Application of CD36 inhibitor in preparation of medicine for treating psoriasis

Abstract

The invention discloses application of a CD36 inhibitor in preparation of a medicine for treating psoriasis, and belongs to the technical field of biological medicine. Experiments prove that the ointment containing the CD36 inhibitor can significantly reduce the expression level of psoriasis related inflammatory factors CXCL5 / CXCL8 and CCL20, and reduce the local infiltration of CCR6 + T cells and neutrophils. The CD36 inhibitor ointment can inhibit secretion of inflammatory chemotactic factors related to keratinocyte psoriasis so as to effectively relieve psoriasis-like dermatitis induced by IMQ, and can be used for preparing local external medicines for treating psoriasis.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the use of a CD36 inhibitor in the preparation of a drug for treating psoriasis. Background Art

[0002] Psoriasis, also known as psoriasis, is a chronic, non-infectious inflammatory skin disease, and its pathophysiological characteristics mainly include the manifestations of epidermal hyperkeratosis and parakeratosis. This disease causes the skin's keratinocytes to be in a state of abnormal proliferation, resulting in an increase in the thickness of the epidermis and the formation of obvious scales. In addition, the skin lesions of psoriasis are often accompanied by the formation of neutrophil microabscesses, which indicates an aggravation of the local inflammatory response. At the same time, the proliferation and expansion of blood vessels in the dermis and the infiltration of a large number of immune cells (such as T cells, B cells, etc.) further promote the aggravation of inflammation, causing the skin to show obvious erythema and scales, and in severe cases may even affect multiple parts of the body. These pathological changes not only affect the appearance of the skin, but may also cause discomfort such as pain and itching, significantly reducing the patient's quality of life.

[0003] The clinical manifestations of psoriasis are usually clearly demarcated scaly erythema or plaques, which are common on the trunk, limbs, scalp and other parts, and often worsen in winter or when stress increases. In some patients, psoriasis may involve large areas of skin and even cause systemic symptoms such as arthritis (psoriatic arthritis), which not only affects the patient's physical health, but may also have a negative impact on their psychological state. Many psoriasis patients feel ashamed, anxious or depressed because of the appearance of skin lesions, and their social activities are restricted, affecting their self-esteem and quality of life. Therefore, the comprehensive impact of psoriasis is multifaceted, seriously affecting the patient's daily life and mental health.

[0004] Currently, there is no cure for psoriasis, and existing treatments mainly focus on controlling symptoms and reducing inflammatory responses. These include topical medications (such as steroids and vitamin D derivatives), systemic treatments (such as immunosuppressants and biological agents), etc. However, these therapies can often only relieve symptoms but not completely cure them, and side effects and drug resistance may occur during use. Based on these challenges, it has become particularly important to develop new treatment options. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention aims to provide a use of a CD36 inhibitor in the preparation of a drug for treating psoriasis, wherein the inhibitor inhibits the secretion of chemokines by inhibiting mitochondrial ROS in keratinocytes, thereby reducing CCR6 + Local infiltration of T cells and neutrophils inhibits inflammatory response in psoriatic skin.

[0006] In order to achieve the above object, the present invention adopts the following technical scheme:

[0007] The present invention provides the use of a CD36 inhibitor in preparing a medicine for treating psoriasis.

[0008] Animal and cell experiments have shown that the increased CD36 in psoriasis promotes mitochondrial ROS in keratinocytes by transporting more palmitic acid, causing increased expression and secretion of psoriasis-related chemokines CXCL5 / CXCL8 and CCL20, and chemotactic CCR6 + T cells and neutrophils aggravate psoriatic skin inflammation.

[0009] Preferably, the structural formula of the CD36 inhibitor is:

[0010] It should be noted that Sulfosuccinimidyl oleate sodium (SSO) is a sodium salt of long-chain fatty acids and an effective and irreversible mitochondrial respiratory chain inhibitor. It can bind to the CD36 receptor on the cell surface and inhibit the transport of fatty acids into cells.

[0011] Preferably, the inhibitor has an inhibitory effect on keratinocytes secreting psoriasis-related inflammatory factors CXCL5 / CXCL8 and CCL20.

[0012] Preferably, the inhibitor is capable of reducing CCR6 + Local infiltration of T cells and neutrophils.

[0013] Preferably, the drug comprises a CD36 inhibitor and a pharmaceutically acceptable excipient.

[0014] The present invention also provides an ointment, comprising the CD36 inhibitor as described above.

[0015] Preferably, the ointment further comprises excipients.

[0016] Preferably, the excipients include white vaseline, ethyl paraben, stearyl alcohol, sodium lauryl sulfate and glycerin.

[0017] The experiment of the present invention proves that the ointment prepared by CD36 inhibitor SSO has an inhibitory effect on the gene expression and secretion level of inflammatory chemokines CXCL5 / CXCL8 and CCL20 in mice with psoriasis-like dermatitis induced by IMQ (imiquimod), and it is found that it can inhibit the mitochondrial ROS of keratinocytes (such as Figure 1 shown).

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention found that the CD36 inhibitor SSO can inhibit the secretion of chemokines by inhibiting mitochondrial ROS in keratinocytes, thereby reducing CCR6 + The local infiltration of T cells and neutrophils inhibits psoriasis-like inflammatory reactions and can be used to prepare drugs for the treatment of psoriasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a diagram of the cellular action mode of the CD36 inhibitor SSO provided by the present invention;

[0021] Figure 2 The figure is a graph showing the expression level of CD36 in HaCaT cells treated with different concentrations of palmitic acid (PA, 100 μM / 500 μM) in Example 1;

[0022] Figure 3 This is a diagram showing changes in lipids in HaCaT cells when SSO blocks CD36 in Example 1;

[0023] Figure 4 This is a diagram showing the effect of PA-CD36 on the expression level of chemokine genes in HaCaT cells detected by qPCR in Example 2;

[0024] Figure 5 This is a diagram showing the effect of PA-CD36 on the secretion of chemokines from the supernatant of HaCaT cells detected by the Cytometric bead array (CBA) method in Example 2;

[0025] Figure 6 This is a diagram showing the effects of PA and SSO treatment on mitochondrial ROS in HaCaT cells in Example 3;

[0026] Figure 7 The expression level of chemokines in HaCaT cells after removing mtROS in Example 3;

[0027] Figure 8 This is a general picture of the skin of IMQ-induced psoriasis mice using the ointment prepared with the topical CD36 inhibitor SSO in Example 4;

[0028] Fig. 9 HE images of the skin of IMQ-induced psoriasis mice of the ointment prepared with the topical CD36 inhibitor SSO in Example 4;

[0029] Fig.10 This is a graph showing the statistical analysis results of local infiltrating immune cells in skin lesions by flow cytometry in Example 4. DETAILED DESCRIPTION

[0030] The present invention is further described in detail below through specific preferred embodiments, but the present invention is not limited to the following embodiments.

[0031] It should be noted that, unless otherwise specified, the chemical reagents involved in the present invention were purchased through commercial channels.

[0032] Example 1

[0033] Effects of different concentrations of PA and CD36 inhibitor SSO on CD36 expression levels and intracellular lipids in HaCaT cells

[0034] HaCaT cells (human immortalized keratinocytes) were inoculated into 12-well plates. When the cell density reached 50-75%, different concentrations of PA (palmitic acid) solution (the group with a final PA concentration of 100 μM was called PA100 group, and the group with a final PA concentration of 500 μM was called PA500 group) and an equal amount of BSA solution were added to the cell culture medium as a control (Control group). The CD36 inhibition group was treated with SSO working solution (200 uM) prepared with SSO preservation solution (SSO was fully dissolved in dimethyl sulfoxide to prepare 120 mM SSO preservation solution, which was divided and stored in a -80°C refrigerator) for 2 h before PA intervention (SSO group). After continuing to culture in a CO2 constant temperature cell culture incubator for 12 h, the effect of PA on the expression level of CD36 in HaCaT cells was detected by qPCR and WB methods; the BODIPY493 / 503 fluorescence of cell neutral lipids was observed by confocal fluorescence microscopy.

[0035] WB and qPCR detection revealed that compared with the control group, the expression level of CD36 in the PA-treated group increased in a dose-dependent manner ( Figure 2 ). Confocal microscopy observations revealed that compared with the control group, the BODIPY 493 / 503 fluorescence in HaCaT cells significantly increased and enhanced after PA solution intervention, while this process was significantly inhibited when SSO blocked CD36 ( Figure 3 ).

[0036] Example 2

[0037] To investigate the effects of PA and CD36 inhibitor SSO on chemokine production in HaCaT cells

[0038] HaCaT cells were inoculated into 12-well plates. When the cell density reached 50-75%, the CD36 inhibition group was treated with 200 μM SSO for 2 h before PA intervention. Then, PA solution (final concentration 500 μM) was added to the cell culture medium. After further culture in a CO2 constant temperature cell culture incubator for 12 h, the effect of PA-CD36 on the expression level of chemokine genes in HaCaT cells was detected by qPCR; the effect of PA-CD36 on the secretion of chemokines by HaCaT cells was detected by Elisa.

[0039] The results showed that after in vitro PA intervention, the mRNA expression levels and serum content of CXCL5 / CXCL8 and CCL20 in HaCaT cells were significantly higher than those in the control group. This phenomenon was inhibited when SSO was used to block CD36 in advance ( Figure 4 Figure 5 ).

[0040] Example 3

[0041] To investigate the effects of PA and SSO on mitochondrial ROS in HaCaT cells and the effects on chemokines of HaCaT cells after removing mtROS

[0042] HaCaT cells were seeded into 12-well plates. When the cell density reached 50-75%, 200 μM SSO was added to the CD36 inhibition group for 2 h before PA solution intervention. MitoTEMPO reagent (10 μM) was added to the culture medium for 3-4 h before PA solution intervention in the mtROS scavenging group. PA solution (final concentration 500 μM) was then added to the cell culture medium. After continued culture in a CO2 constant temperature cell culture incubator for 12 h, mitochondrial ROS levels were detected by flow cytometry. The effect of scavenging mtROS in HaCaT cells on chemokine expression was detected by qPCR.

[0043] The results showed that PA could significantly promote the production of mtROS in HaCaT cells, while the promotion of mtROS by PA was inhibited after blocking CD36 with SSO ( Figure 6 ); After using MitoTEMPO to remove mtROS, the promoting effect of PA on the expression of chemokines (CXCL5 / CXCL8, CCL20) in HaCaT cells was significantly weakened ( Figure 7 ).

[0044] Example 4

[0045] To investigate the effect of an ointment prepared with CD36 inhibitor SSO on IMQ (imiquimod)-induced psoriasis-like dermatitis in mice

[0046] (1) Preparation of SSO ointment

[0047] Weigh 0.3 g of white vaseline, 0.05 g of glyceryl monostearate, 0.005 g of ethyl paraben and 0.2 g of octadecyl alcohol respectively, put them in a clean glass bottle, shake and mix them in a water bath at 80°C, and heat them for 10 minutes; weigh 0.025 g of sodium dodecyl sulfate and put it in another clean glass bottle, add 0.175 g of glycerol and 1.75 mL of double distilled water, shake and mix them in a water bath at 80°C, and heat them for 3 minutes; after heating, mix the two, continue to shake and mix them at 80°C and heat them for 10 minutes, then pour them into a glass container, gradually cool them down, add 86 μL of SSO preservation solution or an equal amount of DMSO at the same time, stir and mix them, and cool them to room temperature to prepare SSO ointment and Vehicle control ointment respectively.

[0048] (2) Establishment of animal model of psoriasis

[0049] Fifteen 6-8 week old C57BL / 6 female mice were randomly divided into 3 groups: Control group (n=5), Vehicle / IMQ group (n=5), SSO ointment / IMQ group (n=5). SSO ointment (2.5 mg / kg body weight / ear) and equal dose of Vehicle ointment were evenly applied to the ears of mice in the Vehicle group and SSO ointment group, respectively; after an interval of 6 hours, IMQ ointment (25 mg / ear) was applied again for 5 consecutive days.

[0050] (3) Experimental results

[0051] The severity of psoriasis-like skin lesions in mice was evaluated by taking pictures of skin lesions with a camera and HE staining. The proliferation activity of epidermal cells was evaluated by Ki67 staining. It was found that the average epidermal thickness of skin lesions in the SSO ointment treatment group was significantly lower than that in the Vehicle control ointment group ( Figure 8 ), the number of Ki67-positive cells decreased significantly ( Fig. 9 ). Flow cytometry was used to detect the local infiltration of immune cells in the skin lesions. It was found that the local infiltration of neutrophils and CCR6 in the SSO ointment treatment group + Decreased number of T cells ( Fig.10 ).

[0052] Finally, it should be noted that the above embodiments do not limit the present invention in any form. For those skilled in the art, some modifications and improvements can be made to the present invention. Therefore, any modification or improvement made without departing from the spirit of the present invention belongs to the scope of protection claimed in the present invention.

Claims

1. Application of CD36 inhibitors in the preparation of drugs for treating psoriasis.

2. The use according to claim 1, characterized in that: The structural formula of the CD36 inhibitor is:

3. The use according to claim 1, characterized in that: The inhibitor has an inhibitory effect on keratinocytes secreting psoriasis-related inflammatory factors CXCL5 / CXCL8 and CCL20.

4. The use according to claim 1, characterized in that: The inhibitor can reduce CCR6 + Local infiltration of T cells and neutrophils.

5. The use according to claim 1, characterized in that: The medicine comprises a CD36 inhibitor and pharmaceutically acceptable excipients.

6. An ointment, characterized in that The ointment comprises the CD36 inhibitor according to claim 1.

7. The ointment according to claim 1, characterized in that The ointment further comprises excipients.

8. The ointment according to claim 7, characterized in that The auxiliary materials include white vaseline, ethyl paraben, stearyl alcohol, sodium lauryl sulfate and glycerin.

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