Application of methyl coptisine in preparation of medicine for preventing and treating atopic dermatitis

Abstract

The invention discloses application of methyl coptisine in preparation of a medicine for preventing and treating atopic dermatitis, and belongs to the technical field of medicines. The invention proves that the methylcoptisine can improve the symptom of atopic dermatitis and reduce the IgE level by reducing the expression of CCL17 and inhibiting the activation of STAT1 and STAT3, and provides a theoretical basis and a technical idea for the treatment of atopic dermatitis and the research of novel drugs.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to application of methylcoptisine in preparing medicines for preventing and treating atopic dermatitis. Background Art

[0002] Atopic dermatitis (AD) is a common chronic, recurrent, inflammatory skin disease with a high incidence, affecting approximately 10% of adults and 20% of children worldwide. The lifetime prevalence of AD worldwide exceeds 15%. The clinical manifestations are varied, with the basic characteristics of dry skin, scaling, chronic eczematous lesions and severe itching. Histopathological manifestations include hyperkeratosis or parakeratosis of the epidermis, thickening of the granular layer and spinous layer, spongiform lesions, and inflammatory cell infiltration. The pathogenesis of atopic dermatitis is very complex and has not yet been fully elucidated. According to current research, it is believed to be the result of the combined effects of genetic factors, immune factors, environmental factors, skin barrier dysfunction, and skin flora disorders. The course of the disease is long and prone to recurrence. Patients are prone to a series of complications such as insomnia, anxiety, and depression, which seriously affect the physical and mental health of patients.

[0003] The main goal of AD treatment is to effectively reduce skin inflammation and itching, restore and strengthen skin barrier function through comprehensive means, and thus achieve an overall improvement in the patient's quality of life. Clinically, the treatment of AD mainly relies on the use of topical drugs, such as glucocorticoids, calcineurin inhibitors (TCIs), and phosphodiesterase 4 (PDE-4) inhibitors, all of which have shown significant anti-inflammatory effects. However, it is worth noting that although these drugs have made an indelible contribution to the control of the disease, long-term use may be accompanied by a series of side effects, which has become a problem that cannot be ignored. Long-term topical glucocorticoids may cause adverse reactions such as local skin atrophy and capillary dilation, affecting skin health, and calcineurin inhibitors have a potential carcinogenic risk. In view of the above limitations, the safety of drugs occupies a pivotal position in the formulation of AD treatment plans. In recent years, traditional Chinese medicine has received widespread attention for its effectiveness and safety in treating skin diseases.

[0004] Coptis chinensis is an important Chinese herbal medicine with multiple biological effects such as antibacterial, antioxidant, anti-inflammatory, lipid regulation, and hypoglycemic effects. Methylcoptis chinensis is an alkaloid extracted from Coptis chinensis and belongs to the isoquinoline alkaloid class. Its activity and function are not well studied. Currently, no studies have proven the application of methylcoptis chinensis in the treatment of AD. Summary of the invention

[0005] The purpose of the present invention is to provide an application of methyl coptisine in the preparation of a drug for preventing and treating atopic dermatitis, so as to solve the problems existing in the above-mentioned prior art. The present invention confirms that methyl coptisine can improve the symptoms of atopic dermatitis and reduce the level of IgE by reducing the expression of CCL17 and inhibiting the activation of STAT1 and STAT3, thus providing a theoretical basis and technical ideas for the treatment of atopic dermatitis and the research of new drugs.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The invention provides application of methyl coptisine in preparing medicine for preventing and treating atopic dermatitis.

[0008] The molecular formula of the methyl coptisine is C 20 H 16 NO4+, molecular weight is 334.35, chemical structure is as follows:

[0009]

[0010] Preferably, the dosage form of the drug includes solution and cream.

[0011] Preferably, when the dosage form of the drug is a cream, the mass concentration of methyl coptisine in the drug is 0.07%.

[0012] Preferably, when the dosage form of the drug is a solution, the concentration of methylcoptisine in the drug is 2-12.5 μM.

[0013] The present invention also provides a medicine for preventing and treating atopic dermatitis, comprising an effective ingredient methyl coptisine and pharmaceutically acceptable excipients.

[0014] Preferably, the excipients include sodium carboxymethylcellulose, hydroxypropyl methylcellulose, maltodextrin, vaseline or glycerin.

[0015] The present invention discloses the following technical effects:

[0016] The present invention uses CCK-8 to measure cell viability and confirms that methyl coptisine has no inhibitory activity on HaCaT cells, and can downregulate the expression of CCL17 in the atopic dermatitis in vivo model, and inhibit the activation of STAT1 and STAT3. The present invention confirms through animal experiments that methyl coptisine can significantly improve the severity of skin lesions in the DNCB-induced atopic dermatitis mouse model, reduce the atopic dermatitis score, reduce the back skin thickness, skin epidermal thickness and dermal inflammatory cell infiltration, and significantly downregulate the expression of IgE in the atopic dermatitis mouse model. Through the above research, it is found that methyl coptisine has a good therapeutic activity for atopic dermatitis, and therefore can be used as an active ingredient for the preparation of a drug for the treatment of atopic dermatitis, providing a theoretical basis for the treatment of atopic dermatitis, and providing a technical idea for the research of new drugs for atopic dermatitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 Design methods for DNCB-induced AD mouse model and drug intervention experiments;

[0019] Figure 2 The skin morphology of mice in each treatment group on the 26th day of modeling;

[0020] Figure 3 The atopic dermatitis score results of mice in each treatment group; n = 6, ### indicates p < 0.001 compared with the Control group; *** indicates p < 0.001 compared with the DNCB group;

[0021] Figure 4 The evaluation results of the back skin thickness of mice in each treatment group; n = 6, ### indicates p < 0.001 compared with the Control group; *** indicates p < 0.001 compared with the DNCB group;

[0022] Figure 5 The results of epidermal thickness evaluation of mice in each treatment group; n = 6, ***p < 0.001;

[0023] Figure 6 The results of HE staining of histopathological sections of mice in each treatment group;

[0024] Figure 7 The results of toluidine blue staining of histopathological sections of mice in each treatment group; the red arrows point to mast cells;

[0025] Figure 8 The number of mast cell infiltration in mice of each treatment group; n = 5, ***p < 0.001;

[0026] Fig. 9 The results of IgE level detection in the serum of mice in each treatment group;

[0027] Fig.10 The effect of different concentrations of methyl coptisine (WOR) on the viability of HaCat cells;

[0028] Fig.11 Effects of different concentrations of methyl coptisine (WOR) on the expression of CCL17 in cell supernatant; n=5, **p<0.01; ***p<0.001;

[0029] Fig.12 The effects of different concentrations of methyl coptisine (WOR) on the activation of STAT1 and STAT3 in AD cell models. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] The methyl coptisine described in the present invention is a chemical substance extracted from Coptis chinensis, which is an isoquinoline alkaloid with a molecular formula of C 20 H 16 NO4+, relative molecular mass 334.3, CAS number 38763-29-0, chemical structure is as follows:

[0036]

[0037] The methyl coptisine (0.07%) cream used in some embodiments of the present invention was prepared by ourselves; the hydrocortisone butyrate cream was purchased from Tianjin Jinyao Pharmaceutical Co., Ltd. (National Medicine Standard H10940095). The preparation method of methyl coptisine (0.07%) cream is as follows:

[0038] Take 1 mg of methylberberine and dissolve it in 20 μL of DMSO, shake and mix, then add 1.427 g of cream base and stir and mix thoroughly to obtain methylberberine cream.

[0039] The cream base formula is composed of: 4.8g of stearic acid, 2.4g of liquid paraffin, 0.4g of white vaseline, 2.0g of lanolin, and 0.16g of triethanolamine. In addition, the cream base can also use the cream base components commonly used in the art.

[0040] The methyl coptisine (CAS No.: 38763-29-0) used in some embodiments of the present invention was purchased from Wuhan Tianzhi Biotechnology Co., Ltd. (purity ≥ 98%).

[0041] Example 1

[0042] The study on the effect of methylcoptisine on reducing the clinical manifestations and pathological changes of atopic dermatitis induced by 1-chloro-2,4-dinitrobenzene (DNCB) was conducted as follows:

[0043] 1. Establishment of an animal model of atopic dermatitis (AD)

[0044] Six-week-old female Balb / c mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. and randomly divided into 4 groups: Control group (CON group, n = 6), DNCB group (n = 6), DNCB + methylcoptisone group (WOR group, n = 6), DNCB + hydrocortisone group (HYD group, n = 6). Mice were housed in a pathogen-free environment with a 12-hour light-dark cycle and unrestricted access to food and water.

[0045] The experimental design methods of DNCB-induced AD mice and drug intervention can be found in Figure 1 The specific operation process is as follows: back hair removal was performed 1 day in advance. From Day 1 to Day 3, cream containing methyl coptisine (0.07%) or hydrocortisone ointment (0.1%) was applied to the back and ears of mice (WOR group and HYD group). 5 hours after application, except for the Control group, the mice in the other treatment groups were sensitized with 150μL and 20μL 1% DNCB solution on the back skin and ears. From Day 8 to Day 26, cream containing methyl coptisine (0.07%) or hydrocortisone ointment (0.1%) was applied to the back and ears of mice (WOR group and HYD group). 5 hours after application, except for the Control group, the mice in the other treatment groups were sensitized with 150μL and 20μL 0.5% DNCB solution on the back skin and ears. The mice were killed on Day 26. After the mice were killed, blood was collected from the eyeballs, and the back and ear tissues were collected for subsequent experiments.

[0046] 2. Observation indicators: On days 0, 7, 14, 21, and 26, the severity of mice in each treatment group was evaluated by evaluating the thickness of the back skin and the atopic dermatitis score. In order to determine the severity of dermatitis, the severity of inflammation of the back skin of mice was scored according to the atopic dermatitis scoring criteria, including edema / exudation, scar / dryness, erythema / hemorrhage, and shedding / erosion. The score indicates the severity: 0 (none), 1 (mild <20%), 2 (moderate 20%-60%); 3 (severe >60%). The final dermatitis score is the sum of the scores of each item. Use a vernier caliper to measure the thickness of the back skin in the same area to monitor the progression of the lesion.

[0047] 3. Histopathological analysis

[0048] 3.1 HE staining

[0049] On the 26th day, the mice were killed by cervical dislocation, and 1×1 cm skin tissue was taken from the back lesions of mice in each treatment group, fixed with 4% formalin solution for 24 hours, embedded in paraffin, and stained with hematoxylin-eosin (H&E) after sectioning. Seven high-power fields were randomly selected from each section to measure the epidermal thickness and the average was taken (n=6).

[0050] 3.2 Toluidine blue staining

[0051] On the 26th day, the mice were killed by cervical dislocation, and 1×1 cm skin tissue was taken from the back lesions of mice in each treatment group, fixed with 4% formalin solution for 24 hours, embedded in paraffin, and stained with toluidine blue (TB) after sectioning. The number of mast cells was detected from 5 random fields of view in each section, and the data were expressed as mean ± standard error (n = 5).

[0052] 4. ELISA detection of IgE expression in mouse serum

[0053] Blood was collected from the eyeball, left to stand at room temperature for 1 hour, centrifuged at 1000×g for 20 minutes, and the supernatant was tested. The serum was diluted 4 times for testing, and the ELISA test was performed using an ELISA kit according to the manufacturer's instructions. 450 / 630nm The absorbance was measured at 400 nm, and a standard curve was drawn using software to calculate the sample concentration in each well.

[0054] 5. Results

[0055] On the 26th day, the back skin morphology of mice in each treatment group was as follows Figure 2 As shown in the figure, it can be seen that compared with the normal group, typical atopic dermatitis features including erythema, dryness, desquamation, and scaly skin lesions appeared in the DNCB-induced skin lesions. The methyl coptisine intervention group (WOR group) significantly inhibited these skin changes, showing only mild symptoms, reduced erythema and scaling, and reduced skin dryness, which was similar to the effect of the hydrocortisone intervention group (HYD group).

[0056] Back skin thickness of mice in each treatment group ( Figure 3 ), atopic dermatitis score ( Figure 4 ), epidermal thickness ( Figure 5 ) showed that compared with the control group (CON group), the back skin thickness, atopic dermatitis score and epidermal thickness of DNCB-induced mice (DNCB group) were significantly increased (###p<0.001, ***p<0.001). Compared with the DNCB group, methyl coptisine intervention (WOR group) could significantly alleviate the skin lesions of DNCB-induced AD-like mice, reduce the atopic dermatitis score, and reduce the back skin thickness and epidermal thickness (***p<0.001).

[0057] HE staining results of histopathological sections of mice in each treatment group ( Figure 6 ) showed that the DNCB group showed epidermal hyperplasia, thickening of the stratum spinosum, hyperkeratosis, and dermal inflammatory cell infiltration. Compared with the DNCB group, the methyl coptisine group (WOR group) showed less epidermal thickening and less dermal inflammatory cell infiltration. The DNCB group showed significantly higher epidermal thickness than the Control group; compared with the DNCB group, the epidermal thickness of the methyl coptisine group (WOR group) and the hydrocortisone group (HYD group) was significantly decreased. Toluidine blue staining results ( Figure 7 ) showed that compared with the CON group, the number of mast cells in the back skin of mice in the DNCB group increased significantly, and the number of mast cell infiltration in the WOR and HYD groups was significantly reduced compared with the DNCB group ( Figure 8 ), and there is a significant difference.

[0058] Results of IgE expression levels in the serum of mice in each treatment group ( Fig. 9 ) showed that compared with the CON group, the DNCB group had a significant increase in IgE, showing obvious characteristics of atopic dermatitis. The IgE of the WOR group and the HYD group was significantly lower than that of the DNCB group, with significant differences.

[0059] Example 2

[0060] The effect and molecular mechanism of methylcoptisine in inhibiting atopic dermatitis were observed in an atopic dermatitis cell model. The specific process is as follows:

[0061] 1. Preparation of methylcoptisine solution in cell experiments

[0062] Methylcopper pine was prepared into a 20 mM stock solution with DMSO, and the stock solution was diluted with DMEM culture medium to prepare methylcopper pine (WOR) solutions of different concentrations.

[0063] 2. CCK-8 method to determine the concentration of 80% HaCat cell survival after the addition of methyl coptisine

[0064] The HaCat cells were digested and counted, and 7000 cells were inoculated per well in a 96-well plate. After 24 hours, 100 μL of methyl coptisine solution of different concentrations (0 μM, 0.4 μM, 2 μM, 10 μM, 50 μM, 100 μM) was added respectively. Six replicate wells were set for each concentration. The blank control group was added with medium without drugs. 24 hours after drug addition, 10 μL of CCK-8 solution was added to each well, shaken to mix, and incubated at 37°C in the dark for 1-4 hours. The absorbance value at a wavelength of 450 nm was measured with an enzyme marker to analyze cell viability. The results are as follows Fig.10 As shown, it was shown that when the concentration of methyl coptisine was below 50 μM, the survival rate of HaCat cells exceeded 80%, and a concentration of 12.5 μM was selected to treat HaCaT cells.

[0065] 3. Effect of methylcoptisine on IFN-γ / TNF-α-induced inflammatory response in HaCaT cells

[0066] IFN-γ / TNF-α was used to stimulate HaCaT cells to simulate the pathological mechanism of atopic dermatitis, so as to detect the effect of methylcoptisine on the inflammatory response of HaCaT cells induced by IFN-γ / TNF-α.

[0067] HaCaT cells were inoculated in a 6-well plate. When the density reached 50-60% cell confluence, 100 μL of methyl coptisine solution of different concentrations (0 μM, 2 μM, 5 μM, 12.5 μM) and different concentrations of TNF-α / IFN-γ (TNF-α and IFN-γ concentrations were the same, 10 ng / mL or 20 ng / mL, respectively) were added according to the grouping for a total of 24 h. The cell supernatant was collected and the level of CCL17 in the cell supernatant was detected by ELISA. The results are shown in Figure 2. Fig.11 As shown in the figure, compared with the control group, the TNF-α / IFN-γ group could significantly increase the expression of CCL17, and the expression of CCL17 was inhibited by methylcoptisine treatment.

[0068] 4. Establish a cell model of atopic dermatitis (AD) and discover the effect and molecular mechanism of methylcoptisine in inhibiting atopic dermatitis

[0069] HaCat cells were inoculated in a 6 cm culture dish. When the cell confluence reached 70%, 100 μl of methyl coptisine at different concentrations was added according to the grouping for pretreatment for 24 h. Then, the cells were incubated with IFN-γ / TNF-α (10 ng / mL each) for 15 minutes, and the samples were collected. The total cell protein was extracted, and the changes in the activity of key molecules in the STAT3 and STAT1 signaling pathways were detected by Western blot. The results are shown in Fig.12 As shown, TNF-α / IFNγ can activate STAT1 (Y701) and STAT3 (Y705), while methylcoptisine can reduce the levels of p-STAT1 and p-STAT3 in a concentration-dependent manner. The above results show that methylcoptisine can inhibit the activation of STAT1 and STAT3 and inhibit the occurrence of atopic dermatitis.

[0070] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. The application of methyl coptisine in the preparation of drugs for preventing and treating atopic dermatitis.

2. The use according to claim 1, characterized in that: The dosage forms of the drug include solutions and creams.

3. The use according to claim 2, characterized in that: When the dosage form of the drug is a cream, the mass concentration of methyl coptisine in the drug is 0.07%.

4. The use according to claim 2, characterized in that: When the dosage form of the drug is a solution, the concentration of methylcoptisine in the drug is 2-12.5 μM.

5. A drug for preventing and treating atopic dermatitis, characterized in that: The invention comprises an effective ingredient of methyl coptisine and pharmaceutically acceptable auxiliary materials.

6. The drug according to claim 5, characterized in that The auxiliary materials include sodium carboxymethylcellulose, hydroxypropyl methylcellulose, maltodextrin, vaseline or glycerin.

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