Application of acidic calcium binding protein S100B in lung injury repair

Abstract

The invention relates to an application of acidic calcium binding protein S100B in lung injury repair, which detects the expression of the S100B protein family member S100B in the lung epithelium injury repair process, and finds that the expression of the S100B protein family member S100B in a lung airway epithelium injury model induced by a Napthelene drug is obviously increased; after the molecular inhibitor is used, the repairing capability of the lung is obviously hindered. In addition, the molecular weight of the S100B protein is only 10 kDa, and the S100B protein is expressed in cells and extracellular cells, can be used as a small molecule for regulating the immune microenvironment around the airway, and can be combined with arginase Arg1 to recruit anti-inflammatory macrophages to participate in the lung tissue regeneration process. The invention also discloses the effect of the S100B micromolecule on mediating the microenvironment to promote the stem cell function in the lung injury repair model, and provides a new detection method and treatment application means for lung injury diseases.

Description

[0001] The present application relates to the field of medical molecular biology, in particular to the application of acidic calcium-binding protein S100B in lung injury repair. BACKGROUND

[0002] The respiratory system provides an important interface for an individual to contact the environment. When the lung performs the function of gas exchange, the airway epithelial cells are inevitably attacked by harmful gases in the air, leading to injury. Non-carcinogenic and carcinogenic lung diseases are important causes of human death. The integrity of epithelial reconstruction after lung tissue injury is crucial for the restoration of lung homeostasis.

[0003] Maintaining a good microenvironment system is essential for the normal growth of lung epithelial cells and surrounding mesenchymal cells and the completion of multiple functions. The microenvironment is an important condition for maintaining normal cell proliferation, differentiation, metabolism and functional activity, and abnormal changes in its components can cause cell lesions, leading to blocked tissue regeneration. In previous studies on lung injury, more attention has been paid to signal factors regulating cell fate, but the study of extracellular regulatory factors is not detailed enough. Therefore, the study of the regulatory effect of extracellular molecule-mediated microenvironment on the lung injury repair process is of great significance for the treatment of lung injury diseases.

[0004] In recent years, a number of studies have found that S100B, a small molecule expressed in both intracellular and extracellular, is widely used in the early diagnosis and treatment of central nervous system infections. SUMMARY

[0005] The purpose of the present application is to provide the application of acidic calcium-binding protein S100B in lung injury repair, and to verify its application in affecting stem cell function. By detecting the expression of S100 protein family member S100B in the process of lung epithelial injury repair, it is found that the expression of S100B is significantly increased in the lung airway epithelial injury model induced by Naphthalene drug. After using the inhibitor of the molecule, the repair ability of the lung is significantly hindered.

[0006] To solve the above technical problems, the technical scheme provided by the present application is: The present application provides the application of acidic calcium-binding protein S100B in lung injury repair, and the amino acid sequence of the S100B protein is as follows: A1) the sequence shown in SEQ ID No. 1; A2) the sequence of a protein with the same biological function obtained by substituting, deleting and / or adding one or more amino acid residues in the sequence shown in SEQ ID No. 1.

[0007] Preferably, The nucleotide sequence of the coding gene of the S100B protein is as follows: B1) the nucleotide sequence as shown in SEQ ID No. 2; B2) the nucleotide sequence as shown in SEQ ID No. 2 which is substituted, deleted and / or increased by one or more nucleotides and expresses the same functional protein.

[0008] Preferably, In the lung epithelial injury repair process, the expression amount and / or activity of S100B protein is increased, thereby enhancing the lung injury repair degree.

[0009] Preferably, In the lung epithelial injury repair process, the expression amount of S100B protein is increased, thereby promoting the differentiation of stem cells into functional cells, thereby enhancing the lung epithelial injury repair degree.

[0010] Preferably, In the lung epithelial injury repair process, the S100B protein directly binds to ARG1 protein, thereby enhancing the lung epithelial injury repair degree. The S100B protein inhibits the expression level of ARG1 in the lung injury repair process.

[0011] Preferably, In the lung epithelial injury repair process, after the S100B protein is inhibited, the proportion of anti-inflammatory macrophages is reduced. The S100B protein interacts with ARG1 to recruit anti-inflammatory macrophages, thereby promoting the epithelial cell repair process.

[0012] The application also provides the use of S100B protein in the preparation of a drug for promoting epithelial cell repair in the treatment of lung injury diseases, wherein the S100B protein interacts with ARG1 to recruit anti-inflammatory macrophages, thereby promoting the epithelial cell repair process.

[0013] The application also provides a drug for treating lung injury diseases, comprising the S100B protein. The amino acid sequence of the S100B protein is as follows: A1) the sequence as shown in SEQ ID No. 1; A2) the sequence of a protein with the same biological function obtained by substituting, deleting and / or adding one or more amino acid residues in the sequence as shown in SEQ ID No. 1.

[0014] The application has the following beneficial effects: The application discloses application of S100B in lung injury repair, and an effect of S100B small molecules in mediating a microenvironment to promote stem cell functions in a lung injury repair model, and provides a new research direction for a therapeutic drug for a drug-induced lung injury disease, and provides a new detection method and treatment application means for a lung injury disease. BRIEF DESCRIPTION OF DRAWINGS

[0015] The accompanying drawings, which form a part of this specification, are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification. The embodiments of the application, and their

[0016] Figure 1 S100B expression results and gray value results in a Naphthalene drug-induced lung epithelial injury model, RAGE is a membrane receptor protein of S100B, and beta-ACTIN is actin as an internal reference index.

[0017] Figure 2 In the Naphthalene drug-induced lung epithelial injury model, the repair of epithelial injury after inhibition of S100B, A is the expression of airway epithelial Clara cells, and B is the expression of neuroendocrine stem cells; CC10 is a marker protein of Clara cells, and PGP9.5 is a marker protein of neuroendocrine cells.

[0018] Figure 3 In the Naphthalene drug-induced lung epithelial injury model, the expression of arginase ARG1 and the proportion of lung anti-inflammatory macrophages after inhibition of S100B, ARG1 is a marker protein of anti-inflammatory macrophages; A is the S100B and ARG1 protein interaction result; B is the expression of ARG1 in the Naphthalene drug-induced lung epithelial injury model; C is the expression of anti-inflammatory macrophages in the Naphthalene drug-induced lung epithelial injury model, F4 / 80 is a marker protein of mature macrophages, and CD11b is a marker of myeloid cells; the red frame encloses the anti-inflammatory macrophage group, and the proportion of the anti-inflammatory macrophage group in the total number is shown in the lower right corner of each graph.

[0019] Figure 4 The result of co-culturing Arg1 positive macrophages and a lung epithelial cell line, MLE-15 is an immortalized mouse lung epithelial cell; A is the clone size of epithelial cells after co-culturing ARG1 positive macrophages and a lung epithelial cell line for 12 days and 20 days, and the larger the clone area, the stronger the cell clone formation and proliferation ability; B is the cell count statistics of 12-day and 20-day clone formation. DETAILED DESCRIPTION

[0020] The materials, reagents and the like used in the following examples are commercially available unless otherwise specified; the technical means used in the examples are conventional means known to those skilled in the art; the reagents and materials used in the following examples are commercially available unless otherwise specified.

[0021] The following non-limiting examples can enable those of ordinary skill in the art to more fully understand the present application. However, the present application is not in any way limited by the examples.

[0022] The present application provides the application of acidic calcium binding protein S100B in lung injury repair, and verifies its application in affecting stem cell function. By detecting the expression of S100 protein family member S100B in the process of lung epithelial injury repair, it is found that the expression of S100B is significantly increased in the lung airway epithelial injury model induced by naphthalene drug; after using the inhibitor of the molecule, the repair ability of the lung is significantly hindered.

[0023] The amino acid sequence of the S100B protein of the present application is shown in SEQ ID No. 1, which consists of 92 amino acid residues; the coding nucleotide sequence of the coding gene is shown in SEQ ID No. 2, which consists of 279 bases, and the gene number is 20203.

[0024] Example 1: Detection of S100B expression in the process of mouse Naphthalene drug-induced lung epithelial injury repair.

[0025] Select 6-week-old female mice as experimental materials, and specifically kill Club cells by intraperitoneal injection of 300 mg / kg NAPH solution.

[0026] First, the lung tissues of 0 days, 3 days, 5 days and 7 days after injury are taken to extract protein, and the expression of S100B and its receptor RAGE is detected. RAGE is a membrane receptor protein of S100B, and β-ACTIN is actin as an internal reference.

[0027] First, take a green bean-sized (about 20-30 mg) tissue mixed sample, add 1 mL of pre-cooled protein lysis solution (RIPA:PMSF=100:1), and use an electric homogenizer to grind on ice until the tissue is completely lysed. After incubating the lysis solution at 4℃ for 30 minutes, centrifuge at 4℃, 12000 rpm for 15 minutes, and carefully transfer the supernatant to a new centrifuge tube.

[0028] Take an appropriate amount of protein supernatant, add 5x protein loading buffer at a volume ratio of 4:1, mix thoroughly, and then boil in a metal bath or boiling water bath for 10 minutes to denature the protein sample. The obtained protein sample can be immediately loaded or stored at -20℃ for standby use.

[0029] The protein sample was separated by SDS-PAGE gel electrophoresis, and the protein was transferred to a PVDF membrane by wet transfer method. The membrane was blocked with 5% skim milk in TBST solution at room temperature for 1 hour. Then S100B and RAGE primary antibodies were added respectively (prepared according to the recommended dilution), and incubated at 4°C overnight. After TBST washing, the corresponding species of HRP labeled secondary antibody was added, and incubated at room temperature for 1 hour. After washing with TBST, ECL chemiluminescence reagent was used for development, and the image was collected under the gel imaging system and analyzed by gray value. The gray value is directly positively correlated with the protein level.

[0030] First, the Western Blot image was opened using ImageJ software, and each target band area was defined by the "Gel Analysis" function or manually drawing a fixed size selection box. The background area gray value was measured and subtracted simultaneously to obtain the integral optical density value of each band. To correct the loading error, the gray ratio of S100B and RAGE protein bands to the internal reference protein β-Actin was calculated to obtain the standardized relative expression. All data were imported into GraphPad Prism for statistical analysis, and t-test was used to compare the differences between groups.

[0031] The analysis results show that Figure 1 The relative expression of S100B in the lung injury repair group was significantly up-regulated (p<0.01) compared with the control group (1.0 ± 0.2), and the gray analysis showed that the expression of RAGE protein was slightly increased at 5 days and 7 days after injury repair, but there was no statistical difference (p>0.05). The results show that the expression of S100B is specifically increased during the repair of lung injury, and this regulation occurs at the ligand level rather than the receptor level.

[0032] Example 2 Effect of S100B on Club cell regeneration and stem cell function during injury repair.

[0033] The expression and localization of S100B, CC10 and PGP9.5 proteins in lung tissues at different injury time points (0 day, 3 days, 5 days, 7 days) were detected by immunofluorescence staining technology. The specific steps are as follows: First, the paraffin-embedded tissue sections were deparaffinized with xylene I and II for 15 minutes, then were hydrated by gradient ethanol (100%, 95%, 85%, 75%) for 5 minutes each, and finally were washed with PBS buffer for 3 times. Microwave high pressure method was used for antigen retrieval, and the sections were immersed in EDTA antigen retrieval solution (pH 8.0), high pressure boiling for 2 minutes, and then cooled to room temperature and washed with PBS. Then, 10% normal goat serum blocking solution was used to block the tissue sections at room temperature for 1 hour to block non-specific binding. After discarding the blocking solution, the prepared primary antibody mixed working solution (containing S100B, CC10 and PGP9.5 antibodies) was directly added, and the sections were incubated in a wet box at 4°C overnight. The next day, after rewarming, the sections were washed with PBST buffer for 3 times, 5 minutes each. The fluorescence secondary antibody mixed working solution (Alexa Fluor 546 red fluorescence secondary antibody for S100B, Alexa Fluor 488 green fluorescence secondary antibody for CC10 and PGP9.5) was added, and the sections were incubated at room temperature for 1 hour, and then were washed with PBST for 3 times. Finally, the sections were stained with DAPI staining solution for 10 minutes, washed with PBS, and then mounted with water-based mounting medium. All samples were observed and imaged under the same exposure parameters by upright fluorescence microscope, in which S100B signal was shown in red (546 nm), CC10 and PGP9.5 signal was shown in green (488 nm), and the cell nucleus was shown in blue (DAPI).

[0034] The experimental results show that the number of CC10 labeled newborn Club cells at 5 days and 7 days is significantly reduced compared with the control group Figure 2 -A), indicating that inhibition of S100B during the repair process hinders the repair of CC10 labeled Club cells.

[0035] At the same time, PGP9.5 labeled neuroendocrine cells as stem cells repair damaged epithelium. The results show that PGP9.5 staining has no obvious difference from the control at 3 days, but the number of PGP9.5 is significantly less than that of the control group at 5 days and 7 days Figure 2-B), indicating that S100B does not affect the initial activation and proliferation of stem cell pool (normal number in early stage), but specifically regulates the differentiation process (blocked differentiation in later stage leads to cell consumption reduction), that is, inhibiting S100B does not affect stem cell proliferation, but inhibits stem cell differentiation process. The main stage of nerve endocrine cell proliferation is 0-3 days after injury, and the main stage of nerve endocrine cell differentiation is 5-7 days after injury. When Club cells (CC10+) are damaged, nerve endocrine cells (PGP9.5+) act as sensors to perceive injury and initiate repair signals to proliferate and differentiate into epithelium.

[0036] PGP9.5, as a marker of nerve endocrine cells, is positively correlated with stem cell activity, representing the size and activation state of the stem cell pool in lung epithelial repair; and The decrease in the number of cells directly reflects the blockage of the differentiation process. The decrease in the number of cells and Together with the insufficient cell regeneration, it indicates that the S100B signaling pathway drives epithelial repair by promoting Differentiation of stem cells to Functional cells, rather than by expanding the number of stem cells.

[0037] Example 3 Detection of ARG1 on epithelial cell proliferation The effect of ARG1 positive macrophages on epithelial cell proliferation was detected by in vitro cell co-culture technology. First, bronchoalveolar lavage was used to obtain mononuclear and macrophage cells, and flow cytometry staining was used to stain the cells with ARG1 antibody. Then, ARG1 positive macrophages were isolated by flow cytometry sorting. The isolated macrophages were added to the Transwell chamber and fixed with Matrigel. MLE-15 mouse lung epithelial cells were plated in the lower layer. Cultured in a 37°C, CO2 incubator for 12 days and 20 days. Observed and photographed under an inverted microscope.

[0038] The experimental results show that the colony formation and proliferation ability of epithelial cells co-cultured with ARG1 positive macrophages is significantly higher than that of ARG1 negative macrophage culture group (P<0.05) Figure 4 -A). And the number of colony formation cells in the experimental group was significantly more than that in the control group (P<0.05) Figure 4 -B). It is shown that ARG1 promotes epithelial cell proliferation.

[0039] Example 4 Detection of S100B on the regulation of microenvironment during airway epithelial injury repair The interaction between S100B and ARG1 proteins in the protein lysate of lung tissue of mice injured by naphthalene (NAPH) for 3 days was verified by co-immunoprecipitation (Co-IP) combined with Western Blot technology. The experimental steps are as follows: first, extract the protein lysate of lung tissue of mice injured by NAPH for 3 days, lyse the tissue at 4°C using pre-cooled RIPA lysis buffer (containing protease inhibitors), obtain the supernatant after centrifugation and determine the protein concentration. Take an equal amount of total protein and anti-S100B antibody to slowly rotate and incubate overnight at 4°C to form a specific immune complex. Then, add Protein A / G agarose beads pre-equilibrated with lysis buffer, continue to incubate for 4 hours to capture the antibody-antigen complex. Precipitate the agarose beads by low-temperature centrifugation, wash 5 times using pre-cooled lysis buffer, and then add 2x protein loading buffer to boil for 10 minutes to dissociate the complex from the beads.

[0040] Subsequently, analyze the immunoprecipitated product by Western Blot: after SDS-PAGE electrophoretic separation, transfer the sample to a PVDF membrane, block with 5% skim milk for 1 hour, add anti-ARG1 primary antibody to incubate overnight at 4°C, wash with TBST, add HRP-labeled secondary antibody to incubate for 1 hour at room temperature, and finally develop by ECL chemiluminescence reagent. In the experiment, set up an input group (Input, 5% of total protein), a negative control (IgG group) and an S100B antibody precipitation group (IP group) for comparative analysis.

[0041] The results show that in the anti-S100B antibody immunoprecipitated group, specific bands of ARG1 protein are detected, while no such bands are detected in the negative control (IgG group) Figure 3 A), indicating that in the protein lysate of lung tissue of mice injured by NAPH for 3 days, there is a specific interaction between S100B and ARG1 protein. This result indicates that S100B may participate in the regulation of the repair process in lung tissue after NAPH injury by directly or indirectly binding to ARG1 protein.

[0042] Subsequently, detect the expression of ARG1 after inhibiting S100B during the repair process, detect the effect of inhibiting S100B function on the expression of ARG1 during the repair process of NAPH-induced lung injury by tissue immunohistochemical staining technology. 8-10-week-old male C57BL / 6 mice were selected for the experiment and randomly divided into two groups: inhibitor group (PTM+NAPH) and control group (NAPH). The PTM inhibitor was dissolved in normal saline, and the dosage was 8 mg / kg. The mice were injected intraperitoneally 3 hours before NAPH injury; the control group was injected with the same volume of normal saline. NAPH was dissolved in corn oil, and both groups were injected intraperitoneally at a dose of 300 mg / kg to establish a lung injury model.

[0043] Firstly, the lung tissues of each group of mice were perfused and fixed with 4% paraformaldehyde, then paraffin-embedded and prepared into 4 μm-thick sections. After the sections were baked at 60°C for 2 hours, they were deparaffinated with xylene I and xylene II for 15 minutes each, then hydrated with gradient ethanol (100%, 95%, 85%, 75%) for 5 minutes each, and finally washed with PBS buffer (pH 7.4) for 3 times. Microwave heat repair was performed using EDTA antigen repair solution (pH 9.0), and the sections were immersed in the repair solution, heated to boiling under medium-high fire, then converted to low fire to keep boiling for 15 minutes, and naturally cooled at room temperature before washing with PBS for 3 times. The tissue sections were blocked with 10% normal horse serum blocking solution at room temperature for 1 hour to block non-specific binding sites. After the blocking solution was poured out, rabbit-derived anti-Argl primary antibody working solution (diluted at a ratio of 1:200) was directly added dropwise, and the sections were incubated in a wet box at 4°C overnight. The next day, after rewarming, the sections were washed with PBST buffer for 3 times, each for 5 minutes. Then, Alexa Fluor 594-labeled donkey anti-rabbit IgG secondary antibody (1:500 dilution) was added dropwise, and the sections were incubated at room temperature in the dark for 1 hour, and then washed with PBST in the dark for 3 times. Finally, the cell nuclei were counterstained with DAPI staining solution (1 μg / mL) in the dark for 10 minutes, washed with PBS, and then mounted with water-based anti-quenching mounting medium. All sections were observed and images were collected under the same parameters by upright fluorescence microscope, and the repair of the damaged tissues was detected by immunohistochemical staining.

[0044] The results showed that the number of ARG1 positive cells in the inhibitor group was significantly reduced compared with the control group Figure 3 B). It is shown that inhibiting the function of S100B can significantly reduce the expression level of ARG1 in the process of lung injury repair, and S100B may participate in the polarization of M2 type macrophages or the process of tissue repair by regulating ARG1.

[0045] The effect of S100B on anti-inflammatory macrophages was further detected. Flow cytometry was used to detect the condition of anti-inflammatory macrophages in the injury repair model. Wild-type C57BL / 6 mice were used as objects, and control group (NAPH injury group) and experimental group (NAPH injury + S100B inhibitor PTM treatment group) were set. PTM inhibitor (8 mg / kg) was administered by intraperitoneal injection 3 hours before NAPH injury, and NAPH (300 mg / kg, dissolved in corn oil) was administered by single intraperitoneal injection to establish a lung epithelial injury model.

[0046] On the 3rd, 5th and 7th day after injury, the mice were sacrificed and the tissues were taken. The whole lung tissue was digested with collagenase IV and DNase I mixed solution (prepared in HBSS buffer) at 37°C for 45 minutes, and the single cell suspension was obtained by filtering through a 70 μm cell strainer. After treatment with red blood cell lysis solution, the cells were resuspended in PBS buffer containing 2% FBS, and the cell concentration was adjusted to . Take 100 μL cell suspension, add anti-mouse CD16 / 32 antibody for Fc receptor blocking, then add fluorescently labeled antibody combination: PerCP anti-mouse F4 / 80 and Alexan Fluor700 anti-mouse ARG1 antibody, 4°C, avoid light, incubate for 30 minutes. Set up isotype control and single positive compensation tubes for flow calibration. After staining, the cells are washed with PBS twice, resuspended in 300 μL PBS, and immediately analyzed by flow cytometry. The following gating strategy is used: First, the nuclear cell population is circled by FSC-A / SSC-A scatter plot, and debris is excluded; by FSC-H / FSC-A, the clumps are excluded; by Double positive gate determines M2 anti-inflammatory macrophage population Figure 3 C is the expression of anti-inflammatory macrophages in the lung epithelial damage model induced by drugs. The experimental results show that after inhibiting S100B, the proportion of anti-inflammatory macrophages is significantly reduced (P < 0.05) Figure 3 -C). It is shown that during the lung damage repair process, the small molecule S100B interacts with ARG1 to recruit anti-inflammatory macrophages to participate in the epithelial repair process.

[0047] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

[0048] Although the present application has been described in detail above with general description, specific embodiments and experiments, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, the modifications or improvements made on the basis of not deviating from the spirit of the present application shall be included in the protection scope of the present application.

Claims

1. Use of the acidic calcium-binding protein S100B in the repair of lung damage, characterized in that, The amino acid sequence of the S100B protein is any one of the following: A1) the sequence shown in SEQ ID No. 1; A2) the sequence of a protein having the same biological function obtained by substitution, deletion and / or addition of one or several amino acid residues of the sequence shown in SEQ ID No.

1.

2. The use according to claim 1, wherein, The nucleotide sequence of the gene encoding the S100B protein is any one of the following: B1) the nucleotide sequence shown in SEQ ID No. 2; B2) the nucleotide sequence obtained by substitution, deletion and / or addition of one or several nucleotides of the nucleotide sequence shown in SEQ ID No. 2 and expressing the same functional protein.

3. The use according to claim 1, wherein, During the repair process of lung epithelial injury, the expression amount of S100B protein is increased to enhance the repair degree of lung injury.

4. The use according to claim 1, wherein, In the lung epithelial damage repair process, the expression of S100B protein is improved, and the differentiation of stem cells to functional cells is promoted, thereby improving the lung epithelial damage repair degree. functional cells is promoted, thereby improving the lung epithelial damage repair degree.

5. The use according to claim 1, wherein, During the repair process of lung epithelial injury, the S100B protein directly binds to ARG1 protein to enhance the repair degree of lung epithelial injury; Inhibiting the S100B protein reduces the expression level of ARG1 during the repair process of lung injury.

6. The use according to claim 1, wherein, During the repair process of lung epithelial injury, the proportion of anti-inflammatory macrophages decreases after inhibiting the S100B protein; The S100B protein interacts with ARG1 to recruit anti-inflammatory macrophages to promote the repair process of epithelial cells.

7. The use of S100B protein in the preparation of a drug for promoting the repair of epithelial cells in the treatment of lung injury diseases, wherein, The S100B protein interacts with ARG1 to recruit anti-inflammatory macrophages to promote the repair process of epithelial cells.

8. A medicament for treating a lung injury disease, characterized by, The S100B protein comprises the S100B protein; The amino acid sequence of the S100B protein is any one of the following: A1) the sequence shown in SEQ ID No. 1; A2) the sequence of a protein having the same biological function obtained by substitution, deletion and / or addition of one or several amino acid residues of the sequence shown in SEQ ID No. 1.

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