Polypeptide for inhibiting fat accumulation and its application

Abstract

The present invention provides a polypeptide for inhibiting fat accumulation and its use. Its amino acid sequence is MNGLTLGSIPKEEAEKERQGWLEAAK. This polypeptide can inhibit CD36 transport of extracellular fatty acids, reducing cellular lipid uptake and accumulation, and effectively inhibiting the accumulation of intracellular triglycerides and other fats caused by free fatty acids.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a polypeptide for inhibiting fat accumulation and an application thereof. Background Art

[0002] NASH typically occurs in individuals without a significant history of alcohol consumption and is closely associated with other symptoms of metabolic syndrome, including obesity, type 2 diabetes, hypertension, and hyperlipidemia. NASH can not only cause liver failure but can also progress to cirrhosis and, in severe cases, hepatocellular carcinoma, posing a serious threat to the patient's life. Non-alcoholic steatohepatitis (NASH) is a serious liver disease and a progressive stage of non-alcoholic fatty liver disease (NAFLD). It is characterized by excessive fat accumulation in the liver, accompanied by inflammation and fibrosis.

[0003] CD36 is a scavenger receptor expressed on a variety of cell types. It plays an important role in lipid metabolism, mediating lipid uptake, immune recognition, inflammation, molecular adhesion, and apoptosis. It is associated with angiogenesis, inflammatory responses, atherothrombotic diseases, and metabolic disorders such as diabetes and obesity. In the immune system, CD36 mediates the acquisition and presentation of dendritic cell antigens and supports the function of regulatory T cells. Studies have shown that targeting CD36 may be an effective strategy to improve the anti-tumor efficacy of CD8+ T cell immunotherapy, providing new ideas and possibilities for clinical treatment.

[0004] The CD36 protein is a transmembrane glycoprotein that plays a crucial pathological role in nonalcoholic steatohepatitis (NASH). As a multifunctional receptor, CD36 primarily mediates the uptake and metabolism of long-chain fatty acids and is involved in the abnormal accumulation of lipids in the liver. One of the hallmarks of NASH is the excessive accumulation of fat in the liver, and CD36 plays a key role in this process. High expression of CD36 increases the uptake of free fatty acids by hepatocytes, leading to lipid metabolism disorders, excessive lipid accumulation, and associated cytotoxicity. Furthermore, CD36 participates in inflammatory responses, binding to oxidized low-density lipoprotein (oxLDL) to activate inflammatory signaling pathways, further exacerbating liver inflammation and fibrosis. These effects make CD36 a key node in the pathogenesis of NASH and a potential therapeutic target.

[0005] Currently, the development of inhibitors targeting CD36 has become a research hotspot. Major CD36 inhibitors include antibodies and small molecules, such as SSO (sulfo-N-succinimidyl oleate) and TSP-1 (thrombospondin-1). SSO is an irreversible CD36 inhibitor that reduces hepatic fatty acid uptake by blocking the binding of CD36 receptors to fatty acids, thereby alleviating hepatic lipid accumulation and improving metabolic disorders. TSP-1, as a ligand for CD36, can also indirectly inhibit CD36 by modulating its activity. Furthermore, several natural compounds have been found to have the potential to inhibit CD36. Although these CD36 inhibitors have shown some efficacy in experimental settings, clinical application still faces challenges, such as drug specificity, toxicity, and long-term safety. Therefore, further research is needed to develop effective and safe CD36 inhibitors. Summary of the Invention

[0006] In view of this, the present invention aims to propose a polypeptide for inhibiting fat accumulation and its application, which can inhibit the transport of extracellular fatty acids by CD36, reduce cellular lipid uptake and accumulation, and prevent and treat non-alcoholic steatohepatitis.

[0007] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0008] A polypeptide that inhibits fat accumulation, its amino acid sequence is:

[0009] MNGLTLGSIPKEEAEKERQGWLEAAK.

[0010] Furthermore, the polypeptide is capable of binding to CD36 protein.

[0011] Furthermore, the polypeptide can target and bind to the CD36 protein, inhibit CD36's transport of extracellular fatty acids, and reduce cellular lipid uptake and accumulation.

[0012] A pharmaceutical composition comprising the above-mentioned polypeptide for inhibiting fat accumulation.

[0013] Use of the above polypeptide for inhibiting fat accumulation in preparing a drug for inhibiting CD36 transport of extracellular fatty acids.

[0014] The application of the above polypeptide for inhibiting fat accumulation in the preparation of drugs for treating or preventing hepatitis, liver cancer, atherosclerosis, diabetes, diabetic nephropathy, obesity or tumors.

[0015] Use of the above-mentioned polypeptide for inhibiting fat accumulation in the preparation of a drug for treating or preventing non-alcoholic steatohepatitis.

[0016] The polypeptide provided by the present invention is obtained through screening, and it can effectively inhibit the transport of extracellular fatty acids by CD36, reduce the lipid uptake and accumulation of cells, inhibit the transport of extracellular fatty acids by CD36, reduce the lipid uptake and accumulation of cells, and thus prevent and / or treat diseases related to its signaling pathway.

[0017] Compared with the prior art, the polypeptide for inhibiting fat accumulation and its application of the present invention have the following advantages:

[0018] The polypeptide provided by the present invention has good solubility and stability.

[0019] The polypeptide provided by the present invention has good biological activity and low toxicity.

[0020] The polypeptide provided by the present invention can effectively inhibit the accumulation of intracellular fat and is used in drugs for treating or preventing hepatitis, liver cancer, atherosclerosis, diabetes, diabetic nephropathy, obesity or tumors.

[0021] The polypeptide provided by the present invention can exert therapeutic effects on both human and mouse cells and has universality and broad spectrum. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings, which constitute part of the present invention, are provided to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are provided to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0023] Figure 1 This is a Pymol visualization result diagram of the binding of the polypeptide of the present invention to the extracellular region of human CD36 protein;

[0024] Figure 2 This is an Oil Red O staining result showing that the polypeptide in Example 1 of the present invention inhibits lipid droplet accumulation in HepG2 human liver cancer cells;

[0025] Figure 3 This is a quantitative result graph showing that the polypeptide in Example 2 of the present invention inhibits triglyceride accumulation in HepG2 human liver cancer cells;

[0026] Figure 4 This is the Oil Red O staining result showing that the polypeptide in Example 3 of the present invention inhibits lipid droplet accumulation in normal hepatocytes of AML12 mice;

[0027] Figure 5 This is a quantitative result graph showing that the polypeptide in Example 4 of the present invention inhibits triglyceride accumulation in normal liver cells of AML12 mice;

[0028] Figure 6 This is a confocal micrograph of the results of BODIPY staining showing that the polypeptide in Example 5 of the present invention alleviates lipid droplet aggregation in normal hepatocytes of AML12 mice;

[0029] Figure 7 This is a quantitative result of BODIPY staining flow cytometry showing that the polypeptide in Example 5 of the present invention alleviates lipid droplet aggregation in normal hepatocytes of AML12 mice. DETAILED DESCRIPTION

[0030] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0031] The present invention will be described in detail below with reference to the accompanying drawings and in conjunction with embodiments.

[0032] The amino acid sequences of the polypeptides provided in this application are:

[0033] T1:MGGLTLGSIPKEEAEAERQGWLAAAK;

[0034] T2: MNGLTLGSIPKEEAEKERQGWLEAAK;

[0035] T3:MNGMQLGTIPKEVAEQERKGFEEAAK;

[0036] Polypeptides can be prepared using methods well known to those skilled in the art, including well-known chemical synthesis methods. In addition to using chemical synthesis methods to prepare polypeptides or their derivatives, they can also be prepared by expressing encoding nucleic acids. This is particularly applicable to the preparation of polypeptides or their derivatives containing only natural amino acids. In this case, well-known methods for preparing nucleic acid encoding polypeptide sequences can be used (see Sambrook et al., Molecular Cloning: A La bora tory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999)). Polypeptides can be expressed in organisms and purified by well-known purification techniques.

[0037] The polypeptides of the present application were prepared and purified using existing technologies.

[0038] The naturally occurring amino acids are alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamic acid (Glu or E), glutamine (Glu or Q), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and the L-isomers of valine (Val or V).

[0039] This application uses AlphaFold to predict the binding modes and abilities of three peptides to CD36, and finds that the interface prediction template modeling (iPTM) scores of T1, T2, and T3 are 0.37, 0.41, and 0.26, respectively.

[0040] The AlphaFold prediction results were visualized using Pymol, and the T2 binding position was most consistent with the original design (see Figure 1 ).

[0041] The binding affinity between T2 and human CD36 protein was detected by BLI technology, with KD=197.9 nM.

[0042] AlphaFold was used again to predict the binding mode and binding ability of T2 and mouse CD36 protein, iPTM=0.6, indicating that T2 can also effectively bind to CD36 protein in mice and mouse cells to prevent lipid transport.

[0043] Based on AlphaFold prediction and solubility analysis, a preferred peptide was screened with the sequence T2:MNGLTLGSIPKEEAEKERQGWLEAAK.

[0044] Example 1: Assessing the therapeutic effect of polypeptides using Oil Red O staining on human cells

[0045] (1) Oil Red O is a fat-soluble dye that is highly soluble in fat and specifically stains neutral fats such as triglycerides in tissues. It is one of the commonly used assessment indicators in NASH disease models.

[0046] (2) Human hepatoma cells HepG2 were seeded into cell culture plates and cultured in a 37°C, 5% CO2 cell culture incubator until the cell confluence reached 80%;

[0047] (3) Divided into 3 groups: control group (Control), induction group (FFA), treatment group T2 (5 μM). The control group was not treated, the induction group was induced with 0.25 mM FFA for 12 h, and the treatment group was pretreated with peptide for 12 h and then induced with 0.25 mM FFA for 12 h.

[0048] (4) Wash the cells twice with PBS, add fixative to fix for 30 minutes, rinse with 60% isopropanol, add Oil Red O staining solution to stain for 20 minutes, rinse with 60% isopropanol, wash three times with water, add hematoxylin staining solution to stain for 2 minutes, wash three times with water, add Oil Red O buffer to incubate for 1 minute, discard, soak in deionized water, and observe under an inverted microscope;

[0049] (5) Experimental results are shown in Figure 2 , polypeptide T2 can alleviate the formation and aggregation of cellular lipid droplets to a certain extent.

[0050] Example 2: Detecting triglyceride levels in human cells to assess the therapeutic effect of polypeptides

[0051] (1) NASH is associated with high levels of cholesterol and triglycerides in the liver. Triglyceride levels are an important evaluation indicator of NASH.

[0052] (2) Human hepatoma cells HepG2 were seeded into cell culture plates and cultured in a 37°C, 5% CO2 cell culture incubator until the cell confluence reached 80%;

[0053] (3) Divided into 5 groups: control group (Control), induction group (FFA), treatment group T2 (5μM), treatment group T2 (25μM), treatment group T2 (50μM). The control group was not treated, the induction group was induced with 0.25 mM FFA for 12 hours, and the treatment group was pretreated with peptide for 12 hours and then induced with 0.25 mM FFA for 12 hours.

[0054] (4) Collect cell pellets, lyse cells, and measure the triglyceride content in cell lysate;

[0055] (5) Experimental results are shown in Figure 3 Treatment of cells with peptide T2 can effectively alleviate the accumulation of triglycerides in cells, but the peptide effect does not show concentration dependence.

[0056] Example 3: Assessing the therapeutic effect of polypeptides using Oil Red O staining on mouse cells

[0057] (1) Mouse normal liver cells AML12 were inoculated into cell culture plates and cultured in a 37°C, 5% CO2 cell culture incubator until the cell confluence reached 80%;

[0058] (2) Divided into 3 groups: control group (Control), induction group (FFA), treatment group T2 (5 μM). The control group was not treated, the induction group was induced with 0.25 mM FFA for 12 h, and the treatment group was pretreated with peptide for 12 h and then induced with 0.25 mM FFA for 12 h.

[0059] (3) Wash the cells twice with PBS, add fixative to fix for 30 minutes, rinse with 60% isopropanol, add Oil Red O staining solution to stain for 20 minutes, rinse with 60% isopropanol, wash three times with water, add hematoxylin staining solution to stain for 2 minutes, wash three times with water, add Oil Red O buffer to incubate for 1 minute, discard, soak in deionized water, and observe under an inverted microscope;

[0060] (4) Experimental results are shown in Figure 4 A large number of red lipid droplets were observed in the induction group, and the peptide treatment group showed a certain degree of relief.

[0061] Example 4: Detection of triglyceride content in mouse cells to assess the therapeutic effect of polypeptides

[0062] (1) Mouse normal liver cells AML12 were inoculated into cell culture plates and cultured in a 37°C, 5% CO2 cell culture incubator until the cell confluence reached 80%;

[0063] (2) Divided into 5 groups: control group (Control), induction group (FFA), treatment group T2 (5μM), treatment group T2 (25μM), treatment group T2 (50μM). The control group was not treated, the induction group was induced with 0.25 mM FFA for 12 hours, and the treatment group was pretreated with peptide for 12 hours and then induced with 0.25 mM FFA for 12 hours.

[0064] (3) Collect cell pellets, lyse cells, and measure the triglyceride content in cell lysate;

[0065] (4) Experimental results are shown in Figure 5 , low concentration of peptide treatment of cells can effectively alleviate the accumulation of triglycerides in cells.

[0066] Example 5: Detecting lipid droplet aggregation in mouse cells to assess the therapeutic effect of polypeptides

[0067] (1) BODIPY, as a green fluorescent probe, can penetrate the cell membrane into the cell interior and locate and specifically stain polar lipids in the cell. It is often used for fatty acid uptake, lipid transport, lipid absorption and accumulation.

[0068] (2) Mouse normal liver cells AML12 were inoculated into cell culture plates and cultured in a 37°C, 5% CO2 cell culture incubator until the cell confluence reached 80%;

[0069] (3) Divided into 3 groups: control group (Control), induction group (FFA), treatment group T2 (5 μM). The control group was not treated, the induction group was induced with 0.25 mM FFA for 12 h, and the treatment group was pretreated with peptide for 12 h and then induced with 0.25 mM FFA for 12 h.

[0070] (4) Labeling intracellular lipid droplets with BODIPY fluorescent probe and observing intracellular lipid droplet aggregation using laser confocal microscopy;

[0071] (5) Confocal experiment results are shown in Figure 6 , a large number of green fluorescent-labeled lipid droplets were observed in the induced group, while the green fluorescence intensity was significantly reduced in the peptide-treated group;

[0072] (6) The cells were divided into 5 groups: control group (Control), induction group (FFA), treatment group T2 (5 μM), treatment group T2 (25 μM), and treatment group T2 (50 μM). The control group was not treated, the induction group was induced with 0.25 mM FFA for 12 h, and the treatment group was pretreated with peptide for 12 h and then induced with 0.25 mM FFA for 12 h.

[0073] (7) Label the intracellular lipid droplets with BODIPY fluorescent probe, collect the cells, and detect the intracellular fluorescence intensity by flow cytometry;

[0074] (8) Flow cytometry results are shown in Figure 7 The fluorescence intensity of the induced group increased significantly, and there was an obvious rightward shift of the peak compared with the control group. The fluorescence intensity of the polypeptide treatment group decreased significantly, that is, the peak shifted to the left.

[0075] This application uses biomembrane interferometry (BLI) technology to analyze the binding effect of peptide T2 and CD36 protein. AlphaFold predicts the binding potential of peptide T2 and mouse CD36 protein. The inhibitory effect of peptide T2 on fat accumulation was verified at the cellular level, confirming that T2 is a peptide that effectively binds to CD36 protein and alleviates cellular fat accumulation.

[0076] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A polypeptide for inhibiting fat accumulation, characterized in that: Its amino acid sequence is: MNGLTLGSIPKEEAEKERQGWLEAAK.

2. A pharmaceutical composition comprising the polypeptide for inhibiting fat accumulation according to claim 1.

3. Use of the polypeptide for inhibiting fat accumulation according to claim 1 in the preparation of a drug for treating or preventing non-alcoholic steatohepatitis.

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