Application of wdr6 gene in preparation of liver aging delaying product

Abstract

This invention relates to the field of biomedicine, specifically to a... Wdr6 Application of genes in the preparation of products that delay liver aging. Validated by in vivo mouse experimental data, liver... Wdr6 Low expression can exacerbate liver aging, while liver-specific... Wdr6 Overexpression significantly downregulated the expression of the aging marker p21 protein in the liver tissue of naturally aging mice, and significantly reduced the number of aging-related β-galactosidase-positive cells, indicating that... Wdr6 Overexpression can effectively maintain liver homeostasis and delay liver aging. Furthermore, an in vitro bleomycin-induced hepatocyte aging model validated that WDR6 protein can increase the expression of the longevity protein SIRT1, downregulate p21 protein expression, and reduce β-galactosidase, thereby delaying liver aging.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a... Wdr6 Application of genes in the preparation of products that delay liver aging. Background Technology

[0002] As the largest metabolic organ in the human body, the liver plays a crucial role in the metabolism of various nutrients, the synthesis and secretion of various proteins, and the decomposition of toxic substances, serving as a key hub for maintaining homeostasis. The liver is also an important endocrine organ; various factors synthesized and secreted by the liver circulate throughout the body via the bloodstream, sending signals and communicating with distant tissues, systematically influencing the physiological state and function of the body's organs and tissues. With age, the liver undergoes degenerative changes, including increased inflammation and fibrosis, weakened regenerative capacity, and reduced ability to repair damage, significantly increasing the risk of various liver diseases such as fatty liver, cirrhosis, and liver cancer. Research on liver aging holds significant importance and value, potentially providing intervention strategies for the prevention and treatment of liver diseases and other related systemic diseases.

[0003] In recent years, interventions targeting aging-related pathways have gradually become a research hotspot. For example, inhibiting the mTOR signaling pathway can promote autophagy and delay cellular senescence; activating the AMPK signaling pathway helps improve energy metabolism and mitochondrial function; in addition, regulating Sirtuins family proteins or supplementing NAD can also help. ＋ Precursor substances are also believed to help slow down the aging process. However, such intervention strategies still have significant limitations in practical applications. On the one hand, the relevant pathways are widely involved in various physiological processes, and systemic regulation can easily lead to side effects such as immunosuppression or metabolic disorders. On the other hand, their targeting in liver tissue is insufficient, making precise intervention difficult. Therefore, elucidating the molecular mechanisms of liver aging and identifying key regulatory targets is of great significance for slowing down the aging process of the liver and preventing and treating related diseases.

[0004] The WD-repeat (WDR) protein family has attracted widespread attention due to its important roles in cell signal transduction, transcriptional regulation, and protein complex assembly. WDR6, a member of this family, was first identified using molecular cloning techniques. This protein contains multiple WD-repeat domains and is widely expressed in various tissues, suggesting its potential involvement in the regulation of multiple fundamental life processes. Previous studies have shown that WDR6, as a protein-protein interaction platform, can participate in cell proliferation, metabolic regulation, and immune-related signaling pathways. However, a systematic study of the mechanism of action of WDR6 in aging, particularly in liver aging, is still lacking. Summary of the Invention

[0005] To address the technical problem that existing intervention strategies for aging-related pathways lack targeting specificity in liver tissue and struggle to achieve precise intervention, this invention provides a... Wdr6 The application, vectors, and drugs of genes in the preparation of products that delay liver aging are described below.

[0006] This invention provides Wdr6 Application of genes in the preparation of products that delay liver aging.

[0007] Furthermore, through overexpression Wdr6 Genes can increase the expression level of WDR6 protein and increase the expression of the longevity protein SIRT1, thereby delaying liver aging. As an upstream regulator, increased WDR6 expression can promote the activation of the longevity protein SIRT1.

[0008] Furthermore, delaying liver aging involves inhibiting β-galactosidase activity and / or downregulating the expression of the liver aging marker p21 protein. Increased expression of the longevity protein SIRT1 can further negatively regulate aging-related phenotypes and aging markers, inhibiting β-galactosidase activity and reducing p21 protein expression levels. This forms a cascade regulatory pathway with WDR6 as the upstream initiator, SIRT1 as the intermediate core regulatory node, and β-galactosidase and p21 as downstream effector targets. Through this upstream-downstream stepwise regulatory model, it collectively blocks the aging process of hepatocytes, ultimately exerting the liver's anti-aging biological role.

[0009] Furthermore, the liver aging referred to here refers to natural liver aging or liver cell aging induced by external factors.

[0010] Furthermore, the external factors include chemically induced factors, oxidative stress factors, or inflammatory stimuli.

[0011] Furthermore, the chemical inducing factor is bleomycin.

[0012] Furthermore, the product for delaying liver aging includes Wdr6 Gene overexpression vectors and products that delay liver aging are among the categories of pharmaceuticals, biological agents, and gene therapy agents.

[0013] Furthermore, the aforementioned Wdr6 The gene overexpression vector is an adeno-associated virus vector, containing Wdr6 Gene sequence and hepatocyte-specific TBG promoter. The TBG promoter mediates liver-specific, persistent overexpression without significant leakage into peripheral tissues.

[0014] Furthermore, Wdr6The capsid protein Cap used for packaging the gene overexpression vector is derived from AAV8. AAV8 has an extremely high affinity for the liver, allowing for efficient targeting of hepatocytes via tail vein injection, and exhibits strong specificity in vivo distribution. Serum-type AAV8, in combination with the TBG promoter, can achieve precise delivery and specific expression in the liver.

[0015] Furthermore, Wdr6 The gene overexpression vector is pAAV2 / 8-TBG- Wdr6 -3xFLAG-tWPA. The gene backbone of this vector plasmid is derived from AAV2, and the 3×FLAG tag enables rapid detection. Wdr6 By controlling expression levels, performing immunoprecipitation and subcellular localization, the tWPA simplified element can effectively control the total fragment length, comply with AAV packaging limitations, and the vector contains no viral replication genes, thus ensuring high safety.

[0016] The beneficial effects of this invention are as follows: 1. This invention provides Wdr6 Application of genes in the preparation of products that delay liver aging. Validated by in vivo mouse experimental data, liver... Wdr6 Low expression can exacerbate liver aging, while liver-specific... Wdr6 Overexpression significantly downregulated the expression of the aging marker p21 protein in the liver tissue of naturally aging mice, and significantly reduced the number of aging-related β-galactosidase-positive cells, indicating that... Wdr6 Overexpression can effectively maintain liver homeostasis and delay liver aging. Furthermore, an in vitro bleomycin-induced hepatocyte aging model validated that WDR6 protein overexpression can increase SIRT1 protein expression, inhibit β-galactosidase activity, and reduce p21 protein expression levels, thereby delaying liver aging.

[0017] 2. The present invention also provides Wdr6 Gene overexpression vectors, through the synergistic effect of the TBG promoter and AAV8 serotype, achieved... Wdr6 This study demonstrates precise, efficient, safe, and sustainable specific overexpression of the gene in mouse liver tissue. Furthermore, the introduction of the 3×FLAG tag greatly facilitates subsequent protein detection, interaction protein screening, and subcellular localization studies. Based on these advantages, products for delaying liver aging are being developed using this gene overexpression vector, providing a new strategy and tool for the intervention and treatment of age-related liver diseases. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a diagram showing the results of β-galactosidase staining in liver tissues of mice of different ages in Example 1.

[0020] Figure 2 This is a diagram showing the expression levels of WDR6 and p21 proteins in the liver tissues of mice of different ages in Example 1.

[0021] Figure 3 In Example 2 Wdr6 Figure showing the expression levels of WDR6 and p21 proteins in the liver tissues of mice with low expression and control mice.

[0022] Figure 4 In Example 2 Wdr6 Image showing the staining results of β-galactosidase in liver tissues of mice with low expression and control mice.

[0023] Figure 5 In Example 3 Wdr6 Figure showing the expression levels of WDR6 and p21 proteins in the liver tissues of overexpressing mice and control mice.

[0024] Figure 6 In Example 3 Wdr6 Image showing the staining results of β-galactosidase in liver tissues of overexpressing mice and control mice.

[0025] Figure 7 This refers to Example 4 with and without bleomycin. Wdr6 Image showing the staining results of β-galactosidase in AML12 overexpressing cell lines and normal AML12 cells.

[0026] Figure 8 This refers to Example 4 with and without bleomycin. Wdr6 Figure showing the expression levels of WDR6 and p21 proteins in AML12 cell lines overexpressing and normal AML12 cells. Detailed Implementation

[0027] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0028] In the following examples, the gene encoding the WDR6 protein is from a mouse ( Mus musculus ) Wdr6 The gene has the NCBI sequence number NM_031392.3 and the Gene ID 83669. Wdr6 The full-length coding sequence (CDS) of the gene transcript (corresponding to NM_031392.3) is 3378 bp.

[0029] Example 1: The expression level of WDR6 protein in the liver of naturally aging mice gradually decreased. The levels of WDR6 protein, p21 protein, and the number of senescent cells in the liver of naturally aging mice were evaluated. The specific experimental procedure is as follows: 1. Construction of a naturally aging mouse model Wild-type C57BL / 6J male mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were fed a normal diet in an SPF environment with a 12h / 12h light / dark cycle, 50% humidity, and a temperature of 22~24℃. They had free access to food and water and were raised to 6, 8, 10, 12, 18, 24 and 27 months of age. They were fasted for 8 hours before being collected.

[0030] 2. Staining of liver aging-related β-galactosidase (SA-β-gal) Fresh liver tissue blocks (0.5cm × 0.5cm) were taken and fixed in 4% paraformaldehyde solution at 4°C for 24 hours. Subsequently, they were dehydrated sequentially in 20% and 30% sucrose solutions at 4°C. The tissues were then embedded in OCT and frozen sectioned. After thawing and drying at room temperature, the sections were washed with PBS, and β-galactosidase staining fixative was added for fixation at room temperature for 15 minutes. After washing with PBS, β-galactosidase staining working solution was added, and the sections were incubated overnight at 37°C. After washing with PBS and mounting, cells expressing β-galactosidase were observed to turn blue under an optical microscope. This staining method is based on the upregulation of SA-β-gal activity levels during senescence and can be used to detect senescent cells. The results are as follows: Figure 1 As shown.

[0031] 3. Assessment of liver WDR6 and p21 protein levels Approximately 30 mg of liver tissue was collected, washed with sterile PBS, and then RIPA buffer containing 1% protease inhibitor and phosphatase inhibitor was added. The mixture was sonicated, centrifuged at 12000 rpm for 15 min at 4°C, and the supernatant was collected as the total protein extract. Protein quantification of the supernatant was performed using a BCA protein assay kit. Subsequently, Western blotting was used to analyze the levels of WDR6 and p21 proteins in the protein sample, with GAPDH protein as a control. The results are as follows: Figure 2 As shown.

[0032] SA-β-gal staining results showed that with increasing age, the number of positively stained cells in the mouse liver increased and the staining became more intense, confirming that with increasing age, the number of senescent cells in the liver increased, and the degree of liver aging gradually increased. During the natural aging process of mice, the expression of the aging marker p21 protein in the liver gradually increased, showing a significant increase from 18 months of age. Simultaneously, the expression level of hepatic WDR6 protein significantly decreased at 10 months of age, earlier than the increase in p21 protein, and further decreased with increasing age.

[0033] Example 2 Wdr6 Effects of low expression on liver aging in mice 1. Liver-specific Wdr6 Construction of low expression mouse model Six-month-old wild-type male C57BL / 6J mice were selected and divided into a model group and a control group. Mice in the model group received a single intravenous injection via tail vein of mice carrying a hepatocyte-specific TBG promoter. Wdr6 Genetic interference recombinant adeno-associated virus AAV8-TBG-sh Wdr6 The target sequence is 5'-AGGTGAAGCTCTTAGAGAA-3' (SEQ ID No:1), and the injection dose is 5×10⁻⁶. 11 vg / each, to achieve the effect of liver parenchymal cells Wdr6 Gene-specific knockdown. Control group mice were injected via tail vein with an equal dose of adeno-associated virus AAV8-TBG-sh. Scramble The target sequence was 5'-GAAGTCGTGAGAAGTAGAA-3' (SEQ ID No:2), which served as a negative control. Three months after injection, liver samples were collected from mice after an 8-hour fast to detect WDR6 protein expression and assess liver p21 protein levels and the number of senescent cells.

[0034] 2. Assessment of liver WDR6 and p21 protein levels Approximately 30 mg of liver tissue was collected, washed with sterile PBS, and then RIPA buffer containing 1% protease inhibitor and phosphatase inhibitor was added. The mixture was sonicated, centrifuged at 12000 rpm for 15 min at 4°C, and the supernatant was collected as the total protein extract. Protein quantification of the supernatant was performed using a BCA protein assay kit. Subsequently, Western blotting was used to analyze the levels of WDR6 and p21 proteins in the protein sample, with GAPDH protein as a control. The results are as follows: Figure 3 As shown.

[0035] 3. Staining of liver aging-related β-galactosidase (SA-β-gal) Fresh liver tissue blocks (0.5cm × 0.5cm) were taken and fixed in 4% paraformaldehyde solution at 4°C for 24 hours. Subsequently, they were dehydrated sequentially in 20% and 30% sucrose solutions at 4°C. The tissues were then embedded in OCT and frozen sectioned. After thawing and drying at room temperature, the sections were washed with PBS, and β-galactosidase staining fixative was added for fixation at room temperature for 15 minutes. After washing with PBS, β-galactosidase staining working solution was added, and the sections were incubated overnight at 37°C. The sections were then washed with PBS, mounted, and observed under a light microscope to reveal cells expressing β-galactosidase that had turned blue. This staining method is based on the upregulation of SA-β-gal activity levels during senescence and can be used to detect senescent cells. The results are as follows: Figure 4 As shown.

[0036] The study results showed that liver-specific Wdr6 Interfering with adeno-associated virus (AAV) injection significantly decreased WDR6 protein expression in the liver, upregulated the expression of the liver aging marker p21 protein, and increased the number of aging-associated β-galactosidase-positive cells. These results indicate that the liver… Wdr6 Low expression exacerbates liver aging, suggesting Wdr6 It plays a key role in maintaining youthful liver homeostasis. Wdr6 Low expression can induce a premature aging phenotype in mouse liver.

[0037] Example 3 Wdr6 Effects of overexpression on the liver of naturally aging mice 1. Liver-specific Wdr6 Construction of overexpression mouse model Wild-type C57BL / 6J mice were fed a normal diet until 9 months of age and then divided into experimental and control groups. Mice in the experimental group received a single intravenous injection of pAAV2 / 8-TBG- via the tail vein. Wdr6 -3xFLAG-tWPA, injection dose is 5×10 11 vg / each, to achieve the effect of liver parenchymal cells Wdr6Gene-specific overexpression. Control mice were injected via tail vein with an equal dose of empty adeno-associated virus pAAV2 / 8-TBG-MCS-3xFLAG-tWPA. Four months after injection, liver tissue was collected after an 8-hour fast to detect the expression level of WDR6 protein, and the level of p21 protein and the number of senescent cells in the liver were assessed.

[0038] 2. Assessment of liver WDR6 and p21 protein levels Approximately 30 mg of liver tissue was collected, washed with sterile PBS, and then RIPA buffer containing 1% protease inhibitor and phosphatase inhibitor was added. The tissue was sonicated, centrifuged at 12000 rpm for 15 min at 4°C, and the supernatant was collected as the total protein extract. Protein quantification was performed on the total protein extract using a BCA protein assay kit. Subsequently, Western blotting was used to analyze the levels of WDR6 and p21 proteins in the protein samples, with GAPDH protein as a control. The results are as follows: Figure 5 As shown.

[0039] 3. SA-β-gal staining Fresh liver tissue blocks (0.5cm × 0.5cm) were taken and fixed in 4% paraformaldehyde solution at 4°C for 24 hours. Subsequently, they were dehydrated sequentially in 20% and 30% sucrose solutions at 4°C. The tissues were then embedded in OCT and frozen sectioned. After thawing and drying at room temperature, the sections were washed with PBS, and β-galactosidase staining fixative was added for fixation at room temperature for 15 minutes. After washing with PBS, β-galactosidase staining working solution was added, and the sections were incubated overnight at 37°C. The sections were then washed with PBS, mounted, and observed under a light microscope to reveal cells exhibiting blue β-galactosidase activity. This staining method is based on the upregulation of SA-β-gal activity levels during senescence and can be used to detect senescent cells. The results are as follows: Figure 6 As shown.

[0040] The study results showed that liver-specific Wdr6 Injection of adeno-associated virus (AAV) with gene overexpression significantly increased the expression of WDR6 protein in the liver, downregulated the expression of p21 protein, a marker of liver aging, and significantly reduced the number of liver aging-associated β-galactosidase-positive cells. These results indicate that the liver… Wdr6 Overexpression improves the degree of liver aging in naturally aging mice, suggesting... Wdr6 It plays a key role in maintaining the youthful homeostasis of the liver.

[0041] Example 4 Wdr6 Effects of overexpression on bleomycin-induced hepatocyte cell lines 1. Build endogenous Wdr6 Stable cell lines overexpressing the gene (1) Lentiviral packaging and collection: Packaging plasmid (psPAX2), envelope plasmid (pMID2.G), and the constructed lentiviral vector ( Wdr6 The full-length overexpression plasmid (pCDH-WDR6-Flag) was co-transfected into human embryonic kidney 293T (HEK293T) cells at a density of 60%-80% at a transfection ratio of 2:2:1. The culture medium was replaced with fresh medium 8 hours after transfection, and the cell supernatant containing viral particles was collected 48 hours later. The supernatant was filtered through a 0.45 μm filter and centrifuged for enrichment before use.

[0042] (2) Cell infection and screening: Mouse hepatocytes AML12 in the logarithmic growth phase were selected, digested, resuspended, and seeded into 6-well plates. The next day, when the cells adhered and reached 50%–60% confluence, freshly collected virus suspension was added directly to each well at a ratio of 100 μL. To improve infection efficiency, an appropriate amount of polybrene could be added simultaneously at this step. After 8 hours, the medium was replaced with fresh, standard complete culture medium. Once the cells had reached confluence, they were digested and passaged to 25 cm⁻¹. 2 Expand the culture in culture flasks. Simultaneously, add puromycin to the culture medium to a final concentration of 2 μg / mL for drug resistance screening. Replace the medium with fresh antibiotic-containing medium every 2-3 days, continuing the screening for 2 weeks. Once surviving cells exhibit stable clonal growth, the desired cells are obtained. Wdr6 Overexpression of stable AML12 cell lines.

[0043] 2. Using bleomycin to induce DNA damage and aging phenotypes in AML12 Those in the logarithmic growth phase Wdr6 Overexpressing stable AML12 cell lines and normal AML12 cells were seeded in cell culture plates. When cell adhesion and confluence reached 70%–80%, complete medium containing 50 μM bleomycin was added for 24 hours to induce DNA damage and senescence phenotypes. (Note: The last sentence appears to be incomplete and possibly refers to a different cell line.) Wdr6 The overexpressing stable AML12 cell line and normal AML12 cells served as solvent control groups.

[0044] 3. SA-β-gal staining Cell culture medium from the model and control groups was discarded. After washing with PBS, β-galactosidase staining fixative was added and the cells were fixed at room temperature for 15 min. After washing with PBS again, β-galactosidase staining working solution was added and incubated overnight at 37°C. After washing with PBS, cells expressing β-galactosidase were observed to turn blue under a light microscope. This staining method is based on the upregulation of SA-β-gal activity levels during senescence and can be used to stain and detect senescent cells. The results are as follows: Figure 7As shown in the figure, it can be seen that the addition of bleomycin to normal AML12 cells significantly increases the number of cells positive for senescence-associated β-galactosidase staining. Compared to cells without bleomycin, Wdr6 After adding bleomycin to the stable AML12 cell line overexpressing bleomycin, the number of cells stained positive for senescence-associated β-galactosidase increased only slightly.

[0045] 4. Assessment of SIRT1 and p21 protein levels in AML12 cells After washing with PBS, RIPA buffer containing 1% protease inhibitor and phosphatase inhibitor was added. Cells were collected, sonicated, and centrifuged at 4°C and 12000 rpm for 15 min. The supernatant was collected. Protein quantification in the supernatant was performed using a BCA protein assay kit. Western blotting was then used to analyze the levels of SIRT1 and p21 proteins in the protein samples, with GAPDH protein as a control. The results are as follows: Figure 8 As shown.

[0046] The research results show that Wdr6 Overexpression significantly reduced the proportion of senescent cells, demonstrating Wdr6 Overexpression has a protective effect against hepatocyte senescence. Furthermore, under bleomycin-induced conditions, Wdr6 Overexpression effectively maintained the expression level of the longevity protein SIRT1 and inhibited the upregulation of p21 protein. Wdr6 Overexpression can effectively improve bleomycin-induced senescence in hepatocyte lines.

[0047] Although the present invention has been described in detail with reference to the accompanying drawings and preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention.

Claims

1. A kind Wdr6 Application of gene overexpression vectors in the preparation of drugs to delay liver aging.

2. The application as described in claim 1, characterized in that, The Wdr6 The gene overexpression vector is an adeno-associated virus vector, containing Wdr6 Gene sequence and hepatocyte-specific TBG promoter.

3. The application as described in claim 1, characterized in that, The Wdr6 The capsid protein Cap used for packaging gene overexpression vectors is derived from AAV8.

4. The application as described in claim 1, characterized in that, The Wdr6 The gene overexpression vector structure includes a gene backbone from AAV2, a 3×FLAG tag, and a tWPA simplified element.

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