CN114366732B - Application of tiamulin in preparation of medicine for treating psoriasis - Google Patents

Abstract

The invention discloses a novel application of tiamulin and analogues thereof in preparing a medicament for treating chronic inflammatory skin disease psoriasis. Experiments prove that the tiamulin has remarkable curative effect on psoriasis, quick response and small toxic and side effects, is a safe, efficient and stable medicament for treating psoriasis with simple preparation process, is suitable for industrial production and is easy to popularize. The invention provides a new medicine source for treating psoriasis.

Description

Application of tiamulin in preparation of medicine for treating psoriasis

Technical Field

The invention relates to a novel application of a compound in preparing a medicament for treating skin diseases, in particular to an application of a compound in preparing a medicament for treating psoriasis, and belongs to the technical field of medicines.

Background

Psoriasis (commonly known as psoriasis) is a common chronic inflammatory skin disease, the worldwide incidence rate is about 1%, psoriasis vulgaris is the most common subtype of psoriasis, typical clinical manifestations are scaling erythema or plaques, skin lesions can be distributed locally or widely, and the repeated clinical characteristics of the skin lesions cause huge economic and psychological stress to patients and also cause great difficulties to clinical medical treatment.

The traditional topical treatment for psoriasis is mainly carried out by adopting external glucocorticoid, however, the long-term external glucocorticoid can cause a series of adverse reactions such as atrophy, hair and the like of skin.

In recent years, development of monoclonal antibody drugs against key inflammatory factors for psoriasis onset, such as tumor necrosis factor α (Tumor necrosis factor alpha, TNF- α), interleukin 17 (Interleukin 17, il-17), has been advanced. However, drugs such as Adalimumab (TNF- α monoclonal antibody) and judicial You Shan anti-IL-17 monoclonal antibody have many inherent drawbacks such as high price, inability to be orally taken and administered externally, and serious side effects for long-term use.

Therefore, the drug development aiming at psoriasis is one of the great problems to be solved in the current experimental research and clinical practice. Recent researches show that the small molecular medicine has the advantages of low cost, flexible administration method and the like, so that the searching and developing of the small molecular medicine for psoriasis is a hot spot of the current researches.

The invention adopts Imiquimod (IMQ) to induce and form an animal model of psoriasis, and tiamulin fumarate (Tiamulin fumarate, TF) is administrated by intragastric administration and external application, and then the treatment effect of the animal model of psoriasis is detected. Experimental results show that the tiamulin can obviously relieve the erythema and the scales induced by imiquimod by intragastric administration or external administration, reduce the expression of the skin proliferation related factor Ki67, and reduce the PASI score and the skin thickening. Immunoblotting results suggest that tiamulin can inhibit TNF- α -induced NF- κB (Nuclear factor- κgene binding) and MAPK (Mitogen activated protein kinase) pathway activation. The results show that tiamulin can improve the psoriasis symptoms induced by the IMQ by inhibiting TNF-alpha and inflammatory pathways downstream of the TNF-alpha, and the small molecule can be externally applied, so that the side effect is relatively small, the influence on the whole body is small, and the tiamulin can be applied to the therapeutic drugs for the psoriasis.

Tiamulin is a pleuromutilin veterinary antibiotic, developed in 1976, and research on the compound is mainly focused on antibacterial aspect, and the research on the compound for treating psoriasis is not reported so far. The invention mainly discovers that tiamulin has remarkable improvement effect on the model of the mice psoriasis induced by imiquimod, and the immunoblotting experiment result shows that the tiamulin has the effect of antagonizing TNF-alpha and the pathway effect thereof, so that the tiamulin can be applied to the therapeutic drugs for the psoriasis.

Disclosure of Invention

Aiming at the technical defects that the prior medicine has a plurality of defects in the prior process of treating psoriasis, the biomacromolecule medicine can not be orally taken and externally used and has serious side effects when being used for a long time, the invention provides the application of the small molecular compound tiamulin in preparing the medicine for treating psoriasis, and the tiamulin can obviously relieve erythema and scales of psoriasis and reduce skin thickening; tiamulin can obviously inhibit the NF-kappa B, MAPK pathway of TNF-alpha downstream inflammation, thereby improving psoriasis symptoms; the tiamulin can also play a role through external administration, and the side effect can be obviously reduced in view of the fact that the external administration has smaller influence on the whole body, so that the tiamulin can be better applied to the therapeutic drugs for psoriasis.

In order to achieve the aim of the invention, the invention provides an application of tiamulin or tiamulin analogues in preparing medicines for treating chronic inflammatory skin diseases.

Wherein the chronic inflammatory skin disease is psoriasis, atopic dermatitis, preferably psoriasis.

In particular, the psoriasis is psoriasis vulgaris.

Wherein the medicine consists of tiamulin and a pharmaceutically acceptable carrier

In particular, pharmaceutically acceptable carriers are commonly accepted by health professionals for this purpose and as inactive ingredients of medicaments. A compilation of relevant pharmaceutically acceptable carriers can be found in the handbook of pharmaceutical excipients (Handbook of Pharmaceutical excipients, 2 nd edition, edited by A. Wade and P.J. Weller; american Pharmaceutical Association publication Washington and The Pharmaceutical Press, london, 1994) et al.

In particular, the carrier includes excipients such as starch, water, etc.; lubricants such as magnesium stearate and the like; disintegrants such as microcrystalline cellulose and the like; fillers such as lactose and the like; binders such as pregelatinized starch, dextrin, etc.; a sweetener; an antioxidant; preservatives, flavoring agents, fragrances, and the like;

wherein the drug is administered by a parenteral route of administration and a parenteral route of administration.

In particular, the parenteral route of administration is selected from injection, respiratory, dermal, mucosal or luminal.

Wherein the medicine is in the form of tablet, capsule, pill, powder, granule, syrup, solution, injection, spray, aerosol, patch, etc.

Wherein parenteral administration is selected from injection, spray, aerosol, patch, tincture, powder, paste, lotion, cream, ointment, etc.

In particular, the preparation for parenteral administration is selected from tablets, capsules, powders, granules, pills, solutions or syrups and the like.

In particular, the tiamulin is tiamulin fumarate.

Wherein, the tiamulin fumarate has the following structure:

in another aspect, the invention provides a medicament for treating psoriasis comprising tiamulin or a tiamulin analog.

Wherein the medicine consists of tiamulin and a pharmaceutically acceptable carrier.

In particular, the tiamulin is tiamulin fumarate.

In particular, the carrier includes excipients such as starch, water, etc.; lubricants such as magnesium stearate and the like; disintegrants such as microcrystalline cellulose and the like; fillers such as lactose and the like; binders such as pregelatinized starch, dextrin, etc.; a sweetener; an antioxidant; preservatives, flavoring agents, fragrances, and the like;

the medicament can be prepared into various dosage forms by methods known in the art, such as tablets, capsules, pills, powder, granules, syrups, solutions, injections, sprays, aerosols, patches, tinctures, powders, pastes, lotions, creams, ointments and the like;

the invention also provides a method of treating psoriasis comprising administering a therapeutically effective amount of tiamulin.

Wherein, the oral treatment effective dose of the animal is 100mg-300mg/kg; the effective concentration for external use is 5%.

As used herein, unless otherwise indicated, the term "therapeutically effective amount" is the amount of a drug that is required to produce an effective effect; the "therapeutically effective amount" is adjustable and variable, ultimately determined by the medical practitioner, and factors considered include the route of administration and the nature of the formulation, the general condition of the recipient's weight, age, etc., and the nature and severity of the condition being treated.

Compared with the prior art, the invention has the following advantages:

the tiamulin can improve the psoriasis symptoms of the mouse model induced by imiquimod, and the small molecule can also be externally applied, has relatively small side effect and small influence on the whole body, so that the tiamulin can be applied to the treatment of psoriasis.

The invention develops a new medicinal value for the known compound tiamulin, is used for treating psoriasis, and can be prepared into a medicament for preventing, relieving and treating psoriasis, thereby developing a new application field for the application of the tiamulin.

The tiamulin has strong pharmacological action for treating psoriasis, can relieve imiquimod-induced psoriasis dermatitis like, reduce the psoriasis skin damage area and severity score (Psoriatic area and severity index, PASI) and abnormal skin proliferation, and inhibit the expression of a skin proliferation related factor Ki 67. Immunoblotting experiments indicate that tiamulin can inhibit TNF-alpha induced NF-kappa B, MAPK pathway activation, thereby treating psoriasis.

The tiamulin has remarkable efficacy for treating psoriasis, small toxic and side effects and good safety, and particularly has good medicinal prospect through external administration.

The tiamulin disclosed by the invention is safe in clinical use, simple in preparation process, and convenient to use, and can be prepared into oral and external preparations, so that the tiamulin is easy to popularize.

Drawings

FIG. 1A is a graph of skin changes on the back, skin lesions and HE staining of the mice in example 1, wherein the left side is the graph of skin changes on the back, and the right side is the graph of skin lesions and HE staining;

FIG. 1B is a chart showing the immunohistochemical staining of the skin Ki67 of the mouse in example 1;

FIG. 1B-1 is a statistical plot of Ki67 positive cells from immunohistochemical staining of mouse skin in example 1;

FIG. 1C is a graph of the PASI score for the degree of back skin damage in the mice of example 1;

FIG. 1D is a graph of the back skin layer thickness measurement of the mice in example 1;

FIG. 2A is a graph of skin changes on the back and skin lesions HE staining of the mice in example 3, wherein the left side is the graph of skin changes on the back and the right side is the graph of skin lesions HE staining;

FIG. 2B is a chart showing the immunohistochemical staining of the skin Ki67 of the mouse in example 3;

FIG. 2B-1 is a statistical plot of mouse skin immunohistochemical staining of Ki67 positive cells in example 3;

FIG. 2C is a graph of the PASI score for the degree of back skin damage in the mice of example 3;

FIG. 2D is a graph of the back skin layer thickness measurement of the mice in example 3;

FIG. 3 is a photograph showing the immunoblot electrophoresis of tiamulin versus TNF- α induced HaCaT cell line of example 4 of the present invention.

Detailed Description

The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.

The beneficial effects of the medicaments according to the invention are further illustrated below by test examples, which include pharmacodynamic tests of the medicaments according to the invention.

Example 1 Effect of gavage administration of tiamulin on imiquimod-induced murine psoriasis model 1, test Material

1.1 laboratory animals, reagents

BALB/c mice, male, 7-8 weeks old, supplied by the collection of medicine and health animal Biotechnology laboratory center, eligibility number: SCXK 2018-0008.

Tiamulin fumarate (purchased from MedChemExpress corporation) was dissolved in water to prepare a tiamulin fumarate solution at a concentration of 25 mg/ml;

levofloxacin (Levofoxacin, available from MedChemexpress corporation) was dissolved in water to prepare a Levofloxacin solution having a concentration of 10 mg/ml;

5% imiquimod cream (available from Sichuan Ming pharmaceutical Co.); petrolatum ointment (ex co-morbidly), ki67 antibody (ex Abcom).

1.2 Experimental methods

30 male BALB/c mice of 7-8 weeks of age were randomly divided into 5 groups of 6, shaved on the back (area: 2 cm. Times.3 cm), and were kept normally, and after three days, different treatments were given to the shaved parts of the mice, respectively, as follows:

blank Control (Control): the back skin of the mouse is smeared with 62.5mg of Vaseline per day for 5 days;

model group (IMQ): the back skin of the mice is smeared with 62.5 mg/day of IMQ (imiquimod) for 5 days;

placebo group (imq+placebo (placebo: levofloxacin)): the back skin of the mouse is smeared with IMQ 62.5 mg/day for 5 days; before IMQ is smeared, 10mg/kg of levofloxacin is administrated by gastric lavage;

low dose treatment group (imq+tf 100 mg/kg): the back skin of the mouse is smeared with IMQ 62.5 mg/day for 5 days; before IMQ is smeared, TF is administrated by lavage at 100mg/kg;

high dose treatment group (imq+tf 300 mg/kg): the back skin of the mouse is smeared with IMQ 62.5 mg/day for 5 days; TF is administered in 300mg/kg by gastric lavage prior to IMQ application.

Each group of mice was fed water normally, observed daily for skin appearance and given a PASI score, sacrificed on day 6 to take the mice skin lesions for HE staining and to measure skin thickness, and immunohistochemical staining of the skin lesions.

The PASI score is provided with 3 evaluation indexes, 0 to 4 points are respectively given according to the severity of the skin damage of the back of the mouse, and the three scores are summarized as the total score of the PASI score. The PASI score is specifically as follows:

1. erythema: the following scores were used for scoring back erythema in mice:

asymptomatic 0; mild 1; a medium degree 2; severe 3; extremely severe 4.

2. Infiltration: the following scores were used for the scoring of back infiltrates in mice:

asymptomatic 0; mild 1; a medium degree 2; severe 3; extremely severe 4.

3. Desquamation: the following scores were used for scoring the back desquamation of mice:

asymptomatic 0; mild 1; a medium degree 2; severe 3; extremely severe 4.

Mice were sacrificed on day 6 and dorsal skin tissues were fixed with 10% formalin for 48h and paraffin embedded;

paraffin sections 5 μm thick were cut for hematoxylin and eosin (H & E) staining, then photographed under 40X field of view and the skin thickness was measured on a vertical cross section of each section using software Image J. Calculating the average value of 5 random points of each mouse to obtain the thickness of the epidermis.

Paraffin sections 5 μm thick were excised and subjected to Ki67 immunohistochemical staining, primary antibody used (Abcam, ab6721,1:400 dilution). The mean of Ki67 positive cells was calculated for 3 random fields per mouse by taking photographs at 40X fields and counting the number of Ki67 positive cells at 100X fields using software Image J.

1.3 experimental results

The change on day 6 of the back skin and skin loss HE staining (40X) of each group of mice gave the results shown in fig. 1A.

As can be seen from fig. 1A: the skin of the back of the mice was visibly marked erythema, scales, and both model (IMQ) and placebo (imq+placebo) groups compared to the placebo group; HE staining revealed a marked hyperkeratosis and hypokeratosis of the epidermis, and a large number of inflammatory cell infiltrates in the superficial dermis. Both the low dose group treatment (IMQ+TF 100 mg/kg) and the high dose group treatment (IMQ+TF 300 mg/kg) suggested significant relief of erythema and scaling following drug gavage treatment, with the high dose group treatment providing more significant relief of skin lesions.

FIG. 1B is a chart of the Ki67 immunohistochemical staining observations of the skin of mice from different treatment groups (40X);

the skin basal layer cells Ki67 immunohistochemical staining of the model group (IMQ) was significantly enhanced compared to the placebo group, and the skin basal layer cells Ki67 immunohistochemical staining of the low dose treatment group (imq+tf 100 mg/kg) and the high dose treatment group (imq+tf 300 mg/kg) were attenuated, wherein the attenuation of the staining was more significant.

FIG. 1B-1 is a graph of the Ki67 immunohistochemical staining of the skin of mice in different treatment groups, i.e., the number of Ki67 positive cells in the field of view was counted at 100X under the microscope field of view of the immunohistochemical staining of Ki67 in mice in different treatment groups using software Image J, the average of the Ki67 positive cells in 3 random fields per mouse was calculated, and statistics were performed using the independent sample t-test.

Blank VS model group: * P <0.0001; low dose treatment VS model group: * P <0.05; high dose treatment VS model group: * P <0.01.

PASI scoring results as in fig. 1C, scoring results show a decrease in scores after TF drug treatment, with a more pronounced decrease in scores in the high dose group. Statistics were performed using Two-way ANOVA (Two-way ANOVA). Low dose treatment VS model group, fourth day: * P <0.05, fifth day: * P <0.01, sixth day: * P <0.01. High dose treatment VS model group, fourth day: * P <0.01, fifth day: * P <0.001, sixth day: * P <0.0001.

The skin thickness measurement results are shown in fig. 1D: the thickness of the skin layer was measured on a vertical cross section of each slice using software Image J under a 40X microscope field of view. The average of 5 random spot epidermis thicknesses per mouse was calculated and counted using the independent sample t-test. Blank VS model group: * P <0.0001; low dose treatment VS model group: * P <0.05; high dose treatment VS model group: * P <0.01.

The experiment of example 1 shows that oral tiamulin can alleviate imiquimod-induced psoriasis-like dermatitis in an imiquimod-induced psoriasis mouse model, indicating that tiamulin has an effect of treating psoriasis.

EXAMPLE 2 preparation of tiamulin cream

1) The materials are prepared according to the following formula:

2) Preparation of tiamulin cream

Weighing tiamulin fumarate (1 g), placing in a beaker, adding 10g of purified water, dissolving the tiamulin by ultrasonic, then adding liquid paraffin, ethylparaben, tween 80 and silicone oil, heating to dissolve, then sequentially adding glyceryl monostearate, stearyl alcohol, glycerol and peregal, stirring while heating to melt, finally adding purified water to make up to 20g, and stirring until solidification to obtain the tiamulin cream.

Example 2A preparation of placebo cream

1) The materials are prepared according to the following formula:

2) Preparation of placebo cream

Placing 10g of purified water into a beaker, adding liquid paraffin, ethylparaben, tween 80 and silicone oil, heating to dissolve, sequentially adding glyceryl monostearate, stearyl alcohol, glycerol and peregal, heating and stirring to melt, adding purified water to make up to 20g, and stirring to solidify to obtain tiamulin soothing agent for later use.

EXAMPLE 2B preparation of tiamulin ointment (oily base)

1) The materials are prepared according to the following formula:

tiamulin fumarate 1.0g

White vaseline 17.0g

Lanolin 2.0g

2) Preparation of tiamulin ointment

Placing white vaseline and lanolin in a beaker, heating to completely melt, adding tiamulin fumarate (1 g), rapidly stirring, stopping heating, and stirring until solidification to obtain tiamulin ointment.

EXAMPLE 2C preparation of tiamulin ointment (aqueous base)

1) The materials are prepared according to the following formula:

2) Preparation of tiamulin ointment (aqueous base)

Weighing tiamulin fumarate (1 g), placing in a beaker, adding 10g of purified water, dissolving the tiamulin by ultrasonic, then adding stearic acid, sorbitan monostearate and tween 60, heating and stirring to melt, adding purified water to make up to 20g, and stirring to solidify to obtain the tiamulin ointment.

Example 3 effects of topical tiamulin administration on imiquimod-induced murine psoriasis model 1, test materials

1.1 laboratory animals, reagents

Tiamulin cream prepared in example 2, 2A, placebo.

BALB/c mice, male, 7-8 weeks old, supplied by the collection of medicine and health animal Biotechnology laboratory center, eligibility number: SCXK 2018-0008.

Imiquimod cream (purchased from the company of the pharmaceutical industry, inc.); petrolatum ointment (ex co-morbidly), ki67 antibody (ex Abcom).

1.2 Experimental methods

24 male BALB/c mice of 7-8 weeks of age were randomly divided into 4 groups of 6, shaved on the back (area: 2 cm. Times.3 cm), and were kept normally, and after three days, different treatments were given to the mice at shaved sites, respectively, as follows:

blank Control (Control): the back skin of the mouse is smeared with 62.5mg of Vaseline per day for 5 days;

building block (IMQ): the back skin of the mouse is smeared with IMQ 62.5 mg/day for 5 days;

placebo group (imq+placebo (placebo prepared in example 2A)): the back skin of the mouse is smeared with IMQ 62.5 mg/day for 5 days; after 6 hours of daily IMQ application, an equal dose of placebo was applied.

Treatment group (imq+tf5%): the back skin of the mouse is smeared with IMQ 62.5 mg/day for 5 days; after IMQ was applied for 6 hours per day, an equal dose of the 5% tiamulin cream prepared in example 2 was applied.

Each group of mice was fed water normally, observed daily for skin appearance and given a PASI score, sacrificed on day 6 to take the mice skin lesions for HE staining and to measure skin thickness, and immunohistochemical staining of the skin lesions.

The PASI scoring criteria were the same as in example 1; mice were sacrificed on day 6 and dorsal skin tissues were fixed with 10% formalin for 48h and paraffin embedded; hematoxylin and eosin (H & E) staining procedure was the same as in example 1; the measurement of the skin thickness was the same as in example 1; ki67 immunohistochemical staining was the same as in example 1.

1.3 experimental results

The change on day 6 of the back skin and HE staining of skin lesions (40X) for each group of mice resulted in the results shown in fig. 2A.

As can be seen from fig. 2A: obvious erythema and scaling were seen in the dorsal skin of the mice in the model group (IMQ) and placebo group (imq+palcebo) compared to the Control group (Control) mice; HE staining revealed a marked hyperkeratosis and hypokeratosis of the epidermis, and a large number of inflammatory cell infiltrates in the superficial dermis. Treatment group (imq+tf5%) suggested significant relief of erythema and scaling following topical drug treatment.

FIG. 2B is a chart of the Ki67 immunohistochemical staining observations of the skin of mice from different treatment groups (40X);

the model group (IMQ) showed significantly enhanced immunohistochemical staining of skin basal layer cells Ki67 compared to the placebo group, and the treatment group (TF 5%) showed reduced immunohistochemical staining of skin basal layer cells Ki 67.

Fig. 2B-1 is a graph of Ki67 immunohistochemical staining statistics of the skin of mice in different treatment groups, i.e. Ki67 positive cells in the fields were counted using software Image J under the field of view of the high-power microscope (100X) for Ki67 immunohistochemical staining of the skin of mice in different treatment groups, the average of 3 random fields Ki67 positive cells per mouse was calculated, statistics were performed using independent sample t-test, VS model group of blank control group: * P <0.0001; treatment VS model group: * P <0.001.

PASI scoring results as in fig. 2C, scoring results show a decrease in score after TF drug treatment. Statistics were performed using Two-way ANOVA (Two-way ANOVA). Treatment VS model group, third day: * P <0.01, fourth day: * P <0.0001, fifth day: * P <0.001, sixth day: * P <0.0001.

The results of the skin thickness measurements for the mice of the different treatment groups are shown in fig. 2D: the thickness of the skin layer was measured on a vertical cross section of each slice using software Image J under a 40X microscope field of view. The average of 5 random spot epidermis thicknesses per mouse was calculated and counted using the independent sample t-test. Blank VS model group: * P <0.0001; treatment VS model group: * P <0.0001.

The experiment of example 3 shows that the externally applied tiamulin can relieve the psoriasis-like dermatitis induced by imiquimod in an imiquimod-induced psoriasis mouse model, and the externally applied administration in skin diseases is more efficient and convenient.

Example 4 Effect of tiamulin on TNF-alpha stimulated HaCaT cell lines

1. Test materials

Taking a human epidermis immortalized cell HaCaT line in a logarithmic growth phase for experiments;

TNF-alpha of human origin is dissolved in purified water at a concentration of 10ng/ml; TF is dissolved in dimethyl sulfoxide to prepare TF solutions with the concentration of 50 mu M and 100 mu M respectively; adalimeumab (Adalimumab, purchased from MedChemexpress corporation) was used as a positive control, and was dissolved in purified water at a concentration of 10. Mu.g/ml. Immunoblots primary antibodies were purchased from CST (Cell Signaling Technology) company as: iκBα (# 4814), p65 (# 8242), p-p65 (# 3033), p-ERK (# 4370), p-JNK (# 4668).

1.2 Experimental methods

The specific operation is as follows:

control, K group: extracting proteins from the HaCaT cell line, namely taking the proteins of the HaCaT cells extracted by using RIPA lysate as blank control;

stimulation group (TNF- α, abbreviated as group C): the HaCaT cell line was stimulated with 10ng/ml TNF- α for 30 min, and then the HaCaT cell protein was extracted using RIPA lysate;

low dose TF group (TNF- α+tf50μm, abbreviated as group D): pretreatment of HaCaT cells with 50 μm TF for 2 hours; then, 10ng/ml TNF-alpha was used to stimulate HaCaT cells for 30 minutes, and then RIPA lysate was used to extract HaCaT cell proteins;

high dose TF group (TNF- α+tf 100 μm, abbreviated G group): pretreatment of HaCaT cells with 100 μm TF for 2 hours followed by stimulation of HaCaT cells with 10ng/ml TNF- α for 30 minutes followed by extraction of HaCaT cell proteins with RIPA lysate;

positive control group (TNF-. Alpha. + Adalimeumab 10. Mu.g/ml, abbreviated as Y group): haCaT cells were pre-treated with 100. Mu.g/ml Adalimeumab for 2 hours, followed by stimulation of HaCaT cells with 10ng/ml TNF-. Alpha.for 30 minutes, followed by extraction of HaCaT cell proteins using RIPA lysate; the specific method for extracting the HaCaT cell protein by using RIPA lysate is as follows:

at room temperature, 100 mu l of RIPA lysate is added into cells, after 2 minutes of lysis, the liquid is collected into a centrifuge tube, the centrifugation is carried out for 10 minutes at 4 ℃ in 10000 r/min by a high-speed centrifuge, and the supernatant is sucked to obtain a protein sample. Electrophoresis was performed at room temperature using a constant pressure method of 150V and 4-20% sodium dodecyl sulfate polyacrylamide gel (Bio-RAD) to separate an equal amount of protein sample; protein transfer to PVDF membrane (Bio-RAD) using semi-dry transfer method; the PVDF membrane was blocked with 5% nonfat dry milk for 1 hour at room temperature; PVDF membranes were incubated with primary antibody (i.e., immunoblotted primary antibody) overnight at 4 ℃ followed by horseradish peroxidase secondary antibody (abclon) for 1 hour at room temperature. Chemiluminescent signals were detected and analyzed using Gel-Doc XR imaging laboratory system (Bio-RAD).

1.3 experimental results

The immunoblotting detection results of NF- κB related proteins IκBα, P-P65 and P65 are shown in FIG. 3, compared with the blank control group and the positive control group, the stimulated group can see that IκBα is significantly down-regulated, while P-P65, P-ERK and P-JNK are significantly up-regulated, and P65 has no obvious change, so that TNF- α can effectively activate NF- κB and MAPK channel related proteins.

In the TF administration group, both low-dose TF and high-dose TF can obviously inhibit the down-regulation of I kappa B alpha and P-P65 and P-JNK induced by TNF-alpha, and the high-dose TF can inhibit the up-regulation of P-ERK induced by TNF-alpha. This result suggests that TF can significantly inhibit activation of TNF- α on its downstream NF-. Kappa. B, MAPK pathway in the HaCaT cell line.

Example 4 the results demonstrate that TF can directly inhibit the TNF- α activated inflammatory pathways of MAPK, NF- κB in an in vitro experiment. Whereas MAPK and NF- κB psoriasis were both significantly upregulated. Therefore, TF can inhibit activation of MAPK and NF- κB, and finally achieves the therapeutic effect on psoriasis.

Claims (4)

1. Use of tiamulin or tiamulin fumarate as sole active ingredient in the manufacture of a medicament for the treatment of chronic inflammatory skin disease, wherein the chronic inflammatory skin disease is psoriasis.

2. The use according to claim 1, wherein the psoriasis is psoriasis vulgaris.

3. The use according to claim 1 or 2, wherein the medicament consists of tiamulin and a pharmaceutically acceptable carrier.

4. The use according to claim 1 or 2, wherein the medicament is in the form of a tablet, capsule, pill, powder, granule, syrup, solution, injection, spray, aerosol, patch, tincture, paste, lotion, cream, ointment.