Application of preparation for regulating and controlling expression level of CSDE1 in preparation of medicine for preventing or treating obesity caused by lipid metabolism disorder

Abstract

The invention relates to the technical field of biological medicine, and provides application of a preparation for regulating and controlling the expression level of CSDE1 in preparation of a medicine for preventing or treating lipid metabolism disorder type obesity. The preparation for regulating and controlling the expression level of the CSDE1 is an overexpression preparation of the CSDE1. Furthermore, the preparation for regulating and controlling the expression level of the CSDE1 is AAV-CSDE1. The invention also provides an application of milrinone or butoconazole in preparation of a medicine for treating obesity caused by lipid metabolism disorder.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of agents that regulate CSDE1 expression levels in the preparation of drugs for the prevention or treatment of lipid metabolism disorder-related obesity. Background Technology

[0002] CSDE1 refers to E1, a protein containing the cold shock domain. It is a protein-coding gene with RNA-binding function and is found in various organisms, including humans and mice. This gene sequence is highly conserved and plays a crucial role in the regulation of gene expression in various life processes, such as hematopoietic stem cell generation and the maintenance of embryonic stem cell pluripotency.

[0003] CSDE1 is involved in post-transcriptional processing of mRNA, including translation and degradation regulation. For example, in zebrafish, the deletion of this gene affects the normal development of hematopoietic stem and progenitor cells; in mice, it can maintain the pluripotency of embryonic stem cells by inhibiting the expression of related genes.

[0004] Obesity is a chronic metabolic disease characterized by abnormal lipid metabolism and accompanied by disordered glucose homeostasis. It is closely related to genetic susceptibility, environment, and lifestyle factors. Its pathological basis mainly lies in the synergistic imbalance in the regulation of lipid and glucose metabolism between the liver and adipose tissue: impaired insulin-mediated gluconeogenesis inhibition in the liver, coupled with persistent hyperglycemia and excessive lipid droplet deposition, leads to steatosis, lipotoxicity, and chronic inflammation; adipose tissue exhibits adipocyte hypertrophy or hyperplasia, an imbalance between lipolysis and re-esterification, accompanied by insulin resistance, decreased glucose uptake, macrophage infiltration, and fibrosis, ultimately resulting in disordered secretion of adipokines and inflammatory factors, further exacerbating the sustained overload of glucose and lipid metabolic flux along the liver-adipose tissue axis. The occurrence of obesity is closely related to the combined effects of multiple factors, including high-fat, high-energy diets, insufficient physical activity, circadian rhythm disorders, genetic susceptibility, and endocrine dysregulation.

[0005] Currently, obesity interventions are still based on lifestyle management (such as controlling energy intake and structured exercise), but long-term adherence is generally insufficient, and weight rebound is a prominent problem. While bariatric surgery can significantly reduce weight and improve hepatic steatosis in the short term, its high cost, postoperative complications, and long-term recurrence risk limit its widespread application. Existing drug treatments mainly work by inhibiting fat absorption, inhibiting lipid synthesis, promoting fat oxidation, or regulating inflammation and endocrine function in adipose tissue (such as pancreatic lipase inhibitors and GLP-1 receptor agonists), but the efficacy of these interventions varies significantly among individuals and is often accompanied by gastrointestinal adverse reactions, limiting their applicability. For example, semaglutide, a typical GLP-1 receptor agonist, primarily reduces weight by suppressing appetite and delaying gastric emptying to decrease energy intake. However, its therapeutic effect is highly dependent on central and gastrointestinal hunger signal regulation, limiting its efficacy in hypometabolic obesity, which is mainly characterized by reduced energy expenditure and inefficient lipid metabolism. Furthermore, these drugs often cause gastrointestinal side effects such as nausea and vomiting in clinical use, affecting long-term adherence, and weight rebound is common after discontinuation. In addition, the weight loss induced by semaglutide is often accompanied by a certain proportion of lean body mass loss, making it difficult to selectively reduce adipose tissue, suggesting significant limitations in its applicability for refined, segmented obesity treatment. The heterogeneity in the efficacy of different obesity treatments stems to some extent from the pathophysiological diversity of obesity itself.

[0006] A classification system based on pathophysiological mechanisms divides obesity into four types: brain hunger, gastrointestinal hunger, emotional hunger, and hypometabolic obesity. These types reflect the dominant roles of central satiety regulation, gastrointestinal hormone signaling, emotional and reward pathways, and differences in basal energy metabolism in the occurrence and maintenance of obesity, respectively. Different phenotypes exhibit fundamental differences in energy intake regulation, metabolic adaptation, and interstitial metabolic flux, making it difficult for intervention strategies targeting a single mechanism to achieve stable efficacy in all obese populations. GLP-1 receptor agonists have shown significant efficacy primarily in obesity phenotypes centered on central satiety regulation and emotional reward (such as brain hunger and gastrointestinal hunger), but have limitations in hypometabolic obesity, which is dominated by energy expenditure restriction.

[0007] Therefore, given the central role of lipid metabolism imbalance and liver-adipose tissue axis dysfunction in various obesity phenotypes, there is an urgent need to develop safer and more precise new treatment strategies based on pathophysiological classification that can restore interstitial lipid homeostasis. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention aims to propose the application of formulations that regulate CSDE1 expression levels in the preparation of drugs for the prevention or treatment of obesity caused by lipid metabolism disorders.

[0009] To achieve the above-mentioned objectives, the technical solution of this invention is as follows:

[0010] This invention provides the application of formulations that regulate CSDE1 expression levels in the preparation of drugs for the prevention or treatment of obesity caused by lipid metabolism disorders.

[0011] Preferably, the formulation that regulates the expression level of CSDE1 is an overexpression formulation of CSDE1.

[0012] Furthermore, the agent that regulates CSDE1 expression level is AAV-CSDE1.

[0013] This invention also provides the use of small molecule compounds that overexpress CSDE1 in the preparation of drugs for treating lipid metabolism disorder-related obesity, wherein the small molecule compounds that overexpress CSDE1 include milrinone or butoconazole.

[0014] Priority is to prepare milrinone or butoconazole into various feasible dosage forms.

[0015] This invention also provides the use of milrinone or butoconazole in the preparation of drugs for the treatment of lipid metabolism disorder-related obesity.

[0016] Compared with the prior art, the beneficial effects of the present invention are:

[0017] 1. This invention provides for the first time the use of CSDE1 overexpression formulations in the preparation of drugs for the prevention and / or treatment of lipid metabolism disorder-related obesity. It demonstrates that increasing CSDE1 expression levels can significantly improve lipid metabolism disorders, enhance energy expenditure and basal metabolic levels, and is particularly suitable for low metabolic obesity.

[0018] 2. The CSDE1 overexpression protocol can achieve precise regulation of pathological fat accumulation in obesity, avoid the risk of muscle loss associated with non-selective weight loss, and has significant technical advantages as a means of preventing or treating obesity and related metabolic abnormalities.

[0019] 3. This invention is the first to discover that milrinone can be used as a drug for treating obesity caused by lipid metabolism disorders.

[0020] 4. This invention is the first to discover that butoconazole can be used as a drug for treating obesity caused by lipid metabolism disorders.

[0021] The detailed structure of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. Attached Figure Description

[0022] Figure 1 Figure showing the results of mouse obesity modeling and model characterization. Figure 1 A: Schematic diagram of the obesity modeling method of 60% high-fat diet (HFD), with the control group being normal diet (CD). Figure 1B: This shows the difference in body size between the CD and HFD groups of mice. Figure 1 C: Weight changes during the modeling process in the CD and HFD groups. Figure 1 D: Morphological differences in various tissues and organs between the CD and HFD groups. **** indicates a statistically significant difference (p<0.0001).

[0023] Figure 2 This is a graph showing the characterization results of serum indicators in a mouse obesity model, where... Figure 2 A represents the triglyceride (TG) results. Figure 2 B is the LDL-C result graph. Figure 2 C represents the results for TC (total cholesterol), HDL-C, and LDL-C (high-density lipoprotein and low-density lipoprotein). Figure 2 D is the GLU result image. Figure 2 E is the LDL-C result graph, where * indicates a significant difference (p<0.05) and ** indicates a significant difference (p<0.01).

[0024] Figure 3 This is a diagram showing the histological characterization results of a mouse obesity model. Figure 3 A represents the H&E results for white fat (eWAT). Figure 3 B represents the H&E results for brown adipose tissue (PAT). Figure 3 This is a quantitative graph of the area of ​​white adipocytes. **** indicates a statistically significant difference (p<0.0001).

[0025] Figure 4 The figure shows the effect of AAV-CSDE1 overexpression on improving body weight in a mouse obesity model. Figure 4 A: Schematic diagram of AAV overexpression CSDE1 method. Figure 4 B: Body size differences in obese mice overexpressing CSDE1 with AAV. Figure 4 C: AAV overexpression of CSDE1 improves body weight in obese mice. Figure 4 D: Morphological differences in various tissues and organs of AAV-overexpressing CSDE1 obese mice. ** indicates statistically significant differences (p<0.01).

[0026] Figure 5 This diagram illustrates the serological improvement effect of AAV overexpression of CSDE1 on an obesity model in mice. Figure 5 A shows the results of aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Figure 5 B is the result of alanine aminotransferase (AST). Figure 5 C represents the triglyceride (TG) results. Figure 5 D is the LDL-C result graph. Figure 5 E is the HDL-C result graph. Figure 5F is the TC (total cholesterol) results graph, where ** indicates a significant difference (p<0.01).

[0027] Figure 6 The effect of AAV-CSDE1 overexpression on liver histology in a mouse obesity model is shown. The top side shows the changes in liver H&E results, and the bottom side shows the changes in Oil Red O histology results.

[0028] Figure 7 The diagram shows the effect of AAV-CSDE1 overexpression on improving adipose tissue in a mouse obesity model. Figure 7 A represents the histological results of white and brown adipose tissue (HE). Figure 7 B is a quantitative graph of the area of ​​white adipocytes. * indicates a statistically significant difference (p<0.05).

[0029] Figure 8 The effects of AAV-CSDE1 overexpression on the hematologic and endothelial (H&E) histological effects on other organs in a mouse obesity model. Figure 8 A stands for Spleen. Figure 8 B stands for lung. Figure 8 C stands for kidney. Figure 8 D stands for Heart.

[0030] Figure 9 The diagram shows the effect of AAV-CSDE1 overexpression on improving glucose metabolism disorder in a mouse obesity model. Figure 9 A: Results of the glucose tolerance test. Figure 9 B: Results of the insulin tolerance test. Figure 9 C: Quantitative results of the area under the insulin tolerance curve (AUC). ** indicates a statistically significant difference (p<0.01).

[0031] Figure 10 AAV-CSDE1 overexpression was shown to alter body composition and improve metabolic levels in mice. Figure 10 A represents the body composition analysis results, where fat mass represents body fat weight and lean mass represents lean body mass. Figure 10 B is the metabolic cage energy expenditure-lean body mass covariate analysis plot (ANCOVA). * indicates a statistically significant difference (p<0.05).

[0032] Figure 11 Figure showing the results of high-throughput screening and protein-level interaction validation of CSDE1-binding drugs using SPR. Figure 11 A: CSDE1 combined with drug SPR high-throughput screening response value ranking results, where red represents the top 50 drugs in the ranking response. Figure 11 B: Quantify the CSDE1 protein levels of the top 50 drugs in response. Figure 11C: Validation and quantification results of the top 10 drugs in response, with red representing the top 2 binding drugs that provide stable CSDE1 protein expression.

[0033] Figure 12 This diagram illustrates the inhibitory effect of CSDE1-binding drugs on lipid droplet formation in primary mouse liver cells. PA / OA represents the palmitic acid / oleic acid induction conditions. Detailed Implementation

[0034] Example 1: Establishment and Characterization of a Mouse Obesity Model

[0035] Male C57BL / 6 mice (20-25 g) purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., license number SCXK(Su)2023-0009) were randomly divided into two groups: a control group (n = 6) and a CSDE1 overexpression group (n = 6). The control group was fed a conventional diet. The model group mice were fed a high-fat diet (Research Diets, D12492). After 14 weeks, the mice were sacrificed and liver, serum, white adipose tissue (eWAT of epididymal fat, scWAT of subcutaneous fat, iWAT of inguinal fat, and PAT of perirenal fat), brown adipose tissue (BAT), and major organs (heart, spleen, lung, kidney, and brain) were collected. Fat accumulation and structural changes were observed histologically (liver Oil Red O staining, H&E staining; various adipose tissues H&E staining). Serological indicators (such as TC, TG, ALT / AST) were used to comprehensively evaluate the effectiveness of the obesity model and the impact of CSDE1 overexpression on metabolic phenotype. Detailed results are available in [link to results]. Figure 1-3 .

[0036] Experimental results:

[0037] like Figure 1-3 As shown, feeding mice with a 60% high-fat diet (HFD) for 14 weeks can establish a mouse obesity model.

[0038] like Figure 1 As shown, assessments of overall body weight and macroscopic organ morphology in mice indicate that feeding them a 60% high-fat diet successfully induced an obesity phenotype. Specifically, as... Figure 1 As shown in B and 1C, the mice in the HFD group were significantly larger than those in the normal diet group (CD). During the 14-week feeding period, the body weight of the HFD group consistently exceeded that of the CD group, and the difference in body weight between the two groups was statistically significant. Figure 1As shown in D and 1E, the volume of adipose tissue in the HFD group mice was significantly larger than that in the CD group, indicating fat hypertrophy. Simultaneously, the liver color was noticeably whiter in the HFD group, suggesting increased lipid deposition in the liver. However, no significant differences were observed in the size or appearance of other organs such as the heart, spleen, lungs, and kidneys. These results demonstrate that the 60% high-fat diet regimen used in this invention can stably induce obesity in mice at both the body weight and organ morphology levels.

[0039] like Figure 2 As shown, serological indicators further validated the reliability of the model. Compared with the CD group, the HFD group mice showed significantly elevated serum triglyceride (TG), total cholesterol (TC), high-density lipoprotein (HDL-C), and low-density lipoprotein (LDL-C) levels, consistent with typical lipid profile changes in high-fat diet-induced obese mice. These serological results, along with changes in body weight and organs, further demonstrate that the mouse obesity model established in this invention is stable and reproducible, suitable for subsequent evaluation of drugs or interventions related to obesity and lipid metabolism disorders.

[0040] like Figure 3 As shown, histological observations were performed on the epididymal white adipose tissue (eWAT), brown adipose tissue (BAT), and liver tissue of mice in the CD and HFD groups. Under magnification, the eWAT tissue of the HFD group showed significantly increased adipocyte volume, marked monovesicularization of cytoplasmic lipid droplets, and thinning of the interstitium, exhibiting typical adipocyte hypertrophy compared to the CD group. The BAT tissue also showed increased lipid droplet volume and a gradual transformation of multilocular lipid droplets into macrovesicles, suggesting decreased energy metabolism activity. HE and Oil Red O staining of the liver revealed numerous lipid droplet vacuoles of varying sizes and diffuse lipid deposition in hepatocytes of the HFD group, while the hepatocytes of the CD group showed intact structure and fewer lipid droplets. These changes, including adipose tissue hypertrophy and significant hepatic steatosis, are consistent with the classic pathological features of obesity and fatty liver disease induced by a high-fat diet, demonstrating the successful establishment of the mouse obesity model in this invention, which can be used for subsequent evaluation of interventions related to obesity and lipid metabolism.

[0041] Example 2: Improvement of morphology, serology, and histology in a mouse obesity model by AAV-CSDE1 overexpression

[0042] Experimental Procedure: Male C57BL / 6J mice (20-25 g) were selected and housed in an SPF-grade animal facility under the following environmental conditions: 22±2℃, relative humidity 50%-60%, 12-hour light-dark cycle, and free access to food and water. After one week of acclimatization, the mice were randomly divided into two groups according to their body weight: a control group (AAV-eGFP, n = 6) and a CSDE1 overexpression group (AAV-CSDE1, n = 6).

[0043] Both groups of mice were fed a high-fat diet (Research Diets, D12492, 60% fat energy) for 16 consecutive weeks to induce a dietary obesity model. During the modeling process, mouse weight changes and general condition were monitored regularly to confirm the gradual formation of the obesity phenotype. At week 8, the control group was injected with AAV-eGFP via tail vein, while the CSDE1 overexpression group was injected with AAV-CSDE1 to achieve systemic overexpression of CSDE1 in mice. AAV-CSDE1 uses a modified adeno-associated virus (AAV) as a vector to efficiently deliver the CSDE1 gene into target cells or experimental animals, thereby artificially and continuously increasing the level of CSDE1 protein in these cells or tissues. The AAV used in this technology was derived from Heyuan Biotechnology; the AAV-CSDE1 vector was pAAV-EF1a-EGFP-T2A-Csde1-3xFLAG-tWPA; the AAV-eGFP control vector was pAAV-EF1a-EGFP-3xFLAG-tWPA. The EF1α promoter is used to drive the stable expression of exogenous genes in multiple tissues, the T2A autoclast peptide sequence is used to achieve co-expression of EGFP and CSDE1, and the 3×FLAG tag is used for subsequent protein detection and expression verification.

[0044] Mice were sacrificed at the end of week 16 after fasting for 6–8 hours following a high-fat diet. Blood samples were obtained via orbital sampling or cardiac sampling, and serum was separated for biochemical analysis. Simultaneously, the following tissues were rapidly dissected and weighed: liver, serum, white adipose tissue (eWAT, scWAT, iWAT, and PAT), brown adipose tissue (BAT), and major organs (heart, spleen, lungs, kidneys, and brain). Some tissues were fixed in 4% paraformaldehyde for histological analysis, while the remaining tissues were flash-frozen in liquid nitrogen for later use.

[0045] Liver tissue sections were frozen and stained with Oil Red O to assess the degree of lipid droplet deposition and steatosis. Paraffin sections were stained with Hematoxylin and eosin (H&E) to observe the overall structure and inflammatory changes of the liver tissue. Various types of adipose tissue (WAT and BAT) were also stained with H&E to assess adipocyte volume, arrangement, and morphological changes. Serum total cholesterol (TC) and triglyceride (TG) levels were measured using commercially available kits (both from Tiandiren Biotechnology), and alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured as liver function indicators. A comprehensive analysis of serological and histological results evaluated the effectiveness of the high-fat diet-induced obesity model and the impact of CSDE1 overexpression on the metabolic phenotype. Results are shown below. Figure 4-8 .

[0046] Experimental results:

[0047] like Figure 4-8 As shown, AAV-CSDE1 overexpression can improve the obesity phenotype in HFD mice.

[0048] like Figure 4 As shown, the obesity phenotype of HFD mice was evaluated based on changes in overall body weight and macroscopic morphology of major organs. The results showed that, compared with the control group (AAV-eGFP), mice treated with AAV-CSDE1 had significantly smaller overall body size and a significantly improved obese appearance.

[0049] Specifically, such as Figure 4 As shown in B-4C, at week 16 of high-fat diet feeding (i.e. week 8 after AAV injection), the body weight of mice in the AAV-CSDE1 group was significantly lower than that in the control group, and the difference between the two groups was statistically significant, suggesting that CSDE1 overexpression can effectively inhibit HFD-induced abnormal weight gain.

[0050] Further macroscopic observation results of organs, such as Figure 4 As shown in D-4E, compared with the control group, the volume of white adipose tissue in the AAV-CSDE1 group mice was significantly reduced, and the degree of fat accumulation was significantly decreased. Simultaneously, the color of their livers changed from the pale yellow or whitish of the control group to a bright red color closer to normal, indicating a significant improvement in liver lipid deposition and fatty degeneration. No significant differences were observed in the size and morphology of major non-metabolic organs such as the heart, spleen, lungs, and kidneys, indicating that the technical solution of this invention did not cause significant non-specific organ abnormalities while improving obesity.

[0051] The above results indicate that the AAV-CSDE1 overexpression technology used in this invention can specifically improve the obesity phenotype induced by HFD at the levels of overall body weight and key metabolic organs such as fat and liver.

[0052] like Figure 5As shown, the effect of AAV-CSDE1 overexpression on improving obesity and lipid metabolism disorders was further evaluated by detecting serological indicators. The results showed that compared with the control group, the serum triglyceride (TG) and total cholesterol (TC) levels in the AAV-CSDE1 group were significantly lower, with statistically significant differences, indicating that CSDE1 overexpression can effectively improve the dyslipidemia induced by a high-fat diet. Simultaneously, the serum ALT and AST levels, reflecting the degree of liver function damage, were also significantly lower in the AAV-CSDE1 group than in the control group, suggesting that CSDE1 overexpression can significantly alleviate hepatocellular damage and lipotoxicity burden caused by HFD. These serological results were highly consistent with the changes in body weight and macroscopic observations of organs, further demonstrating from a systemic metabolic perspective that this invention, through CSDE1 overexpression, can simultaneously improve obesity-related dyslipidemia and liver function damage, and has a clear therapeutic effect on obesity caused by lipid metabolism disorders.

[0053] like Figure 6 As shown in Figure 8, histological analysis was performed on the liver tissue, epididymal white adipose tissue (eWAT), and brown adipose tissue (BAT) of HFD mice to further verify the effect of AAV-CSDE1 overexpression on key target tissues of obesity.

[0054] Regarding the liver, the overall color of the livers in the AAV-CSDE1 group mice was closer to normal blood red. HE and Oil Red O staining results showed that, compared with the control group, the number and volume of lipid droplets in hepatocytes in the AAV-CSDE1 group were significantly reduced, diffuse lipid deposition was significantly alleviated, and the hepatocyte structure was more intact, suggesting that the pathological changes related to fatty liver disease were effectively reversed.

[0055] Regarding adipose tissue, in WAT, the AAV-CSDE1 group showed a significant reduction in adipocyte volume and hypertrophy, with a relative thickening of the interstitial structure, consistent with typical pathological features of adipose tissue remodeling during obesity improvement. In BAT, the AAV-CSDE1 group showed a significant reduction in lipid droplet volume and number, suggesting that the energy metabolism activity of brown adipose tissue was restored, which is conducive to improving overall energy consumption.

[0056] Meanwhile, histological observation of non-major metabolic organs such as the heart, spleen, lungs, and kidneys revealed no obvious pathological abnormalities, further demonstrating that the CSDE1 overexpression scheme used in this invention has good tissue selectivity and safety.

[0057] In summary, this invention, through AAV-mediated CSDE1 overexpression, demonstrates significant and consistent improvements in multiple key aspects closely related to the development of obesity, including weight control, adipose tissue remodeling, improvement of hepatic lipid deposition, and recovery of blood lipids and liver function. The aforementioned multi-level evidence fully demonstrates that the technical solution of this invention has a clear, stable, and targeted therapeutic effect on high-fat diet-induced obesity and lipid metabolism disorders.

[0058] Example 3: Improvement of glucose metabolism disorder in a mouse obesity model by AAV-CSDE1 overexpression

[0059] Experimental procedure:

[0060] Abnormal glucose metabolism is a core cause of obesity due to lipid metabolism disorders, stemming from excessive adipose tissue accumulation and endocrine dysfunction. To assess the overall glucose metabolism capacity of mice, a glucose tolerance test (GTT) was performed on each group of mice before the experimental endpoint. The mice in both groups were fasted for 16 hours starting the evening before the experiment (approximately 17:00–18:00), with free access to water during this period to minimize dietary interference with blood glucose levels. On the morning of the experiment, after the mice were placed in a quiet environment for 30 minutes to acclimatize, blood was collected via a slight tail clipping, and fasting blood glucose levels (0 min) were measured using a portable blood glucose meter. Subsequently, a 20% (w / v) glucose solution was administered to the mice via gavage at a dose calculated based on body weight, at a dose of 2 g / kg body weight. After the glucose load, blood was collected via tail clipping at 15 min, 30 min, 60 min, 90 min, and 120 min to measure blood glucose concentrations at each time point. Consistency in procedure was maintained throughout the experiment to avoid repeated stimulation and the influence of stress responses on blood glucose fluctuations. Results are as follows: Figure 9 As shown in Figure A.

[0061] To evaluate the effect of this invention's technical solution on improving the insulin resistance phenotype associated with obesity, especially lipid metabolism disorder-related obesity, from the perspective of overall metabolic function, the insulin sensitivity test (ITT) was used to test experimental animals. Mice with a high-fat diet-induced obesity model were selected as experimental subjects. Before the experiment, mice were housed under uniform environmental conditions and fasted for 6 hours before the test, allowing only free access to water to eliminate the influence of recent food intake on insulin sensitivity measurement. After fasting, blood was collected from the tail tip of each mouse, and fasting blood glucose levels were measured using a glucometer as baseline blood glucose levels. Subsequently, the injection dose was calculated based on the mouse's body weight, and insulin solution was administered intraperitoneally at a dose of 0.75 IU / kg. After insulin injection, blood was collected from the tail tip of each mouse at 15 min, 30 min, 60 min, 90 min, and 120 min, and blood glucose levels were measured to continuously monitor the changes in blood glucose levels under the action of insulin. Blood glucose values ​​at each time point were recorded, and blood glucose change curves over time were plotted to reflect the mice's response to exogenous insulin stimulation.

[0062] The differences in systemic insulin sensitivity in mice were evaluated by comparing the rate of glucose decline, the lowest glucose level, and the area under the curve (AUC) among different treatment groups. A faster, greater decrease in glucose and a lower AUC after insulin stimulation indicated higher insulin sensitivity; conversely, a lower AUC suggested insulin resistance, an important functional phenotype of obesity and lipid metabolism disorders. Results are as follows: Figure 9 BC.

[0063] Experimental results:

[0064] like Figure 9 As shown, the metabolic improvement effect of the technology of the present invention in a high-fat diet-induced obesity model was systematically evaluated from two complementary dimensions: glucose tolerance and insulin sensitivity.

[0065] like Figure 9 As shown in Figure A, in the glucose tolerance test (GTT), compared with the control group (AAV-eGFP), the AAV-CSDE1 overexpression group showed a smaller overall increase in blood glucose after gavage, and a significantly lower peak blood glucose level. At the key time points (15–60 min) after glucose loading, the blood glucose levels in the AAV-CSDE1 group were lower than those in the control group, and gradually recovered to near baseline levels in the later stages. These results indicate that AAV-CSDE1 overexpression can significantly improve the clearance capacity of exogenous glucose load in HFD mice, suggesting that it effectively alleviates the typical metabolic abnormality of impaired glucose tolerance in obese individuals.

[0066] like Figure 9 As shown in B, the effect of CSDE1 overexpression on obesity-related insulin resistance was further evaluated using an insulin tolerance test (ITT). After normalizing blood glucose levels at multiple time points, the normalized blood glucose levels in the AAV-CSDE1 group were lower than those in the control group at all time points after insulin injection, with particularly significant differences in the 30-90 min range, indicating a more sensitive response to insulin stimulation.

[0067] like Figure 9 As shown in Figure C, area under the curve (AUC) analysis of the ITT curves revealed that the ITT-AUC value of the AAV-CSDE1 group was significantly lower than that of the AAV-eGFP control group, with a statistically significant difference (**P < 0.01). This significant decrease in AUC indicates that AAV-CSDE1 overexpression significantly enhanced insulin-mediated glucose clearance throughout the experiment.

[0068] In summary, this invention systematically demonstrates the significant metabolic improvement effects of CSDE1 on two key metabolic dimensions: glucose tolerance and insulin sensitivity, by upregulating CSDE1 expression in a high-fat diet-induced obesity model. CSDE1 overexpression effectively promotes glucose clearance, reduces post-glucose load peak blood glucose levels, and significantly enhances the body's response to insulin stimulation, thereby comprehensively alleviating obesity-related glucose intolerance and insulin resistance. This demonstrates the application potential of this invention in intervening in lipid metabolism disorder-related obesity and its abnormal glucose metabolism.

[0069] Example 4: Effects of AAV-CSDE1 overexpression on changes in body composition and improved metabolic levels in mice

[0070] Experimental procedure:

[0071] To further evaluate the ameliorative effect of CSDE1 overexpression on an obesity model, this invention used a body composition analyzer (Bruker, Germany) to detect body composition in experimental mice. Low-frequency nuclear magnetic resonance (NMR) technology was used for non-invasive and quantitative determination of the content of adipose tissue, lean body tissue, and free fluid in the mice. By comparing the changes in adipose tissue and lean body tissue content before and after CSDE1 overexpression treatment, the regulatory effect of this technology on obesity-related abnormal fat accumulation was evaluated, and it was used to determine whether weight changes were mainly due to a reduction in fat mass rather than a loss of lean body tissue, thereby verifying the effectiveness of this invention in specifically improving the obesity phenotype. Results are as follows: Figure 10 A.

[0072] To evaluate the effect of CSDE1 overexpression on obesity-related energy metabolism, an integrated metabolic cage system was used to measure energy expenditure in mice. Mice induced by a high-fat diet and injected with either AAV-eGFP or AAV-CSDE1 were selected and housed individually in the metabolic cage system for 24 hours before the experiment to allow for environmental acclimatization and minimize the impact of environmental changes on metabolic parameters. During the metabolic cage experiment, constant environmental conditions were maintained, including temperature (22–24 °C), humidity (40%–60%), and a 12-hour light / dark cycle, with free access to food and water. Formal collection of metabolic parameters was conducted after the acclimatization period, with continuous monitoring for 72 hours, recording indicators such as oxygen consumption (VO2), carbon dioxide production (VCO2), and activity levels. Energy expenditure (EE) was calculated using indirect calorimetry. To eliminate the influence of body size differences on energy metabolism and highlight the specificity of obesity-related lipid metabolism regulation, lean mass in mice was used as a covariate. Analysis of covariance (ANCOVA) was employed to standardize energy expenditure data, allowing for comparison of energy expenditure levels in different treatment groups under unit lean mass conditions. Results are as follows: Figure 10 B.

[0073] Experimental results:

[0074] like Figure 10 As shown in Figure A, low-frequency nuclear magnetic resonance (NMR) compositional analysis was used to quantitatively analyze adipose tissue and lean body tissue in mice. The results showed that compared with the control group (AAV-eGFP), the fat mass of mice in the AAV-CSDE1 overexpression group was significantly reduced (*P < 0.05), while there was no significant difference in lean body mass between the two groups (ns). These results indicate that the weight loss in mice induced by the CSDE1 overexpression technology used in this invention mainly stems from a reduction in adipose tissue, without causing significant loss of skeletal muscle or other lean body tissues, demonstrating a selective intervention effect on pathological fat accumulation in obesity.

[0075] like Figure 10 As shown in Figure B, an analysis of covariance (ANCOVA) was performed on daily energy expenditure in mice with lean body weight as a covariate. The results showed that, under the same lean body weight conditions, the energy expenditure level of mice in the AAV-CSDE1 overexpression group was significantly higher than that of the control group (P < 0.05). This result suggests that CSDE1 overexpression not only reduces fat accumulation but also increases the overall energy expenditure level of the body independently of changes in lean body weight, thus inhibiting excessive energy storage in obese states from a metabolic perspective.

[0076] In summary, this invention demonstrates through body composition analysis and energy metabolism assessment that CSDE1 overexpression selectively reduces obesity-related fat accumulation without affecting the integrity of lean body tissue, and significantly increases the body's energy expenditure level per unit of lean body mass. This provides targeted intervention in the core pathological basis of obesity from two aspects: reducing fat storage and enhancing energy expenditure, highlighting the significant advantages and application value of this technology in the safe and effective treatment of obesity and its metabolic abnormalities.

[0077] Example 5: CSDE1 Combined with High-Throughput Drug Screening and Efficacy

[0078] To screen candidate drugs that can directly act on the CSDE1 protein and thus be used to intervene in lipid metabolism disorder-related obesity, this invention uses surface plasmon resonance (SPR) technology to systematically and in a high-throughput screening of an FDA-approved small molecule drug compound library.

[0079] Experimental Procedure: First, the recombinant CSDE1 protein was purified in vitro and stably immobilized on the surface of the detection channel of a CM7 chip (Cytiva, 28953828) via amino-coupling. The immobilization level was controlled within a range that ensured signal sensitivity without causing mass transfer limitations. A blank channel containing uncoupled protein was simultaneously set up as a reference channel to eliminate non-specific binding and interference from background signals. The selected compounds were derived from the FDA-approved drug library (MCE, HY-L022M), which contains 2774 small molecule compounds with established safety and clinical use backgrounds. This library covers a wide range of known metabolic, cardiovascular, endocrine, and anti-infective drugs, possessing high drug repurposing value. During the screening process, candidate compounds were sequentially injected at a uniform concentration using an automated injection system, and the binding response signal (Response Unit, RU) between the compounds and the immobilized CSDE1 protein on the chip surface was monitored in real time. The blank flow channel signal was used as a reference for difference correction, and chlorfolcitol, a compound known to have a weak binding ability with CSDE1, was set as a positive control to verify the stability and reliability of the screening system.

[0080] Based on the binding response value (RU) generated after each compound injection, the CSDE1 affinity of 2774 candidate drugs was ranked. Figure 9As shown in Figure A, a batch of candidate compounds that showed a significant binding response to CSDE1 were screened, and the top 50 compounds with the highest response values ​​were defined as the CSDE1 high-affinity candidate drug set. This screening result indicates that CSDE1, as a lipid metabolism regulation-related protein, can be directly bound by a variety of marketed small molecule drugs, providing a clear compound basis for the subsequent development of targeted intervention strategies for lipid metabolism disorder-related obesity.

[0081] To further verify whether the screened CSDE1-binding drugs can exert biological effects at the protein level, in vitro cell-level validation experiments were conducted on the top 50 high-affinity candidate drugs. Specifically, cells were treated with the candidate drugs respectively, changes in CSDE1 protein expression levels were detected, and the CSDE1 band intensity was quantitatively analyzed using Western blot to assess the effects of different compounds on CSDE1 protein stability or degradation. Figure 9 As shown in B–C, the regulatory effects of different candidate drugs on CSDE1 protein levels varied significantly. Milrinone and butoconazole both showed significant increases in CSDE1 protein levels in multiple replicate experiments, with significantly higher gray values ​​of the CSDE1 band compared to the control group. This suggests that these two compounds can enhance CSDE1 protein stability or inhibit its degradation by directly binding to CSDE1.

[0082] Example 6: Improvement of lipid droplet formation in primary mouse liver cells by CSDE1 stabilizer

[0083] To further validate the application potential of candidate compounds obtained through CSDE1-targeted screening in lipid metabolism disorder-related obesity at the cellular function level, this invention constructed a hyperlipidemia-induced model based on primary mouse hepatocytes and validated the screened candidate drugs.

[0084] Experimental Procedure: Healthy male C57BL / 6 mice aged 8–12 weeks were selected and processed according to standard methods for primary liver cell isolation. Specifically, after anesthesia, mice were pre-perfused via the portal vein with HBSS-EDTA perfusion solution (calcium and magnesium-free) to thoroughly remove blood components and disrupt intercellular junctions. Subsequently, the liver was perfused with HBSS digestion solution containing type I collagenase IV for in situ digestion. After the liver tissue was sufficiently softened, it was removed and gently dispersed. Undigested tissue fragments and impurities were removed by low-speed centrifugation, and the hepatocyte pellet was collected and resuspended in DMEM medium containing 10% fetal bovine serum. The obtained primary hepatocytes were then cultured at 2 × 10⁻⁶ cells / year. 5Cells were seeded per well in a six-well plate and cultured at 37°C and 5% CO2. Once the cells were stably adhered, they were used for subsequent experiments.

[0085] To simulate the pathological process of excessive lipid deposition in hepatocytes under conditions of obesity and lipid metabolism disorders, a primary hepatocyte hyperlipidemia model was constructed using a free fatty acid induction method. Specifically, palmitic acid (PA) and oleic acid (OA) were respectively compounded with free bovine serum albumin (BSA) to prepare a mixed fatty acid solution simulating a hyperlipidemia environment, which was then added to the cell culture system. This PA / OA compound induction system effectively simulates the pathological characteristics of elevated blood lipids and increased lipid load in hepatocytes under obesity, leading to significant accumulation of neutral lipids and lipid droplets in the cells. It is a classic in vitro model for studying obesity-related and lipid metabolism abnormalities.

[0086] Under hyperlipidemic induction conditions, primary hepatocytes were treated with drug-containing culture media prepared with butoconazole nitrate and milrinone at predetermined concentrations. The following control groups were also included: a normal culture group without PA / OA (normal control); a hyperlipidemic model group with PA / OA but without the drug; a PA / OA + milrinone treatment group; and a PA / OA + butoconazole treatment group. All treatment groups were cultured under the same conditions for 24 hours to evaluate the effect of the candidate drugs on lipid deposition in hepatocytes in a hyperlipidemic environment. After culture, Oil Red O staining was performed on each group to detect the deposition of neutral lipids and lipid droplets within the cells. The number, size, and distribution of lipid droplets were observed and recorded under a microscope. Differences in lipid deposition among different treatment groups were compared and analyzed. The results are shown below. Figure 12 As shown.

[0087] Compared with the PA / OA-induced hyperlipidemia model group, the milrinone and butoconazole treatment groups showed a significant reduction in lipid droplet deposition in primary hepatocytes, with both droplet volume and density decreasing, suggesting that the aforementioned candidate drugs can improve the hyperlipidemia-induced lipid accumulation phenotype at the cellular level. These results demonstrate that the candidate compounds obtained through CSDE1-targeted high-throughput screening have a clear functional ameliorative effect in an obesity-related hepatocyte lipid metabolism disorder model, validating their regulatory ability on lipid metabolism disorder phenotypes at the cellular level.

[0088] One of the key pathological features of obesity and lipid metabolism disorder-related obesity is excessive lipid deposition in hepatocytes and the development of fatty liver disease. This embodiment demonstrates, through the construction of a primary hepatocyte hyperlipidemia model, that milrinone and butoconazole, as small molecule compounds that bind to and stabilize the CSDE1 protein, can effectively improve lipid deposition in hepatocytes, suggesting their clear application potential in the prevention or treatment of obesity and related lipid metabolism abnormalities. The above cellular level experimental results corroborate the aforementioned CSDE1 overexpression animal experiments and SPR drug screening results, further demonstrating the good reliability and specificity of the CSDE1-targeted drug screening and validation system established in this invention.

[0089] The above description is a specific 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 concept of the present invention, should be covered within the scope of protection of the claims of the present invention.

Claims

1. Use of a preparation for regulating expression level of CSDE1 in the preparation of a drug for preventing or treating obesity with lipid metabolism disorder.

2. Use according to claim 1, characterized in that, The preparation for regulating expression level of CSDE1 is a preparation for overexpression of CSDE1.

3. Use according to claim 1 or 2, characterized in that, The preparation for regulating expression level of CSDE1 is AAV-CSDE1.

4. Use of a small molecule compound for overexpressing CSDE1 in the preparation of a drug for treating obesity with lipid metabolism disorder, wherein the small molecule compound for overexpressing CSDE1 comprises milrinone or broconazole.

5. Use according to claim 4, characterized in that, The milrinone or broconazole is prepared into various feasible dosage forms.

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