Application of secretory glucose regulatory protein 78 in preparation of medicine for treating allergic contact dermatitis

Abstract

The invention belongs to the technical field of biological medicines, discloses an application of secreting type glucose regulatory protein 78 in preparation of a medicine for treating allergic contact dermatitis, and proves that the secreting type Grp78 protein reduces inflammatory reaction by inducing macrophage M2 polarization and reducing expression of cytokines IL-6, INF-gamma and TNF-alpha, so that symptoms of ACD are improved, and the application of the secreting type Grp78 protein in preparation of the medicine for treating allergic contact dermatitis is promoted. And a new theoretical basis and a new technical method are provided for treatment of ACD and exploration of novel drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of secreted glucose-regulated protein 78 in the preparation of drugs for treating allergic contact dermatitis. Background Art

[0002] Allergic Contact Dermatitis (ACD) is an inflammatory skin disease caused by various substances contacting the skin, belonging to delayed-type hypersensitivity. Patients often show symptoms such as itching, erythema, rash, and in severe cases, blisters or exudate may occur. The allergens of ACD are very extensive, including metals, spices, preservatives, hair dyes, pollen, dyes, and various industrial chemicals. The prevalence of ACD in the general population is as high as 20%, and the incidence rate is even higher in occupational populations (such as the medical, chemical, construction industries, etc.). When ACD is mild, it will cause discomfort, and when severe, it will affect daily life and working ability. Currently, the main treatments for ACD at home and abroad are topical corticosteroids, topical calcineurin inhibitors, systemic immunosuppressants, and phototherapy. However, long-term use of corticosteroids, calcineurin inhibitors, and systemic immunosuppressants has adverse reactions such as skin atrophy, telangiectasia, skin striae, burning sensation and stinging sensation at the application site, liver and kidney function damage, metabolic disorders, and increased risk of infection. Therefore, it is of great significance to find new drugs for treating ACD.

[0003] Secreted glucose-regulated protein 78 (secreted Grp78 protein) is a stress-induced molecular chaperone mainly present in the endoplasmic reticulum and plays a very important role in maintaining intracellular protein homeostasis. Secreted Grp78 can exert its anti-inflammatory properties through multiple mechanisms such as inhibiting the expression of inflammatory factors, regulating the function of immune cells, promoting the resolution of inflammation, and protecting cells from stress damage.

[0004] The application of secreted Grp78 protein in enteritis, tumors, and autoimmune diseases has been reported, but there is currently no study proving the application of secreted Grp78 protein in ACD. Summary of the Invention

[0005] In view of the current many side effects and adverse reactions in the treatment of allergic contact dermatitis (ACD), the present invention provides the application of secreted glucose-regulated protein 78 in the preparation of drugs for treating allergic contact dermatitis. The present invention has confirmed that secreted Grp78 protein can improve the symptoms of ACD by inducing macrophage M2 polarization, reducing the expression of cytokines IL-6, INF-γ, and TNF-α, and reducing the inflammatory response, providing a new theoretical basis and technical method for the treatment of ACD and the exploration of new drugs.

[0006] To achieve the above object, the present application adopts the following technical solutions:

[0007] In a first aspect, the present invention provides an application of a secreted glucose-regulated protein 78 (secreted Grp78 protein) in the preparation of a drug for treating allergic contact dermatitis.

[0008] In the above technical solution, the secreted Grp78 protein induces macrophage M2 polarization, reduces the expression of cytokines IL-6, INF-γ, and TNF-α, and reduces the inflammatory response.

[0009] In the above technical solution, the preparation method of the secreted Grp78 protein includes the following steps:

[0010] S1. Transform the plasmid encoding the full length of mouse Grp78 into BL21 Escherichia coli to produce glutathione-S-transferase-Grp78;

[0011] S2. Purify the glutathione-S-transferase-Grp78 using a GST purification column, and further use thrombin to digest the GST tag to obtain a GST-tag-free purified protein;

[0012] S3. Remove endotoxin in the GST-tag-free purified protein through a high-capacity endotoxin removal column to obtain the secreted Grp78 protein.

[0013] In a second aspect, the present invention provides an application of a secreted Grp78 protein in the preparation of a drug for inducing macrophage M2 polarization, reducing the expression of cytokines IL-6, INF-γ, and TNF-α, or reducing the inflammatory response.

[0014] In a third aspect, the present invention provides a drug for treating allergic contact dermatitis, which consists of a secreted Grp78 protein at an effective dose, and can be a single component or a composition containing an effective dose of the secreted Grp78 protein. The composition may include a pharmaceutically acceptable carrier.

[0015] In a fourth aspect, the present invention provides a preparation method of a secreted Grp78 protein, including the following steps:

[0016] S1. Transform the plasmid encoding the full length of mouse Grp78 into BL21 Escherichia coli to produce glutathione-S-transferase-Grp78;

[0017] S2. Purify the glutathione-S-transferase-Grp78 using a GST purification column, and further use thrombin to digest the GST tag to obtain a GST-tag-free purified protein;

[0018] S3. Remove endotoxin from the GST-tag-free purified protein through a high-capacity endotoxin removal column, thereby obtaining the secreted Grp78 protein.

[0019] In the above technical solution, it further includes

[0020] S4. Identify the secreted Grp78 protein by sodium dodecyl sulfate polyacrylamide gel electrophoresis.

[0021] The beneficial effects of the present invention are as follows:

[0022] (1) Efficient protein preparation technology

[0023] The present invention discloses a method for preparing secreted Grp78 protein with high efficiency in expression, purification and endotoxin removal. The prepared protein has high purity, strong activity and low endotoxin level (<2.5 EU / mg), meeting the requirements of subsequent therapeutic research.

[0024] (2) Innovative treatment strategy

[0025] The present invention discloses for the first time the application of secreted Grp78 in the treatment of ACD. Secreted Grp78 can effectively reduce the expression of inflammatory factors IL-6, IFN-γ and TNF-α by inducing M2 polarization of macrophages, thereby significantly improving the symptoms of ACD and providing a new immunomodulatory pathway for the treatment of ACD.

[0026] (3) Significant anti-inflammatory effect

[0027] Experiments have confirmed that the secreted Grp78 protein can effectively relieve the symptoms of ACD, significantly reduce the ear swelling degree of mice, and improve pathological changes.

[0028] (4) Higher safety

[0029] The secreted Grp78 protein purified and endotoxin-removed does not show obvious side effects during the treatment process. Compared with traditional immunosuppressants or glucocorticoids, it has better safety and applicability.

[0030] (5) Broad application prospects

[0031] This method and technical solution can not only be applied to the treatment research of ACD, but also provide a new theoretical basis and experimental method for the treatment development of other immune-related diseases (such as rheumatoid arthritis, inflammatory bowel disease, etc.).

[0032] (6) Drug development potential

[0033] The present invention provides a clear direction for the further development and application of the secreted Grp78 protein, has the potential to be used as a new anti-inflammatory drug, and provides the possibility for clinical translation and personalized treatment. Brief Description of the Drawings

[0034] Figure 1 It is the partial sequencing result of the secreted Grp78 of the present invention.

[0035] Figure 2 It is the result of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the secreted Grp78 protein of the present invention.

[0036] Figure 3 It is the standard curve of the concentration of the secreted Grp78 protein of the present invention.

[0037] Figure 4 It is the ACD model establishment and experimental grouping of the present invention. Normal group: No treatment was given. On day 1, acetone + olive oil was applied to the abdomen, and on day 6, the matrix was applied to the left and right ears. Model group: Model establishment + continuous intraperitoneal injection of 200 μl of normal saline for 5 days from day 2 to day 6. On day 1 and day 2, 0.5% DNFB was applied to the abdomen, and on day 6, 0.2% DNFB was applied to the right ear and the matrix was applied to the left ear. Grp78 treatment group: Model establishment + continuous intraperitoneal injection of Grp78 (10 μg / g) for 5 days from day 2 to day 6. On day 1 and day 2, 0.5% DNFB was applied to the abdomen, and on day 6, 0.2% DNFB was applied to the right ear and the matrix was applied to the left ear. Dexamethasone treatment group: Model establishment + intraperitoneal injection of dexamethasone (5 mg / kg) on day 6. On day 1 and day 2, 0.5% DNFB was applied to the abdomen, and on day 6, 0.2% DNFB was applied to the right ear and the matrix was applied to the left ear.

[0038] Figure 5 It is the evaluation of ear swelling degree in the ACD treatment of different treatment groups of the present invention; where A: Ear swelling degree (μm) = Thickness of the right ear after stimulation - Thickness of the right ear before stimulation; B: Ear swelling degree (%) = (Thickness of the right ear after stimulation - Thickness of the right ear before stimulation) / Thickness of the right ear before stimulation * 100%; C: Ear swelling degree (μm) = Thickness of the right ear after stimulation - Thickness of the left ear after stimulation; D: Ear swelling degree (μm) = (Thickness of the right ear after stimulation - Thickness of the left ear after stimulation) - (Thickness of the right ear before stimulation - Thickness of the left ear before stimulation); n = 5 for all groups, ***p < 0.001; **p < 0.01; *p < 0.05.

[0039] Figure 6 It is the ear tissue pathological result in the ACD treatment of different treatment groups of the present invention; where A: Normal group; B: Model group; C: Grp treatment group; D: Dexamethasone treatment group; n = 5 for all groups.

[0040] Figure 7CBA detection results of ear tissues in different treatment groups during ACD treatment in the present invention; where A: IL-6 expression level; B: IFN-γ expression level; C: TNF-α expression level; all groups n = 5, ***p < 0.001; **p < 0.01; *p < 0.05.

[0041] Figure 8 CBA detection results of blood in different treatment groups during ACD treatment in the present invention; where A: IL-6 expression level; B: IFN-γ expression level; C: TNF-α expression level; all groups n = 5, ***p < 0.001; **p < 0.01; *p < 0.05.

[0042] Figure 9 Phenotype results of DC cells and macrophages in ascites during ACD treatment in different treatment groups in the present invention; where A: percentage of DC cells in ascites; B: percentage of CD80 + DC cells in ascites; C: percentage of macrophages in ascites; D: CD80 + percentage of M cells in ascites; E: CD206 + percentage of M cells in ascites; all groups n = 5, ***p < 0.001; **p < 0.01; *p < 0.05. Detailed implementation manners

[0043] To better illustrate the purpose, technical solution and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. The present invention can be implemented in many different forms and should not be construed as limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the present invention to those skilled in the art. The present invention will be defined only by the claims.

[0044] Research idea of the present invention: First, prepare secreted Grp78 protein by oneself, then design different experimental groups through mouse animal experiments to explore the effect of secreted Grp78 in ACD treatment, and finally explore the mechanism of secreted Grp78 protein in ACD treatment by CBA and flow cytometry.

[0045] The present invention provides a method for preparing secreted Grp78 protein, including the steps of expression, purification and endotoxin removal, specifically:

[0046] 1. Prepare secreted Grp78 protein: Transform the plasmid encoding the full length of mouse Grp78 into BL21 Escherichia coli to produce glutathione-S-transferase (GST)-Grp78.

[0047] 2. Purify the secreted Grp78 protein: Use a GST purification column to purify the secreted Grp78 protein, and further use thrombin to cleave the GST tag to obtain a purified protein without the GST tag.

[0048] 3. Remove endotoxin from the secreted Grp78 protein: Use a high-capacity endotoxin removal column to remove endotoxin from the secreted Grp78 protein, and the final concentration of endotoxin in the protein is < 2.5 EU / ml.

[0049] 4. Identify the secreted Grp78 protein: Use Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) to identify the secreted Grp78 protein.

[0050] 5. Detect the concentration of the secreted Grp78 protein: Use a protein quantification kit based on the bicinchoninic acid assay to detect the concentration of the secreted Grp78 protein, draw a standard curve, and calculate the protein concentration.

[0051] The present invention also provides an application of the secreted Grp78 protein in allergic contact dermatitis (ACD). The specific research method is as follows:

[0052] 1. Establish an ACD model: Apply 0.5% dinitrofluorobenzene (DNFB) to the abdomen and ears of female BALB / c mice for induction and challenge respectively to establish an ACD model.

[0053] 2. Experimental design:

[0054] (1) Normal group: No treatment was given. On day 1, apply acetone + olive oil to the abdomen, and apply the matrix to the left and right ears around day 6.

[0055] (2) Model group: Establish the model and intraperitoneally inject 200 μl of normal saline continuously for 5 days from day 2 to day 6. Apply 0.5% DNFB to the abdomen on days 1 and 2, and apply 0.2% DNFB to the right ear and the matrix to the left ear on day 6.

[0056] (3) Grp78 treatment group: Establish the model and intraperitoneally inject Grp78 (10 μg / g) continuously for 5 days from day 2 to day 6. Apply 0.5% DNFB to the abdomen on days 1 and 2, and apply 0.2% DNFB to the right ear and the matrix to the left ear on day 6.

[0057] (4) Dexamethasone treatment group: Establish the model and intraperitoneally inject dexamethasone (5 mg / kg) on day 6. Apply 0.5% DNFB to the abdomen on days 1 and 2, and apply 0.2% DNFB to the right ear and the matrix to the left ear on day 6.

[0058] 3. Evaluation of the effect of secreted Grp78 protein in the treatment of ACD:

[0059] (1) Ear swelling degree: Measure the ear thickness of mice before and after stimulation, and calculate the ear swelling degree by four different methods.

[0060] (2) Histological detection: Observe the pathological changes of the ears of mice in different groups through HE staining of pathological sections.

[0061] 4. Explore the mechanism of secreted Grp78 protein in the treatment of ACD

[0062] (1) Use the Cytometric Bead Array (CBA) technology to detect the expression levels of cytokines such as IL-6, IFN-γ, and TNF-α in the ear tissues of mice.

[0063] (2) Use the CBA technology to detect the expression levels of cytokines such as IL-6, IFN-γ, and TNF-α in the blood of mice.

[0064] (3) Use flow cytometry to detect the phenotypes of dendritic cells (DC) and macrophages (M).

[0065] The secreted Grp78 protein prepared by the present invention has high purity, strong activity, and a low endotoxin level (<2.5 EU / mg), meeting the requirements of treatment research, and for the first time, the secreted Grp78 is applied to the treatment of contact dermatitis (ACD).

[0066] The present invention confirms through animal experiments that secreted Grp78 can significantly reduce the expression of inflammatory factors IL-6, IFN-γ, and TNF-α by inducing the polarization of macrophage M2 type, thereby alleviating the symptoms of ACD, reducing ear swelling, and improving pathological changes, showing a significant anti-inflammatory effect.

[0067] Secreted Grp78 has a good anti-inflammatory effect, can effectively improve the symptoms of ACD, can be used as an active ingredient for drugs for ACD, lays a theoretical foundation for the treatment of ACD, and provides a new direction for the research and clinical transformation of new drugs for ACD.

[0068] The preparation method and application of the secreted Grp78 protein of the present invention will be described in detail below through specific examples.

[0069] Example 1

[0070] This example relates to the preparation of secreted Grp78 protein, including the steps of expression, purification, and endotoxin removal, as follows:

[0071] 1. Preparation of secreted Grp78 protein:

[0072] Extract the plasmid PGEX-4T-3-GPR78 encoding the full length of Grp78, transform it into Escherichia coli BL-21, and select monoclonal colonies for expansion culture. Further inoculate the bacterial solution into LB medium containing 100 μg / ml ampicillin, culture overnight at 37 °C, and then add ampicillin and the overnight cultured bacterial solution to fresh medium and culture at 37 °C for 3 hours. Add isopropyl-β-D-thiogalactoside for induction of expression, collect the bacterial solution after culturing at 25 °C for 7 hours, centrifuge to obtain cell pellets, resuspend with pre-cooled phosphate buffer, add lysozyme to lyse the cells, centrifuge after ultrasonic disruption, and collect the supernatant to obtain secreted Grp78 protein for subsequent purification.

[0073] 2. Purification of secreted Grp78 protein:

[0074] Use a GST purification column (16105; Thermo Fisher scientific, Waltham, MA, USA) for protein purification. First, equilibrate the column with phosphate buffer twice, and then add the secreted Grp78 protein extract to the column at a flow rate of 1 ml / min and repeat 3 times. Wash the column with phosphate buffer until the protein content in the effluent detected by Bradford is zero. Then centrifuge to remove the residual liquid, add phosphate buffer containing 80 U thrombin, and perform enzymatic digestion at 16 °C for 18 hours to complete protein purification.

[0075] 3. Removal of endotoxin from secreted Grp78 protein

[0076] Use an endotoxin removal column (88274; Thermo Fisher scientific, Rockford, IL, USA) to treat the purified GRP78 protein sample. First, incubate the column bed with 0.2N NaOH solution overnight, and then wash the column bed multiple times with 2M NaCl solution and endotoxin-free ultrapure water. Add the protein sample to the column, gently shake and incubate at 4 °C for 1 hour, then centrifuge to collect the effluent, filter to remove bacteria, and aliquot into endotoxin-free centrifuge tubes and store at -80 °C. Finally, use the limulus reagent to detect the endotoxin content in the protein extracted by the above steps (semi-gel method), and the endotoxin content < 2.5 EU / ml.

[0077] 4. Identification of secreted Grp78 protein

[0078] Send the plasmid PGEX-4T-3-GPR78 encoding the full length of Grp78 to the company (Wuhan Yunclon Technology Co., Ltd., Wuhan, Hubei) for sequencing. The sequencing result is correct, and part of the sequencing result is shown in Figure 1 .

[0079] The secreted Grp78 protein extracted by the above steps was identified by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). The identification result was correct. The protein on the band was located at 78 kDa. The identification result is shown in Figure 2 .

[0080] 5. Detect the concentration of secreted Grp78 protein

[0081] The concentration of secreted Grp78 protein was detected using a protein quantification kit based on the dithiol colorimetric method, and a standard curve was plotted. The result is shown in Figure 3 .

[0082] Example 2

[0083] This example relates to the application of secreted Grp78 protein in ACD, including establishing an ACD model, experimental design, evaluating the effect of secreted Grp78 protein in the treatment of ACD, and exploring the mechanism of secreted Grp78 protein in the treatment of ACD. The specific process is as follows:

[0084] 1. Establish an ACD model

[0085] SPF-grade female BALB / c mice aged 6 - 8 weeks were purchased from Beijing Huafukang Bioscience Co., Ltd.; DNFB was purchased from Sigma Chemical Co. (USA). A 0.5% and 0.2% DNFB (original density: 1.48 g / ml) solution was prepared with a mixture of acetone and olive oil (volume ratio 4:1), and it was used immediately after preparation.

[0086] Modeling protocol: On Day 0, the abdomen was shaved (2 * 1.5 = 3 cm) 2 ; on Day 1, 25 μl of 0.5% DNFB was applied to the abdomen; on Day 2, 25 μl of 0.5% DNFB was applied to the abdomen; on Day 6, 10 μl of 0.2% DNFB was applied to each of the ventral and dorsal sides of the right ear, and 10 μl of the acetone and olive oil solution was applied to the ventral and dorsal sides of the left ear in total; on Day 7, it was terminated 24 hours after induction.

[0087] 2. Experimental design

[0088] The experimental design for DNFB-induced ACD mice and drug intervention is shown in Figure 4 . In this invention, the experimental design has a total of 4 groups; the normal group is the negative control group; the model group is the blank control group without treatment after the ACD model is established; the Grp78 treatment group is the experimental group treated with secreted Grp78; the dexamethasone treatment group is the experimental group treated with dexamethasone.

[0089] Experimental grouping:

[0090] (1) Normal group: No treatment was given. On day 1, acetone + olive oil was applied to the abdomen, and on around day 6, the matrix was applied to the right and left ears.

[0091] (2) Model group: Modeling + continuous intraperitoneal injection of 200 μl of normal saline for 5 consecutive days from day 2 to day 6. On days 1 and 2, 0.5% DNFB was applied to the abdomen, and on day 6, 0.2% DNFB was applied to the right ear and the matrix was applied to the left ear.

[0092] (3) Grp78 treatment group: Modeling + continuous intraperitoneal injection of Grp78 (10 μg / g) for 5 consecutive days from day 2 to day 6. On days 1 and 2, 0.5% DNFB was applied to the abdomen, and on day 6, 0.2% DNFB was applied to the right ear and the matrix was applied to the left ear.

[0093] (4) Dexamethasone treatment group: Modeling + intraperitoneal injection of dexamethasone (5 mg / kg) on day 6. On days 1 and 2, 0.5% DNFB was applied to the abdomen, and on day 6, 0.2% DNFB was applied to the right ear and the matrix was applied to the left ear.

[0094] 3. Evaluation of the effect of secreted Grp78 protein in the treatment of ACD

[0095] Ear swelling degree: Before and after challenge, the mice were anesthetized with isoflurane, and then the thickness of the middle parts of the right and left ears of the mice was measured using a micrometer / screw gauge (Nanjing Sucheng Measuring Instruments Co., Ltd.). We used 4 calculation methods for ear swelling degree, and the results all showed that compared with the normal group, the ears of the mice in the model group were significantly swollen and there were statistical differences; compared with the model group, the ear swelling of the mice in the Grp78 treatment group and the dexamethasone treatment group was significantly reduced and the differences were statistically significant ( Figure 5 A - D).

[0096] Ear tissue pathology: The right ear tissue of the mice was fixed with paraformaldehyde, embedded in paraffin, sectioned, stained with HE, and observed under a Nikon optical microscope at 10× magnification. It could be clearly seen from the tissue section results that compared with the normal group, the thickness of the ear tissue of the mice in the model group increased and the inflammatory exudation increased ( Figure 6 A - B); compared with the model group, the thickness of the ear tissue of the mice in the Grp78 treatment group and the dexamethasone treatment group decreased and the inflammatory reaction was significantly weakened ( Figure 6 B - D).

[0097] The results of ear swelling degree and ear tissue pathology indicate that in ACD mice treated with secreted Grp78, the ear swelling symptoms are alleviated and the inflammatory symptoms are also relieved. Secreted Grp78 has achieved good curative effects in the treatment of ACD and can be used as a new biological agent for the treatment of ACD.

[0098] 4. Explore the mechanism of secreted Grp78 protein in the treatment of ACD

[0099] The mice were sacrificed 24 hours after stimulation. Eyeball blood, ear tissues, and peritoneal effusion were collected to explore the mechanism of secreted Grp78 protein in the treatment of ACD.

[0100] CBA detection of ear tissues: The right ear tissues of mice were homogenized, and the supernatant was taken. The expressions of inflammatory factors IL-6, IFN-γ, and TNF-α were detected using CBA (BD, USA). The results are shown in Figure 7 . Compared with the normal group, the expressions of IL-6, IFN-γ, and TNF-α in the ear tissues of mice in the model group were significantly increased, and the differences in IL-6 and IFN-γ between groups were statistically significant. Compared with the model group, the expressions of IL-6, IFN-γ, and TNF-α in the ear tissues of mice in the Grp78 treatment group and dexamethasone treatment group were significantly decreased, and the difference in IFN-γ between groups was statistically significant.

[0101] CBA detection in blood: Blood was taken from the eyeballs and left to stand on ice. After centrifugation at 4°C and 7500 rpm for 25 minutes, the expressions of cytokines IL-6, IFN-γ, and TNF-α in the serum were detected using CBA (BD, USA). The results are shown in Figure 8 . Compared with the normal group, the expressions of IL-6, IFN-γ, and TNF-α in the ear tissues of mice in the model group were significantly increased and the differences were all statistically significant. Compared with the model group, the expressions of IL-6, IFN-γ, and TNF-α in the ear tissues of mice in the Grp78 treatment group and dexamethasone treatment group all showed a downward trend.

[0102] The CBA detection results of ear tissues and blood indicate that secreted Grp78 treatment can reverse the Th1-response induced by DNFB antigen by reducing the expressions of inflammatory factors IL-6, IFN-γ, and TNF-α, thereby alleviating the inflammatory symptoms of ACD mice.

[0103] 5. Phenotypes of DC cells and macrophages in peritoneal effusion

[0104] Mouse peritoneal cells were taken out in a laminar flow hood (Suzhou Purification Equipment Company, China), and the phenotypes of DC and macrophages were detected by flow cytometry. The results are shown in Figure 9 . Compared with the model group, the percentage of CD80 + DC in the peritoneal effusion of mice in the Grp78 treatment group and dexamethasone treatment group showed a significant decrease; compared with the model group, the proportion of macrophages in the peritoneal effusion of mice in the Grp78 treatment group and dexamethasone treatment group increased significantly, the percentage of CD80 + M cells decreased, and the percentage of CD206 + M cells increased, and the differences were statistically significant.

[0105] Phenotypic results of DC cells and macrophages in ascites showed that macrophages polarized towards the M2 type. It is generally believed that M2 macrophages have the effect of inhibiting inflammatory responses, suggesting that secreted Grp78 may promote macrophage polarization towards the M2 type, thereby playing an anti-inflammatory role and alleviating ACD symptoms.

[0106] In summary, for the secreted Grp78 protein extracted in the present invention, the sequencing results and identification results are correct. Evaluation of the effect of the secreted Grp78 protein in the treatment of ACD in the present invention:

[0107] The ear swelling degree and ear tissue pathology results showed that in ACD mice treated with secreted Grp78, the ear swelling symptoms were alleviated, and the inflammatory symptoms were also relieved. Secreted Grp78 achieved good curative effects in the treatment of ACD and can be used as a new biological agent for the treatment of ACD.

[0108] Mechanism of the secreted Grp78 protein in the treatment of ACD in the present invention:

[0109] CBA test results in ear tissues and blood showed that treatment with secreted Grp78 could reverse the Th1-response induced by DNFB antigen by reducing the expression of inflammatory factors IL-6, IFN-γ, and TNF-α, thereby alleviating the inflammatory symptoms of ACD mice.

[0110] Phenotypic results of DC cells and macrophages in ascites showed that macrophages polarized towards the M2 type. It is generally believed that M2 macrophages have the effect of inhibiting inflammatory responses, suggesting that secreted Grp78 may promote macrophage polarization towards the M2 type, thereby playing an anti-inflammatory role and alleviating ACD symptoms.

[0111] Obviously, the above embodiments are merely examples for clear illustration and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. Use of secreted glucose-regulated protein 78 (secreted Grp78 protein) in the preparation of a medicament for treating allergic contact dermatitis.

2. The application according to claim 1, wherein: The secreted Grp78 protein induces macrophage M2 polarization, reduces the expression of cytokines IL-6, INF-γ and TNF-α, and reduces the inflammatory response.

3. The application according to claim 1, wherein: The preparation method of the secreted Grp78 protein comprises the following steps: S1. Transform the plasmid encoding the full length of mouse Grp78 into Escherichia coli BL21 to produce glutathione-S-transferase-Grp78; S2. Purify the glutathione-S-transferase-Grp78 using a GST purification column, and further digest the GST tag with thrombin to obtain a GST-tag-free purified protein; S3. Remove endotoxin in the GST-tag-free purified protein through a high-capacity endotoxin removal column to obtain the secreted Grp78 protein.

4. Use of secreted Grp78 protein in the preparation of a medicament for any one of inducing macrophage M2 polarization, reducing the expression of cytokines IL-6, INF-γ and TNF-α, and reducing the inflammatory response.

5. A drug for treating allergic contact dermatitis, characterized in that: It is composed of the secreted Grp78 protein at an effective dose, which can be a single component or a composition containing an effective dose of the secreted Grp78 protein, and the composition may include a pharmaceutically acceptable carrier.

6. A method for preparing a secreted Grp78 protein, characterized in that: Comprises the following steps: S1. Transform the plasmid encoding the full length of mouse Grp78 into Escherichia coli BL21 to produce glutathione-S-transferase-Grp78; S2. Purify the glutathione-S-transferase-Grp78 using a GST purification column, and further digest the GST tag with thrombin to obtain a GST-tag-free purified protein; S3. Remove endotoxin in the GST-tag-free purified protein through a high-capacity endotoxin removal column to obtain the secreted Grp78 protein.

7. According to the preparation method described in claim 6, characterized in that: Also includes S4. Identify the secreted Grp78 protein by sodium dodecyl sulfate polyacrylamide gel electrophoresis.

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