Use of an exogenous phospholipid liposome in the preparation of a product for inducing the browning transition of white adipose tissue

Abstract

This invention discloses the application of exogenous phospholipid liposomes in the preparation of products for inducing the browning transformation of white adipose tissue. By providing exogenous phospholipids to white adipocytes, the lipid droplet size is significantly reduced, and the expression of coupling protein 1 (UCP1) and peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) is upregulated, enhancing mitochondrial oxidative metabolism and promoting the transformation of white adipocytes to a brown-like phenotype. The exogenous phospholipids are preferably in liposome form and may contain one or more of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and sphingomyelin. This invention provides a novel strategy for achieving white adipose tissue browning by utilizing exogenous phospholipid-mediated lipid droplet remodeling, achieving metabolic reprogramming of adipose tissue without relying on gene editing or drug receptor activation. This strategy can be used for the development of drugs or products for metabolic diseases such as obesity, insulin resistance, type 2 diabetes, non-alcoholic fatty liver disease, and metabolic syndrome.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of an exogenous phospholipid liposome in the preparation of products for inducing the browning transformation of white fat. Background Technology

[0002] Obesity is a chronic metabolic disease characterized by abnormal expansion of white adipose tissue and imbalance in energy metabolism. With changes in lifestyle and an increase in high-calorie diets, the incidence of obesity and related diseases (including type 2 diabetes, non-alcoholic fatty liver disease, insulin resistance, and metabolic syndrome) continues to rise, becoming a significant public health problem that seriously threatens human health.

[0003] Adipose tissue is mainly divided into white adipose tissue (WAT) and brown adipose tissue (BAT). White adipocytes are primarily responsible for energy storage and typically contain one or a few large lipid droplets; brown adipocytes are rich in mitochondria and contain multiple small lipid droplets, exhibiting strong fatty acid oxidation and energy expenditure capabilities. In recent years, inducing white adipocytes to acquire a brown-like phenotype (i.e., white adipose browning) to enhance the body's energy expenditure has been considered an important strategy for treating obesity and related metabolic diseases. Currently available methods for white adipose browning mainly include: β3-adrenergic receptor agonists; PPARγ agonists; cold stimulation; gene editing; transcription factor regulation; and drug intervention. While these methods can promote white adipose browning to some extent, they generally suffer from side effects, insufficient long-term safety, difficulties in clinical translation, or reliance on complex molecular regulation.

[0004] Lipid droplets are important organelles within cells responsible for storing neutral lipids, and their size, number, and distribution are closely related to cellular metabolic status. Current research mainly focuses on regulating lipid droplet formation, fusion, or degradation through transcription factors, metabolic enzymes, hormones, and signaling pathways, thereby indirectly affecting adipocyte function. However, a method for directly regulating lipid droplet morphology using exogenous materials to achieve adipocyte phenotypic regulation is currently lacking. Therefore, developing a safe, effective, and highly operable novel adipocyte regulation strategy is of great significance for improving energy metabolism disorders and developing novel metabolic regulatory products. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0006] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0007] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a method for inducing the browning transformation of white fats by exogenous phospholipid-mediated lipid droplet remodeling.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution: the application of exogenous phospholipid liposomes in the preparation of products for inducing the browning transformation of white fats, characterized in that: the product uses exogenous phospholipid liposomes as the active ingredient; The phospholipid components of the exogenous phospholipid liposomes, by mass percentage, include: 5%~100% phosphatidylcholine, 10%~100% phosphatidylethanolamine, 2%~50% phosphatidylinositol, 2%~50% phosphatidylserine, 2%~50% sphingomyelin, and the liposome particle size is 50~500 nm. The exogenous phospholipid liposomes integrate into the lipid droplet membrane to reshape the lipid droplet membrane structure, mediating morphological remodeling of lipid droplets within white adipocytes, thereby enabling white adipocytes to acquire a thermogenic phenotype.

[0009] In a preferred embodiment of the application described in this invention, the mass ratio of phosphatidylcholine: phosphatidylethanolamine: phosphatidylinositol: phosphatidylserine: sphingomyelin is 57:24:13:3:3.

[0010] In a preferred embodiment of the application described in this invention, the liposomes have a particle size of 100–300 nm, a polydispersity index (PDI) ≤ 0.2, and a zeta potential of -40–-70 mV.

[0011] As a preferred embodiment of the application described in this invention, the lipid droplet morphology reshaping includes one or more of the following variations: The average size of lipid droplets decreased; The number of lipid droplets decreased; The average area of ​​lipid droplets decreases; The spatial distribution of lipid droplets changes.

[0012] As a preferred embodiment of the application described in this invention, the heat-generating phenotype includes one or more of the following variations: Increased expression of uncoupling protein 1, or UCP1; Increased expression of peroxisome proliferator-activated receptor gamma coactivator 1α, i.e., PGC-1α; Increased cellular oxygen consumption; Increased mitochondrial proton leakage; An increase in the number of mitochondria; The degree of mitochondrial networking is enhanced.

[0013] In a preferred embodiment of the application described in this invention, the white adipocytes are selected from mature white adipocytes, adipocytes formed during the differentiation of adipocyte precursor cells, primary adipocytes, human adipocytes, and adipocytes derived from 3T3-L1.

[0014] Another objective of this invention is to overcome the shortcomings of the prior art and provide a liposome formulation for inducing the browning transformation of white adipocytes, characterized in that: the liposome formulation is prepared from exogenous phospholipids, the components of which include phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and sphingomyelin; the particle size of the liposome formulation is 50-500 nm, which can mediate the morphological remodeling of lipid droplets in white adipocytes, enabling white adipocytes to acquire a thermogenic phenotype.

[0015] As a preferred embodiment of the liposome formulation of the present invention, the liposome formulation can further induce one or more of the following changes in white adipocytes: enhanced mitochondrial fusion, enhanced selective mitophagy, increased oxidative phosphorylation level, enhanced fatty acid oxidation capacity, and enhanced endoplasmic reticulum-lipid droplet-mitochondrial interaction.

[0016] Another objective of this invention is to overcome the shortcomings of the prior art and provide the application of exogenous phospholipid liposomes in the preparation of drugs for the treatment or prevention of metabolic diseases, characterized in that: the drug uses exogenous phospholipid liposomes as the active ingredient; The components of the exogenous phospholipid include phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and sphingomyelin; The metabolic diseases mentioned include obesity, insulin resistance, type 2 diabetes, non-alcoholic fatty liver disease, and metabolic syndrome.

[0017] Beneficial effects of this invention: (1) No gene editing required: No need to construct transgenic animals or perform gene manipulation; (2) No receptor agonist required: Not dependent on drugs such as β3 receptor agonists; (3) Good biocompatibility: The material is constructed using natural phospholipids, which has good biocompatibility; (4) It can directly act on mature adipocytes: it can change the size of lipid droplets in mature white adipocytes; (5) It can promote the acquisition of thermogenic phenotype by adipocytes: it can reshape mitochondrial morphology and function and enhance thermogenesis; (6) It has good scalability and can be applied to different types of lipid droplet-related cells. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein: Figure 1 A schematic diagram illustrating the process by which exogenous phospholipid liposomes reshape the lipid droplet morphology of white adipocytes and induce their transformation to a brownish phenotype. Figure 2 Size distribution and TEM images of exogenous phospholipid liposomes; Figure 3 exogenous phospholipids ER The effect of LPO on in vitro lipid droplet models; Figure 4 exogenous phospholipids ER The effect of LPO on lipid droplet morphology in mature white adipocytes; Figure 5 Adding exogenous phospholipids during the differentiation process ER The effect of LPO on lipid droplet morphology in white adipocytes; Figure 6 It is a single-component exogenous phospholipid PC The effect of LPO on lipid droplet morphology in white adipocytes; Figure 7 exogenous phospholipids ER The effect of LPO on the morphology of adipocyte mitochondria; Figure 8 exogenous phospholipids ER The effect of LPO on adipocyte mitochondrial function; Figure 9 A comparison of the expression of browning marker proteins in white adipocytes after the action of exogenous phospholipids. Detailed Implementation

[0019] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0020] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0021] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0022] The raw materials used in this invention were: dexamethasone, indomethacin, rosiglitazone, triiodothyronine, phosphatidylethanolamine, sphingomyelin, lecithin, 1,2-dipalmitoyl-sn-glycerol-3-phosphate-L-serine (sodium salt), and phosphatidylinositol, all purchased from Aladdin Biotech Co., Ltd. (Shanghai, China). Insulin, MitoTracker RedCMXRos, and Hoechst 33342 were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Recombinant Anti-UCP1 antibody was purchased from Abcam (Cambridge, UK). High-efficiency RIPA tissue lysis buffer and recombinant Anti-β-tubulin antibody were purchased from Solarbio (Beijing, China). PGC-1α monoclonal antibody was purchased from Proteintech (Wuhan, China). 3-Isobutyl-1-methylxanthine (IBMX) was purchased from Yuye Biotechnology Co., Ltd. (Shanghai, China). BODIPY 493 / 503 were purchased from Cayman Chemical (Ann Arbor, USA). Seahorse XF 100 mM pyruvate solution, XFe96 / XF Pro probe plate, XFe96 / XF Pro cell culture microplate, Seahorse XF 200 mM glutamine solution, Seahorse XF DMEM medium, Seahorse XF calibration solution (500 mL), Seahorse XF 1.0 M glucose solution, and Seahorse XF mitochondrial stress assay kit were all purchased from Agilent Technologies (Santa Clara, USA).

[0023] The instruments used in the embodiments of this invention are: dynamic light scattering particle size analyzer (Malvern, Zetasizer Nano ZS), transmission electron microscope (JEOL JEM-1400), rotary evaporator (Shanghai Yarong, RE-52AA), ultrasonic disruptor (Ningbo Xinzhi, SB-5200D), fluorescence biological microscope (Mingmei, MF53-M), laser confocal microscope (Zeiss, LSM800 with Airyscan), cell energy metabolism analysis system (Agilent, Seahorse XFe 96), WB power supply (Shanghai Tianneng, ESP300), WB electrophoresis tank (Shanghai Tianneng, EPS300), and multicolor gel imaging analyzer (Shanghai Tianneng, Tanon Chemi Dog ULTRA).

[0024] Figure 1 The schematic diagram illustrating the process by which exogenous phospholipid liposomes reshape the morphology of white adipocyte lipid droplets and induce their transformation to a brown phenotype, provided by this invention, visually presents the core mechanism of action and the entire process of phenotypic change. The structure of each part and the corresponding technical principle are as follows: The left-hand region illustrates the basic morphology of a typical mature white adipocyte: the cell is characterized by large, unilocular or few-locular lipid droplets as its core, with each large droplet occupying the majority of the cytoplasmic space; mitochondria are sparse, distributed in a fragmented, dotted pattern, and confined to a narrow region around the nucleus and inside the plasma membrane; the cell as a whole is in a low metabolic state of energy storage, with extremely low expression levels of heat-related proteins such as uncoupling protein 1 (UCP1). This part corresponds to the intervention target of this invention and is also a classic morphological feature of white adipose tissue in the prior art.

[0025] The central area illustrates the process of exogenous phospholipid liposomes entering the cell, which is the core innovation of this invention that distinguishes it from the prior art: exogenous phospholipid liposomes interact with intracellular lipid droplets, and their phospholipid components integrate into the monolayer phospholipid membrane of the lipid droplets. By changing the phospholipid composition and interfacial tension of the lipid droplet membrane, the original interfacial homeostasis of the large lipid droplets is broken, directly driving the large lipid droplets to undergo fragmentation and splitting, and actively completing the remodeling of lipid droplet morphology.

[0026] This regulatory pathway is completely different from traditional browning schemes: existing technologies indirectly induce lipid droplet fragmentation through biological pathways such as receptor activation and transcriptional regulation, and lipid droplet morphology changes are a downstream consequence of the browning process; however, this invention takes physical interface regulation as the starting point, and uses lipid droplet morphology reshaping as an upstream triggering factor for browning, achieving a technological breakthrough in reverse regulation.

[0027] The right-hand area shows the brown-like thermogenic phenotype obtained by cells after lipid droplet remodeling, demonstrating the final technical effect of this invention: At the lipid droplet level: the original large lipid droplets are broken down into small lipid droplets, which effectively promotes the transformation of white adipocytes into brown thermogenic phenotypes; At the organelle level: the number of mitochondria increases significantly, forming a highly branched network structure that is widely distributed in the cytoplasm; at the same time, the interaction between the endoplasmic reticulum, lipid droplets, and mitochondria is enhanced, providing a structural basis for oxidative metabolism; Functionally: The expression of thermogenic marker proteins such as UCP1 and PGC-1α is significantly upregulated, and the cells’ ability to oxidative phosphorylation and fatty acid oxidation is enhanced, ultimately transforming them from energy storage cells to energy-consuming thermogenic cells.

[0028] The following description, based on the results of exogenous phospholipid liposomes, physicochemical characterization, and cell experiments, further illustrates the implementation scheme of the present invention.

[0029] Example 1: Exogenous phospholipid liposomes ( ER Preparation of LPO This embodiment provides an exogenous phospholipid liposome that simulates the lipid composition of the endoplasmic reticulum membrane. ER Preparation method of LPO. Phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), and sphingomyelin (SM) were selected as lipid components and mixed in a mass ratio of 57:24:13:3:3. Liposomes were prepared using a thin-film hydration-extrusion method, and the specific steps are as follows: Weigh each lipid component according to the above mass ratio and dissolve it in chloroform to make the total lipid concentration 10 mg / mL. Transfer the solution to a round-bottom flask and rotary evaporate it in a 37°C water bath (50 rpm) until a uniform, transparent lipid film is formed. Continue vacuuming for 30 min to completely remove residual organic solvent. Add preheated phosphate-buffered saline (PBS, pH 7.4) to the flask to make the final lipid concentration 5 mg / mL, and hydrate it by shaking in an ultrasonic cleaner for 30 min to obtain a multilayer liposome suspension. Extrude the liposome suspension through a 200 nm polycarbonate membrane 21 times using an extruder to obtain monolayer liposomes with uniform particle size. ER LPO should be stored at 4°C for later use.

[0030] Example 2: ER Physicochemical properties characterization of LPO This embodiment describes the physicochemical characterization of the exogenous phospholipid liposomes prepared in Example 1, specifically as follows: The average hydrated particle size, polydispersity index (PDI), and zeta potential of liposomes ERLPO were determined using a dynamic light scattering particle size analyzer (Malvern Zetasizer Nano ZS). The average particle size of the liposomes was measured to be 163.87 ± 5.50 nm, the PDI was 0.15 ± 0.05, and the potential was -60.9 ± 1.61 mV.

[0031] The morphology of liposomes was observed using transmission electron microscopy (TEM). Liposome suspensions were dropped onto a copper grid, negatively stained with 2% phosphotungstic acid, dried, and observed under a JEOL JEM-1400 electron microscope. Results are as follows: Figure 2 As shown, the liposomes are spherical or ellipsoidal with clear boundaries, and no obvious aggregation was observed.

[0032] The above results indicate that ER LPO exhibits good stability and uniformity.

[0033] Example 3: Establishment of an in vitro lipid droplet model To evaluate the effect of exogenous phospholipids on lipid droplet structure, an in vitro lipid droplet model was first constructed: using triglycerides as the oil phase and phosphatidylcholine (PC) as the surfactant, an in vitro lipid droplet model was established using ultrasonic emulsification. Specifically: 2 mg of PC was dissolved thoroughly in 1 mL of chloroform using ultrasound. The chloroform was then evaporated using a rotary evaporator. 400 μL of triglycerides was added and the solution was dissolved by vigorous vortexing. 100 μL of the solution was then diluted with 1 mL of 25 mM Tris (pH 7.4) buffer containing 1 mM MgCl2 and emulsified thoroughly using an ultrasonic homogenizer to obtain triglyceride-dispersed oil droplets. The morphology of the droplets was observed using an optical microscope, yielding uniformly sized and morphologically stable droplet structures, which can be used to evaluate the effect of exogenous phospholipids on droplet morphology.

[0034] Example 4: Effect of exogenous phospholipid liposomes on in vitro lipid droplet morphology The preparation obtained in Example 1 ER LPO was added to the lipid droplet model established in Example 3 at different concentrations (0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL), while the control group received an equal volume of PBS buffer. The mixtures were incubated at room temperature, and the concentrations were recorded using an inverted microscope. ER Morphological changes after LPO treatment were observed, and the size of lipid droplets was measured using ImageJ software. After a certain treatment time, the morphological changes of lipid droplets were observed using an optical microscope.

[0035] The results are as follows Figure 3 As shown, where, Figure 3 (a) Construction of lipid droplets in vitro with different concentrations EROptical microscope images showing morphological changes after LPO treatment; Figure 3 (b) is a comparison of lipid droplet size changes with ERLPO concentration.

[0036] The results showed that the lipid droplets in the control group remained spherical, with no significant size change throughout the incubation period. The liposome-treated group showed a concentration-dependent reduction in lipid droplet size. However, with… ER As LPO concentration increases, the average size of lipid droplets gradually decreases. This indicates that exogenous phospholipid liposomes can effectively reduce the size of lipid droplets in vitro and reshape their morphology.

[0037] Example 5: Establishment of a 3T3-L1 white adipocyte model Mouse 3T3-L1 preadipocytes were induced to differentiate into mature white adipocytes. Cells were seeded in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin and cultured at 37°C and 5% CO2. Once cell confluence reached 80%, differentiation was initiated using an induction culture medium.

[0038] The induction culture medium consisted of: 0.5 mmol / L 3-isobutyl-1-methylxanthine; 1 μmol / L dexamethasone; 10 μg / mL insulin; 10% fetal bovine serum; and DMEM basal medium. After 48 h of induction, the medium was replaced with maintenance medium containing 10 μg / mL insulin. The culture medium was changed every 2 days. After approximately 8–10 days of continuous culture, numerous lipid droplet structures appeared within the cells. Intracellular lipid droplets were observed using BODIPY staining.

[0039] The results showed that a large number of large lipid droplets were formed inside the cells, indicating that a mature white adipocyte model was successfully established.

[0040] Example 6: Effects of exogenous phospholipids on the lipid droplet structure of mature white adipocytes Different concentrations (0, 0.1, 0.2, 0.3, 0.4, 0.5 mg / mL) of liposomes were added to the culture medium of mature white adipocytes containing large lipid droplets. ER After LPO treatment and co-culturing for 3 days, changes in lipid droplet morphology were observed. Lipid droplets were labeled with the lipid droplet-specific dye BODIPY (green fluorescent dye), and cell nuclei were labeled with Hoechst. The morphology of lipid droplets was observed using fluorescence confocal microscopy. ER Changes in LPO concentration after treatment.

[0041] The results are as follows Figure 4 As shown, where, Figure 4 (a) Fluorescence microscopy images of white adipocytes treated with different concentrations of ERLPO (lipid droplets: green, cell nuclei: blue); Figure 4 (b) represents the change in cell lipid droplet size with... ERBar chart comparing LPO concentration changes; Figure 4 (c) represents the area of ​​cell lipid droplets as a function of... ER Bar chart comparing changes in LPO concentration.

[0042] The results show that ER LPO treatment significantly reduced the size of lipid droplets within adipocytes in a concentration-dependent manner; the droplet size decreased significantly with increasing concentration. This indicates that... ER LPO can act directly on mature white adipocytes, effectively reducing the number and size of internal lipid droplets.

[0043] Example 7: Effects of exogenous phospholipids on adipocyte differentiation To investigate whether the addition of exogenous phospholipid liposomes leads to changes in lipid droplet morphology during the differentiation of preadipocytes into mature white adipocytes, starting from day 1 of 3T3-L1 cell differentiation induction, different concentrations (0, 0.1, 0.2, 0.3, 0.4, 0.5 mg / mL) of liposomes from Example 1 were added to the differentiation medium. Fresh liposomes were added each time the medium was changed until differentiation matured. No liposomes were added to the control group throughout the process. When a large number of large lipid droplets appeared in the control group adipocytes (i.e., differentiated into mature adipocytes), the lipid droplets were labeled with the lipid droplet-specific dye BODIPY (green fluorescence), and the cell nuclei were labeled with Hoechst. The morphology of the lipid droplets was observed using fluorescence confocal microscopy. ER Changes in LPO concentration after treatment.

[0044] The results are as follows Figure 5 As shown, it can be seen ER LPO promoted a small lipid droplet phenotype in a concentration-dependent manner: as the treatment concentration increased from 0 to 0.5 mg / mL, the size and number of intracellular lipid droplets significantly decreased. Furthermore, compared to mature adipocytes, the addition of LPO during differentiation... ER LPO has a better effect on reducing lipid droplet size.

[0045] Example 8: Validation of single-component phospholipid liposomes To verify the universality of the effects of exogenous phospholipids, lipids were prepared alone using phosphatidylcholine (PC). PC LPO). The preparation method is the same as in Example 1, except that the lipid composition is only PC, excluding PE, PI, PS, and SM. The specific steps are as follows: Weigh 100 mg of PC and prepare liposomes according to the thin-film hydration-extrusion method in Example 1 to obtain a pure PC liposome suspension. PC LPO. Starting from day 1 of 3T3-L1 cell differentiation induction, add LPO to the differentiation medium. PC LPO, final concentration 0~0.5 mg / mL, replenish with fresh solution each time the medium is changed.PC LPO liposomes were used, while the control group received PBS until differentiation matured. Lipid droplets were labeled with the lipid droplet-specific dye BODIPY (green fluorescence), and cell nuclei were labeled with Hoechst. The morphology of lipid droplets was observed using fluorescence confocal microscopy. PC Changes in LPO concentration after treatment.

[0046] The results are as follows Figure 6 As shown, liposomes PC LPO can also induce a small lipid droplet phenotype, but its cytotoxicity is higher than that of liposomes. ER The maximum tolerated concentration of LPO was only 0.2 mg / mL (higher concentrations led to cell death). This indicates that liposome-mediated lipid droplet size regulation has a certain degree of universality, but liposome components significantly affect its cellular safety window.

[0047] Example 9: Effects of exogenous phospholipids on mitochondrial morphology Mito-Tracker fluorescent probes were used to label mitochondria in adipocytes to detect whether treatment with exogenous phospholipid liposomes affected the morphology of intracellular mitochondria.

[0048] Figure 7 The effect of exogenous phospholipids on the morphology of adipocyte mitochondria (mitochondria: red, nucleus: blue, lipid droplets: green), in the control group without added phospholipids. ER In LPO-treated white adipocytes, mitochondria are mainly distributed in narrow regions around the nucleus and inside the plasma membrane, exhibiting a fragmented or restricted punctate structure morphologically; ER LPO treatment increased the number of mitochondria, and their morphology exhibited a more distinct, branched network structure, more closely resembling the mitochondrial morphology of metabolically active 3T3-L1 preadipocytes. This may be because in mature white adipocytes, lipid droplets are large and numerous, occupying most of the cytoplasmic space, leading to significant spatial compression of the mitochondrial network; while ER LPO treatment significantly reduced intracellular lipid droplets and reshaped mitochondrial morphology.

[0049] Example 10: Effects of exogenous phospholipids on mitochondrial function The oxygen consumption rate (OCR) of cells was measured using a Seahorse analyzer to assess mitochondrial respiration capacity. Undifferentiated 3T3-L1 adipocyte precursor cells, mature white adipocytes, and brown adipocytes were compared. ER LPO-treated mature white adipocytes (W-ER), and those added during differentiation ER LPO-treated white adipocytes (T-ER).

[0050] The results are as follows Figure 8As shown, the results indicated that undifferentiated 3T3-L1 preadipocytes exhibited the lowest OCR curve, reflecting their resting metabolic state. Brown adipocytes showed a significantly higher OCR than white adipocyte controls, reflecting their high oxidative metabolic capacity. In contrast, both W-ER and T-ER treatments significantly increased the OCR level of white adipocytes, with the T-ER group showing a more pronounced increase. Quantitative analysis further revealed that basal respiration, ATP synthase-coupled oxygen consumption, and proton leakage levels significantly increased after both treatments, with the T-ER group showing a more significant increase. The increased proton leakage level indicates enhanced UCP1-mediated uncoupling activity. This suggests that liposomes... ER LPO treatment, especially intervention throughout the differentiation process, can reshape the respiratory capacity of white adipocytes and transform them into brown adipocytes.

[0051] Example 11: Exogenous phospholipids promote the acquisition of a thermogenic phenotype in white adipocytes Detected using Western blot technology ER LPO treatment of white adipocytes altered the expression of browning marker proteins such as peroxidase proliferator-activated receptor PPAR-γ, peroxidase proliferator-activated receptor γ coactivator 1α (PGC-1α), and uncoupling protein 1 (UCP1). The expression levels of undifferentiated 3T3-L1 adipocyte precursor cells, mature white adipocytes, and brown adipocytes were compared. ER LPO-treated mature white adipocytes (W-ER), and those added during differentiation ER Expression of PPAR-γ, PGC-1α and UCP1 in LPO-treated white adipocytes (T-ER).

[0052] The results are as follows Figure 9 As shown, the results indicated that the expression levels of UCP1 and PGC-1α were extremely low in untreated white adipocytes. After T-ER and W-ER treatment, the expression of both was significantly upregulated, and their expression levels were close to those in the brown adipocyte group. In contrast, PPAR-γ, which was highly expressed in white adipocytes, showed only a slight increase after T-ER and W-ER treatment. UCP1 is a mitochondrial uncoupling protein responsible for converting the proton gradient into heat and is the most specific marker of thermogenic adipocytes; PGC-1α is a master regulator of mitochondrial biogenesis and oxidative metabolism; and PPAR-γ is a key transcription factor in adipocyte differentiation and browning processes. The synergistic upregulation of these three factors suggests the presence of exogenous phospholipids. ER LPO can effectively promote the transformation of white adipocytes into brown thermogenic phenotypes while regulating lipid droplet size.

[0053] This invention proposes a novel technical approach that differs from existing technologies: lipid droplets are neutral lipid droplets encased in a single phospholipid membrane, and their morphological stability is directly determined by the interfacial tension of the membrane. By regulating the interfacial properties of the lipid droplet membrane, the size and distribution of the droplets can be altered. Since the phospholipid components of the outer membrane of lipid droplets mainly originate from phospholipid molecules secreted by the endoplasmic reticulum, supplementing the corresponding phospholipid components exogenously can intervene in the interfacial homeostasis of the lipid droplet membrane, achieving active remodeling of the lipid droplet morphology. Currently, classic white adipose tissue browning induction protocols often employ small molecule drugs such as rosiglitazone and triiodothyronine, which work by activating PPAR-γ receptors or thyroid hormone pathways. Although the browning effect is clear, small molecule drugs generally have potential off-target effects and systemic side effects. This invention does not pursue a browning induction intensity superior to existing protocols, but rather provides a novel strategy based on endogenous phospholipid molecules to regulate lipid droplet morphology. It induces white adipocytes to transform into a brown-like phenotype in a non-receptor activation and non-gene editing manner, thus avoiding the potential side effects of small molecule drugs at the mechanism of action level.

[0054] The exogenous phospholipid liposomes provided by this invention have a wide range of raw material sources, mature preparation processes, and excellent biocompatibility, and can be produced on a large scale. They can be applied in multiple fields such as metabolic regulation drugs, cell culture reagents, functional foods, and medical aesthetic products, to promote the browning of white adipose tissue, improve lipid metabolism disorders, and regulate the body's energy balance. They have good industrial application prospects in scenarios such as obesity and related metabolic disease intervention and basic research on adipocytes.

[0055] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. The application of an exogenous phospholipid liposome in the preparation of a product for inducing the browning transformation of white adipose tissue, characterized in that: The product uses exogenous phospholipid liposomes as its active ingredient; The phospholipid components of the exogenous phospholipid liposomes, by mass percentage, include: 5%~100% phosphatidylcholine, 10%~100% phosphatidylethanolamine, 2%~50% phosphatidylinositol, 2%~50% phosphatidylserine, 2%~50% sphingomyelin, and the liposome particle size is 50~500 nm. The exogenous phospholipid liposomes integrate into the lipid droplet membrane to reshape the lipid droplet membrane structure, mediating morphological remodeling of lipid droplets within white adipocytes, thereby enabling white adipocytes to acquire a thermogenic phenotype.

2. The application as described in claim 1, characterized in that: The mass ratio of phosphatidylcholine: phosphatidylethanolamine: phosphatidylinositol: phosphatidylserine: sphingomyelin is 57:24:13:3:

3.

3. The application as described in claim 1 or 2, characterized in that: The liposomes have a particle size of 100–300 nm, a polydispersity index (PDI) ≤ 0.2, and a zeta potential of -40–-70 mV.

4. The application as described in claim 1, characterized in that: The lipid droplet morphology remodeling includes one or more of the following changes: The average size of lipid droplets decreased; The number of lipid droplets decreased; The average area of ​​lipid droplets decreases; The spatial distribution of lipid droplets changes.

5. The application as described in claim 1, characterized in that: The heat-producing phenotype includes one or more of the following variations: Increased expression of uncoupling protein 1, or UCP1; Increased expression of peroxisome proliferator-activated receptor gamma coactivator 1α, i.e., PGC-1α; Increased cellular oxygen consumption; Increased mitochondrial proton leakage; An increase in the number of mitochondria; The degree of mitochondrial networking is enhanced.

6. The application as described in claim 1, characterized in that: The white adipocytes were selected from mature white adipocytes, adipocytes formed during the differentiation of adipocyte precursor cells, primary adipocytes, human adipocytes, and adipocytes derived from 3T3-L1.

7. The application as described in claim 1, characterized in that: The product is also used to improve lipid metabolism and regulate the body's metabolism.

8. A liposomal formulation for inducing the browning transformation of white adipose tissue, characterized in that: The liposome formulation is prepared from exogenous phospholipids, the components of which include phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and sphingomyelin; the liposome formulation has a particle size of 50-500 nm and can mediate the remodeling of lipid droplets in white adipocytes, enabling white adipocytes to acquire a thermogenic phenotype.

9. The liposome formulation according to claim 8, characterized in that: The liposome formulation can further induce one or more of the following changes in white adipocytes: enhanced mitochondrial fusion, enhanced selective mitophagy, increased oxidative phosphorylation, enhanced fatty acid oxidation capacity, and enhanced endoplasmic reticulum-lipid droplet-mitochondrial interaction.

10. The use of an exogenous phospholipid liposome in the preparation of a medicament for the treatment or prevention of metabolic diseases, characterized in that: The drug uses exogenous phospholipid liposomes as its active ingredient; The components of the exogenous phospholipid include phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and sphingomyelin; The metabolic diseases mentioned include obesity, insulin resistance, type 2 diabetes, non-alcoholic fatty liver disease, and metabolic syndrome.

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