Cholesterol synthesis pathway suppressing and inhibiting agent

Abstract

[Problem] The present invention has been made in response to the need to elucidate the mechanism of action underlying a previously confirmed reduction in plasma and liver total cholesterol levels. The present invention relates to producing and providing an inhibitor that contains 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient and is highly effective for directly suppressing and inhibiting a cholesterol synthesis pathway by acting on HMGCR via SREBP2. [Solution] The present invention is characterized by having suppressive and inhibitory effects on a cholesterol synthesis pathway on the basis of the action of 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient on HMGCR via SREBP2.

Description

Cholesterol synthesis pathway suppression and inhibitor

[0001] The present invention relates to a cholesterol synthesis pathway suppressor or inhibitor that inhibits the cholesterol synthesis pathway in order to reduce the amount of cholesterol in the liver and plasma.

[0002] Nonalcoholic steatohepatitis (NASH) is caused by the accumulation of fat in the liver due to factors other than alcohol consumption, and increases the risk of developing cirrhosis and liver cancer. Therefore, there is always a lot of interest in measures to prevent liver fat accumulation.

[0003] The DHMBA of the present invention, i.e., 3,5-dihydroxy-4-methoxybenzyl alcohol, is a food ingredient obtained primarily from oyster extract. The applicant has discovered that DHMBA has the effect of reducing cholesterol levels in the liver and plasma, and has already filed a patent application.

[0004] However, the detailed mechanism of action has not been fully elucidated. Therefore, in this application, we focused on the possibility that DHMBA has an inhibitory effect on the cholesterol synthesis pathway, and clarified the changes in gene or protein expression levels of enzymes involved in cholesterol synthesis, mainly HMGCR and SREBP2.

[0005] We found that DHMBA acts on HMGCR via SREBP2, suppressing and inhibiting the cholesterol synthesis pathway.

[0006] DHMBA is currently sold as a functional food, and its value as a food with effects such as preventing NASH is expected to increase even further.

[0007] As mentioned above, DHMBA is a compound obtained mainly from oyster extracts. The present inventors have found that feeding a DHMBA-rich diet to mice with a nonalcoholic steatohepatitis (NASH) model accompanied by fatty liver not only protects the liver but also reduces total cholesterol levels in plasma and liver. From these results, it is inferred that DHMBA leads to a decrease in cholesterol metabolism, but the mechanism of action for reducing total cholesterol levels in plasma and liver has not been clearly elucidated.

[0008] NASH is caused by the accumulation of fat in the liver due to overnutrition, obesity, and insulin resistance, and increases the risk of progressing to cirrhosis and liver cancer. The prevalence of NASH in the United States is estimated to have increased from 1.51% to 2.79% over the 10-year period from 2010 to 2020, and the number of patients continues to increase both domestically and internationally. However, effective treatments for NASH have yet to be established. Given this background, the prevention and improvement of ectopic fat accumulation are of particular interest.

[0009] Cholesterol synthesis in the liver is thought to be catalyzed by multiple enzymes. Among these, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) is the rate-limiting enzyme in this synthesis pathway and is known to be the target of statin drugs, which are used internationally to inhibit cholesterol synthesis. Sterol Regulatory Element Binding Transcription Factor 2 (SREBP2) is a factor that globally regulates cholesterol synthesis-related enzymes.

[0010] The low density lipoprotein receptor (LDLR) is under the control of SREBP2 and plays a role in regulating plasma cholesterol levels through LDL uptake.

[0011] Based on these findings, we focused on the possibility that the food compound DHMBA may have an inhibitory effect on the cholesterol synthesis pathway as one of the strategies to prevent fat accumulation in the liver. That is, in this application, we added DHMBA to cultured human hepatocytes and clarified the changes in gene or protein expression levels of enzymes involved in cholesterol synthesis, mainly HMGCR and SREBP2.

[0012] JP 2010-193756 A

[0013] The present invention has been made in view of the need for a clear elucidation of the mechanism of action for reducing total cholesterol levels in plasma and liver, which has hitherto existed. The present invention aims to provide an inhibitor containing 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient, which has the excellent effect of acting on HMGCR via SREBP2 and suppressing and inhibiting the cholesterol synthesis pathway itself.

[0014] The present invention is characterized by: having 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient, acting on HMGCR via SREBP2 to suppress and inhibit the cholesterol synthesis pathway; or having 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient, regulating via SREBP2 to reduce genes and SREBP2, and acting on HMGCR via the reduced SREBP2 to suppress and inhibit the cholesterol synthesis pathway; or having 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient, regulating via several SREBP2 including HMGCR to reduce genes and SREBP2, and acting on HMGCR via the reduced SREBP2 to suppress and inhibit the cholesterol synthesis pathway.

[0015] The present invention was made in consideration of the need for a clear elucidation of the mechanism of action for reducing total cholesterol levels in plasma and liver, which has hitherto been the case. The present invention produces an inhibitor containing 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient, which acts on HMGCR via SREBP2 and has the excellent effect of suppressing and inhibiting the cholesterol synthesis pathway itself.

[0016] This table shows the primer sequences used in real-time PCR. This graph shows the results of gene expression analysis. Figure 3(a) shows the control group as 1-4 and the DHMBA group as 5-8. The lower panel shows the membrane immersed in CBB solution and stained for total protein for correction. Figure 3(b) shows the HMGCR luminescence intensity corrected for total protein color intensity, quantified using ImageJ. This diagram shows the mechanism by which DHMBA inhibits the cholesterol synthesis pathway. This diagram shows the relationship between enzymes involved in cholesterol synthesis in the liver and SREBP2, which regulates them. The decreased expression levels of several genes, including SREBP2 and HMGCR, suggest that DHMBA acts on HMGCR via SREBP2 to suppress and inhibit cholesterol synthesis. Furthermore, one possible reason for the increased LDLR gene expression level is that it may have increased expression to compensate for the decrease in intracellular cholesterol levels associated with the suppression of cholesterol synthesis. Abbreviations used in Figure 4 are as follows: SOAT1: Sterol O-Acyltransferase 1, HMGCS: 3-Hydroxy-3-Methylglutaryl-CoA Synthase, HMGCR: 3-Hydroxy-3-Methylglutaryl-CoA Reductase, MVK: Mevalonate Kinase, FDPS: Farnesyl Diphosphate Synthase, MVD: Mevalonate Diphosphate Decarboxylase, FDFT1: Farnesyl-Diphosphate Farnesyltransferase 1, SC5D: Sterol-C5-Desaturase, DHCR7: 7-Dehydrocholesterol Reductase, LDLR: Low-density lipoprotein receptor. The down arrow to the right of the enzyme name indicates a decrease in gene expression due to the action of DHMBA, and the up arrow indicates an increase.

[0017] In the present invention, human cultured hepatocytes C3A were cultured, stimulated with DHMBA, and then collected. The collected cells were then subjected to gene expression analysis and protein expression analysis. In other words, changes in gene expression and protein expression were clarified.

[0018] As a result, it was revealed that DHMBA acts on HMGCR via SREBP2, suppressing and inhibiting the cholesterol synthesis pathway, based on the reduction of several SREBP2-regulated genes, including HMGCR, and SREBP2 (see Figure 4).

[0019] (Subjects and Methods) 1. Cell Culture: Human cultured hepatocytes, C3A, purchased from the American Type Culture Collection were used. The culture medium was Gibco Minimum Essential Media (MEM) containing GlutaMAX, supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin-neomycin. The cells were cultured in an incubator (37°C, 5% CO2).

[0020] 2. DHMBA stimulation and cell recovery. 2.0 × 10 C3A cells were added to a 24-well plate. 5 Cells were seeded at 1000 cells / well and allowed to settle for 24 hours. Afterwards, the cells were divided into a control group (PBS) and a DHMBA group (final concentration: 250 μM), and stimulated with either of these dissolved in culture medium for 24 hours.

[0021] 3. Gene Expression Analysis After stimulation as described above, the cells were harvested (n = 8 per group). TMRNA (ribonucleic acid) was extracted from the harvested cells using an RNA Mini Kit (Thermo Fisher Scientific). After quantification of RNA purity and concentration using a NanoDropOne (Thermo Fisher Scientific), 1000 ng of RNA was converted to cDNA using ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo).

[0022] The cholesterol synthesis-related genes HMGCR, SREBP2, sterol O-acyltransferase 1 (SOAT1), 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1), mevalonate kinase (MVK), mevalonate diphosphate decarboxylase (MVD), farnesyl diphosphate synthase (FDPS), farnesyl-diphosphate farnesyltransferase 1 (FDFT1), sterol-C5-desaturase (SC5D), 7-dehydrocholesterol reductase (DHCR7), LDLR related to LDL uptake, and proprotein convertase subtilisin / kexin type 9 (PCSK9) were measured as targets (all corrected for β-actin).

[0023] The primer sequences used in this study are shown in Figure 1. Real-time PCR measurements were performed using THUNDERBIRD SYBR qPCR Mix (TOYOBO) as the reagent and CFX Connect Real-times System (Bio-Rad Laboratories, Inc.) as the measurement equipment.

[0024] Protein expression analysis: After stimulation as described above, cells were harvested from three wells (n = 4 per group). Proteins were extracted from the cells using RIPA buffer (Fujifilm) supplemented with protease inhibitor cocktail (P8340, Sigma-Aldrich), phosphatase inhibitor cocktail (P5726, Sigma-Aldrich), and 10 ng / mL phenylmethylsulfonyl fluoride (PMSF).

[0025] And Pierce TM Protein concentration was measured using a BCA Protein Assay Kit (Thermo Fisher Scientific). Samples containing 1.25 μg of protein were loaded onto a 5-20% gradient gel and subjected to polyacrylamide gel electrophoresis. The electrophoresis equipment used was a pageRun Ace (Hi mode, WSE-1150, ATTO).

[0026] After electrophoresis, the gel, a wetted PVDF membrane, and filter paper were layered, and the gel proteins were transferred to the PVDF membrane. A PoweredBLOT-One (standard mode, 60 minutes, WSE-4110, ATTO) was used for transfer. After blocking the PVDF membrane with EzBlock Chemi (AE-1475, ATTO), an antigen-antibody reaction was performed using an anti-human HMGCR antibody (Abcam, ab242315, dilution 1:2000) as the primary antibody and a goat anti-mouse IgG HRP (Santacruz, sc-2005, dilution 1:2500) as the secondary antibody. Washing was performed with Tris-Buffered Saline with Tween 20 (T9142, Takara Bio Inc.). After the reaction, luminescence intensity was measured using the luminescent substrate EzWestLumi plus (WSE-7120S, ATTO). The total amount of protein transferred to the PVDF membrane was stained with CBB using EzStainAQua (AE-1340, ATTO), and the luminescence intensity of HMGCR was corrected based on the color intensity.TM MP Imaging System (BIO-RAD) and ImageJ were used for analysis.

[0027] 5. Statistical analysis: Statistical analysis was performed using an unpaired t-test. The significance level was set at 5% (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant).

[0028] (Results) 1. Changes in gene expression levels. Among cholesterol synthesis-related enzymes, HMGCR, SREBP2, HMGCS1, FDFT1, MVD, and DHCR7 were significantly decreased in the DHMBA group compared to the control group (Figure 2). LDLR was also significantly increased in the DHMBA group. There were no significant differences between the groups in the expression levels of SOAT1, MVK, FDPS, SC5D, and PCSK9.

[0029] 2. Changes in protein expression levels At the protein level, a statistically significant decrease in HMGCR was observed in the DHMBA group compared to the control group (see Figure 3).

[0030] (Discussion) The reduction of several SREBP2-regulated genes, including HMGCR, and SREBP2 indicated that DHMBA acts on HMGCR via SREBP2 to suppress and inhibit the cholesterol synthesis pathway (see Figure 4).

[0031] Here, the HMGCR (3-hydroxy-3-methylglutaryl-coenzyme A reductase) is also called HMG-CoA reductase and belongs to the HMG-CoA reductase family.

[0032] Cholesterol synthesis is thought to occur through approximately 30 enzymatic reactions, starting from acetyl-CoA as a starting substrate. It is estimated that approximately 1 gram of a compound with a complex 27-carbon structure is synthesized in the human liver per day from a compound with only two carbon atoms. Therefore, blocking this cholesterol synthesis pathway is effective in reducing the amount of cholesterol in the body. Based on this idea, the inventors of this invention have invented and developed DHMBA, which functions as an inhibitor of the cholesterol synthesis pathway.

[0033] Cholesterol synthesis is regulated by a sophisticated negative feedback mechanism, in which the expression of genes encoding all enzymes involved in cholesterol synthesis, including HMG-CoA reductase, is reduced with increasing cholesterol levels.

[0034] The HMG-CoA reductase protein is rapidly degraded when the amount of intracellular cholesterol increases. HMG-CoA reductase activity is precisely controlled as a regulator of intracellular cholesterol levels, and DHMBA functions as one of the regulatory mechanisms, i.e., as an inhibitor of the cholesterol synthesis pathway.

[0035] Figure 4 shows the relationship between enzymes involved in cholesterol synthesis in the liver and SREBP2, which controls them.

[0036] Administration of DHMBA reduced the expression levels of several genes, including SREBP2 and HMGCR, suggesting that DHMBA acts on HMGCR via SREBP2 to suppress cholesterol synthesis.

[0037] In addition, the increased expression level of the LDLR gene may be due to the increase in expression level to compensate for the decrease in intracellular cholesterol amount caused by the suppression of cholesterol synthesis.

[0038] The full spelling of the spelling abbreviations in Figure 4 is as follows: SOAT1: Sterol O-Acyltransferase 1, HMGCS: 3-Hydroxy-3-Methylglutaryl-CoA Synthase, HMGCR: 3-Hydroxy-3-Methylglutaryl-CoA Reductase, MVK: Mevalonate Kinase, FDPS: Farnesyl Diphosphate Synthase, MVD: Mevalonate Diphosphate Decarboxylase, FDFT1: Farnesyl-Diphosphate Farnesyltransferase 1, SC5D: Sterol-C5-Desaturase, DHCR7: 7-Dehydrocholesterol Reductase, LDLR: Low-density lipoprotein receptor.

[0039] In FIG. 4, the downward arrow to the right of the enzyme name indicates a decrease in gene expression due to the action of DHMBA, and the upward arrow indicates an increase in gene expression due to the action of DHMBA.

[0040] It is believed that this inhibitory effect of DHMBA administration led to the reduction in total cholesterol levels in plasma and liver, as observed in in vivo studies.

[0041] On the other hand, LDLR expression increased. It is speculated that the increased expression level may have compensated for the decrease in intracellular cholesterol levels due to the suppression of cholesterol synthesis.

[0042] Since LDLR plays an important role in lowering plasma cholesterol, a more detailed understanding of the mechanism is considered a future challenge.

[0043] Statins are known to have the effect of inhibiting cholesterol synthesis. Their mechanism of action is to competitively inhibit HMGCR, leading to a decrease in intracellular cholesterol levels, and to promote SREBP2 synthesis through a feedback mechanism.

[0044] As a result, the expression of LDLR, a downstream gene of SREBP2, increases, reducing plasma cholesterol.

[0045] It has been revealed that DHMBA of the present invention acts on HMGCR via SREBP2 and suppresses and inhibits the cholesterol synthesis pathway itself, and therefore has an action that is partially different from that of statin drugs.

[0046] Soystatin, a food-derived cholesterol-lowering substance with a different mechanism of action from statin drugs, is derived from soybeans. DHMBA of the present invention is derived from oysters, so it can also be called "oystatin."

[0047] DHMBA is already on the market as a food with functional claims. By adding the new knowledge revealed in this invention that it has the ability to suppress and inhibit the cholesterol synthesis pathway, it is expected that its value as a food that can prevent NASH and improve high cholesterol levels will be further enhanced.

Claims

1. A cholesterol synthesis pathway inhibitor containing 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient, which acts on HMGCR via SREBP2 to suppress and inhibit the cholesterol synthesis pathway.

2. A cholesterol synthesis pathway suppressor / inhibitor, characterized by having 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient, which is regulated by SREBP2 to reduce genes and SREBP2, and acts on HMGCR via the reduced SREBP2 to suppress and inhibit the cholesterol synthesis pathway.

3. A cholesterol synthesis pathway suppressor / inhibitor, characterized by having 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient, which reduces genes and SREBP2 by regulating several SREBP2s including HMGCR, and acts on HMGCR via the reduced SREBP2 to suppress and inhibit the cholesterol synthesis pathway.

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