CN113462770B

CN113462770B - Chemotactic factor as molecular marker for diagnosing rosacea

Info

Publication number: CN113462770B
Application number: CN202110978624.0A
Authority: CN
Inventors: 孙艳; 吴严; 陈良宏; 乔帅; 肖碧环; 王敬玉; 褚海涛; 程铭; 高兴华
Original assignee: First Hospital of China Medical University
Current assignee: First Hospital of China Medical University
Priority date: 2021-08-25
Filing date: 2021-08-25
Publication date: 2023-03-31
Anticipated expiration: 2041-08-25
Also published as: CN113462770A

Abstract

The invention belongs to the field of biological medicines, and particularly relates to application of a chemokine serving as a rose acne related molecular marker in diagnosis and treatment of the rose acne related molecular marker. LL-37 induces the increased expression of TLR2 and chemokines CCL5, CXCL9, CXCL10 and CXCL11 in rosacea animals, and the AhR agonist benvynimod can reduce erythema and inflammatory cell infiltration and reduce the expression of TLR2 and the chemokines. LL-37 induces the expression of TLR2 and chemokines CCL5, CXCL9, CXCL10 and CXCL11 in the rosacea cell model, and the AhR agonist, i.e. the iguratimod, can reduce the expression of TLR2 and the chemokines; up-regulation of TLR2 may cause increased expression of the chemokine in the cell model, while the AhR agonist siponimod may decrease expression of the chemokine by down-regulating TLR2 expression. The chemokines CCL5, CXCL9, CXCL10 and CXCL11 are expected to become new diagnostic indexes or treatment targets of the rosacea disease.

Description

Chemotactic factor as molecular marker for diagnosing rosacea

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to application of a chemotactic factor as a molecular marker related to rosacea in diagnosis and treatment.

Background

Chemokines are secreted proteins with small molecular weights (8-14 kd), have chemotactic activities, and play an important role in the maintenance of immune homeostasis and in inflammatory response. Chemokines are divided into 4 subtypes, including CC, CXC, C and CX3C, based on structural differences and exert biological effects by interacting with cognate cell surface chemokine receptors. About 50 kinds of chemokines are found at present, are mainly involved in development, differentiation and migration of leukocytes, and also play important roles in inflammatory diseases, angiogenesis and wound repair.

The aromatic hydrocarbon receptor (AhR) can bind to polycyclic aromatic hydrocarbons, dioxins, and the like in the environment, and causes oxidative stress by generating a large number of reactive oxygen species. AhR is also a receptor for many endogenous and exogenous ligands such as phototrops, phytochemicals, microbial byproducts, etc., and many AhR ligands can produce an antioxidant effect by activating the antioxidant transcription factor NRF 2. AhR-CYP1A1 mediated oxidative stress can promote the production of various proinflammatory cytokines such as IL1, IL6 and IL 8.

Rosacea is a chronic inflammatory skin disease occurring in the middle of the face, with a global population morbidity of about 5.5%. The precise etiology and pathogenesis of the disease are unknown, and may be related to the dysfunction of innate immunity, the dysfunction of adaptive immunity, the dysfunction of neurovascular regulation and the dysfunction of skin barrier. The disease not only has certain influence on the physiology of patients due to the occurrence of the disease on the face, but also can cause serious psychological problems for some patients. The pathological manifestations of rosacea are mainly infiltration of inflammatory cells in blood vessels, hair follicles and areas around the sebaceous glands. The traditional treatment method comprises systemic application of macrolides, tetracyclines, nitroimidazoles, tretinoin and other medicaments, external application of metronidazole, azelaic acid, ivermectin and the like, and treatment of various light and laser and operation treatment. At present, the diagnosis of the rosacea lacks of specific laboratory indexes, and the rosacea is difficult to treat and easy to repeat, so that the rosacea is widely concerned by experts and scholars all the time, and a new diagnosis and treatment means needs to be developed urgently. In the early period, through bioinformatics analysis, the different genes of skin lesions of patients with rosacea and normal skin tissues, including TLR2, chemokines CCL5, CXCL9, CXCL10 and CXCL11, are found, and the expression of the genes is increased in the skin lesions of the patients with rosacea. In addition, studies have shown that TLR2 is highly expressed in skin lesions of patients with rosacea, but it is unclear whether TLR2 promotes the onset of rosacea by up-regulating the expression of the above chemokines.

Disclosure of Invention

Aiming at the fact that no specific laboratory diagnosis index exists for rosacea at present, the invention aims to provide application of a chemotactic factor as a molecular marker for rosacea diagnosis. In vitro experiments show that the AhR agonist, iguratimod, can alleviate the condition and reduce the expression of the chemokines by down-regulating the expression of TLR 2. Therefore, the chemokines CCL5, CXCL9, CXCL10 and CXCL11 are expected to become new diagnostic indexes or treatment targets of the rosacea disease.

In order to achieve the above object, the present invention provides the following technical solutions.

The invention provides an application of a detection reagent for the expression level of a chemokine gene or an encoding protein thereof in preparing a product for the auxiliary diagnosis of rosacea, which is characterized in that the chemokine comprises one or more of the following combinations: CCL5, CXCL9, CXCL10 and CXCL11.

Further, the chemokine gene or the encoded protein thereof exhibits high expression in rosacea.

Further, the product detects the expression level of chemokine genes or coding proteins thereof in a sample through reverse transcription PCR, real-time quantitative PCR, a chip, a high-throughput sequencing platform, immunohistochemical staining or enzyme-linked immunosorbent.

Furthermore, the product contains specific primers for amplifying chemokine genes, probes hybridized with chemokine gene nucleotide sequences or antibodies specifically bound with chemokine proteins.

More further, the antibody is a monoclonal antibody or a polyclonal antibody.

Further, the product is a chip, a preparation or a kit.

Further, the sample is a tissue, serum or cell.

The invention also provides application of an inhibitor of chemokine gene expression level in preparing a medicament for treating rosacea, which is characterized in that the chemokine comprises one or more of the following combinations: CCL5, CXCL9, CXCL10 and CXCL11.

Further, the inhibitor of chemokine gene expression level comprises an AhR agonist.

Further, the medicine comprises pharmaceutically acceptable carriers and/or excipients, and is prepared into an internal preparation for oral administration, or an injection for non-oral administration, or an external preparation according to a conventional method.

Compared with the prior art, the invention has the beneficial effects.

The invention firstly provides application of the chemotactic factor as a rose acne related molecular marker in diagnosis and treatment, determines the effective functions of the chemotactic factors CCL5, CXCL9, CXCL10 and CXCL11 in the aspect of diagnosing the rose acne, and determines the effective treatment function of the AhR agonist in the aspect of treating the rose acne. Fills the blank that no specific laboratory diagnosis index exists for the current rosacea, and provides a new target point for the treatment of the rosacea.

Drawings

FIG. 1 is a rough photograph of LL-37-induced rosacea-like skin rash in mice alleviated by AhR agonist.

FIG. 2.AhR agonist relieves HE staining of LL-37-induced inflammatory cell infiltration in mice.

Figure 3. Effect of ahr agonist on mouse rosacea model TLR2 and chemokines.

FIG. 4.QRT-PCR detects the expression of TLR2 and chemokines in LL-37 induced HaCaT cells.

FIG. 5 WesternBlot detects LL-37-induced expression of TLR2 and chemokines in HaCaT cells.

FIG. 6. Effect of AhR agonist on LL-37 induced TLR2 overexpression in HaCaT cells with TLR2 and chemokines.

FIG. 7. Effect of AhR agonists on LL-37-induced TLR2 overexpression in HaCaT cells and chemokines.

Detailed Description

The present invention will be described in detail with reference to specific examples. The following examples will help to understand the present invention, but they are only for illustrative purposes and the present invention is not limited to these contents. The methods of operation in the examples are conventional in the art.

Example 1 mouse experiments.

Balb/c mice female for 6-8 weeks were selected and randomly divided into control, building and treatment groups. The control group was not treated, and the model set 40. Mu.L LL-37 (320. Mu.M) was injected subcutaneously into the back to produce an animal model of rosacea at 12 h/time for 4 times; treatment group was treated with 0.5% ahr agonist this vismod, 30min after the second LL-37 injection, for 3 consecutive days; finally, pictures are taken 24 hours after the medicine is taken, and the condition of erythema is evaluated. And (3) changing under an HE staining observation mirror, and detecting the levels of TLR2 and chemokines CCL5, CXCL9, CXCL10 and CXCL11 by qRT-PCR.

1. The erythema scoring method comprises the following steps: score 1-5 (5 scores are highest) according to erythema severity; measuring the area of skin lesion erythema of the shot digital photo by using Image J software, and carrying out quantitative analysis on the area.

2. The dyeing and observation method comprises the following steps: the tissue blocks were fixed with 10% formalin at room temperature for 48h and then taken out, and dehydrated with gradient in 70%, 80%, 90%, 95%, 100% ethanol for 1h each. Taking out the tissue block, and adding wax-soaking transparent agent 1 and 2 for 2h respectively to obtain transparent tissue block. The transparent tissue block is immersed in paraffin solution for embedding. The wax block is cut into 5 mu m slices by a paraffin tissue slicer, absorbed on a glass slide and dried for 1h by a dryer at 60 ℃. Soaking the slices in 100%, 95%, 90%, 80%, and 70% ethanol in sequence for 5min respectively, and washing with tap water for 5min. Staining with hematoxylin for 1-3min, and washing with tap water for 5-10min. Eosin staining for 1-3min, and washing with tap water for 5-10min. Soaking the slices in 70%, 80%, 90%, 95%, 100%, and 100% ethanol for 10min respectively, dehydrating, soaking in wax and clearing agent 1, 2 for 10min respectively, sealing with neutral resin, and covering with glass slide. After 24h drying, the mixture was observed microscopically. 3 high power lens fields with obvious inflammatory infiltration are selected for each section to take pictures, and the number of inflammatory cells in the pictures is counted by using Image J software. Graphpad software compares plots and P <0.05 is statistically significant.

3. And (3) a PCR method.

3.1 Extracting total RNA:

(1) The mouse skin tissue specimens were thawed at room temperature and the surface embedding agent was wiped off with paper in the kitchen. Rinsing with PBS 3 times;

(2) Each sample was placed into a 1.5mL polypropylene centrifuge tube, 600. Mu.L of RLT was added to each tube and then inserted on ice, and the grinder was shaken to grind thoroughly until no visible tissue was present. Transferring the liquid in the centrifuge tube into a new centrifuge tube, adding 70% ethanol with the same volume (300-600 μ L), fully blowing, beating and mixing uniformly;

(3) mu.L of the sample was transferred to RNeasy spin column and placed in a 2mL collection tube. Centrifugation at 10000Xg for 30s discarded the liquid in the tube. If the liquid in (2) is remained, transferring all the liquid into RNeasy spin column, and repeating the operation;

(4) RW 1700. Mu.L was added to RNeasy spin column and centrifuged at 10000Xg for 30s. Washing the membrane, and discarding liquid in the tube;

(5) 500. Mu.L of RPE was added to RNeasy spin column and centrifuged at 10000Xg for 30 seconds. Washing the membrane, and discarding liquid in the tube;

(6) Adding RPE500 μ L into RNeasy spin column, and centrifuging at 10000Xg for 2min. Washing the membrane, and discarding the lower collecting pipe;

(7) The RNeasy spin column was centrifuged at full speed for 1min, the membrane was dried and the RNeasy spin column was put in a new 1.5mL collection tube, 15-50. Mu.L of RNase-free water was added to the membrane and centrifuged at 10000Xg for 1min. RNeasy spin column was discarded, leaving 1.5mL collection tube;

(8) Determination of RNA concentration: ND-1000-nucleic acid (DNA, RNA) -1. Mu.L of RNase-free water-ok-wiping-1. Mu.L of RNase-free water (zero-set) -RNA-40-BLANK (0.0 ng/mL) -1. Mu.L of sample-measure-preservation (-80 ℃).

3.2 A reverse transcription step:

(1) Preparation of a reaction system 1:

；

(2) Preparation of a reaction system 2:

the required volume is calculated from the sample concentration. (sample loading amount 1. Mu.g, reaction system 20. Mu.L)

RNA+H ₂ O=8μL；

(3) Reaction conditions are as follows: 42 ℃ for 15 min; 15min at 70 ℃;4 ℃ and infinity.

3.3 qPCR reaction system and reaction conditions (384 well plate, reaction system 10 μ L):

(1) The PCR primer sequence:

；

(2) Primer dilution:

centrifuging a primer test tube at 10000xg for 30s, respectively adding different amounts of dd water according to the instruction to prepare storage solution (100 mu mol/L), storing for a long time at 20 ℃ and diluting to 10 mu mol/L before use;

(3) Preparation of a reaction system 1:

；

(4) Preparation of a reaction system 2:

；

(5) The reaction conditions are as follows:

at 95 ℃ for 2 min; (95 ℃,15 s, 60 ℃,1 min) X40 cycles;

dissolution curves were added for each index at the first run.

3.4 data processing:

and (3) leading the CT values of different indexes of each sample to an Excel table, and respectively calculating the average value of the target gene and the reference gene of each index of each sample. Calculated according to the following formula:

rCT = CT value of target gene-CT value of reference gene

rrCT = treatment group rCT-control group rCT

Gene expression level =2 ^-rrCT 。

3.5 Experimental results for the effect of AhR agonists on chemokines in an animal model of rosacea.

(1) General observation: in general, the model group showed localized skin erythema with roseo-acneiform skin rash, while the treatment group reduced erythema and reduced erythema area (fig. 1). It was suggested that rosacea-like skin rash can occur in the rosacea animal model, and that AhR agonists can alleviate this.

(2) Pathological changes: HE staining suggested increased dermal and subdermal tissue inflammatory cell infiltration in the model and that the treatment group could alleviate this condition (figure 2). Inflammatory cell infiltration was suggested in an animal model of rosacea, and AhR agonists alleviated this.

(3) Alterations in TLR2 and chemokines: the qRT-PCR results indicate that the expressions of TLR2 and chemotactic factors CCL5, CXCL9, CXCL10 and CXCL11 in the skin lesion of the model are all increased, and the difference has statistical significance (P < 0.05). Compared with the model group, the treatment group can inhibit the expression of TLR2 and chemokines CCL5, CXCL9, CXCL10 and CXCL11 (P <0.05, figure 3). The expression of TLR2 and chemokines CCL5, CXCL9, CXCL10 and CXCL11 in the rose acne animal model is increased, and the AhR agonist can inhibit the increase of the indexes.

Example 2 cell experiments.

LL-37 was added to HaCaT cells to create a model of acne rosacea cells, followed by treatment with the AhR agonist, benvyimod. Detecting the levels of TLR2 and chemokines CCL5, CXCL9, CXCL10 and CXCL11 by using qRT-PCR and WesternBlot/ELISA methods; lentivirus transfection over-expresses TLR2, and the levels of chemokines CCL5, CXCL9, CXCL10 and CXCL11 are detected.

1. Cell culture:

HaCaT cells were cultured in DMEM medium containing 10% FBS and 1% streptomycin double antibody, and placed in a medium containing 5% CO ₂ At 37 ℃ in a humidified incubator. Changing the culture solution for 1 time in 1-2 days, observing the growth condition of the cells under a microscope, and passaging when the coverage rate reaches 80-90%. Operating in the cell cleaning station, the vial/dish is first aspirated by pipette suspension, and the cells are rinsed with 2mL of sterile PBS for a total of 2 washes. Then, the product is processedAdding 1-2mL pancreatin into culture flask, heating at 37 deg.C and 5% CO ₂ Incubating in incubator for 5-10min. Digestion was stopped when the cells became round and bright. Equal amounts of culture medium were added to the flasks to stop digestion. The digested cells were transferred to a centrifuge tube and centrifuged at 1200r/min at 37 ℃ for 3min. The supernatant from the centrifuge tube was discarded, and the cells in the lower layer were left, added with an appropriate amount of medium, thoroughly aspirated and mixed, and passaged at a ratio of 1. When the cells are frozen, the frozen stock solution is FBS, 10% DMSO is added, and the cells are stored in a refrigerator at minus 80 ℃ in a gradient cooling manner.

2. TLR2 and chemokine levels were detected.

2.1 Cell processing

The HaCaT cells were plated at 5x 10 ⁵ One/well was seeded in 6-well plates. After the cells are cultured for 24 hours, LL-37 with different concentrations and time is added, and after a period of action, the Vitinomod with different concentrations is added for treatment. And extracting RNA at different time points for qRT-PCR detection.

2.2 Extraction of Total RNA

The liquid in the well is sucked and flicked against the wall of the plate to loosen the cells. Adding 350 μ L of RLT (3-5 min) into each well, thoroughly pipetting, mixing, transferring into polypropylene centrifuge tube, adding 70% ethanol with equal volume (350 μ L), and thoroughly pipetting. The procedure is as in 3.1 of example 1.

2.3 reverse transcription:

the procedure is as in 3.2 of example 1.

2.4 qRT-PCR

The method is the same as 3.3 in example 1.

2.5 Data processing

The procedure is as in 3.4 of example 1.

3. Detection of TLR2 level by Western Blot

3.1 cell treatment

HaCaT cells were cultured at 5X 10 ⁵ One/well was seeded in 6-well plates. After the cells are cultured for 24 hours, LL-37 with proper concentration and time and the vacuummod are added in sequence for treatment. And extracting protein and carrying out Western Blot detection.

3.2 Preparation of lysate:

per 100 μ L lysate: 98. Mu.L of RIPA + 1. Mu.L of PMSF + 1. Mu.L of protease inhibitor (6-well plate: 60-100. Mu.L/well).

3.3 extraction of Total cellular proteins (the whole procedure was performed on ice as much as possible):

(1) Completely sucking the culture solution, and washing with precooled PBS for 2 times;

(2) Sucking PBS, adding lysis solution (ice surface lysis for 15-30min, and precooling with centrifuge);

(3) Respectively scraping off the protein scrapers and sucking the protein into a precooled EP tube;

(4) 15000rpm,15-30min,4 deg.C, and collecting supernatant.

3.4 Measurement of protein concentration

(1) Preparing a protein standard: adding 10 μ L protein standard into 90 μ L LRIPA to obtain standard (concentration of 0.5 mg/mL), and storing at 20 deg.C in refrigerator;

(2) The standards were loaded into 96-well plates according to the following table, and 20. Mu.L/well (2. Mu.L sample + 18. Mu.LRIPA) was added after 10-fold dilution of the samples. Preparing a standard curve;

；

(3) Add 200. Mu.L BCA working solution per well (A: B =50, stable within 24 hours), blow well with a pipette, mix well, incubate for 20-30min at 37 ℃;

(4) Detecting the absorbance at 562nm at 37 ℃ by using an enzyme labeling instrument, and recording data;

(5) Drawing a standard curve by using the concentration of the protein standard substance and the corresponding absorbance, calculating the protein concentration of each sample through a regression equation, and multiplying by 10 to obtain the concentration;

(6) Protein samples were made up to 1 μ g/μ L (100 μ g, 100 μ L), protein + RIPA =80 μ L, and trimmed with a loading buffer (5 ×);

(7) Boiling at 100 deg.C for 5-10min, rapidly cooling to 4 deg.C for more than 5min, and storing at-80 deg.C without further test.

3.5 SDS-PAGE electrophoresis

(1) Cleaning a glass plate, a comb (1.0 mm/1.5mm,10 holes/15 holes) and an electrophoresis tank, sucking surface water with kitchen paper, and drying at room temperature;

(2) After the glass plate is dried, the bottom edges are aligned and clamped on the glue making frame. Adding dd water into the glass plate, standing, and observing whether the glass plate leaks;

(3) And (5) preparing separation gel. Selecting polyacrylamide gel with corresponding concentration according to the molecular weight of the target protein. 1.0 mm. When in preparation, TEMED (solidification) is not added temporarily;

3 mL A +3 mL B +30 μ L10% APS +3 μ L TEMED (1 plate);

(4) Observing the glass plate, pouring out the water after the glass plate is free from liquid leakage, and sucking the water by using filter paper;

(5) And adding TEMED into the separation glue prepared in the front, fully blowing, beating and uniformly mixing, and quickly pouring glue along one side of the glass plate. Sealing the upper layer with ethanol to promote gel polymerization;

(6) And when the separation gel is solidified, preparing the concentrated gel. (again, TEMED was not added first);

1 mL A +1 mL B +10 μ L10% APS +2 μ L TEMED (1 plate);

(7) When an obvious limit appears between the separation gel and the ethanol, the separation gel is solidified, the ethanol is poured out, and the paper for kitchen is sucked dry;

(8) Filling the concentrated glue, and inserting a 1.0mm comb after filling;

(9) Preparing an electrophoresis solution when waiting for the solidification process of the concentrated gel;

the formula of the electrophoretic fluid is as follows: 200 mL 5 XTris Glycine electrophoresis buffer +800 mL dd H ₂ O =1L, shake up;

(10) After the concentrated gel is solidified, the glass plate is taken down from the gel making frame and is placed into an electrophoresis tank, the electrophoresis liquid is filled, and a comb is pulled out (vertical, slow). After the comb is pulled out, the bubbles in the sample loading hole are expelled by electrophoretic liquid;

(11) Before loading, the samples were centrifuged by shaking. And (4) loading, recording and replacing a gun head, markers on two sides and a middle sample. Balancing the vacant holes by a marker;

(12) And filling the electrophoresis tank with electrophoresis liquid and performing electrophoresis after the sample loading is finished. Conditions are as follows: 100 V (about 10 min), which is changed to 150V (about 1 h) after the sample is aligned, and attention is paid to low current caused by poor contact;

(13) And in the electrophoresis process, making ice and preparing a transfer liquid. The formula of the transfer printing liquid is as follows: 100 mL 10X electrotransfer +200 mL methanol +700 mL dd water =1L, mix well. And (3) soaking a plastic plate, filter paper and sponge in the transfer solution soon after electrophoresis is finished, washing the plate for cutting the gel, and drying. Taking ice;

(14) And after electrophoresis, taking out the glass plate, washing with small water, opening the glass plate, and cutting the gel. Transferring the cut glue into a transfer printing liquid;

(15) Using a ruler to measure the glue, cutting the PVDF membrane, and marking the name at the upper right corner to distinguish the proteins;

(16) Soaking the membrane in methanol for 30s, and transferring the membrane into a transfer liquid;

(17) After the film is completely immersed into the transfer liquid, making a sandwich (black-sponge-filter paper (new) -glue-film-filter paper-sponge-black) (from bottom to top);

(18) The transfer printing plate is placed in a transfer printing groove, and black is used for black and white is used for red. The transfer process was performed on ice. 200 mA,30-50 kd:30-40 minutes; 70-90kd:60min; >130kd:90min;

(19) During the transfer process, collecting articles, cleaning the glass plate and drying;

(20) After the transfer printing is finished, taking out the membrane, cutting a small opening at the upper left corner of the membrane to distinguish the front side from the back side, sealing the membrane for 30 minutes by using an antigen sealing liquid, and performing the sealing process on a shaking table;

(21) In the sealing process, the sealing box is taken and matched with primary antibody, the proportion of the primary antibody is referred to the specification, and the primary antibody is prepared by using primary antibody diluent. Internal reference GAPDH (35.9 KD), 1;

(22) The blocking solution was decanted off, washed with TBST for 10min 3 times, and primary antibody was added to drive out the air bubbles. 4 ℃ overnight, typically 16 hours;

(23) Taking out the membrane on the next day, and shaking on a shaking bed for about 15-30 min;

(24) Recovering primary antibody, washing with TBST for 3 times, and washing for 10 min/time;

(25) A secondary antibody, 1;

(26) Adding a secondary antibody. Shaking on a shaking bed for 1-2h, generally one and a half hours;

(27) Recovering the secondary antibody, and washing for 3 times and 10 min/time by PBST;

(28) Preparing luminous liquid (prepared at present), wherein A + B liquid 1:1. generally, one film needs 200-300 μ L of luminous liquid;

(29) The ECL gel imaging system glows and takes a picture.

4. ELISA for chemokine levels (CXCL 9 as an example, operating according to the instructions)

(1) Collecting samples: carefully collecting cell supernatant in 3.1, immediately centrifuging at 4 deg.C, 10000rpm,20min, and preserving at-80 deg.C;

(2) And (3) configuring a standard product: the reagent kit was prepared as 20000pg/mL stock solution with dd water added to Human MIG Standard. Gently shaking for at least 15min to dissolve it completely, and diluting. 100 u L stock solution +900 u LRD5P, after mixing, suction 500 u L to the next tube, adding 500 u LRD5P dilution. Performing gradient dilution, and sequentially preparing 2000, 1000, 500, 250, 125, 62.5, 31.3 and 0pg/mL of standard substances;

(3) Adding 100 mu L of RD1W into each hole;

(4) Adding 100 μ L of standard or sample (2 multiple wells for standard and 3 multiple wells for sample) into each well, covering with film, and incubating at room temperature for 2 hr;

(5) The liquid in the wells was aspirated and washed 4 times with Wash Buffer (400. Mu.L/well). Care is taken to suck all the liquid out of the hole each time. The 96-well plate is inverted and patted dry on a paper towel;

(6) Adding 200 mu L of Human MIG Conjugate into each well, covering a film, and incubating for 2h at room temperature;

(7) Repeating the step in 5), and washing for 4 times;

(8) Adding 200 mu L of Substrate Solution into each hole, incubating for 30min at room temperature, and keeping out of the sun;

(9) Adding 50 mu L of stop solution into each hole;

(10) Detecting the absorbances at 540nm and 450nm by a spectrophotometer within 30 minutes;

(11) And exporting the absorbances of the standard substance and the sample into an Excel table, drawing a standard curve according to the absorbances corresponding to the standard substances with different concentrations, and calculating the concentrations of the different samples by using a multiple regression method.

5. Lentiviral transfection

The company provided the following virus and control concentrations:

HaCaT cells were transfected at MOI =100 according to the instructions and preliminary experiments. Virus dose = number of cells + MOI/virus concentration. HaCaT cells were cultured at 5X 10 ⁴ Density per well was inoculated into 12-well plates and the concentration was 5% CO at 37% ₂ Was incubated in the incubator for 24h. Respectively infecting HaCaT cells with TLR2 over-expressed lentivirus (LV-TLR 2) or empty vector (LV-null), (500 mu L system containing 20 mu L infection enhancing fluid P), changing fluid after 12-16 h, taking pictures by an inverted fluorescence microscope after 48-72h, and transferring to a culture flask for expanding culture after trypsinization. The growth of the cells was observed by regular replacement of the medium with DMEM containing 1ug/mL puromycin.

6. Statistical analysis

All measurements were expressed as mean. + -. SEM and quantified using Image J1.52 a software. Statistical analysis was performed using a one-way analysis of variance (ANOVA) and multiple comparisons with Graphpad Prism 7.0a software, with P <0.05 being statistically significant for differences.

Experimental results for the effect of agonists on chemokines in the rosacea cell model.

(1) qRT-PCR detection of the expression of TLR2 and chemokines in LL-37-induced HaCaT cells: after 8 mu MLL-37 acts on HaCaT cells for 24h, the HaCaT cells are respectively treated by 5, 10 and 20 mu MAhR agonist vacuummod, RNA is respectively extracted after 4, 8, 12 and 24h, and qRT-PCR is used for detecting the levels of TLR2 and chemokines CCL5, CXCL9, CXCL10 and CXCL11. The results show that after 8h of action of the AhR agonist, the LL-37-induced expression levels of genes of TLR2 and chemokines CCL5, CXCL9, CXCL10 and CXCL11 in HaCaT cells are all increased, and the AhR agonist can relieve the situation, and the difference has statistical significance (P < 0.05). 5. The effect of 10 μ M of this vismod relief was most pronounced (P < 0.05) (fig. 4). Therefore, we chose 10 μ M of this vitamin mod for the subsequent experiments. The gene level prompts that the expressions of TLR2 and chemokines CCL5, CXCL9, CXCL10 and CXCL11 in a rose acne cell model are all increased, and the AhR agonist can inhibit the expressions of TLR2 and the chemokines.

(2) WesternBlot and ELISA detect the expression of TLR2 and chemokines in LL-37-induced HaCaT cells: after 8 mu MLL-37 acts on HaCaT cells for 24h, 10 mu MAhR agonist is added to treat the HaCaT cells for 24h, cell extract protein WesternBlot detects the level of TLR2, and cell supernatant ELISA detects the level of the chemotactic factors. The results show that TLR2 and the chemokines CCL5, CXCL9, CXCL10, CXCL11 protein level expression are all elevated in LL-37 induced HaCaT cells, and this condition can be alleviated by sumitomod with statistically significant differences (P < 0.05) (fig. 5). The protein level indicates that the expressions of TLR2 and chemokines CCL5, CXCL9, CXCL10 and CXCL11 in the rosacea cell model are all increased, and the AhR agonist can inhibit the expressions of TLR2 and the chemokines.

(3) Effect of AhR agonists on TLR2 and chemokines in LL-37-induced HaCaT cells overexpressing TLR 2.

(1) qRT-PCR for TLR2 and chemokine levels: 8 mu MLL-37 acts on HaCaT cells transfected by lentivirus for 8h, then RNA is extracted and qRT-PCR is carried out to detect the level of TLR2 and chemokines CCL5, CXCL9, CXCL10 and CXCL11, and the result shows that the expression of the chemokines is increased after TLR2 is over-expressed, while the yivinmod can inhibit the expression of the chemokines by down-regulating TLR2 (figure 6). It is suggested that in the rosacea cell model, TLR2 can promote the expression of chemokines CCL5, CXCL9, CXCL10 and CXCL11 at the gene level, and AhR agonists can inhibit the expression of the chemokines by down-regulating TLR 2.

(2) WesternBlot and ELISA detect TLR2 and chemokine levels: 8 mu MLL-37 was applied to Lentiviral transfected HaCaT cells 24h and cell extract WesternBlot was used to measure TLR2 levels and cell supernatant ELISA was used to measure the above chemokine levels. The results show that the expression of the chemokines CCL5, CXCL9, CXCL10, CXCL11 is increased after TLR2 overexpression, and the protein level suggests that TLR2 can promote the expression of the chemokines CCL5, CXCL9, CXCL10, CXCL11 (fig. 7). It is suggested that in the rosacea cell model, TLR2 can promote the expression of chemokines CCL5, CXCL9, CXCL10 and CXCL11 at the protein level, and AhR agonist can inhibit the expression of the chemokines by down-regulating TLR 2.

In a word, the research result indicates that after the LL-37 induction, the mice have rosacea-like skin rash, the inflammatory cell infiltration is increased, the expressions of TLR2 and chemokines CCL5, CXCL9, CXCL10 and CXCL11 are increased, and the AhR agonist iguimod can relieve the condition. On a cellular level, after LL-37 induction, the expressions of TLR2 and chemokines CCL5, CXCL9, CXCL10 and CXCL11 are increased, and the AhR agonist siponimod can relieve the condition and reduce the expression of the chemokines by down-regulating the expression of TLR 2. The chemokines CCL5, CXCL9, CXCL10 and CXCL11 are expected to become new diagnostic indexes or therapeutic targets of the rosacea disease.

Claims

Use of an AhR agonist in the manufacture of a medicament for the treatment of rosacea, wherein the AhR agonist is yivinmod; the drug acts by inhibiting the expression of the chemokines CCL5, CXCL9, CXCL10 and CXCL11.
2. The use of an AhR agonist according to claim 1 in the preparation of a medicament for the treatment of rosacea, wherein the medicament comprises a pharmaceutically acceptable carrier and/or excipient in a form for topical administration.