New application of two procyanidins
By studying the mechanism of action of Procyanidin C1 and procyanidin C2, it was found that it can inhibit lipid accumulation and oxidative stress, improve mitochondrial function, solve the problems of lipid accumulation and oxidative stress in NAFLD, and achieve the effect of anti-non-alcoholic fatty liver.
Patent Information
- Application Number
- CN202410630492.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-05-21
- Publication Date
- 2025-03-11
AI Technical Summary
The pathogenesis of non-alcoholic fatty liver disease (NAFLD) is unclear, and the prior art is difficult to effectively inhibit lipid accumulation and oxidative stress.
The study found that Procyanidin C1 and procyanidin C2 have the activity of inhibiting lipid accumulation and oxidative stress in HepG2 cells induced by oleic acid. By inhibiting the expression of FAS, ACC, SCD-1, and SREBP-1c, it upregulates the expression of PPARα and CTP1A proteins, activates the AMPK/mTOR signaling pathway, and reduces fat production.
Procyanidin C1 and procyanidin C2 significantly reduce lipid accumulation, improve redox levels, improve mitochondrial function, and have the effect of anti-non-alcoholic fatty liver.
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Figure CN119587529A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of natural medicines. Background Art
[0002] Non-alcoholic fatty liver disease (NAFLD) is one of the most common health problems worldwide, and its main feature is the accumulation of lipids in the absence of heavy alcohol consumption. Clinically, NAFLD can be histologically divided into non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH). NAFLD is closely related to metabolic disorders such as type 2 diabetes, obesity, dyslipidemia, atherosclerosis, and hypertension. The pathogenesis of NAFLD is still unclear, and the "two-hit" hypothesis (recently updated to "multiple hits") has always been the mainstream theory. The "first hit" is the excessive accumulation of triglycerides (TG) and free fatty acids, while the "second hit" refers to inflammation, oxidative stress, and apoptosis after the "first hit."
[0003] The liver, diet, de novo lipogenesis, and fatty acids released from adipose and other tissues are the three main sources of free fatty acids. De novo lipogenesis during fasting accounts for 30% of the liver fatty acid pool. Enhanced efflux of free fatty acids to hepatocytes leads to lipotoxicity, lipid metabolism disorders, and excessive lipid accumulation, which promote the pathogenesis of NAFLD. Hepatic lipid metabolism may be regulated by lipogenesis-related proteins such as SREBP1, FAS, and SCD-1. In addition, peroxisome proliferator-activated receptors (PPARs) play a key role in regulating lipid synthesis, storage, fatty acid oxidation, and lipogenesis. C / EBPβ induces the expression of PPARγ and C / EBPα, forming a positive feedback loop that helps to induce and maintain the expression of adipocyte-specific genes. Accumulated fat is mainly stored in lipid droplets of adipose tissue in the form of TG.
[0004] Procyanidin C1 and procyanidin C2 are B-type trimers of proanthocyanidins. The present invention intends to study whether the above two proanthocyanidin trimers have the activity of inhibiting lipid accumulation and resisting non-alcoholic fatty liver disease. Summary of the invention
[0005] The present invention studies and finds the effects of procyanidin C1 and procyanidin C2 on inhibiting lipid accumulation and oxidative stress in HepG2 cells induced by oleic acid (OA).
[0006] The structural formulas of the two proanthocyanidins are as follows:
[0007]
[0008] Based on this, the present invention provides the use of procyanidin Procyanidin C1 or\ and procyanidin C2 in the preparation of a medicament for preventing or treating non-alcoholic fatty liver disease.
[0009] Furthermore, the drug is a drug that inhibits lipid accumulation.
[0010] Furthermore, the drug is a drug that inhibits hepatic lipid accumulation.
[0011] Furthermore, the lipid is selected from TG and / or TC.
[0012] TG: triglyceride, TC: total cholesterol.
[0013] Furthermore, the drug is an antioxidant drug.
[0014] Furthermore, the drug is a drug that increases the SOD content and reduces the MDA content.
[0015] Superoxide dismutase (SOD) is an important member of the antioxidant enzyme system in biological systems.
[0016] MDA, malondialdehyde, is one of the commonly used indicators to measure the degree of oxidative stress.
[0017] Lipid droplets are spherical organelles composed of a neutral lipid core, a monolayer phospholipid membrane and surface proteins. Cellular oxidative stress leads to an increase in the number of lipid droplets, and the increase in lipid droplets alleviates cellular oxidative stress, but the molecular mechanism by which lipid droplets regulate cellular oxidative stress is still unclear. The main cause of cellular oxidative stress is mitochondrial oxidative damage, which causes mitochondria to release more reactive oxygen, thereby causing mitochondrial damage, endoplasmic reticulum stress, etc., and triggering cell apoptosis. Studies have shown that lipid droplets and mitochondria can contact each other, and it is speculated that the interaction between lipid droplets and mitochondria will regulate mitochondrial damage, thereby regulating cellular oxidative stress.
[0018] Furthermore, the drug is a drug that improves mitochondrial function.
[0019] Furthermore, the drug is a drug for preventing or repairing mitochondrial damage.
[0020] Furthermore, the drug is a drug that reduces the expression of lipogenic protein via the AMPK / mTOR signaling pathway.
[0021] AMPK is a highly conserved metabolic regulator that maintains energy balance under metabolic stress at both the cellular and physiological levels. Cells continuously adapt their metabolism to meet energy demands and respond to the availability of nutrients. In general, AMPK is activated by sensing changes in the ratio of AMP:ATP and ADP:ATP, restoring energy balance by inhibiting anabolic processes that consume ATP and promoting catabolic processes that produce ATP. In addition, AMPK activity is regulated by multiple upstream signals, making AMPK a central node that cells utilize to coordinate their metabolism with specific energy needs. In addition, the therapeutic potential of AMPK has been widely recognized and used to treat metabolic diseases such as diabetes, obesity, inflammation, and cancer. mTOR is an integrator of growth factor and nutrient signals, integrating multiple upstream signals to regulate cell growth. The main function of mTORC1 is to regulate protein synthesis and cell cycle progression, while mTORC2 can play an important role in actin cytoskeleton organization and cell survival.
[0022] AMPK and mTORC1 are key cellular nutrient indicators and cell growth regulators. Therefore, dysregulation of this pathway is associated with many human diseases. For example, uncontrolled mTORC1 activation through mutations in TSC1 / TSC2 or constitutive activation of the PI3K pathway can lead to tumorigenesis. Therefore, mTORC1 inhibitors have been shown to have therapeutic effects in TSC diseases. In addition, mTORC1 inhibitors, rapamycin analogs, have been approved as drugs for the treatment of advanced renal cancer, and many clinical trials are using mTOR inhibitors for cancer treatment. mTORC1 can also regulate protein metabolism through autophagy, targeting synthesis and degradation; therefore, inhibition of mTORC1 may help develop drugs for proteinopathies, including neurodegenerative diseases such as Alzheimer's disease. Similarly, defects in AMPK activation may be associated with tumorigenesis, as the upstream kinase LKB1 has long been considered a tumor suppressor. Metformin, the most widely used diabetes drug, has been reported to suppress cancer, as the incidence of cancer in patients using metformin was significantly reduced. In addition, the prominent roles of AMPK and mTOR in energy metabolism make them drug targets for metabolic diseases such as diabetes and obesity.
[0023] Furthermore, the drug is a drug that reduces the expression levels of FAS, ACC, SCD-1, and SREBP-1c, and upregulates the expression levels of PPARα and CTP1A proteins. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1.Changes in HepG2 cell viability after incubation with procyanidin C1 and procyanidin C2; (A) Chemical structure of procyanidin C1; (B) Chemical structure of procyanidin C2; (C) HepG2 cell viability after incubation with procyanidin C1. (D) HepG2 cell viability after incubation with procyanidin C2. (E) HepG2 cell viability after incubation with orilistat; (g) There was no change in HepG2 cell viability by incubating OA with procyanidin C1, procyanidin C2, and orilistat for 48 hours.
[0025] Figure 2 Procyanidin C1 and procyanidin C2 inhibit OA-induced lipid accumulation in HepG2 cells. HepG2 cells were incubated with procyanidin C1 and procyanidin C2 for 48 h in the presence of 0.5 mM OA. (A) Oil red O staining in HepG2 cells; (B) Nile red staining of lipid droplets in HepG2 cells; (C) Quantification of intracellular TG content in HepG2 cells; (D) Quantification of intracellular TC content in HepG2 cells.
[0026] Figure 3 Procyanidin C1 and procyanidin C2 inhibited OA-induced redox levels in HepG2 cells. (A) SOD content; (B) MDA content; (C) ROS level; (D) mitochondrial ROS level. Figure 4 .Procyanidin C1 and procyanidin C2 improve mitochondrial function. (A) SOD content; (B) MDA content; (C) ROS level.
[0027] Figure 5 Effects of procyanidin C1 and procyanidin C2 on lipid synthesis proteins. (A) Expression levels of SREBP-1c, ACC, FAS, SCD-1, and PPARγ proteins. (B) Expression levels of CPT1A and PPARα proteins. (C) Expression levels of Nrf2 and HO-1 proteins. (D) Expression levels of AMPK, mTOR, and JNK proteins. Figure 6 Effects of procyanidin C1 and procyanidin C2 on high-fat-induced non-alcoholic fatty liver disease in zebrafish. (A) Levels of biochemical indicators TG, TC, LDL-C, HDL-C, GSH and SOD; (B) HE staining of zebrafish liver. DETAILED DESCRIPTION
[0028] Example 1
[0029] 1. Cell activity study
[0030] 1.1. Chemicals and reagents
[0031] Procyanidin C1 and procyanidin C2 were purchased from Shanghai Yuanye. OA, Oil Red O, and MTT were purchased from SigmaAldrich. Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Gibco. Fetal bovine serum (FBS) was from Zhejiang Sijiqing. Triglyceride (TG) and superoxide dismutase (SOD) assay kits were from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). BCA assay kit and protein lysis buffer were purchased from Beyotime (Shanghai, China). Antibodies to FAS, SCD-1, SREBP1, PPARγ, C / EBPα, Nrf2, HO-1, and β-actin were purchased from Cell Signaling Technology.
[0032] 1.2.HepG2 cell culture
[0033] HepG2 is a human hepatoma cell line purchased from the Cell Resource Center, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). Cells were cultured at 37°C, 5% CO2 in DMEM supplemented with 10% FBS.
[0034] Cell viability assay
[0035] The cell viability was detected by MTT method. HepG2 cells in logarithmic growth phase were taken and the concentration was adjusted to 1×10 5 150 μL per well was inoculated in a 96-well plate and cultured for 24 hours. The original culture medium was replaced with a solution containing different concentrations of compounds (Procyanidin C1, procyanidin C2), and a blank control (Con) was set. After continuing to culture for 48 hours, the absorbance value (A) at a wavelength of 490 nm was detected by MTT method. The cell survival rate was calculated according to the formula. Cell survival rate / % = A treatment group / A blank group × 100%.
[0036] 1.4. Oil Red O staining
[0037] Oil red O staining was used to measure lipid droplet content in HepG2 cells. HepG2 cells were plated in 6-well plates at 5 × 10 4The cells were cultured at a density of 10 cells / mL, and 0.5 mM OA was added to the culture medium. The cells were treated with 10 μM Procyanidin C1 and procyanidin C2 for 48 hours. After treatment, the cells were washed twice with PBS and then fixed with 4% paraformaldehyde in the dark for 30 minutes. Subsequently, the treated cells were stained with Oil Red O solution for 1 hour. Lipid droplets were observed and photographed using an inverted optical microscope.
[0038] 1.5. Measurement of TG content
[0039] The kit was used to measure the intracellular TG content. In the presence of 0.5 mM OA, HepG2 cells were grown at a rate of 5 × 10 4 The cells were cultured in 6-well plates at a density of 1:1. The cells were incubated for 48 h in the presence or absence of 10 μM procyanidin C1, procyanidin C2. The treated cells were digested by trypsin. The digested centrifuged cells were sonicated on ice for 5 min before disruption.
[0040] 1.6. Measurement of SOD levels
[0041] The kit was used to determine the level of SOD in cells. In the presence of 0.5 mM OA, HepG2 cells were grown at a rate of 5 × 10 4 The cells were cultured in 6-well plates at the same density. The cells were incubated with 10 μM procyanidin C1 and procyanidin C2 for 48 h. The cell morphology was observed under an inverted microscope. In addition, the level of SOD was measured using a commercial kit according to the manufacturer's protocol.
[0042] Western blot analysis
[0043] The cells were washed with cold PBS and homogenized in RIPA buffer containing protease inhibitors. The homogenate was centrifuged at 12,000g for 15 minutes at 4°C, and the supernatant was collected. 20μg of protein samples were separated on 10% SDS polyacrylamide gels and transferred to PVDF membranes (Millipore. Billerica, MA, USA). The membrane was blocked with skim milk solution for 1 hour at room temperature and then incubated with primary antibodies overnight at 4°C. The membrane was washed twice with TBST buffer and incubated with secondary antibodies for 1 hour. β-actin was used as an internal control. The blots were visualized using 5200 Multi Luminescent imaging systems (Tanon, Shanghai, China), and the band intensity was analyzed using ImageJ (NIH).
[0044] 1.8. Establishment of zebrafish nonalcoholic fatty liver disease model and compound treatment
[0045] Juvenile zebrafish 20 days after fertilization of comparable size and weight were selected, with 30 zebrafish in each group. The model group was fed with a high-fat diet for 14 days, and was fed with a high-fat diet and high-protein brine shrimp once in the morning and evening to establish the model. After 14 days, the drug was immersed in drugs (Procyanidin C1, procyanidin C2, positive drug orilistat, the treatment concentration of each compound was 5 μM). After one week, the whole tissues of the juveniles were taken to measure the levels of triglycerides, total cholesterol, high-density lipoprotein, low-density lipoprotein, SOD and GSH.
[0046] Statistical analysis
[0047] Data analysis was performed using GraphPad Prism 7.0 (GraphPad Software, Inc., San Diego, CA, USA). Results are expressed as the mean standard deviation (SD) of three independent experiments. Unpaired t-test (two groups) or one-way ANOVA (three or more groups) were used for group comparisons. p<0.05 was considered statistically significant.
[0048] 2. Results
[0049] 2.1 Effects of Procyanidin C1 and Procyanidin C2 on Cell Viability
[0050] The MTT assay was initially used to determine the cytotoxicity of compounds to HepG2 cells. Figure 1 As shown, compared with the control group, the compounds Procyanidin C1 and procyanidin C2 had no cytotoxic effect on the viability of HepG2 cells at concentrations between 1 and 100 μM. In addition, the positive drug orilistat had no effect on cell viability. Therefore, the optimal treatment concentration of the compounds Procyanidin C1, procyanidin C2 and orilistat was 10 μM.
[0051] 2.2 Compounds Procyanidin C1 and Procyanidin C2 inhibit lipid accumulation in OA-treated HepG2 cells
[0052] To investigate whether procyanidin C1 and procyanidin C2 affect intracellular lipid accumulation, Oil Red O staining was performed. Compared with the control group, 0.5 mM OA led to a significant increase in lipid droplet accumulation in HepG2 cells ( Figure 2A, 2B). Compared with the model group, intracellular lipid droplets were significantly reduced after incubation with procyanidin C1 and procyanidin C2 for 48 hours. In addition, compared with the model group, the TG and TC contents were significantly reduced after procyanidin C1 and procyanidin C2 treatment ( Figure 2 C, 2D).
[0053] 2.3 Effects of Procyanidin C1 and Procyanidin C2 on Redox Levels
[0054] Under oxidative stress conditions, cells respond to cellular damage by activating their antioxidant defense mechanisms. We investigated the effects of compounds procyanidin C1 and procyanidin C2 on oxidative stress by analyzing changes in SOD and MDA levels and reactive oxygen species. Figure 3 A, 3B, Compared with the model group, the compounds Procyanidin C1 and procyanidin C2 can increase the SOD content and reduce the MDA content in HepG2 cells after treatment. In addition, the compounds Procyanidin C1 and procyanidin C2 can also reduce the levels of total reactive oxygen species and mitochondrial reactive oxygen species ( Figure 3 C, 3D).
[0055] 2.4 Effects of Procyanidin C1 and Procyanidin C2 on Mitochondrial Function
[0056] In order to evaluate the effects of compounds procyanidin C1 and procyanidin C2 on mitochondrial function, we conducted mitochondrial membrane potential, mitochondrial mass number and electron microscopy to observe mitochondrial structure. Figure 4 Compared with the model group, procyanidin C1 and procyanidin C2 could increase the membrane potential of HepG2 cells ( Figure 4 A), and can also increase mitochondrial mass number ( Figure 4 B). In addition, the structure of mitochondria was significantly improved after treatment with procyanidin C1 and procyanidin C2, with an increase in the number of mitochondria and mitochondrial ridges ( Figure 4 C). The results showed that procyanidin C1 and procyanidin C2 significantly improved mitochondrial function and thus improved lipid accumulation.
[0057] 2.5. Effects of Procyanidin C1 and Procyanidin C2 on Lipid Synthesis Proteins
[0058] To determine the potential mechanism of the inhibitory effects of compounds procyanidin C1 and procyanidin C2 on lipid accumulation, the expression levels of proteins involved in lipogenesis, fatty acid oxidation, and related signaling pathways were analyzed by Western blotting. Figure 5 Compared with the model group, Procyanidin C1 and procyanidin C2 could significantly reduce the expression levels of FAS, ACC, SCD-1, and SREBP-1c in HepG2 cells. At the same time, the expression levels of PPARα and CTP1A proteins related to fatty acid oxidation were upregulated. In addition, Procyanidin C1 and procyanidin C2 could also significantly increase the expression levels of antioxidant proteins Nrf2 and HO-1. Finally, Procyanidin C1 and procyanidin C2 could also significantly upregulate the expression levels of phosphorylated AMPK and downregulate the expression levels of phosphorylated mTOR. These results indicate that Procyanidin C1 and procyanidin C2 reduce lipid accumulation in OA-treated HepG2 cells by reducing the expression of lipogenic proteins through the AMPK / mTOR signaling pathway, thereby improving non-alcoholic fatty liver disease.
[0059] Effects of Procyanidin C1 and Procyanidin C2 on High-fat-induced Non-alcoholic Fatty Liver Disease in Zebrafish
[0060] In order to determine the effects of compounds Procyanidin C1 and procyanidin C2 on high-fat-induced non-alcoholic fatty liver disease in zebrafish, we evaluated the effects of compounds Procyanidin C1 and procyanidin C2 on improving non-alcoholic fatty liver disease by biochemical indices and HE staining. Figure 6 Compared with the model group, procyanidin C1 and procyanidin C2 could significantly reduce the content of TG and TC in zebrafish, and also significantly increase the levels of HDL-C, GSH and SOD ( Figure 6 A). In addition, HE staining results showed that procyanidin C1 and procyanidin C2 could reduce lipid accumulation in zebrafish liver ( Figure 6 B).
[0061] 3. Conclusion
[0062] Based on the above experimental results, it was found that the proanthocyanidin compounds Procyanidin C1 and procyanidin C2 have the effects of reducing lipid accumulation and increasing redox levels, thereby having the effect of resisting non-alcoholic fatty liver disease.
Claims
1. Use of procyanidin C1 or procyanidin C2 in the preparation of a medicament for preventing or treating non-alcoholic fatty liver disease.
2. The use according to claim 1, characterized in that The drug is a drug that inhibits lipid accumulation.
3. The use according to claim 1 or 2, characterized in that The drug is a drug that inhibits lipid accumulation in the liver.
4. The use according to claim 2, characterized in that: The lipid is selected from TG and / or TC.
5. The use according to claim 1 or 2, characterized in that: The drug is an antioxidant drug.
6. The use according to claim 1 or 2, characterized in that: The medicine is a medicine for increasing the SOD content and reducing the MDA content.
7. The use according to claim 1 or 2, characterized in that: The drug is a drug that improves mitochondrial function.
8. The use according to claim 1 or 2, characterized in that: The drug is a drug that prevents or repairs mitochondrial damage.
9. The use according to claim 1 or 2, characterized in that: The drug is a drug that reduces the expression of lipogenic proteins through the AMPK / mTOR signaling pathway.
10. The use according to claim 1 or 2, characterized in that: The drug is a drug that reduces the expression levels of FAS, ACC, SCD-1, and SREBP-1c, and increases the expression levels of PPARα and CTP1A proteins.