Application of buspirone hydrochloride in preparation of anti-psoriasis medicine

Abstract

The invention belongs to the technical field of medicines and disease treatment, and particularly relates to application of buspirone hydrochloride in preparation of an anti-psoriasis medicine. Research results show that buspirone hydrochloride can effectively increase expression of BCL-2 in DC, inhibit the phagocytic function of DC and maturation of DC, relieve skin injury of psoriasis model mice and inhibit infiltration and maturation of DC in tissues. Moreover, buspirone hydrochloride can inhibit the function of the DC by increasing the expression of BCL-2 in the DC, thereby playing a role in resisting psoriasis.

Description

Technical Field

[0001] The present invention belongs to the technical field of drugs and disease treatment, and specifically relates to the application of buspirone hydrochloride in the preparation of drugs for treating psoriasis. Background Art

[0002] Psoriasis, as a common chronic disease with an incidence rate of about 2% - 3% globally and about 0.47% in China, affects the quality of life of more than 6.5 million patients. It is a recurrent disease caused by the combined action of genetic factors and environmental factors, leading to immune system disorders. Even if the symptoms are relieved through standardized treatment, it may relapse due to inducements such as infection, mental stress, and seasonal alternation. From the pathological mechanism, immune cells such as T lymphocytes in the patient's body are abnormally activated, releasing a large number of inflammatory factors, which promote the excessive proliferation of skin keratinocytes, causing the progression of the disease and even leading to complications such as joint lesions and metabolic syndrome in severe cases.

[0003] Dendritic cells are the most powerful professional antigen - presenting cells in the body's immune system. In patients with psoriasis, dendritic cells in the skin can uptake, process foreign antigens or self - antigens, and present antigen information to T lymphocytes, thereby activating the immune response mediated by T lymphocytes. For example, when dendritic cells recognize certain damage - associated molecular patterns or microbial antigens in the skin, they will initiate an immune response, prompting T lymphocytes to differentiate into cell subsets such as Th1 and Th17. These cells will secrete a large number of cytokines, such as interferon - γ, interleukin - 17, etc., triggering an inflammatory response and leading to the formation of psoriasis skin lesions.

[0004] Currently, the treatment methods for psoriasis include topical application of psoriasis medications to relieve local inflammation, local ultraviolet phototherapy, and the use of local anti - inflammatory inhibitors. These methods have the problem of high recurrence rate and inability to cure when applied. Summary of the Invention

[0005] To solve the problem that the existing treatment methods for psoriasis have a high recurrence rate and inability to cure when applied, the present invention provides an application of buspirone hydrochloride in the preparation of drugs for treating psoriasis. To achieve the above - mentioned purpose, the present invention adopts the following technical solutions.

[0006] The object of the present invention is to provide an application of buspirone hydrochloride in the preparation of a medicament for treating psoriasis. The buspirone hydrochloride provided by the present invention inhibits the function of dendritic cells by increasing the expression level of Bcl-2 protein in dendritic cells, inhibits the abnormal activation of local dendritic cells in psoriasis and reduces the activation of T cells at the same time, so as to fundamentally improve the local immune condition of psoriasis lastingly, reduce the recurrence risk after drug withdrawal, and the price of buspirone hydrochloride is very low compared with the current price of biological agent treatment. The application provided by the present invention can solve the problem of high recurrence rate and inability to cure in the treatment methods of psoriasis in the prior art.

[0007] Preferably, the medicament is used for inhibiting the phagocytic function and maturation of dendritic cells.

[0008] Preferably, the medicament is used for increasing the expression of BCL-2 in dendritic cells.

[0009] BCL-2 is an important anti-apoptotic protein and plays various roles in the maturation process of dendritic cells (DC). Research shows that BCL2 is an endogenous inhibitor of DC function, and overexpression of BCL-2 will inhibit the function of DC. Therefore, by promoting the expression of BCL-2 in DC, it is possible to be used as a medicament for treating psoriasis. The above-mentioned medicament provided by the present invention can inhibit the function of DC by increasing the expression level of Bcl-2 protein in DC cells, inhibit the abnormal activation of local DC in psoriasis and reduce the activation of T cells at the same time, so as to fundamentally improve the local immune condition of psoriasis lastingly, and can be used as a medicament for treating psoriasis.

[0010] Preferably, the medicament takes the buspirone hydrochloride as an active ingredient and is supplemented with pharmaceutically acceptable excipients.

[0011] Preferably, the medicament includes any one of tablets, solution agents and injections.

[0012] Preferably, the excipients include any one or more of water, ethanol, glycerol, PBS buffer solution and physiological saline.

[0013] Preferably, the concentration of the PBS buffer solution is 0.01 mol / L and the pH is 7.2 - 7.4.

[0014] Preferably, the medicament is a solution agent.

[0015] The solution agent is obtained by compounding the buspirone hydrochloride and a solvent.

[0016] The solvent includes any one of water, ethanol and glycerol.

[0017] Preferably, the concentration of buspirone hydrochloride in the solution is 0.625 μg / mL to 10 μg / mL. Solution dosage forms of drugs have a large degree of dispersion, rapid absorption, quick action, and good bioavailability; the active ingredients are evenly dispersed, can be accurately measured for use, and are particularly suitable for pediatric and elderly patients; the dosage size is easy to adjust and control; it can reduce the local irritation of some highly soluble drugs; it can increase the stability and safety of some drugs. Therefore, the dosage form of the above-mentioned drug provided by the present invention is a solution.

[0018] Compared with the prior art, the present invention has the following beneficial effects: 1. The object of the present invention is to provide an application of buspirone hydrochloride in the preparation of drugs for treating psoriasis, and to provide a new method for treating psoriasis. Buspirone hydrochloride inhibits the function of dendritic cells (DCs) by increasing the expression level of Bcl-2 protein in DCs, inhibits the abnormal activation of local DCs in psoriasis and reduces the activation of T cells at the same time to fundamentally improve the local immune situation of psoriasis persistently, and reduces the recurrence risk after drug withdrawal. At the same time, the price of buspirone hydrochloride is much lower than that of the current biological agent treatment. The application provided by the present invention can solve the problem of high recurrence rate and inability to cure in the treatment methods of psoriasis in the prior art.

[0019] 2. The research results of the present invention show that buspirone hydrochloride can inhibit the function of DCs by increasing the expression of BCL-2 in DCs, and thus play an anti-psoriasis role.

[0020] 3. The present invention proves that an antidepressant drug, buspirone hydrochloride, can effectively increase the expression of BCL-2 in DCs, inhibit the phagocytic function and maturation of DCs, reduce the skin damage of psoriasis model mice, and inhibit the infiltration and maturation of DCs in tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 shows the effect of buspirone hydrochloride in the present invention on the expression of Bcl-2 protein in DC 2.4 cells; wherein, Figure 1 Figure A in shows that the expression level of Bcl-2 protein expressed by DC2.4 cells under the action of buspirone hydrochloride increases significantly; Figure 1 Figure B in shows the semi-quantitative result mapping analysis of the protein expression levels of the bands in the three western Blot experimental results.

[0022] Figure 2 shows the effect of buspirone hydrochloride on the expression of Bcl-2 protein in DC 2.4 cells detected by the cell smear experiment; wherein, "+" represents the experimental group treated with buspirone hydrochloride at a concentration of 5 μg / mL, and "-" represents the control group without buspirone hydrochloride added.

[0023] Figure 3 Effect of buspirone hydrochloride on the phagocytic function of DC 2.4 cells in the present invention: Among them, Figure 3 Figure A in shows the phagocytosis of FITC-OVA by DC2.4 cells detected by flow cytometry. It can be seen that under the action of buspirone hydrochloride, the phagocytosis rate of OVA gradually decreases, showing an obvious inhibitory effect; Figure 3 Figure B in is the statistical graph and analysis of multiple experiments.

[0024] Figure 4 Expression of CD11c on the surface of primary DC cells in mouse bone marrow cells in the present invention.

[0025] Figure 5 Effect of buspirone hydrochloride on the expression of Bcl-2 protein in primary DCs in the present invention; among them, Figure 5 Figure A in shows that the expression level of Bcl-2 protein in the extracted primary DC cells is significantly increased under the action of buspirone hydrochloride; Figure 5 Figure B in is the semi-quantification result graph analysis of the protein expression levels of the bands in the results of three western Blot experiments.

[0026] Figure 6 Effect of buspirone hydrochloride on the phagocytic function of primary DCs in the present invention; among them, Figure 6 Figure A in shows the phagocytosis of FITC-OVA by primary DC cells detected by flow cytometry. It can be seen that under the action of buspirone hydrochloride, the phagocytosis rate of OVA gradually decreases, showing an obvious inhibitory effect; Figure 6 Figure B in is the statistical graph and analysis of multiple experiments.

[0027] Figure 7 Effect of buspirone hydrochloride on the maturation of primary DC cells under the action of LPS in the present invention; among them, Figure 7 Figure A in shows the proportion of CD86 expressed by primary DC cells treated with buspirone hydrochloride under the treatment of LPS as an activator detected by flow cytometry, indirectly reflecting the effect on the maturation of primary DC cells. The results show that buspirone hydrochloride can significantly inhibit the maturation of primary DC cells under LPS stimulation; Figure 7 Figure B in is the statistical graph and analysis of multiple experiments.

[0028] Figure 8 Effect of buspirone hydrochloride on the maturation of primary DC cells under the action of OVA in the present invention; among them, Figure 8Figure A in it shows the proportion of CD86 expressed by primary DC cells treated with buspirone hydrochloride under the activator OVA detected by flow cytometry, indirectly reflecting the effect on the maturation of primary DC cells. The results show that buspirone hydrochloride can significantly inhibit the maturation of primary DC cells stimulated by OVA; Figure 8 Figure B in it is for the statistical plotting and analysis of multiple experiments.

[0029] Figure 9 This is the effect of buspirone hydrochloride on skin damage in psoriasis model mice in the present invention. Figure 9 A in it is mouse No. 1 in the model group; Figure 9 B in it is mouse No. 2 in the model group; Figure 9 C in it is mouse No. 3 in the model group; Figure 9 D in it is mouse No. 4 in the model group; Figure 9 E in it is mouse No. 5 in the model group. Figure 9 F in it is mouse No. 1 in the buspirone hydrochloride treatment group; Figure 9 G in it is mouse No. 2 in the buspirone hydrochloride treatment group; Figure 9 H in it is mouse No. 3 in the buspirone hydrochloride treatment group; Figure 9 I in it is mouse No. 4 in the buspirone hydrochloride treatment group; Figure 9 J in it is mouse No. 5 in the buspirone hydrochloride treatment group.

[0030] Figure 10 This is the effect of buspirone hydrochloride on the infiltration of activated DCs in the skin tissue of psoriasis model mice in the present invention. Specific Embodiments

[0031] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0032] Example 1 0. Method 1. Experimental drugs and experimental animals Buspirone hydrochloride: The buspirone hydrochloride used in the example is Yishu, buspirone hydrochloride tablets (5 mg / tablet) from Enhua.

[0033] Cytokines: The cytokines in the example are used for the extraction and culture of mouse primary bone marrow-derived dendritic cells (BMDC); both IL-4 and GM-CSF are purchased from PeproTech.

[0034] The DC 2.4 cells were derived from the Key Laboratory of New Cancer Vaccines and Immunotherapy of Xinxiang Medical University, located in Xinxiang, Henan 453000, China.

[0035] The bone marrow cells of mice were obtained from the femur marrow of wild-type C57BL-6 (purchased from Skbex) in this research group after extracting and lysing red blood cells.

[0036] The BALB / C mice were purchased from Skbex (Henan Sibex Biotechnology Co., Ltd.).

[0037] 2. Detection of Bcl-2 expression in DC 2.4 cells by Western blot DC 2.4 cells were seeded in six-well plates at a cell density of 3.5×10 5 cells / well and incubated in a 37°C, 5% CO2 incubator for 24 hours. Then, different concentrations of buspirone hydrochloride (0, 0.625 μg / mL, 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, and 10 μg / mL) were used to treat the DC 2.4 cells for 24 hours, and then cell proteins were extracted. SDS protein samples were prepared and protein samples were separated by 10% SDS-PAGE gel electrophoresis. After that, the proteins were transferred to a PVDF membrane (EMD Millipore, Billerica, MA, USA) by electrotransfer. The PVDF membrane was incubated with a TBST solution containing 0.05 g / mL skim milk powder and 0.5 mL / L Tween for 2 hours. Then, the PVDF membrane was incubated overnight at 4°C with the primary antibody Bcl-2 (Cell Signaling Technology, USA) diluted according to the instructions. After washing the PVDF membrane, the membrane was incubated with the corresponding secondary antibody (ZSGB-BIO 1:5000) at room temperature for 1 hour. Finally, chemiluminescent reagent (Beyotime) was added to the membrane, and specific protein bands were detected using a chemiluminescent imager (Vilber). The Image J software was used to semi-quantify the images.

[0038] Among them, overnight refers to a time ≥ 12 h.

[0039] 3. Detection of the effect of buspirone hydrochloride on Bcl-2 expression in DC 2.4 cells by cell smear A sterilized glass slide was placed in the center of a small culture dish, and a cell suspension of DC 2.4 cells was dropped in the center of the slide at a density of 3.5×10 5Seed cells at a density of [number] cells per well. After the cells adhered to the well surface, add buspirone hydrochloride (0 and 5 μg / mL respectively), and continue culturing for 24 h. Then, add 2 mL of fixative solution and incubate for 20 min, followed by washing the cells. Next, add blocking solution (containing 0.0005 mL / mL Triton X-100, 0.03 g / mL BSA, and 0.05 mL / mL FBS) onto the glass slides, incubate for another two hours, then add Bcl-2 antibody and incubate overnight at 4 °C. On the next day, take out the sections, place them at room temperature for 2 h, wash the glass slides, add secondary antibody (Abways) labeled with corresponding fluorescent marker, incubate at room temperature for 30 min, then add DAPI solution (Beyotime) and stain at room temperature for 5 min. Finally, seal the glass slides with anti-fluorescence quencher and take images using a confocal microscope (AR1+, Nikon).

[0040] Among them, overnight incubation means the time is ≥ 12 h.

[0041] 4. Detect the effect of buspirone hydrochloride on the phagocytic ability of DC2.4 Seed DC 2.4 cells into a six-well plate at a density of 4×10 5 . After incubating for 12 h, add buspirone hydrochloride at different concentrations (0, 0.625 μg / mL, 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, and 10 μg / mL), and culture for 24 h. Then, add FITC-OVA, incubate for 4 h, collect the cells, and detect the phagocytic function of DC 2.4 cells in different groups by measuring the intensity of FITC fluorescence using flow cytometry.

[0042] 5. Isolation of mouse primary DC cells Culture mouse bone marrow cells in a 10-cm culture dish, and add RPMI-1640 medium containing 0.1 mL / mL fetal bovine serum, GM-CSF (20 ng / mL), and IL-4 (10 ng / mL). Replace half of the medium every two days. On the 9th day, remove the suspended and semi-suspended cells, and collect BMDCs for subsequent experiments.

[0043] 6. Detect the effect of buspirone hydrochloride on Bcl-2 expression in primary DC cells by Western blot Use the isolated primary DC cells, seed them at a density of 2.5×10 5Cells per well were seeded in 6-well plates and incubated in an incubator at 37°C with 5% CO2 for 16 hours. After that, primary DC cells were treated with different concentrations of buspirone hydrochloride (0, 0.625 μg / mL, 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL). After 24 hours, cell proteins were extracted. Proteins were separated by 10% SDS-PAGE gel electrophoresis and then transferred to a PVDF membrane (EMD Millipore, Billerica, MA, USA) by electrotransfer. Subsequently, the PVDF membrane was incubated in a TBST solution containing 0.05 g / mL skim milk powder and 0.5 mL / L Tween for 2 hours, and then incubated overnight at 4°C with the diluted primary antibody Bcl-2. The PVDF membrane was washed and then incubated with the corresponding secondary antibody (ZSGB-BIO 1:5000) at room temperature for 1 hour. Finally, enhanced chemiluminescence reagent (Beyotime) was added to the membrane, and specific protein bands were detected using a chemiluminescence imager (Vilber). The images were semi-quantified using Image J software.

[0044] 7. Effect of buspirone hydrochloride on the phagocytic ability of immature primary DC cells Primary DC cells were seeded in 6-well plates at a density of 2.5×10 5 . After incubation for 16 hours, different concentrations of buspirone hydrochloride (0, 0.625 μg / mL, 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, and 10 μg / mL) were added, and after continued culture for 24 hours, FITC-OVA was added and incubated for 4 hours. Cells were collected, and flow cytometry was used to detect the intensity of FITC fluorescence to reflect the phagocytic ability of DC cells in different groups.

[0045] 8. Effect of buspirone hydrochloride on the maturation of primary DC cells Primary DC cells were seeded in 6-well plates at a density of 2.5×10 5 . After incubation for 16 hours, different concentrations of buspirone hydrochloride (0, 0.625 μg / mL, 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, and 10 μg / mL) and LPS (5 μg / mL) or OVA (50 μg / mL) were added. After incubation for 24 hours, cells were collected, and CD86 antibody was added for staining for 30 minutes. Finally, flow cytometry was used to detect the expression of CD86 in DC cells to clarify the effect of buspirone hydrochloride on the maturation of primary DC cells.

[0046] 9. Effect of buspirone hydrochloride on the mouse psoriasis model In this invention, imiquimod (IMQ) was used to induce the establishment of a mouse psoriasis model. IMQ was purchased from Sichuan Mingxin Lidi. The specific process is as follows:

[0047] First, depilate the back of BALB / C mice in advance, and then apply IMQ at a dose of 62.5 mg per mouse every morning and inject pimozide hydrochloride subcutaneously at a dose of 400 μg per mouse every afternoon. At the same time, inject 100 μL of PBS subcutaneously in the control group for 4 consecutive days, and record the skin lesion conditions of the mice. On the 7th day, sacrifice the mice and isolate the psoriatic skin lesion tissues.

[0048] Among them, the concentration of PBS is 0.01 M, the pH is 7.4, and it is purchased from Servicebio.

[0049] 10. Immunofluorescence detection of DC cell infiltration in psoriatic skin tissues Prepare mouse psoriatic skin tissue sections with a thickness of 5 μm. Place the sections at room temperature for 10 min, then continue to place them in an oven at 55 °C for 30 min, and wash them 3 times with PBS buffer for 5 min each time. Then, add blocking solution (containing 0.0005 mL / mL TritonX-100, 0.03 g / mL BSA, and 0.05 mL / mL FBS) dropwise onto the tissue sections, incubate at room temperature for 1 h, and then add diluted antibodies (CD11c, Abmart, 1:500, China; CD86, Affinity, 1:500, USA) dropwise to the section tissues and incubate overnight at 4 °C. The next day, take out the wet box storing the tissue sections, rewarm for 40 min, then after washing, add the corresponding secondary antibodies with fluorescent labels (R, Abways, 1:200, China; M, Abways, 1:200, China) dropwise, incubate in the dark at room temperature for 30 min, wash again, incubate with DAPI staining solution for 10 min, wash and then add anti-fluorescence quenching agent, and take pictures with a confocal fluorescence microscope.

[0050] Among them, overnight refers to a time ≥ 12 h.

[0051] The concentration of PBS buffer is 0.01 mol / L, and the pH is 7.2 - 7.4.

[0052] II. Results 1. Pimozide hydrochloride significantly increased the expression level of Bcl-2 protein in DC 2.4 cells As Figure 1 shown, the Western blot results showed that after treating DC 2.4 cells with pimozide hydrochloride for 24 h, it could effectively increase the expression of Bcl-2 protein in DC 2.4 cells.

[0053] As Figure 2 shown, the immunofluorescence results of cell smears also showed that pimozide hydrochloride significantly increased the expression of Bcl-2 in DC 2.4 cells.

[0054] 2. Buspirone hydrochloride significantly reduced the phagocytic function of DC 2.4 cells As seen from the results of the FITC-OVA phagocytosis experiment Figure 3 , buspirone hydrochloride significantly inhibited the phagocytic function of DC 2.4 cells.

[0055] 3. Isolation of primary DC cells from mouse bone marrow cells After isolating cells according to the method of isolating primary DC cells from mouse bone marrow cells, the results of detecting CD11c on the cell surface by flow cytometry are as Figure 4 shown. The expression of CD11c in the cells extracted by the present invention is above 80%, and the proportion is relatively high, meeting the requirements of subsequent experiments.

[0056] 4. Buspirone hydrochloride can significantly increase the expression of Bcl-2 protein in primary DC The results of Western blot are as Figure 5 shown. After acting on primary DC cells for 24 hours, buspirone hydrochloride can effectively increase the expression of Bcl-2 protein in DC cells.

[0057] 5. Buspirone hydrochloride significantly reduced the phagocytic function of primary DC As Figure 6 shown, it was confirmed by detecting the phagocytic ability of primary DC cells through the FITC-OVA phagocytosis experiment that buspirone hydrochloride has an obvious inhibitory effect on the phagocytic function of primary DC cells.

[0058] 6. Buspirone hydrochloride significantly inhibited the maturation of primary DC cells As Figure 7 and Figure 8 shown, by adding LPS or OVA to promote the maturation of primary DC cells and simultaneously adding different concentrations of buspirone hydrochloride, the effect of buspirone hydrochloride on the maturation of primary DC cells was detected. The results showed that whether LPS stimulation or OVA stimulation was added, buspirone hydrochloride could significantly inhibit the expression of the mature marker molecule CD86 on the surface of primary DC cells, suggesting that buspirone hydrochloride has the effect of inhibiting the maturation of primary DC cells.

[0059] 7. Buspirone hydrochloride significantly alleviated the skin damage of psoriasis model mice The present invention established a psoriasis mouse model by applying IMQ and detected the effect of buspirone hydrochloride on the skin damage of model mice. The results are as Figure 9 shown. Treatment with buspirone hydrochloride significantly alleviated the skin damage of model mice.

[0060] 8. Buspirone hydrochloride significantly reduced the infiltration of activated DC in the skin tissue of psoriasis mice The present invention detected the infiltration of activated DC cells in the skin tissues of psoriasis mice, and the results are as Figure 10 shown. Buspirone hydrochloride significantly reduced the infiltration of activated DCs in the skin tissues of mice.

[0061] From the above experimental results, it can be seen that buspirone hydrochloride significantly improves the pathogenesis of psoriasis by inhibiting the function of DC cells by significantly increasing the expression level of Bcl-2 protein in DC cells.

[0062] It should be noted that when the present invention involves numerical ranges, it should be understood that any value between the two endpoints of each numerical range and the two endpoints can be selected. To avoid repetition, the present invention describes preferred embodiments.

[0063] Although the preferred embodiments of the present invention have been described, once those skilled in the art learn the basic creative concept, additional changes and modifications can be made to these embodiments, and all such changes and modifications fall within the scope of the present invention.

Claims

1. Use of buspirone hydrochloride in the preparation of a drug for treating psoriasis.

2. The application according to claim 1, characterized in that The drug is used to inhibit the phagocytic function and maturation of dendritic cells.

3. The application according to claim 1, wherein The drug uses buspirone hydrochloride as the active ingredient and is supplemented with pharmaceutically acceptable excipients.

4. The application according to claim 3, wherein The drug includes any one of tablets, solutions, and injections.

5. The application according to claim 3, characterized in that, The excipients include any one or more of water, ethanol, glycerol, PBS buffer solution, and physiological saline.

6. The application according to claim 5, characterized in that, The concentration of the PBS buffer solution is 0.01 mol / L, and the pH is 7.2 - 7.

4.

7. The application according to claim 3, characterized in that The drug is a solution; The solution is obtained by compounding buspirone hydrochloride and a solvent; The solvent includes any one of water, ethanol, and glycerol.

8. The application according to claim 7, characterized in that, The concentration of buspirone hydrochloride in the solution is 0.625 μg / mL to 10 μg / mL.

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