Natural plant agonist of PPAR gamma and application thereof

Abstract

The invention discloses a natural plant agonist of PPAR gamma and application of the natural plant agonist, the agonist is a bibenzyl derivative Gigantol separated from dendrobium plants, and the agonist can be used as a unique effective component for preparing a preparation for activating PPAR gamma to treat skin aging and can also be prepared into a composition containing the agonist. The traditional Chinese medicine composition is used for treating skin aging caused by skin barrier injury (including physical injury and ultraviolet sunburn), can also be used for preparing anti-aging masks, and can be prepared into various dosage forms such as injections, pills and the like by being matched with auxiliary materials.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of new medical uses of active ingredients of traditional Chinese medicinal materials, and relates to a natural plant agonist of PPARγ (peroxisome proliferator-activated receptor gamma) and application thereof, in particular to application of gigantol as an effective component of the agonist in preparation of a medicine for treating skin aging. BACKGROUND

[0002] Skin, as the largest organ of the human body, its aging is not only accompanied by an increase in wrinkles and age spots, but also brings different degrees of anxiety to people. There is evidence that skin aging can lead to loss of skin function, increase the risk of skin cancer, and also cause physical aging and related dysfunction, affecting the health of patients. How to prevent and delay skin aging has become a hot and important topic in current medical research.

[0003] “Molecular Mechanism Research on PPAR Pathway Regulating Chronic Skin Barrier Damage Induced Aging” (Jia Xiaorong, Master's Thesis, Kunming Medical University, May 1, 2023) discloses that long-term chronic skin barrier damage can lead to more severe skin aging, and verifies that long-term barrier destruction can exacerbate skin aging through the EGF-EGFR-JNK / ERK-PPARγ-NLRP3 pathway; it is suggested that the application of PPARγ agonists can alleviate the phenomenon of skin aging caused by barrier damage.

[0004] Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that regulate the expression of many target genes associated with cellular functions, including cell proliferation, differentiation, and immune, inflammatory responses. More and more studies have shown that PPARγ plays an important role in the skin, involved in a variety of pathophysiological processes of the skin. PPARγ may play an important role in skin barrier permeability, inhibition of epidermal cell growth, promotion of epidermal terminal differentiation and regulation of skin inflammatory response through various mechanisms. These characteristics point to PPARγ can play an important role in the treatment of atopic dermatitis, psoriasis, scleroderma, skin wound healing, etc. Different animal, cell models confirmed that PPARγ plays a crucial role in the balance of epidermal function. The expression and activity of PPARγ decrease during aging, and the decrease of PPARγ signal leads to accelerated aging of rat kidney and age-related functional loss in mouse adipose tissue. Dan Shen et al. found that PPARγ agonist pioglitazone can effectively regulate the aging of multiple organs including skin aging in apoE− / − mice, and the use of PPARγ agonists may be a promising anti-aging therapy. The three recognized PPARγ agonists currently available include synthetic agonist pioglitazone (thiazolidinedione drugs); endogenous ligand 15d-PGJ2 and natural product phellopterin, but due to the side effects of pioglitazone, the potential mechanism of endogenous ligand 15d-PGJ2 for various diseases is unknown, and the safety of long-term use of phellopterin is considered, another natural PPARγ targeting agonist with low toxicity and side effects is planned to be found, which is used as an effective component to prepare a tool for activating PPARγ, and further regulates the problem of skin aging caused by chronic skin barrier damage through PPARγ signaling pathway. SUMMARY

[0005] In view of the non-targeting and toxic side effects of the current agonists, the inventors screened a natural agonist targeting PPARγ from a self-built database of Yunnan Agricultural University Gigantol , which is used to overcome the technical problems of existing PPARγ agonists and solve the problem of skin aging.

[0006] An object of the present application is to provide a natural plant agonist of PPARγ, which is a bibenzyl derivative isolated from Dendrobium plants Gigantol , the structural formula of which is Gigantol .

[0007] Use of PPARγ agonist in preparation of a preparation for treating skin aging.

[0008] Further, the preparation for treating skin aging contains the natural plant agonist of PPARγ as the only effective componentGiganto The composition of l.

[0009] Further, the use of the composition in the preparation of a medicament for treating skin aging caused by skin barrier damage.

[0010] Further, the skin barrier damage includes physical damage and ultraviolet sunburn.

[0011] Further, the use of the composition in the preparation of an anti-aging mask.

[0012] Further, the composition is used for Giganto l is formulated with excipients of the corresponding dosage form into injections, pills, granules, solutions, suspensions, emulsions, microcapsules or gels.

[0013] The working principle of the application: through pharmacological screening, the application identifies a natural compound Gigantol extracted from Dendrobium is a highly specific and potent PPARγ agonist (Kd = 1.76 10 6 M). In a mouse aging model induced by barrier damage, Gigantol treatment significantly improves key aging phenotypes and typical aging markers, showing comparable PPARγ activation efficacy to pioglitazone and significantly lower side effects than pioglitazone at high concentrations.

[0014] Compared with the prior art, the beneficial effects of the application are: (1) A new ingredient Gigantol based on natural products is provided for anti-aging therapy; (2) Compared with pioglitazone, the cytotoxicity of Gigantol is significantly reduced at a higher concentration (≥100 μM), highlighting its superior safety in potential therapeutic applications; (3) Gigantol does not cause allergic or irritating reactions, and biological toxicity evaluation shows that there is no pathological change in the heart, liver, spleen, lung and kidney of mice, including histological changes or cell necrosis shown by hematoxylin and eosin (HE) staining. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The construction and characterization of the barrier-damaged skin aging mouse model of the application; wherein: (A) Flow chart illustrating the construction of a barrier damage model in BALB / C mice; (B) Severe pathological changes in the back skin of two groups of mice; (C) Histological analysis of the back skin by HE, Masson, VG and Weigert staining (200 ); (D) The levels of ROS, SOD, MDA, CAT and GSH-PX in blood of two groups; (E) Western blot analysis of skin tissue proteins with corresponding quantitative data; (F) Measurement of transepidermal water loss (TEWL) and water content in the back skin; (G) ROS staining of the back skin tissue (200 ); (H) Statistical analysis of the back skin epidermis thickness, n = 10; *p<0.05, ** p<0.01, *** p<0.001. n = 10, *p<0.05, ** p<0.01, *** p<0.001.

[0016] Figure 2 is the transcriptome analysis of the tape-stripped mice; wherein: (A) keGG enrichment analysis shows that the PPAR pathway is significantly enriched in the back skin of the tape-split group compared with the control group; (B) GSEA plot of the skin transcriptome; (C) Volcano plot showing differentially expressed genes; (D) PPARy immunofluorescence with quantification (200 ); (E) Western blot quantification of PPARy protein expression. n = 3; *** p<0.001.

[0017] Figure 3 Barrier protein expression in skin cells of the tape-stripped model of barrier disruption.

[0018] Figure 4 Tape-stripping induces skin aging markers in mice; wherein: A is the immunohistochemical staining and quantification of the senescence markers p16, p21, p53 and β-gal (200x); B is the immunofluorescence of col-i, col-iii, lamin b1 and γ-H2AX with collagen expression (200x); C is the expression levels of TNF-a and IL-6 in the back skin tissue of control and tape-split mice. n = 10; *p<0.05, ** p<0.01, *** p<0.001.

[0019] Figure 5Western blot analysis and quantification of EGF-EGFR-JNK / ERK-PPARy-NLRP3-NF-KB / COX2 / TNF-a / IL-1b signaling pathway components in tape-stripped mice and control mice.

[0020] Figure 6 For in vivo and in vitro PPARy knockdown experiments; wherein: (A) β-GAL staining in HACAT cells transfected with SINC and Sipparγ; (B) Western blot analysis of PPARy protein expression in HACAT cells transfected with SINC and Sipparγ; (C) Analysis of PPARy RNA expression and Western blot quantification in HACAT cells transfected with SINC and Sipparγ; (D) Histological representation of the dorsal skin in mice treated with AAV-NC and AAV-PPARy-shRNA; (E) Western blot analysis of mouse skin tissue using the indicated antibodies; (F) Analysis of PPARy RNA expression and Western blot quantification in mice treated with AAV-NC and AAV-PPARy-shRNA.

[0021] Figure 7 Structural formula of the PPARy natural plant agonist of the present invention Gigantol and sensorgrams and molecular docking model of the interaction of Peroxisome Proliferator-Activated Receptor Gamma (PPARG) with Gigantol

[0022] Figure 8 Effect of Gigantol and pioglitazone on HaCaT cell viability; wherein: A is the effect of Gigantol Effect of 24h, 48h and 72h treatment at 0, 100nM, 1pM, 10pM, 20pM, 100pM and 1mM concentrations on HaCaT cell viability, ****P<0.0001; B is the effect of pioglitazone 24h, 48h and 72h treatment at 0, 100nM, 1pM, 10pM, 20pM, 100pM and 1mM concentrations on HaCaT cell viability, ****P<0.0001.

[0023] Figure 9 Representative images of the dorsal skin of mice in different experimental groups; wherein:​ A: Pathological manifestations of the back skin of mice in different experimental groups; B: HE staining of the back skin of mice in different experimental groups (40 ); C: Immunofluorescence staining of the back skin of mice in different experimental groups (40 ).

[0024] Figure 10 Western blot analysis of PPARγ expression in the dorsal skin of mice from different experimental groups.

[0025] Figure 11 Quantitative analysis of PPARγ expression in the dorsal skin of mice in different experimental groups.

[0026] Figure 12 Figure 5. Transepidermal water loss (TEWL) values ​​of the back skin of mice in different experimental groups.

[0027] Figure 13 HE staining of the heart, liver, spleen, lung and kidney tissues of BALB / c mice in different experimental groups after back skin treatment (400 ). DETAILED DESCRIPTION

[0028] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the present invention is further described below in conjunction with specific embodiments and with reference to the accompanying drawings. It should be understood that these descriptions are merely exemplary and are not intended to limit the scope of the present invention. In addition, in the following invention, descriptions of well-known structures and technologies are omitted to avoid unnecessary confusion of the concepts of the present invention.

[0029] Using a database built by Yunnan Agricultural University and based on the principle of structural docking, we screened for natural PPARγ agonists and obtained Gigantol. We then purchased this natural product extract from Glip Biotech Co., Ltd. We then performed the following steps.

[0030] The results are as follows Figure 1 The chemical structure of Gigantol is shown in Figure 7 A, SPR analysis using the Biacore S200 system (GE Healthcare) showed PPARγ binding to Gigantol (KD = 1.76 × 10 -6 M)( Figure 7 B), Molecular docking simulations using EVPL and SBSN algorithms showed that gigantol formed a stable interaction with PPARγ through multiple hydrogen bonds and hydrophobic contacts ( Figure 7C), exhibiting excellent shape complementarity with the ligand-binding pocket of PPARγ. These computational predictions were experimentally validated by surface plasmon resonance (SPR) analysis, which quantified direct binding of gigantol to PPARγ with a dissociation constant (KD) of 1.76 × 10 -6 M, confirmed the feasibility of Gigantol as a PPARγ agonist.

[0031] Example 2: Gigantol Effects on a mouse model of skin aging with impaired barrier function 1. Experimental Animals: Female BALB / c mice (8 weeks old) were purchased from the Laboratory Animal Center of Kunming Medical University. Mice were housed under controlled environmental conditions (relative humidity = 45%-55%; ambient temperature = 23°C ± 2°C; 12:12-h light / dark cycle) with free access to water and food. Mice were acclimated for 1 week before experiments. All animal experiments were approved by the Institutional Animal Care and Use Committee of Kunming Medical University (approval number: KMMU20221545) and conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the U.S. National Institutes of Health. Minimize animal suffering during experiments.

[0032] 2. Experimental methods (1) Modeling: Tape stripping-induced skin barrier damage mouse model (Figure 1A). To establish the skin barrier damage mouse model, the shaved mice were subjected to tape stripping tests every day for three months. The model mice showed characteristic barrier damage manifestations, including mild exudation and capillary dilation, as well as aging phenotypes such as deepened wrinkles, roughness, keratinization, and desquamation (Figure 1B). Figure 1-2 As shown, ① Barrier integrity quantification: Transepidermal water loss (TEWL) and stratum corneum hydration measurements—established indicators of skin barrier function—showed a significant increase in TEWL and decrease in stratum corneum hydration in tape-stripped mice compared with the control group (Figure 1F), confirming impaired barrier function. Immunofluorescence analysis of key barrier homeostasis regulators (β-Catenin, E-Cadherin, Zonula Occludens-1, Filaggrin, and Involucrin) demonstrated significant protein reduction in the experimental group (Figures 4A-C), further validating the model's effectiveness.

[0033] ②Histopathological and ultrastructural changes: HE staining showed that the stratum corneum of the tape-stripped mice was hyperplastic, and the epidermis thickness increased from 20 pm (control group) to 60 pm (Fig. 1C, H). Connective tissue histochemistry (Masson, VG, Weigert staining) showed that collagen bundles were fragmented and dissolved (Fig. 1C), indicating that the extracellular matrix was denatured. These findings provided strong pathological evidence that mechanical barrier disruption would lead to skin aging.

[0034] ③Oxidative stress and aging biomarkers: Compared with the control group, the barrier-damaged skin showed increased oxidative stress, with 2.2-fold and 1.7-fold increases in ROS and MDA levels, respectively, and significant decreases in SOD (52%), CAT (61%), and GSH-PX activities (46%) (Fig. 1D). Immunofluorescence confirmed the accumulation of ROS (Fig. 1G), consistent with redox imbalance.

[0035] ④Disorder of aging-related markers at the protein level: Western blotting showed upregulation of P53, P21, P16, γ-H2AX, and MMP1, and downregulation of LaminB1, Col-I, and Col-III (Fig. 1E). Immunofluorescence confirmed the increase in P21 / P16 / P53 positivity (Fig. 2A) and the accumulation of β-gal+ cells in the perifollicular region (Fig. 3A). The immunofluorescence patterns of LaminB1, collagen, and γ-H2AX (Fig. 3A) were consistent with the Western blotting data. Figure 4 A), while β-gal+ cells accumulated in the perifollicular region (Fig. 3A). The immunofluorescence patterns of LaminB1, collagen, and γ-H2AX (Fig. 3A) were consistent with the Western blotting data.

[0036] ⑤Systemic propagation of aging phenotypes: Barrier disruption led to an increase in circulating oxidative stress markers, exacerbating systemic aging (Fig. 2D). The expression levels of TNF-a, IL-6, and IL-1β in the dorsal skin tissues of the model mice were significantly increased, consistent with the upregulation of components of the senescence-associated secretory phenotype (SASP) Figure 4 C、 Figure 5 ), thus extending previous findings on the inter-tissue propagation of cellular senescence.

[0037] Consistent with the evidence that skin aging would propagate senescence to distant organs, our data showed that barrier disruption would also exacerbate systemic aging by increasing blood oxidative stress (Fig. 2D).

[0038] The above data proved the success of modeling, and then the grouping treatment was carried out.

[0039] (2) Grouping and treatment: The mice with successful modeling were randomly divided into 3 groups (6-8 mice per group): model group (Tape stripping), Gigantolgroup, and a positive drug control group (pioglitazone group or pioglitazone PGZ group). In addition, mice that were not modeled and raised normally were selected as the control group (6-8 mice). Gigantol The drug solution dissolved in the group and the pioglitazone group was applied to the skin twice a day, topically applied to the back skin, 30 minutes apart, and 1 hour each time. A homemade solution of dimethyl sulfoxide and normal saline was used as the drug dissolution and delivery vehicle. The results are shown in Table 1 and Figure 8-12 shown.

[0040] Table 1 Characterization results of different experimental groups Control Taped stripping Taped stripping+Gigantol Tapedstripping+PGZ Macroscopic skin phenotype Skin smooth, no damage, hair uniform (healthy state) Skin redness, damage, bleeding Damage significantly reduced (redness range, damage reduction) Damage repair effect similar to Gigantol group, Skin histopathology (HE staining) Epidermis thin and regular, dermal collagen fibers arranged in order (healthy skin structure) Epidermis thickening, disorder (compensatory hyperplasia of epidermis after injury), inflammatory cells infiltrate in dermis Epidermis thickness close to normal, inflammatory cells significantly reduced, collagen fibers arranged more regularly Pathological changes consistent with Gigantol group P21 Weak fluorescence (normal skin few senescent cells) Fluorescence significantly enhanced (damage induced a large number of senescent cells) Fluorescence significantly reduced Fluorescence similar to Gigantol group The results are as follows Figure 8-12 As shown, Gigantol Administration significantly alleviated the clinical manifestations of skin barrier dysfunction, including reduced erythema and improved skin roughness, while reversing the aging phenotype, reducing skin epidermal wrinkles and promoting the healing of epidermal lesions ( Figure 9A ).

[0041] Figure 9 The HE staining results shown in B are as follows: Tape stripping causes macroscopic damage to mouse skin, disordered pathological structure, and cell senescence (increased P21). Gigantol can promote the healing of damaged skin (macroscopic phenotype), repair tissue pathological structure (HE staining), and inhibit cell senescence (downregulation of P21), with an effect comparable to that of the positive drug PGZ.

[0042] Combined with PPARγ protein experiments, we know that Gigantol By activating the PPARγ pathway, it can repair the skin barrier and resist cell aging, providing new drug targets and strategies for the treatment of skin aging.

[0043] By administering the drug on the skin Gigantol, Its epidermal morphology was significantly improved, and the improvement effect was no less than that of the positive drug group. Figure 8 The cytotoxicity experiments shown in the figure show that the cytotoxicity of pioglitazone and Gigantol Comparative evaluation of the cytotoxicity and proliferation of the two compounds showed that both compounds were non-toxic at lower concentrations and could promote cell proliferation. However, compared with pioglitazone at higher concentrations (≥100 μM), Gigantol The cytotoxicity of Gigantol It can be used as an active ingredient to prepare an agonist that activates PPARγ and can be used to prepare a preparation for treating skin aging. The results of Masson's trichrome staining showed (9C) that applying the drug solution to the skin can inhibit the expression of the aging marker p21. Figure 10-11Western blot analysis and quantitative analysis of PPARγ expression in the dorsal skin of mice revealed that skin barrier damage (such as physical tape stripping) inhibits PPARγ protein expression, while Gigantol can restore PPARγ expression (the PPARγ band in the "Taped Stripping + Gigantol" group became darker, and expression levels in the histogram were significantly higher than those in the "Taped Stripping + Gigantol" group). This suggests that Gigantol can activate the PPARγ pathway and alleviate the suppression of PPARγ expression caused by skin barrier damage, indicating its potential to promote skin repair and anti-aging.

[0044] In addition, there was no significant difference between the positive drug group and the Gigantol group (ns), indicating that the effect of Gigantol is similar to that of the classic PPARγ agonist PGZ, further verifying the function of Gigantol as a PPARγ agonist; from the perspective of drug action, the activation effect of Gigantol on PPARγ is equivalent to that of PGZ, providing a basis for its alternative application.

[0045] pass Figure 12 The TEWL values ​​of the dorsal skin of mice in each group showed that tape stripping could damage the skin barrier and lead to increased TEWL; Gigantol could effectively repair the damaged skin barrier and reduce TEWL, and the effect was consistent with that of the classic PPARγ agonist PGZ.

[0046] Combined with previous PPARγ protein expression experiments, it's clear that Gigantol activates the PPARγ pathway to repair the skin barrier, reduce water loss, and thus combat skin aging. This functional (TEWL) validation of Gigantol's repairing effect on skin barrier damage complements the molecular (PPARγ expression) experiments, further strengthening the evidence base for Gigantol's role in treating skin aging.

[0047] It should be understood that the above-described specific embodiments of the present invention are merely illustrative of the invention or explanation of the principles of the present invention, and do not constitute limitations of the present invention. Therefore, any modifications, equivalent substitutions, improvements, etc. made without departing from the spirit and scope of the present invention should be included within the scope of protection of the present invention. In addition, the appended claims are intended to cover all variations and modifications that fall within the scope and metes and bounds of the appended claims, or equivalents of such scope and metes and bounds.

Claims

1. A natural plant agonist of PPARγ, characterized in that The natural plant agonist of PPARγ is a bibenzyl derivative isolated from Dendrobium plants. Gigantol , Gigantol The structural formula is: 。 2. Use of the natural plant agonist of PPARγ according to claim 1 in the preparation of a preparation for treating skin aging.

3. The use according to claim 2, characterized in that The skin aging treatment preparation contains Giganto l composition.

4. The use according to claim 3, characterized in that The composition is used in preparing a medicine for treating skin aging caused by skin barrier damage.

5. The use according to claim 3, characterized in that The skin barrier damage includes physical damage and ultraviolet sunburn.

6. The use according to claim 5, characterized in that Application of the composition in preparing an anti-aging facial mask.

7. The use according to any one of claims 2 to 6, characterized in that: Will Giganto l It can be prepared into injection, pill, granule, solution, suspension, emulsion, microcapsule or gel with the corresponding dosage form excipients.

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