Application of Eepd1 expression regulating reagent in medicine for treating obesity and related metabolic diseases thereof

Abstract

The invention discloses application of an expression regulating reagent of Eepd1 in preparation of a medicine for treating obesity and related metabolic diseases of the obesity. The expression regulating reagent of the Eepd1 is retigabine. It is verified for the first time that retigabine promotes DNA repair and mitochondrial function recovery by up-regulating Eepd1 expression, so that obesity-related metabolic disorders are improved, and a new treatment strategy is provided for treating obesity and related metabolic diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the application of an Eepd1 expression regulating agent in a drug for treating obesity and related metabolic diseases. Background Art

[0002] Obesity refers to the excessive accumulation of fat tissue in the body, a condition that has a negative impact on health. It is usually measured by the body mass index (BMI), which is calculated as weight (kg) divided by height (m) squared. According to global statistics, more than 650 million adults were diagnosed with obesity (BMI ≥ 28 kg / m 2 ), more than 1.9 billion adults are overweight (24kg / m 2 <BMI<28kg / m 2 ). Over the past four decades (1975-2016), the prevalence of obesity has nearly tripled, and the trend continues.

[0003] Large-scale population studies have shown that increased BMI is positively correlated with obesity-related health risks and mortality. 2 ) is associated with more serious health problems. The 2015 Global Burden of Disease Study estimated that overweight and obesity caused more than 4 million deaths worldwide, of which 70% were related to cardiovascular disease. Clinical studies have confirmed that obesity is an important cause of many chronic diseases, including cardiovascular and cerebrovascular diseases, type 2 diabetes, chronic kidney disease, and various cancers.

[0004] Obesity has become a global epidemic, significantly increasing the risks associated with cardiovascular and cerebrovascular diseases and placing tremendous pressure on global health systems. Therefore, effective prevention and management strategies are urgently needed. Summary of the invention

[0005] The purpose of the present invention is to address the deficiencies in the prior art and provide an application of an Eepd1 expression regulating agent in the preparation of a drug for treating obesity and related metabolic diseases.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] Provided is the use of an Eepd1 expression regulating reagent in preparing a drug for treating obesity and related metabolic diseases.

[0008] Furthermore, the Eepd1 expression regulating agent is retigabine.

[0009] Furthermore, the dosage of retigabine is 25 mg / kg.

[0010] Furthermore, the retigabine improves obesity-related metabolic disorders by upregulating Eepd1 expression, promoting DNA repair and recovery of mitochondrial function.

[0011] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0012] The present invention verifies for the first time that retigabine improves obesity-related metabolic disorders by upregulating Eepd1 expression, providing a new treatment strategy for treating obesity and its related metabolic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Figure 3 shows that Eepd1 is highly expressed in normal adipose tissue but downregulated in obese adipose tissue; (a) Venn diagram depicting DNA repair genes from the intersection of differentially expressed genes from RNA-Seq datasets GSE70353 (human) and GSE167264 (mouse), and the PBmices database of obesity-related genes; (B) Immunoblotting analysis of Eepd1 protein expression in tissues of C57BL / 6JWT mice, with β-actin as a loading control; (C) Immunoblotting analysis of Eepd1 in eWAT of young and middle-aged mice, with β-actin as a loading control; (D) Immunoblotting of Eepd1 in iWAT and eWAT from DIO mice and WT controls of the same age, with β-actin as a loading control; (E) Immunoblotting of Eepd1 and Fabp4 in mature adipocytes and SVF of iWAT and eWAT of C57BL / 6J mice, with GAPDH as a loading control; (F) flox / flox Mice and Eepd1 AKO Representative photos of mice; (G) Eepd1 flox / flox Mice and Eepd1 AKO Body weight of mice (n=7); (H) Eepd1 flox / flox Mice and Eepd1 AKO Representative photos of fat pad morphology in mice; (IK) Eepd1 flox / flox Mice and Eepd1 AKO Histological examination results of H&E staining of iWAT, eWAT, and BAT of mice. Scale bar, 50 μm.

[0014] Figure 2The overexpression of Eepd1 enhances the thermogenic capacity of adipose tissue and improves obesity; (A) Experimental flow chart of adenovirus injection in iWAT of DIO mice (N=7); (B) Representative photos of fat pad morphology; (C) Representative images of H&E staining of anatomical tissues, scale bar 100 μm; (D) Quantitative analysis of iWAT adipocyte diameter in Figure (C); (E) Immunoblotting of indicated proteins in iWAT injected with Adv-Ctl or Adv-Eepd1, with β-Actin as a loading control; (F) Percentage of weight gain in DIO mice injected with Adv-Ctl or Adv-Eepd1 ; (G) Core body temperature of Adv-Eepd1 injected mice; (H) Energy expenditure under basal conditions of light and dark cycles; (I) Immunoblotting of Eepd1 in iWAT treated with Vehicle or retigabine, with β-actin as a loading control; (J) Body weight changes of mice treated with Vehicle or retigabine at the indicated time points; (K) Representative photos of fat pad morphology; (L) Representative H&E staining pictures of dissected tissues, scale bar is 100 μm; (M) Immunoblotting of the indicated proteins in iWAT treated with Vehicle or retigabine, with β-actin as a loading control. DETAILED DESCRIPTION

[0015] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but is not intended to be limiting of the present invention. It should be noted that the embodiments and features in the embodiments of the present invention may be combined with each other without conflict.

[0016] The present invention provides, and its specific experimental method and results are as follows.

[0017] 1. Experimental Methods

[0018] 1.1 Animal experiments

[0019] Animal experiments were performed in accordance with regulations and general recommendations and approved by the Animal Care Committee of Shanghai Sixth People's Hospital, Shanghai Jiao Tong University School of Medicine (DWLL2022-0599). All mice were housed on a C57BL / 6J background. flox / flox Generation of mice by crossing with Eepd1 flox / - By crossing Eepd1, mice with adipose tissue-specific knockout of Eepd1 were obtained. flox / floxAdiponectin promoter-driven Cre recombinase transgenic mice. C57BL / 6J male mice (SPF grade) were purchased from Biocell. The animals were housed at room temperature (24-26°C) in a humidity-controlled (40%-70%) room with a 12-h light / dark cycle. Unless otherwise specified, standard normal chow (SLACOM, P1101F-25) and sterile water were given ad libitum. All mice used in the experiments throughout the study showed normal health. Mice in different groups were assigned according to their genotype or randomly assigned. For studies on specific diets, 6-week-old C57BL / 6J mice were given a high-fat (60% fat, research diet, D12492) diet for 8-12 weeks before other experiments to generate DIO mice.

[0020] exist Figure 2 In the experiment:

[0021] DIO mice were injected orally with adenovirus: pAdEasy-EF1-MCS-3xFlag-CMV-EGFP (labeled as Adv-Ctl) and pAdEasy-EF1-MCS-Eepd1-3xFlag-CMV-EGFP (labeled as Adv-Eepd1). The injection dose per mouse was 3×10^ 8 Plaque forming units (pfu) were injected twice a week for one week. DIO mice were given regabine hydrochloride (25 mg / kg) daily for 11 consecutive days.

[0022] 1.2 Immunoblotting

[0023] Adipose tissue, SVF or adipocytes were lysed with lysis buffer (50 mM Tris-HCl, 1% SDS, pH 7.4), and the lysate was supplemented with EDTA-free protease inhibitor cocktail (Thermo Scientific Pierce, A32955) and phosphatase inhibitor cocktail (Thermo Scientific Pierce, A32961). Protein content was determined using the BCA protein concentration assay kit (Thermo Scientific Pierce, 23225). Equal amounts of protein samples were separated by SDS-PAGE and incubated with the specified primary antibodies overnight. Immunoreactions were detected by enhanced chemiluminescence autoradiography (Millipore, WBKLS0500), and chemiluminescent signals were determined by the AI600 system (GE Healthcare).

[0024] The following antibodies were used: anti-Eepd1 (24310-1-AP) antibody (purchased from ProteinTech) and anti-β-actin (8457), anti-GAPDH (5174), anti-p-HSL (4126), anti-HSL (4107), anti-PKA (9624) antibodies (purchased from Cell Signaling Technology).

[0025] 1.3 Histological analysis

[0026] iWAT, eWAT, and BAT were fixed in 4% (v / v) paraformaldehyde solution and embedded in paraffin. The sections were 5 μm thick and stained with hematoxylin-eosin (H&E). The cell diameters in H&E-stained sections of iWAT and eWAT of 7 mice in each group were quantitatively analyzed using AdipoCount software.

[0027] 1.4 Metabolic cage experiment

[0028] Mice were acclimated to the system for 48 h before formal testing, and had free access to food and water during the test. Whole-body energy expenditure was measured using the Comprehensive Laboratory Animal Monitoring System (CLAMS; Columbus, USA). Energy expenditure was calculated as: (3.815 + 1.232 × RER) × VO2.

[0029] 1.5 Cold resistance test

[0030] The body temperature of mice was measured using a rectal probe (FT3400, China). The mice were exposed to a 4°C environment, and their rectal temperature was measured by a digital thermometer at 0, 30, 60, 90, 120, 150, and 240 minutes. During the test, mice had free access to water and food.

[0031] 1.6 Statistical analysis

[0032] Statistical analysis was performed using GraphPad Prism software (version 9.0). Unpaired two-tailed Student's t test, Mann-Whitney U test, one-way ANOVA or two-way ANOVA and Before multiple comparison tests, the assumption of normal distribution was assessed using the Sharpiro-Wilk test and the Kolmogorov-Smirnov test. Statistical details of the experiments are shown in the figure legends. Data are presented as mean ± standard error (SEM). Statistical significance is defined as *p<0.05, **p<0.01, ***p<0.001. For mouse experiments, “n” indicates the number of mice used.

[0033] 2. Experimental Results and Analysis

[0034] 2.1 Eepd1 is highly expressed in normal adipose tissue but downregulated in obese adipose tissue

[0035] To identify DNA repair genes that may be associated with obesity and adipose thermogenesis, we retrieved the GSE70353 dataset (human samples: lean vs. obese) from the Gene Expression Omnibus (GEO) and identified 137 differentially expressed DNA repair genes. In addition, we found 144 differentially expressed DNA repair genes in the GSE167264 dataset (mouse samples: lean vs. obese), which were also from the GEO database. We also analyzed the screening results of 37 obesity-related genes from the piggyBac mouse (PBMictes) database. At the intersection of these three databases, Eepd1, a DNA damage repair enzyme, emerged as a key candidate gene that may play a key role in the development of obesity ( Figure 1 A). We observed that among different tissue types, Eepd1 expression was mainly localized in eWAT and iWAT ( Figure 1 B). The expression level of Eepd1 in eWAT of middle-aged mice was significantly reduced compared with that of young mice ( Figure 1 C). Consistent with these observations, Eepd1 levels were also downregulated in iWAT and eWAT of obesity models such as diet-induced obese (DIO) mice compared with their respective control groups ( Figure 1 D).

[0036] To further investigate, we isolated mature adipocytes and stromal vascular fraction (SVF) cells from iWAT and eWAT of C57BL / 6J mice. Our results confirmed that Eepd1 is mainly expressed in mature adipocytes ( Figure 1 E). Taken together, these findings point to a potential metabolic role for Eepd1 in adipocytes, which is critically involved in the development of obesity.

[0037] 2.2 Eepd1 ablation in adipose tissue promotes the development of middle-aged obesity

[0038] The prominent expression of Eepd1 in mature adipocytes and its downregulation in obesity prompted us to speculate that Eepd1 in adipose tissue plays a key role in the development of obesity. To verify this, we used adipocyte-specific Eepd1 knockout mice (Eepd1 flox / flox To investigate whether downregulation of Eepd1 expression contributes to the weight gain in middle-aged mice, we monitored Eepd1. flox / flox and Eepd1 AKOMice were fed a standard diet until 14 months of age. flox / flox In comparison, Eepd1 AKO The mice had a significant increase in body weight ( Figure 1 F and 1G). In addition, our analysis showed that Eepd1 AKO The volume of all three adipose tissue types increased significantly ( Figure 1 H). Histological examination by hematoxylin and eosin (H&E) staining showed that Eepd1 AKO The adipocytes in both iWAT and eWAT of mice were enlarged, accompanied by lipid accumulation in BAT ( Figure 1 I-1K).

[0039] 2.3 Intervention of Eepd1 overexpression can enhance the thermogenesis of adipose tissue and improve obesity

[0040] Given the Eepd1 deficiency observed in obese mice ( Figure 1 C and 1D), we further explored whether Eepd1 overexpression has the potential to alleviate obesity. We enhanced the expression of Eepd1 by in situ injection in the white adipose tissue (iWAT) of DIO (diet-induced obesity) mice using the adenovirus system. In the experiment, we injected adenovirus overexpressing Eepd1 (Adv-Eepd1) into the right fat pad and control adenovirus (Adv-Ctl) into the left fat pad for one week ( Figure 2 A). The results showed a significant reduction in fat mass and adipocyte volume ( Figure 2 BD). Immunoblotting analysis showed that Eepd1 overexpression significantly increased the phosphorylation levels of Ucp1 and PKA substrates (including HSL) ( Figure 2 E). Next, we injected Adv-Eepd1 bilaterally into the iWAT of DIO mice, with Adv-Ctl-injected mice as a control group. These mice also showed enhanced cold tolerance ( Figure 2 G). At the same time, their energy consumption increased significantly ( Figure 2 H).

[0041] We performed a screen using a library of FDA-approved drugs and identified retigabine as a small molecule that can restore Eepd1 protein levels. Here, we detected that retigabine also significantly increased Eepd1 protein levels in adipocytes ( Figure 2 I). Considering that the reduction of Eepd1 promotes the development of obesity, we injected retigabine into the adipose tissue pads of DIO mice daily. Notably, the body weight of DIO mice was significantly reduced in just 11 days after continuous administration of retigabine ( Figure 2 J). In addition, the fat pads of treated mice appeared red ( Figure 2 K), H&E staining showed that the adipocytes of these mice were smaller ( Figure 2 L). Immunoblotting analysis further showed that phosphorylated PKA substrates were significantly increased, especially HSL ( Figure 2 M). In summary, our results suggest that retigabine alleviates obesity progression by reversing the reduction of Eepd1.

[0042] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the specification and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of Eepd1 expression regulating reagents in the preparation of drugs for the treatment of obesity and related metabolic diseases.

2. The use according to claim 1, characterized in that: The Eepd1 expression regulating agent is retigabine.

3. The use according to claim 2, characterized in that: The dose of retigabine was 25 mg / kg.

4. The use according to claim 2, characterized in that: The retigabine improves obesity-related metabolic disorders by upregulating Eepd1 expression, promoting DNA repair and mitochondrial function recovery.

Images