Use of kif13b gene in drug

Abstract

The present invention relates to the technical field of drugs for gene therapy, in particular to the use of the Kif13b gene as a drug target in drugs. The use comprises the use in screening drugs for preventing, alleviating and / or treating fatty liver, atherosclerosis, abdominal aortic aneurysm and / or macrophage senescent inflammation.

Description

Application of Kif13b gene in medicine Technical Field

[0001] This invention belongs to the field of gene function and application technology, and particularly relates to the application of the Kif13b gene in drugs, including its application in drugs for treating fatty liver, atherosclerosis, abdominal aortic aneurysm and / or macrophage senescent inflammation. Background Technology

[0002] Kif13b, a member of the kinin family, is the largest member of the kinin-3 family. As a scaffold protein, it effectively promotes the recruitment of low-density lipoprotein receptor-associated protein 1 (LRP1) to caveolae, thereby enhancing LRP1's endocytosis on the lipid membrane. LRP1 achieves endocytosis via the Clathrin and Caveolin pathways, thereby completing the transport and regulation of various ligands such as lipoproteins, proteases, protease inhibitor complexes, bacterial toxins, and viruses. Kif13b also participates in the circulation and transport of vascular endothelial growth factor receptor (VEGFR). For example, under PI3K-dependent conditions, Kif13b binds to microtubules and transports VEGFR2 from the Golgi membrane to the plasma membrane via anterograde transport, participating in the internalization and recycling of VEGFR2 and regulating the localization of VEGFR2 on the lipid membrane to achieve receptor phosphorylation. Studies have shown that Kif13b knockout mice exhibit significantly higher levels of total cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and coagulation factor FVIII compared to the control group. Simultaneously, Kif13b knockout mice show reduced LDL uptake in embryonic fibroblasts. For example, Hirokawa et al. (The Journal of Cell Biology, 204(2014)395-408) found these changes in Kif13b knockout mice, suggesting a correlation with abnormal LRP1 transport. Chishti et al. (Biochim Biophys Acta Mol Cell Res, 1866(2019)118552) also observed elevated cholesterol levels in a specific knockout model, but FVIII showed no significant change. These findings indicate that Kif13b plays a crucial role in lipid metabolism and vascular homeostasis, but its association with specific diseases requires further investigation.

[0003] Metabolic dysfunction-associated fatty liver disease (MAFLD) is highly heterogeneous and represents a complex metabolic disorder. Currently, only one thyroid receptor agonist is available clinically for treating MAFLD complicated by liver fibrosis, but its efficacy is poor and its target population is quite narrow. Low-density lipoprotein receptor-associated protein 1 (LRP1), an important lipoprotein receptor, is highly expressed in the liver and participates in the regulation of 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 a new therapeutic approach for clinical diagnosis and treatment. Unfortunately, previous studies have not reported whether Kif13b gene deletion is associated with hepatic lipid metabolism disorders and MAFLD, and it remains unclear whether the Kif13b gene and its functional domains can serve as drug targets for the development of treatments for MAFLD.

[0004] The incidence of atherosclerotic cardiovascular disease (ASCVD) is steadily rising and remains a leading cause of death worldwide. Rupture of unstable atherosclerotic plaques is a major direct cause of myocardial infarction (MI) and ischemic stroke. Atherosclerosis is a chronic inflammatory disease whose pathological processes typically involve endothelial damage, lipid accumulation in the arterial wall, inflammatory cell infiltration, smooth muscle cell proliferation, and extracellular matrix remodeling. Generally, abnormal lipid metabolism and inflammation are key factors contributing to the occurrence and development of atherosclerosis. Excessive uptake of low-density lipoprotein (LDL) and / or insufficient lipid output mediated by high-density lipoprotein (HDL) by different types of cells in the arterial wall triggers the formation of lipid-rich plaques. Simultaneously, the inflammatory response activates immune cells to secrete cytokines and chemokines, further promoting plaque formation and instability. However, despite extensive research into these processes, the exact molecular mechanisms remain incompletely understood, and the incidence of atherosclerosis has remained high over the past few decades. Currently, lipid-lowering drugs, especially statins, are the mainstay of treatment for atherosclerosis. However, lipid-lowering therapy has limited effect on reducing cardiovascular risk because patients receiving lipid-lowering drugs still have a high residual risk of ASCVD. This indicates that the exact molecular mechanisms of atherosclerosis pathogenesis are not widely documented, and potential therapeutic targets remain to be identified. The kinin-3 family, classified as N-terminal motor proteins, has eight members and was initially discovered to facilitate rapid transport in nerve cells. Independent studies have shown that kinin defects can lead to neurological and metabolic diseases. Kif13b may be a key regulator of vascular homeostasis; however, the relationship between Kif13b and ASCVD has not been established, and information on the molecular mechanisms by which Kif13b regulates the development of atherosclerosis remains lacking. Currently, in clinical practice, only antilipid-lowering drugs or anti-inflammatory drugs combined with antithrombotic drugs can delay the development of atherosclerosis, but they cannot effectively reverse atherosclerosis caused by different factors.

[0005] Abdominal aortic aneurysm (AAA) is a fatal vascular disease characterized by a permanent localized dilation of the abdominal aorta exceeding 1.5 times its normal size. It is primarily influenced by multiple risk factors, including hypertension, high plasma cholesterol, atherosclerosis, smoking, age, and sex. Importantly, once an aneurysm ruptures, the mortality rate exceeds 80%. However, to date, no drug therapies have been approved to inhibit AAA development or reduce the risk of aneurysm rupture. Treatment for AAA is primarily considered through surgical repair or stent placement, but this is suitable for less than 10% of the eligible population, indicating that a large number of AAA patients remain at high risk. The main pathological processes in AAA involve the infiltration of inflammatory cells into the vessel wall, apoptosis of vascular smooth muscle cells, and degradation of extracellular matrix elastin. Increasing new evidence suggests a positive correlation between the incidence of AAA and abnormal lipid metabolism, particularly triglyceride-rich lipoproteins (TRLs). Recently, a genome-wide association meta-analysis of AAA identified 121 independent risk loci and highlighted the important role of a series of lipoprotein receptor regulators (such as PCSK9) in AAA. Furthermore, both human and experimental animal studies have confirmed that lipoprotein receptors such as low-density lipoprotein receptor-associated protein 1 (LRP1) are associated with the occurrence and development of AAA, indicating that lipoproteins and their receptors play an indispensable role in AAA. However, it is noteworthy that the detailed molecular mechanisms by which abnormal lipoprotein metabolism induces AAA, and how lipoprotein receptors participate in the pathogenesis of AAA, remain largely unclear, thus limiting the further application of targeted lipoproteins and their receptors in the treatment of AAA. Existing research suggests that Kif13b may be an important factor involved in maintaining vascular cell homeostasis, thereby treating vascular injury diseases. However, the fundamental role of Kif13b in other vascular cell components has not been extensively studied, and whether targeting Kif13b will become a potential therapeutic approach for AAA remains to be confirmed. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention proposes the application of the Kif13b gene in pharmaceuticals, including its application in drugs for treating atherosclerosis, abdominal aortic aneurysm, and fatty liver.

[0007] To address the aforementioned issues, this invention provides the application of the Kif13b gene as a drug target in pharmaceuticals, including its use in screening drugs for the prevention, relief, and / or treatment of fatty liver, atherosclerosis, abdominal aortic aneurysm, and / or macrophage senescent inflammation.

[0008] Furthermore, the nucleotide sequence of the Kif13b gene is shown in SEQ ID NO.24.

[0009] Furthermore, the drugs for preventing, alleviating, and / or treating fatty liver, atherosclerosis, abdominal aortic aneurysm, and / or macrophage senescent inflammation are drugs that promote the overexpression of the Kif13b gene.

[0010] Furthermore, the drugs for preventing, alleviating, and / or treating fatty liver, atherosclerosis, abdominal aortic aneurysm, and / or macrophage senescent inflammation enhance macrophage burial by promoting Kif13b gene overexpression, thereby reducing the formation of necrotic arterial cores and treating atherosclerosis.

[0011] Furthermore, the use of the drug overexpressing the Kif13b gene as described in claim 1 in the preparation of drugs for the prevention, relief and / or treatment of atherosclerosis, abdominal aortic aneurysm, macrophage senescent inflammation, and fatty liver is promoted.

[0012] The present invention also provides a medicament for preventing, alleviating and / or treating fatty liver, atherosclerosis, abdominal aortic aneurysm and / or macrophage senescent inflammation, including a medicament that promotes the overexpression of the Kif13b gene.

[0013] The present invention also provides the use of a protein containing a CAP-Gly functional peptide or a reagent 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, and the protein includes Kif13b.

[0014] Furthermore, the drug for treating fatty liver is a drug for treating metabolic-related fatty liver disease, a drug for reducing lipid accumulation in liver cells, or a drug for increasing the phosphorylation of protein kinase α activated by adenosine monophosphate.

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

[0016] Furthermore, the reagent for increasing the expression level of CAP-Gly functional peptide includes a recombinant vector that overexpresses CAP-Gly, wherein CAP-Gly is the gene encoding the CAP-Gly functional peptide;

[0017] Furthermore, reagents for increasing the expression level of proteins containing CAP-Gly functional peptides include recombinant vectors that overexpress encoding genes, wherein the encoding gene is a gene encoding the protein containing the CAP-Gly functional peptide;

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

[0019] Compared with the prior art, the present invention has the following advantages and technical effects:

[0020] (1) This invention also provides the application of CAP-Gly functional peptides or reagents for increasing the expression of CAP-Gly functional peptides in the preparation of drugs for treating fatty liver, wherein the amino acid sequence of the CAP-Gly functional peptide is shown in SEQ ID NO.23. This invention has found that overexpression of Kif13b protein or its CAP-Gly functional peptide in mice can effectively reduce lipid accumulation in hepatocytes and increase phosphorylation of adenosine monophosphate-activated protein kinase α, providing a new target and strategy for the precision treatment of patients with fatty liver (especially metabolic-related fatty liver disease).

[0021] (2) Given the important role of the Kif13b gene in maintaining normal vascular function and its various cellular activities, this invention hypothesizes that Kif13b may be involved in the pathogenesis of atherosclerosis. This invention investigates the specific role and mechanism of the Kif13b gene in atherosclerosis, elucidating the molecular mechanism by which the Kif13b gene integrates lipid metabolism and inflammatory response through macrophage phagocytosis, providing a new theoretical basis and intervention strategy for the prevention and treatment of atherosclerosis.

[0022] (3) This invention discovers that macrophage homeostasis can be regulated by targeting the expression of the Kif13b gene in macrophages. Overexpression of the Kif13b gene can effectively reverse macrophage senescent inflammation, thereby alleviating abdominal aortic aneurysm, providing a new target and strategy for the precise treatment of abdominal aortic aneurysm patients in clinical practice. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0024] Figure 1 shows the genotype identification results of wild-type, heterozygous, and Kif13b knockout mice;

[0025] Figure 2 shows the relative expression levels of Kif13b mRNA in the livers of wild-type and Kif13b knockout mice;

[0026] Figure 3 shows the weight measurement results of wild-type and Kif13b knockout mice;

[0027] Figure 4 shows the plasma cholesterol levels in wild-type and Kif13b knockout mice;

[0028] Figure 5 shows the plasma triglyceride levels in wild-type and Kif13b knockout mice;

[0029] Figure 6 shows the plasma alanine aminotransferase (ALT) levels in wild-type and Kif13b knockout mice;

[0030] Figure 7 shows the plasma aspartate aminotransferase (AST) levels in wild-type and Kif13b knockout mice;

[0031] Figure 8 shows the results of H&E staining and Oil Red O staining of the livers of wild-type and Kif13b knockout mice;

[0032] Figure 9 shows the KEGG enrichment analysis results of liver transcriptome sequencing of wild-type and Kif13b knockout mice;

[0033] Figure 10 shows the results of liver protein content detection in wild-type and Kif13b knockout mice;

[0034] Figure 11 shows the Oil Red O staining results of HepG2 cells transfected with scrRNA or siKif13b;

[0035] Figure 12 shows the Oil Red O staining results of HepG2 cells transfected with Kif13b overexpression plasmid or pcDNA3.1 expression vector;

[0036] Figure 13 shows the immunoprecipitation experiment of HepG2 cells transfected with Kif13b overexpression plasmid or different functional domain overexpression plasmids;

[0037] Figure 14 shows the results of Western blot experiments on HepG2 cells transfected with Kif13b overexpression plasmid or different functional domain overexpression plasmids.

[0038] Figure 15 shows the Oil Red O staining results of HepG2 cells transfected with Kif13b overexpression plasmid or different functional domain overexpression plasmids;

[0039] Figure 16 is a flowchart of the construction process for Kif13b;Ldlr double gene knockout mice. WT represents wild-type C57 mice. - / - Representing Kif13b knockout mice, Ldlr - / - Kif13b represents the Ldlr knockout mouse. - / - ;Ldlr - / - Represents Kif13b and Ldlr double gene knockout mice;

[0040] Figure 17 shows the diet of Kif13b and Ldlr double gene knockout mice. - / - Kif13b represents the Ldlr knockout mouse. - / - ;Ldlr - / - Represents Kif13b and Ldlr double gene knockout mice;

[0041] Figure 18 shows the statistical distribution of total cholesterol and plasma triglyceride levels in Kif13b knockout mice. In this figure, A represents total cholesterol, B represents plasma triglyceride, and LDLR... - / - Representing Ldlr knockout mice, LDLR - / - Kif13b - / -Represents Kif13b and Ldlr double gene knockout mice;

[0042] Figure 19 shows the gross Oil Red O staining of blood vessels in Kif13b;Ldlr double gene knockout mice. - / - Kif13b represents the Ldlr knockout mouse. - / - ;Ldlr - / - Represents Kif13b and Ldlr double gene knockout mice;

[0043] Figure 20 shows HE, Oil Red O, Bodipy, and CD68 staining patterns in the outflow tract of Kif13b and Ldlr double gene knockout mice. The scale bar is 100 μm. LDLR - / - Kif13b represents the Ldlr knockout mouse. - / - ;LDLR - / - Represents Kif13b and Ldlr double gene knockout mice;

[0044] Figure 21 shows the BMDM Oil Red O staining image of Kif13b knockout mice. WT represents wild-type C57 mice. - / - The Kif13b gene knockout mice are represented by Control, which represents the control group (without stimulation), and oxLDL represents oxidized low-density lipoprotein.

[0045] Figure 22 shows the process of constructing bone marrow transplantation from Kif13b knockout mice and their dietary regimen. (Ldlr) - / - Kif13b represents the Ldlr knockout mouse. - / - ;Ldlr - / - Representing the Kif13b; Ldlr double gene knockout mouse, Kif13b BM△WT ;Ldlr - / - Represents Ldlr - / - Kif13b, a bone marrow transplant mouse derived from this source. BM△KO ;Ldlr - / - Represents Kif13b - / - ;Ldlr - / - Mice from which bone marrow transplantation originated;

[0046] Figure 23 shows the gross Oil Red O staining of blood vessels in Kif13b myeloid-specific deletion mice. BM△WT ;Ldlr - / - Represents Ldlr - / - Kif13b, a bone marrow transplant mouse derived from this source. BM△KO ;Ldlr - / - Represents Kif13b - / - ;Ldlr - / - Mice from which bone marrow transplantation originated;

[0047] Figure 24 shows HE, Oil Red O, and CD68 staining of the outflow tract in Kif13b myeloid-specific deletion mice. The scale bar is 100 μm. BM△WT ;Ldlr - / - Represents Ldlr - / - Kif13b, a bone marrow transplant mouse derived from this source. BM△KO ;Ldlr - / - Represents Kif13b - / - ;Ldlr - / - Mice from which bone marrow transplantation originated;

[0048] Figure 25 shows the transcriptomic results of macrophages from Kif13b knockout mice and WT mice;

[0049] Figure 26 shows the results of Kif13b knockout affecting the expression of receptors related to macrophage phagocytosis. WT Mφ represents wild-type mouse primary macrophages, and Kif13b... - / - Mφ represents Kif13b - / - Mouse primary macrophages;

[0050] Figure 27 shows the effect of Kif13b overexpression on the expression of macrophage phagocytic function-related receptors. WT Mφ+LV-GFP represents wild-type mouse primary macrophages overexpressing control lentivirus GFP, and WT Mφ+LV-Kif13b represents wild-type mouse primary macrophages overexpressing control lentivirus Kif13b.

[0051] Figures 28A and 28B show the effects of Kif13b overexpression on macrophages. Figure 28A shows the effect on macrophage lipophagus. WT Mφ+LV-GFP represents wild-type mouse primary macrophages overexpressing the control lentiviral GFP, and KO Mφ+LV-GFP represents Kif13b. - / - Mouse primary macrophages overexpressing control lentivirus GFP, WT Mφ+LV-Kif13b represents wild-type mouse primary macrophages overexpressing control lentivirus Kif13b, KO Mφ+LV-Kif13b represents Kif13b - / - Mouse primary macrophages overexpressing control lentivirus Kif13b (scale bar: 50 μm). Figure 28B shows the effect on foam cell formation. WT+GFP represents wild-type mouse primary macrophages overexpressing control lentivirus GFP, and KO+GFP represents Kif13b. - / - Mouse primary macrophages overexpressing control lentivirus GFP, WT+Kif13b represents wild-type mouse primary macrophages overexpressing control lentivirus Kif13b, KO+Kif13b represents Kif13b - / - Overexpression of control lentivirus Kif13b in mouse primary macrophages.

[0052] Figure 29 shows the process of constructing Kif13b conditional knockout mice. In the figure, WT represents wild-type mice, Floxed represents conditional gene knockout mice, and Myeloid Specific deletion represents myeloid conditional Kif13b gene knockout mice.

[0053] Figure 30 shows the mRNA and protein expression assays in macrophages of Kif13b conditional knockout mice. In this figure, A represents mRNA expression, WT represents wild-type mice, and Kif13b... - / - This represents Kif13b macrophage conditional knockout mice. "****" indicates significant difference, and B represents protein expression assay. f / f Representing wild-type mice, Lyz2Kif13b f / f Represents a conditional knockout mouse of Kif13b macrophages;

[0054] Figure 31 is a schematic diagram of the construction of the Kif13b conditional knockout mouse AAA model. f / f Representing wild-type mice, Lyz2Kif13b f / f Represents a conditional knockout mouse of Kif13b macrophages;

[0055] Figure 32 shows the gross appearance and diameter statistics of the Kif13b conditional knockout mouse AAA model, where A represents the gross appearance and Kif13b diameter. f / f Representing wild-type mice, Lyz2Kif13b f / f Representing Kif13b macrophage conditional knockout mice, scale bar is 10 mm, B represents diameter statistics, Kif13b f / f Representing wild-type mice, Lyz2Kif13b f / f Representing Kif13b macrophage conditional knockout mice, "***" indicates significant difference;

[0056] Figure 33 shows the HE staining image of the Kif13b conditional knockout mouse AAA model. f / f Representing wild-type mice, Lyz2Kif13b f / f Represents a conditional knockout mouse of Kif13b macrophages;

[0057] Figure 34 shows the EVG staining map of the Kif13b conditional knockout mouse AAA model. In Figure 34, A represents the EVG staining map, and B represents the proportion of the tunica media area with positive elastic fiber staining. f / f Representing wild-type mice, Lyz2Kif13b f / f Representing Kif13b macrophage conditional knockout mice, "*" indicates significant difference;

[0058] Figure 35 shows the transcriptomic enrichment analysis of primary BMDM in mice, Kif13b f / f Representing wild-type mice, Lyz2Kif13b f / f Represents a conditional knockout mouse of Kif13b macrophages;

[0059] Figure 36 is a heatmap of differentially expressed genes in the aging pathway of primary BMDM mice, Kif13b f / f Representing wild-type mice, Lyz2Kif13b f / f Represents a conditional knockout mouse of Kif13b macrophages;

[0060] Figure 37 shows the primary BMDM SA-β-gal staining of mice, where A is the staining pattern, WT represents wild-type mice, and Kif13b... - / - This represents Kif13b macrophage conditional knockout mice. The scale bar is 50 μm. B is the chromosomal count. WT represents wild-type mice. - / - Kif13b macrophage conditional knockout mice are represented by WT+LPS, which represents lipopolysaccharide-treated wild-type mice. - / - +LPS represents lipopolysaccharide treatment in conditional knockout mice of Kif13b macrophages;

[0061] Figure 38 shows primary BMDM ROS staining in mice. A is the staining pattern, WT represents wild-type mice, Kif13b. - / - This represents Kif13b macrophage conditional knockout mice. The scale bar is 100 μm. B represents the count of reactive oxygen species (ROS) positive cells. WT represents wild-type mice. - / - Kif13b macrophage conditional knockout mice are represented by WT+LPS, which represents lipopolysaccharide-treated wild-type mice. - / - +LPS represents lipopolysaccharide treatment in conditional knockout mice of Kif13b macrophages;

[0062] Figure 39 shows the detection of primary BMDM SASP mRNA in mice. WT represents wild-type mice, Kif13b - / - Kif13b macrophage conditional knockout mice are represented by WT+LPS, which represents lipopolysaccharide-treated wild-type mice. - / - +LPS represents lipopolysaccharide treatment in conditional knockout mice of Kif13b macrophages;

[0063] Figure 40 shows the staining of primary BMDMγ-H2A.X mice, where A is the staining plot, WT represents wild-type mice, and Kif13b - / -Kif13b macrophage conditional knockout mice, scale bar is 10 μm, B is the average γ-H2A.X density per cell nucleus, WT represents wild-type mice, Kif13b - / - Kif13b macrophage conditional knockout mice are represented by WT+LPS, which represents lipopolysaccharide-treated wild-type mice. - / - +LPS represents lipopolysaccharide treatment in conditional knockout mice of Kif13b macrophages;

[0064] Figure 41 shows the staining of THP1 cells overexpressing Kif13b SA-β-gal. In the figure, A is the staining pattern, GFP-HA represents THP1 macrophages infected with LV-GFP-HA, Kif13b-HA represents THP1 macrophages infected with LV-Kif13b-HA lentivirus, and the scale bar is 200 μm. B is the count of SA-β-gal positive cells. NC represents the group transfected with the control lentivirus LV-GFP-HA group, OE represents the group transfected with lentivirus LV-Kif13b-HA group, and * represents the difference between genotypes.

[0065] Figure 42 shows the detection of Kif13b SASP mRNA overexpression in THP1 cells. GFP+PBS represents LV-GFP-HA infection of THP1 macrophages, Kif13b OE +PBS represents LV-Kif13b-HA lentiviral infection of THP1 macrophages, and GFP+LPS represents LPS-treated LV-GFP-HA infection of THP1 macrophages. OE +LPS represents LPS-treated LV-Kif13b-HA lentivirus infection of THP1 macrophages;

[0066] Figure 43 shows the gross appearance and diameter statistics of the mouse Kif13b overexpression AAA model. In this figure, A is the gross appearance with a scale bar of 10 mm, B is the diameter statistics, LV-GFP-HA represents mice injected with LV-GFP-HA via tail vein, and LV-Kif13b-HA represents mice injected with LV-Kif13b-HA lentivirus via tail vein.

[0067] Figure 44 shows HE and EVG staining of a mouse model overexpressing Kif13b AAA. LV-GFP-HA represents mice injected with LV-GFP-HA via the tail vein, and LV-Kif13b-HA represents mice injected with LV-Kif13b-HA lentivirus via the tail vein.

[0068] In the attached figures, * represents differences between genotypes, # represents differences between treatment groups, "ns" represents no statistically significant difference, * represents P<0.05, ** represents P<0.01, *** represents P<0.001, **** represents P<0.0001, # represents P<0.05, ## represents P<0.01, ### represents P<0.001, and ##### represents P<0.0001. Detailed Implementation

[0069] This invention provides the application of CAP-Gly functional peptides or reagents for increasing the expression of CAP-Gly functional peptides in the preparation of drugs for treating fatty liver. The amino acid sequence of the CAP-Gly functional peptide is shown in SEQ ID NO.23, and is as follows:

[0070] GEFVTVGAHKTGVVRYVGPADFQEGTWVGVELDLPSGKNDGSIGGKQYFRCNPGYGLLVRPSR.

[0071] This invention also provides the use of a protein containing a CAP-Gly functional peptide or a reagent that increases the expression level of a protein containing a CAP-Gly functional peptide in the preparation of a medicament for treating fatty liver, wherein the amino acid sequence of the CAP-Gly functional peptide is shown in SEQ ID NO. 23. As one embodiment, the protein may be a member of the kinin family 13B. As one embodiment, the medicament for treating fatty liver is a medicament for treating metabolic-associated fatty liver disease. As one embodiment, the medicament for treating fatty liver is a medicament for reducing lipid accumulation in hepatocytes. As one embodiment, the medicament for treating fatty liver is a medicament for increasing phosphorylation of protein kinase α activated by adenosine monophosphate. As one embodiment, the nucleotide sequence of the gene encoding the CAP-Gly functional peptide is shown in SEQ ID NO. 20. As one embodiment, the reagent that increases the expression level of the CAP-Gly functional peptide may be a recombinant vector overexpressing CAP-Gly, wherein CAP-Gly is the gene encoding the CAP-Gly functional peptide. As one implementation method, the reagent for increasing the expression level of a protein containing the CAP-Gly functional peptide can be a recombinant vector overexpressing a gene encoding the protein containing the CAP-Gly functional peptide. As another implementation method, when the protein containing the CAP-Gly functional peptide is kinin family member 13B (Kif13b), the nucleotide sequence of the gene encoding Kif13b is shown in SEQ ID NO. 24, specifically as follows:

[0072] As one implementation, the starting vector of the recombinant vector can be a pcDNA3.1 expression vector.

[0073] This invention reveals that knocking out the Kif13b gene in mice leads to obesity, elevated plasma cholesterol levels, liver damage, and hepatic steatosis. Conversely, overexpression of the Kif13b gene in mice reduces lipid accumulation in adipocytes and increases monophosphate-activated protein kinase α-phosphorylation. Furthermore, by overexpressing different functional domains of the Kif13b gene (RILP, CAP-Gly, FHA, and Motor) in mice, it was found that overexpression of the CAP-Gly domain further reduces lipid accumulation in adipocytes and increases monophosphate-activated protein kinase α-phosphorylation. Therefore, the CAP-Gly functional peptide is the domain responsible for reducing lipid accumulation in adipocytes. This invention provides a new target and strategy for the precision treatment of patients with fatty liver (especially metabolic-related fatty liver disease).

[0074] To further illustrate the present invention, the application of the CAP-Gly functional peptide provided by the present invention in the preparation of a drug for treating fatty liver is described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0075] Example 1

[0076] Construction of Kif13b knockout mice: Kif13b knockout mice were constructed by the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences and Peking Union Medical College using CRISPR / Cas9 targeting exon 6 of Kif13b. Wild-type mice were 8-week-old C57BL / 6J mice. The method included mixing Cas9 mRNA and sgRNA to final concentrations of 50 ng / μL and 25 ng / μL, respectively, and then microinjecting them into fertilized eggs. The sgRNA consisted of an equimolar mixture of sgRNA1 and sgRNA2, and their specific sequences are shown in Table 1.

[0077] Table 1 Sequence information of sgRNA

[0078] Genotyping: Mouse DNA was extracted using a one-step mouse genotyping kit (Vazyme, PD101-01). The extracted mouse DNA was used as a template for PCR amplification, followed by sequence analysis by agarose gel electrophoresis. The primer sequences for mouse genotyping are as follows:

[0079] F: 5'-ATTGTTCCTGCACTATTACAA-3', SEQ ID NO.5;

[0080] R: 5'-GGGAAGTGTAGGTCTGAGCTAG-3', SEQ ID NO. 6.

[0081] The results are shown in Figure 1, where Kif13b + / + Wild-type mice, Kif13b + / - Kif13b is a heterozygous knockout mouse. - / - These are homozygous knockout mice.

[0082] Determining wild-type mice and constructed Kif13b - / - The relative expression level of Kif13b mRNA in mouse liver was determined, with β-actin as the internal reference gene. The primers used were as follows:

[0083] Kif13b-F: 5'-AACGAACCCAGAAAGAGGA-3', SEQ ID NO.7;

[0084] Kif13b-R: 5'-GCTTGTGACAGCCAGTTTA-3', SEQ ID NO.8;

[0085] β-actin-F: 5'-TGTGCTGTCCCTGTATGCCTCT-3', SEQ ID NO.9;

[0086] β-actin-R: 5'-GGAACCGCTCGTTGCCAATAGT-3', SEQ ID NO. 10.

[0087] The relative expression levels of Kif13b mRNA are shown in Figure 2, where *** indicates p < 0.001.

[0088] As shown in Figure 2, compared with wild-type mice, Kif13b - / - Kif13b mRNA was almost undetectable in mouse livers.

[0089] Example 2

[0090] The Kif13b knockout mice constructed in Example 1 (Kif13b - / - ) and purchased wild-type mice (Kif13b + / + They were divided into 4 groups of 5 each, and labeled as: Kif13b + / + +CD, Kif13b - / - +CD, Kif13b + / + +HFD and Kif13b - / - +HFD. The specific processing is as follows:

[0091] Kif13b + / ++CD: Wild-type mice on a normal diet for 16 weeks;

[0092] Kif13b - / - +CD: Kif13b knockout mice were fed a normal diet for 16 weeks;

[0093] Kif13b + / + +HFD: Wild-type mice were fed a normal diet for 8 weeks, then switched to a high-fat diet (purchased from Research Diets, Inc., catalog number D12492) and continued to be fed for 8 weeks.

[0094] Kif13b - / - +HFD: Kif13b knockout mice were fed a normal diet for 8 weeks, then switched to a high-fat diet for another 8 weeks.

[0095] After feeding, the body weight, plasma cholesterol content (total cholesterol kit, Zhong Sheng Bei Kong Biotechnology Co., Ltd.), plasma triglyceride content (total triglyceride kit, Zhong Sheng Bei Kong Biotechnology Co., Ltd.), plasma alanine aminotransferase (ALT) content (ALT kit, Nanjing Jiancheng Biotechnology Institute), and plasma aspartate aminotransferase (AST) content (AST kit, Nanjing Jiancheng Biotechnology Institute) of the four groups of mice were measured. The results are shown in Figures 3 to 7. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and results without a significance indicator indicate no significant difference.

[0096] After feeding, the livers of the four groups of mice were stained with H&E and Oil Red O, as follows:

[0097] H&E staining: Paraffin-embedded tissue samples were sectioned, dewaxed with xylene and dehydrated with ethanol, then stained with Harry hematoxylin (HHS128-4L, Sigma) for 5 minutes and stained with eosin aqueous solution (HT110232-1L, Sigma) for 3 minutes. After gradient dehydration with ethanol and clearing with xylene, the sections were mounted with resin.

[0098] Oil Red O staining: Liver samples were embedded in OCT (4583, Sakura, American) and flash-frozen in liquid nitrogen. Frozen tissue was sectioned to a thickness of 7 μm. After air-drying at room temperature for 30 minutes, the sections were soaked in 60% isopropanol for 10 seconds, stained with 0.3% Oil Red O working solution for 30 minutes, counterstained with hematoxylin for 5 minutes, and mounted with glycerol. The results are shown in Figure 8. H&E represents the H&E staining results, and ORO represents the Oil Red O staining results. The scale bar is 100 μm.

[0099] After feeding, Beijing Novogene Technology Co., Ltd. was entrusted to process Kif13b. + / + +CD, Kif13b - / -Liver transcriptome sequencing and KEGG enrichment analysis were performed on mice in both the +CD groups. The results are shown in Figure 9.

[0100] After feeding, the protein content of the livers of the four groups of mice was detected by immunoblotting as follows: Total protein was extracted from cells or tissues using RIPA buffer (P0013B, Beyotime) pre-chilled at 4°C and supplemented with a mixture of protease inhibitors and phosphatase inhibitors. Protein concentration was quantified using a BCA protein assay kit (23225, Thermo Fisher Scientific). The lysate was mixed with loading buffer (P0015L, Beyotime) and denatured at 95°C for 10 min. The samples were then analyzed by SDS-PAGE and nitrocellulose membrane transfer. Membranes were blocked for 1 hour at room temperature with 5% skim milk in TBST buffer (2 mM Tris, 137 mM NaCl, 2.7 mM KCl, 0.075% Tween-20), and then incubated overnight at 4°C with the specified primary antibody (see Table 2). The membranes were then washed and incubated for 1 hour at room temperature with horseradish peroxidase-conjugated secondary antibody (ZSGB-BIO, China). Developed using ECL chemiluminescence. The results are shown in Figure 10, where n in Srebp1(n) and Srebp1(p) is the mature Srebp1 and p is the precursor of Srebp1.

[0101] Table 2 Source information for different primary antibodies

[0102] As shown in Figure 3, Kif13b varies depending on whether the diet is normal or high-fat. - / - The mice had significantly higher body weights compared to wild-type mice.

[0103] As shown in Figure 4, Kif13b in a normal diet - / - Mice with a high-fat diet showed significantly higher plasma cholesterol levels compared to wild-type mice, but Kif13b... - / - There was no significant difference in plasma cholesterol levels between mice and wild-type mice.

[0104] As shown in Figure 5, Kif13b varies depending on whether the diet is normal or high-fat. - / - There was no significant difference in plasma triglyceride levels between mice and wild-type mice.

[0105] As shown in Figure 6, Kif13b varies depending on whether the diet is normal or high-fat. - / - The plasma alanine aminotransferase levels in mice were significantly higher than those in wild-type mice.

[0106] As shown in Figure 7, Kif13b varies depending on whether the diet is normal or high-fat. - / - The plasma aspartate aminotransferase (AST) levels in mice were significantly higher than those in wild-type mice.

[0107] As shown in Figure 8, Kif13b varies depending on whether the diet is normal or high-fat. - / - Compared to wild-type mice, mice showed increased lipid accumulation in their livers.

[0108] As shown in Figure 9, Kif13b - / - The SREBP pathway in mouse liver was significantly activated, while the AMPK pathway was significantly inhibited.

[0109] As shown in Figure 10, Kif13b - / - Increased nuclear translocation of Srebp1 and Srebp2 in mouse liver, and inhibition of AMPKα phosphorylation.

[0110] Example 3

[0111] siRNA transfection: scrRNA and siKif13b were synthesized by Sangon Biotech (Shanghai) Co., Ltd. and their knockdown efficiency was tested. siKif13b with good knockdown efficiency was selected for subsequent experiments. The siKif13b consisted of equimolar amounts of siKif13b-1, siKif13b2, and siKif13b-3. The specific siRNA sequences are shown in Table 3. Among them, scrRNA was the negative control group and Kif13b was not knocked down.

[0112] Table 3 Sequence information of different siRNAs

[0113] 3×10 5 HepG2 cells were seeded in 6-well plates using Opti-MEM medium and transfected with scrRNA and siKif13b using the Invitrogen Lipofectamine RNAiMAX (13778150) kit, following the manufacturer's instructions. After 48 hours of transfection, the HepG2 cells were divided into three groups: scrRNA+PA, siKif13b+PA, and siKif13b+PA+Met, with the following treatments:

[0114] scrRNA+PA: Add palmitic acid (PA) to the culture medium with a final concentration of 300 μM after 48 hours of scrRNA transfection, and continue culturing for another 24 hours;

[0115] siKif13b+PA: Add palmitic acid to the medium 48 hours after siKif13b transfection to a final concentration of 300 μM and continue culturing for another 24 hours;

[0116] siKif13b+PA+Met: Add palmitic acid to the culture medium with a final concentration of 300 μM and metformin (AMPKα agonist, denoted as Met) with a final concentration of 2 mM after siKif13b transfection for 48 hours, and continue culturing for 24 hours.

[0117] Oil Red O staining was performed on the three groups of HepG2 cells after 24 hours of culture. The results are shown in Figure 11.

[0118] The results showed that knocking down Kif13b aggravated lipid accumulation in HepG2 cells, while metformin treatment reduced lipid accumulation in HepG2 cells.

[0119] Example 4

[0120] We commissioned Sangon Biotech (Shanghai) Co., Ltd. to construct the Kif13b overexpression plasmid. The construction was carried out 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.

[0121] 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. After 48 hours of transfection, the HepG2 cells were divided into three groups: NC+PA, Kif13b+PA, and Kif13b+PA+IN3, with the following treatments:

[0122] NC+PA: Add palmitic acid to the culture medium with a final concentration of 300 μM 48 hours after transfection with empty vector, and continue culturing for another 24 hours;

[0123] Kif13b+PA: Add palmitic acid to the culture medium with a final concentration of 300 μM 48 hours after transfection with Kif13b overexpression plasmid, and continue culturing for another 24 hours;

[0124] Kif13b+PA+IN3: Add palmitic acid to the culture medium 48 hours after Kif13b overexpression plasmid transfection to a final concentration of 300 μM and AMPK-IN3 (AMPKα inhibitor) to a final concentration of 110 nM, and continue culturing for 24 hours.

[0125] Oil Red O staining was performed on the three groups of HepG2 cells after 24 hours of culture. The results are shown in Figure 12.

[0126] The results showed that overexpression of Kif13b reduced lipid accumulation in HepG2 cells, while inhibition of AMPKα increased lipid accumulation in HepG2 cells.

[0127] Example 5

[0128] Overexpression vectors of different functional domains (RILP, CAP-Gly, FHA, and Motor) of Kif13b were constructed by Sangon Biotech (Shanghai) Co., Ltd., using a method similar to that in Example 4. The nucleotide sequences of the different functional domains are as follows:

[0129] Functional domain RILP (SEQ ID NO.19):

[0130] Functional domain CAP-Gly (SEQ ID NO.20):

[0131] Functional domain FHA (SEQ ID NO.21):

[0132] Functional domain Motor (SEQ ID NO.22):

[0133] 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. After 48 hours of transfection, HepG2 cells were subjected to co-immunoprecipitation and Western blotting experiments. The co-immunoprecipitation experiment included:

[0134] 1×10 7 Cells were lysed on ice with 1 mL RIPA buffer for 30 minutes. After vortexing for 15 seconds, the lysate was centrifuged at 12,000 rpm for 30 minutes at 4°C. 800 μL of the supernatant was incubated with 10 μL of anti-HA antibody at 4°C for 6 hours. Then, bound protein A / G agarose beads were added, and the mixture was incubated overnight at 4°C. After centrifugation at 12,000 rpm for 1 minute, the supernatant was discarded, and the cells were washed 5 times with lysis buffer, then centrifuged at 12,000 rpm for 1 minute and the supernatant was discarded. 120 μL of 1× loading buffer was added, and the mixture was boiled at 95°C for 10 minutes. 20 μL of the sample was used for Western blot analysis. The results are shown in Figure 13. The results show that overexpression of Kif13b or its functional domain CAP-Gly significantly increased its binding to AMPKα.

[0135] The immunoblotting assay was performed according to Example 2, and the results are shown in Figure 14. The primary antibody corresponding to p-AMPKα was p-AMPKαThr172. The results show that overexpression of Kif13b or its functional domain CAP-Gly significantly increases the level of pAMPKα.

[0136] Example 6

[0137] Using the method described in Example 5, HepG2 cells were transfected with Kif13b overexpression plasmid, overexpression vectors of different functional domains (RILP, CAP-Gly, FHA, and Motor), or an empty vector (denoted as NC). After 48 hours of transfection, HepG2 cells were divided into 6 groups: NC+PA, Kif13b+PA, PILP+PA, CAP-Gly+PA, FHA+PA, and Motor+PA. Specific treatments are as follows:

[0138] NC+PA: Add palmitic acid to the culture medium with a final concentration of 300 μM 48 hours after transfection with empty vector, and continue culturing for another 24 hours;

[0139] Kif13b+PA: Add palmitic acid to the culture medium with a final concentration of 300 μM 48 hours after transfection with Kif13b overexpression plasmid, and continue culturing for another 24 hours;

[0140] PILP+PA: Add palmitic acid to the culture medium with a final concentration of 300 μM 48 hours after transfection with the functional domain PILP overexpression plasmid, and continue culturing for another 24 hours;

[0141] CAP-Gly+PA: Add palmitic acid to the culture medium with a final concentration of 300 μM 48 hours after transfection with the CAP-Gly overexpression plasmid, and continue culturing for another 24 hours;

[0142] FHA+PA: Add palmitic acid to the culture medium with a final concentration of 300 μM 48 hours after transfection with the functional domain FHA overexpression plasmid, and continue culturing for another 24 hours.

[0143] Motor+PA: Add palmitic acid to the culture medium with a final concentration of 300 μM 48 hours after transfection with the Motor overexpression plasmid, and continue culturing for another 24 hours.

[0144] Six groups of HepG2 cells were cultured for 24 hours and then stained with Oil Red O. The results are shown in Figure 15.

[0145] The results showed that overexpression of Kif13b or its functional domain CAP-Gly significantly reduced lipid accumulation in HepG2 cells.

[0146] In conclusion, Kif13b protein or CAP-Gly functional peptide can effectively reduce lipid accumulation in hepatocytes, providing new targets and strategies for the precision treatment of patients with metabolic disorder-related fatty liver disease in clinical practice.

[0147] Example 7

[0148] The Kif13b gene synthesis sequence (CDS sequence: an abbreviation for coding sequence. DNA is transcribed into mRNA, and mRNA is processed through splicing and other processes to translate into proteins. A CDS is a DNA sequence that corresponds one-to-one with the protein sequence, and this sequence does not contain any other sequences that do not correspond to that protein. Sequence changes during mRNA processing are not considered; in short, it is a complete correspondence between the codons of the protein. A CDS is a sequence that encodes a protein product and is a term in structural genomics.)

[0149] I. Laboratory Animals and Their Care

[0150] Laboratory animal species, sex, age, and origin: Ldlr gene (gene number 16835, gene function: involved in lipid transport; regulation of inflammatory responses; and regulation of lipid metabolism) knockout (Ldlr) - / - ) and Kif13b / Ldlr double gene knockout (Kif13b - / - ;Ldlr - / - Male mice, 8 weeks old, weighing 19–25 g. Ldlr gene knockout mice (Ldlr...) - / - The Kif13b gene knockout mice were purchased from GemPharmatech (Nanjing, China). Kif13b gene knockout was generated using CRISPR / Cas9 at the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, and Peking Union Medical College (Beijing, China). Figure 16 shows the Kif13b / Ldlr double gene knockout mouse construction strategy, representing sgRNAs targeting exon 6 flanking sites. Specifically, two loxP sites were inserted flanking exon 6, and a donor template was constructed based on the mouse genome sequence (GRCm39). After knocking out exon 6, the mice were crossed with Ldlr gene knockout mice to obtain the aforementioned Kif13b / Ldlr double gene knockout mice. - / - ;Ldlr - / - Mice.

[0151] II. Laboratory Animal Feed Formulation

[0152] The Western Diet (WD) was purchased from Research Diet, product number: D12108C.

[0153] III. Animal husbandry and environmental conditions

[0154] All experiments were conducted in accordance with the principles of laboratory animal care (NIH publication No. 85-23, revised in 1996) and approved by the Laboratory Animal Ethics Committee of Peking University (LA2023460). All animals were bred and raised in the SPF-class animal facility of Peking University Health Science Center (License number: SYXK (Beijing) 2022-0037). A 12-hour light-dark cycle was maintained daily, and the temperature was kept at 25 ± 2 °C and the humidity at 40 ± 5%. All mice had free access to food and water unless otherwise specified. As shown in Figure 17, the diet of Ldlr gene knockout mice and Kif13b / Ldlr double gene knockout mice is illustrated. Eight-week-old mice on a normal diet (CD) were given the above Western diet (WD) for 22 weeks.

[0155] IV. Determination of plasma total cholesterol and triglyceride levels

[0156] After 22 weeks of Western diet (WD) in eight-week-old mice on a normal diet (CD), the mice were fasted for 4 hours, and anticoagulant or non-anticoagulant blood samples were collected from the orbital vein. The samples were centrifuged at 4 °C for 10 minutes to separate plasma or serum. Plasma total cholesterol (TC) and triglyceride (TG) concentrations were measured using commercially available kits from中生北控(Beijing, China).

[0157] As shown in Figures 18A and 18B, relatively speaking, the levels of total cholesterol and triglyceride in the plasma of Kif13b / Ldlr double gene knockout mice were both elevated.

[0158] V. Gross oil red O staining of blood vessels

[0159] After 22 weeks of Western diet (WD) in eight-week-old mice on a normal diet (CD), after sampling, the entire length of the mouse aorta was taken, fixed with paraformaldehyde, dehydrated with 20% sucrose, and stained with oil red O.

[0160] The results are shown in Figure 19. Kif13b - / - ; Ldlr - / - The aortic plaques in double gene knockout mice were more severe than those in Ldlr - / - single gene knockout mice.

[0161] VI. HE, oil red O, Bodipy, and CD68 staining of the aortic outflow tract

[0162] After 22 weeks of Western diet (WD) in eight-week-old mice on a normal diet (CD), frozen samples of the aortic outflow tract (aortic root) were selected with a section thickness of 7 μm, and HE, oil red O staining, as well as Bodipy and CD68 immunofluorescence staining were performed.

[0163] The results are shown in Figure 20. Kif13b - / - ; Ldlr - / -Double gene knockout mice compared to Ldlr - / - In single-gene knockout mice, aortic outflow tract plaques showed increased lipid accumulation, expanded necrotic core area, increased inflammatory infiltration, and significant macrophage changes.

[0164] VII. Oil Red O staining of primary macrophages (BMDM)

[0165] WT and Kif13b - / - Eight-week-old male mice had primary macrophages extracted and stained with 100 ng / mL oxidized low-density lipoprotein (oxLDL) before Oil Red O staining.

[0166] The results are shown in Figure 21, Kif13b - / - Primary BMDM in mice showed increased lipid phagocytosis and increased macrophage foaming.

[0167] VIII. Construction of Kif13b knockout mouse bone marrow transplantation

[0168] Ldlr knockout recipient mice were subjected to lethal irradiation (8.5 Gy X-ray irradiation) followed by intravenous (tail vein) injection of Ldlr from donor mice. - / - Knockout and Kif13b; Ldlr - / - 1×10⁻⁶ of double gene knockout mice 7 One bone marrow cell line was used to construct myeloid Kif13b-specific deletion mice and feed them a Western diet (WD) for 16 weeks (as shown in Figure 22).

[0169] IX. Gross Oil Red O staining of blood vessels in myeloid Kif13b-specific deletion mice

[0170] After feeding myeloid Kif13b-specific deletion mice a Western diet (WD) for 16 weeks, the entire length of the mouse aorta was harvested, fixed with paraformaldehyde, dehydrated with 20% sucrose, and stained with Oil Red O.

[0171] The results are shown in Figure 23. Myeloid Kif13b-specific deletion mice (Kif13b BM△KO ;Ldlr - / - The accumulation of aortic plaque worsens.

[0172] 10. HE, Oil Red O, and CD68 staining of the aortic outflow tract in myeloid Kif13b-specific deletion mice

[0173] After myeloid Kif13b-specific deletion mice were fed a Western diet (WD) for 16 weeks, frozen samples of the aortic outflow tract (aortic root) with a thickness of 7 μm were selected and subjected to HE, Oil Red O staining, and Bodipy and CD68 immunofluorescence staining.

[0174] The results are shown in Figure 24. Myeloid Kif13b-specific deletion mice (Kif13b BM△KO ;Ldlr - / - The aortic outflow tract plaque showed increased lipid accumulation, expanded necrotic core area, increased inflammatory infiltration, and significant macrophage changes.

[0175] 11. WT and Kif13b - / - Primary BMDM transcriptome sequencing in mice

[0176] Methods for primary BMDM transcriptome sequencing: Primary BMDM from mice was extracted and sent for transcriptome sequencing (BGI) analysis.

[0177] The results are shown in Figure 25, WT and Kif13b - / - Primary BMDM transcriptome sequencing results in mice showed that Kif13b - / - Significant changes were observed in pathways related to phagocytosis in mouse macrophages, and macrophage burial pathways were significantly inhibited.

[0178] 12. WT and Kif13b - / - Detection of phagocytosis and cell burial-related protein expression in primary mouse BMDM macrophages

[0179] Detection method: Primary mouse BMDM was extracted, cell proteins were extracted, and Western blotting was used to detect the expression of phagocytosis and cell burial-related proteins.

[0180] The results are shown in Figure 26, Kif13b - / - The expression of MerTK, a receptor related to phagocytosis function, was significantly reduced in mouse BMDM. Kif13b knockout mainly affected the expression of MerTK, a receptor related to phagocytosis function in macrophages.

[0181] XIII. Detection of expression of phagocytic and cell burial-related proteins in primary BMDM mice after Kif13b overexpression.

[0182] The detection methods are the same as those for "12, WT and Kif13b". - / - The expression of proteins related to phagocytosis and burial in primary mouse BMDM macrophages was the same.

[0183] As shown in Figure 27, the expression of the burial function-related receptor MerTK in BMDM cells overexpressing Kif13b was significantly increased, and the expression of the macrophage phagocytic function-related receptor MerTK was restored by Kif13b overexpression.

[0184] XIV. Oil Red O staining after Kif13b overexpression in primary BMDM mice of WT and Kif13b- / - mice

[0185] Specific method: Extract BMDM from mice. After lentiviral infection for overexpression of Kif13b or control GFP, stain with Oil Red O (sigma O0625) and take pictures under the microscope.

[0186] As shown in Figures 28A and 28B, overexpression of the Kif13b gene can reduce lipid accumulation in macrophages of WT mice and can also restore lipid accumulation in macrophages of KO mice to the WT level.

[0187] Experimental Example 8

[0188] Synthetic sequence of the Kif13b gene (CDS sequence: Abbreviation for Coding sequence. DNA is transcribed into mRNA, and after processes such as splicing, mRNA is translated into protein. The CDS is the DNA sequence that corresponds one-to-one with the protein sequence, and this sequence does not contain other sequences that do not correspond to this protein, without considering sequence changes during mRNA processing, etc. In short, the CDS sequence corresponds exactly to the codons of the protein. The CDS is the sequence encoding a protein product and is a term in structural genomics).

[0189] I. Construction of conditional knockout mice of the Kif13b gene in macrophages

[0190] Wild-type (Kifi3b f / f ) mice were constructed by the Institute of Laboratory Animals of Peking Union Medical College. Lyz2-cre mice were from the Institute of Model Animals of Wuhan University and were kindly donated by Professor Li Hongliang of the Department of Cardiovascular Medicine of Renmin Hospital. The two were crossed to obtain Lyz2Kif13b f / f mice (as shown in Figure 29). Littermate Kif13b f / f mice were used as controls in the experiment. All animals were bred in the SPF-class animal house of Peking University Health Science Center (License number: SYXK (Beijing) 2022 - 0037), ensuring 12h cyclic light per day, maintaining 24 ± 2 °C and 40 ± 5 humidity. Without special instructions, all mice could freely intake food and water.

[0191] Select 8-week-old male wild-type (Kif13b f / f ) and conditional knockout of Kif13b in macrophages (Lyz2Kif13b f / f ) mice, extract primary bone marrow-derived macrophages (BMDM), and detect the relative expression levels of Kif13b mRNA and protein by RT-qpcr and Western Blot: Compared with Kif13b f / f mice, Lyz2Kif13b f / fKif13b mRNA and protein were almost undetectable in mouse macrophages, proving that myeloid conditional knockout Kif13b mice were successfully constructed (as shown in Figure 30A and Figure 30B).

[0192] II. Building Kif13b f / f Mice and Lyz2Kif13b f / f Mouse AAA model

[0193] Male mice aged 8-10 weeks, weighing 22-26g, were selected and incubated in situ with 10mg / mL porcine pancreatic elastase (PPE, MCE, HY-P2974) on the subrenal abdominal aortic segment for 40 minutes. Samples were collected two weeks later (as shown in Figure 31). All animal experiments were conducted in the SPF-grade animal facility of Peking University School of Medicine, following the principles of laboratory animal care (NIH Publication No. 85Y23, 1996, revised) and approved by the Peking University Laboratory Animal Ethics Committee (LA2023460).

[0194] III. Measurement of Abdominal Aortic Diameter in Mice

[0195] Aortas from mice with PPE-induced abdominal aortic aneurysms were collected, fixed in 4% paraformaldehyde for 24 h, dehydrated in 20% sucrose for 24 h, and then the aortic enface was performed to obtain Kif13b. f / f and Lyz2Kif13b f / f In the mouse AAA model, the ratio of the maximum diameter of the abdominal aortic aneurysm to the diameter of the adjacent normal abdominal aorta was statistically analyzed using ImageJ.

[0196] The results are shown in Figure 32A and Figure 32B, Lyz2Kif13b f / f Mouse abdominal aortic diameter compared to Kif13b f / f The aortic diameter dilation in mice was significantly increased.

[0197] IV. Observation of aortic characterization in mice with abdominal aortic aneurysms

[0198] The area of ​​the abdominal aortic aneurysm with significant diameter dilation was harvested, paraffin-embedded, and sectioned (5 μm thickness), followed by Kif13b analysis. f / f and Lyz2Kif13b f / f HE staining of mouse AAA model.

[0199] The results are shown in Figure 33, where Lyz2Kif13b was observed. f / f Compared to Kif13b, the abdominal aortic wall of mice f / f Increased thickness of the abdominal aortic wall in mice, and Lyz2Kif13b f / f The number of inflammatory cells with large, deeply stained nuclei increased in the wall of the abdominal aorta of mice.

[0200] V. Observation of the characterization of the aortic media in mice with aortic aneurysms

[0201] Paraffin sections were prepared in accordance with "IV. Observation of Aortic Characterization in Mouse Abdominal Aortic Aneurysms" and Kif13b was analyzed. f / f and Lyz2Kif13b f / f EVG staining was performed on a mouse AAA model, and ImageJ was used to statistically analyze the proportion of the tunica media area in which elastic fibers were positively stained.

[0202] The results are shown in Figure 34A and Figure 34B. In comparison, Lyz2Kif13b f / f The degree of elastic fiber disintegration in the abdominal aorta of mice was aggravated.

[0203] VI. High-throughput sequencing of transcriptomics

[0204] From 8-week-old male Kif13b f / f and Lyz2Kif13b f / f Primary bone marrow-derived macrophages (BMDM) were extracted from mice. Specifically, cells were obtained from the tibia and femur of mice and cultured in DMEM supplemented with 10% FBS (FS301-02, TransGen, China) and 1% penicillin-streptomycin. The cells were then stimulated with 25 ng / mL mouse macrophage colony-stimulating factor 1 (M-CSF; 315-02, Peprotech, USA) for 6 days. Transcriptomic high-throughput sequencing was performed, and differentially expressed genes were enriched for pathway analysis.

[0205] The result is shown in Figure 35. Lyz2Kif13b f / f Mice compared to Kif13b f / f The BMDM aging pathway in mice was significantly upregulated.

[0206] VII. Analysis of Transcriptome Sequencing Results

[0207] Analysis of Kif13b f / f and Lyz2Kif13b f / f Transcriptome sequencing results of mouse primary bone marrow-derived macrophages (BMDM), as shown in Figure 36, Lyz2Kif13b f / f In mice, BMDM upregulates pro-aging genes and downregulates anti-aging genes.

[0208] VIII. Determination of Senescent Cell Content in Primary Mouse Bone Marrow-Derived Macrophages

[0209] Kif13b f / f and Lyz2Kif13b f / fPrimary mouse bone marrow-derived macrophages (BMDM) were treated with 10 ng / mL LPS (Sigma, L2630) for 24 h, followed by SA-β-gal staining.

[0210] The results are shown in Figure 37A and Figure 37B, Lyz2Kif13b f / f The number of senescent cells (SA-β-gal staining positive) in mice with BMDM increased.

[0211] IX. Oxidative Stress Assay of Primary Mouse Bone Marrow-Derived Macrophages

[0212] Kif13b f / f and Lyz2Kif13b f / f Primary mouse bone marrow-derived macrophages (BMDM) were treated with 10 ng / mL LPS for 24 h and then stained with ROS.

[0213] The results are shown in Figure 38A and Figure 38B, Lyz2Kif13b f / f Oxidative stress was aggravated in BMDM mice.

[0214] 10. Detection of Primary BMDM SASP mRNA in Mice

[0215] Kif13b f / f and Lyz2Kif13b f / f Primary mouse bone marrow-derived macrophages (BMDM) were treated with 10 ng / mL LPS for 24 h, and the mRNA level of aging-associated secretory phenotype (SASP) was detected by qPCR.

[0216] The results are shown in Figure 39. Lyz2Kif13b f / f BMDM in mice activates SASP.

[0217] XI. Mouse primary BMDM γ-H2A.X staining

[0218] Kif13b f / f and Lyz2Kif13b f / f Primary mouse bone marrow-derived macrophages (BMDM) were treated with 10 ng / mL LPS for 24 h and stained with γ-H2A.X.

[0219] The results are shown in Figure 40A and Figure 40B, Lyz2Kif13b f / f Increased levels of γ-H2A.X in the nucleus of mouse BMDM indicate aggravated DNA damage.

[0220] 12. THP1 cells overexpressing Kif13b SA-β-gal staining

[0221] THP1 cells (from the Institute of Cardiovascular Medicine, Peking University) were treated with 100 ng / mL PMA (Solepro, P6741) for 48 h to induce them to become macrophages. LV-GFP-HA or LV-Kif13b-HA lentiviruses were constructed using the following method: lentiviral vector (15 mg) and pMDLg / pRRE, RSV / Rev, and VSV-G (5 mg each) were co-transfected into 293T cells in 10 cm dishes using the calcium phosphate (Ca3(PO4)2) method. The culture medium was changed after 16 h of cell culture. Two days after transfection, the viral supernatant was collected and filtered through a 0.45 μm filter (SLHP033RB, Millipore, USA). The filtered supernatant was concentrated to 1e8 TU by ultracentrifugation for in vivo and in vitro experiments. THP1 macrophages were infected with 1e5 TU of lentivirus and treated with 1 μg / mL LPS for 24 h, followed by SA-β-gal staining.

[0222] The results are shown in Figure 41A and Figure 41B, indicating a decrease in senescent cells overexpressing Kif13b (SA-β-gal staining positive).

[0223] 13. Detection of Kif13b SASP mRNA overexpression in THP1 cells

[0224] THP1 cells were treated with 100 ng / mL PMA (Solepro, P6741) for 48 h to induce them to become macrophages. THP1 macrophages were then infected with LV-GFP-HA or LV-Kif13b-HA lentivirus (1e5 TU) and treated with 1 μg / mL LPS for 24 h. The level of senescence-associated secretory phenotype (SASP) mRNA was detected by qPCR.

[0225] The results are shown in Figure 42. Expressing Kif13b can significantly suppress SASP.

[0226] XIV. Gross appearance and diameter statistics of the mouse Kif13b overexpression AAA model

[0227] Eight-week-old WT mice were selected and injected intravenously with LV-GFP-HA and LV-Kif13b-HA lentiviruses (1e7 TU). One week later, a PPE-induced AAA model was established (method as in "II. Construction of Kif13b"). f / f Mice and Lyz2Kif13b f / fA mouse AAA model was established, and samples were collected two weeks later. The aortas of mice with PPE-induced abdominal aortic aneurysms were collected, fixed in 4% paraformaldehyde for 24 h, dehydrated in 20% sucrose for 24 h, and the aorta enface was performed to obtain the gross appearance of the Kif13b overexpressing mice and the control group AAA model. The ratio of the maximum diameter of the abdominal aortic aneurysm to the diameter of the adjacent normal abdominal aorta was calculated using ImageJ.

[0228] The results are shown in Figure 43A and Figure 43B. The abdominal aortic diameter dilation was significantly reduced in mice overexpressing Kif13b.

[0229] 15. HE and EVG staining of a mouse Kif13b overexpression AAA model.

[0230] The aortic aneurysm with significant diameter dilation was cut, paraffin-embedded and sectioned (5 μm thickness), and then HE and EVG staining was performed in a WT mouse model of Kif13bAAA overexpression. ImageJ was used to count the proportion of the tunica media area in which the elastic fiber staining was positive.

[0231] As shown in Figure 44, mice overexpressing Kif13b showed reduced abdominal aortic wall thickness, fewer large, deeply stained inflammatory cells, and reduced elastic fiber breakdown.

[0232] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

The application of the Kif13b gene as a drug target in pharmaceuticals is characterized by: The applications include screening for drugs to prevent, alleviate and / or treat fatty liver, atherosclerosis, abdominal aortic aneurysm and / or macrophage senescent inflammation. The application according to claim 1 is characterized in that: The nucleotide sequence of the Kif13b gene is shown in SEQ ID NO.

24. The application according to claim 1 is characterized in that: The drugs mentioned for the prevention, relief and / or treatment of fatty liver, atherosclerosis, abdominal aortic aneurysm and / or macrophage senescent inflammation are drugs that promote the overexpression of the Kif13b gene. The application according to claim 1 is characterized in that: The drugs described for the prevention, relief, and / or treatment of fatty liver, atherosclerosis, abdominal aortic aneurysm, and / or macrophage senescent inflammation enhance macrophage burial by promoting Kif13b gene overexpression, thereby reducing the formation of necrotic arterial cores and treating atherosclerosis. Promote the use of the drug for overexpression of the Kif13b gene as described in claim 1 in the preparation of drugs for the prevention, relief and / or treatment of fatty liver, atherosclerosis, abdominal aortic aneurysm and / or macrophage senescent inflammation. A drug for preventing, alleviating, and / or treating fatty liver, atherosclerosis, abdominal aortic aneurysm, and / or macrophage senescent inflammation, characterized in that: Including drugs that promote the overexpression of the Kif13b gene as described in claim 1. The use of a protein containing a CAP-Gly functional peptide or an agent that increases 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, and the protein includes Kif13b. The application according to claim 7 is characterized in that: The drugs mentioned for treating fatty liver are drugs for treating metabolic-related fatty liver disease, drugs for reducing lipid accumulation in liver cells, or drugs for increasing the phosphorylation of protein kinase α activated by adenosine monophosphate. The application according to claim 7 or 8 is characterized in that: The nucleotide sequence of the gene encoding the CAP-Gly functional peptide is shown in SEQ ID NO.

20. The application according to claim 7 or 8 is characterized in that: The reagent for increasing the expression level of the CAP-Gly functional peptide includes a recombinant vector that overexpresses CAP-Gly, wherein CAP-Gly is the gene encoding the CAP-Gly functional peptide; the reagent for increasing the expression level of the protein containing the CAP-Gly functional peptide includes a recombinant vector that overexpresses the encoding gene, wherein the encoding gene is the gene encoding the protein containing the CAP-Gly functional peptide; the starting vector of the recombinant vector includes a pcDNA3.1 expression vector.

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