Application of kinsenoside in preparation of medicine for treating skin inflammatory diseases

Abstract

The invention belongs to the technical field of medicines for treating inflammatory skin diseases, and discloses application of kinsenoside in preparation of medicines for treating skin inflammatory diseases, the structural formula of kinsenoside is shown in the specification, and kinsenoside can be particularly used for preparing medicines for treating psoriasis. According to the application, the kinsenoside is applied to the preparation of the medicine for treating the skin inflammatory diseases, particularly the medicine for treating the psoriasis for the first time, the kinsenoside can up-regulate arachidonate 15-lipoxygenase (ALOX15) in the skin so as to intervene the psoriasis, and a candidate medicine is provided for clinically developing natural products for treating the skin inflammatory diseases including the psoriasis.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology for treating inflammatory skin diseases, and more specifically, relates to the application of Anoectochilus roxburghii glycoside in the preparation of drugs for treating inflammatory skin diseases. Background Technology

[0002] Inflammatory skin diseases plague many patients. Psoriasis, a common chronic inflammatory skin disease, affects approximately 60 million people worldwide. The prevalence in Europe is about 1%–3%, while in Asia it is about 0.5%. Clinically, psoriasis can be classified into several types, including plaque psoriasis, erythrodermic psoriasis, pustular psoriasis, and arthropathic psoriasis, with plaque psoriasis being the most common. Typical pathological features of different types of psoriasis include hyperkeratosis, keratinocyte proliferation, dermal capillary dilation, and inflammatory cell infiltration. The pathogenesis of psoriasis is closely related to genetic susceptibility and environmental factors. Recent immunological and genetic studies have shown that the IL-23 / IL-17 pathway plays a crucial role in the development and progression of psoriasis. During this process, dendritic cells and Th17 cells are activated, releasing various inflammatory cytokines, promoting abnormal proliferation and chemotaxis of keratinocytes, further forming a positive feedback loop that promotes the recruitment of immune cells, leading to persistent chronic inflammation.

[0003] Taking psoriasis, an inflammatory skin disease, as an example, current treatment options are primarily individualized based on the severity of the condition and whether it is accompanied by psoriatic arthritis. Common methods include topical corticosteroids, vitamin D3 derivatives, calcineurin inhibitors, systemic drugs such as methotrexate and cyclosporine, as well as phototherapy and biologics. Although these treatments can alleviate symptoms to some extent, limitations such as local skin irritation and adverse reactions remain. Therefore, developing safer and more effective new therapeutic drugs remains an important direction for current psoriasis research.

[0004] *Anoectochilus roxburghii* is a traditional Chinese herbal medicine widely used in folk medicine. It possesses various pharmacological activities, including anti-obesity, hypoglycemic, anti-osteoporosis, and hepatoprotective effects. Kinsenoside, a bioactive glycoside extracted from *Anoectochilus roxburghii*, has been extensively studied and has shown important roles in protecting the liver, promoting bone formation, combating hyperglycemia, regulating lipid metabolism, and inhibiting infection.

[0005] The research group of the inventors of this invention previously reported compounds with anti-autoimmune hepatitis activity and their preparation methods (see Chinese patent application CN106317142A). Using Kinsenoside (KD) with the following structural formula, its application in the preparation of drugs for anti-autoimmune hepatitis was achieved. Currently, there are no studies on the association between Kinsenoside and inflammatory skin diseases.

[0006]

[0007] KD Summary of the Invention To address the limited efficacy of existing treatments for inflammatory skin diseases (especially psoriasis), this invention aims to provide the application of *Anoectochilus roxburghii* glycosides in the preparation of drugs for treating inflammatory skin diseases. This invention marks the first application of *Anoectochilus roxburghii* glycosides in the preparation of drugs for treating inflammatory skin diseases, particularly psoriasis. It upregulates arachidonic acid 15-lipoxygenase (ALOX15) in the skin, thereby intervening in psoriasis, and provides a candidate drug for the clinical development of natural products for treating inflammatory skin diseases, including psoriasis.

[0008] To achieve the above objectives, the present invention provides the application of *Anoectochilus roxburghii* glycoside in the preparation of drugs for treating inflammatory skin diseases, characterized in that the structural formula of *Anoectochilus roxburghii* glycoside is as follows: .

[0009] As a further preferred embodiment of the present invention, the inflammatory skin disease is psoriasis.

[0010] As a further preferred embodiment of the present invention, the anolysin is used to treat psoriasis by upregulating arachidonic acid 15-lipoxygenase (ALOX15) in the skin.

[0011] As a further preferred embodiment of the present invention, the psoriasis is imiquimod-induced psoriasis-like skin inflammation.

[0012] As a further preferred embodiment of the present invention, the genistein can reduce the PASI score of the skin, the transcription level of pro-inflammatory factors in the skin, and the ratio of neutrophils, Th17 cells and γδT cells in lymph nodes, improve the immune balance ratio of γδT / Treg and Th17 / Treg in lymph nodes, reduce the proportion of dendritic cells in the spleen and the secretion level of their pro-inflammatory factors, and alleviate the appearance of psoriatic dermatitis.

[0013] As a further preferred embodiment of the present invention, the psoriasis-like dermatitis appearance includes at least one of skin erythema, skin scaling, and skin thickening.

[0014] As a further preferred embodiment of the present invention, the pro-inflammatory factors in the skin include at least one of IL-6, IL-23, CXCL1, and CCL20.

[0015] As a further preferred embodiment of the present invention, the pro-inflammatory factors in the spleen include at least one of TNF-α, IL-1β and IL-23.

[0016] As a further preferred embodiment of the present invention, the dose of roximate glycoside applied per kg of organism per day is 40-80 mg, preferably 80 mg.

[0017] Compared with existing technologies, this invention provides a novel drug for treating inflammatory skin diseases. Taking psoriasis as an example, this invention is the first to discover that roxithrin can significantly improve psoriasis-like skin inflammation, demonstrating good therapeutic effects. Previous literature and early studies have mostly focused on the pharmacological effects of keratinogen (KD) in areas such as liver protection and blood sugar reduction, but its effects and molecular targets in inflammatory skin diseases, especially psoriasis, have not been reported. This invention, by constructing an imiquimod (IMQ)-induced mouse model of psoriasis-like dermatitis, demonstrates for the first time that roxithrin can effectively alleviate psoriasis-like skin inflammation in mice, exploring the role of roxithrin in intervening in inflammatory skin diseases, including psoriasis. Taking psoriasis as an example, this invention reveals that Anoectochilus glycoside can upregulate the expression of ALOX15 in the skin and thus intervene in psoriasis-like dermatitis. In addition, Anoectochilus glycoside can significantly downregulate the transcriptional expression levels of typical psoriasis-related pro-inflammatory factors and chemokines such as IL-23, IL-6, IL-1β, CXCL1, CXCL2 and CCL20.

[0018] Establishing animal models that closely resemble human diseases is crucial. This invention employs IMQ-induced psoriasis-like dermatitis in mice to mimic the typical skin lesions of clinical psoriasis vulgaris. Through establishing an imiquimod-induced mouse psoriasis-like dermatitis model, this invention found that intervention with roxithromycin significantly alleviated pathological manifestations such as erythema, scaling, and thickening of the mouse skin, resulting in a significant decrease in PASI scores. Furthermore, the efficacy of roxithromycin was superior to that of the commonly used clinical drug methotrexate (MTX), validating the potential of roxithromycin in treating psoriasis.

[0019] Anoectochilin significantly downregulated the transcriptional expression levels of multiple pro-inflammatory and chemokine factors in the skin of psoriatic mice, including IL-6, IL-23, CXCL1, and CCL20; reduced the proportion of neutrophils, Th17 cells, and γδT cells in lymph nodes; decreased the proportion of dendritic cells (i.e., the proportion of live cells) and the expression levels of their pro-inflammatory factors (such as TNF-α, IL-1β, and IL-23) in the spleen; and upregulated the expression of ALOX15 in psoriatic skin.

[0020] As illustrated in the examples below, HE and immunohistochemical results revealed that roxithromycin can alleviate pathological thickening of the skin and inhibit keratinocyte proliferation (Ki67⁺), angiogenesis (CD31⁺), and T cell infiltration (CD3⁺). Simultaneously, qPCR results showed that it can downregulate the transcriptional expression of key pro-inflammatory and chemokine factors such as IL-6, IL-23, CXCL1, and CCL20, confirming its significant anti-inflammatory effect. Furthermore, flow cytometry analysis showed that roxithromycin treatment significantly reduced the proportions of neutrophils, γδT cells, and Th17 cells in lymph nodes, thereby improving the imbalance between Th17 / Treg and γδT / Treg, and its immunomodulatory effect was close to or superior to that of MTX.

[0021] Taking the IL-23 / IL-17 pathway as an example, this invention not only discovered that *Anoectochilus roxburghii* glycosides can downregulate the transcriptional levels of IL-23 / IL-17 in psoriatic-like skin tissue, but also further explored how KD affects the IL-23 / IL-17 pathway. Through high-throughput transcriptome sequencing and protein level validation, it was found that *Anoectochilus roxburghii* glycosides can significantly upregulate ALOX15 expression in the skin, suggesting that it is likely an important target for the therapeutic effect of *Anoectochilus roxburghii* glycosides. Intervention combined with the ALOX15-specific inhibitor ML351 revealed that ML351 can significantly weaken the alleviating effect of *Anoectochilus roxburghii* glycosides on psoriatic-like dermatitis, including improved skin appearance, decreased PASI score, and upregulation of ALOX15 protein expression. Further research found that ML351 intervention can partially reverse the downregulation of neutrophils, γδT cells, and Th17 cells in the lymph nodes of psoriatic-like mice by *Anoectochilus roxburghii* glycosides, as well as the regulation of the Th17 / Treg and γδT / Treg imbalance. The above results suggest that ALOX15 plays a key role in the regulation of psoriasis-like immune inflammation in mice by Anoectochilus roxburghii glycosides.

[0022] Furthermore, immunofluorescence co-staining showed that ALOX15 was mainly expressed in CD11c⁺ dendritic cells. Flow cytometry results indicated that ALOX15 could inhibit the increase in the number of dendritic cells and reduce the secretion of pro-inflammatory factors TNF-α, IL-1β, and IL-23, while ML351 could weaken this regulatory effect. This further suggests that ALOX15 can regulate the number and function of psoriatic immune cells, thereby exerting an anti-psoriatic effect.

[0023] Furthermore, the drug for treating inflammatory skin diseases prepared based on Anoectochilus roxburghii glycosides in this invention can avoid local irritation and adverse reactions. Local irritation is generally an adverse reaction of topical medications; the Anoectochilus roxburghii glycosides in this invention, when taken orally, will not cause local irritation. Taking psoriasis as an example, systemic drugs for psoriasis such as methotrexate and cyclosporine can cause liver and kidney toxicity problems. However, the oral administration of Kinsenoside in this invention is preferred at a dose of 40-80 mg / kg. Studies have shown that the oral administration of KD is 20-100 mg / kg (for example, see: [1] Gao L, Chen X, Fu Z, Yin J, Wang Y, SunW, Ren H, Zhang Y. Kinsenoside Alleviates Alcoholic Liver Injury by Reducing Oxidative Stress, Inhibiting Endoplasmic Reticulum Stress, and Regulating AMPK-Dependent Autophagy. Front Pharmacol. 2022 Jan 18;12:747325. [2] Xiang M, Liu T, Tian C, Ma K, Gou J, Huang R, Li S, Li Q, Xu C, Li L, Lee CH, Zhang Y. Kinsenoside attenuates liver fibro-inflammation by suppressing dendritic cells via the PI3K-AKT-FoxO1 pathway. Pharmacol Res. 2022 Mar;177:106092. [3]Lee YG, Sue YM, Lee CK, Huang HM, He JJ, Wang YS, Juan SH. Synergisticeffects of cAMP-dependent protein kinase A and AMP-activated protein kinase on lipolysis in kinsenoside-treated C3H10T1 / 2 adipocytes. Phytomedicine. 2019Mar 1;55:255-263.), there is no systemic toxicity mentioned above. Attached Figure Description

[0024] Figure 1This study demonstrates the effects of low-dose roxithromycin (KD-L, 40 mg / kg; denoted as "IMQ+KD-L"), high-dose roxithromycin (KD-H, 80 mg / kg; denoted as "IMQ+KD-H"), and methotrexate (MTX, 1 mg / kg; denoted as "IMQ+MTX") interventions on the appearance of skin lesions and PASI scores in an imiquimod (IMQ)-induced mouse psoriasis-like dermatitis model. Figure 1 In Figure A, the images show the appearance of skin lesions on the backs of mice in different treatment groups. The results showed that the skin erythema, scaling and thickening in the roxithromycin treatment group were significantly reduced compared with the imiquimod group. Figure 1 B in the figure represents the dynamic changes in PASI total score and scores for erythema, scaling, and skin thickness in each group during the 6-day treatment period. It can be seen that the indicators in the roximate treatment group are significantly lower than those in the imiquimod group, and the treatment effect is close to or better than that in the methotrexate group, suggesting that roximate can effectively relieve the symptoms of imiquimod-induced psoriasis-like dermatitis.

[0025] Figure 2 The study aimed to evaluate the pathological changes in the skin tissue on the back of mice. HE staining showed that treatment with Anoectochilus roxburghii significantly reduced typical pathological changes of psoriasis, such as epidermal thickening and dermal infiltration. Immunohistochemical staining results showed that the number of Ki67-positive cells in the skin of the Anoectochilus roxburghii group was reduced, indicating that cell proliferation was inhibited. The number of CD31-positive blood vessels and CD3-positive T cells also decreased.

[0026] Figure 3 This study compared the mRNA expression levels of pro-inflammatory and chemokine factors in the skin of mice from different groups. qPCR analysis revealed that roxithrin significantly downregulated the transcriptional levels of typical psoriasis-related pro-inflammatory factors such as IL-23, IL-17, IL-6, IL-1β (corresponding to IL-1b in the figure), CXCL1, CXCL2, and CCL20, suggesting its anti-inflammatory effect. n=5, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0027] Figure 4 Analysis of the regulation of immune cell subset ratios in lymph nodes of psoriasis-like mice by anolyse glycosides; among which: Figure 4 A in the figure shows the results of flow cytometry, displaying the proportions of neutrophils (CD11b⁺Ly6G⁺), γδT cells (TCRγδ⁺RORγt⁺), Th17 cells (CD4⁺RORγt⁺), and Treg cells (CD4⁺Foxp3⁺) in the lymph nodes of different treatment groups; Figure 4 Figures B and C in the table present statistical results. The results show that, compared to the imiquimod group, the roxithromycin group significantly reduced the proportions of neutrophils, γδT cells, and Th17 cells. Its effect in improving the γδT / Treg and Th17 / Treg immune balance was comparable to or superior to that of the methotrexate group. Figure 4 In B and C, n=5, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0028] Figure 5 Detection of key target genes for the improvement of psoriasis-like dermatitis by Anoectochilus roxburghii glycosides; among which: Figure 5 A in the figure represents a volcano plot of differential transcriptome expression analysis, showing that ALOX15 was significantly upregulated in the Anoectochilin intervention group. The upregulated genes in the figure are: Soat1, Krt77, Alox12e, Far2, Cyp2j12, Elovl3, Alox8, Awat2, Cyp2j8, and Alox15; the downregulated genes are: Hoxb5, Hoxb6, Hoxb7, Lhx8, Hoxc8, Col12a1, Cd177, Sox11, Ly6g, and P4ha3. Figure 5 B in the diagram represents the interaction network of upregulated genes, with ALOX15 located at one of the core nodes, suggesting that it may play a key role in the intervention process. Figure 5 In the figure, C represents the Western blot results and quantitative analysis of ALOX15 protein in skin tissue, showing that IMQ induction significantly inhibits ALOX15 expression, while anolysin can significantly restore its expression level. n=5, **p < 0.01, ****p < 0.0001.

[0029] Figure 6 The effects of roximate glycoside and the ALOX15 inhibitor ML351 on IMQ-induced psoriatic dermatitis; among which: Figure 6 In the figure, A represents the appearance of the skin lesions on the back of mice in each treatment group and HE staining. Compared with the IMQ group, the appearance of skin inflammation and epidermal thickness were significantly improved in the roximate group (referred to as "IMQ+KD" in the figure), while the improvement effect was significantly weakened after roximate group was combined with ML351 (referred to as "IMQ+KD+ML351" in the figure). Figure 6B in the figure represents the dynamic change curves of the PASI total score and the scores for erythema, thickness, and scaling. It shows that lanoxetine can significantly reduce each score, while the addition of subcutaneous administration of ML351 (10 mg / kg) weakens the therapeutic effect of lanoxetine, further supporting ALOX15 as the key target of lanoxetine in alleviating psoriasis. Figure 6 In the figure, C represents the Western blot results and quantitative analysis of ALOX15 protein in skin tissue. Compared with the IMQ group (corresponding to IMQ being +, KD being -, and ML351 being - in the figure), ALOX15 expression was upregulated in the IMQ+KD group (corresponding to IMQ being +, KD being +, and ML351 being - in the figure), while ALOX15 expression was inhibited in the IMQ+ML351 group (corresponding to IMQ being +, KD being -, and ML351 being + in the figure) and the IMQ+KD+ML351 group (corresponding to IMQ being +, KD being +, and ML351 being + in the figure). n=5, *p < 0.05, **p < 0.01.

[0030] Figure 7 Analysis of immune cell subsets in lymph nodes of psoriasis-like mice treated with roxithromycin and the ALOX15 inhibitor ML351; among which: Figure 7 A in the figure represents the results of flow cytometry, showing the proportions of neutrophils (CD11b⁺Ly6G⁺), γδT cells (TCRγδ⁺RORγt⁺), Th17 cells (CD4⁺RORγt⁺), and Treg cells (CD4⁺Foxp3⁺) in each group; Figure 7 B and C in the figure represent the statistical analysis results of the above-mentioned cell subset proportions. The results showed that compared with the imiquimod group (corresponding to IMQ+, KD-, and ML351- in the figure), the anabolin group (corresponding to IMQ+, KD+, and ML351- in the figure) significantly reduced the proportion of inflammation-related cells (neutrophils, γδT, and Th17), as well as the γδT / Treg and Th17 / Treg ratios. When combined with ML351 (corresponding to IMQ+, KD+, and ML351+ in the figure), the above-mentioned regulatory effects of anabolin were partially weakened, suggesting that ALOX15 is a key target for anabolin in regulating psoriatic-like inflammation. n=5, *P<0.05, ***P<0.001, ****P<0.0001.

[0031] Figure 8 The results showed that roximate can reduce the number of dendritic cells (DCs) and the secretion of pro-inflammatory factors in the spleen of psoriasis-like mice; among which: Figure 8A in the figure represents the results of immunofluorescence co-staining, which shows that ALOX15 is mainly expressed in CD11c⁺ dendritic cells, suggesting that it may affect the number or function of dendritic cells; Figure 8 B in the figure represents the changes in the number of dendritic cells analyzed by flow cytometry. IMQ treatment (corresponding to IMQ being +, KD being -, and ML351 being - in the figure) significantly increased the proportion of dendritic cells, while Anoectochilus glycoside intervention (corresponding to IMQ being +, KD being +, and ML351 being - in the figure) significantly reduced the proportion of dendritic cells. After ML351 was used in combination (corresponding to IMQ being +, KD being +, and ML351 being + in the figure), the inhibitory effect was weakened. Figure 8 C represents the detection of secretion levels of pro-inflammatory cytokines TNF-α, IL-1β, and IL-23 in dendritic cells. The results showed that the roxithromycin group (corresponding to IMQ +, KD +, and ML351 - in the figure) significantly downregulated the secretion of these factors in dendritic cells. However, after combination with the ALOX15 inhibitor ML351 (corresponding to IMQ +, KD +, and ML351 + in the figure), the secretion levels of these inflammatory factors rebounded, suggesting that roxithromycin negatively regulates the number of dendritic cells and the inflammatory response through the ALOX15 pathway. n=5, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0033] Example 1: Pharmacological verification of the efficacy of roxithromycin in alleviating imiquimod-induced psoriatic dermatitis The roximate powder was prepared by our research group (the preparation method can be found in Chinese Patent ZL201610685644.8, US Patent US1115456, or the journal article Pharmacol Res. 2022;177:106092.), with a purity >98%, and the structural formula is shown below:

[0034] KD This embodiment establishes an imiquimod (IMQ)-induced psoriatic dermatitis model in mice to verify the therapeutic effect of anesthetic in animals. C57BL / 6 mice were randomly divided into five groups of five mice each (n=5): a control group, an imiquimod model group, a low-dose anesthetic group (KD-L, 40 mg / kg; i.e., 40 mg of KD per kg of body weight per day), a high-dose anesthetic group (KD-H, 80 mg / kg; i.e., 80 mg of KD per kg of body weight per day), and a methotrexate positive control group (MTX, 1 mg / kg; i.e., 1 mg of MTX per kg of body weight per day). Except for the control group, the remaining mice received 62.5 mg of imiquimod applied to the neck daily for 6 consecutive days, along with oral administration of the drug. The experimental groups were denoted as follows: Control group: "Control" group; Imiquimod model group: "IMQ" group; Low-dose group of Anoectochilus roxburghii glycosides: "IMQ+KD-L" group; High-dose group of Anoectochilus roxburghii glycosides: "IMQ+KD-H" group; Methotrexate positive control group: "MTX" group.

[0035] The results showed that, compared with the model group, the mice in the roxithromycin treatment group had significantly reduced typical psoriasis-like changes such as skin erythema, scaling, and thickening, and the PASI score decreased significantly. Moreover, the therapeutic effect of the roxithromycin treatment group was close to or better than that of the methotrexate group. Figure 1 Among them, the high-dose group of roxithromycin showed better performance than the low-dose group. HE staining results indicated that roxithromycin treatment could reduce excessive epidermal proliferation, and immunohistochemical results showed a significant decrease in the number of Ki67, CD3, and CD31 positive cells, suggesting that it has anti-epidermal proliferation, anti-T cell infiltration, and anti-angiogenic effects. Figure 2 Among them, the high-dose group of roxithromycin showed better performance than the low-dose group. qPCR results showed that roxithromycin could downregulate the mRNA expression of various pro-inflammatory factors and chemokines such as IL-6, IL-17, IL-23, CXCL1, and CCL20 in skin tissue, indicating that it has significant anti-inflammatory activity. Figure 3Among them, the high-dose group of roxithromycin showed better performance than the low-dose group. Flow cytometry analysis of the proportions of neutrophils (CD11b⁺Ly6G⁺), γδT cells (TCRγδ⁺RORγt⁺), Th17 cells (CD4⁺RORγt⁺), and Treg cells (CD4⁺Foxp3⁺) in mouse lymph nodes revealed that roxithromycin significantly reduced the proportions of neutrophils, γδT cells, and Th17 cells. The effect of roxithromycin in improving the γδT / Treg and Th17 / Treg immune balance was similar to or greater than that of the methotrexate group. Figure 4 Among them, the high-dose group of anoectochilin showed better performance than the low-dose group of anoectochilin.

[0036] Example 2: Study on the mechanism by which anolyse glycosides regulate immune inflammation in psoriasis by upregulating ALOX15 expression. To investigate the mechanism of action of roxithromycin in alleviating psoriatic dermatitis, this study performed transcriptome sequencing analysis on skin tissues from mice in the imiquimod model group and the high-dose roxithromycin treatment group to screen for potential key targets. Differential transcriptome analysis showed that roxithromycin significantly upregulated the expression of the arachidonic acid metabolite ALOX15, and was located at a core node in the upregulated gene interaction network (e.g., ...). Figure 5 As shown in A and B; where, Figure 5 In this context, B represents the top ten hub genes obtained from a protein-protein interaction map of upregulated genes using Cytoscape. Western blot results further confirmed that ALOX15 expression was significantly downregulated in the skin of mice in the imiquimod model group, but its expression was restored after intervention with roxithromycin. Figure 5 The C in the figure suggests that ALOX15 is likely a key target for its therapeutic effect.

[0037] To further verify the role of ALOX15 in this mechanism, its specific inhibitor ML351 was introduced and administered subcutaneously to mice, which were divided into the following experimental groups: Control group: Vaseline was applied (Vaseline was applied to the neck of mice daily for 6 consecutive days). IMQ group: IMQ was applied only (62.5 mg of imiquimod was applied to the neck of mice daily for 6 consecutive days); IMQ+KD group: IMQ and KD were administered (mice were treated with 62.5 mg imiquimod applied to the neck daily for 6 consecutive days, and KD was administered by gavage at a dose of 80 mg / kg). IMQ+KD+ML351 group: IMQ, KD and ML351 were administered (mice were treated with 62.5 mg imiquimod applied to the neck daily for 6 consecutive days, while KD was administered by gavage at a dose of 80 mg / kg, and ML351 was administered subcutaneously at a dose of 10 mg / kg). IMQ+ML351 group: IMQ and ML351 were administered (62.5 mg of imiquimod was applied to the neck of mice daily for 6 consecutive days, while ML351 was administered subcutaneously at a dose of 10 mg / kg).

[0038] The results showed that subcutaneous administration of ML351 (10 mg / kg; i.e., a daily dose of 10 mg of ML351 per kg of organism) significantly reversed the ameliorative effects of roxithromycin on the appearance of psoriasis-like skin lesions, PASI scores, and ALOX15 expression in mice (e.g., Figure 6 (As shown in A, B, and C). At the immune cell level, ALOX15 significantly reduces the proportions of neutrophils, γδT cells, and Th17 cells in lymph nodes, thereby correcting the immune imbalance between γδT / Treg and Th17 / Treg. However, this regulatory effect is significantly weakened after combined intervention with ML351, further supporting the key role of ALOX15 in the immunomodulatory function of ALOX15. Figure 4 and Figure 7 ).

[0039] Furthermore, immunofluorescence co-staining showed that ALOX15 was mainly distributed in CD11c⁺ dendritic cells (such as...). Figure 8 As shown in Figure A), this suggests that ALOX15 has a regulatory effect on the number or function of dendritic cells. Further experiments revealed that, compared with the control group (Control group), the number of dendritic cells and the expression of pro-inflammatory factors (TNF-α, IL-1β, IL-23) in the spleen of mice in the imiquimod model group (IMQ group) were significantly increased. Intervention with roxithromycin significantly inhibited these indicators (corresponding to the IMQ+KD group), while combined intervention with ML351 reversed this effect (corresponding to the IMQ+KD+ML351 group). This suggests that roxithromycin negatively regulates the number of dendritic cells and pro-inflammatory activation through the ALOX15 pathway, thereby alleviating psoriasis-like inflammatory responses (such as...). Figure 8 (As shown in B and C).

[0040] The above embodiments involved daily topical application of 62.5 mg IMQ to the dorsal skin of mice. Histological and inflammation-related tests of the skin lesions were performed after 6 days of application, consistent with clinical practice. This model can rapidly develop psoriatic-like lesions (erythema, scaling, and epidermal thickening) and is consistent with the mechanisms related to the human IL-23 / IL-17 pathway. To comprehensively evaluate efficacy and mechanism, the above embodiments employed phenotypic evaluation (PASI score, lesion appearance), histological indicators (HE, IHC: Ki67, CD3, CD31, etc.), molecular detection (qPCR for IL-6, IL-23, CXCL1, CCL20, etc.), and protein level validation (Western blot). Flow cytometry was also used to assess the proportions of immune cell subsets (neutrophils, γδT, Th17, Treg, DC, etc.) in lymph nodes and spleen. Sampling time for each endpoint was primarily on day 6 of IMQ induction to ensure reliable and comparable biological signals were obtained during the peak of the disease.

[0041] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of Anoectochilus roxburghii glycoside in the preparation of drugs for treating inflammatory skin diseases, characterized in that, The structural formula of the genistein is as follows: 。 2. The application as described in claim 1, characterized in that, The inflammatory skin disease mentioned is psoriasis.

3. The application as described in claim 2, characterized in that, The genistein mentioned treats psoriasis by upregulating arachidonic acid 15-lipoxygenase (ALOX15) in the skin.

4. The application as described in claim 2, characterized in that, The psoriasis described is a psoriasis-like skin inflammation induced by imiquimod.

5. The application as described in claim 2, characterized in that, The aforementioned genistein can reduce the PASI score of the skin, the transcription level of pro-inflammatory factors in the skin, and the ratio of neutrophils, Th17 cells and γδT cells in lymph nodes, improve the immune balance ratio of γδT / Treg and Th17 / Treg in lymph nodes, reduce the proportion of dendritic cells in the spleen and the secretion level of their pro-inflammatory factors, and alleviate the appearance of psoriatic dermatitis.

6. The application as described in claim 5, characterized in that, The appearance of psoriatic dermatitis includes at least one of skin erythema, skin scaling, and skin thickening.

7. The application as described in claim 5, characterized in that, The pro-inflammatory factors in the skin include at least one of IL-6, IL-23, CXCL1, and CCL20.

8. The application as described in claim 5, characterized in that, Pro-inflammatory factors in the spleen include at least one of TNF-α, IL-1β, and IL-23.

9. The application as described in claim 1, characterized in that, The daily dose of anoectochilin applied per kg of organism is 40-80 mg, preferably 80 mg.

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