Composition for promoting browning of white adipose cells and preparation method thereof

Abstract

The invention discloses a composition for promoting browning of white fat cells and a preparation method thereof, and belongs to the technical field of biological medicine, the composition is prepared from the following raw materials by weight: 5-8 parts of 2-BrO, 2-5 parts of anacaric acid, 1-2 parts of 8-cyclopentyl-1, 3-dipropyl xanthine (DPCPX), 1-2 parts of endothelin-31, 0.1-0.5 part of S-4-nitrobenzyl-6-thioinosine (NBTI), and 1-2 parts of catecholamine. The liposome of which the surface is modified by the CKGGRAKDC is used as a carrier for targeting white fat cells, and the carrier for targeting the white fat cells is coated with 2-BrO, anacardic acid, S-4-nitrobenzyl-6-thioinosine, endothelin-3 and catecholamine. The finally prepared composition for promoting browning of white adipocytes promotes fat in adipose tissues to be decomposed into fatty acid, enhances the beta oxidation effect of the fatty acid, improves heat production efficiency, maintains normal reactivity of fat decomposition, and effectively promotes the white adipocytes to be browned into brown adipocytes.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a composition for promoting browning of white adipocytes and a preparation method thereof. Background Art

[0002] The prevalence of obesity severely affects every region and population group worldwide and shows no sign of decreasing. Obesity greatly increases the risk of several chronic diseases, including cardiovascular diseases, fatty liver, diabetes, insulin resistance, and cancer. Current obesity treatment strategies focus on inhibiting adipogenesis, increasing energy expenditure, and reducing adipose tissue inflammation. Therefore, there is an urgent need to develop new therapeutic targets and a wide range of treatment methods for treating obesity and its related complications. Obesity is characterized by a significant increase in adipocyte volume (hypertrophy) and an increase in adipocyte number (hyperplasia).

[0003] Since obesity develops from excess energy stored in adipose tissue, treatment methods that reduce energy intake or increase energy expenditure, or both, would provide an attractive approach to combat obesity and related diseases. Although thermogenic adipocytes and their precursors consist of various different cell populations (3, 4), adipose tissue populations mainly consist of white adipose tissue (WAT) and brown adipose tissue (BAT), which play opposite metabolic roles in regulating energy balance. WAT is specialized for energy storage and can be induced to undergo browning through genetic or pharmacological means. This browning, also known as "beige" or "white-brown," is associated with increased expression of mitochondrial uncoupling protein 1 (UCP1) in response to external stimuli, including chronic cold exposure, treatment with the β-adrenergic agonist CL316,243, exercise, and endocrine factors (8, 9). This type of adipose browning is also associated with an increase in cellular thermogenic capacity because activation of UCP1 decouples mitochondrial respiration from ATP production, providing a significant metabolic benefit comparable to BAT (8).

[0004] Fats are generally classified into thermogenic fat and non-thermogenic fat, which determines their thermogenic capacity. Non-thermogenic fat is represented by white adipose tissue (WAT), which is characterized by large unilocular lipid droplets (lipid droplets) and a low range of mitochondria and low levels of uncoupling protein 1 (UCP1). White adipose tissue is responsible for the body's energy storage, stored in unilocular lipid droplets in the form of triglycerides. In contrast, thermogenic fat includes brown adipocytes and beige adipocytes, which are characterized by multilocular lipid droplets and mitochondria with a high-density cristae structure, express UCP1, and are capable of effectively consuming energy in the form of heat. Mammals maintain a constant core body temperature through adaptive thermogenesis, including shivering and non-shivering thermogenesis, in response to cold stimuli. The energy sources for non-shivering thermogenesis are diverse, including glucose, fatty acids (FAs), succinate, and branched-chain amino acids. Although there are multiple energy sources available during thermogenesis, fatty acids are the main molecules selected during fat thermogenesis. In response to cold stimuli, the thermogenic program in adipose tissue is activated, and lipid droplets in adipocytes release fatty acids through lipases, which are burned by mitochondria as fuel to generate heat.

[0005] An increase in the basal lipolysis rate can lead to an imbalance in lipid turnover in adipocytes, a decrease in the ability of adipocytes to store and release fatty acids, and further exacerbate adipose tissue dysfunction. Circulating FAs are the main substrate source for the liver to generate triglyceride-rich lipoproteins, and adipose tissue lipolysis damage will inhibit VLDL synthesis. Changes in lipolysis are usually associated with obesity, including an increase in the basal lipolysis rate that can lead to insulin resistance, and impaired responsiveness of lipolysis to stimuli. The main characteristics of obesity are excessive WAT and an increase in adipocyte size, which is caused by an increase in triglyceride storage. Summary of the Invention

[0006] In order to overcome the deficiencies of the prior art, one of the objectives of the present invention is to provide a composition for promoting the browning of white adipocytes. The composition for promoting the browning of white adipocytes promotes the decomposition of fat in adipose tissue into fatty acids, enhances fatty acid β-oxidation, improves thermogenic efficiency, maintains normal lipolytic reactivity, and effectively promotes the browning of white adipocytes into brown adipocytes.

[0007] Another objective of the present invention is to provide a method for manufacturing a composition for promoting the browning of white adipocytes, which has simple steps and low costs.

[0008] One of the objectives of the present invention is achieved by adopting the following technical solution:

[0009] It is prepared from raw materials with the following weight ratios: 2-BrO 5-8 parts, anacardic acid 2-5 parts, 8-cyclopentyl-1,3-dipropylxanthine 1-2 parts, endothelin-3 1-2 parts, S-4-nitrobenzyl-6-thioinosine 0.1-0.5 parts, catecholamine 1-2 parts; a liposome modified with surface CKGGRAKDC is used as a carrier targeting white adipocytes, and the carrier targeting white adipocytes is coated with 2-BrO, anacardic acid, S-4-nitrobenzyl-6-thioinosine, endothelin-3, and catecholamine.

[0010] Further, it is prepared from raw materials with the following weight ratios: 2-BrO 5 parts, anacardic acid 3 parts, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) 2 parts, endothelin-3 1 part, S-4-nitrobenzyl-6-thioinosine (NBTI) 0.1 part, catecholamine 2 parts.

[0011] Further, the surface of the liposome is modified with maleimide-modified polyethylene glycol, and the specific preparation method includes the following steps:

[0012] S1. Maleimide-modified polyethylene glycol-phospholipid is dissolved in an organic solvent, and the maleimide-modified polyethylene glycol-phospholipid accounts for 1 mol% to 1.25 mol% of the total lipids.

[0013] S2. 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and cholesterol are dissolved in an organic solvent to form a lipid solution, and then the solution of step S1 is added. The molar ratio of the maleimide-modified polyethylene glycol-phospholipid to the total lipids in the lipid solution is 1:100 to 1.25:100;

[0014] S3. The solution of step S2 is mixed with an equal volume of diisopropyl ether, 10 mM HEPES buffer is added, and stirred evenly;

[0015] S4. The solution of step S3 is treated with ultrasonic waves to promote the formation of liposomes, and the ultrasonic treatment time is 15 s to 45 s;

[0016] S5. The organic solvent is removed by nitrogen evaporation to obtain a preliminary liposome solution;

[0017] S6. The preliminary liposome solution is ultrasonically treated to ensure uniform dispersion of the liposomes, and the ultrasonic treatment time is 45 s;

[0018] S7. The solution of step S6 is dialyzed in a buffer to remove the unreacted maleimide-modified polyethylene glycol-phospholipid and other impurities. The dialysis time is 2 h to obtain maleimide-modified polyethylene glycol liposomes.

[0019] Further, in step S3, the specific steps after mixing the solution of step S2 with an equal volume of diisopropyl ether are as follows: Weigh 2-BrO, anacardic acid, S-4-nitrobenzyl-6-thioinosine, 1-2 parts of purine, endothelin-3, and catecholamine according to the formula amount, mix them together, add them to the 10 mM HEPES buffer, and mix them together. Finally, a liposome of maleimide-modified polyethylene glycol coated with 2-BrO, anacardic acid, S-4-nitrobenzyl-6-thioinosine, endothelin-3, and catecholamine is obtained.

[0020] Further, the preparation method of the liposome with surface modification of CKGGRAKDC is as follows: Dissolve 10 mM inhibin-targeting peptide CKGGRAKDC in distilled water, add the liposome of maleimide-modified polyethylene glycol coated with 2-BrO, anacardic acid, S-4-nitrobenzyl-6-thioinosine, endothelin-3, and catecholamine according to a molar ratio of 1:1, and react with shaking at 30 °C for 24 h to obtain the liposome with surface modification of CKGGRAKDC.

[0021] The second object of the present invention is achieved by the following technical solution:

[0022] A preparation method of a composition for promoting browning of white adipocytes includes the following steps: Mix the liposome with surface modification of CKGGRAKDC and 8-cyclopentyl-1,3-dipropylxanthine together to obtain the composition for promoting browning of white adipocytes.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] A composition for promoting the browning of white adipocytes provided by the present invention, adding 2-BrO, blocks the last step of triacylglycerol synthesis by inhibiting DGAT activity, blocks the process of converting diacylglycerol and fatty acyl-CoA into triacylglycerol, increases the interaction between CIDEC and HDAC6, enhances the deacetylation of HDAC6, thereby making HDAC6 unstable and reducing lipid droplet fusion and fat storage; adding anacardic acid, anacardic acid is a histone acetyltransferase (HAT) inhibitor with an IC50 value of about 5 μM for PCAF, promotes the binding of CIDEC to HDAC6 by inhibiting the activity of PCAF, resulting in the deacetylation of HDAC6, thereby being unstable and reducing lipid droplet fusion, and synergistically acting with 2-BrO; adding endothelin 3 to activate the cAMP-EPAC1-ERK pathway, promotes the proliferation of preadipocytes and the upregulation of UCP1, and then differentiates into thermogenic beige adipocytes in white adipose tissue; adding 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), 8-cyclopentyl-1,3-dipropylxanthine is a highly selective adenosine A1 receptor (A1R) antagonist, which can inhibit the signal transduction of adenosine by blocking the A1 receptor, thereby avoiding its inhibitory effect on lipolysis and thermogenesis; S-4-nitrobenzyl-6-thioinosine as an adenosine transport inhibitor can inhibit the transport of adenosine, reduce its accumulation outside white adipocytes, and inhibit the signal transduction of extracellular adenosine, and synergistically act with 8-cyclopentyl-1,3-dipropylxanthine; adding catecholamine, under the stimulation of catecholamine, PLIN1 is phosphorylated, resulting in the dissociation of CGI-58 from PLIN1, enabling it to interact with ATGL, thereby activating the TG hydrolysis activity of ATGL. Under basal conditions, CGI-58 binds to lipid droplet coating proteins (such as PLIN1) and inhibits the activity of ATGL. Under the stimulation of catecholamine, PLIN1 is phosphorylated, resulting in the dissociation of CGI-58 from PLIN1 and its interaction with ATGL, thereby activating ATGL, and CGI-58 also dissociates from perilipin-1, promoting the binding of CGI-58 to the lipase ATGL to form a CGI-58 / ATGL complex, effectively promoting lipolysis and hydrolyzing triacylglycerol into diacylglycerol.Liposomes with surface-modified CKGGRAKDC serve as carriers targeting white adipocytes. The carrier targeting white adipocytes encapsulates 2-BrO, anacardic acid, S-4-nitrobenzyl-6-thioinosine, endothelin-3, catecholamine. The short peptide (CKGGRAKDC) can specifically bind to prohibitin (PHB) mainly expressed on the surface of vascular endothelial cells and mature white adipocytes in white adipose tissue. After the liposomes enter the cells, ATGL may act on the triglyceride component in the liposome membrane, leading to the instability of the liposomes and releasing the composition that promotes the browning of white adipocytes. It has strong targeting, so as to improve the safety and effectiveness of the drug; Brief Description of the Drawings

[0025] To more clearly illustrate the embodiments of the present invention, the drawings required for the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on the provided drawings.

[0026] Figure 1 It is a result diagram of HE staining and Oil Red O staining for evaluating the content of white adipose tissue in the lower abdominal adipose tissue in Example 1;

[0027] Figure 2 It is a result diagram of the size of white adipocytes in the lower abdominal adipose tissue in Example 1;

[0028] Figure 3 It is a result diagram of the number of white adipocytes in the lower abdominal adipose tissue in Example 1. Detailed Embodiments

[0029] Combined with specific embodiments below, the present invention will be further described. It should be noted that, on the premise of no conflict, any combination of the following-described embodiments or technical features can form a new embodiment.

[0030] The inhibin targeting peptide (NH2-GKGGRAKDGGC-Amide) was purchased from SCRUM.

[0031] Example 1

[0032] A composition for promoting the browning of white adipocytes provided by this embodiment is prepared from raw materials with the following weight ratios: 2-BrO 5 parts, anacardic acid 3 parts, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) 2 parts, endothelin-3 1 part, S-4-nitrobenzyl-6-thioinosine (NBTI) 0.1 part, and catecholamine 2 parts; liposomes modified with CKGGRAKDC are used as carriers targeting white adipocytes, and the carriers targeting white adipocytes are coated with 2-BrO, anacardic acid, S-4-nitrobenzyl-6-thioinosine, endothelin-3, and catecholamine.

[0033] In this embodiment, the EDN3 / EDNRB signal activates the cAMP-EPAC1-ERK pathway, promotes the proliferation of preadipocytes and the upregulation of UCP1, and then differentiates into thermally active beige adipocytes in white adipose tissue. Among them, EPAC1 has recently been considered a key molecule that, independent of the PKA pathway, plays a role in the proliferation and differentiation of thermogenic preadipocytes under β-adrenergic signaling or cold stimulation. In the white adipose tissue niche, EDN3 is produced by the stromal vascular fraction of white adipose tissue. Although the basal level of EDN3 in white adipose tissue is low, cold exposure can further induce it.

[0034] Role in promoting the browning of white adipose tissue (WAT). Overexpression of EDNRB stimulates the thermogenic differentiation of human white preadipocytes by activating cAMP-EPAC1-ERK

[0035] Endothelin 3 (EDN3): By activating the EDNRB signaling pathway, it promotes the generation and thermogenesis of brown adipocytes in white adipose tissue, demonstrating that EDNRB activation promotes the browning of human white preadipocytes through cAMP-EPAC1-ERK activation.

[0036] In white adipose tissue, histone deacetylase 6 (HDAC6) deacetylates CIDEC, leading to the instability of CIDEC, thereby reducing lipid droplet fusion. On the contrary, FAs can prevent the deacetylation of CIDEC by promoting the dissociation of CIDEC from HDAC6, thereby promoting lipid droplet fusion.

[0037] Regulatory role of fatty acids: The metabolic state of fatty acids (whether they are converted into triglycerides, triacylglycerols) affects the interaction between PCAF and CIDEC. The interaction between PCAF and CIDEC induced by fatty acids mainly occurs on the endoplasmic reticulum, and this process is affected by the triglyceride synthesis pathway.

[0038] Lipid droplets are organelles that store lipids. By regulating the size and number of lipid droplets, it is possible to affect the release and transport of fatty acids, thereby promoting thermogenesis. These compounds can activate the lipase activity on the surface of lipid droplets and promote lipolysis of lipid droplets.

[0039] In this example, Anacardic Acid: Anacardic acid is a natural compound extracted from cashew nut shells and is a histone acetyltransferase (HAT) inhibitor. Its IC50 value for PCAF is approximately 5 μM. Anacardic acid promotes the binding of CIDEC to HDAC6 by inhibiting the activity of PCAF, resulting in its deacetylation, thereby being unstable and reducing lipid droplet fusion.

[0040] In white adipocytes, the acetylation of CIDEC is mediated by PCAF and occurs in the endoplasmic reticulum. After acetylation, CIDEC is released from the endoplasmic reticulum and transferred to the surface of lipid droplets. PCAF (p300 / CBP-associated factor) is an important histone acetyltransferase (HAT) that can not only acetylate histones but also non-histones such as CIDEC. The acetylation process of CIDEC mainly occurs in the endoplasmic reticulum because the endoplasmic reticulum is the site of triglyceride synthesis and lipid droplet formation. PCAF acetylates CIDEC to make it stable and transfer it to the surface of lipid droplets, thereby promoting lipid droplet fusion and fat storage. After being released from the endoplasmic reticulum, acetylated CIDEC is transferred to the surface of lipid droplets, promoting the clustering and fusion of lipid droplets, increasing the size of lipid droplets, which is beneficial to fat storage.

[0041] In this example, regulating the lipid synthesis pathway: By inhibiting the conversion of fatty acids to triacylglycerols, it is possible to reduce the acetylation of CIDEC, thereby inhibiting lipid droplet fusion. 2-BrO blocks the last step of triacylglycerol synthesis by inhibiting DGAT activity, blocking the process of converting diacylglycerol (DAG) and fatty acyl-CoA to triacylglycerol (TAG). When triacylglycerol synthesis is blocked by 2-BrO, the level of acetylated CIDEC and the association between PCAF and CIDEC will be significantly reduced. However, the interaction between CIDEC and HDAC6 increases, resulting in the deacetylation of HDAC6, thereby making HDAC6 unstable and reducing lipid droplet fusion and fat storage. Lipid droplet proteins are wrapped by various proteins, and their roles in thermogenic lipid droplets include members of the PLIN family and lipases that are crucial for lipid droplet function. The most important role of lipid droplets in thermogenic lipids is to maintain body temperature by rapidly generating the fatty acids required for mitochondrial combustion and thermogenesis under cold stimulation. The rapid mobilization of lipids depends on lipases adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) on the surface of lipid droplets.

[0042] cAMP further activates protein kinase A (PKA), leading to the phosphorylation of Plin1, which in turn activates ATGL. ATGL hydrolyzes triglycerides stored in lipid droplets into diacylglycerol and fatty acids, which is the rate-limiting step of triglyceride hydrolysis. HSL is phosphorylated by PKA and then hydrolyzes diacylglycerol into fatty acids and monoacylglycerol, which can be hydrolyzed by HSL, MGL, and ABHD5 into glycerol and fatty acids. The fatty acids released in the above steps are transported to the mitochondria by FABP, activate UCP1, and serve as combustion substrates, ultimately dissipating the H+ potential energy through proton leakage to generate heat.

[0043] In this example, the separation of CGI-58 from Plin1 enables it to interact with ATGL, thereby activating the hydrolytic activity of ATGL for triacylglycerol. Under basal conditions, CGI-58 binds to lipid droplet coating proteins (such as PLIN1), inhibiting the activity of ATGL. Upon catecholamine stimulation, PLIN1 is phosphorylated, resulting in the dissociation of CGI-58 from PLIN1 and its interaction with ATGL, thereby activating ATGL. CGI-58 also dissociates from perilipin-1, promoting the binding of CGI-58 to the lipase ATGL to form a CGI-58 / ATGL complex, effectively promoting lipolysis and hydrolyzing triacylglycerol into diacylglycerol.

[0044] In this example, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) is a highly selective adenosine A1 receptor (A1R) antagonist that can inhibit adenosine signaling by blocking the A1 receptor, thereby avoiding its inhibitory effect on lipolysis and thermogenesis.

[0045] Adenosine can regulate the function of adipocytes by acting on different subtypes of adenosine receptors (such as A1, A2A, A2B). Among them, the activation of the A1 receptor can affect the expression of genes related to lipolysis and thermogenesis by inhibiting the production of cAMP.

[0046] In this example, S-4-nitrobenzyl-6-thioinosine (NBTI), as an adenosine transport inhibitor, can inhibit the transport of adenosine, reduce its accumulation outside white adipocytes, and inhibit the signaling of extracellular adenosine.

[0047] In this example, the surface of the liposome is modified with maleimide-modified polyethylene glycol. The specific preparation method includes the following steps:

[0048] S1. Maleimide-modified polyethylene glycol-phospholipid is dissolved in an organic solvent, and maleimide-modified polyethylene glycol-phospholipid accounts for 1 mol% to 1.25 mol% of the total lipids.

[0049] S2. Dissolving 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and cholesterol in an organic solvent to form a lipid solution, and then adding the solution of step S1, wherein the molar ratio of maleimide-modified polyethylene glycol-phospholipid to total lipid in the lipid solution is 1:100 to 1.25:100;

[0050] S3. Mix the solution from step S2 with an equal volume of diisopropyl ether, add 10 mM HEPES buffer, and stir evenly;

[0051] S4, treating the solution in step S3 with ultrasound to promote the formation of liposomes, the ultrasound treatment time being 15s to 45s;

[0052] S5, removing the organic solvent by nitrogen evaporation to obtain a preliminary liposome solution;

[0053] S6. Ultrasonic treatment of the preliminary liposome solution to ensure uniform dispersion of the liposomes, the ultrasonic treatment time is 45 s;

[0054] S7. The solution of step S6 is dialyzed in a buffer solution to remove unreacted maleimide-modified polyethylene glycol-phospholipid and other impurities. The dialysis time is 2 h to obtain maleimide-modified polyethylene glycol liposomes.

[0055] In this embodiment, in step S3, the step after mixing the solution of step S2 with an equal volume of diisopropyl ether is specifically as follows: 2-BrO, anacardic acid, S-4-nitrobenzyl-6-thioinosine, 1 to 2 parts of purine, endothelin-3, and catecholamines are weighed according to the formula, mixed together, added to 10 mM HEPES buffer, and mixed together to finally obtain maleimide-modified polyethylene glycol liposomes coated with 2-BrO, anacardic acid, S-4-nitrobenzyl-6-thioinosine, endothelin-3, and catecholamines.

[0056] In this example, the preparation method of liposomes surface-modified with CKGGRAKDC is as follows: 10 mM of the inhibin targeting peptide CKGGRAKDC is dissolved in distilled water, and maleimide-modified polyethylene glycol liposomes coated with 2-BrO, anacardic acid, S-4-nitrobenzyl-6-thioinosine, endothelin-3, and catecholamine are added in a molar ratio of 1:1, and the reaction is shaken at 30°C for 24 hours to obtain liposomes surface-modified with CKGGRAKDC.

[0057] This embodiment also provides a method for preparing a composition for promoting browning of white adipocytes, comprising the following steps: mixing liposomes surface-modified with CKGGRAKDC and 8-cyclopentyl-1,3-dipropyl yellow to obtain a composition for promoting browning of white adipocytes.

[0058] Example 2

[0059] A composition for promoting the browning of white adipocytes provided in this example is prepared from raw materials with the following weight ratios: 2-BrO 6 parts, anacardic acid 2 parts, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) 1 part, endothelin-3 2 parts, S-4-nitrobenzyl-6-thioinosine (NBTI) 0.3 part, catecholamine 1 part; liposomes surface-modified with CKGGRAKDC are used as carriers targeting white adipocytes, and the carriers targeting white adipocytes are coated with 2-BrO, anacardic acid, S-4-nitrobenzyl-6-thioinosine, endothelin-3, and catecholamine.

[0060] Example 3

[0061] A composition for promoting the browning of white adipocytes provided in this example is prepared from raw materials with the following weight ratios: 2-BrO 8 parts, anacardic acid 5 parts, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) 1.5 parts, endothelin-3 1.5 parts, S-4-nitrobenzyl-6-thioinosine (NBTI) 0.5 part, catecholamine 1.5 part; liposomes surface-modified with CKGGRAKDC are used as carriers targeting white adipocytes, and the carriers targeting white adipocytes are coated with 2-BrO, anacardic acid, S-4-nitrobenzyl-6-thioinosine, endothelin-3, and catecholamine.

[0062] Experimental Example

[0063] 1. Animal Experiment

[0064] C57BL / 6J male mice at 5 weeks old and weighing 16 - 20 g were purchased from Top-Bio Technology Co., Ltd. in Shenzhen, China. The mice were housed in a temperature-controlled animal facility with humidity control, and had free access to food and water. The temperature was 25°C, and there was a 12-hour light-dark cycle. After one week of adaptive feeding, the mice were divided into a normal control group, a model group, and Example 1 - 3 groups, with 10 mice in each group. The 6-week-old mice in the model group and Example 1 - 3 groups were fed a high-fat diet with a mass percentage of 60% for 12 weeks. The body weight of the mice was recorded daily, and a weight gain of more than 30% was set as the standard for successful establishment of the obesity model. An obesity model of mice was established. The normal control group continued to be fed a standard mouse diet. After the obesity model of mice was successfully established after 12 weeks, the mice in Example 1 - 3 groups were fed the composition for promoting the browning of white adipocytes provided in Example 1 - 3, and the mice in the normal control group and the model group were fed the standard mouse diet. After feeding for 3 weeks, the following subsequent tests were carried out after 15 weeks. The above animal experiment protocol has passed the review and approval of the Animal Ethics Committee.

[0065] 2. Collect the adipose tissue under the abdomen and preserve it in a 4% paraformaldehyde solution by volume. The samples are dehydrated through an ethanol gradient and fixed in paraffin to make paraffin sections. The paraffin sections are stained with hematoxylin for 5 minutes, dehydrated with graded alcohol, then stained with eosin for 5 minutes. After dehydration with alcohol, the sections are sealed with neutral resin. Prepare an Oil Red O dye solution by mixing the original Oil Red O solution and distilled water at a volume ratio of 3:2. Stain the sections with the Oil Red O staining solution, and stain the cells in the dark at room temperature for 20 min. Subsequently, wash the cells with PBS, differentiate with 60% isopropanol by volume, counterstain with hematoxylin, and mount with glycerin gelatin. Figure 1 Randomly capture images of the Oil Red O-stained samples under an optical microscope, and then use ImageJ software to measure the stained area.

[0066] 3. Estimation of the size and number of adipocytes

[0067] Digitally process the prepared adipose tissue sections with panoramic software and perform detailed image analysis using a Scan 150 system. Use ImageJ software to obtain the average diameter (d) of adipocytes. The weight of a single adipocyte can be estimated based on its volume (approximated as a sphere) and the density of adipose tissue (0.9 g / ml). The volume of a sphere is πd 3 / 6. The QMR06-090H analyzer is a low-field nuclear magnetic resonance instrument for small animal body composition analysis, which can quickly and non-destructively measure the fat content of mice. Use the QMR06-090H analyzer to measure the weight of adipose tissue in mouse tissues, and the measurement results are as Figure 2 shown. The total number of adipocytes in vivo is estimated by calculating the body fat weight and the adipocyte volume distribution. Divide the body fat weight by the average volume of adipocytes to obtain the total number of adipocytes, and the measurement results are as Figure 2 shown.

[0068] Experimental results: It can be seen from Figure 1 that through Oil Red O and HE staining, the fat deposition in the mice of Example 1 group is significantly lower than that in the model control group; it can be seen from Figure 2 that the size and number of white adipocytes in Examples 1-3 are lower than those in the model control group.

[0069] The above embodiments are only the preferred embodiments of the present invention, and the scope of protection of the present invention cannot be limited thereby. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention fall within the scope of protection required by the present invention.

Claims

1. A composition for promoting the browning of white adipocytes, characterized in that, It is prepared from the following raw materials in the following weight ratios: 2-BrO 5-8 parts, anacardic acid 2-5 parts, 8-cyclopentyl-1,3-dipropylxanthine 1-2 parts, endothelin-3 1-2 parts, S-4-nitrobenzyl-6-thioinosine 0.1-0.5 parts, catecholamine 1-2 parts; liposomes modified with CKGGRAKDC on the surface are used as carriers targeting white adipocytes, and the carriers targeting white adipocytes are coated with 2-BrO, anacardic acid, S-4-nitrobenzyl-6-thioinosine, endothelin-3, and catecholamine.

2. The composition for promoting browning of white adipocytes according to claim 1, wherein It is prepared from the following raw materials in the following weight ratios: 2-BrO 5 parts, anacardic acid 3 parts, 8-cyclopentyl-1,3-dipropylxanthine 2 parts, endothelin-3 1 part, S-4-nitrobenzyl-6-thioinosine 0.1 part, catecholamine 2 parts.

3. The composition for promoting browning of white adipocytes according to claim 1, characterized in that, The surface of the liposome is modified with maleimide-modified polyethylene glycol, and the specific preparation method includes the following steps: S1. Maleimide-modified polyethylene glycol-phospholipid is dissolved in an organic solvent, and the maleimide-modified polyethylene glycol-phospholipid accounts for 1 mol% to 1.25 mol% of the total lipids. S2. 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and cholesterol are dissolved in an organic solvent to form a lipid solution, and then the solution in step S1 is added. The molar ratio of maleimide-modified polyethylene glycol-phospholipid to total lipids in the lipid solution is 1:100 to 1.25:100; S3. The solution in step S2 is mixed with an equal volume of diisopropyl ether, and 10 mM HEPES buffer is added and stirred evenly; S4. The solution in step S3 is treated with ultrasonic waves to promote the formation of liposomes, and the ultrasonic treatment time is 15 s to 45 s; S5. The organic solvent is removed by nitrogen evaporation to obtain a preliminary liposome solution; S6. The preliminary liposome solution is ultrasonically treated to ensure uniform dispersion of the liposomes, and the ultrasonic treatment time is 45 s; S7. The solution in step S6 is dialyzed in a buffer to remove unreacted maleimide-modified polyethylene glycol-phospholipid and other impurities. The dialysis time is 2 h to obtain liposomes of maleimide-modified polyethylene glycol.

4. The composition for promoting browning of white adipocytes according to claim 3, wherein In step S3, the specific steps after mixing the solution in step S2 with an equal volume of diisopropyl ether are: Weigh 2-BrO, anacardic acid, S-4-nitrobenzyl-6-thioinosine, purine 1-2 parts, endothelin-3, and catecholamine according to the formula amount, mix them together, add them to the 10 mM HEPES buffer, and mix them together. Finally, liposomes of maleimide-modified polyethylene glycol coated with 2-BrO, anacardic acid, S-4-nitrobenzyl-6-thioinosine, endothelin-3, and catecholamine are obtained.

5. The composition for promoting browning of white adipocytes according to claim 4, wherein The preparation method of the liposome with surface modification of CKGGRAKDC is as follows: Dissolve 10 mM inhibin targeting peptide CKGGRAKDC in distilled water, add the liposome of maleimide-modified polyethylene glycol coated with 2-BrO, anacardic acid, S-4-nitrobenzyl-6-thioinosine, endothelin-3, and catecholamine according to a molar ratio of 1:1, and react with shaking at 30 °C for 24 h to obtain the liposome with surface modification of CKGGRAKDC.

6. A method for preparing a composition for promoting browning of white adipocytes according to any one of claims 1-5, characterized in that, It includes the following steps: Mix the liposome with surface modification of CKGGRAKDC and 8-cyclopentyl-1,3-dipropylxanthine together to obtain the composition for promoting browning of white adipocytes.

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