Application of Reg4 gene in treatment of metabolism-related fatty liver

Abstract

The invention discloses application of a Reg4 gene in treatment of metabolism-related fatty liver, and relates to the technical field of biomedicine. The Reg4 gene is applied to the aspect of treating obesity and metabolism-related fatty liver; according to the application, by increasing expression of the Reg4 gene, liver lipid deposition, liver cell ballooning and inflammatory infiltration related to the metabolism-related fatty liver are inhibited; the application is realized by inhibiting the processes of de novo synthesis of fat in the liver, liver lipid uptake and fatty acid beta oxidation. The step of inhibiting the de novo synthesis of fat in the liver comprises the step of inhibiting the expression of genes of acetyl coenzyme A carboxylase 1, fatty acid synthase and / or stearoyl coenzyme A desaturase 1. The invention definitely proves that the Reg4 gene has an obvious effect of improving obesity and metabolism-related fatty liver, and can relieve hepatic fatty degeneration and inflammation.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of the Reg4 gene in the treatment of metabolic-related fatty liver. Background Technology

[0002] With socioeconomic development and improved living standards, the prevalence of metabolic diseases such as obesity and non-alcoholic fatty liver disease (NAFLD) has risen rapidly, gradually becoming a major public health problem threatening human health worldwide. Obesity, as a major risk factor for many chronic diseases such as cardiovascular disease, stroke, type 2 diabetes mellitus (T2DM), hypertension, and cancer, has a significant impact.

[0003] Regenerating gene family member 4 (Reg4) is a member of the Reg gene family and a subset of the calcium-dependent (C-type) lectin superfamily. The Reg protein family, as multifunctional secretory molecules, possesses nutritional, anti-apoptotic, anti-inflammatory, antimicrobial, and immunomodulatory functions. Currently, increasing evidence suggests that the Reg family plays a crucial role in the development and progression of various gastrointestinal inflammations and inflammation-related diseases. Studies have shown that Reg4 can regulate the efficiency of intestinal absorption of dietary fat. When Reg4 is deficient in the intestine, the AMPK signaling pathway in intestinal epithelial cells is overactivated, leading to upregulation of the expression of lipid transport proteins and triglyceride synthesis-related enzymes, resulting in a significant increase in intestinal fat absorption. Excess fat absorbed by the intestine is transported to the liver, directly promoting hepatic steatosis and fatty degeneration.

[0004] Based on this, Reg4, as a hormone mainly secreted by intestinal endocrine cells, has a significant protective effect on the intestine, but its specific effects on the liver are still unclear, as are its specific effects on liver lipid metabolism. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by proposing the application of the Reg4 gene in the treatment of metabolic-related fatty liver.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: Application of Reg4 gene in the treatment of obesity and metabolic-related fatty liver.

[0007] Preferred: The application involves increasing the expression of the Reg4 gene to inhibit hepatic lipid deposition, hepatocyte ballooning degeneration, and inflammatory infiltration associated with metabolic-related fatty liver.

[0008] Preferably, the application is achieved by inhibiting de novo fat synthesis, hepatic lipid uptake, and fatty acid β-oxidation in the liver.

[0009] Preferably, the inhibition of de novo fat synthesis in the liver includes inhibiting the expression of the genes for acetyl-CoA carboxylase 1, fatty acid synthase, and / or stearoyl-CoA desaturase 1.

[0010] Preferably, the inhibition of hepatic lipid uptake includes inhibiting the expression of lipid transporter and / or differentiation cluster 36 gene.

[0011] Preferably, the regulation of fatty acid β-oxidation includes inhibiting the expression of the medium-chain acyl-CoA dehydrogenase gene.

[0012] Preferably, the inhibition of inflammatory infiltration includes reducing the expression of tumor necrosis factor-α.

[0013] Preferably, the inhibition of inflammatory infiltration also includes reducing the expression of interleukin-1 and / or interleukin-6.

[0014] A drug for the prevention and treatment of metabolic-associated fatty liver includes an effective dose of the Reg4 gene, the protein it encodes, or a vector that can promote the expression of the Reg4 gene.

[0015] Preferably, the vector is a viral vector used to specifically overexpress the Reg4 gene in the liver.

[0016] The beneficial effects of this invention are as follows: 1. This invention clearly demonstrates that the Reg4 gene has a significant effect on improving obesity and metabolic-related fatty liver, and can reduce hepatic steatosis and inflammation.

[0017] 2. This invention discloses that the Reg4 gene has a multi-target mechanism of action, which can simultaneously regulate lipid metabolism and inflammation.

[0018] 3. This invention reveals that the Reg4 gene can simultaneously inhibit dendritic lipogenesis (DNL) and liver inflammation. In regulating lipid metabolism, the Reg4 gene can specifically inhibit key gene clusters in the hepatic dendritic lipogenesis (DNL) pathway (including ACC1, Fasn, and Scd1) and downregulate the expression of fatty acid uptake and storage-related genes (Fatp1, CD36), providing a precise molecular target explanation for its therapeutic effect.

[0019] 4. In terms of inhibiting inflammation, this invention shows that the Reg4 gene can significantly reduce the level of the key pro-inflammatory factor TNF-α mRNA. This indicates that the Reg4 gene can reduce the liver recruitment and activation of inflammatory cells by inhibiting the production of inflammatory factors at their source, thereby breaking the vicious cycle of inflammation in metabolic-related fatty liver.

[0020] 5. This invention provides a novel therapeutic target for obesity and metabolic-related fatty liver. Attached Figure Description

[0021] Figure 1 This is a diagram showing the experimental results of overexpressing the Reg4 gene in AML-12 cells (a normal mouse hepatocyte cell line) in this invention. Figure 2 This is a diagram showing the verification results of liver-specific overexpression of the Reg4 gene in C57 mice in this invention; Figure 3 This is a diagram illustrating the gross effects of liver-specific overexpression of the Reg4 gene on high-fat fed C57 mice in this invention. Figure 4 This figure shows the effect of liver-specific overexpression of the Reg4 gene on the liver of C57 mice fed a high-fat diet, as described in this invention. Detailed Implementation

[0022] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0023] Example 1: Effects of liver-specific overexpression of the Reg4 gene on obesity and metabolic-related fatty liver in high-fat fed male C57 rats I. Laboratory Animals Six-week-old male C57BL / 6J mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were randomly divided into two groups of 10 mice each. After one week of acclimatization feeding, the mice in both groups were injected with the corresponding virus via the tail vein.

[0024] The control group mice were injected with GFP control virus, while the experimental group mice were injected with Reg4 gene overexpression virus. After the injection, the mice were fed a normal diet for 2 weeks. One mouse from each group was taken to verify the protein and mRNA levels of gene overexpression.

[0025] Both groups were fed a high-fat diet (Research Diets, with approximately 60% of energy coming from fat) for 12 consecutive weeks.

[0026] II. Relevant Indicator Testing Mouse weight monitoring: The weight of the mice was monitored weekly, and the growth curve was recorded.

[0027] Mouse food intake test: Before the end of the experiment, each mouse was isolated and its 24-hour food intake was tested individually.

[0028] Mouse liver weight test: At the end of the experiment, the mice were euthanized, the livers were removed, and the liver weight was measured.

[0029] Serum biochemical marker detection: Blood was collected from the fundus venous plexus and allowed to stand for 1 hour to allow for full serum separation. The serum was then centrifuged (3000 rpm, 20 min) and the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides (TG), and total cholesterol (TC) were detected using a kit (Nanjing Jiancheng Bioengineering Institute).

[0030] Liver histological analysis: Mouse liver tissue was immediately fixed with 4% paraformaldehyde, then dehydrated in a gradient manner, embedded in paraffin, and sectioned to a thickness of 5 μm. Oil Red staining and H&E staining were performed.

[0031] Real-time fluorescence PCR (RT-qPCR): Total RNA was extracted from the obtained mouse liver tissue using Trizol reagent (ABclonal Technology, Wuhan, China), and cDNA was synthesized by reverse transcription (ABclonal Technology). The mRNA levels of each gene were detected using the SYBR Green real-time fluorescence quantitative PCR kit (ABclonal Technology). β-actin was used as an internal control, and relative quantification was performed using the 2-ΔΔCT method.

[0032] III. Treatment Results Figure 1 In the diagram: A is a validation diagram of the protein level of Reg4 gene overexpression; B is a validation diagram of the mRNA level of Reg4 gene overexpression; C is the Oil Red staining result of treating control group cells (transformed into GFP empty vector plasmid) and overexpressing cells with FFA (free fatty acids).

[0033] Figure 2 In the diagram: A shows the protein level verification of Reg4 gene overexpression; B shows the mRNA level verification of Reg4 gene overexpression.

[0034] Figure 3 In the table: A compares the body weight of mice with liver-specific Reg4 gene overexpression with that of the control group; B compares the food intake of mice with liver-specific Reg4 gene overexpression with that of the control group; C compares the rectal temperature of mice with liver-specific Reg4 gene overexpression with that of the control group; D compares the serum parameters of mice with liver-specific Reg4 gene overexpression with that of the control group, including TC, TG, ALT, and AST.

[0035] Figure 4In the first two sections: A) Comparison of liver morphology between mice with liver-specific Reg4 gene overexpression and those in the control group; B) Comparison of liver weight between mice with liver-specific Reg4 gene overexpression and those in the control group; C) Comparison of liver pathological morphology between mice with liver-specific Reg4 gene overexpression and those in the control group, including Oil Red staining and H&E staining (hematoxylin & eosin staining); D) Comparison of mRNA levels of liver inflammation-related indicators between mice with liver-specific Reg4 gene overexpression and those in the control group; D) Comparison of mRNA levels of lipid regulation-related indicators between mice with liver-specific Reg4 gene overexpression and those in the control group.

[0036] After liver-specific overexpression of the Reg4 gene, compared with the control group, the experimental group significantly improved obesity induced by high-fat diet, and the mice in the experimental group showed a significant reduction in body weight. Figure 3 A), and there was no significant change in food intake ( Figure 3 B).

[0037] After liver-specific overexpression of the Reg4 gene, serum TC, TG, ALT, and AST levels were significantly decreased compared to the control group. Figure 3 C).

[0038] Liver-specific overexpression of the Reg4 gene resulted in a significant reduction in liver size and weight compared to the control group.

[0039] Hepatic lipid deposition and inflammation were assessed between the two groups using HE staining and Oil Red staining. Liver-specific overexpression of the Reg4 gene significantly reduced lipid deposition, intralobular inflammation, and ballooning degeneration scores in the livers of high-fat fed mice. Figure 4 C), the area of ​​positive oil red staining in the liver was significantly reduced ( Figure 4 C).

[0040] After liver-specific overexpression of the Reg4 gene, compared with the control group, the levels of inflammatory response factors IL-1 and IL-6 in liver tissue were significantly decreased, and the core pro-inflammatory factor tumor necrosis factor TNF-α in metabolic-related fatty liver was also significantly downregulated. Figure 4 (D) indicates that Reg4, after liver-specific overexpression, can significantly inhibit the inflammatory response in the liver, thereby alleviating the experimental environment created by metabolic-related fatty liver.

[0041] After liver-specific overexpression of the Reg4 gene, compared with the control group, the genes related to de novo fat synthesis (Scd-1), fat uptake (CD36), and fatty acid β-oxidation (MCAD) in liver tissue were significantly downregulated. Figure 4 These results suggest that specific overexpression of Reg4 in the liver can regulate lipid metabolism in multiple ways, modulating lipid synthesis and lipid accumulation to reduce lipid accumulation in the liver.

[0042] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. Application of Reg4 gene in the treatment of obesity and metabolic-related fatty liver.

2. The application according to claim 1, characterized in that, The application works by increasing the expression of the Reg4 gene to inhibit hepatic lipid deposition, hepatocyte ballooning degeneration, and inflammatory infiltration associated with metabolic-related fatty liver.

3. The application according to claim 1, characterized in that, The application is achieved by inhibiting de novo fat synthesis, hepatic lipid uptake, and fatty acid β-oxidation in the liver.

4. The application according to claim 3, characterized in that, The inhibition of de novo fat synthesis in the liver includes inhibiting the expression of genes for acetyl-CoA carboxylase 1, fatty acid synthase, and / or stearoyl-CoA desaturase 1.

5. The application according to claim 3, characterized in that, The inhibition of hepatic lipid uptake includes inhibiting the expression of lipid transporters and / or the differentiation cluster 36 gene.

6. The application according to claim 3, characterized in that, The regulation of fatty acid β-oxidation includes inhibiting the expression of the medium-chain acyl-CoA dehydrogenase gene.

7. The application according to claim 2, characterized in that, The inhibition of inflammatory infiltration includes reducing the expression of tumor necrosis factor-α.

8. The application according to claim 7, characterized in that, The inhibition of inflammatory infiltration also includes reducing the expression of interleukin-1 and / or interleukin-6.

9. A drug for the prevention and treatment of metabolic-related fatty liver, characterized in that, It contains an effective dose of the Reg4 gene, the protein it encodes, or a vector that can promote Reg4 gene expression.

10. The medicament according to claim 9, characterized in that, The vector is a viral vector used to specifically overexpress the Reg4 gene in the liver.

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