Polypeptide for inhibiting fat accumulation and application thereof

Abstract

The invention provides a polypeptide for inhibiting fat accumulation and application of the polypeptide. The amino acid sequence of the polypeptide is MNGLTLGSIPKEEAEKERQGWLEAAK. The polypeptide can inhibit transport of extracellular fatty acids by CD36, reduce lipid uptake and accumulation of cells, and effectively inhibit accumulation of intracellular triglyceride and other fats caused by free fatty acids.

Description

Technical Field

[0001] The 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 usually occurs in individuals without a significant history of drinking and is closely related to other symptoms of metabolic syndrome, including obesity, type 2 diabetes, hypertension, hyperlipidemia, etc. NASH can not only cause liver failure, but may also progress to cirrhosis, and in severe cases, even 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 accumulation of fat in the liver, accompanied by inflammatory response and the appearance of fibrosis.

[0003] CD36 is a scavenger receptor expressed in 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 response, atherosclerotic thrombotic 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 regulatory T cell function. 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] CD36 protein is a transmembrane glycoprotein that plays an important pathological role in non-alcoholic steatohepatitis (NASH). As a multifunctional receptor, CD36 mainly mediates the uptake and metabolism of long-chain fatty acids and participates in the abnormal accumulation of lipids in the liver. One of the characteristics 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 absorption of free fatty acids by hepatocytes, leading to lipid metabolism disorders, excessive lipid accumulation and its related cytotoxicity. In addition, CD36 can also participate in inflammatory responses, activate inflammatory signaling pathways by binding to oxidized low-density lipoprotein (oxLDL), and further aggravate liver inflammation and fibrosis. These effects make CD36 a key node in the pathogenesis of NASH and one of the potential targets for treatment.

[0005] At present, the development of inhibitors against CD36 has become one of the research hotspots. The main CD36 inhibitors include antibodies and small molecule compounds, such as SSO (sulfo-N-succinimidyl oleate) and TSP-1 (thrombospondin-1). SSO is an irreversible CD36 inhibitor that reduces the liver's uptake of fatty acids by blocking the binding of CD36 receptors to fatty acids, thereby alleviating liver fat accumulation and improving metabolic disorders. As one of the ligands of CD36, TSP-1 can also indirectly exert an inhibitory effect by regulating the activity of CD36. In addition, some natural compounds have also been found to have the potential to inhibit CD36. Although these CD36 inhibitors have shown certain efficacy in the experimental stage, they still face challenges in clinical application, such as drug specificity, toxicity, and safety of long-term use. Therefore, the development of effective and safe CD36 inhibitors still requires in-depth research. 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 CD36 transport of extracellular fatty acids, reduce cellular lipid uptake and accumulation, and prevent and treat non-alcoholic fatty liver disease.

[0007] To achieve the above object, the technical solution of the present invention is achieved as follows: The amino acid sequence of the polypeptide that inhibits fat accumulation is: MNGLTLGSIPKEEAEKERQGWLEAAK.

[0008] Furthermore, the polypeptide is capable of binding to CD36 protein.

[0009] Furthermore, the polypeptide can target and bind to CD36 protein, inhibit CD36's transport of extracellular fatty acids, and reduce cellular lipid uptake and accumulation.

[0010] A pharmaceutical composition comprising the above-mentioned polypeptide for inhibiting fat accumulation.

[0011] Use of the above polypeptide for inhibiting fat accumulation in preparing a drug for inhibiting CD36 transport of extracellular fatty acids.

[0012] The polypeptide for inhibiting fat accumulation is used in the preparation of drugs for treating or preventing hepatitis, liver cancer, atherosclerosis, diabetes, diabetic nephropathy, obesity or tumors.

[0013] Use of the above polypeptide for inhibiting fat accumulation in the preparation of a drug for treating or preventing non-alcoholic fatty hepatitis.

[0014] 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, thereby preventing and / or treating diseases related to its signaling pathway.

[0015] Compared with the prior art, the polypeptide for inhibiting fat accumulation and its application described in the present invention have the following advantages: The polypeptide provided by the invention has good solubility and stability.

[0016] The polypeptide provided by the invention has good biological activity and low toxicity.

[0017] The polypeptide provided by the present invention can effectively inhibit the accumulation of intracellular fat and can be used in drugs for treating or preventing hepatitis, liver cancer, atherosclerosis, diabetes, diabetic nephropathy, obesity or tumors.

[0018] 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

[0019] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings: Figure 1 It is a Pymol visualization result diagram of the binding of the polypeptide of the present invention to the extracellular region of human CD36 protein; Figure 2 This is the Oil Red O staining result of the polypeptide in Example 1 of the present invention inhibiting lipid droplet accumulation in HepG2 human liver cancer cells; Figure 3 is a quantitative result diagram of the polypeptide in Example 2 of the present invention inhibiting triglyceride accumulation in HepG2 human liver cancer cells; Figure 4 This is the Oil Red O staining result of the polypeptide in Example 3 of the present invention inhibiting lipid droplet accumulation in normal hepatocytes of AML12 mice; Figure 5 is a quantitative result diagram of the polypeptide in Example 4 of the present invention inhibiting triglyceride accumulation in normal liver cells of AML12 mice; Figure 6 This is a confocal result of BODIPY staining showing that the polypeptide in Example 5 of the present invention alleviates lipid droplet aggregation in normal liver cells of AML12 mice; 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 liver cells of AML12 mice. DETAILED DESCRIPTION

[0020] 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.

[0021] The present invention will be described in detail below with reference to the accompanying drawings and in conjunction with embodiments.

[0022] The amino acid sequences of the polypeptides provided in this application are: T1:MGGLTLGSIPKEEAEAERQGWLAAAK; T2: MNGLTLGSIPKEEAEKERQGWLEAAK; T3:MNGMQLGTIPKEVAEQERKGFEEAAK; The polypeptide 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 encoding nucleic acid expression. This is particularly applicable to the preparation of polypeptides or their derivatives containing only natural amino acids, in which case the well-known method 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)). The polypeptide can be expressed in an organism and purified by known purification techniques.

[0023] The polypeptides of the present application are prepared and purified using existing technologies.

[0024] The naturally occurring amino acids are the L-isomers of 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 valine (Val or V).

[0025] This application uses AlphaFold to predict the binding modes and abilities of three polypeptides with 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.

[0026] The AlphaFold prediction results were visualized using Pymol, and the T2 binding position was most consistent with the original design (see Figure 1 ).

[0027] The binding affinity between T2 and human CD36 protein was detected by BLI technology, KD=197.9 nM.

[0028] 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 rat cells to prevent lipid transport.

[0029] Based on AlphaFold prediction and solubility analysis, a preferred peptide was screened, with the sequence T2:MNGLTLGSIPKEEAEKERQGWLEAAK.

[0030] Example 1: Evaluation of the therapeutic effect of polypeptides using Oil Red O staining on human cells (1) Oil Red O is a fat-soluble dye that is highly soluble in fat and specifically colors neutral fats such as triglycerides in tissues. It is one of the commonly used evaluation indicators in NASH disease models. (2) Human liver cancer cells HepG2 were inoculated into cell culture plates and placed at 37°C and 5% CO 2 Culture in a cell culture incubator until the cell confluence reaches 80%; (3) Divided into 3 groups: control group (Control), induced group (FFA), treatment group T2 (5 μM). The control group was not treated, the induced 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. (4) Wash the cells twice with PBS, add fixative to fix for 30 min, rinse with 60% isopropanol, add Oil Red O staining solution to stain for 20 min, rinse with 60% isopropanol, wash with water three times, add hematoxylin staining solution to stain for 2 min, wash with water three times, add Oil Red O buffer to incubate for 1 min, discard, soak in deionized water, and observe under an inverted microscope; (5) Experimental results are shown in Figure 2 , polypeptide T2 can alleviate the formation and aggregation of cellular lipid droplets to a certain extent.

[0031] Example 2: Detection of triglyceride content in human cells to assess the therapeutic effect of polypeptides (1) NASH is associated with high levels of cholesterol and triglycerides in the liver. Triglyceride levels are an important evaluation indicator of NASH. (2) Human liver cancer cells HepG2 were seeded in a cell culture plate and cultured in a 37°C, 5% CO2 cell culture incubator until the cell confluence reached 80%; (3) Divided into 5 groups: control group (Control), induced group (FFA), treatment group T2 (5μM), treatment group T2 (25μM), treatment group T2 (50μM). The control group was not treated, the induced 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. (4) Collect cell pellets, lyse cells, and measure the triglyceride content of cell lysate; (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 effect of the peptide does not show concentration dependence.

[0032] Example 3: Evaluation of the therapeutic effect of polypeptides using Oil Red O staining on mouse cells (1) Mouse normal liver cells AML12 were inoculated into a cell culture plate and placed at 37°C and 5% CO 2 Culture in a cell culture incubator until the cell confluence reaches 80%; (2) Divided into 3 groups: control group (Control), induced group (FFA), and treatment group T2 (5 μM). The control group was not treated, the induced 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. (3) Wash the cells twice with PBS, add fixative to fix for 30 min, rinse with 60% isopropanol, add Oil Red O staining solution to stain for 20 min, rinse with 60% isopropanol, wash with water three times, add hematoxylin staining solution to stain for 2 min, wash with water three times, add Oil Red O buffer to incubate for 1 min, discard, soak in deionized water, and observe under an inverted microscope; (4) Experimental results are shown in Figure 4 A large number of red lipid droplets were observed in the induced group, and the peptide treatment group showed a certain degree of relief.

[0033] Example 4: Detection of triglyceride content in mouse cells to evaluate the therapeutic effect of polypeptides (1) Mouse normal liver cells AML12 were inoculated into a cell culture plate and placed at 37°C and 5% CO 2 Culture in a cell culture incubator until the cell confluence reaches 80%; (2) Divided into 5 groups: control group (Control), induced group (FFA), treatment group T2 (5μM), treatment group T2 (25μM), treatment group T2 (50μM). The control group was not treated, the induced 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. (3) Collect cell pellets, lyse cells, and measure the triglyceride content of cell lysate; (4) Experimental results are shown in Figure 5 , low concentration peptide treatment of cells can effectively alleviate the accumulation of triglycerides in cells.

[0034] Example 5: Detection of lipid droplet aggregation on mouse cells to evaluate the therapeutic effect of polypeptides (1) BODIPY is a green fluorescent probe that can penetrate the cell membrane into the cell, locate and specifically stain polar lipids in the cell, and is often used for fatty acid uptake, lipid transport, lipid absorption and accumulation; (2) Mouse normal liver cells AML12 were inoculated into a cell culture plate and placed at 37°C and 5% CO 2 Culture in a cell culture incubator until the cell confluence reaches 80%; (3) Divided into 3 groups: control group (Control), induced group (FFA), treatment group T2 (5 μM). The control group was not treated, the induced 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. (4) Labeling intracellular lipid droplets with BODIPY fluorescent probe and observing the aggregation of intracellular lipid droplets using laser confocal microscopy; (5) Confocal experiment results are shown in Figure 6 , a large number of green fluorescently labeled lipid droplets were observed in the induced group, and the green fluorescence intensity was significantly reduced in the peptide treatment group; (6) The cells were divided into 5 groups: control group (Control), induced 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 induced 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. (7) Label the intracellular lipid droplets with BODIPY fluorescent probe, collect the cells, and detect the intracellular fluorescence intensity by flow cytometry; (8) Flow cytometry results are shown in Figure 7 The fluorescence intensity of the induced group increased significantly, and there was an obvious peak rightward shift compared with the control group. The fluorescence intensity of the peptide treatment group decreased significantly, that is, the peak shifted to the left.

[0035] 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 effect of peptide T2 in inhibiting fat accumulation is verified at the cellular level, and T2 is determined to be a peptide that can effectively bind to CD36 protein and alleviate cellular fat accumulation.

[0036] 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 principle of the present invention should be included in the protection scope of the present invention.

Claims

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

2. The polypeptide for inhibiting fat accumulation according to claim 1, characterized in that: The polypeptide is capable of binding to the CD36 protein.

3. The polypeptide for inhibiting fat accumulation according to claim 1, characterized in that: The peptide can target and bind to CD36 protein, inhibit CD36's transport of extracellular fatty acids, and reduce cellular lipid uptake and accumulation.

4. A pharmaceutical composition comprising the polypeptide for inhibiting fat accumulation as claimed in claim 1.

5. Use of the polypeptide for inhibiting fat accumulation according to claim 1 in the preparation of a drug for inhibiting CD36 transport of extracellular fatty acids.

6. Use of the polypeptide for inhibiting fat accumulation according to claim 1 in the preparation of drugs for treating or preventing hepatitis, liver cancer, atherosclerosis, diabetes, diabetic nephropathy, obesity or tumors.

7. Use of the polypeptide for inhibiting fat accumulation according to claim 1 in the preparation of a drug for treating or preventing non-alcoholic fatty liver disease.

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