Application of KLF4 in the preparation of drugs for the prevention or treatment of glucose and lipid metabolism diseases

Abstract

This invention relates to the application of KLF4 in the preparation of drugs for the prevention or treatment of glucose and lipid metabolism disorders. This invention elucidates for the first time the key pathogenic role of the transcription factor KLF4 in excessive gluconeogenesis, abnormal lipid accumulation, and inflammatory necrosis in the liver, filling a target gap in this field at the transcriptional regulatory level and providing a novel theoretical framework for the study of the molecular mechanisms of type 2 diabetes and non-alcoholic steatohepatitis (NASH).

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to the application of KLF4 in the preparation of drugs for the prevention or treatment of glucose and lipid metabolism diseases. Background Technology

[0002] With changes in modern lifestyles and dietary structures, the incidence of glucose and lipid metabolism disorders, represented by obesity, type 2 diabetes mellitus (T2DM), and non-alcoholic fatty liver disease (NAFLD), is rising year by year, becoming a major global public health problem. The liver, as the body's most important metabolic center, plays a central role in maintaining systemic glucose and lipid metabolism homeostasis.

[0003] In the pathological progression of diabetes, abnormally active hepatic gluconeogenesis is a significant cause of fasting hyperglycemia (Petersen MC, Nature Reviews Endocrinology, 2017, DOI:10.1038 / nrendo.2017.80). Similarly, in the development of fatty liver disease, abnormal uptake and excessive accumulation of lipids such as free fatty acids by hepatocytes are the core pathological features driving hepatic steatosis and even the progression to non-alcoholic fatty liver disease (NASH) (Samuel VT, Cell, 2012, DOI: 10.1016 / j.cell.2012.02.017). Currently, while clinically used drugs for treating these metabolic disorders (such as metformin, insulin sensitizers, or lipid-lowering drugs) can control symptoms to some extent, they often have side effects such as gastrointestinal reactions and hepatotoxicity / nephrotoxicity, and some drugs have poor response rates. In the current technology, the precise transcriptional regulation mechanism of hepatic gluconeogenesis and lipid metabolism networks has not been fully elucidated, and there is still a lack of precise therapeutic targets in clinical practice that can simultaneously target and regulate abnormal glucose metabolism and lipid distribution.

[0004] KLF4 (Kruppel-like factor 4) is a transcription factor containing a zinc finger structure. Previous studies have focused on its role in cell proliferation, differentiation, and maintenance of stem cell pluripotency (Ghaleb AM, Comprehensive Physiology, 2017, DOI: 10.1002 / cphy.c160015). In recent years, the regulatory potential of the KLF transcription factor family in systemic metabolic homeostasis has begun to attract attention (Sweet DR, Frontiers in Cardiovascular Medicine, 2018, DOI: 10.3389 / fcvm.2018.00069). However, current technology lacks in-depth and systematic research on the specific metabolic regulatory role of KLF4 in liver physiological starvation and pathological states such as obesity and diabetes.

[0005] Specifically, no studies have yet clearly confirmed whether KLF4 directly participates in driving hepatic gluconeogenesis (e.g., amino acid-driven or cAMP-stimulated pathways), and there is a lack of research into its direct regulatory mechanism on the transcriptional activity of key metabolic target genes such as GLUD1. Furthermore, the molecular pathway by which KLF4 mediates the expression of lipid transporters such as CD36 to affect hepatic lipid uptake and induce hepatocyte inflammation and necrosis is also unknown in current technologies. Therefore, in-depth elucidation of the key molecular mechanisms of KLF4 in hepatic glucose and lipid metabolism will provide extremely important theoretical basis and novel intervention targets for developing new and highly effective drugs for the prevention, screening, or treatment of metabolic diseases (especially type 2 diabetes and fatty liver). Summary of the Invention

[0006] The technical problem to be solved by the present invention is to address the shortcomings of existing drugs for treating disorders of glucose and lipid metabolism, such as diabetes and fatty liver, which have inaccurate target points and cannot simultaneously and effectively address abnormal glucose production and lipid accumulation. The present invention provides the application of KLF4 in the preparation of drugs for the prevention or treatment of disorders of glucose and lipid metabolism.

[0007] This invention provides the application of KLF4 in the preparation of drugs for the prevention or treatment of glucose and lipid metabolism diseases.

[0008] Furthermore, inhibitors were used to specifically downregulate or inhibit the expression and transcriptional activity of the KLF4 gene or protein in the liver. Experiments showed that in obese and diabetic mouse models, the expression of KLF4 mRNA and protein in the mouse liver was significantly increased. In addition, physiological starvation also promoted the expression of KLF4 mRNA and protein in the liver.

[0009] Furthermore, the glucose and lipid metabolism disorder is one or more of type 2 diabetes mellitus, hyperglycemia, or non-alcoholic fatty liver disease. In an in vitro model, knockdown of KLF4 in primary hepatocytes attenuated the gene expression of key enzymes involved in cAMP-stimulated hepatocyte glucose production and gluconeogenesis. Simultaneously, knockdown of KLF4 in primary hepatocytes also attenuated amino acid-driven hepatic gluconeogenesis.

[0010] Furthermore, the mechanism by which KLF4 regulates glucose and lipid metabolism is as follows: KLF4 binds to the promoter region of the metabolic enzyme GLUD1 and regulates its transcriptional expression. Overexpression and knockdown of KLF4 can increase or decrease GLUD1 expression. In pathological models, there is a positive correlation between the expression of KLF4 and GLUD1.

[0011] Furthermore, the pathway by which KLF4 induces steatohepatitis is as follows: KLF4 participates in hepatocyte lipid uptake and inflammation by upregulating CD36 protein expression. In mouse primary liver cultures under low-glucose or high-glucose conditions, simple overexpression of KLF4 leads to upregulation of CD36 protein expression. In in vivo experiments, the AAV-OE KLF4 5E11 group of mice showed significantly upregulated CD36 expression in the liver, increased liver TC (total cholesterol), and tissue sections (H&E and F4 / 80 staining) revealed increased hepatocyte necrosis and macrophage infiltration in the overexpression group.

[0012] The present invention also provides a pharmaceutical composition for the prevention and / or treatment of glucose and lipid metabolism disorders, the pharmaceutical composition comprising an effective dose of a KLF4 inhibitor and pharmaceutically acceptable excipients.

[0013] Preferably, the pharmaceutically acceptable excipients include at least one of diluents, binders, surfactants, humectants, adsorbents, lubricants, fillers, and disintegrants.

[0014] Preferably, the dosage form of the pharmaceutical composition includes tablets, capsules, granules, oral liquids, injections, lyophilized powder injections, lipid nanoparticle formulations, sustained-release formulations, or controlled-release formulations.

[0015] Beneficial effects

[0016] (1) Discovery of a new key driver of glucose and lipid metabolism disorders: For the first time, the key pathogenic role of transcription factor KLF4 in excessive gluconeogenesis and abnormal lipid accumulation and inflammation and necrosis in the liver was elucidated, filling the target gap in the transcriptional regulation level in this field and providing a new theoretical framework for the study of the molecular mechanisms of type 2 diabetes and non-alcoholic steatohepatitis (NASH).

[0017] (2) Providing a novel and precise dual-effect therapeutic target: Hepatocyte-specific knockdown of KLF4 (si-KLF4) showed a significant metabolic protective effect in an in vitro gluconeogenesis model, effectively reducing cAMP stimulation and amino acid-driven glucose production in hepatocytes; while in vivo AAV-mediated KLF4 overexpression accelerated hepatic lipid accumulation and macrophage infiltration. The above data from both positive and negative aspects confirm that KLF4 is a highly promising and operable dual-effect therapeutic target for lowering blood sugar and reducing inflammation, providing a precise targeting technical approach for the subsequent development of targeted nucleic acid drugs (such as siRNA) or small molecule inhibitors.

[0018] (3) Discovery of a novel intervention node for anti-glycemic and lipid disorders—the KLF4-GLUD1 and KLF4-CD36 dual axis: For the first time, it was revealed that KLF4 can directly bind to and activate the GLUD1 promoter to drive hepatic gluconeogenesis, while promoting hepatic lipid uptake and inflammatory response by upregulating CD36 protein expression. Intervention on this dual axis can effectively and simultaneously block abnormal glucose production and steatosis. Compared with existing clinical hypoglycemic or lipid-lowering drugs that target a single pathway, this intervention strategy is more precise and comprehensive.

[0019] (4) Revealing specific expression associations in pathological models: It was clarified that in physiological starvation and pathological mouse models such as obesity and diabetes, the mRNA and protein expression of liver KLF4 and GLUD1 showed significant positive correlation and abnormally increased levels. The expression levels of both can serve as effective molecular reference indicators reflecting the imbalance of glucose and lipid metabolism and the degree of pathological activity, which can help to make up for the deficiencies of existing preclinical drug screening and efficacy evaluation tools. Attached Figure Description

[0020] Figure 1 The study showed that KLF4 expression was abnormally elevated in the livers of obese and diabetic mouse models. Specifically, the expression levels of KLF4 mRNA (A) and protein (B) in the livers of (AB) db / m control and db / db diabetic mice were detected; the expression levels of KLF4 mRNA (C) and protein (D) in the livers of (CD) obese mice induced by normal diet (NCD) and high-fat diet (HFD) were detected; and the expression levels of KLF4 mRNA (E) and protein (F) in the livers of (EF) wild-type (WT) control and Ob / Ob hereditary obese mice were detected.

[0021] Figure 2The study showed that KLF4 expression was significantly increased in the liver of mice under physiological starvation. Specifically, (A) the relative expression level of KLF4 mRNA in the liver of mice after ad libitum feeding and after fasting for 8 hours (Fast 8h), 16 hours (Fast 16h), and 24 hours (Fast 24h), respectively; and (B) the Western blot analysis of KLF4 protein expression level in the liver of mice after refeeding and after fasting for 16 hours (Fast 16hr) and 24 hours (Fast 24hr), respectively.

[0022] Figure 3 The study showed that KLF4 overexpression promoted glucose production and the expression of key gluconeogenesis enzyme genes in cAMP-stimulated primary liver cells. Among them, (A) verification of KLF4 overexpression efficiency in primary liver cells; (B) glucose concentration (μM) in the culture medium of primary liver cells in the NC group and the OE-KLF4 group under culture conditions containing only basal substrate (Sub) or cAMP stimulation (cAMP); (CE) detection of the relative mRNA expression levels of key gluconeogenesis enzyme genes Pck1 (C), G6pc (D) and Fbp (E) in primary liver cells in the NC group and the OE-KLF4 group under basal substrate (Sub) and cAMP stimulation conditions.

[0023] Figure 4 This study demonstrates how KLF4 overexpression promotes the expression of key genes involved in amino acid-driven hepatocyte gluconeogenesis and amino acid metabolism. Specifically, (A) verification of KLF4 overexpression efficiency in mouse primary hepatocytes; (B) glucose concentration (μM) in the culture medium of primary hepatocytes in the NC and OE-KLF4 groups under conditions of only basal medium (Medium), added alanine (Ala), or added glutamine (Gln) as gluconeogenesis substrates; and (CG) relative mRNA expression levels of key amino acid metabolism-related genes Gpt2 (C), Tat (D), Hgd (E), Cbs (F), and Mat1a (G) in primary hepatocytes under conditions of only basal substrate (NC-Sub), added cAMP stimulation (Nc-cAMP), and KLF4 overexpression combined with cAMP stimulation (OE-KLF4-cAMP).

[0024] Figure 5This study demonstrates that knocking down KLF4 weakens cAMP-induced glucose production and expression of key gluconeogenesis enzymes in primary liver cells. Specifically, (A) verification of the KLF4 knockdown efficiency in mouse primary liver cells; (B) detection of the relative mRNA expression levels of key gluconeogenesis enzyme genes Pck1 and G6pc in primary liver cells of the negative control group (NC-cAMP) and the KLF4 knockdown group (si-KLF4-cAMP) under conditions containing only the basal substrate (NC-Sub) and with added cAMP stimulation; and (C) glucose concentration (μM) in the culture medium of primary liver cells in each group under the corresponding basal substrate and cAMP stimulation conditions.

[0025] Figure 6 The diagram shows that KLF4 directly binds to the Glud1 promoter region and positively regulates its transcription and expression. Among them, (A) is a KLF4 transcription factor binding motif (containing the MA0039.1 and MA0039.2 sequence patterns) predicted based on the database; (B) is a schematic diagram of the mouse Glud1 gene promoter region (-2000 to transcription start site), with yellow circles indicating the predicted potential KLF4 binding sites; (C) is a dual-luciferase reporter gene assay to detect Glud1 promoter activity; (DE) is the detection of the relative mRNA expression level of the target gene Glud1 after overexpression of KLF4 (D) and knockdown of KLF4 (E) in mouse primary liver cells; (F) is a Western blot analysis of the effects of KLF4 overexpression (top) and knockdown (bottom) on the expression level of glutamate dehydrogenase (GDH) protein encoded by Glud1.

[0026] Figure 7 The study showed that liver-specific overexpression of KLF4 led to yellowing of gross tissues and atrophy of adipose tissue in mice.

[0027] Figure 8 The study showed that liver-specific overexpression of KLF4 induced liver tissue necrosis and inflammatory cell infiltration in mice.

[0028] Figure 9 The results showed that persistent liver-specific overexpression of KLF4 exacerbated liver tissue damage and inflammatory response.

[0029] Figure 10 The study showed that liver-specific overexpression of KLF4 promotes macrophage infiltration in liver tissue.

[0030] Figure 11The study showed that KLF4 overexpression promotes lipid accumulation in primary hepatocytes and upregulates CD36 protein expression. Among them, (A) verification of KLF4 overexpression efficiency in primary hepatocytes; (BC) effect of KLF4 overexpression on the expression level of lipid transporter CD36; (DE) detection of lipid content in primary hepatocytes. Detailed Implementation

[0031] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0032] Example 1

[0033] KLF4 expression characteristics in physiological starvation and metabolic disease models:

[0034] This embodiment aims to verify the expression changes of the target KLF4 under abnormal glucose and lipid metabolism conditions. Mice were fasted for 8 h, 16 h, and 24 h under physiological conditions, and liver tissue was extracted for analysis. The results showed that physiological starvation significantly promoted the expression of Klf4 mRNA and protein in mouse liver. Figure 2 Under pathological conditions, obese and diabetic mouse models were used for testing. The results confirmed that, compared to the control group, the mRNA and protein expression levels of KLF4 in the liver of obese and diabetic mice also showed a significantly increased trend. Figure 1 This indicates that abnormally high expression of KLF4 is closely related to the pathological process of glucose and lipid metabolism disorders.

[0035] Example 2

[0036] In vitro validation of KLF4 regulation of gluconeogenesis in primary liver cells:

[0037] This embodiment uses an in vitro mouse primary liver cell model to verify the direct regulatory effect of KLF4 on hepatic gluconeogenesis.

[0038] In a constructed primary hepatic cell gluconeogenesis model, gluconeogenesis pathways were stimulated by adding cAMP or specific amino acids. Experimental results showed that overexpression of KLF4 (OE-KLF4) further promoted cAMP-stimulated glucose production in hepatocytes and the expression of key gluconeogenesis enzymes. Figure 3 Simultaneously, overexpression of KLF4 also significantly promoted the expression of genes involved in gluconeogenesis and its metabolism, driven by amino acids such as alanine, methionine, and aromatic amino acids. Figure 4Conversely, knocking down KLF4 specifically using small interfering RNA in primary hepatocytes (Si-KLF4) effectively reduced cAMP-stimulated glucose production and key enzyme gene expression, while also weakening amino acid-driven gluconeogenesis. Figure 5 ).

[0039] Example 3

[0040] The molecular mechanism by which KLF4 binds to and regulates the downstream metabolic target gene GLUD1:

[0041] This embodiment reveals a core downstream pathway by which KLF4 promotes gluconeogenesis. Bioinformatics combined with motif prediction revealed a KLF4 binding site in the Glud1 promoter region. Reporter gene experiments further confirmed that KLF4 overexpression directly promotes the transcriptional activity of the Glud1 promoter. At the cellular level, overexpression and knockdown of KLF4 respectively increased or decreased Glud1 expression. Furthermore, in the livers of obese and diabetic mice, KLF4 and Glud1 expression showed a significant positive correlation (…). Figure 6 ).

[0042] Example 4

[0043] AAV-mediated liver-specific overexpression of KLF4 induces lipid accumulation and inflammation:

[0044] This embodiment validated the promoting effect of KLF4 overexpression on hepatic lipid uptake and liver injury at the in vivo level. Liver-specific overexpression was achieved by intravenous injection of AAV-TBG-KLF4 into mice (dose of 2E11 and 5E11). Gross observation showed that 3 weeks after viral injection, the livers of mice in the 5E11 dose group showed significant yellowing, indicating lipid accumulation. Figure 7 The tests revealed that the levels of total cholesterol (TC) and triglycerides (TG) in the liver of the OE-KLF4 group (5E11) were higher than those in the control group. At the molecular level, both in vitro primary liver cells and in vivo AAV-injected mice showed that overexpression of KLF4 significantly upregulated the protein expression level of the lipid transporter CD36. Histological examination (H&E staining) revealed multiple focal necrosis and inflammatory cell infiltration in the liver of AAV-OE KLF4 5E11 mice, and immunohistochemical staining for the macrophage marker F4 / 80 showed a significant increase in macrophage infiltration in this group. Figure 8 ).

[0045] Furthermore, to evaluate the effect of sustained KLF4 overexpression on liver tissue damage and inflammatory response, the observation period for AAV-OEKLF4 5E11 mice was extended to 3 weeks after viral injection. H&E staining results showed that, compared with the AAV control group, liver tissue damage and inflammatory cell infiltration were further aggravated in the AAV-OEKLF4 group mice; the mRNA expression levels of liver inflammation-related genes IL-1β and CCL2 were significantly increased. Figure 9 Simultaneously, F4 / 80 immunohistochemical staining showed a significant increase in F4 / 80 positive cells in the liver tissue of the AAV-OE KLF4 group, indicating increased macrophage infiltration. Figure 10 ).

[0046] Furthermore, KLF4 was overexpressed in primary mouse liver cells in vitro, and the lipid transporter CD36 and intracellular lipid content were detected. The results showed that KLF4 overexpression efficiency was significantly increased, CD36 protein expression was upregulated, and intracellular triglyceride and cholesterol levels were significantly increased. Figure 11 The above results indicate that KLF4 overexpression can promote lipid accumulation in hepatocytes and exacerbate liver tissue damage by enhancing inflammatory responses and macrophage infiltration.

[0047] Example 5

[0048] Nucleic acid drug delivery and administration strategies targeting KLF4 inhibition:

[0049] Based on the above findings, this invention proposes the use of KLF4 inhibitors to prepare therapeutic drugs for metabolic diseases. In a preferred embodiment of this invention, the inhibitor is an siRNA targeting the KLF4 gene. To achieve efficient liver-targeted delivery and metabolic regulation, lipid nanoparticles (LNPs) are used as the delivery carrier for this siRNA. In the intervention protocol for a mouse model of metabolic disorders, the preferred dosing strategy for LNP-siRNA is set at twice a week (i.e., once every 3 days), for a total of 8 consecutive doses. This dosing frequency and cycle design can effectively maintain the continuous and stable knockdown of the KLF4 gene in liver tissue in vivo, thereby maximally blocking GLUD1-mediated abnormal gluconeogenesis and CD36-mediated lipid accumulation and necrosis inflammation, achieving excellent preclinical therapeutic effects.

Claims

1. Application of KLF4 in the preparation of drugs for the prevention or treatment of glucose and lipid metabolism diseases.

2. The application according to claim 1, characterized in that, Inhibitors are used to specifically downregulate or inhibit the expression and transcriptional activity of the KLF4 gene or protein in the liver.

3. The application according to claim 1, characterized in that, The glucose and lipid metabolism disorders mentioned are one or more of type 2 diabetes, hyperglycemia, or non-alcoholic fatty liver disease.

4. The application according to claim 1, characterized in that, The mechanism by which KLF4 regulates glucose and lipid metabolism is as follows: KLF4 binds to the promoter region of the metabolic enzyme GLUD1 and regulates its transcriptional expression.

5. The application according to claim 1, characterized in that, The mechanism by which KLF4 induces fatty liver disease is as follows: KLF4 participates in hepatocyte lipid uptake and inflammation by upregulating the expression of CD36 protein.

6. A pharmaceutical composition for the prevention and / or treatment of glucose and lipid metabolism disorders, characterized in that, The pharmaceutical composition comprises an effective dose of a KLF4 inhibitor and pharmaceutically acceptable excipients.

7. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutically acceptable excipients include at least one of the following: diluents, binders, surfactants, humectants, adsorbents, lubricants, fillers, and disintegrants.

8. The pharmaceutical composition according to claim 6, characterized in that, The dosage forms of the pharmaceutical composition include tablets, capsules, granules, oral liquids, injections, lyophilized powder injections, lipid nanoparticle formulations, sustained-release formulations, or controlled-release formulations.

Images