Method for extracting lipid components of dermal adipocytes and use thereof
By extracting lipid components from mouse dermal fat cells and combining them with bee sponge spicules, a therapeutic composition was prepared that can effectively inhibit psoriasis inflammation. This solves the problems of long treatment cycles, significant side effects, and easy recurrence in existing treatment methods, and achieves effective relief of psoriasis.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- XIAMEN UNIV
- Filing Date
- 2025-01-09
- Publication Date
- 2026-07-03
AI Technical Summary
Existing treatments for psoriasis have problems such as long treatment cycles, significant side effects, easy relapse, and drug resistance. The use of biological agents has a strong targeting effect on inflammatory mediators, which may lead to changes in immunological characteristics.
By extracting lipid components from mouse dermal adipocytes and combining them with bee sponge spicules, a therapeutic composition was prepared that can effectively inhibit macrophage inflammation and keratinocyte proliferation.
It significantly inhibits the inflammatory state of macrophages and the proliferation of keratinocytes, alleviating psoriatic skin inflammation, and has good market application value.
Smart Images
Figure CN119792351B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for extracting lipid components from dermal fat cells and its application. Background Technology
[0002] Psoriasis is a common chronic inflammatory skin disease caused by a combination of genetic and environmental factors, leading to autoimmune-mediated pathological changes. Its typical morphological feature is well-defined, thickened, red plaques covered with silvery scales. Psoriasis patients often have multiple comorbidities, such as psoriatic arthritis, hypertension, obesity, type 2 diabetes, dyslipidemia, and cardiovascular disease, which place a significant burden on their lives and mental well-being. It is estimated that approximately 125 million people worldwide (about 1.7% of the world's population) are affected by psoriasis.
[0003] The histological features of psoriasis are mainly reflected in three aspects: First, epidermal thickening and excessive proliferation of keratinocytes leading to scale accumulation; second, parakeratosis resulting in cell nuclei within the keratinized layer; and finally, prominent dermal vascular dilation, with dilated and tortuous vessels extending to the tips of the dermal papillae. In addition, there is extensive infiltration of T lymphocytes, macrophages, mast cells, and neutrophils in both the dermis and epidermis. These cells communicate through cytokines such as TNFα, IFNγ, and IL-1, mediating the interaction between the innate and adaptive immune systems, activating keratinocytes, driving excessive epidermal proliferation, and producing more inflammatory factors, thus forming an inflammatory cycle that sustains the continuous development of psoriasis.
[0004] Currently, there is no specific drug for psoriasis. Commonly used treatments include topical therapy, phototherapy, traditional therapies, and biologics. Topical therapies often use corticosteroids and vitamin D derivatives, but these have side effects such as long treatment cycles and skin irritation. For patients with moderate to severe psoriasis, phototherapy and photochemotherapy are effective, but they are time-consuming and usually only used for short-term disease control. In recent years, the development of biologics has provided new methods for the treatment of psoriasis. Currently approved biologics are mainly recombinant monoclonal antibodies or receptor fusion proteins. There are four main classes of biologics used to treat moderate to severe psoriasis: anti-TNFα, anti-IL17, anti-IL12 / 23p40, and anti-IL23p19. However, the use of biologics carries risks, including the potential for drug resistance, relapse after discontinuation, and alterations in clinical and immunological characteristics due to their highly specific targeting of inflammatory mediators. Summary of the Invention
[0005] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a method for extracting lipid components from dermal fat cells.
[0006] Another object of the present invention is to provide the use of the lipid components of dermal adipocytes obtained by the above extraction method.
[0007] The technical solution of the present invention is as follows:
[0008] A method for extracting lipid components from dermal adipocytes, comprising the following steps:
[0009] (1) Primary dermal fibroblasts were extracted from mice on day 0 of birth;
[0010] (2) The above primary dermal fibroblasts were plated and cultured in DMEM++ medium until the cell adhesion density reached 100%. Then, they were induced to undergo adipogenic differentiation in vitro in culture using adipogenic differentiation medium, and then maintained in culture using maintenance medium until the cells were in an early adipogenic state. The supernatant of the early differentiated cells was then separated.
[0011] (3) The supernatant obtained in step (2) is extracted and separated using the Matyash method to obtain the lipid components of the dermal fat cells.
[0012] In a preferred embodiment of the present invention, the formulation of the DMEM+++ culture medium is shown in the table below:
[0013]
[0014] More preferably, the formulation of the adipogenic differentiation culture medium is shown in the table below:
[0015] reagents Dosage Dexamethasone 2μM 3-Isobutyl-1-methylxanthine 250μM Indomethacin 200μM insulin 10 μg / mL DMEM+++ .
[0016] More preferably, the formulation of the maintenance culture medium is shown in the table below:
[0017] reagents Dosage insulin 10 μg / mL DMEM+++ .
[0018] The use of the dermal adipocyte lipid components obtained by the above extraction method in the preparation of psoriasis treatment compositions.
[0019] In a preferred embodiment of the invention, the psoriasis treatment composition further includes bee sponge bone needles.
[0020] A psoriasis treatment composition comprising, as an effective component, dermal fat cell lipid components obtained by the above extraction method.
[0021] In a preferred embodiment of the invention, a bee sponge spicule is also included.
[0022] More preferably, its effective components are dermal fat cell lipid components and bee sponge spicules obtained by the extraction method.
[0023] The beneficial effects of this invention are:
[0024] 1. This invention extracts and separates lipid and protein components from the culture supernatant of early differentiated adipocytes. The lipid components obtained have significant immunomodulatory functions and can significantly inhibit the inflammatory state of macrophages and the proliferation of keratinocytes.
[0025] 2. The serum-free culture medium used in this invention is used for the maintenance culture of adipocytes, which does not affect the production and secretion of anti-inflammatory substances, thus ensuring the maintenance of anti-inflammatory activity during the culture process.
[0026] 3. This invention mixes the lipid components in the culture supernatant of early differentiated adipocytes with bee sponge spicules to prepare a composition that can effectively alleviate skin inflammation on the back of psoriatic mice, thus achieving a therapeutic effect on psoriasis and having good market application value. Attached Figure Description
[0027] Figure 1 This shows the timeline of adipogenic differentiation of dermal fibroblasts and the time points of cell culture supernatant collection in Example 1 of the present invention.
[0028] Figure 2 This invention demonstrates the RNA expression levels of adipocyte characteristic genes during adipogenic differentiation of dermal fibroblasts in Example 1 of the present invention.
[0029] Figure 3 This shows the expression level of inflammatory factor RNA after co-culturing macrophage and adipocyte culture supernatant in Example 2 of the present invention.
[0030] Figure 4 This shows the phenotype of psoriasis mice treated with eAd_CM in Example 2 of the present invention.
[0031] Figure 5 This invention demonstrates a comparison of the in vitro anti-inflammatory effects of adipocyte culture supernatants collected from cultures with different fetal bovine serum concentrations in Example 3 of this invention.
[0032] Figure 6 This shows the expression levels of inflammatory factor RNA after macrophages were co-cultured with each component of eAd_CM in Example 5 of the present invention.
[0033] Figure 7 This shows the phenotype of psoriasis mice after combined treatment with eAd_lipids and bee sponge bone needles in Example 6 of the present invention.
[0034] Figure 8This invention presents a lipidomics analysis of the culture supernatant of adipocytes at different differentiation stages in Example 7 of the present invention. Detailed Implementation
[0035] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.
[0036] Example 1: Extraction, adipogenic differentiation, and collection of culture supernatant from primary dermal fibroblasts
[0037] Mice born on day 0 were euthanized by decapitation, their limbs were removed, and the skin was girdled off. Excess fat and mucous membrane tissue were removed, and the skin was then immersed in 2 mL of DMEM containing 2 mg / mL Dispase II and incubated overnight at 4°C with rotation. The next day, HBSS buffer (HBSS++, formulation shown in Table 1) containing 20 mg / mL bovine serum albumin and antibiotics was prepared. The epidermis was carefully removed, washed with 1×PBS, and HBSS++ was added. The tissue was then chopped into small pieces (1–2 mm wide strips). The chopped tissue was transferred to centrifuge tubes. Each newborn mouse's dermis required 10 mL of digestion solution (HBSS + 1 mg / mL Dispase I + 2 mg / mL collagenase IV). After adding the digestion solution, the tubes were heated in a 37°C water bath for 5 min, and then digested on a shaker at 37°C for 2 h. After digestion, the centrifuge tubes were placed on ice for 5 min to stop the digestion. After digestion, the tissue suspension was filtered through 100μm and 40μm screens to remove hair, tissue and other incompletely digested tissue. The filtrate was collected and centrifuged at 4℃ and 1400rpm for 5min. The supernatant was removed, red blood cells were lysed, and after centrifugation, the cells were resuspended in preheated DMEM+++ medium (the formula of DMEM+++ medium is shown in Table 2) and cultured at 37℃ and 5%CO2.
[0038] Table 1. Recipes for HBSS++
[0039]
[0040] After cell attachment, replace the medium with fresh DMEM+++ medium (formulation shown in Table 2) 8-12 hours later and continue culturing at 37℃ and 5% CO2. Subsequently, replace the medium with the same fresh DMEM+++ medium every 2 days and continue culturing at 37℃ and 5% CO2 until the cell density reaches 100%. Two days later, replace the medium with adipogenic differentiation medium (as shown in Table 3, adding 2 μM dexamethasone, 250 μM 3-isobutyl-1-methylxanthine, 200 μM indomethacin, and 10 μg / mL insulin to DMEM+++) and continue differentiation culturing at 37℃ and 5% CO2. Figure 1As shown, on the second day of differentiation culture, fresh adipogenic differentiation medium was used, and on the third day of differentiation culture, fresh maintenance medium (formulation shown in Table 4, with 10 μg / mL insulin added to DMEM+++) was used for maintenance culture at 37℃ and 5% CO2. Fresh maintenance medium was then used every 3 days thereafter.
[0041] Table 2: Formulation of DMEM++
[0042]
[0043] Table 3. Formulation of adipogenic differentiation medium
[0044] reagents Dosage brand model Dexamethasone 2μM Sigma D4902 3-Isobutyl-1-methylxanthine 250μM Sigma I5879 Indomethacin 200μM Sigma I8280 insulin 10 μg / mL Sigma 91077C DMEM+++
[0045] Table 4. Formulation of maintenance culture medium
[0046] reagents Dosage brand model insulin 10 μg / mL Sigma 91077C DMEM+++
[0047] At the time of no adipogenic differentiation, at the beginning of adipogenic differentiation, and at 6 h, 1 d, 2 d, 3 d, 6 d and 9 d after induction of adipogenic differentiation, the corresponding culture medium was removed, trizol was added and the cells were slowly pipetted to lyse the cells, and the cells were collected into 1.5 mL centrifuge tubes to extract RNA. qRT-PCR experiments were performed to detect the expression levels of adipocyte characteristic genes, thereby distinguishing adipocytes at different differentiation stages.
[0048] The results are as follows Figure 2As shown, compared to undifferentiated cells and other differentiation time points, adipocytes on day 3 of differentiation culture were in an early adipate (eAd) state, characterized by the peak expression of the antimicrobial peptide Camp; while as the number of days of adipocyte differentiation increased, cells on day 9 of differentiation were in a mature adipate (mAd) state, characterized by a significant loss of Camp expression (less than 10% of eAd) and maintenance of Fap4 expression (maintained at 50% to 100% of eAd). Therefore, this embodiment uses high expression of Camp as the defining indicator of eAd. Related research on defining the stage of adipocyte differentiation can be found in the research paper: Zhang LJ, Guerrero-Juarez CF, Chen SX, Zhang X, Yin M, Li F, Wu S, Chen J, Li M, Liu Y, Jiang SIB, Hata T, Plikus MV, Gallo RL. Diet-induced obesity promotes infection by impairment of the innate antimicrobial defense function of dermal adipocyte progenitors. SciTransl Med. 2021 Jan 20; 13(577):eabb5280. In this paper, early adipocyte state (eAd) is equivalent to Early adipocytes, and mature adipocyte state (mAd) is equivalent to Adipocytes (AD).
[0049] Cell culture supernatants were collected from differentiation days -2 to 0, 3 to 6, and 9 to 12, and denoted as undif_CM, eAd_CM, and mAd_CM, respectively. Figure 1 (As shown). Simultaneously, cell-free blank maintenance medium, stored under the same culture conditions for 3 days, was collected as a control and denoted as BM. The collected cell culture supernatant was centrifuged at 4°C and 3000 rpm for 5 minutes to remove dead cells or cell debris, and then frozen at -80°C for subsequent analysis.
[0050] Example 2: Anti-inflammatory properties test of dermal adipocyte culture supernatant
[0051] In vitro anti-inflammatory performance test: Peritoneal macrophages were extracted from 2-3 month old C57BL / 6 mice and cultured in DMEM++ medium (as shown in Table 5) at 37℃ and 5% CO2. Fresh DMEM++ medium was added every other day to remove non-adherent cells. After 24 hours of culture, the medium was changed again, and BM, undif_CM, eAd_CM, and mAd_CM prepared in Example 1 were added respectively. The co-culture system was DMEM++:CM = 1:1 (volume ratio). After 2 hours of co-culture, LPS was added to activate macrophage inflammation. After 12 hours of stimulation, cells were collected, RNA was extracted, and qRT-PCR was performed to detect the expression levels of inflammatory factors in macrophages. The results are shown in Table 5. Figure 3 As shown, eAd_CM can significantly downregulate the RNA levels of Il1b, Nos2, and Cxcl2, indicating that only early adipocytes can secrete large amounts of anti-inflammatory substances to exert unique anti-inflammatory effects.
[0052] Table 5. Formulation of DMEM++
[0053]
[0054] In vivo anti-inflammatory performance test: 7-8 week old female C57BL / 6 mice were randomly divided into two groups. Hair was removed from their backs, and then 40 mg imiquimod cream was applied to a 2 cm × 2 cm area to induce psoriasis-like lesions for six consecutive days. Starting from the second day of application, 100 μL of BM or eAd_CM was injected intradermally into the back skin of both groups of mice twice daily, morning and evening. On the seventh day, non-lesioned and lesioned areas on the back of the mice were harvested. PASI scoring and qRT-PCR experiments were used to analyze the therapeutic effect of eAd_CM on psoriasis-like inflammation in mice. Results are as follows: Figure 4 As shown, after eAd_CM injection, the levels of inflammatory factor Il1b and epidermal cell proliferation (Krt6a) decreased, indicating that the severity of psoriasis-like skin lesions in mice was reduced, demonstrating the good therapeutic effect of eAd_CM on psoriasis.
[0055] Example 3: Test of the culture effect of serum-free culture medium
[0056] Dermal fibroblasts were induced to differentiate into adipocytes in vitro according to the method described in Example 1. On day 3 of differentiation, cells were cultured in maintenance media containing 10% fetal bovine serum (FBS), 5% FBS, 2% FBS, 1% FBS, and serum-free media, respectively. The concentration of FBS did not affect the differentiation efficiency or growth of adipocytes. On day 6 of differentiation, the adipocyte culture supernatant was collected, and the differences in in vitro anti-inflammatory effects of the supernatant containing different FBS concentrations were compared according to the method described in Example 2. The results are as follows: Figure 5As shown, the concentration of fetal bovine serum does not alter the ability of adipocytes to secrete anti-inflammatory substances. Therefore, in this embodiment, serum-free medium was used to culture early adipocytes differentiated on days 3 to 6, and the adipocyte culture supernatant was collected for subsequent experiments in Examples 4 to 7.
[0057] Example 4: Lipid Extraction from Early Adipocyte Culture Supernatant
[0058] Lipids, polar metabolites, and proteins were extracted and separated from serum-free BM and eAd_CM using the Matyash method. The specific implementation plan is as follows:
[0059] Serum-free BM and eAd_CM were thawed from a -80℃ freezer. Methyl tert-butyl ether (MTBE) and methanol were added in a volume ratio of 10:3:2.5. The mixture was vortexed for 1 min and then sonicated in an ice-water bath for 10 min. After centrifugation at 4℃ and 4000 rpm for 15 min, the solution separated into two layers with a white precipitate. The upper layer was a lipid layer, and the white precipitate was protein. These were collected separately into new centrifuge tubes. The liquid in each component was removed by nitrogen blowing at room temperature, and then freeze-dried to completely remove any remaining liquid. Finally, each component was resuspended in serum-free maintenance medium to its original volume before extraction, fully thawed, and then stored at -80℃ for subsequent analysis.
[0060] Example 5: In vitro efficacy verification of lipids
[0061] The anti-inflammatory efficacy of each component extracted and separated from eAd_CM was verified according to the method in "In Vitro Anti-inflammatory Performance Test" in Example 2. The specific implementation plan is as follows:
[0062] Peritoneal macrophages were extracted from 2-3 month old C57BL / 6 mice. After plating, the medium was replaced with fresh DMEM++ medium the following day, and non-adherent cells were removed. After 24 hours of further culture, the medium was changed again, and BM, eAd_lipids, and eAd_proteins were added respectively. The co-culture system was DMEM++:eAd_lipids (or eAd_proteins) = 1:1. After 2 hours of co-culture, LPS was added to activate macrophage inflammation. After 12 hours of stimulation, cells were collected, RNA was extracted, and qRT-PCR was performed to detect the expression levels of inflammatory factors in macrophages. The results are as follows: Figure 6 As shown, eAd_lipids can significantly downregulate the RNA levels of Il1b and Cxcl2, indicating that only lipids secreted by early adipocytes can stably exert significant anti-inflammatory effects.
[0063] Example 6: Preparation and in vivo efficacy verification of the lipid-bee sponge bone needle composition.
[0064] To further verify the in vivo efficacy of eAd_lipids, this embodiment uses bee sponge spicules and lipids to prepare a composition and applies it to a psoriasis-like mouse model. The bee sponge spicules can help the lipids penetrate the skin better, thereby exerting a therapeutic effect. The specific preparation method is as follows: Take 1 mg of bee sponge spicule powder and resuspend it in 100 μL of BM_lipids solution or eAd_lipids solution.
[0065] In vivo anti-inflammatory performance test: 7-8 week old female C57BL / 6 mice were randomly divided into two groups. Hair was removed from their backs, and 25 mg imiquimod cream was applied to a 1.77 cm² circular area on the back to induce psoriasis-like lesions for six consecutive days. Starting from the second day of application, 100 μL of a mixture of BM_lipids or eAd_lipids and bee sponge spicules was dripped into the modeled area on the back of each group of mice daily, followed by manual massage for 2 minutes to aid absorption. On the seventh day, non-lesioned and lesioned areas on the back of the mice were collected, and the PASI score and qRT-PCR experiments were used to analyze the therapeutic effect of eAd_CM on psoriasis-like inflammation in mice. Results are as follows: Figure 7 As shown, eAd_lipids can reduce the expression of inflammatory factor Il1b and the proliferation of epidermal cells (Krt6a), indicating that the severity of psoriasis-like skin lesions in mice is reduced, demonstrating the good therapeutic effect of eAd_lipids on psoriasis.
[0066] Example 7: Lipidomics analysis of culture supernatant from adipocytes at different differentiation stages
[0067] To further clarify the specific lipid components that exert anti-inflammatory effects in early adipocytes, this example performed lipidomics analysis on the culture supernatant of adipocytes at different differentiation stages. The results are as follows: Figure 8 As shown, eAd_CM is primarily enriched with glycerophospholipids, such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE), as well as lysophospholipids, including lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE). In contrast, the culture supernatant of mature adipocytes (mAd-CM) is primarily enriched with triglycerides (TG) and free fatty acids (FFAs). These results suggest that early adipocytes may exert anti-inflammatory effects by secreting glycerophospholipids and lysophospholipids.
[0068] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.
Claims
1. The use of dermal adipocyte lipid components in the preparation of psoriasis treatment compositions, characterized in that: The method for extracting lipid components from dermal adipocytes includes the following steps: (1) Primary dermal fibroblasts were extracted from mice on day 0 of birth; (2) The above primary dermal fibroblasts were plated and cultured in DMEM+++ medium until the cell adhesion density reached 100%. Then, they were cultured in vitro in adipogenic differentiation medium and then in maintenance medium until the cells were in an early adipogenic state. The supernatant of the early differentiated cells was then separated. (3) The supernatant obtained in step (2) is extracted and separated using the Matyash method to obtain the lipid components of the dermal adipocytes; The formulation of the DMEM+++ culture medium is shown in the table below: 。 2. The use as described in claim 1, characterized in that: The formulation of the adipogenic differentiation medium is shown in the table below: 。 3. The use as described in claim 1, characterized in that: The formulation of the maintenance culture medium is shown in the table below: 。 4. The use as described in any one of claims 1 to 3, characterized in that: The psoriasis treatment composition also includes bee sponge bone needles.
5. A psoriasis treatment composition, wherein the effective component comprises dermal adipocyte lipid components, and the extraction method of the dermal adipocyte lipid components comprises the following steps: (1) Primary dermal fibroblasts were extracted from mice on day 0 of birth; (2) The above primary dermal fibroblasts were plated and cultured in DMEM+++ medium until the cell adhesion density reached 100%. Then, they were cultured in vitro in adipogenic differentiation medium and then in maintenance medium until the cells were in an early adipogenic state. The supernatant of the early differentiated cells was then separated. (3) The supernatant obtained in step (2) is extracted and separated using the Matyash method to obtain the lipid components of the dermal adipocytes; The formulation of the DMEM+++ culture medium is shown in the table below: 。 6. The psoriasis treatment composition according to claim 5, characterized in that: The formulation of the adipogenic differentiation medium is shown in the table below: ； The formulation of the maintenance culture medium is shown in the table below: 。 7. A psoriasis treatment composition as described in claim 5 or 6, characterized in that: It also includes bee sponge spicules.
8. The psoriasis treatment composition according to claim 7, characterized in that: Its effective components are the lipid components of the dermal fat cells and the spicules of the bee sponge.