Application of CAP-Gly functional peptide in preparation of medicine for treating fatty liver

Abstract

The invention relates to the technical field of drugs for gene therapy, in particular to application of CAP-Gly functional peptide in preparation of drugs for treating fatty liver. The invention provides application of a CAP-Gly functional peptide or a reagent for improving the expression quantity of the CAP-Gly functional peptide in preparation of a medicine for treating fatty liver. The amino acid sequence of the CAP-Gly functional peptide is shown as SEQ ID NO.23. The invention further provides a preparation method of the CAP-Gly functional peptide. The invention finds that the overexpression of the KIF13B protein or the CAP-Gly functional peptide thereof in mice can effectively reduce lipid accumulation in liver cells and increase phosphorylation of protein kinase alpha activated by adenosine monophosphate, and provides a new target and a new strategy for precise treatment of patients with fatty liver (especially metabolism-related fatty liver diseases) clinically.

Description

Technical Field

[0001] The present invention relates to the technical field of gene therapy medicines, in particular to the application of CAP-Gly functional peptide in the preparation of medicines for treating fatty liver. Background Art

[0002] Metabolic dysfunction-associated fatty liver disease (MAFLD) is a highly heterogeneous metabolic disease with an extremely complex etiology. Currently, only one thyroid receptor agonist is clinically used to treat MAFLD-associated fatty liver disease with liver fibrosis, but this drug has poor efficacy and a very narrow target population.

[0003] Low-density lipoprotein receptor-related protein 1 (LRP1), a key lipoprotein receptor, is highly expressed in the liver and participates in regulating glucose and lipid metabolism. Increasing LRP1 receptor activity or enhancing LRP1-mediated signaling pathways can effectively prevent and treat the adverse consequences of glucose and lipid metabolism disorders. This novel molecular mechanism provides new therapeutic approaches for clinical diagnosis and treatment.

[0004] Kinesin family member 13B (KIF13B) acts as a scaffold protein to effectively promote the recruitment of LRP1 to caveolae, thereby enhancing the endocytosis of LRP1 on lipid membranes. LRP1 relies on the Clathrin and Caveolin pathways to achieve cellular endocytosis, thereby completing the transport and regulation of various ligands such as lipoproteins, proteases, protease inhibitor complexes, bacterial toxins, and viruses. Hirokawa et al. [1] A study found that the plasma total cholesterol, low-density lipoprotein cholesterol LDL-C, high-density lipoprotein cholesterol HDL-C and coagulation factor FVIII levels of Kif13b gene (i.e. KIF13B gene) knockout mice were significantly higher than those in the control group; at the same time, the LDL uptake of embryonic fibroblasts of Kif13b gene knockout mice was only 60% of that of embryonic fibroblasts of wild-type mice, suggesting that this phenomenon may be related to abnormal LRP1 transport process. In another independent study, Chishti et al. [2]No significant changes in FVIII levels were found in peripheral blood of Kif13b-specific knockout mice, but elevated plasma total cholesterol and LDL-C levels were observed compared to wild-type mice. Unfortunately, none of the previous studies reported whether Kif13b gene deletion is associated with liver lipid metabolism disorders and metabolic-related fatty liver disease. Furthermore, it remains unclear whether the Kif13b gene and its functional domains can be used as drug targets for the development of treatments for metabolic-related fatty liver disease.

[0005] [1] Y. Kanai, D. Wang, N. Hirokawa, KIF13B enhances the endocytosis of LRP1 by recruiting LRP1 to caveolae, The Journal of cell biology, 204 (2014) 395-408.

[0006] [2]J.Mills,T.Hanada,Y.Hase,L.Liscum,AHChishti,LDL receptor relatedprotein 1requires the I(3)domain of discs-large homolog 1 / DLG1 for interaction with the kinesin motor protein KIF13B,Biochim Biophys Acta MolCell Res,1866(2019)118552. Summary of the Invention

[0007] To address the above issues, the present invention provides the use of a CAP-Gly functional peptide in the preparation of a drug for treating fatty liver disease. The present invention discovered that the CAP-Gly functional peptide of the KIF13B protein can effectively reduce lipid accumulation in hepatocytes, providing a new target and strategy for the precise treatment of patients with clinical fatty liver disease (especially metabolic-related fatty liver disease).

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] The present invention provides use of a CAP-Gly functional peptide or a reagent for increasing the expression of a CAP-Gly functional peptide in preparing a drug for treating fatty liver. The amino acid sequence of the CAP-Gly functional peptide is shown in SEQ ID NO.23.

[0010] The present invention provides the use of a protein containing a CAP-Gly functional peptide or an agent for increasing the expression of a protein containing a CAP-Gly functional peptide in preparing a drug for treating fatty liver. The amino acid sequence of the CAP-Gly functional peptide is shown in SEQ ID NO. 23.

[0011] Preferably, the protein comprises kinesin family member 13B.

[0012] Preferably, the drug for treating fatty liver is a drug for treating metabolic associated fatty liver disease (MAFLD).

[0013] Preferably, the drug for treating fatty liver is a drug that reduces lipid accumulation in liver cells.

[0014] Preferably, the drug for treating fatty liver is a drug that increases the phosphorylation of adenosine monophosphate-activated protein kinase α.

[0015] Preferably, the nucleotide sequence of the gene encoding the CAP-Gly functional peptide is shown as SEQ ID NO.20.

[0016] Preferably, the reagent for increasing the expression level of the CAP-Gly functional peptide comprises a recombinant vector that overexpresses CAP-Gly, and the CAP-Gly is a gene encoding the CAP-Gly functional peptide.

[0017] Preferably, the reagent for increasing the expression level of the protein containing the CAP-Gly functional peptide comprises a recombinant vector for overexpressing a coding gene, wherein the coding gene is a gene encoding a protein containing the CAP-Gly functional peptide.

[0018] Preferably, the starting vector of the recombinant vector includes a pcDNA3.1 expression vector.

[0019] Beneficial effects:

[0020] The present invention provides the use of a CAP-Gly functional peptide or an agent for increasing the expression of a CAP-Gly functional peptide in the preparation of a drug for treating fatty liver disease. The amino acid sequence of the CAP-Gly functional peptide is shown in SEQ ID NO. 23. The present invention has found that overexpressing the KIF13B protein or its CAP-Gly functional peptide in mice can effectively reduce lipid accumulation in hepatocytes and increase the phosphorylation of adenosine monophosphate-activated protein kinase α, providing a new target and new strategy for the precise treatment of patients with clinical fatty liver disease (especially metabolic-related fatty liver disease). BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0022] Figure 1 Genotyping results of wild-type, heterozygous, and Kif13b knockout mice;

[0023] Figure 2 is the relative expression level of Kif13b mRNA in the liver of wild-type and Kif13b knockout mice;

[0024] Figure 3 The results of body weight measurement of wild-type and Kif13b knockout mice are shown;

[0025] Figure 4 is the plasma cholesterol content of wild-type and Kif13b knockout mice;

[0026] Figure 5 is the plasma triglyceride content of wild-type and Kif13b knockout mice;

[0027] Figure 6 is the plasma alanine aminotransferase level in wild-type and Kif13b knockout mice;

[0028] Figure 7 is the plasma aspartate aminotransferase level of wild-type and Kif13b knockout mice;

[0029] Figure 8 H&E staining and Oil Red O staining results of the liver of wild-type and Kif13b knockout mice;

[0030] Figure 9 KEGG enrichment analysis results of liver transcriptome sequencing of wild-type and Kif13b knockout mice;

[0031] Figure 10 Results of liver protein content detection in wild-type and Kif13b knockout mice;

[0032] Figure 11 The results of Oil Red O staining of HepG2 cells transfected with scrRNA or siKIF13B;

[0033] Figure 12 The results of Oil Red O staining of HepG2 cells transfected with KIF13B overexpression plasmid or pcDNA3.1 expression vector;

[0034] Figure 13 For the co-immunoprecipitation experiment, HepG2 cells were transfected with KIF13B overexpression plasmid or plasmids overexpressing different functional domains;

[0035] Figure 14 The results of immunoblotting experiments are shown in HepG2 cells transfected with KIF13B overexpression plasmids or plasmids overexpressing different functional domains.

[0036] Figure 15 Oil Red O staining results of HepG2 cells transfected with KIF13B overexpression plasmid or different functional domain overexpression plasmids. DETAILED DESCRIPTION

[0037] The present invention provides the use of a CAP-Gly functional peptide or an agent for increasing the expression of a CAP-Gly functional peptide in the preparation of a drug for treating fatty liver. The amino acid sequence of the CAP-Gly functional peptide is shown in SEQ ID NO. 23, which is as follows:

[0038] GEFVTVGAHKTGVVRYVGPADFQEGTWVGVELDLPSGKNDGSIGGKQY FRCNPGYGLLVRPSR.

[0039] The present invention also provides the use of a protein containing a CAP-Gly functional peptide or an agent for increasing the expression of a protein containing a CAP-Gly functional peptide in the preparation of a drug for treating fatty liver disease. The amino acid sequence of the CAP-Gly functional peptide is shown in SEQ ID NO. 23. In one embodiment, the protein may be kinesin family member 13B. In one embodiment, the drug for treating fatty liver disease is a drug for treating metabolic-related fatty liver disease. In one embodiment, the drug for treating fatty liver disease is a drug for reducing lipid accumulation in liver cells. In one embodiment, the drug for treating fatty liver disease is a drug for increasing phosphorylation of adenosine monophosphate-activated protein kinase α. In one embodiment, the nucleotide sequence of the gene encoding the CAP-Gly functional peptide is shown in SEQ ID NO. 20. In one embodiment, the agent for increasing the expression of the CAP-Gly functional peptide may be a recombinant vector that overexpresses CAP-Gly, and the CAP-Gly is the gene encoding the CAP-Gly functional peptide. In one embodiment, the reagent for increasing the expression of a protein containing a CAP-Gly functional peptide can be a recombinant vector that overexpresses a gene encoding the protein containing the CAP-Gly functional peptide. In one embodiment, when the protein containing the CAP-Gly functional peptide is kinesin family member 13B (KIF13B), the nucleotide sequence of the gene encoding KIF13B is as shown in SEQ ID NO. 24, specifically as follows:

[0040]

[0041] As an embodiment, the starting vector of the recombinant vector may be a pcDNA3.1 expression vector.

[0042] The present invention found that knocking out the Kif13b gene in mice can lead to obesity, increased plasma cholesterol levels, liver damage and hepatic steatosis in mice, while overexpressing the Kif13b gene in mice can reduce lipid accumulation in adipocytes and increase adenosine monophosphate-activated protein kinase α phosphorylation. Finally, by overexpressing different functional domains (RILP, CAP-Gly, FHA and Motor) of the Kif13b gene in mice, it was found that overexpressing the functional domain CAP-Gly can reduce lipid accumulation in adipocytes and increase adenosine monophosphate-activated protein kinase α phosphorylation. It can be seen that the CAP-Gly functional peptide is a functional domain that plays a role in reducing lipid accumulation in adipocytes. The present invention provides new targets and new strategies for the precise treatment of patients with fatty liver (especially metabolic-related fatty liver disease) in clinical practice.

[0043] To further illustrate the present invention, the application of the CAP-Gly functional peptide provided by the present invention in preparing a drug for treating fatty liver is described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0044] Example 1

[0045] Kif13b knockout mice were constructed by CRISPR / Cas9 targeting Kif13b exon 6 at the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, and Peking Union Medical College. Wild-type mice were 8-week-old C57BL / 6J mice. The method involved mixing Cas9 mRNA and sgRNA to final concentrations of 50 ng / μL and 25 ng / μL, respectively, and microinjecting the mixture into fertilized eggs. The sgRNAs consisted of an equimolar mixture of sgRNA1 and sgRNA2. The specific sequences are shown in Table 1.

[0046] Table 1 Sequence information of sgRNA

[0047]

[0048] Genotyping: Mouse DNA was extracted using a one-step mouse genotyping kit (Vazyme, PD101-01). PCR amplification was performed using the extracted mouse DNA as a template, followed by sequence analysis by agarose gel electrophoresis. The primer sequences for mouse genotyping are as follows:

[0049] F: 5'-ATTGTTCCTGCACTATTACAA-3', SEQ ID NO.5;

[0050] R: 5'-GGGAAGTGTAGGTCTGAGCTAG-3', SEQ ID NO. 6.

[0051] See the results Figure 1 , among which Kif13b + / + For wild-type mice, Kif13b + / - Heterozygous knockout mice, Kif13b - / - These are homozygous knockout mice.

[0052] Determination of Kif13b in wild-type mice and constructed - / - Relative expression of Kif13b mRNA in mouse liver, with β-actin as the internal reference gene; the primers used are as follows:

[0053] Kif13b-F: 5'-AACGAACCCAGAAAGAGGA-3', SEQ ID NO.7;

[0054] Kif13b-R: 5'-GCTTGTGACAGCCAGTTTA-3', SEQ ID NO.8;

[0055] β-actin-F: 5'-TGTGCTGTCCCTGTATGCCTCT-3', SEQ ID NO.9;

[0056] β-actin-R: 5'-GGAACCGCTCGTTGCCAATAGT-3', SEQ ID NO. 10.

[0057] The relative expression of Kif13b mRNA is shown in Figure 2 , where *** means p < 0.001.

[0058] Depend on Figure 2 It can be seen that compared with wild-type mice, Kif13b - / - Kif13b mRNA was barely detectable in mouse liver.

[0059] Example 2

[0060] The Kif13b knockout mice (Kif13b) constructed in Example 1 were - / - ) and purchased wild-type mice (Kif13b + / + ), divided into 4 groups, 5 in each group, respectively denoted as: Kif13b + / + +CD, Kif13b - / - +CD, Kif13b + / + +HFD and Kif13b - / - +HFD. Specific treatment is as follows:

[0061] Kif13b + / + +CD: wild-type mice on a normal diet for 16 weeks;

[0062] Kif13b - / - +CD: Kif13b knockout mice ate a normal diet for 16 weeks;

[0063] Kif13b + / + +HFD: Wild-type mice were fed a normal diet for 8 weeks and then switched to a high-fat diet (purchased from Research Diets, Inc., catalog number D12492) for another 8 weeks;

[0064] Kif13b - / - +HFD: Kif13b knockout mice were fed a normal diet for 8 weeks and then switched to a high-fat diet for another 8 weeks.

[0065] After the feeding, the body weight, plasma cholesterol content (total cholesterol kit, Zhongsheng Beikong Biotechnology Co., Ltd.), plasma triglyceride content (total triglyceride kit, Zhongsheng Beikong Biotechnology Co., Ltd.), plasma alanine aminotransferase content (alanine aminotransferase kit, Nanjing Jiancheng Bioengineering Institute), and plasma aspartate aminotransferase content (aspartate aminotransferase kit, Nanjing Jiancheng Bioengineering Institute) of the four groups of mice were measured. The results are shown in Figures 3 to 7 Among them, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and the results without significant difference indicate no significant difference.

[0066] After the feeding, the livers of the four groups of mice were stained with H&E and Oil Red O as follows:

[0067] H&E staining: Paraffin-embedded tissue samples were sectioned, dewaxed with xylene, and dehydrated with ethanol. The sections were then stained with Harry's hematoxylin (HHS128-4L, Sigma) for 5 minutes and stained with eosin aqueous solution (HT110232-1L, Sigma) for 3 minutes. The sections were dehydrated with gradient ethanol, cleared with xylene, and mounted with gum.

[0068] Oil Red O staining: Liver samples were embedded in OCT (4583, Sakura, American) and quickly frozen in liquid nitrogen. The frozen tissue was sliced ​​at 7 μm thickness. After drying at room temperature for 30 minutes, the sections were soaked in 60% isopropanol for 10 seconds and stained with 0.3% Oil Red O working solution for 30 minutes. They were then counterstained with hematoxylin for 5 minutes and mounted with glycerol. The results are shown in Figure 8 , H&E is the result of H&E staining, ORO is the result of Oil Red O staining, and the scale bars are 100 μm.

[0069] After the feeding was completed, Beijing Novogene Technology Co., Ltd. was commissioned to + / + +CD, Kif13b - / - Liver transcriptome sequencing and KEGG enrichment analysis were performed for mice in the +CD groups. The results are shown in Figure 9 .

[0070] After the feeding was completed, the protein content of the livers of the four groups of mice was detected by immunoblotting as follows: the total protein of the cells or tissues was extracted using RIPA buffer (P0013B, Beyotime) pre-cooled at 4°C and supplemented with a protease inhibitor cocktail and a phosphatase inhibitor cocktail. The protein concentration was quantified using a BCA protein detection kit (23225, Thermo Fisher Scientific). The lysate was mixed with a loading buffer (P0015L, Beyotime) and denatured at 95°C for 10 minutes. The samples were then analyzed by SDS-PAGE and nitrocellulose membrane transfer. The membrane was blocked with 5% skim milk in TBST buffer (2mMTris, 137mM NaCl, 2.7mM KCl, 0.075% Tween-20) at room temperature for 1 hour, and then incubated with the designated primary antibody (see Table 2) at 4°C overnight. The membrane was then washed and incubated with a horseradish peroxidase-conjugated secondary antibody (ZSGB-BIO, China) at room temperature for 1 hour. ECL luminescent solution was used for color development. The results are shown in Figure 10 , where n in Srebp1(n) and Srebp1(p) is the mature form of Srebp1 and p is the precursor of Srebp1.

[0071] Table 2 Source information of different primary antibodies

[0072]

[0073] Depend on Figure 3 It can be seen that Kif13b in normal diet or high-fat diet - / - The body weight of mice was significantly increased compared with wild-type mice.

[0074] Depend on Figure 4 It can be seen that Kif13b in normal diet - / - The plasma cholesterol level of mice was significantly increased compared with wild-type mice, but Kif13b was expressed in high-fat diet mice. - / - There was no significant difference in plasma cholesterol levels between mice and wild-type mice.

[0075] Depend on Figure 5 It can be seen that Kif13b in normal diet or high-fat diet - / - There was no significant difference in plasma triglyceride levels between mice and wild-type mice.

[0076] Depend on Figure 6It can be seen that Kif13b in normal diet or high-fat diet - / - The plasma alanine aminotransferase levels of mice were significantly increased compared with wild-type mice.

[0077] Depend on Figure 7 It can be seen that Kif13b in normal diet or high-fat diet - / - The plasma aspartate aminotransferase levels of mice were significantly increased compared with wild-type mice.

[0078] Depend on Figure 8 It can be seen that Kif13b in normal diet or high-fat diet - / - The liver lipid accumulation of mice was increased compared with wild-type mice.

[0079] Depend on Figure 9 It can be seen that Kif13b - / - The SREBP pathway in mouse liver was significantly activated, and the AMPK pathway was significantly inhibited.

[0080] Depend on Figure 10 It can be seen that Kif13b - / - In mouse liver, Srebp1 and Srebp2 were increased in the nucleus and AMPKα phosphorylation was inhibited.

[0081] Example 3

[0082] siRNA transfection: scrRNA and siKif13b were synthesized by Sangon Biotech (Shanghai) Co., Ltd. and tested for knockdown efficiency. siKif13b with good knockdown efficiency was selected for subsequent experiments; the siKif13b was composed of equimolar siKif13b-1, siKif13b2, and siKif13b-3. The specific siRNA sequences are shown in Table 3, where scrRNA was a negative control group and Kif13b was not knocked down.

[0083] Table 3 Sequence information of different siRNAs

[0084]

[0085] Note: The "TT" bases at the 3' end in Table 3 are all thymine, which can increase the stability of siRNA and prevent it from being degraded by nucleases in cells.

[0086] 3×10 5HepG2 cells were seeded in 6-well plates and transfected with scrRNA and siKIF13B using Opti-MEM medium. The Lipofectamine RNAiMAX kit (13778150) from Invitrogen (USA) was used according to the manufacturer's instructions. After 48 hours of transfection, HepG2 cells were divided into three groups: scrRNA + PA, siKIF13B + PA, and siKIF13B + PA + Met. The treatments were as follows:

[0087] scrRNA+PA: Palmitic acid (PA) was added to the culture medium after 48 hours of scrRNA transfection at a final concentration of 300 μM and cultured for another 24 hours;

[0088] siKIF13B+PA: Palmitic acid was added to the culture medium after siKif13b transfection for 48 hours at a final concentration of 300 μM and culture was continued for 24 hours;

[0089] siKIF13B+PA+Met: Palmitic acid at a final concentration of 300 μM and metformin (AMPKα agonist, denoted as Met) at a final concentration of 2 mM were added to the culture medium after siKif13b transfection for 48 hours, and the culture was continued for 24 hours.

[0090] After culturing for 24 hours, the three groups of HepG2 cells were stained with Oil Red O. The results are shown in Figure 11 .

[0091] The results showed that knockdown of KIF13B aggravated lipid accumulation in HepG2 cells, while metformin treatment reduced lipid accumulation in HepG2 cells.

[0092] Example 4

[0093] Sangon Biotech (Shanghai) Co., Ltd. was commissioned to construct the KIF13B overexpression plasmid using standard molecular cloning techniques, including amplifying the coding sequence (excluding the stop codon) of KIF13B (NCBI accession number: NM_015254.4) by PCR and inserting it into the pcDNA3.1 expression vector.

[0094] 3×10 5 HepG2 cells were seeded in 6-well plates using Opti-MEM medium and transfected with 2 μg of KIF13B overexpression plasmid or pcDNA3.1 expression vector (empty vector) using Lipo8000 (C0533, Beyotime) according to the manufacturer's instructions. HepG2 cells transfected for 48 hours were divided into three groups: NC+PA, KIF13B+PA, and KIF13B+PA+IN3. The specific treatments were as follows:

[0095] NC+PA: palmitic acid was added to the culture medium after 48 hours of empty vector transfection at a final concentration of 300 μM and cultured for another 24 hours;

[0096] KIF13B+PA: palmitic acid was added to the culture medium after KIF13B overexpression plasmid transfection for 48 hours at a final concentration of 300 μM and culture was continued for 24 hours;

[0097] KIF13B+PA+IN3: Palmitic acid at a final concentration of 300 μM and AMPK-IN3 (AMPKα inhibitor) at a final concentration of 110 nM were added to the culture medium after KIF13B overexpression plasmid transfection for 48 hours, and the culture was continued for 24 hours.

[0098] After culturing for 24 hours, the three groups of HepG2 cells were stained with Oil Red O. The results are shown in Figure 12 .

[0099] The results showed that overexpression of KIF13B reduced lipid accumulation in HepG2 cells, while inhibition of AMPKα increased lipid accumulation in HepG2 cells.

[0100] Example 5

[0101] Sangon Biotech (Shanghai) Co., Ltd. was commissioned to construct overexpression vectors of different functional domains of KIF13B (RILP, CAP-Gly, FHA, and Motor). The method was similar to that in Example 4. The nucleotide sequences of the different functional domains are as follows:

[0102] Functional domain RILP (SEQ ID NO. 19):

[0103] 5'-atgatattaaacagcttagaacaacagcatgaagaagaaaaacgatctgcactggagcgccagaggcttatgtatgagcacgaattggagcagctccggagaaggctgtctcctgagaagcagaactgccggagcatggacaggttttctttc cactcgcccagcgctcagcaacgcttaagacagtgggctgaggagagagaagcaacgttgaataacagcctgatgaggctgagggaacaaattgttaaggccaatctattggtgagagaagctaattacattgctgaggagctggataaaaga-3';

[0104] Functional domain CAP-Gly (SEQ ID NO. 20):

[0105] 5'-gttcgtcaccgtgggcgcccacaaaacgggcgtggtgagatacgtggggcctgccgacttccaagagggcacgtgggtcggcgtggagctcgacctgccctcaggtaagaatgacggttccatcggcgggaagcagtacttcaggtgtaaccctggctacgggctgctggtcaggcccagccgggtccg-3';

[0106] Functional domain FHA (SEQ ID NO.21):

[0107] 5'-atgaaatgcttccttgtgaatctgaatgctgacccagctctgaatgagcttctggtgtactatttaaaggaacatacattgatagggtcagcaaattcccaagatatccaactgtgcggcatgggaattcttcctgaacactgtattatagacatcacgtcagaaggccaggttatgctgactcctcagaagaacaccagaacatttgtaaatgggtcatctgtctccagtccaatacagctacaccatggggacaggatattatggggaaacaatcatttcttcagactcaatttg-3';

[0108] Functional domain Motor (SEQ ID NO.22):

[0109] 5'-atggtgaaagtggcggtgcggatacgacccatgaaccggcgagagactgacttgcataccaaatgtgtggtggatgtggatgcaaacaaggttattcttaatcctgtaaatacgaatctttccaaaggagatgcccggggccagccgaaggtgtttgcttatgatcattgtttctggtctatggatgaatctgtcaaagaaaagtatgcaggtcaagatattgttttcaagtgccttggagagaatatcctgcagaatgcttttgatggctacaatgcatgtatctttgcctatggacagactggctctggaaaatcttataccatgatgggcacagctgaccaacctggattaatcccaagactttgcagtggactctttgaacgaactcagaaagaggaaaatgaagaacagagttttaaagtagaagtgtcctacatggaaatttataatgaaaaagttcgagaccttcttgatcccaaaggaagccgtcagacgttgaaagtcagagagcatagtgtgttgggaccttatgtcgacggactttctaaactggctgtcacaagctacaaggatattgagtcgttgatgtctgagggtaacaaatctcgcacagttgctgcaaccaacatgaacgaggagagtagccgatcccatgcagttttcaaaatcaccctcacacatactctctacgatgtgaagtctgggacatctggagagaaagtgggcaaactcagcctggtggatttagctggcagtgaacgagcaacgaagacaggcgctgcaggggacaggctgaaggaagggagcaacattaacaagtccctcacaaccctcggtctggttatctcagctcttgcagatcagagtgctggcaaaaacaagaataaatttgttccatatcgtgactcagttctcacttggctgctcaaagacagcctcgggggtaacagcaagaccgccatggtggctactgtgagtcctgcagctgataactatgatgaaaccctctcaactctgcggtatgcagatcgagccaagcacattgtaaaccacgctgtggtgaat-3'。

[0110] 3×10 5 HepG2 cells were seeded in 6-well plates using Opti-MEM medium and transfected with 2 μg of KIF13B overexpression plasmid, overexpression vectors of different functional domains (RILP, CAP-Gly, FHA, and Motor), or empty vector (denoted as NC) using Lipo8000 (C0533, Beyotime) according to the manufacturer's instructions. The HepG2 cells were transfected for 48 hours and subjected to immunoprecipitation and immunoblotting experiments, respectively. The immunoprecipitation method includes:

[0111] 1×10 7 The cells were lysed on ice with 1 mL of RIPA buffer for 30 minutes. After shaking for 15 seconds, the lysate was centrifuged at 12,000 rpm for 30 minutes at 4°C. 800 μL of supernatant was incubated with 10 μL of anti-HA antibody at 4°C for 6 hours. Bound protein A / G agarose beads were then added and incubated overnight at 4°C. Centrifuged at 12,000 rpm for 1 minute, the supernatant was discarded, and after washing 5 times with lysis buffer, the supernatant was discarded after centrifugation at 12,000 rpm for 1 minute; 120 μL of 1× loading buffer was added and boiled at 95°C for 10 minutes. 20 μL of sample was taken for immunoblot analysis. Results are shown in Figure 13 The results showed that overexpression of Kif13b or its functional domain CAP-Gly significantly increased its binding to AMPKα.

[0112] The method of the immunoblotting experiment is shown in Example 2. Figure 14 Among them, the primary antibody corresponding to p-AMPKα is p-AMPKαThr172. The results show that overexpression of KIF13B or its functional domain CAP-Gly significantly increases the level of pAMPKα.

[0113] Example 6

[0114] The method of Example 5 was used to transfect HepG2 cells with KIF13B overexpression plasmids, overexpression vectors of different functional domains (RILP, CAP-Gly, FHA, and Motor), or an empty vector (denoted as NC). After 48 hours of transfection, the HepG2 cells were divided into 6 groups, namely NC+PA, KIF13B+PA, PILP+PA, CAP-Gly+PA, FHA+PA, and Motor+PA. The specific treatments were as follows:

[0115] NC+PA: palmitic acid was added to the culture medium after 48 hours of empty vector transfection at a final concentration of 300 μM and cultured for another 24 hours;

[0116] KIF13B+PA: palmitic acid was added to the culture medium after KIF13B overexpression plasmid transfection for 48 hours at a final concentration of 300 μM and culture was continued for 24 hours;

[0117] PILP+PA: Palmitic acid was added to the culture medium of cells transfected with the functional domain PILP overexpression plasmid for 48 hours at a final concentration of 300 μM and cultured for another 24 hours;

[0118] CAP-Gly+PA: Palmitic acid was added to the culture medium of cells transfected with the functional domain CAP-Gly overexpression plasmid for 48 hours at a final concentration of 300 μM and cultured for another 24 hours;

[0119] FHA+PA: Palmitic acid was added to the culture medium after 48 hours of transfection with the functional domain FHA overexpression plasmid at a final concentration of 300 μM and cultured for another 24 hours;

[0120] Motor+PA: Palmitic acid at a final concentration of 300 μM was added to the culture medium after 48 hours of transfection with the functional domain Motor overexpression plasmid, and the culture was continued for 24 hours.

[0121] After culturing for 24 hours, the 6 groups of HepG2 cells were stained with Oil Red O. The results are shown in Figure 15 .

[0122] The results showed that overexpression of KIF13B or its functional domain CAP-Gly significantly reduced lipid accumulation in HepG2 cells.

[0123] In summary, KIF13B protein or CAP-Gly functional peptide can effectively reduce lipid accumulation in hepatocytes, providing new targets and new strategies for the precise treatment of patients with metabolic disorder-related fatty liver disease in clinical practice.

[0124] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. Use of a CAP-Gly functional peptide or an agent for increasing the expression of a CAP-Gly functional peptide in the preparation of a drug for treating fatty liver, wherein the amino acid sequence of the CAP-Gly functional peptide is shown in SEQ ID NO.

23.

2. Use of a protein containing a CAP-Gly functional peptide or an agent for increasing the expression of a protein containing a CAP-Gly functional peptide in the preparation of a drug for treating fatty liver, wherein the amino acid sequence of the CAP-Gly functional peptide is shown in SEQ ID NO.

23.

3. The use according to claim 2, characterized in that: The proteins include kinesin family member 13B.

4. The use according to claim 1 or 2, characterized in that: The drug for treating fatty liver is a drug for treating metabolism-related fatty liver disease.

5. The use according to claim 1 or 2, characterized in that: The drug for treating fatty liver is a drug for reducing lipid accumulation in liver cells.

6. The use according to claim 1 or 2, characterized in that: The drug for treating fatty liver is a drug that increases the phosphorylation of adenosine monophosphate-activated protein kinase α.

7. The use according to claim 1 or 2, characterized in that: The nucleotide sequence of the gene encoding the CAP-Gly functional peptide is shown in SEQ ID NO.

20.

8. The use according to claim 1, characterized in that: The reagent for increasing the expression amount of the CAP-Gly functional peptide comprises a recombinant vector for overexpressing CAP-Gly, and the CAP-Gly is a gene encoding the CAP-Gly functional peptide.

9. The use according to claim 2, characterized in that: The reagent for increasing the expression amount of the protein containing the CAP-Gly functional peptide comprises a recombinant vector for overexpressing a coding gene, wherein the coding gene is a gene encoding the protein containing the CAP-Gly functional peptide.

10. The use according to claim 8 or 9, characterized in that: The starting vector of the recombinant vector includes a pcDNA3.1 expression vector.

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