Application of total glucosides of paeony in treatment of psoriasis by combining with judiciespecially and calcipotriol

Abstract

The invention discloses an application of total glucosides of paeony in treatment of psoriasis in combination with Jikukiespecially and calcipotriol, and relates to the technical field of medical treatment, a psoriasis-like mouse model induced by imiquimod is established, and the synergistic treatment effect of a triple therapy of total glucosides of paeony, Jikukiespecially and calcipotriol is verified; the application comprises the step of jointly applying the three medicines according to a specific dosage. The triple therapy can improve erythema, scale and infiltration symptoms of psoriasis-like skin lesion, effectively reduce the area and severity index score of psoriasis, and inhibit the protein and mRNA expression level of key proinflammatory cytokines in skin lesion tissues; compared with a single drug or a combination of two drugs, the triple therapy provided by the invention shows a better treatment effect, and a new and effective drug combination scheme is provided for clinical treatment of psoriasis.

Description

Technical Field

[0001] This invention belongs to the field of medical technology, and in particular relates to the application of total glucosides of paeony in combination with secukinol and calcipotriol in the treatment of psoriasis. Background Technology

[0002] Psoriasis is a common, chronic, inflammatory, immune-mediated skin disease. Its main pathological features include excessive proliferation and abnormal differentiation of epidermal keratinocytes, as well as the infiltration and activation of immune cells in the dermis. The typical clinical manifestation is well-defined red plaques covered with silvery-white scales, often accompanied by itching or discomfort. Its pathogenesis involves a complex interaction of multiple factors, including genetics, environment, and immune system abnormalities. In particular, dysregulation of pro-inflammatory cytokine signaling pathways such as IL-17, TNF-α, and IL-23 plays a central role in the development and progression of the disease. Psoriasis has a prolonged course and is prone to recurrence, significantly impacting the physical and mental health and quality of life of patients.

[0003] Currently, biologics targeting specific cytokines have achieved significant results in clinical treatment. However, single-pathway blocking strategies may have insufficient efficacy or limited long-term effects for some patients. This is mainly because the pathological process of psoriasis is driven by multiple cytokine pathways in synergy, making it difficult to completely inhibit with a single target. Therefore, the following approach is proposed to address these issues. Summary of the Invention

[0004] The purpose of this invention is to provide the application of total paeoniflorin combined with secukinur and calcipotriol in the treatment of psoriasis. By combining total paeoniflorin, secukinur and calcipotriol, a synergistic effect is produced, which can effectively inhibit the key inflammatory factor network of psoriasis on multiple targets, thereby improving skin lesion symptoms and reducing the severity of the disease. This solves the problem that the efficacy of existing single-target therapies is insufficient or limited due to their inability to fully inhibit the complex cytokine network.

[0005] To solve the above-mentioned technical problems, the present invention is achieved through the following technical solution: This invention relates to the application of total paeoniflorin combined with secukinol and calcipotriol in the treatment of psoriasis, wherein the application includes administering total paeoniflorin, secukinol and calcipotriol in combination to an individual suffering from psoriasis.

[0006] Furthermore, the dosage of the total paeoniflorin is 50 mg / kg–150 mg / kg daily, preferably 100 mg / kg.

[0007] Furthermore, the dosage of the secukinic acid is 10 mg / kg–30 mg / kg daily, preferably 20 mg / kg.

[0008] Furthermore, the dosage of calcipotriol is 20 mg / cm²–40 mg / cm² daily, preferably 30 mg / cm².

[0009] Furthermore, the total glucosides of paeony are administered via gavage, secukinol is administered via intraperitoneal injection, and calcipotriol is administered via topical application.

[0010] Furthermore, the combined medication is used to reduce the PASI score of psoriatic lesions.

[0011] Furthermore, the combined medication is used to inhibit the expression of psoriasis-related inflammatory factors, including one or more of IL-17, IL-21, IL-23, TNF-α, and IFN-γ.

[0012] Furthermore, the combined medication is used to improve the erythema, scaling, and infiltration symptoms of psoriatic lesions.

[0013] A pharmaceutical composition comprising therapeutically effective amounts of total paeoniflorin, secukinol, and calcipotriol, as well as a pharmaceutically acceptable carrier.

[0014] Furthermore, the composition is used to treat psoriasis.

[0015] The present invention has the following beneficial effects: This invention utilizes the synergistic effects of total glucosides of paeony, secukinol, and calcipotriol to improve typical clinical symptoms of psoriasis and effectively reduce the severity of skin lesions. This combination therapy reduces the Disease Activity Index score and shows a trend of superiority over single or dual-drug therapies. Mechanistically, this regimen synergistically inhibits the expression and release of key pro-inflammatory cytokines closely related to the pathogenesis of psoriasis at multiple targets, thereby effectively controlling the inflammatory response. This synergistic effect is expected to provide a new and effective strategy for clinical treatment while achieving better therapeutic effects.

[0016] Of course, any product implementing this invention does not necessarily need to achieve all of the advantages described above at the same time. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying 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.

[0018] Figure 1 This is a statistical chart showing the clinical manifestations of skin lesions on the back of mice in this invention. Figure 2 The image shows the PASI score and skin lesion on the back of the mouse in this invention. Figure 3 Statistical chart of the protein expression level of inflammatory factors in the skin lesion tissue of mice in the present invention; Figure 4 Statistical chart of the mRNA expression level of inflammatory factors in the skin lesion tissue of mice in the present invention; Specific embodiments

[0019] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

[0020] Please refer to Figure 1-4 As shown, the present invention is the application of total glucosides of paeony combined with secukinumab and calcipotriol in the treatment of psoriasis, including: 1. Materials and methods 1.1. Experimental animals 24 male C57 mice, weighing 20±2 g, SPF grade. Purchased from Henan Skbeshi Biotechnology Co., Ltd. [Experimental animal production license number SCXK (Henan) 2020-0005], the feeding environment temperature is (23±2) °C, and the relative humidity is (60±10)%. They are fed and watered freely, and the drinking water is sterilized secondary ultrapure water, and the quality of the drinking water meets the requirements of the national standard of the People's Republic of China "Hygienic Standard for Drinking Water" (GB5749-2006). Adapt to the animal house environment for one week before the experiment.

[0021] 1.2. Main drugs and reagents Vaseline (New Joy Company, Product No.: SAN10707A); 5% Imiquimod Cream (Tianfang Pharmaceutical Co., Ltd., National Drug Approval Number: H20031230); Total Glycosides of Paeoniae Radix Alba (Ningbo Lihua Pharmaceutical Co., Ltd., National Drug Approval Number: H20055058); Secukinol (Novartis, Switzerland, National Drug Approval Number: SJ20225001); Calcipotriol (Leo Pharmaceutical, National Drug Approval Number: HJ20160070); IL-21 antibody, IL-17 antibody, IL-23 antibody (Affinity Pharmaceuticals, Catalog Nos.: DF6127, DF4818, DF13760); IFNγ antibody, TNFα antibody (Proteintech Pharmaceuticals, Catalog Nos.: 15365-1-AP, 17590-1-AP); Horseradish enzyme-labeled goat anti-mouse IgG, horseradish enzyme-labeled goat anti-rabbit IgG (Zhongshan Jinqiao Pharmaceuticals, Catalog Nos.: ZB-2305, ZB-2301); TRI Reagent, SYBR Green qPCR Mix (Biosharp Pharmaceuticals, Catalog Nos.: BS258A, BL697A); Hifair II 1st StrandcDNA Synthesis Kit (YEASEN Pharmaceuticals, Catalog No.: 11121ES60).

[0022] 1.3 Main Instruments Real-time PCR instrument (Roche); benchtop high-speed refrigerated centrifuge (Shanghai Luxiangyi); electrophoresis system (Bio-RAD); gel imaging system (Bio-RAD); chemiluminescence imaging system (Hangzhou Shenhua Technology Co., Ltd.); shaker (SCILOGEX).

[0023] 1.4 Animal Grouping After 24 days of acclimatization feeding, male C57 mice were randomly divided into a control group, a model group, a model + total paeoniflorin + secukinol group, and a model + total paeoniflorin + calcipotriol group, with 6 mice in each group.

[0024] 1.5 Establishment of a psoriasis-like mouse model A classic topical imiquimod method was used to establish a mouse model of psoriasis-like skin lesions. The day before the experiment, the back of the mouse was dehaired using an electric shaver, followed by the application of depilatory cream to remove any remaining hair, creating an exposed skin area of ​​approximately 2 cm × 3 cm. The following day, 62.5 mg of 5% imiquimod cream was evenly applied to the exposed skin. This intervention was continued for 5 days, during which the mouse had free access to food and water. Successful modeling was indicated by the appearance of psoriasis-like symptoms in the local lesions, including erythema, scaling, and infiltration.

[0025] 1.6 Administration Control group: No modeling was performed; 62.5 mg of petroleum jelly was applied topically to the back; 0.2 mL of normal saline was administered by gavage daily. Model group: 62.5 mg of 5% imiquimod was applied topically to induce modeling (in clinical practice, imiquimod is used to simulate the symptoms of skin diseases in mice (such as psoriasis), and the specific modeling process is mentioned in section 1.5, "Establishment of Psoriasis-like Mouse Model"). 0.2 mL of physiological saline was administered by gavage daily. Model + total paeoniflorin + secukinur group: 62.5 mg of 5% imiquimod was applied topically to induce modeling. At the same time, 0.2 mL of total paeoniflorin solution was administered by gavage at a dose of 100 mg / kg daily; and 0.2 mL of secukinur solution was injected intraperitoneally at a dose of 20 mg / kg daily.

[0026] Model + Paeonia lactiflora total glycosides + calcipotriol group: 62.5 mg of 5% imiquimod was applied topically to induce modeling. At the same time, 0.2 mL of Paeonia lactiflora total glycosides solution was administered by gavage at a dose of 100 mg / kg daily; calcipotriol was applied topically at a dose of 30 mg / cm2 daily.

[0027] Model + Paeonia lactiflora total glycosides group: 62.5 mg of 5% imiquimod was applied topically to induce the model, and at the same time, 0.2 mL of Paeonia lactiflora total glycosides solution was administered by gavage at a dose of 100 mg / kg per day.

[0028] Model + total paeoniflorin + secukinol + calcipotriol group: 62.5 mg of 5% imiquimod was applied topically to induce modeling. At the same time, 0.2 mL of total paeoniflorin solution was administered by gavage at a dose of 100 mg / kg daily; 0.2 mL of secukinol solution was injected intraperitoneally at a dose of 20 mg / kg daily; and calcipotriol was applied topically at a dose of 30 mg / cm² daily.

[0029] Each group underwent intervention for 5 consecutive days, during which they had free access to food and water. Samples were collected on the 6th day.

[0030] 1.7. Collection of materials After the last gavage, mice were fasted for 12 hours but allowed free access to water. They were then anesthetized with an intraperitoneal injection of 0.3% sodium pentobarbital. Local skin lesion images were acquired, and skin samples were collected from C57 mice. Three mice from each group were soaked in 4% paraformaldehyde for pathological examination, and the remaining samples were frozen at -80°C for Western blotting and PCR detection.

[0031] 1.8 Observe the skin lesions and the Psoriasis Area and Severity Index (PASI) score. The Psoriasis Area and Severity Index (PASI) score was used to measure the severity of the condition. The area and severity of psoriasis were measured in all samples, and images of the affected skin areas were taken under stable and uniform lighting conditions; the scoring criteria are shown in Table 1.

[0032] Table 1. PASI Scoring Table for Mice 1.9 Western blotting to detect the expression of related proteins Total protein was extracted from mouse skin tissues from each group, and protein concentration was determined using the BCA method. After adjusting the protein concentrations of each group to be consistent, the samples were boiled in a 100℃ metal bath for 5 min. 30 μg of total protein was loaded into each well, and after 10% SDS-PAGE electrophoresis, the intermediate membrane was transferred to a polyvinylidene fluoride (PVDF) membrane using a steady-current transfer at 200 mA. After transfer, the membrane was blocked with 5% skim milk at 37℃ for 1 h. The blocked PVDF membrane was then removed and immersed in 1×TBST buffer, and slowly washed on a shaker for 5 min. It was then transferred to a small bag containing primary antibody and incubated overnight at 4℃. The next day, the membrane was washed three times with TBST (15 min each time), and the corresponding HRP-labeled secondary antibody was added. The membrane was incubated at room temperature for 2 h, washed three times with TBST (15 min each time), and a chemiluminescent developing solution (A:B = 1:1) was prepared by mixing the solutions in a specific ratio. The solution was then transferred to a gel imaging analyzer and exposed for development in chemiluminescent mode.

[0033] 1.10. Detection of relevant mRNA expression using real-time quantitative reverse transcription polymerase chain reaction (qRTPCR). Total RNA was extracted from mouse skin tissue using the TRIzol method, and its concentration and purity were determined. Reverse transcription was performed according to the instructions of the reverse transcription kit. The reaction program was: 95℃, 3 min; (95℃, 15 s; 60℃, 30 s), for a total of 40 cycles. The experimental results were statistically analyzed and calculated using the real-time PCR software LightCycler480, and the corresponding raw data files were generated in Excel format. The primer sequences used are detailed in Table 2.

[0034] Table 2 Primer Sequences 1.11 Statistical Analysis SPSS version 26.0 was used for statistical analysis. Experimental data were analyzed using... ±s indicates that the first step is to test the normality and homogeneity of variance of all groups. If the data meet the normality test (P>0.05) and the homogeneity of variance test (P>0.05), subsequent comparisons between groups are performed using one-way ANOVA, and comparisons between two groups are performed using independent samples t-test. If the variances are not homogeneous, Dunnet's T3 test is used. If there are cases where the data do not meet the normality test, the rank-sum test is used. When P<0.05, the difference between groups is considered statistically significant.

[0035] 2. Results 2.1 Clinical observation and comparison of dorsal skin lesions in mice of different groups: After 6 days, compared with the normal control group, mice in the imiquimod-induced model group showed psoriasis-like symptoms on their backs, including erythema, scaling, and infiltration (P < 0.05). Compared with the IMQ model group, the dorsal skin lesions in the combined treatment group (optimal content ratio: total paeoniflorin: secukinol: calcipotriol = 100 mg / kg: 20 mg / kg: 30 mg / cm2) were significantly improved (P < 0.05). Furthermore, the results also indicated that the triple therapy of total paeoniflorin + secukinol + calcipotriol, compared with the use of total paeoniflorin alone and the commonly used dual therapy, can reduce the clinical severity of IMQ-induced psoriasis. For details, please refer to [link to relevant documentation]. Figure 1 As shown, Figure 1 A represents the erythema score on the mouse's back; Figure 1 B represents the scale score on the mouse's back; Figure 1 C represents the dorsal infiltration score of the mouse.

[0036] 2.2 Comparison of PASI scores among different groups of mice: Compared with the IMQ model group, the severity of skin lesions in the PASI scores of the total paeoniflorin + secukinol group, the total paeoniflorin + calcipotriol group, the total paeoniflorin + secukinol + calcipotriol group, and the total paeoniflorin + secukinol + calcipotriol group were all significantly reduced (P < 0.05). Among them, the total paeoniflorin + secukinol + calcipotriol combined treatment group had the lowest PASI score, indicating that the combined application of the three drugs has a better therapeutic effect on experimental psoriasis in mice. (See details...) Figure 2 As shown, Figure 2 A represents the PASI score of the mouse's back. Figure 2 B shows photographs of skin lesions on the backs of mice in each group from Day 1 to Day 6.

[0037] 2.3 Comparison of IL-17, IL-21, IL-13, TNF-α, and IFN-γ expression levels in skin lesions of mice in different groups: The levels of IL-17, IL-21, IL-13, TNF-α, and IFN-γ in the model group were significantly higher than those in the normal control group (P < 0.05). The levels of IL-17, IL-21, IL-13, TNF-α, and IFN-γ in the combined administration group of total paeoniflorin + secukinol + calcipotriol were significantly lower than those in the model group (P < 0.05). This indicates that the combined administration of total paeoniflorin + secukinol + calcipotriol can significantly inhibit the cytokine levels in the skin tissue of the IMQ-induced psoriasis-like mouse model. (See details...) Figure 3 As shown.

[0038] 2.4 Comparison of IL-17, IL-21, IL-13, TNF-α, and IFN-γ mRNA expression levels in skin lesions of mice in different groups: The model group showed significantly higher levels of IL-17, IL-21, IL-13, TNF-α, and IFN-γ mRNA compared to the normal control group (P < 0.05). The combined administration group of total paeoniflorin + secukinol + calcipotriol showed significantly lower levels of IL-17, IL-13, and TNF-α mRNA compared to the model group (P < 0.05). These results indicate that the combined administration group of total paeoniflorin + secukinol + calcipotriol has a significant intervention effect on the mRNA expression of cytokines in skin lesions of IMQ-induced psoriasis-like mouse models. (See details...) Figure 4 As shown, Figure 4 A represents the expression level of IL-17 mRNA in mouse skin lesions; Figure 4 B represents the expression level of IL-21 mRNA in mouse skin lesions; Figure 4 C represents the expression level of IL-13 mRNA in mouse skin lesions; Figure 4 D represents the expression level of IFN-γ mRNA in mouse skin lesions; Figure 4 E represents the expression level of TNF-α mRNA in mouse skin lesions.

[0039] Conclusion: Previous studies have shown that the combined administration of total paeoniflorin, secukinur, and calcipotriol significantly improved dorsal skin lesions in mice, and significantly reduced PASIP scores and expression of inflammation-related cytokines. Furthermore, the triple therapy of total paeoniflorin, secukinur, and calcipotriol is more effective in alleviating the clinical symptoms of IMQ-induced psoriasis compared to total paeoniflorin alone or the commonly used dual therapy.

[0040] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0041] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. The application of total glucosides of paeony combined with secukinol and calcipotriol in the treatment of psoriasis, characterized by: The application includes the combined administration of total paeoniflorin, secukinol, and calcipotriol to individuals with psoriasis.

2. The application of total paeoniflorin combined with secukinol and calcipotriol in the treatment of psoriasis according to claim 1, characterized in that: The dosage of the total glucosides of paeony is 50 mg / kg–150 mg / kg daily, preferably 100 mg / kg.

3. The application of total paeoniflorin combined with secukinol and calcipotriol in the treatment of psoriasis according to claim 1, characterized in that: The dosage of the secukinic acid is 10 mg / kg–30 mg / kg daily, preferably 20 mg / kg.

4. The application of total paeoniflorin combined with secukinol and calcipotriol in the treatment of psoriasis according to claim 1, characterized in that: The dosage of calcipotriol is 20 mg / cm²–40 mg / cm² daily, preferably 30 mg / cm².

5. The application of total paeoniflorin combined with secukinol and calcipotriol in the treatment of psoriasis according to claim 1, characterized in that: The total glucosides of paeony are administered by gavage, secukinol by intraperitoneal injection, and calcipotriol by topical application.

6. The application of total paeoniflorin combined with secukinol and calcipotriol in the treatment of psoriasis according to claim 1, characterized in that: The combined medication is used to reduce the PASI score of psoriatic lesions.

7. The application of total paeoniflorin combined with secukinol and calcipotriol in the treatment of psoriasis according to claim 1, characterized in that: The combined medication is used to inhibit the expression of psoriasis-related inflammatory factors, including one or more of IL-17, IL-21, IL-23, TNF-α, and IFN-γ.

8. The application of total paeoniflorin combined with secukinol and calcipotriol in the treatment of psoriasis according to claim 1, characterized in that: The combined medication is used to improve the erythema, scaling, and infiltration symptoms of psoriatic lesions.

9. A pharmaceutical composition, characterized in that: It contains therapeutically effective amounts of total paeoniflorin, secukinol, and calcipotriol, as well as pharmaceutically acceptable carriers.

10. The pharmaceutical composition according to claim 9, characterized in that: The composition is used to treat psoriasis.

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