Application of acetyl-CoA promoter in the preparation of drugs promoting liver regeneration

Abstract

The present invention discloses the use of an acetyl-CoA (Ac‑CoA) promoter in the preparation of a drug for promoting liver regeneration. The present invention utilizes a mouse partial hepatectomy model and administers GS‑0976 before hepatectomy to increase Ac‑CoA in the liver. The results show that increased Ac‑CoA in the liver has the ability to improve liver function after liver injury and promote hepatocyte proliferation. The mechanism involves Ac‑CoA promoting MSL1 phase separation, increased STAT3 and H4K16 acetylation, which manifests as significantly upregulated cell cycle-related genes and increased hepatocyte proliferation. The experimental results show that increasing liver Ac‑CoA levels has the effect of promoting liver regeneration, providing a new solution for the recovery and treatment of hepatectomy and its complications.

Description

Technical Field

[0001] The invention relates to application of an acetyl-CoA promoter in preparing a drug for promoting liver regeneration, and belongs to the field of pharmaceuticals. Background Art

[0002] According to the World Health Organization, primary liver cancer is the sixth most common cancer worldwide, with 80% of cases diagnosed in patients aged 70 and above. 1 . Liver resection is a method of treating primary liver cancer with good efficacy and low recurrence rate. In addition to being used to treat liver cancer, liver resection is also used to treat a variety of liver diseases such as hepatic hemangioma, liver cysts, and hepatobiliary stones. However, postoperative complications and even liver failure have an important impact on patient recovery and postoperative survival. The incidence of complications after liver resection is as high as 45%, including infection or sepsis, bleeding, leakage or cardiopulmonary events, etc. The mortality rate is about 10%, and when liver resection involves more than 4 lobes of the liver, the mortality rate is as high as 30%. 2 The rapid proliferation of hepatocytes and the restoration of liver function after liver resection are crucial to reducing complications and improving survival rates. Elderly patients often suffer from more severe complications and higher mortality after liver resection due to the weakened liver regeneration capacity. 3 Therefore, developing a drug to promote liver regeneration after hepatectomy is of great clinical significance.

[0003] Acetyl-Coenzyme A (Ac-CoA), as a central metabolite and second messenger, is widely involved in cellular life activities. Evidence suggests that Ac-CoA has anti-aging effects by regulating epigenetic modifications. 4,5 Ac-CoA-sensitive genes include many genes involved in cell cycle, DNA replication, cell adhesion and migration. 6 , suggesting that Ac-CoA may play a role in promoting liver regeneration. As the only acetyl donor for acetylation, Ac-CoA can promote the acetylation of proteins. 7 GS-0976 is an acetyl-CoA carboxylase inhibitor (ACC) that increases acetyl-CoA (Ac-CoA) levels in the liver. 8 .

[0004] Liquid-liquid phase separation can separate proteins, nucleic acids or small molecules in cells to form aggregates, and is widely involved in the regulation of cell signaling. 9 Proteins with large intrinsically disordered regions (IDRs) are more prone to phase separation. 10 Phase separation can increase the rate of an enzyme reaction by increasing the concentrations of both the enzyme and the substrate (mass action). 11 Small molecule metabolites such as ATP can not only participate in the regulation of phase separation 12,13 , and can also be enriched in the phase separation condensate as a substrate 14Ac-CoA has a similar structure to ATP and may also have the ability to regulate protein phase separation. Male-specific lethal protein (MSL1) contains a large number of IDRs and is capable of phase separation. Histone H4 Lys16 (H4K16) is a target of the MSL acetyltransferase complex, which promotes the acetylation of H4K16 (H4K16ac). H4K16ac is believed to increase chromatin accessibility and promote gene expression. 15 CHIP-seq data showed that H4K16ac was widely enriched on the promoters of cell cycle-related genes, and cell cycle-related proteins play an important role in cell cycle progression and cell proliferation. 16 Signal transducer and activator of transcription 3 (STAT3) is a key transcription factor that initiates hepatocyte proliferation and liver regeneration. After liver resection, cytokines released by macrophages activate STAT3 through the JAK2 pathway, initiating liver regeneration. 17 The post-translational function-related modifications of STAT3 under the action of cytokines mainly include phosphorylation of tyrosine 705. However, STAT3 acetylation is also affected by cytokines and participates in the regulation of STAT3 phosphorylation and transcriptional activation. For example, STAT3 acetylation can not only promote its formation of dimers and enhance downstream gene expression, but also promote its phosphorylation. 18,19 The Ac-CoA level is positively correlated with the acetylation level of the protein and may regulate phase separation. 7 Therefore, increasing the level of Ac-CoA in the liver may be a potential method to promote liver regeneration after hepatectomy.

[0005] ACC plays an important role in the synthesis of fatty acids in the human body and is widely involved in the occurrence and development of metabolic diseases such as obesity, diabetes, and non-alcoholic fatty liver disease (NASH) by regulating lipid metabolism. Among them, the upregulation of de novo fat synthesis is the main cause of the pathogenesis of NASH, and reducing the fat content in the liver and thus reducing liver tissue fibrosis is the main direction of treatment for NASH patients. Currently, studies have reported that GS-0976, as an acetyl-CoA carboxylase inhibitor, can reduce lipid synthesis or accelerate its decomposition, thereby reducing the hepatic triglyceride content and liver fibrosis indicators in patients with fatty liver disease. It has been used clinically to treat NASH. 20 After searching, there is no literature report on the use of GS-0976 or acetyl-CoA promoter for regenerative treatment after liver resection. Summary of the Invention

[0006] The purpose of the present invention is to provide an acetyl-CoA promoter for use in the preparation of a drug for promoting liver regeneration, which can be used for the clinical treatment of liver cell proliferation and regeneration in patients undergoing liver resection surgery, thereby reducing complications caused by liver resection.

[0007] The above objectives are achieved through the following technical solutions:

[0008] First, the applicant studied the mechanism of action of acetyl-CoA in promoting liver regeneration and found that male-specific lethal protein (MSL1) plays an important role in liver regeneration. It can promote liver regeneration after liver resection, and Ac-CoA can promote the phase separation of MSL1 and enrichment in aggregates. Not only that, Ac-CoA can also promote the phase separation of MSL1 and STAT3 or H4, and the MSL1 phase separation can promote the acetylation of STAT3 and H4. Given that the activation of STAT3 signaling is crucial for liver regeneration, and H4K16ac is the switch for transcriptional activation, therefore, by increasing the level of Ac-CoA in the liver, it is possible to promote the phase separation of MSL1 and provide an acetylation donor, promote the acetylation of STAT3 and H4K16, lead to STAT3 transcriptional activation and enrichment of H4K16ac on the promoters of cell cycle-related genes, enhance the expression of cell cycle-related genes, and thereby promote hepatocyte proliferation and liver regeneration.

[0009] Next, the applicant used hepatectomized mice as test subjects to investigate the efficacy of acetyl-CoA promoters in treating liver injury and promoting liver regeneration. The results showed that oral administration of GS-0976 increased Ac-CoA levels in the livers of mice before and after hepatectomy. Further testing of serum ALT and AST levels revealed that hepatectomy led to significant increases in ALT and AST levels, while GS-0976 treatment reduced serum ALT and AST levels, thereby alleviating liver injury. Furthermore, GS-0976 treatment increased the number of BrdU- and Ki67-positive hepatocytes in the mouse livers, indicating that increasing Ac-CoA levels in the liver promoted cell proliferation in the mouse livers.

[0010] Molecular biological analysis revealed that GS-0976 treatment increased STAT3 acetylation and phosphorylation in the livers of post-hepatectomy mice, promoted H4K16 acetylation, and elevated H4K16ac levels on the promoters of CyclinA2, B1, and D1. Cell cycle-related genes play an important role in promoting cell proliferation. GS-0976 treatment also upregulated CyclinA2, B1, and D1 mRNA expression in the livers of post-hepatectomy mice, further promoting hepatocyte proliferation.

[0011] The above results confirm that by increasing the level of Ac-CoA in the liver, it is possible to promote the phase separation of MSL1 and provide an acetylation donor, promote the acetylation of STAT3 and H4K16, lead to STAT3 transcriptional activation and H4K16ac enrichment on the promoters of cell cycle-related genes, enhance the expression of cell cycle-related genes, promote hepatocyte proliferation and liver regeneration, indicating that increasing the level of Ac-CoA in the liver can be used in the treatment of liver regeneration after clinical hepatectomy.

[0012] The chemical structure of GS-0976 is as follows:

[0013]

[0014] Explanation of related terms:

[0015] Male-specific lethal protein (MSL1): Male-specific lethal 1 (NCBI Reference Sequence: NM_028722.3), a component of the MSL acetyltransferase complex.

[0016] Signal transducer and activator of transcription 3 (STAT3): Signal transducer and activator of transcription 3, mediates the response of cells to a variety of cytokines and growth factors. STAT3 is a key transcriptional mediator that initiates hepatocyte proliferation and plays an important role in liver regeneration.

[0017] Histone H4 Lys16 acetylation (H4K16ac): refers to the acetylation of the 16th amino acid residue (lysine) of histone H4, which is mainly mediated by the MSL complex. H4K16ac can maintain chromatin in a loose state, increase chromatin accessibility, and promote gene expression.

[0018] ALT and AST: Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are mainly present in the cytoplasm of hepatocytes and are usually released into the blood after acute liver injury. They are important detection indicators of liver function damage.

[0019] BrdU and Ki67: BrdU is a thymidine nucleoside analog that can replace thymidine (T) and incorporate into replicating DNA molecules during cell proliferation. Detection of BrdU labeling can accurately reflect cell proliferation. Ki67 is an antigen associated with proliferating cells. Its function is closely related to mitosis and is an important indicator reflecting cell proliferation.

[0020] CyclinA2, CyclinB1, CyclinD1: CyclinA2 (NCBI Reference Sequence: NM_009828.3), CyclinB1 (NCBI Reference Sequence: NM_172301.3), and CyclinD1 (NCBI Reference Sequence: NM_001379248.1) are three different cell cycle proteins that can promote cell mitosis.

[0021] Liquid-liquid phase separation: In biology, it refers to the phenomenon in which macromolecules in cells interact to form "droplets" with high fluidity.

[0022] Aggregates: refers to the "droplets" formed by liquid-liquid phase separation of macromolecules in cells, also known as aggregates.

[0023] Acetyl-CoA enhancer: refers to drugs or reagents that can increase the level of acetyl-CoA in animals, including acetyl-CoA carboxylase inhibitors such as GS-0976.

[0024] The beneficial effects of the present invention are:

[0025] The present invention provides a new strategy for promoting liver regeneration therapy, which can be used for clinical liver cell proliferation and regeneration treatment in patients undergoing liver resection, thereby reducing complications caused by liver resection. The results of the examples show that increasing the level of Ac-CoA in the liver by using an acetyl-CoA promoter can promote the phase separation of MSL1 and STAT3 or H4 in hepatocytes, promote the acetylation of STAT3 and H4K16 in the liver after liver resection, increase the abundance of H4K16ac on the promoters of cell cycle-related genes, upregulate the expression of cell cycle-related genes, promote liver regeneration and liver cell proliferation, and thus provide a safe and effective alternative drug option for the treatment of patients after liver resection.

[0026] For more detailed technical solutions, please refer to the specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 : Effects of MSL1 on BrdU- and Ki67-positive cells after mouse hepatectomy. In the figure: (A) Liver tissues of WT and LKO mice were stained with BrdU antibody; (B) BrdU-positive cell statistics; (C) Liver tissues of WT and LKO mice were stained with Ki67 antibody; (D) Ki67-positive cell statistics.

[0028] Figure 2 : Effects of MSL1 on serum AST and ALT levels in mice after hepatectomy.

[0029] Figure 3 : Effect of MSL1 on liver-to-weight ratio after hepatectomy in mice.

[0030] Figure 4 : Effects of CMS-121 or SB-204990 on the formation of MSL1 aggregates in cells. In the figure: (A) After treatment with 10 μM CMS-121 or SB-204990 for 24 hours, primary hepatocytes from WT mice were stained with MSL1 antibody; (B) Aggregates formed by MSL1 in each group of cells were quantitatively analyzed.

[0031] Figure 5 :Ac-CoA at different concentrations was added to the GFP-MSL1 protein buffer, and the phase separation phenomenon and its statistical results were observed under a microscope.

[0032] Figure 6 Ac-CoA can promote the phase separation of MSL1 and STAT3 or H4. In the figure: (A) 293T cells were co-transfected with GFP-MSL1 and mCherry-STAT3 for 24 h, then treated with 10 ng / ml IL-6 for 30 min, and the cells were fixed and observed; (B) 293T cells were co-transfected with GFP-MSL1 and H4-mCherry for 24 h, and the cells were fixed and observed.

[0033] Figure 7 :MSL1 phase separation can promote the acetylation of STAT3 and H4. In the figure: (A) After Hep1-6 cells were transfected with MSL1 and MSL1-ΔIDR3, the levels of STAT3 acetylation and phosphorylation were detected by WB; (B) After Hep1-6 cells were transfected with MSL1 and MSL1-ΔIDR3, the level of H4K16 acetylation was detected by WB.

[0034] Figure 8 : qPCR and WB detection of MSL1 protein expression levels in mouse livers. In the figure: (A) qPCR detection of MSL1 mRNA expression levels in the livers of young and old mice; (B) WB detection of MSL1 protein expression levels in the livers of young and old mice.

[0035] Figure 9 : The Ac-CoA detection kit was used to detect the Ac-CoA content in the liver of each group of mice before and after hepatectomy.

[0036] Figure 10 : The levels of ALT and AST in the serum of mice in each group before and after hepatectomy were detected using detection kits.

[0037] Figure 11Effects of GS-0976 on BrdU- and Ki67-positive hepatocytes in mouse livers. Figure: (A) Immunohistochemical staining of the livers of mice after hepatectomy in each group. (B) Quantification of positive cells. Five fields of view were selected for each mouse, and the proportion of positive cells was calculated.

[0038] Figure 12 : Western blot was used to detect the acetylation and phosphorylation levels of STAT3 in the livers of young and old mice after GS-0976 treatment.

[0039] Figure 13 : Western blot analysis was used to detect H4K16 acetylation levels in the livers of young and old mice after GS-0976 treatment.

[0040] Figure 14 : Results of H4K16ac level detection on the promoters of Cyclin A2, B1, and D1. In the figure: (A) CHIP-PCR detection of H4K16ac level on the promoters of Cyclin A2, B1, and D1; (B) CHIP-qPCR detection of H4K16ac level on the promoters of Cyclin A2, B1, and D1.

[0041] Figure 15 : qPCR was used to detect the expression of Cyclin A2, B1, and D1 mRNA in the liver of young and old mice after GS-0976 treatment.

[0042] In the above figures, conventional one-way ANOVA test was performed using GraphPad Prism 8 software, * indicates P < 0.05, ** indicates P < 0.05, and *** indicates P < 0.001). DETAILED DESCRIPTION

[0043] To facilitate those skilled in the art to implement the present invention, the present invention is described in detail below with reference to specific examples. It should be understood that the following specific examples are only used to explain the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. The materials and reagents used in the examples, unless otherwise specified, can be obtained from commercial sources. Experimental methods for which specific conditions are not specified in the examples are generally implemented according to conventional conditions or the methods recommended in the operating manuals provided by the instrument manufacturers.

[0044] Given the important role of cell cycle-related proteins in hepatocyte proliferation and liver regeneration, some specific embodiments of the present invention provide a method for increasing the expression of cell cycle-related genes to promote liver regeneration after hepatectomy, providing a new strategy for the treatment of liver regeneration after hepatectomy in patients, especially elderly patients. Due to the effects of aging, the expression of cell cycle-related genes in the liver of elderly patients after hepatectomy decreases, and liver regeneration is weakened. Therefore, by increasing Ac-CoA levels in the liver and promoting the phase separation of MSL1 and STAT3 or H4, enhancing the acetylation of STAT3 and H4K16, promoting the transcriptional activation of STAT3 and the level of H4K16ac on the promoters of cell cycle-related genes and promoting their mRNA expression, it is of great significance to promote hepatocyte proliferation.

[0045] The present invention demonstrates that GS-0976 can increase Ac-CoA levels in the livers of aged mice. High Ac-CoA levels can reduce serum AST and ALT levels in aged mice after hepatectomy, while also increasing the proportion of BrdU- and Ki67-positive cells in the liver. This suggests that increasing Ac-CoA levels in the liver can promote liver function recovery and hepatocyte proliferation. Further studies have shown that increasing Ac-CoA levels in the liver can enhance STAT3 and H4K16 acetylation levels in the liver after hepatectomy, promoting STAT3 transcriptional activation, and increasing H4K16ac levels on the promoters of Cyclin A2, B1, and D1 genes, thereby promoting their mRNA expression, thereby promoting hepatocyte proliferation.

[0046] Example 1 Mechanism of action of acetyl-CoA in promoting liver regeneration

[0047] Hepatocyte-specific MSL1 deletion impairs liver regeneration after hepatectomy in mice

[0048] Wild-type (WT) and hepatocyte-specific MSL1 knockout (LKO) mice underwent hepatectomy, and samples were collected at different time points after surgery to analyze the role of MSL1 in liver regeneration. The results showed that compared with WT mice, LKO mice had fewer BrdU and Ki67-positive cells after hepatectomy ( Figure 1 ), AST and ALT levels increased ( Figure 2 ), liver-to-weight ratio was reduced, and liver regeneration was attenuated after hepatectomy in LKO mice ( Figure 3 ), the above results indicate that MSL1 plays an important role in liver regeneration after liver resection in mice.

[0049] 2. Ac-CoA can promote phase separation of MSL1 and enrichment in aggregates

[0050] Analysis of the MSL1 protein sequence revealed that it contains many intrinsically disordered regions and may undergo phase separation. MSL1 staining in primary hepatocytes showed that MSL1 formed small aggregates in the cell nucleus. In vitro purified MSL1 was also able to form aggregates in solution, indicating that MSL1 can undergo phase separation. When primary hepatocytes were treated with CMS-121 (an Ac-CoA carboxylase inhibitor that increases the level of Ac-CoA in cells), the number of aggregates formed by MSL1 in the cells increased, while when primary hepatocytes were treated with SB-204990 (an ATP citrate lyase inhibitor that reduces the level of Ac-CoA in cells), the number of aggregates formed by MSL1 in the cells decreased ( Figure 4 ); and the addition of Ac-CoA promoted the formation of MSL1 aggregates in vitro ( Figure 5 ), indicating that Ac-CoA can promote phase separation of MSL1. Furthermore, in vitro centrifugation analysis showed that Ac-CoA promoted the formation of MSL1 aggregates. Measurement of Ac-CoA concentration in the precipitated phase revealed that Ac-CoA was enriched in MSL1 aggregates. These results demonstrate that Ac-CoA can promote phase separation of MSL1 and enrich it in aggregates.

[0051] 3. Ac-CoA can promote the phase separation of MSL1 and STAT3 or H4, and MSL1 phase separation can promote the acetylation of STAT3 and H4

[0052] The activation of STAT3 signaling after hepatectomy is crucial for liver regeneration. H4K16ac is considered to be a switch for transcriptional activation, which can maintain the open state of chromatin, allowing transcription factors to bind to gene promoters and promote gene transcription. Therefore, we tested whether MSL1 is involved in the regulation of STAT3 and H4. In 293T cells, GFP-MSL1 and mCherry-STAT3 were co-transfected. GFP-MSL1 and mCherry-STAT3 were localized in the nucleus and cytoplasm, respectively. mCherry-STAT3 did not undergo phase separation. When IL-6 was added to the cells, mCherry-STAT3 entered the nucleus and phase-separated with GFP-MSL1 ( Figure 6 A); Similarly, when H4-mCherry was transfected alone, H4-mCherry did not phase separate. However, when co-transfected with GFP-MSL1, H4-mCherry and GFP-MSL1 phase separated together ( Figure 6B). In vitro experiments also showed that GFP-MSL1 phase separated from mCherry-STAT3 or H4-mCherry proteins, and that Ac-CoA promoted the formation of phase-separated aggregates. These results indicate that MSL1 can drive the phase separation of STAT3 or H4, and that Ac-CoA can promote this phase separation.

[0053] To investigate whether phase separation can promote the acetylation of STAT3 or H4, we first constructed the MSL1 mutant MSL1-ΔIDR3, which cannot undergo phase separation. We transfected Hep1-6 cells with MSL1 and MSL1-ΔIDR3, respectively, and found that MSL1 could promote the acetylation and phosphorylation of STAT3 after IL-6 treatment ( Figure 7 A), while MSL1-ΔIDR3, which cannot undergo phase separation, does not have this function. Similarly, MSL1-ΔIDR3 cannot promote the acetylation of H4K16 ( Figure 7 B), indicating that MSL1 phase separation plays an important role in promoting STAT3 and H4K16 acetylation.

[0054] Example 2 In vivo animal experiment on the treatment of liver injury and promotion of liver regeneration by acetyl-CoA promoter

[0055] 1. Test methods

[0056] Animal Handling

[0057] Male C57BL / 6J young (3 months old) and aged (14 months old) mice were purchased from the Experimental Animal Center of Huazhong Agricultural University. For this experiment, mice were divided into the young (Young) group, the aged (Aged) group, and the aged (Aged + GS-0976) treatment group, with six mice in each group. The young and aged groups were gavaged with a vehicle containing 1% Tween 80 and 0.5% methylcellulose in normal saline. The aged treatment group was gavaged with the same volume of vehicle containing GS-0976 at a dose of 10 mg / kg / day for 28 days. Mice were housed at the Experimental Animal Center of Huazhong Agricultural University at a room temperature of 22–26°C, a relative humidity of 40%–60%, and a 12-hour light-dark cycle. They had free access to food and water. On the last day of treatment, mice were anesthetized with tribromoethanol, their tails were snipped, and a small amount of blood was collected and centrifuged for serum. The abdomen was then disinfected with ethanol. A laparotomy was performed along the midline below the xiphoid process. The liver was excised, the hepatic pedicle ligated, and the left lateral lobe, caudate lobe, and median lobe, representing approximately 70% of the liver volume, were aseptically resected along the lateral side of the ligature. The abdominal incision was sutured, and the mouse was placed in a 35°C incubator to allow recovery. The excised liver was immediately frozen in liquid nitrogen for subsequent analysis. 34 hours after hepatectomy, the mouse was intraperitoneally injected with 50 mg / kg of BrdU. 36 hours after hepatectomy, the mouse was euthanized, and blood and liver were collected. The blood was centrifuged and serum was obtained. A portion of the liver was frozen in liquid nitrogen, while the remaining portion was fixed in paraformaldehyde.

[0058] 1.2 Detection

[0059] 1.2.1 Detection of MSL1 expression in mouse liver by qPCR and WB

[0060] 1.2.1.1 qPCR detection

[0061] Total RNA from liver tissue was extracted using the Trizol method. The RNA was reverse transcribed according to the Takara Bio reverse transcription instructions to obtain cDNA. qPCR detection primers for MSL1 were designed, and the obtained cDNA was used as a template for qPCR detection.

[0062] 1.2.1.2 WB detection

[0063] Mouse liver was lysed using RIRP buffer, boiled in loading buffer, and loaded onto an SDS-PAGE gel. Protein samples were separated by electrophoresis and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk powder and incubated with primary antibodies overnight at 4°C. The membrane was then incubated with corresponding secondary antibodies for 60 minutes at room temperature and developed with ECL colorimetric solution. Antibodies used included MSL1 (1:1000), GAPDH (1:1000), HRP-goat anti-mouse (1:5000), and HRP-goat anti-rabbit (1:5000). GAPDH was used as a loading control.

[0064] 1.2.2 Biochemical detection of Ac-CoA levels in mouse liver tissue

[0065] The Ac-CoA detection kit was purchased from Abcam. The samples were processed according to the kit instructions, and the corresponding reagents were added. The detection was performed on a multifunctional microplate reader using a wavelength of Ex / Em = 535 / 587 nm. The test results were substituted into the standard curve for calculation to obtain the final results.

[0066] 1.2.3 Biochemical detection of ALT and AST levels in mouse serum

[0067] Alanine aminotransferase (ALT / GPT) and aspartate aminotransferase (AST / GOT) detection kits were purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd. Samples were processed and the corresponding reagents were added according to the kit instructions. The assay was performed using a microplate reader at an OD of 510 nm, and the assay results were applied to the standard curve for calculation to obtain the final results.

[0068] 1.2.4 Immunohistochemical detection of BrdU and Ki67 positive hepatocytes in the liver of mice

[0069] The liver tissue was removed from the paraformaldehyde solution, dehydrated, embedded, and then sectioned. The following steps were then performed: 1) baking the sections; 2) deparaffinizing and hydrating; 3) antigen retrieval; 4) peroxidase removal; 5) application of primary antibody; 6) incubation with secondary antibody; 7) development with DAB; 8) counterstaining with hematoxylin; 9) hydrochloric acid differentiation; 10) dehydration and clearing; 11) mounting. Antibodies used included: BrdU (1:100), Ki67 (1:100), HRP-goat anti-mouse (1:500), and HRP-goat anti-rabbit (1:500).

[0070] 1.2.5 Western blotting to detect STAT3 acetylation and phosphorylation levels in mouse liver

[0071] Western blotting (WB) was performed using the same method as described in 1.2.1.2. Antibodies used included STAT3 (1:1000), p-STAT3 Y705 (1:1000), ac-STAT3 K685 (1:1000), HRP-goat anti-mouse (1:5000), and HRP-goat anti-rabbit (1:5000). STAT3 was used as an internal control.

[0072] 1.2.6 Molecular Biology Detection of H4K16ac Levels in Mouse Liver

[0073] Western blotting (WB) was performed using the same method as in 1.2.1.2. Antibodies used included: H4K16ac (1:1000), H3 (1:1000), HRP-goat anti-mouse (1:5000), and HRP-goat anti-rabbit (1:5000). H3 served as the internal control protein.

[0074] 1.2.7 Molecular Biological Detection of H4K16ac Levels on CyclinA2, B1, and D1 Promoters in Mouse Liver

[0075] The CHIP assay kit was purchased from Beyotime Biotechnology Co., Ltd. Liver samples were processed according to the kit instructions. Primers were designed using the cyclinA2, B1, and D1 promoter sequences as templates, and the resulting samples were analyzed by PCR and qPCR. Antibodies used included H4K16ac (1:50) and rabbit IgG (1:50).

[0076] 1.2.8 Molecular Biology Detection of CyclinA2, B1, and D1 mRNA Expression in Mouse Liver

[0077] The detection method is the same as 1.2.1.1. Design qPCR detection primers for CyclinA2, B1, and D1, and use the obtained cDNA as a template for qPCR detection.

[0078] 2. Test results

[0079] 2.1 Increasing Ac-CoA levels in the liver alleviated liver damage after hepatectomy in aged mice and promoted hepatocyte proliferation.

[0080] The liver regeneration capacity of aged mice is impaired, and MSL1 plays an important role in liver regeneration. Therefore, the impaired liver regeneration in aged mice may be related to the changes in MSL1 expression in the liver. qPCR and WB results showed that the expression of MSL1 in the liver of aged mice was reduced ( Figure 8). To determine whether Ac-CoA can promote liver regeneration, aged mice were treated with GS-0976, an acetyl-CoA carboxylase inhibitor that effectively inhibits acetyl-CoA carboxylase from catalyzing acetyl-CoA into malonyl-CoA, thereby increasing Ac-CoA levels in cells. First, we examined the effect of GS-0976 on Ac-CoA levels in the livers of mice before and after hepatectomy. Before hepatectomy, there was no difference in Ac-CoA levels in the livers of young and aged mice, while Ac-CoA levels were significantly increased in the aged mice + GS-0976 group; 36 hours after hepatectomy, there was no change in Ac-CoA levels in the livers of aged mice, while Ac-CoA levels were significantly increased in the young and aged mice + GS-0976 groups ( Figure 9 ), indicating that GS-0976 treatment increased Ac-CoA levels in the livers of elderly mice before and after hepatectomy. Serum ALT and AST levels were further tested. Before hepatectomy, serum ALT and AST levels remained unchanged among the three groups of mice and were within the normal range. Thirty-six hours after hepatectomy, ALT and AST levels increased significantly in all three groups, with the most significant increase in the elderly mouse group, followed by the elderly mouse + GS-0976 group and the young mouse group ( Figure 10 ), indicating that after liver resection, the liver damage in old mice was more severe than that in young mice, and increasing the liver Ac-CoA level alleviated the liver damage in old mice. In addition, the number of BrdU and Ki67 positive hepatocytes in the liver of young mice was significantly higher than that of old mice, while GS-0976 treatment increased the number of BrdU and Ki67 positive hepatocytes in the liver of old mice ( Figure 11 ), indicating that increasing the Ac-CoA level in the liver promoted the proliferation of hepatocytes in the liver of aged mice.

[0081] 2.2 Increasing Ac-CoA levels in the liver enhanced the acetylation levels of STAT3 and H4K16 in the liver of aged mice after hepatectomy and increased the acetylation of H4K16 and mRNA expression on the promoters of Cyclin A2, B1, and D1 genes

[0082] STAT3 activation plays an important role in liver regeneration after hepatectomy. WB results showed that GS-0976 treatment increased the acetylation and phosphorylation levels of STAT3 in the livers of aged mice after hepatectomy ( Figure 12 H4K16ac is considered a switch for transcriptional activation, which maintains an open chromatin state, allowing transcription factors to bind to gene promoters and promote gene transcription. After hepatectomy, H4K16ac levels in the livers of young mice increased significantly, while those in old mice were relatively low. However, GS-0976 treatment increased H4K16ac levels in the livers of old mice ( Figure 13); CHIP-PCR and CHIP-qPCR results showed that the H4K16ac levels on the CyclinA2, B1, and D1 promoters in the livers of old mice after hepatectomy were lower than those in young mice, but GS-0976 treatment increased the H4K16ac levels on the CyclinA2, B1, and D1 promoters in the livers of old mice ( Figure 14 ), indicating that GS-0976 promoted H4K16 acetylation in the livers of aged mice after hepatectomy and increased H4K16ac levels on the promoters of CyclinA2, B1, and D1. Cell cycle-related genes play an important role in promoting cell proliferation. After hepatectomy, CyclinA2, B1, and D1 mRNA expression in the livers of aged mice was less upregulated than that in young mice, but GS-0976 treatment upregulated CyclinA2, B1, and D1 mRNA expression ( Figure 15 ). This indicates that increasing Ac-CoA levels promotes hepatocyte proliferation by upregulating the expression of CyclinA2, B1, and D1.

[0083] The results of the above examples demonstrate that Ac-CoA can promote phase separation between MSL1 and STAT3 or H4, promote acetylation of STAT3 or H4K16, and increase Ac-CoA levels in the livers of elderly mice after hepatectomy. Furthermore, Ac-CoA can promote acetylation of STAT3 or H4K16, promote H4K16ac enrichment at the CyclinA2, B1, and D1 promoters, promote CyclinA2, B1, and D1 mRNA expression, and promote hepatocyte proliferation and liver regeneration. The method provided by the present invention for increasing Ac-CoA levels in the liver has excellent efficacy in promoting hepatocyte proliferation in the treatment of liver regeneration after hepatectomy in elderly patients. Furthermore, the method has no significant toxic side effects or adverse reactions to the liver, making it suitable for the treatment of hepatectomy and its complications in elderly patients.

[0084] The above specific embodiments are only used to explain the present invention and are not intended to limit the present invention. In addition, those skilled in the art may make various modifications or equivalent substitutions to the present invention within the essence and scope of protection of the present invention, and such modifications or equivalent substitutions should also be deemed to fall within the scope of protection of the present invention.

[0085] References

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Claims

1. Application of acetyl-CoA promoter GS-0976 in the preparation of drugs to promote liver regeneration.

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