Application of curcumin in preparation of medicine taking TKFC as therapeutic target

Abstract

The invention provides an application of curcumin in preparation of a medicine taking TKFC as a therapeutic target. The invention finds and verifies that curcumin can be directly combined with Val136 and Glu538 sites of TKFC to enhance the enzymatic activity of the TKFC, so that a downstream gene GPAT3 and a mediated metabolic process of the downstream gene GPAT3 are regulated and controlled, and meanwhile, curcumin also inhibits a metabolic pathway of triglyceride and reduces biosynthesis of the triglyceride. The curcumin is of great significance to reveal the mechanism of curcumin in treatment of MASLD, and a brand new angle is provided for follow-up research to solve the treatment problem of MASLD.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to the application of curcumin in preparing a medicine with TKFC as a therapeutic target. Background Art

[0002] Metabolic dysfunction-associated fatty liver disease (MASLD) is a spectrum of diseases characterized by excessive fat accumulation in the liver, ranging from the relatively benign nonalcoholic fatty liver disease (NAFL) to the more severe nonalcoholic steatohepatitis (NASH). Although simple hepatic steatosis is often considered a benign disease, its relationship with liver fibrosis can progress to cirrhosis and even hepatocellular carcinoma.

[0003] Current studies have shown that the exact mechanism of progression of MASLD and its related complications is still not fully understood. There is a lack of reliable biomarkers for early diagnosis and monitoring of disease progression in the clinic, and no drugs have been approved for management and treatment. Therefore, early intervention can effectively prevent the malignant progression of MASLD. Summary of the invention

[0004] The purpose of the present invention is to provide an application of curcumin in the preparation of a drug with TKFC as a therapeutic target, which is of great significance for revealing the mechanism of curcumin in the treatment of MASLD and helps to provide a new angle for subsequent research to solve the treatment problem of MASLD.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] The present invention provides application of curcumin in preparing a medicine taking TKFC as a therapeutic target.

[0007] Furthermore, the binding sites of curcumin and TKFC are Val136 and Glu538 residues of TKFC.

[0008] Furthermore, the drug enhances the enzyme activity of TKFC through curcumin.

[0009] Furthermore, the drug improves lipid accumulation in HepG2 cells by targeting TKFC via curcumin.

[0010] Furthermore, the drug regulates GPAT3 expression by targeting TKFC through curcumin, thereby improving lipid metabolism disorders.

[0011] Furthermore, the drug regulates glucose metabolism by targeting TKFC via curcumin.

[0012] Furthermore, the drug inhibits the metabolic pathway of triglycerides through curcumin and reduces the biosynthesis of triglycerides.

[0013] Furthermore, the medicine includes a medicine for treating fatty liver disease associated with metabolic dysfunction.

[0014] The beneficial effects of the present invention are:

[0015] The present invention discovered and verified that curcumin can directly bind to the Val136 and Glu538 sites of TKFC, enhance its enzyme activity, and thus regulate the expression of the downstream gene GPAT3 and its mediated metabolic process. At the same time, curcumin also inhibits the metabolic pathway of triglycerides and reduces the biosynthesis of triglycerides. This is of great significance for revealing the mechanism of curcumin in the treatment of MASLD, and helps to provide a new angle for subsequent research to solve the treatment problem of MASLD. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 (A) Staining of Bodipy and Oil Red O in OA-treated HepG2 cells. (B) Determination of TG content in OA-treated HepG2 cells. (C) Effect of Cur on HepG2 cell viability. (D)-(E) Quantification of fluorescence values ​​and Oil Red O area of ​​HepG2 cells. Data are expressed as mean ± standard error (SEM) and compared using one-way ANOVA. *p<0.05, **p<0.01, ***p<0.001.

[0017] Figure 2 .Curcumin directly binds to the Glu538 and Val136 sites of TKFC. (A) The binding sites of curcumin and TKFC amino acid residues were identified by LC-MS / MS. (B) The effect of curcumin on triglyceride (TG) content was detected by overexpression of mutant plasmids (n=3). Data are expressed as mean ± standard error (SEM); *P < 0.05, **P < 0.01. (C) The docking results of TKFC protein and curcumin molecule.

[0018] Figure 3 . Detection results of the effect of curcumin on TKFC enzyme activity. (A) Enzyme activity of TKFC in HepG2 cells. (B) Enzyme activity of human recombinant TKFC protein. Data are expressed as mean ± standard error (SEM) and compared using one-way ANOVA. *P<0.05, **P<0.01, ***P<0.001.

[0019] Figure 4.Curcumin improves lipid accumulation in HepG2 cells by targeting TKFC. (A) and (C) Effects of Cur on lipid accumulation in oleic acid (OA)-treated HepG2 cells with TKFC knockdown. (D) Effects of Cur on triglyceride (TG) levels in OA-treated HepG2 cells with TKFC knockdown. (B) and (E) Effects of Cur on lipid accumulation in OA-treated HepG2 cells with TKFC overexpression. (F) Effects of Cur on triglyceride levels in OA-treated HepG2 cells with TKFC overexpression. Data are expressed as mean ± standard error and compared by one-way ANOVA. *P<0.05, **P<0.01, ***P<0.001.

[0020] Figure 5 .The regulation of lipid metabolism disorders by curcumin depends on its interaction with TKFC. (A) Experimental flow of AAV8 transfection in C57BL / 6 mice. (B) Effect of curcumin on body weight of TKFC knockdown mice (n=8). (C) Liver size and hepatic steatosis measured by hematoxylin-eosin (H&E) staining and Oil Red O staining (n=3). (D) Effect of curcumin on triglyceride (TG) and total cholesterol (TC) in the liver of TKFC knockdown mice (n=8). (E)-(F) Effect of curcumin on liver and white adipose tissue (WAT) weight and index of TKFC knockdown mice (n=8). (G) Effect of curcumin on blood lipid levels in TKFC knockdown mice (n=8). Data are expressed as mean ± standard error (SEM) and compared using one-way ANOVA. *P<0.05, **P<0.01, ***P<0.001.

[0021] Figure 6 Curcumin regulates glucose metabolism in TKFC knockdown mice by directly interacting with TKFC. (A) The transfection efficiency of AAV was detected by observing the fluorescence of liver tissue sections of C57BL / 6 mice. (B) The expression of TKFC mRNA in mouse liver tissue. (C) Changes in mouse kidney weight. (D) Serum free fatty acid (NEFA) level in mice. (E) Effects of curcumin on fasting blood glucose and glucose tolerance in TKFC knockdown mice (n=8). (F) Effects of curcumin on insulin level and HOMA-IR in TKFC knockdown mice (n=8). (G) Ratio of serum AST and ALT in mice. (H) Quantitative analysis of Oil Red O staining area in mouse liver. Data are expressed as mean ± standard error (SEM) and compared using one-way ANOVA. *P<0.05, **P<0.01, ***P<0.001.

[0022] Figure 7Curcumin regulates GPAT3 expression by targeting TKFC, thereby improving lipid metabolism disorders. (A) Effects of curcumin on GPAT3 and TPI1 expression in oleic acid (OA)-treated HepG2 cells with TKFC knockdown. (B) Effects of curcumin on GPAT3 and TPI1 expression in OA-treated HepG2 cells with TKFC overexpression. (C) Spearman correlation analysis. (D) mRNA expression of glycerol phosphate metabolic pathway in TKFC knockdown mice (n=4). (E)-(F) Effects of curcumin on protein expression in glycerol phosphate metabolic pathway in TKFC knockdown mice (n=4). Data are expressed as mean ± standard error (SEM) and compared using one-way ANOVA. *P<0.05, **P<0.01, ***P<0.001. DETAILED DESCRIPTION

[0023] In order to explain the present invention more clearly, the present invention is further described in detail below in conjunction with embodiments and with reference to the accompanying drawings. It should be understood by those skilled in the art that the content described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0024] Example

[0025] The present invention is mainly achieved through the following steps:

[0026] 1. A lipid accumulation model was established using oleic acid-induced human liver cancer HepG2 cells. The cells were treated with curcumin for 24 h. The fluorescence value and area of ​​lipid droplets were detected using BODIPY and Oil Red O. The intracellular triglyceride content was determined using a triglyceride assay kit.

[0027] 1. TG content determination

[0028] The lipid accumulation model was established by treating human liver cancer HepG2 cells (National Biomedical Experimental Cell Resource Bank) with oleic acid (OA, 0.5 mM) for 24 h. The cells were treated with 10 μM curcumin for 24 h, and the intracellular triglyceride content was measured using a triglyceride assay kit.

[0029] 2. Lipid Staining

[0030] Human hepatocellular carcinoma HepG2 cells were fixed with 4% paraformaldehyde, stained with 0.5% Oil Red O isopropanol solution (3:2, v / v), induced to differentiate with 75% ethanol for 2 min, and imaged after rinsing 3 times with 100 μL distilled water. The lipid droplet area was quantitatively analyzed using ImageJ software. HepG2 cells were incubated with PBS buffer (including 5 μM BODIPY and Hoechst 33342 fluorescent dye) in an opaque 96-well plate for 30 min. Fluorescence intensity was analyzed using a high-content cell analyzer.

[0031] The results are as follows Figure 1 As shown, in HepG2 cells treated with OA, there was no obvious cytotoxicity when the concentration of curcumin was lower than 10 μM ( Figure 1 C). BODIPY and Oil Red O staining also showed that 10 μM Cur could significantly improve the cellular lipid accumulation caused by OA ( Figure 1 A, D and E). Meanwhile, Cur significantly reduced the intracellular TG level ( Figure 1 B). 2. The binding site of Cur on TKFC was determined by mass spectrometry identification and construction of amino acid mutants, and the function of the site in HepG2 cells was verified.

[0032] 1. Molecular docking

[0033] AutoDock Vina (version 1.1.2) was used for semi-flexible docking, with TKFC as the receptor and Cur as the ligand. The protein structure was obtained from AlphaFold and the Cur structure was obtained from PubChem. PyMOL software (version 4.3.0) was used for ligand-protein separation, dehydration, and removal of organic compounds. It was used to add hydrogen, check the charge, assign the atom type to the AD4 type, calculate the gasteiger, and build the docking grid box of the protein structure. In addition, the chemical composition (small molecule ligand) should determine the Root, and the torsional bond of the ligand should be selected in AutodockTools. After docking with Vina, the score of the combination of protein and small molecule curcumin was calculated, and the force analysis and visualization of three-dimensional and two-dimensional angles were performed using Pymol and Discovery Studio software.

[0034] 2. Determination of Cur binding sites on TKFC

[0035] rhTKFC was incubated with Cur at 4°C overnight, and then treated with TCEP solution at 60°C for 1 hour for reduction. Then it was reacted with MMTS solution in the dark for 45 minutes, ultrafiltration and centrifugation, treated with 8M urea solution, washed with 0.25M TEAB, and centrifuged. Finally, the filtrate was collected after centrifugation and dried under low temperature vacuum. The peptides were dissolved in 0.1% formic acid and 2% acetonitrile solution for mass spectrometry identification. The mass spectrometry data were analyzed using the MASCOT search engine.

[0036] The results are as follows Figure 2 To determine whether Cur modified the amino acid residues, recombinant TKFC was incubated with Cur and then subjected to LC-MS / MS analysis. The results showed that Val136 and Glu538 were modified by Cur ( Figure 2A). To further investigate the binding mode of Cur to TKFC, we performed molecular docking simulations using AutoDock Vina. We found that the three-dimensional conformation of Cur matched the binding site around the active valine residue (Val136), and Cur bound to the valine 136 of TKFC through a hydrophobic interaction of 3.8A ( Figure 2 C) To verify the binding site of Cur, we mutated Val136 and Glu538 of TKFC to Val136Ala and Glu538Ala, respectively. Figure 2 As shown in B, Val136Ala and Glu538Ala mutations reversed the decrease in TG levels induced by Cur in OA-treated HepG2 cells, indicating that these mutations blocked the binding of TKFC to Cur.

[0037] 3. Verify the effect of curcumin on TKFC enzyme activity by detecting kinase activity.

[0038] Using Kinase-lumi TM The enzyme activity of TKFC was detected by the luminescent kinase assay kit. The TKFC activity reaction system was as follows: 50 mM Tris-HCl solution, 10 mM DL-glyceraldehyde, 0.2 mM ATP, and 0.125 μg of human recombinant TKFC protein or 10 μL of cell lysate (total protein concentration of 5 mg / mL) were added to the enzyme reaction system. The reaction was carried out at 25°C for 10 min and the assay was performed within 20 min according to the manufacturer's instructions. The enzyme activity was expressed as the reduction of ATP per minute.

[0039] The results are as follows Figure 3 As shown in Figure 2, Cur not only promoted the enzymatic activity of TKFC in cells ( Figure 3 A), and also promoted the enzymatic activity of human recombinant TKFC protein ( Figure 3 B). This indicates that the lipid-lowering effect of Cur may be related to the activation of TKFC.

[0040] 4. TKFC gene was knocked down or overexpressed in HepG2 cells by transfecting siRNA and recombinant plasmid, and the regulation of GPAT3 expression was detected to study its role in lipid accumulation model and curcumin treatment.

[0041] 1. siRNA transient transfection

[0042] use HepG2 cells were transfected with TKFC siRNA (GenePharma, Jiangsu, China) using 2000 transfection reagent for 6 h before further study. The siRNA sequences are shown in the table below.

[0043]

[0044] 2. Construction of plasmids and amino acid mutants

[0045] HepG2 cells were transfected with human TKFC plasmid vector (CMV enhancer-MCS-3flag-polyA-EF1A-zsGreen-sv40-puromycin) and transfected when the cells reached 70-80% fusion. Site-directed mutant TKFC (Val136Ala or Glu538Ala) was cloned into GV657 vector (CMV enhancer-MCS-3flag-polyA-EF1A-zsGreen-sv40-puromycin). GeneChem (Shanghai, China) was commissioned to construct plasmids, and all constructed plasmids were verified by DNA sequencing. Before constructing the lipid accumulation model and Cur treatment, according to The overexpression was performed by transfection for 8 h.

[0046] The results are as follows Figure 4 As shown, in HepG2 cells, TKFC knockdown reversed the Cur-mediated decrease in TG content and lipid droplet area ( Figure 5 A, C, and D). However, Cur treatment significantly reduced the TG content and lipid droplet area in HepG2 cells with high expression of TKFC ( Figure 5 B, E and F).

[0047] Meanwhile, siRNA-mediated knockdown of TKFC in HepG2 cells significantly inhibited the decrease in GPAT3 expression and increase in TPI1 expression regulated by Cur. In contrast, TKFC overexpression plasmid transfection was used in HepG2 cells. Compared with the group without Cur addition, GPAT3 expression was significantly downregulated, while TPI1 expression was significantly upregulated, as shown in Figure 2. Figure 7 As shown in A and B.

[0048] 5. The TKFC gene in mice was knocked down by adeno-associated virus (AAV8) transfection and combined with curcumin treatment to study the role of TKFC in curcumin-treated mice with lipid metabolism disorders, and the changes in lipid metabolism, gene and protein expression were evaluated by biochemical analysis, histological analysis, qRT-PCR and Western blot.

[0049] 1. Adeno-associated virus (AAV) knockdown

[0050] C57BL / 6 mice were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. and were raised in a constant temperature (24±2℃) 12h light-dark cycle environment. The mice were fed a high-fat diet (20% protein + 20% carbohydrates + 60% fat, D12492) for 4 weeks. TKFC was knocked down in C57BL / 6 mice using the AAV auxiliary system. The three plasmids GV687, pAAV-RC and pHelper were transfected into AAV-293 cells and purified by CsCl density gradient centrifugation and ultrafiltration to obtain the AAV8 vector carrying EGFP. Finally, AAV particles (1.896×1011v.g) were injected into the tail vein of mice. The transfection experiment process is as follows Figure 5 As shown in A. The mice were divided into 5 groups: Control (normal diet), AAV8-NC (high-fat diet + control siRNA), AAV8-NC-Cur (high-fat diet + control siRNA + 140 mg / kg curcumin), AAV8-TKFC (high-fat diet + siTKFC) and AAV8-TKFC-Cur (high-fat diet + siTKFC + 140 mg / kg curcumin). The treatment was continued for 4 weeks, and the body weight and physiological parameters were recorded every week. The results showed that the body weight of mice in the AAV8-NC-Cur group was significantly lower than that of mice in the AAV8-NC group, see Figure 5 B. Obvious green fluorescence can be seen in the liver tissue sections of the AAV group. Figure 6 A, The mRNA level of TKFC was significantly decreased, indicating that the expression of TKFC in mouse liver tissue was inhibited. Figure 6 B. However, after TKFC knockdown, the weight loss effect of curcumin was weakened. After the experiment, the liver, kidney and white adipose tissue were weighed. Figure 5 As shown in E, F, and 6C, Cur reduced the relative liver weight and relative white adipose tissue (WAT) weight in AAV8-NC mice, but the effect was weaker in TKFC knockout mice. HFD is usually accompanied by kidney tissue damage. Cur effectively reduced the weight and index of kidney tissue, but had no obvious effect in TKFC knockout mice. Liver tissue samples were subjected to RNA / protein assays and stored at -80°C.

[0051] 2. Biochemical Analysis

[0052] Serum TC, TG, LDL-C, HDL-C, AST, ALT, NEFA and INS levels were detected using commercially available kits. Figure 5 G. Figure 6As shown in D, F, and G. TKFC knockdown reversed Cur-mediated downregulation of serum TC, TG, LDL-C, and NEFA and upregulation of HDL-C / TC ratio in mice. At the same time, TKFC knockdown significantly reversed Cur-regulated decrease in insulin levels and increase in HOMA-IR. The ratio of ALT to AST indicated that Cur- and AAV-mediated TKFC knockdown was not significantly toxic to the liver.

[0053] Liver TG and TC determinations were performed using anhydrous ethanol homogenization to prepare tissue extracts. Figure 5 D. The determination of TG and TC contents in liver tissue showed that Cur could significantly reduce the levels of TG and TC in liver in AAV8-NC mice, and the therapeutic effect of Cur was significantly weakened after TKFC knockdown.

[0054] Mice were subjected to an intraperitoneal glucose tolerance test (IPGTT) after fasting for 16 h to assess blood glucose levels at 0, 15, 30, 60, 90, and 120 min, and blood glucose concentrations were measured using a glucometer. Figure 6 E. In terms of blood glucose, TKFC knockdown significantly reversed the Cur-regulated decrease in fasting blood glucose and the increase in glucose tolerance.

[0055] 3. Histological Analysis

[0056] Liver samples were fixed in 4% paraformaldehyde for 24 h, dehydrated using an ethanol gradient, infiltrated with xylene, and embedded in paraffin. 5 μm thick sections were taken for hematoxylin-eosin (H&E) staining. Other liver samples were quickly frozen in liquid nitrogen, stored at -80°C, and embedded in Tissue- OCT and cut into 10 μm thick sections for Oil Red O staining. Figure 5 C and 6H. The results of liver tissue HE staining and Oil Red O staining showed that the lipid droplet area of ​​AAV8-NC mice was significantly reduced after Cur treatment, while Cur treatment did not show a significant inhibitory effect on hepatocyte lipid accumulation in TKFC knockdown mice.

[0057] 4.qRT-PCR

[0058] RNA was extracted from liver tissue using an RNA extraction kit and then purified using PrimeScript TM The RT kit was used to reverse transcribe into cDNA. qRT-PCR was performed on the Applied Biosystems 7500 system using SYBR Green Master Mix. β-actin was used as the internal reference gene and the data were calculated according to the 2-ΔΔCt method. The primer sequences are as follows.

[0059]

[0060] The results are as follows Figure 7 C and D. Spearman correlation coefficient analysis was used to compare the relationship between TKFC (or GPAT3) mRNA expression and serum TG levels in mice. There was no significant correlation between TKFC mRNA levels and serum TG levels, while GPAT3 mRNA levels were positively correlated with serum TG levels. In order to further clarify the relationship between TKFC and GPAT3 in vivo, RT-qPCR was used to analyze the effect of TKFC gene knockdown on GPAT3 and related pathway genes at the mRNA level. The results showed that in the Cur-treated control group, the mRNA expression of GPAT3, DGAT1 and Lipin1 was significantly downregulated, while after TKFC knockdown, the expression of GPAT3 and DGAT1 was not regulated by Cur.

[0061] 5. Western blot analysis

[0062] Lyse tissue or cells with RIPA lysis buffer containing 1 mM protease inhibitor cocktail. Centrifuge the lysate and adjust the concentration to 2 mg / mL. Separate the lysate on a 6-15% SDS-PAGE gel and perform immunoblotting using standard protocols.

[0063] The results are as follows Figure 7 As shown in E and F, Western blotting showed that Cur inhibited the glycerophosphate pathway (GPAT3 / AGPAT1 / Lipin1 / DGAT1), thereby reducing the generation of TAG. However, the inhibitory effect of Cur on the glycerophosphate pathway was weakened in TKFC knockdown mice. These findings suggest that the effect of Cur in improving lipid metabolism disorders may be achieved by targeting TKFC to regulate the expression of GPAT3.

[0064] Obviously, the above embodiments of the present invention are merely examples to more clearly illustrate the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. Application of curcumin in the preparation of drugs with TKFC as therapeutic target.

2. The use according to claim 1, characterized in that: The binding sites of curcumin and TKFC are Val136 and Glu538 residues of TKFC.

3. The use according to claim 1, characterized in that: The drug enhances the enzymatic activity of TKFC through curcumin.

4. The use according to claim 1, characterized in that: The drug improves lipid accumulation in HepG2 cells by targeting TKFC via curcumin.

5. The use according to claim 1, characterized in that: The drug targets TKFC via curcumin to regulate GPAT3 expression, thereby improving lipid metabolism disorders.

6. The use according to claim 1, characterized in that: The drug regulates glucose metabolism by targeting TKFC via curcumin.

7. The use according to claim 1, characterized in that: The drug inhibits the metabolic pathway of triglycerides through curcumin and reduces the biosynthesis of triglycerides.

8. The use according to any one of claims 1 to 7, characterized in that: The medicine includes a medicine for treating fatty liver disease associated with metabolic dysfunction.

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