Use of ITGA3 preparation in the preparation of a drug for preventing or treating liver metabolic abnormalities associated with type 2 diabetes

Abstract

The application discloses use of an ITGA3 preparation in preparation of a medicine for preventing or treating liver metabolic abnormalities related to type 2 diabetes. The application is based on a key role of ITGA3 in maintaining insulin signal in a T2DM liver and stabilizing sugar and lipid metabolism, and proposes a preparation capable of up-regulating ITGA3 expression or enhancing signal transduction mediated by the preparation, for preparation of a medicine for preventing and / or treating T2DM, insulin resistance, and liver sugar and lipid metabolism disorders or non-alcoholic fatty liver disease related thereto, thereby providing a new target and technical path for drug replacement or enhancement of a sports effect.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and drug development, specifically to the use of ITGA3 formulations in the preparation of drugs for the prevention or treatment of liver metabolic disorders associated with type 2 diabetes. Background Technology

[0002] Type 2 diabetes mellitus (T2DM) is a metabolic disease characterized by insulin resistance and relative insufficiency of pancreatic β-cell function, often accompanied clinically by hepatic glucose and lipid metabolism disorders and non-alcoholic fatty liver disease (NAFLD). In the context of T2DM, impaired hepatic insulin signaling can further promote glucose output and ectopic lipid deposition, forming a mutually reinforcing pathological cycle of insulin resistance and fatty liver. Current clinical interventions primarily focus on lifestyle management and hypoglycemic drugs. Aerobic exercise is considered to improve hepatic insulin sensitivity and reduce lipid accumulation; however, its key molecular targets and the mechanisms underlying individual differences in benefit are not fully understood, and there is a lack of precise targets that can be used to replace drugs or amplify the effects of exercise.

[0003] Integrins are transmembrane α / β heterodimer receptors that mediate cell-extracellular matrix interactions and regulate pathways such as PI3K-AKT, AMPK, and FAK-Src through bidirectional signal transduction, participating in the maintenance of energy metabolism homeostasis. Previous studies have suggested that some integrin isoforms are associated with insulin sensitivity, but systematic research on the isoform-specific roles of integrins in hepatic metabolic disorders of type 2 diabetes mellitus (T2DM) and their relationship with exercise-induced improvement is still lacking.

[0004] ITGA3 (Integrin α3) is one of the important α subunits of the laminin receptor. Current research mainly focuses on its role in liver development, damage repair, and fibrosis. There are no reports on the expression characteristics and functional role of ITGA3 in obesity or T2DM-related liver insulin resistance and glucose and lipid metabolism disorders. Furthermore, there is no public information on its use as a drug intervention target for T2DM treatment. This is mainly reflected in the following aspects: (1) The metabolic function and pathway association of ITGA3 are still blank: The tissue expression profile, metabolic regulatory function, and causal relationship of ITGA3 in hepatocytes and insulin signal transduction pathway have not been systematically elucidated to date. There is a lack of evidence in this field regarding ITGA3's involvement in T2DM-related liver insulin resistance and glucose and lipid metabolism disorders, and it is also impossible to determine whether it has the feasibility as a "motor effect mimic / substitute" level intervention target, resulting in a lack of clear theoretical basis and directional guidance for the precise drug design targeting this target. (2) Insufficient liver-specific positive models and therapeutic validation: Current technologies lack liver-specific T2DM animal models with ITGA3 overexpression or functional activation and corresponding control validation systems. Therefore, it is difficult to directly confirm whether "enhancing ITGA3 signaling" can replicate or amplify the metabolic benefits of aerobic exercise, nor can its reversal effect on diabetic fatty liver and its sustained effect on long-term improvement of insulin sensitivity be determined. The lack of the above-mentioned models and validation methods makes the drug development evaluation and clinical translation of ITGA3-targeted intervention lack key empirical support. (3) Existing intervention methods have limited adherence and lack alternative / enhancing drug options: Although exercise therapy can improve metabolism, its efficacy is limited by long-term adherence and the limited exercise population. Existing hypoglycemic / metabolic drugs are also difficult to replace or amplify the effect of exercise in a mechanistic way. Therefore, it is urgent to develop multi-modal agents (such as gene delivery, protein / peptide, agonist antibodies, small molecule expression promoters and post-translational modification enhancers, etc.) that can upregulate or enhance ITGA3 function, so as to break through the treatment bottleneck of non-adherence and exercise contraindications, and form a more accessible, personalized and precise T2DM and related fatty liver intervention strategy.

[0005] Therefore, clarifying the role of ITGA3 in T2DM liver lesions and its feasibility as a therapeutic target is of great significance. Summary of the Invention

[0006] The purpose of this invention is to provide the use of ITGA3 formulations in the preparation of medicaments for the prevention or treatment of liver metabolic disorders associated with type 2 diabetes.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] This invention first provides the use of an ITGA3 formulation in the preparation of a medicament for the prevention or treatment of liver metabolic disorders associated with type 2 diabetes. The ITGA3 formulation is one capable of upregulating ITGA3 expression or enhancing ITGA3-mediated signal transduction.

[0009] Further, the ITGA3 formulation is at least one of the following: the ITGA3 gene, the ITGA3-encoded protein, a splice variant of ITGA3, an ITGA3 fusion protein, a functional polypeptide containing ITGA3, an ITGA3 overexpression vector, an agonist antibody or antibody fragment of ITGA3, a small molecule compound capable of activating ITGA3, a polypeptide that mimics the extracellular matrix (ECM) to enhance ITGA3 activity, or a promoter capable of promoting post-translational modification of ITGA3 to enhance its activity.

[0010] Furthermore, the aforementioned type 2 diabetes-related liver metabolic abnormalities include one or more of hepatic insulin resistance, hepatic glucose and lipid metabolism disorders, and non-alcoholic fatty liver disease.

[0011] The present invention also provides a pharmaceutical composition for the prevention or treatment of type 2 diabetes-related liver metabolic disorders, comprising a pharmaceutically effective dose of an agent capable of upregulating ITGA3 expression or enhancing ITGA3-mediated signal transduction, and a pharmaceutically acceptable carrier, diluent or excipient.

[0012] Further, the formulation is at least one of the following: the ITGA3 gene, the ITGA3-encoded protein, a splice variant of ITGA3, a fusion protein of ITGA3, a functional polypeptide containing ITGA3, an ITGA3 overexpression vector, an agonist antibody or antibody fragment of ITGA3, a small molecule compound capable of activating ITGA3, a polypeptide that mimics the extracellular matrix (ECM) to enhance ITGA3 activity, or a promoter capable of promoting post-translational modifications of ITGA3 to enhance its activity.

[0013] Furthermore, the dosage form of the pharmaceutical composition is an injection, an oral preparation, or a transdermal preparation.

[0014] To address the lack of key molecular targets in existing technologies that can be used to simulate or amplify the metabolic benefits of exercise, and the difficulty in effectively intervening in hepatic insulin resistance and steatosis associated with type 2 diabetes mellitus (T2DM), this invention provides a new pharmaceutical use for formulations targeting ITGA3. Specifically, based on the crucial role of ITGA3 in maintaining hepatic insulin signaling and glucose and lipid metabolism homeostasis in T2DM, this invention proposes formulations that can upregulate ITGA3 expression or enhance its mediated signal transduction. These formulations can be used to prepare drugs for the prevention and / or treatment of T2DM, insulin resistance, and related hepatic glucose and lipid metabolism disorders or non-alcoholic fatty liver disease, thereby providing new targets and technical pathways for the pharmacological substitution or enhancement of exercise effects.

[0015] Compared with the prior art, the present invention has at least the following beneficial effects:

[0016] (1) Establishing the key regulatory role of ITGA3 in hepatic insulin signaling and glucose metabolism homeostasis: Through experiments on a T2DM animal model with hepatocyte-specific ITGA3 knockdown, it was found that the absence of ITGA3 can significantly reduce the phosphorylation levels of key nodes in the hepatic insulin signaling pathway, such as PI3K, AKT and insulin receptor INSR, and lead to a significant deterioration in the results of oral glucose tolerance test (OGTT) and insulin tolerance test (ITT), thus proving that ITGA3 plays an important role in maintaining hepatic insulin sensitivity and glucose homeostasis.

[0017] (2) Revealing the regulatory significance of ITGA3 on hepatic lipid deposition, liver injury and oxidative stress related to T2DM: In the same model, ITGA3 deficiency can significantly aggravate hepatic lipid deposition and histopathological damage, induce serum TG and TC elevation and ALT and AST abnormalities, and significantly amplify the level of reactive oxygen species (ROS) in liver tissue; at the same time, ITGA3 deficiency will significantly weaken the aerobic exercise effect on blood lipids, liver injury and oxidative stress, suggesting that ITGA3 is a key molecular node in the regulation of hepatic glucose and lipid metabolism disorders and oxidative stress in T2DM.

[0018] (3) Provide sufficient evidence for new uses of ITGA3-targeted enhancement / activation agents: From the perspective of functional loss, it is confirmed that there is a clear causal relationship between ITGA3 and T2DM and insulin resistance-related liver pathological phenotypes, indicating that ITGA3 can be used as a potential drug intervention target for the prevention and / or treatment of T2DM, insulin resistance and related fatty liver; accordingly, the use of related agents that can upregulate or enhance ITGA3 function in the preparation of drugs for the above indications has a clear experimental basis and feasibility. Attached Figure Description

[0019] Figure 1 The figures show the body weight (A) and blood glucose (B) of mice in each group after 16 weeks of feeding in Example 1.

[0020] Figure 2 This serves as a verification of the fluorescence effect of mouse virus using adenovirus aav8 to knock down the ITGA3 gene in Example 2.

[0021] Figure 3 The immunohistochemical staining in Example 2 shows the protein localization and expression level of the ITGA3 gene in mouse liver.

[0022] Figure 4 In Example 2, PCR detection (A) and Western blot detection (B and C) were performed using ITGA3 to knock down the expression level of ITGA3 protein in mice.

[0023] Figure 5 The results of OGTT and ITT tests in mice of each group after 16 weeks of high-fat diet feeding in Example 4 are shown.

[0024] Figure 6 In Example 4, Western blot was used to detect the phosphorylation levels of PI3K, AKT, and INSR in the liver tissue of mice in each group after 16 weeks of high-fat diet feeding.

[0025] Figure 7 The images show the liver vacuoles (A) and lipid deposition (B) of mice in each group after 16 weeks of high-fat diet feeding in Example 5.

[0026] Figure 8 The values ​​represent the serum TC (A) and TG (B) levels of mice in each group after 16 weeks of high-fat diet feeding in Example 1.

[0027] Figure 9 The values ​​represent the serum ALT and AST levels of mice in each group after 16 weeks of high-fat diet feeding in Example 1.

[0028] Figure 10 The ROS content of mice in each group after 16 weeks of high-fat diet feeding in Example 1 is shown. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0030] In this example, SPF-grade C57BL / 6J mice were purchased from Shanghai Slack Laboratory Animal Co., Ltd., serial number 20220004058123.

[0031] Example 1: Grouping and Construction of a T2DM Mouse Model

[0032] Five-week-old C57BL / 6J male mice were acclimatized for one week and then randomly divided into five groups (n=8 per group): control group, model group, model exercise group, knockdown model group, and knockdown exercise model group. The specific grouping schemes are as follows: (1) Control group: wild-type mice, fed with normal diet throughout the entire process; (2) Model group: wild-type mice, fed with high-fat diet with 60% fat energy throughout the entire process; (3) Model exercise group: wild-type mice, fed with high-fat diet throughout the entire process, and regular exercise intervention was carried out from the 9th week of high-fat diet (see Example 3 for the scheme); (4) Knockdown model group: fed with high-fat diet throughout the entire process, and ITGA3 specific knockdown was carried out after 8 weeks of feeding (see Example 2 for the construction method); (5) Knockdown exercise model group: fed with high-fat diet throughout the entire process, and ITGA3 specific knockdown mice were constructed after 8 weeks of feeding, and regular exercise intervention was carried out in the same way as the model exercise group from the 9th week.

[0033] Except for the control group, all other groups were fed a high-fat diet for 8 consecutive weeks to induce a type 2 diabetes model. During the modeling period, the body weight and fasting blood glucose levels of mice in each group were monitored weekly to assess the establishment of the model.

[0034] like Figure 1 As shown, to assess the successful establishment of the T2DM model, this study systematically monitored changes in body weight and fasting blood glucose in mice during a high-fat diet. The results showed that, compared to the control group, the model group mice exhibited significantly higher body weight and fasting blood glucose at the end of the experiment, indicating that a high-fat diet (HFD) successfully induced obesity and a persistent hyperglycemic state, preliminarily confirming the establishment of the T2DM model.

[0035] To further assess glucose metabolism function, an oral glucose tolerance test (OGTT) and an insulin tolerance test (ITT) were performed after feeding. Figure 5 As shown, compared with the control group, the model group, knockdown model group, model exercise group, and knockdown exercise model group all exhibited typical T2DM metabolic characteristics. In the OGTT experiment, the area under the blood glucose curve (AUC) of each group increased, the blood glucose peak increased, and the recovery was delayed, indicating impaired glucose tolerance. In the ITT experiment, the rate of blood glucose decline in each group was significantly slowed, indicating insulin resistance. These results indicate that T2DM models were successfully established in all high-fat diet feeding groups in this study.

[0036] Example 2: Liver-specific ITGA3 knockout model

[0037] 1. Construct an AAV8 vector containing an ITGA3-specific shRNA sequence and add a liver-specific TBG promoter to ensure that ITGA3 knockdown only occurs in hepatocytes;

[0038] 2. The vector was transfected into 293T cells for virus packaging, the viral supernatant was collected, and the virus titer was determined by ultracentrifugation.

[0039] 3. The purified AAV8-shRNA-ITGA3 virus was injected into T2DM model mice via tail vein injection (dose: 1.5 × 10⁻⁶). 11 vg / each);

[0040] 4. Mouse liver tissue was collected, and the expression levels of ITGA3 mRNA and protein were detected by immunohistochemistry, qPCR and Western blot to verify its specific knockdown efficiency in hepatocytes.

[0041] like Figure 2 As shown, fluorescence signal detection confirmed that the virus successfully infected liver cells, indicating that the delivery system was effective. Figure 3Immunohistochemical results showed that, compared with the control group, the positive expression signal of ITGA3 in the liver of model group mice was weakened, while the signal was enhanced in the model exercise group; the positive signal of ITGA3 in the knockdown model group was significantly lower than that in the model group, indicating that both high-fat diet and gene intervention affected the expression of ITGA3 in the liver. Further verification of the expression level of ITGA3 in liver tissue was performed using real-time quantitative PCR (qPCR) and Western blot (WB), and the results are as follows: Figure 4 As shown, the expression of ITGA3 was significantly downregulated at both the gene and protein levels in the knockdown model group, confirming that ITGA3 was effectively knocked down in this group. These results collectively validate, from different perspectives, the changes in ITGA3 expression in the liver and its association with the intervention.

[0042] Example 3 Construction of the motion model

[0043] Treadmill exercise was used as an aerobic exercise intervention, with a specific program divided into two phases: an adaptation period (6 days) and a training period (8 weeks). Adaptation period: Exercise speeds were successively increased to 5 m / min, 8 m / min, and 10 m / min, with each speed trained for 2 days; exercise time was 15 minutes / day on days 1-2, 20 minutes / day on days 3-4, and 30 minutes / day on days 5-6; the treadmill incline was 0°. Training period: Exercise speed was 10 m / min, exercise time was 60 minutes / day; exercise frequency was 5 days / week; the treadmill incline was 0°.

[0044] Example 4: ITGA3 gene knockdown reduces insulin signaling pathway activation and exacerbates insulin resistance.

[0045] 1. Glucose tolerance tests were conducted on mice in each group. Mice were first starved overnight (12 hours), and blood glucose levels were measured by collecting blood from the tail tip. Mice were then orally administered a glucose solution (2 g / kg body weight of 20% glucose solution prepared with physiological saline). Blood samples were collected periodically to measure blood glucose levels at 0, 15, 30, 60, 90, and 120 minutes. As shown in Figure 5, the blood glucose levels of the ITGA3 gene knockdown mice were significantly higher than those of the other four groups.

[0046] 2. Insulin tolerance tests were conducted on mice in each group. Mice were fasted for 4 hours, and blood glucose levels were measured by collecting blood from the tail tip. Insulin (0.75 mU / g body weight) was injected intraperitoneally. Blood samples were collected periodically to measure blood glucose levels at 0, 15, 30, 60, 90, and 120 minutes. As shown in Figure 5, the blood glucose levels of ITGA3 knockdown mice were significantly higher than those of the other four groups.

[0047] 3. After starving the mice in each group overnight, liver tissue was taken and the levels of p-PI3K, p-AKT and p-INSR were detected. The results are shown in Figure 6. The levels of ITGA3 knockdown mice were significantly lower than those of the other four groups of mice.

[0048] Example 5: ITGA3 gene knockdown exacerbates lipid deposition and liver damage

[0049] 1. The extracted liver tissue was fixed in 4% paraformaldehyde, embedded in paraffin, and serially sectioned to a thickness of 5 μm. The sections were dewaxed twice with xylene, hydrated with gradients of anhydrous ethanol, washed with tap water, and stained with hematoxylin and eosin (HE). Finally, the sections were dehydrated, mounted with neutral resin, and observed under a 200x light microscope for pathological changes. As shown in Figure 7(A), the ITGA3 gene knockdown mice had significantly more fat cavities than the other four groups.

[0050] 2. Fresh liver tissue was frozen and fixed into serial sections with a thickness of 10 μm. After rewarming and drying, the sections were fixed in 4% neutral formaldehyde fixative for 15 min, washed with tap water, and air-dried. The sections were then immersed in Oil Red O staining solution for 8–10 min (covered to protect from light). After removing the sections, they were left to stand for 3 s, and then immersed sequentially in two tanks of 60% isopropanol for differentiation, for 3 s and 5 s respectively. The sections were then immersed sequentially in two tanks of pure water for 10 s each. After that, hematoxylin staining was performed, and the sections were mounted with glycerol-gelatin mounting medium. The changes in liver lipids were observed under a 200x light microscope. After image acquisition, the area of ​​Oil Red O stained positive regions was analyzed and statistically analyzed using ImageJ software. Figure 7 As shown in (B), the number of lipid droplets in the ITGA3 gene knockdown mice was significantly higher than in the other four groups.

[0051] 3. Serum total cholesterol (TC) and triglycerides (TG) were measured according to the instructions of the Ellanet Biochemical Kit. As shown in Figure 8, after 8 weeks of high-fat induction, TG and TC in the model group mice were significantly higher than those in the control group, and simple aerobic exercise significantly reversed this trend. Hepatocyte-specific ITGA3 knockdown further aggravated lipid abnormalities; even with the same treadmill training, the knockdown exercise group showed some reduction but was still significantly higher than the exercise group alone.

[0052] 4. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected according to the instructions of the Ellanet Biochemical Kit. As shown in Figure 9, after 8 weeks of high-fat induction, the serum AST and ALT activities of the model group mice were significantly increased compared with the control group; simple 8 weeks of aerobic exercise could significantly inhibit the upward trend of transaminase. Hepatocyte-specific ITGA3 knockdown further aggravated liver damage. Even with the same treadmill training, although AST and ALT in the knockdown exercise group decreased, they were still significantly higher than those in the exercise-only group, indicating that ITGA3 deficiency weakened the protective effect of exercise on liver function.

[0053] Example 6: ITGA3 gene knockdown amplifies oxidative stress

[0054] The BB-460522 tissue reactive oxygen species (ROS) detection kit was used. 50 mg of liver tissue was weighed and mixed with 500 μL of pre-chilled homogenization buffer A. The tissue was homogenized on ice (6000 rpm, 30 s × 3), centrifuged at 100 × g at 4℃ for 5 min, and the supernatant was collected. 190 μL of the supernatant and 10 μL of 10 × 008 probe were added to each well of a 96-well black plate, and incubated at 37℃ in the dark for 30 min. Detection was performed using a fluorescence microplate reader: Ex / Em = 516 / 606 nm. Results are expressed as RFU·mg. -1 Protein representation. BCA-based protein concentrations were determined by taking 30 μL of supernatant in parallel. As shown in Figure 10, the ROS level in liver tissue increased to 1.9 times that of the control group in the high-fat-induced T2DM model group (P<0.001). Eight weeks of aerobic exercise alone significantly inhibited ROS production, with the fluorescence value in the exercise model group decreasing by 44% compared to the model group. However, hepatocyte-specific ITGA3 knockdown further increased ROS levels, approximately 2.3 times that of the control group. In conclusion, ITGA3 is a key node in maintaining hepatic redox homeostasis, and its expression level directly determines the ROS clearance effect of exercise on T2DM.

Claims

1. Use of ITGA3 formulations in the preparation of medicaments for the prevention or treatment of liver metabolic disorders associated with type 2 diabetes.

2. The use according to claim 1, characterized in that, The aforementioned ITGA3 formulation is one that can upregulate ITGA3 expression or enhance ITGA3-mediated signal transduction.

3. The use according to claim 1, characterized in that, The ITGA3 formulation is at least one of the following: the ITGA3 gene, the ITGA3-encoded protein, a splice variant of ITGA3, an ITGA3 fusion protein, a functional peptide containing ITGA3, an ITGA3 overexpression vector, an agonist antibody or antibody fragment of ITGA3, a small molecule compound capable of activating ITGA3, a peptide that mimics ECM to enhance ITGA3 activity, or a promoter capable of promoting post-translational modification of ITGA3 to enhance its activity.

4. The use according to claim 1, characterized in that, The aforementioned type 2 diabetes-related liver metabolic abnormalities include one or more of the following: hepatic insulin resistance, hepatic glucose and lipid metabolism disorders, and non-alcoholic fatty liver disease.

5. A pharmaceutical composition for the prevention or treatment of liver metabolic disorders associated with type 2 diabetes, characterized in that, It includes pharmaceutically effective doses of formulations that upregulate ITGA3 expression or enhance ITGA3-mediated signal transduction, as well as pharmaceutically acceptable carriers, diluents, or excipients.

6. A pharmaceutical composition according to claim 5 for the prevention or treatment of liver metabolic disorders associated with type 2 diabetes, characterized in that, The dosage form of the pharmaceutical composition is an injection, an oral preparation, or a transdermal preparation.

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