Medicine box for treating psoriasis
By combining lupene ketone with regulatory T lymphocytes, the treatment approach addresses the limitations of existing psoriasis treatments in terms of efficacy and side effects, achieving significant improvement in psoriasis symptoms and reduced treatment costs.
Patent Information
- Application Number
- CN202510939036.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-08
- Publication Date
- 2025-11-18
AI Technical Summary
The existing technologies for treating psoriasis have not yet effectively solved or have failed to solve the technical problems of how to solve them. Existing treatments for psoriasis also have issues such as side effects, limited therapeutic effects, and individual patient differences.
This treatment approach utilizes the synergistic effect of lupene ketone and regulatory T lymphocytes (Tregs). Lupene ketone and Tregs are administered orally and intravenously in combination to treat psoriasis, reducing the dosage of Tregs to lower treatment costs and the risk of complications, while simultaneously improving treatment efficacy.
It significantly improves the symptoms of psoriatic lesions, reduces the infiltration of inflammatory cells in the lesion tissue, reduces the expression of inflammatory factors, lowers treatment costs and reduces the risk of complications such as infection and bleeding, and improves treatment efficacy.
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Figure CN120960258A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of biological medicine, and particularly relates to a medicine box for treating psoriasis. BACKGROUND
[0002] Psoriasis is a common chronic inflammatory skin disease, mainly manifested as red plaques, scales and itching on the skin, and can cause complications such as arthritis in severe cases. The pathogenesis of the disease is complex, involving the immune system, genetic factors and environmental factors. Psoriasis has always been a hot and difficult point in the field of dermatology, and its pathogenesis has not been understood so far. Current research suggests that T cell-mediated cellular immunity plays an important role in it. Psoriasis is pathologically related to the autoimmune system and inflammatory response, and changes in certain immune cells and inflammatory factors in the body are important reasons for the onset, aggravation and recurrence of the disease. At present, glucocorticoids, keratolytic agents, biological agents, immunosuppressants and the like are common treatments for psoriasis in clinical practice. Although existing treatments such as topical medications, phototherapy and immunosuppressants can alleviate symptoms to some extent, due to side effects, limited treatment effectiveness and individual differences among patients, psoriasis still presents a great challenge in treatment. Therefore, it is of great clinical and social significance to develop new and effective treatments for psoriasis.
[0003] Infiltrating T cells play an important role in the occurrence and development of psoriasis, significantly affecting the progression and treatment prognosis of the disease. Studies have found that Anti-CD25, a Treg inhibitor, can exacerbate psoriasis symptoms, suggesting that regulatory T lymphocytes (Tregs) are involved in the occurrence of psoriasis. Tregs are a type of T cells with immunosuppressive effects, maintaining immune tolerance and balance by secreting anti-inflammatory cytokines and directly inhibiting the function of effector T cells. In psoriasis, Treg function may be impaired or its number reduced, leading to immune imbalance and promoting the persistence and exacerbation of skin inflammation.
[0004] Lupenone is a pentacyclic triterpenoid compound with a chemical formula of C 30 H 48 O, a molecular weight of 424.7, a CAS number of 1617-70-5, and a structural formula as shown below:
[0005] Lupenone is widely present in various plants and has a variety of complex activities, such as anti-inflammatory, anti-diabetic, anti-tumor and the like. SUMMARY
[0006] The main purpose of the present application is to provide a kit for treating psoriasis. The kit provided by the present application treats psoriasis by adopting the method of treating psoriasis by combining the treatment of adoptive transfer of regulatory T lymphocytes (Tregs) with the treatment of spirocyclic ketone. The spirocyclic ketone and the Tregs work synergistically, which not only can effectively reduce the dosage of Tregs, thereby reducing the treatment cost and the risk of complications such as infection and bleeding, but also can significantly improve the treatment effect of psoriasis.
[0007] According to one aspect of the present application, a kit for treating psoriasis is provided, which consists of a first pharmaceutical composition with spirocyclic ketone as the main effective component and a second pharmaceutical composition containing Tregs, which exist independently from each other.
[0008] In some embodiments, the Tregs are CD4 + CD25 + T cells.
[0009] In the present application, the Tregs can be autologous Tregs or allogeneic Tregs. The Tregs can be directly applied to treat psoriasis after being isolated from peripheral blood in vitro by a conventional extraction and separation method, or can be used after being expanded in vitro. The Tregs can also be stored in a cell bank or purchased on the market.
[0010] In some embodiments, the purity of the autologous Tregs can be greater than or equal to 85%, and the purity of the allogeneic Tregs can be greater than or equal to 85%.
[0011] In some embodiments, the spirocyclic ketone can be administered by a method commonly used in the art, for example, can be administered orally. When administered orally, the therapeutically effective amount of the spirocyclic ketone can be (1-11) mg / kg.
[0012] In some embodiments, the Tregs can be administered by a method commonly used in the art, for example, can be administered intravenously. The therapeutically effective amount of the Tregs can be (1x10 6 ~5x10 6 ) cells / kg.
[0013] In some embodiments, the components of the first pharmaceutical composition can further include a pharmaceutically acceptable excipient.
[0014] In some embodiments, the pharmaceutically acceptable excipient can be selected from at least one of a filler, a binder, a disintegrant, a lubricant, a thickening agent, a pigment, a flavoring agent, a solvent, a surfactant, a preservative, an antioxidant, and a coating agent.
[0015] Exemplarily, the filler can be selected from one or more of starch, sucrose, maltodextrin, resistant dextrin, lactose, microcrystalline cellulose, pregelatinized starch, sorbitol, xylitol, erythritol; the binder can be selected from one or more of starch paste, sodium carboxymethyl cellulose, hypromellose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, povidone, gelatin, polyethylene glycol, ethanol, water; the disintegrant can be selected from one or more of dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose; the lubricant can be selected from one or more of magnesium stearate, silicon dioxide, talc, polyethylene glycol; the thickening agent can be selected from one or more of hydrogenated castor oil, glycerin monostearate, aluminum stearate; the pigment can be selected from one or more of sappan, betacyanin, cochineal carmine, curcumin, carotene, amaranth, tartrazine, carmine, alizarin blue, and sunset yellow.
[0016] In some embodiments, the dosage form of the first pharmaceutical composition can be a tablet, a capsule, a powder, an oral solution, a pill, or a granule. The relevant dosage forms can be prepared according to the conventional processes in the field of pharmaceutical preparations.
[0017] In some embodiments, the components of the second pharmaceutical composition can further include a Treg preservation solution.
[0018] In some embodiments, the Treg preservation solution can be a short-term preservation solution or a cryopreservation solution.
[0019] The short-term preservation solution can allow the Tregs to be stored at room temperature or refrigerated conditions (e.g., 4°C) for a short period of time without significantly affecting the cell viability and function. Exemplarily, the components of the short-term preservation solution can include serum-free medium 90-95% v / v, human serum albumin 2-5% v / v, HEPES buffer 10-25 mM, clinical-grade IL-2 100-300 IU / mL, and antibiotics in an appropriate amount.
[0020] The cryopreservation solution can be used for long-term, low-temperature storage of Tregs. The Treg cryopreservation solution can use commercialized clinical-grade cryopreservation solutions such as CryoStor ® CS5 / CS10 (BioLife Solutions), CTS™ Synth-a-Freeze™ (Gibco), etc., or can be self-made, and the components thereof can include serum-free medium, human serum albumin, and DMSO, etc.
[0021] In some embodiments, the kit can further include a pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solution, dispersion, suspension, or emulsion, which is mainly used for preparing a cell suspension for injection.
[0022] In some embodiments, the sterile isotonic aqueous or non-aqueous solutions can be balanced salt solutions or physiological saline.
[0023] The Tregs placed in the short-term preservation solution and stored at 4℃ can be used to prepare a cell suspension with a balanced salt solution or physiological saline and then intravenously injected, or can be expanded in vitro and then used to prepare a cell suspension for treating psoriasis. The cryopreserved Tregs can be directly used to prepare a cell suspension with a balanced salt solution or physiological saline for treating psoriasis, or can be expanded in vitro and then used.
[0024] When the kit provided by the application is used to treat a patient with psoriasis, the spiroketal can be orally administered at a dose of 1 mg / kg to 11 mg / kg, once a day for 7 times; the Tregs can be prepared into a cell suspension and administered by intravenous injection at a dose of 1×10 6 to 5×10 6 per kg, once a day for 3 times.
[0025] Compared with the prior art, the application has the following advantages: (1) The kit provided by the application can significantly improve the symptoms of psoriasis lesions and reduce the PASI score, reduce the infiltration of inflammatory cells in psoriasis lesion tissues, and reduce the expression of IL-6, TNF-α and IL-17A inflammatory factors in skin tissues, thereby effectively treating psoriasis.
[0026] (2) The kit provided by the application can significantly inhibit the proliferation of psoriasis-related keratinocytes (hacat cells), and reduce the expression of IL-6, TNF-α, IL-8 and IL-1β inflammatory factors in hacat cells, thereby effectively treating psoriasis.
[0027] (3) The content of regulatory T lymphocytes in peripheral blood is low, and it is generally difficult to directly separate a large number of regulatory T lymphocytes from peripheral blood. The in vitro expansion efficiency of regulatory T lymphocytes is low, and the GMP-level Treg production process is complex, requiring sorting, expansion, QC detection, etc., and the cost is high. In the application, the spiroketal and the regulatory T lymphocytes can synergistically exert a therapeutic effect, which can effectively reduce the amount of regulatory T lymphocytes, thereby effectively reducing the treatment cost and the risk of complications such as infection and bleeding caused by the collection and adoptive transfer of regulatory T lymphocytes. BRIEF DESCRIPTION OF DRAWINGS
[0028] Figure 1Flow cytometry plots of CFSE-labeled CD4 + CD25 + Flow cytometry plots of CD4 Figure 2 Flow cytometry plots of CD4 + CD25 + Bar graphs of proliferation ratio of CD4 ** Indicate P <0.01 Figure 3 Photographs of psoriatic lesions of mice after different treatments Figure 4 PASI scores of mice after different treatments * Indicate P <0.05 ** Indicate P <0.01 Figure 5 HE staining photographs of psoriatic lesions of mice after different treatments Figure 6 Ki67 immunohistochemical staining photographs of psoriatic lesions of mice after different treatments Figure 7 Bar graphs of Ki67 expression levels in tissues of psoriatic lesions of mice after different treatments ** Indicate P <0.01 Figure 8 Bar graphs of IL-6 expression levels in tissues of psoriatic lesions of mice after different treatments ** Indicate P <0.01 Figure 9 Bar graphs of IL-17A expression levels in tissues of psoriatic lesions of mice after different treatments * Indicate P <0.05 ** Indicate P <0.01 Figure 10 Bar graphs of TNF-α expression levels in tissues of psoriatic lesions of mice after different treatments ** Indicate P <0.01 Figure 11 Bar graphs of IL-6 mRNA expression levels in tissues of psoriatic lesions of mice after different treatments ** Indicate P <0.01 Figure 12 Bar graphs of IL-17A mRNA expression levels in tissues of psoriatic lesions of mice after different treatments ** IndicateP <0.01; Figure 13 Figure 7 is a column chart showing the expression level of TNF-α mRNA in the tissue of the skin lesion of the mice after different treatments; * P <0.05; ** P <0.01; Figure 14 Figure 8 is a column chart showing the cell viability of the hacat cells after different treatments by CCK-8 detection; ** P <0.01; Figure 15 Figure 9 is a column chart showing the expression level of IL-6 in the cells of the hacat cells after different treatments; ** P <0.01; Figure 16 Figure 10 is a column chart showing the expression level of TNF-α in the cells of the hacat cells after different treatments; * P <0.05; ** P <0.01; Figure 17 Figure 11 is a column chart showing the expression level of IL-8 in the cells of the hacat cells after different treatments; * P <0.05; ** P <0.01; Figure 18 Figure 12 is a column chart showing the expression level of IL-1β in the cells of the hacat cells after different treatments; * P <0.05; ** P <0.01. DETAILED DESCRIPTION
[0029] The application will be further described in conjunction with the embodiments. The embodiments are only used for explanation and do not limit the application in any way. If no special description is made, the raw materials and reagents used in the embodiments are conventional products which can be obtained by market purchase; the experimental methods not specified in the embodiments are usually according to the conventional conditions in the art or according to the conditions suggested by the manufacturers.
[0030] The spathulenone used in the embodiments of the application is purchased from Nanjing Daosi Biological Technology Co., Ltd., and the purity is ≥98%.
[0031] Example 1, kit for treating psoriasis including the sparteine powder and the regulatory T lymphocyte (CD4 + CD25 + T cell) suspension each independently present in the kit without mixing with each other.
[0032] The regulatory T lymphocyte (CD4 + CD25 + T cell) suspension can be prepared extemporaneously, specifically, autologous peripheral blood of a patient can be collected before administration, and CD4 + CD25 + T cells are isolated from the peripheral blood by a conventional extraction and separation method, and then resuspended in normal saline to obtain the regulatory T lymphocyte (CD4 + CD25 + T cell) suspension, which is then administered by intravenous injection; the dosage is generally 1 x 10 6 ~ 5 x 10 6 6 / kg, and the administration cycle is 2 days once, for a total of 3 times.
[0033] When used for treating psoriasis patients, the sparteine powder can be dissolved in normal saline for oral administration; the dosage is generally 1 mg / kg ~ 11 mg / kg, and the administration cycle is 1 day once, for a total of 7 times.
[0034] If the number of isolated regulatory T lymphocytes (CD4 + CD25 + T cells) is small, the cells can be cultured and expanded in vitro before being applied to treat psoriasis.
[0035] In other embodiments, the regulatory T lymphocytes in the kit can be stored in a short-term storage solution or a cryopreservation solution.
[0036] In other embodiments, the sparteine in the kit can be in the form of a tablet, a capsule, an oral solution, a pill, or a granule.
[0037] Experimental Example 1: Preparation and isolation and purification of Balb / c mouse CD4 + CD25 + T cells Balb / c mouse CD4 + CD25 + T cells were isolated and purified from the spleen using EasySep™ Mouse CD4 + CD25 + Regulatory T Cell Isolation Kit II (Cat # 18783, STEMCELL), including the following steps: (1) After the BALB / c mice were sacrificed, the mice were soaked with 75% ethanol for 5 min; (2) The mouse spleen was taken and placed on a 70 µm filter screen, a small amount of 1640 culture medium was added to the culture dish, and after grinding, it was uniformly blown and collected in a 15 mL centrifuge tube. 20 µL of cell suspension was added to 200 µL of red blood cell lysis solution, and after lysis on ice, it was directly counted. The remaining cells were synchronously centrifuged (300g, 5min), and after the supernatant was discarded, 3mL of red blood cell lysis solution was added and lysed on ice for 5min, and centrifuged (300g, 5-10min); (3) After the supernatant was poured, 1mL of EasySep buffer was added for resuspension; (4) CD4 + T cell antibody cocktail (CD4 + T Cell Isolation Cocktail) (50 L / mL) was added and incubated at room temperature for 10min; (5) Streptavidin (75 µL / mL) was added and incubated at room temperature for 2.5min; Streptavidin (streptavidin RapidSpheres) was vortexed for 30s before use; (6) EasySep buffer was added to 2.5mL (the tip was gently blown 2-3 times), the tube (without cover) was placed in the magnet and incubated, and incubated at room temperature for 2.5min; (7) The enriched cell suspension was poured into a new test tube and centrifuged (200g, 10min); the supernatant was discarded, and the cells were resuspended (0.5mL); (8) FcR blocker (FcR Blocker) (25 µL) was added to the sample and incubated at room temperature for 5min; (9) CD25 regulatory T cell positive selection cocktail (CD25 Regulatory T Cell Positive Selection Cocktail) (25 µL) was added and incubated at room temperature for 10min; (10) PE selection cocktail (PE Selection Cocktail) (10 µL) was added and incubated at room temperature for 5min; (11) Dextran (30 µL) was added and incubated at room temperature for 5min; Dextran (Vortex Dextran RapidSpheres) was vortexed for 30s before use; (12) EasySep buffer was added to 2.5mL (the tip was gently blown 2-3 times), and each time was incubated at room temperature for 5min (this step was repeated 4-5times); (13) The magnet was resuspended with 1mL of medium to obtain CD4+ CD25 + T cells (Tregs); (14) Take 20 µL of cell suspension and use a flow cytometer to detect the purity of Treg cells according to the standard operating procedure. The purity of Tregs was found to be greater than 96%. (15) Collect CD4 + CD25 + After T lymphocyte counting, centrifuge at 300g for 5 min, and resuspend in 1640 complete culture medium to a final volume of 2×10⁶ cells / mL. 6 The concentration of cells / mL was mixed and added to a 6-well plate; then incubated at 37°C in a 5% CO2 incubator.
[0038] Experiment Example 2: Detection of the effect of lupinone on Tregs cell proliferation using CFSE labeling (1) The isolated and purified CD4 + CD25 + T cells were centrifuged at 300 g for 5 min, washed twice with PBS preheated to 37°C, and counted. (2) Adjust the T cell concentration to 2×10⁻⁶ using PBS. 6 cells / mL, add 1 μL CFSE (10mM) to each milliliter of cell suspension, and incubate at room temperature in the dark for 15 min; (3) Add 1640 complete medium pre-cooled at 4℃ to terminate the reaction, react at 4℃ for 5 min, centrifuge at 300 g for 5 min, discard the supernatant, and then wash 3 times with 5 times the volume of complete medium. (4) Resuspend the above cells in complete culture medium to 2×10⁻⁶. 6 Cells were collected at a concentration of 100 μL / mL and then divided into blank, Anti-CD3 / CD28, Anti-CD3 / CD28+IL-2, and Anti-CD3 / CD28+IL-2+lupe ketone groups. Anti-CD3 (4 µg / mL), Anti-CD28 (2 µg / mL), rm IL-2 (10 ng / μL), or lupe ketone (10 µM) were added to each well of a 96-well plate. The plates were then incubated at 37°C in a 5% CO2 incubator for 48 h, and the proliferation of Tregs cells was subsequently detected by flow cytometry.
[0039] The results are as follows Figures 1-2 As shown in the figure. The results indicate that lupinone can significantly promote CD4. + CD25 + T cell proliferation ( P <0.01).
[0040] Experimental Example 3: Adoptive infusion of lupene combined with Tregs to treat psoriatic mice 1. Establishment of a mouse model of psoriasis Purchase 6-8 week old male BALB / c mice and house them in an SPF-grade animal facility. All mice have free access to food and drink. After one week of acclimatization, remove a 2×4 cm area from the back of each mouse. Keep the mouse in this area for one day, and initiate the psoriasis model every other day. For 7 consecutive days, apply 62.5 mg of 5% imiquimod cream (IMQ) per mouse to the prepared area on the back daily to establish the psoriasis animal model. Successful modeling is indicated by the appearance of erythema with white scales.
[0041] 2. Animal grouping and drug dosage The BALB / c mice that had been successfully modeled were numbered in ascending order of body weight. Using Excel's random number function (=RANDBETWEEN(1, 100)), random numbers were generated for each corresponding body weight. These numbers were grouped into four groups of four based on body weight, and then further divided into groups 1, 2, 3, and 4 in ascending order of the corresponding random number. Group 1 was the control group, group 2 was the model group, group 3 was the lupene group, group 4 was the regulatory T lymphocyte adoptive infusion group (Tregs group), and group 5 was the lupene combined with regulatory T lymphocyte (Tregs) adoptive infusion treatment group (Tregs + lupene group).
[0042] The dosage of regulatory T lymphocyte therapy (tail vein injection) is 5 × 10 Treg cells. 6 Animals / day, the lupene group received lupene (100 mg / kg / day) via gavage for 7 days. Lupene combined with adoptive infusion of regulatory T lymphocytes (Tregs) consisted of lupene 100 mg / kg via gavage + 1 × 10 Tregs cells via tail vein injection. 6 Cells were administered at 3 doses per day (number per cell), with lupene ketone administered for 7 consecutive days and Tregs administered on days 1, 3, and 5. Both the normal and model groups received equal volumes of physiological saline. Lupene ketone was dissolved in physiological saline and administered via gavage, while Tregs were mixed with 1640 complete culture medium (containing 10% FBS) and the cell suspension was injected via tail vein.
[0043] 3. Experimental methods and results analysis (1) Exterior symptoms and PASI scores of skin lesions in psoriasis mice Mice were treated with different drugs, and the appearance of skin lesions was recorded daily by digital photography. After 7 days, the appearance of skin lesions was scored (erythema, scaling, and infiltration / thickness) (scored from 0 to 4, from mild to severe). The scores of the three factors were added together to obtain the total score. The PASI scoring criteria are as follows: 0, none; 1, mild; 2, moderate; 3, severe; 4, very severe.
[0044] The results are as follows Figures 3-4 As shown, from Figures 3-4 It can be seen that after 7 days of different treatments, the dorsal skin of the model group mice showed significant thickening at the site of the skin lesion, along with symptoms such as erythema and scaling, similar to human psoriasis-like lesions. Compared with the model group, the lupene group, the regulatory T lymphocyte adoptive infusion treatment group, and the lupene combined with regulatory T lymphocyte treatment group showed reduced skin thickening at the dorsal skin lesion, lighter erythema color, thinner scales, and decreased PASI scores. Among these, the lupene combined with regulatory T lymphocyte treatment group showed the most significant improvement in symptoms, with a statistically significant difference compared to the lupene group and the regulatory T lymphocyte adoptive infusion treatment group. P The result was <0.01, indicating that lupene combined with regulatory T lymphocyte therapy can significantly improve the superficial symptoms of skin lesions on the back of psoriatic mice, and that the two have a synergistic effect.
[0045] (2) Infiltration of inflammatory cells in the skin lesions of psoriatic mice Mice were treated with different drugs, and after 7 days, tissue samples from the skin lesions were taken for HE staining and detection.
[0046] The results are as follows Figure 5 As shown, from Figure 5 It can be seen that after 7 days of different treatments, compared with the model group (Vehicle), the number of parakeratotic cells in the skin lesions of mice in the lupene group, the adoptive infusion treatment group of regulatory T lymphocytes, and the lupene group combined with regulatory T lymphocytes treatment group was significantly reduced, and the thickness of the epidermal spinous layer and the epidermal layer was significantly reduced. Among them, the reduction of parakeratotic cells, epidermal spinous layer and epidermal layer thickness in the skin lesions of mice in the lupene group combined with regulatory T lymphocytes treatment group was more significant.
[0047] (3) Ki67 expression in skin lesions of psoriatic mice After 7 days of treatment with different drugs, tissue samples were taken from the skin lesions of mice and the expression of Ki67 in the lesions was detected by immunohistochemistry.
[0048] The results are as follows Figures 6-7 As shown, from Figures 6-7As can be seen: compared with the model group, the expression of Ki67 in the skin lesion tissue of the mouse in the spiroketal group, the regulatory T lymphocyte adoptive transfer treatment group and the spiroketal combined with regulatory T lymphocyte treatment group was significantly reduced after 7 days of treatment with different drugs, and the expression of Ki67 in the mouse skin lesion in the spiroketal combined with regulatory T lymphocyte treatment group was more significantly reduced.
[0049] (4) Expression of inflammatory factors in psoriasis mouse skin lesion tissue The mice were treated with different drugs, and after 7 days, the psoriasis skin lesion tissue of the mice in each group was taken for Elisa detection.
[0050] The detection results are shown in Figures 8-10 As can be seen: compared with the model group, the expression of IL-6, IL-17A and TNF-α in the mouse skin lesion of the spiroketal group, the regulatory T lymphocyte adoptive transfer treatment group and the spiroketal combined with regulatory T lymphocyte treatment group was significantly decreased, and the effect of the spiroketal combined with regulatory T lymphocyte treatment on the down-regulation of IL-6, IL-17A and TNF-α expression level in the mouse skin lesion was more significant, and the difference was significant compared with the spiroketal group and the regulatory T lymphocyte adoptive transfer treatment group (P<0.01), which indicated that the spiroketal combined with regulatory T lymphocyte treatment could significantly reduce the inflammatory factor level of the psoriasis mouse skin lesion, and the spiroketal combined with regulatory T lymphocyte treatment had a synergistic effect. P <0.01), which indicated that the spiroketal combined with regulatory T lymphocyte treatment could significantly reduce the inflammatory factor level of the psoriasis mouse skin lesion, and the spiroketal combined with regulatory T lymphocyte treatment had a synergistic effect.
[0051] (5) mRNA expression of skin-related inflammatory factors in psoriasis mice The mice were treated with different drugs, and after 7 days, the relative expression of IL-6, IL-17A and TNF-α mRNA in the skin lesion tissue of the mice in each group was detected by RT-PCR, and the primer sequences of IL-6, IL-17A, TNF-α gene and internal reference gene GAPDH are shown in Table 1.
[0052] Table 1 RT-PCR primers
[0053] The results are shown in Figures 11-13 As can be seen from Figures 11-13 : compared with the model group, the expression of IL-6, IL-17A and TNF-α mRNA in the mouse skin lesion of the spiroketal group, the regulatory T lymphocyte adoptive transfer treatment group and the spiroketal combined with regulatory T lymphocyte treatment group was significantly decreased, and the effect of the spiroketal combined with regulatory T lymphocyte treatment on the down-regulation of IL-6, IL-17A and TNF-α mRNA level in the mouse skin lesion was more significant, and the difference was significant compared with the spiroketal group and the regulatory T lymphocyte adoptive transfer treatment group (P<0.01).P <0.05 or P The result was <0.01, indicating that lupene ketone combined with regulatory T lymphocyte therapy has a synergistic effect.
[0054] 4. Experimental Conclusions Lucene combined with adoptive infusion of regulatory T lymphocytes significantly improved the symptoms of psoriatic lesions in mice and reduced PASI scores. It also reduced the infiltration of inflammatory cells in psoriatic lesions, decreased the expression level of Ki67 in the lesions, and reduced the expression of inflammatory factors in the psoriatic lesions, demonstrating a significant therapeutic effect on psoriasis. Furthermore, lucene combined with regulatory T lymphocyte therapy had a synergistic effect.
[0055] Experimental Example 4: Lupenone combined with Tregs to treat IL-17A in vitro induced psoriasis-like cell model 1. Cell sorting and culture According to the reagent EasySep™ Mouse CD4 + CD25 + Regulatory T Cell Isolation Kit II (Cat #18783, STEMCELL) describes the use of CD4 cells isolated from the spleen of Balb / c mice. + CD25 + T cells were used in in vitro experiments. The sorted Tregs had a purity greater than 96%.
[0056] To obtain mouse keratinocytes (hacat), skin was isolated from 4-7 day old Balb / c pups and incubated at 4°C for 16-21 hours in a skin dissociation enzyme solution (Sigma, C4-28-100MG). After epidermal separation, TrypLE™ Express (Gibco) was added, and the cells were incubated at 37°C with shaking for 30 minutes. Primary keratinocyte culture medium (PriMed-iCell-010, iCell, China) was added to terminate the digestion. After filtration and centrifugation, the cells were resuspended and collected.
[0057] Primary keratinocytes were cultured in this cell-specific medium (PriMed-iCell-010, iCell, China). 2. In vitro induction of a psoriasis-like model and drug administration treatment Keratinocytes were randomly divided into a control group, a model group, a lupene group, a regulatory T lymphocyte group (Tregs group), and a lupene combined with regulatory T lymphocyte group (Tregs+lupene group).
[0058] Control group: Normal culture, no other treatment.
[0059] Model group: add recombinant mouse IL-17A (10 ng / mL) and culture for 48 hours.
[0060] Hepatanone group: add recombinant mouse IL-17A (10 ng / mL) and culture for 48 hours, and further treat the cells with 10 μM heptanone for 48 hours.
[0061] Tregs group: add recombinant mouse IL-17A (10 ng / mL) and culture for 48 hours, and further add 2×10 5 CD4 + CD25 + T cells and co-culture for 48 hours.
[0062] Tregs+heptanone group: add recombinant mouse IL-17A (10 ng / mL) and culture for 48 hours, and further add 2×10 5 CD4 + CD25 + T cells and 10 μM heptanone and co-culture for 48 hours.
[0063] All cells were maintained in a humidified incubator at 37℃ and 5% CO2.
[0064] 3. Cell viability detection Cell counting kit (CCK-8) (B34304, Selleck, Shanghai, China) was used to analyze cell viability. The above treated keratinocytes were inoculated in 96-well plates, respectively. Then 10 μL CCK-8 solution was added to each well, and the culture was incubated at 37℃ for 45 minutes to 1 hour. The absorbance at 450 nm was measured using a microplate reader.
[0065] 4. Experimental method and result analysis (1) Proliferation of psoriasis-related keratinocytes Different drug treatments of hacat cells were subjected to cell viability detection.
[0066] The detection results are shown in Table 1: Figure 14 Compared with the model group, the cell viability of the heptanone group, the regulatory T lymphocyte adoptive transfer treatment group, and the heptanone combined with regulatory T lymphocyte treatment group was significantly decreased, and the cell viability of the heptanone combined with regulatory T lymphocyte treatment group was more significantly decreased, and the difference was significant compared with the heptanone group (P<0.01), indicating that heptanone combined with regulatory T lymphocyte treatment can significantly reduce the viability of hacat cells, and heptanone combined with regulatory T lymphocyte treatment has a synergistic effect. P <0.01), indicating that heptanone combined with regulatory T lymphocyte treatment can significantly reduce the viability of hacat cells, and heptanone combined with regulatory T lymphocyte treatment has a synergistic effect.
[0067] (2) Inflammatory factor expression in psoriasis-related keratinocytes Different drug treatment of hacat cells, Elisa detection was performed on each group of cells.
[0068] The detection results are shown in Figures 15-18 Compared with the model group, the expression levels of IL-6, TNF-α, IL-8 and IL-1β in the spiroketal group, the regulatory T lymphocyte adoptive transfer treatment group and the spiroketal combined with regulatory T lymphocyte treatment group were significantly decreased, and the down-regulation effect of IL-6, TNF-α, IL-8 and IL-1β expression levels in the spiroketal combined with regulatory T lymphocyte treatment group was more significant, and the difference was significant compared with the spiroketal group (P<0.01), indicating that spiroketal combined with regulatory T lymphocyte treatment can significantly reduce the inflammatory factor level of hacat cells, and spiroketal combined with regulatory T lymphocyte treatment has a synergistic effect. P <0.01), indicating that spiroketal combined with regulatory T lymphocyte treatment can significantly reduce the inflammatory factor level of hacat cells, and spiroketal combined with regulatory T lymphocyte treatment has a synergistic effect.
[0069] The above only describes some embodiments of the present application. For those skilled in the art, without departing from the inventive concept, several modifications and improvements can be made, which are all within the scope of protection of the present application.
Claims
1. A medicine box for treating psoriasis, characterized in that, include: The primary active ingredient is a first pharmaceutical composition consisting of lupene ketone and a second pharmaceutical composition containing regulatory T lymphocytes.
2. The medicine box for treating psoriasis according to claim 1, characterized in that, The regulatory T lymphocytes are CD4. + CD25 + T cells.
3. The medicine box for treating psoriasis according to claim 1 or 2, characterized in that, The lupene ketone is administered orally, while the regulatory T lymphocytes are administered by injection.
4. The medicine box for treating psoriasis according to claim 3, characterized in that, The therapeutically effective dose of the lupene ketone is (1~11) mg / kg.
5. The medicine box for treating psoriasis according to claim 4, characterized in that, The therapeutically effective dose of the regulatory T lymphocytes is (1×10⁻⁶). 6 ~5×10 6 ) pieces / kg.
6. The medicine box for treating psoriasis according to claim 3, characterized in that, The first pharmaceutical composition further includes pharmaceutically acceptable excipients.
7. The medicine box for treating psoriasis according to claim 6, characterized in that, The pharmaceutically acceptable excipients are selected from at least one of the following: fillers, binders, disintegrants, lubricants, thickeners, colorants, flavoring agents, solvents, surfactants, preservatives, antioxidants, and coating agents.
8. The medicine box for treating psoriasis according to claim 7, characterized in that, The dosage form of the first pharmaceutical composition is tablets, capsules, powders, oral liquids, pills, or granules.
9. The medicine box for treating psoriasis according to claim 3, characterized in that, The second pharmaceutical composition also includes a regulatory T lymphocyte preservation solution.
10. The medicine box for treating psoriasis according to any one of claims 4 to 9, characterized in that, It also includes pharmaceutically acceptable sterile isotonic or non-aqueous solutions, dispersions, suspensions or emulsions.