin A pretreatment prevented Aβ-induced mtROS accumulation in SHSY5Y and iPSC-ND cells (Figures 46 and 47). Moreover, urolithin A pretreatment blocked LDH release from SHSY5Y cells treated with Aβ for 72 h (Figure 48). We further investigated the effect of TGM2 silencing on Aβ-treated neurons and APP Swedish mutant SK-N-MC cells (APPSwe) secreting Aβ. TGM2 silencing reduced Aβ reduced mitochondrial calcium levels in SH-SY5Y cells (Figure 49). TGM2 silencing or urolithin A pretreatment reduced Aβ increased mitochondrial calcium levels in APPSwe cells (Figure 50). Additionally, TGM2 silencing suppressed Aβ-induced mtROS accumulation in SH-SY5Y (Figure 51) and APPSwe cells transfected with TGM2 siRNA showed lower mtROS levels than APPSwe cells transfected with NT siRNA (Figure 52). Urolithin A pretreatment also inhibited Aβ-induced mtROS accumulation in APPSwe cells (Figure 52). TGM2 silencing significantly dephosphorylated Aβ-stimulated Tau phosphorylation (Figure 53). In APPSwe cells, both TGM2 silencing and urolithin A pretreatment reduced Tau phosphorylation (Figure 54). The level of LDH release from SH-SY5Y cells treated with Aβ was also reduced by TGM2 silencing (Figure 55). Taken together, the results of the above examples show that urolithin A pretreatment and TGM2 silencing prevent Aβ-induced mitochondrial calcium influx, mtROS accumulation, Tau phosphorylation, and cell death in neuronal cells.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical idea or essential features. In this regard, the embodiments described above should be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed as including the meaning and scope of the patent claims described below rather than the detailed description above, and all changes or modified forms derived from the equivalent concept thereof are included in the scope of the present invention.

Claims (6)

delete In in vitro conditions, treatment of neuroblastoma cell lines SH-SY5 or iPSC-ND under high glucose conditions with urolithin A reduced LDH production in these cells; Reduce mitochondrial reactive oxygen species generation; Inhibition of mitochondrial calcium accumulation; Reduced expression of amyloid beta; Inhibition of TGM2 expression; and promoting the formation of an AIP-AhR complex.
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