Application of secretory protein AGR2 in improvement of MASLD mouse liver fatty degeneration

Abstract

The invention discloses application of secretory protein AGR2 in improvement of liver fatty degeneration of MASLD mice, and relates to the technical field of biological pharmacy, and the key points of the technical scheme are as follows: expression down-regulation and secretion reduction of AGR2 in MASLD related models are found by constructing MASLD disease mouse models including high fat diet (HFD) induced obese mice and genetic ob / ob obese mouse models. Animal level experiments show that after the AGR2 recombinant protein treats MASLD mice for two weeks, the mice are placed in a metabolism cage to monitor the metabolic level change of the mice, and it is found that the energy consumption level of the mice treated by the AGR2 is remarkably increased. Liver tissue is subjected to lipid content determination, the content of triglyceride, cholesterol and free fatty acid NEFA is remarkably reduced, and it is indicated that by means of AGR2 recombinant protein injection, the liver lipid content can be remarkably reduced, and liver fatty degeneration is relieved. The protein / polypeptide medicine capable of remarkably improving liver fatty degeneration is found, and a new thought and a new strategy are expected to be provided for diagnosis and treatment of MASLD related diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and more particularly to application of secretory protein AGR2 in improving hepatic fatty degeneration in MASLD mice. Background Art

[0002] Anterior gradient protein 2 (AGR2) is a protein disulfide isomerase (PDI) composed of 175 amino acids and approximately 19 kDa in size. AGR2 primarily comprises three classical domains: a signal peptide sequence (amino acids 1-20), a thioredoxin domain (CPHS, amino acids 81-84), and an endoplasmic reticulum retention sequence (KTEL, amino acids 172-175). The signal peptide sequence directs AGR2 to the endoplasmic reticulum (ER); the thioredoxin domain confers its oxidoreductase function, catalyzing the oxidation, reduction, and isomerization of substrate disulfide bonds, maintaining ER homeostasis; and the ER retention sequence regulates its intracellular trafficking. AGR2 is widely expressed in gastric, intestinal, mammary, lung, and tracheal tissues. Based on its domain structure, AGR2 exists in two main forms: AGR2 localized in the ER and secreted extracellularly. Under physiological conditions, AGR2, located in the endoplasmic reticulum, is closely associated with glandular secretion, influencing the secretion of mucus proteins in the airways and intestines. Mouse models have shown that transgenic mice lacking the AGR2 gene are more susceptible to experimental colorectal inflammation induced by dextran sulfate sodium (DSS). Currently, much research on AGR2 has focused on the field of cancer. Studies have shown that AGR2 is highly expressed in various tumor tissues, such as breast cancer, pancreatic cancer, and colorectal cancer, and is associated with poor patient prognosis.

[0003] In recent years, a growing number of studies have focused on secretory AGR2. Secretory AGR2 can be detected in urine, serum, and other body fluid specimens from cancer patients, and serum AGR2 can also be used as a tumor diagnostic marker. Secretory AGR2 also plays an important role in tumor proliferation and metastasis. For example, secretory AGR2 can affect angiogenesis and fibroblast function, promoting tumor progression. In pancreatic ductal carcinoma, secretory AGR2 promotes tumor cell growth and migration by binding to the functional receptor C4.4A. However, research on AGR2 in metabolic diseases is very scarce, especially the role of secretory AGR2 in metabolic diseases has not been reported. Therefore, it is urgent to explore the role and molecular mechanisms of AGR2 in the progression of metabolic diseases such as metabolic dysfunction-associated fatty liver disease (MASLD) to provide new ideas and strategies for the diagnosis and treatment of MASLD-related diseases. Summary of the Invention

[0004] The purpose of the present invention is to provide the use of secretory protein AGR2 in improving liver fatty degeneration in MASLD mice, which can provide new ideas and strategies for the diagnosis and treatment of MASLD-related diseases.

[0005] The above technical objectives of the present invention are achieved through the following technical solutions: a composition for improving fatty degeneration of the liver in MASLD mice, comprising secretory protein AGR2 and pharmaceutically acceptable excipients.

[0006] The present invention is further provided that: the pharmaceutically acceptable excipient is selected from at least one of pharmaceutically acceptable solvents, solubilizers, cosolvents, emulsifiers, osmotic pressure regulators, stabilizers, suspending agents, coating materials, anti-adhesives, integrators, penetration enhancers, pH regulators, buffers, surfactants, absorbents, diluents, filter aids, and sustained-release materials.

[0007] The present invention further provides the use of secretory protein AGR2 in improving hepatic fatty degeneration in MASLD mice.

[0008] The present invention is further provided as follows: the secretory protein AGR2 can be prepared into a therapeutic preparation for metabolic dysfunction-related fatty liver disease, and the therapeutic preparation for metabolic dysfunction-related fatty liver disease comprises the secretory protein AGR2 and a pharmaceutically acceptable excipient, wherein the pharmaceutically acceptable excipient is selected from at least one of a pharmaceutically acceptable solvent, a solubilizer, a cosolvent, an emulsifier, an osmotic pressure regulator, a stabilizer, a suspending agent, a coating material, an anti-adhesive, an integrator, a penetration enhancer, a pH regulator, a buffer, a surfactant, an absorbent, a diluent, a filter aid, and a sustained-release material.

[0009] The present invention is further configured as follows: the secretory protein AGR2 improves the fatty degeneration of the liver of MASLD mice by regulating the content of triglycerides, cholesterol and free fatty acids NEFA in liver lipids.

[0010] The present invention is further configured as follows: the drug is a drug that can reduce liver lipid content and alleviate liver fatty degeneration.

[0011] In summary, the present invention has the following beneficial effects:

[0012] The present invention constructs MASLD disease mouse models, including obese mice induced by a high-fat diet (HFD) and a genetic ob / ob obese mouse model, and finds that AGR2 expression is downregulated and secretion is reduced in MASLD-related models. Animal level experiments show that after two weeks of treatment of MASLD mice with AGR2 recombinant protein, the mice were placed in metabolic cages to monitor changes in their metabolic levels, and it was found that their energy consumption levels increased significantly after AGR2 treatment. Lipid content in liver tissue was measured, and it was found that the levels of triglycerides, cholesterol, and free fatty acids (NEFA) were significantly reduced, indicating that injection of AGR2 recombinant protein can significantly reduce the lipid content in the liver and alleviate liver fatty degeneration. The present invention has discovered a protein / peptide drug that can significantly improve liver fatty degeneration, which is expected to provide new ideas and strategies for the diagnosis and treatment of MASLD-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 AGR2 expression is downregulated and secretion is reduced in MASLD-related models in the present invention. (A) Changes in Agr2 expression in the intestines of obese patients; (B-C) Western Blot analysis of Agr2 levels in the plasma of HFD and ob / ob obese mice;

[0014] Figure 2 AGR2 improves hepatic lipid accumulation and insulin resistance in HFD mice according to the present invention. (A) Body weight; (B) Food intake; (C) Liver HE and Oil Red O staining (scale bar: 50 μm); (E) Liver lipid content; (F) Metabolic cage analysis of energy expenditure in mice two weeks after PBS and rAGR2 injection.

[0015] Figure 3 AGR2 alleviates hepatic lipid accumulation in ob / ob mice, as shown in this example. (A) Body weight; (B) Food intake; (C) Liver HE and Oil Red O staining (scale: 50 μm); (D) Liver triglyceride, cholesterol, and NEFA levels; (E) Metabolic cage analysis of energy expenditure. DETAILED DESCRIPTION

[0016] The following is combined with Figure 1-3 The present invention is described in further detail.

[0017] Example: Expression and purification of AGR2 recombinant protein

[0018] The recombinant protein expression strain used in this experiment is BL21(DE3). DE3 is a lysogenic λDE3 with a copy of T7 RNA polymerase, suitable for expressing non-toxic proteins. The experimental steps are as follows:

[0019] (1) Transformation: Take 10 ng of the plasmid with successful sequencing sequence and transfer it into 50 ul BL21 (DE3) and place it in an ice bath.

[0020] 10min, heat shock at 42℃ for 90s, ice bath for 2min, add 600ul LB medium, recover at 37℃ for 10min, take 200ul liquid and spread on LB plate containing kanamycin resistance, and incubate at 37℃ for about 15hr.

[0021] (2) Shake culture: Pick a single clone and place it in a 5 ml LB culture tube containing kanamycin resistance. Incubate the tube in a shaker at 37°C for about 8 hours. Transfer the tube to 1 L LB liquid culture medium for expansion. Continue incubating the tube at 37°C until the OD600 reaches about 0.6. Add IPTG at a final concentration of 0.5 mM and induce the tube in a shaker at 16°C for about 16 hours.

[0022] (3) Ni column purification:

[0023] 1) Centrifuge at 4,000 rpm for 10 minutes in a Thermo Fisher Scientific floor-standing centrifuge to collect E. coli cells containing the target protein.

[0024] 2) Discard the supernatant, resuspend the cells in buffer (20 mM Tris, 500 mM NaCl, 10 mM Imidazole, pH 8.0), and lyse the E. coli using a high-pressure homogenizer.

[0025] 3) Centrifuge the lysate at 15,000 rpm for 30 min at 4°C in a Thermo Fisher Scientific floor-standing centrifuge to separate the precipitate and supernatant, and collect the supernatant.

[0026] 4) Wash the gravity column containing 5 ml of Ni-NTA medium with ultrapure water, and then equilibrate the medium with 20 ml of buffer (20 mM Tris, 500 mM NaCl, 10 mM Imidazole, pH 8.0);

[0027] 5) The lysate supernatant was repeatedly passed through the Ni-NTA medium by gravity three times;

[0028] 6) Wash the solution with a buffer solution (20 mM Tris, 500 mM NaCl, 30 mM Imidazole, pH 8.0) to remove contaminating proteins. Detect the flow-through with Coomassie Brilliant Blue G-250 until the solution turns blue.

[0029] 7) Elute the target protein with a buffer solution (20 mM Tris, 500 mM NaCl, 250 mM Imidazole, pH 8.0). Detect the flow-through with Coomassie Brilliant Blue G-250 until the color stops turning blue. Keep the target protein eluate on ice.

[0030] 8) Wash the Ni-NTA medium with 1 M imidazole, then wash again with ultrapure water, and finally store the medium in 20% ethanol solution; take 20 μl of samples from each of the above stages and analyze the results (including protein molecular weight, expression level, purity, etc.) by denaturing polyacrylamide gel electrophoresis (12%-SDS-PAGE)

[0031] Mouse energy metabolism assay:

[0032] Oxygen consumption (VO2), carbon dioxide production (VCO2), respiratory exchange ratios (RER), and heat production were measured using a Sable Promethion small animal metabolic measurement and analysis system. During the experiment, the metabolic chamber temperature was maintained at 22°C, with intelligent control ensuring a 12-hour day / night cycle (8:00 am–8:00 pm daytime; 8:00 pm–8:00 am nighttime). Each mouse was housed in an individual metabolic cage with free access to food and water. Metabolic parameters were recorded for 5–6 days, and raw data were analyzed 48 hours later and normalized to body weight. Dynamic curves and bar graphs were plotted. Dynamic curves represent the mean ± standard error of the mean (SEM) for wild-type (WT), knockout (KO), and UBE2O-overexpressing mice at each time point. Bar graphs represent the mean ± SEM for each time point during the day or night.

[0033] The specific monitoring situation is as follows:

[0034] 1. AGR2 expression and secretion are decreased in MASLD-related models

[0035] In order to establish the correlation between AGR2 and MASLD, the expression of AGR2 was detected in the intestinal tissues of MASLD patients. The results showed that AGR2 was decreased in the intestinal tissues of MASLD patients ( Figure 1 In addition, we also tested the secretion of AGR2 in MASLD model mice. In HFD and ob / ob mouse models, the content of AGR2 in serum was significantly reduced ( Figure 1 (B, C).

[0036] 2. Intraperitoneal injection of AGR2 recombinant protein improves hepatic steatosis in HFD mice

[0037] To clarify the regulatory role of AGR2 on animal metabolic levels under pathological conditions, purified recombinant protein was intraperitoneally injected into HFD mice. 8-week-old male C57BL / 6 mice were fed an HFD diet for 8 weeks and then intraperitoneally injected with AGR2 recombinant protein at 1 μg / g once a day for two consecutive weeks. During this period, body weight changes and food intake were monitored. The results showed that there was no significant difference in body weight and food intake between the two groups ( Figure 2 After the mice were sacrificed, their livers were harvested for H&E staining and Oil Red O staining. The results showed that AGR2 treatment significantly improved the degree of liver lipid degeneration in mice ( Figure 2 C, D). The lipid content of liver tissue was measured and it was found that the content of triglyceride, cholesterol and free fatty acid NEFA was significantly reduced ( Figure 2 E), indicating that AGR2 recombinant protein injection into HFD mice significantly reduced liver steatosis. In addition, when mice were placed in metabolic cages for monitoring, it was found that the energy expenditure of mice in the rAGR2 treatment group was significantly increased ( Figure 2 Middle F).

[0038] 3. AGR2 recombinant protein improves insulin resistance and hepatic steatosis in ob / ob mice

[0039] In addition to HFD obese mice, we also used ob / ob mice for verification. After one week of adaptation, 8-week-old male ob / ob mice were intraperitoneally injected with AGR2 recombinant protein at 1 μg / g once a day for two consecutive weeks. During this period, body weight changes and food intake were monitored. The results showed that there was no significant difference in body weight and food intake between the two groups ( Figure 3 After the mice were sacrificed, their livers were harvested for H&E staining and Oil Red O staining. The results showed that AGR2 treatment significantly improved the degree of liver lipid degeneration in mice ( Figure 3 C). Lipid content in liver tissue was measured and it was found that the content of triglycerides, cholesterol and free fatty acid NEFA were significantly reduced ( Figure 3 Middle D) shows that AGR2 recombinant protein injection into HFD mice significantly reduced liver lipid content and alleviated liver steatosis. In addition, the mice were placed in metabolic cages to monitor changes in their metabolic levels, and it was found that their energy consumption levels increased significantly at night after AGR2 treatment ( Figure 3 Middle E).

[0040] This specific embodiment is merely an explanation of the present invention and is not intended to limit the present invention. After reading this specification, those skilled in the art may make non-creative modifications to this embodiment as needed. However, as long as such modifications are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A composition for improving hepatic steatosis in MASLD mice, characterized by: The composition comprises secretory protein AGR2 and pharmaceutically acceptable excipients.

2. A composition for improving hepatic steatosis in MASLD mice according to claim 1, characterized in that: The pharmaceutically acceptable excipient is selected from at least one of pharmaceutically acceptable solvents, solubilizers, cosolvents, emulsifiers, osmotic pressure regulators, stabilizers, suspending agents, coating materials, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, surfactants, absorbents, diluents, filter aids, and sustained-release materials.

3. Application of secretory protein AGR2 in the preparation of drugs to improve liver fatty degeneration in MASLD mice.

4. Use of the secretory protein AGR2 according to claim 3 in the preparation of a drug for improving hepatic steatosis in MASLD mice, characterized in that: The secretory protein AGR2 can be prepared into a therapeutic preparation for metabolic dysfunction-related fatty liver disease, which comprises the secretory protein AGR2 and a pharmaceutically acceptable excipient, wherein the pharmaceutically acceptable excipient is selected from at least one of a pharmaceutically acceptable solvent, a solubilizer, a cosolvent, an emulsifier, an osmotic pressure regulator, a stabilizer, a suspending agent, a coating material, an anti-adhesive, an integrant, a penetration enhancer, a pH regulator, a buffer, a surfactant, an absorbent, a diluent, a filter aid, and a sustained-release material.

5. Use of the secretory protein AGR2 according to claim 3 in the preparation of a drug for improving hepatic steatosis in MASLD mice, characterized in that: The secretory protein AGR2 improves the fatty degeneration of the liver of MASLD mice by regulating the content of triglycerides, cholesterol and free fatty acids NEFA in liver lipids.

6. Use of the secretory protein AGR2 according to claim 3 in the preparation of a drug for improving hepatic steatosis in MASLD mice, characterized in that: The medicine is a medicine that can reduce liver lipid content and alleviate liver fatty degeneration.

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