CN115282260A - Application of KGF-2 in the preparation of drugs for treating skin dysfunction - Google Patents

Abstract

The invention discloses the application of KGF-2 in preparing a medicine for treating skin dysfunction. It belongs to the field of medical technology. The invention discloses and provides the application of KGF-2 in preparing a medicine for treating skin dysfunction. Scar tissue is characterized by dense collagen fibers, strong secretion of extracellular matrix and low expression of KGF-2. KGF-2 significantly inhibits mechanical stress-induced scarring in mice and reduces the expression of extracellular matrix in primary fibroblasts. STAP2 plays a key role in the process of scarring. KGF-2 regulates the expression of STAP2 through P38, and further regulates the level of p-STAT3, thereby affecting the expression of downstream fibrosis-related proteins and reducing scar formation. KGF-2 holds promise as an effective drug for preventing scarring.

Description

Application of KGF-2 in preparation of medicine for treating skin dysfunction

technical field

The invention relates to the technical field of medicine, and more specifically relates to the application of KGF-2 in the preparation of medicines for treating skin dysfunction.

Background technique

Hypertrophic scar (HS) is a hyperfibrotic disorder of the skin and is a common complication of trauma such as burns, skin trauma, or surgery. The essence of hypertrophic scar is the excessive proliferation and continuous activation of fibroblasts, which then synthesize a large amount of extracellular matrix (ECM), resulting in the deposition of a large number of collagen fibers and affecting the normal physiological functions of the skin.

Although interventions such as skin grafts, compression therapy, steroids, lasers, and silicone dressings have achieved some suppression of scarring, surgical resection remains the only effective treatment for hypertrophic scars so far. Therefore, there is an urgent need to develop drugs for the prevention and treatment of scars to reduce or avoid the scars caused by injuries and the resulting skin dysfunction.

Therefore, how to provide a medicine for treating skin fibrosis and scar is an urgent problem to be solved by those skilled in the art.

Contents of the invention

In view of this, the present invention provides the application of KGF-2 in the preparation of medicines for treating skin dysfunction. Keratinocyte growth factor-2 (KGF-2) is a growth factor that plays an important role in regulating wound healing. The present invention uses clinical samples, animal models and cell models to explore the role of KGF-2 in scar formation in vivo and in vitro, and to explore the molecular mechanism of KGF-2 down-regulation of scar formation, providing a basis for the application of KGF-2 in the treatment of hypertrophic scars. in accordance with.

In order to achieve the above object, the present invention adopts the following technical solutions:

An application of KGF-2 in the preparation of medicines for treating skin dysfunction.

Preferably: the skin dysfunction is skin fibrosis and hypertrophic scarring.

Preferably: the drug down-regulates the expression of STAP2 and reduces the phosphorylation level of STAT3, thereby affecting the expression of downstream fibrosis-related proteins.

Preferably: the protein includes COLI, COLIII and α-SMA.

Preferably: KGF-2 down-regulates the expression of collagen in mouse skin tissue.

Preferred: KGF-2 inhibits mechanical stress-induced scar formation in mice.

Preferred: KGF-2 reduces the expression of extracellular matrix in primary fibroblasts.

The present invention also provides a drug for inhibiting scars, which is characterized in that it includes KGF-2, and the dosage of KGF-2 is 250-500 μg/kg.

Further, the present invention specifically uses HE and Masson staining to observe the structure of the hyperplastic region of clinical scar tissue, and detects the expression of extracellular matrix, α-SMA, collagen I and collagen III by WB, and simultaneously detects α-SMA by RT-PCR , Collagen I and Collagen III protein mRNA levels, so as to conduct a comprehensive analysis of clinical scar tissue from the histopathological and molecular perspectives, and determine the pathological characteristics of scar tissue. At the same time, the expression changes of common growth factors in scar tissue were detected by WB and RT-qPCR. Establish a mechanical stress-induced scar mouse model and extract primary fibroblasts, and verify the effects of KGF-2 on ECM expression and The effects of scarring. Further use proteomics methods to analyze and find the key protein regulated by KGF-2, and inhibit or overexpress it, study the effect of this protein on the phosphorylation of downstream STAT3 and the expression of scar-related proteins, and explore its role in KGF-2 regulation of scar role played in. Finally, the MAPK pathway inhibitor was used to study the regulatory pathway on which KGF-2 depends.

The beneficial effect lies in that the collagen fibers of the clinical scar skin tissue are dense, there is highly expressed collagen, and proteins related to extracellular matrix synthesis are highly expressed. In addition, the mRNA and protein levels of KGF-2 were downregulated in scar tissue, and p-STAT3 protein level was upregulated in scar tissue. Animal experiments showed that KGF-2 can reduce mechanical stress-induced scar formation and reduce the area of hypertrophic scars. And KGF-2 can down-regulate the expression of extracellular matrix and phosphorylated STAT3 in animal skin tissue. Research on the extracted primary cells showed that KGF-2 could not only reduce the expression of collagen I, collagen III, and α-SMA in primary hypertrophic scar fibroblasts, but also down-regulate the expression of phosphorylated STAT3. Through the proteomic analysis of animal tissues, the results pointed out that STAP2 is a key protein that affects KGF-2 regulation of scar formation. The up-regulation of STAP2 and phosphorylated STAT3 in skin tissue and extracted primary fibroblasts from a mouse model of mechanical stress-induced scar just confirmed this result. Further verification experiments showed that inhibiting the expression of STAP2 could reduce the expression of collagen I, collagen III, α-SMA and p-STAT3 in primary fibroblasts. Overexpression of STAP2 resulted in increased expression of p-STAT3 and accumulation of ECM. By inhibiting the P38 pathway, it is shown that KGF-2 cannot regulate the expression of collagen I, collagen III, and α-SMA, which further shows that KGF-2 regulates the expression of STAP2 through P38, and further regulates the level of p-STAT3, thereby affecting downstream fibrosis-related proteins expression, reducing scar formation.

It can be seen from the above-mentioned technical solutions that compared with the prior art, the present invention discloses the application of KGF-2 in the preparation of medicines for treating skin dysfunction. Compared with the prior art, the present invention shows that the scar tissue is dense with collagen fibers, and the extracellular The secretion of matrix is strong, and the expression of KGF-2 is low. KGF-2 can significantly inhibit mechanical stress-induced scar formation in mice and reduce the expression of extracellular matrix in primary fibroblasts. STAP2 plays a key role in the process of scar formation. KGF-2 regulates the expression of STAP2 through P38, and further regulates the level of p-STAT3, thereby affecting the expression of downstream fibrosis-related proteins and reducing scar formation. Therefore, STAP2 is expected to become a new target for scar prevention and treatment, and KGF-2 is expected to be an effective drug for preventing scar formation.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only It is an embodiment of the present invention, and those skilled in the art can also obtain other drawings according to the provided drawings without creative work.

Figure 1 is an accompanying drawing of the immunofluorescence analysis diagram of p-STAT3 expression in clinical skin samples provided by the present invention, wherein, red: vimentin, green: p-STAT3.

Fig. 2 is the figure of immunofluorescence analysis of STAT3 expression in clinical skin samples provided by the present invention, wherein, red: vimentin, green: STAT3.

Figure 3 is a statistical analysis diagram of p-STAT3 in IF provided by the present invention.

Figure 4 is the IF statistical analysis diagram of STAT3 provided by the present invention.

Figure 5 is a process diagram of the mouse model of mechanical stress-induced scar formation provided by the present invention.

Figure 6 is a diagram of the appearance of mouse skin scars before the end of administration provided by the present invention.

Figure 7 is the HE and Masson staining diagram provided by the present invention.

Figure 8 is a statistical analysis diagram of mouse skin hypertrophic scar area provided by the present invention.

FIG. 9 is a diagram of statistical analysis of p-STAT3 in Western blot provided by the present invention.

Fig. 10 is a diagram of statistical analysis of type I collagen provided by the present invention in Western blot.

Fig. 11 is a diagram of statistical analysis of type III collagen provided by the present invention in Western blot.

Fig. 12 is a diagram of statistical analysis of α-SMA provided by the present invention in Western blot.

Fig. 13 is the figure of immunofluorescence analysis of the expression of α-SMA and vimentin in mouse skin provided by the present invention, wherein red: vimentin and green: α-SMA.

Figure 14 is an immunofluorescence analysis diagram of the expression of p-STAT3 and vimentin in mouse skin provided by the present invention, wherein red: vimentin and green: p-STAT3.

Fig. 15 is the figure of immunofluorescence identification of primary human hypertrophic scar fibroblasts provided by the present invention.

Figure 16 is a statistical analysis diagram of α-SMA provided by the present invention in Western blot analysis.

Figure 17 is a statistical analysis diagram of p-STAT3 provided by the present invention in Western blot analysis.

Figure 18 is a statistical analysis diagram of type I collagen provided by the present invention in Western blot analysis.

Figure 19 is a statistical analysis diagram of type III collagen provided by the present invention in Western blot analysis.

Fig. 20 is a graph showing the clustering analysis of cellular components of differential proteins provided by the present invention.

Figure 21 is a diagram of cluster analysis of extracellular matrix-related cellular components provided by the present invention.

Figure 22 is a co-immunoprecipitation diagram of STAT3 and STAP2 provided by the present invention.

Figure 23 is the figure of immunofluorescence analysis of the expression of STAT2 and p-STAT3 in clinical skin samples provided by the present invention, in which, red: STAP2, green: p-STAT3.

Figure 24 is a statistical analysis diagram of STAP2 provided by the present invention in clinical skin sample IF.

Figure 25 is a statistical analysis diagram of p-STAT3 provided by the present invention in clinical skin sample IF.

Figure 26 is a diagram showing the experimental results of STAP2 in western blot in mouse skin tissue and primary fibroblasts provided by the present invention.

Figure 27 is a statistical analysis diagram of STAP2 provided by the present invention in Western blot analysis of clinical samples.

Figure 28 is a statistical analysis diagram of STAP2 provided by the present invention in Western blot analysis of mouse skin tissue.

Figure 29 is a statistical analysis diagram of STAP2 in Western blot analysis of primary fibroblasts provided by the present invention.

Figure 30 is a Western blot analysis diagram of KGF-2 in overexpressed STAP2 cell line provided by the present invention.

Figure 31 is a statistical analysis diagram of STAP2 provided by the present invention in Western blot analysis.

Figure 32 is a statistical analysis chart of α-SMA mRNA level provided by the present invention.

Figure 33 is a statistical analysis chart of type I collagen mRNA level provided by the present invention.

Figure 34 is a statistical analysis diagram of type III collagen mRNA levels provided by the present invention.

Figure 35 is the figure of immunofluorescence analysis of STAT2 and p-STAT3 in the cell line overexpressing STAP2 provided by the present invention, in which red: STAP2 and green: p-STAT3.

Detailed ways

The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

The embodiment of the invention discloses the application of KGF-2 in the preparation of medicines for treating skin dysfunction.

488)、驴抗鼠IgG H&L(Alexa

555)、驴抗羊IgG H&L(Alexa

647)购自Abcam公司；PPIC蛋白磷酸酶抑制剂、PIC蛋白酶抑制剂购自北京全式金生物技术有限公司等。The necessary instruments, equipment, and experimental materials in the examples are obtained from commercially available channels, for example: CCC-HSF-1 was purchased from Shanghai Baili Biotechnology Co., Ltd., DMEM high-glucose liquid medium, DMEM-F12 liquid medium were purchased from Gibco Company; penicillin-streptomycin solution (double antibody) was purchased from Hyclone Company; KGF-2 lyophilized powder and FGF-21 lyophilized powder were purchased from Zhejiang Key Laboratory of Biotechnology Pharmaceutical Engineering; MTT was purchased from Sigma Company; anti-fluorescence Quenched PVP mounting solution, RIPA lysate (strong), protease inhibitor (PMSF), and Western blot hypersensitive chemiluminescent substrate were purchased from Beyond Biotechnology Company; DMSO was purchased from Amresco Company; siRNA was purchased from Gemma Biotechnology Co., Ltd. Company; Lipo3000 was purchased from Sigma; total RNA extraction kit and reverse transcription kit were purchased from Beijing Solaibao Technology Co., Ltd; overexpression lentivirus was purchased from Heyuan Biotechnology Co., Ltd; qPCRSYBR Green MasterMix was purchased from Shanghai Yisheng Biotechnology Technology Co., Ltd.; STAT3 Rabbit Polyclonal Antibody was purchased from Proteintech; STAP-2Goat Polyclonal Antibody was purchased from Abcam; p-STAT3 Rabbit Polyclonal Antibody was purchased from SAB (signalway Antibody) Company; COLⅢRabbit Polyclonal antibodyMMP-9 Rabbit Polyclonalantibody购自proteintech公司；KGF-2Rabbit Polyclonal antibody、p-ERK1/2Polyclonal Antibody、ERK1/2Polyclonal Antibody、FGF2 Rabbit Polyclonalantibody、P38 Rabbit Polyclonal antibody、p-P38 Rabbit Polyclonal antibody购自SAB(signalway Antibody) company; FGF1 Rabbit Polyclonal antibody was purchased from SANTA CRUZ company; AKT Mouse Monoclonal antibody, p-AKT Mouse Monoclonal anti body, GAPDH antibody, goat anti-rabbit IgG-HRP, donkey anti-goat IgG-HRP were purchased from proteintech company; donkey anti-rabbit IgG H&L (Alexa

488), donkey anti-mouse IgG H&L (Alexa

555), donkey anti-goat IgG H&L (Alexa

647) were purchased from Abcam; PPIC protein phosphatase inhibitors and PIC protease inhibitors were purchased from Beijing Quanshijin Biotechnology Co., Ltd., etc.

Electrophoresis buffer

Weigh 18.90g of glycine, 3.02g of Tris-Base and 1g of SDS powder, dissolve in double-distilled water and make up to 1000mL, make 1× electrophoresis buffer, and store it at room temperature for later use.

Electroporation buffer: Weigh 14.42g glycine, dissolve 3.02g Tris-Base powder in double-distilled water and set the volume to 800mL, finally add 200mL methanol solution, adjust the pH to 8.2-8.3, and configure 1× electroporation buffer, Store at room temperature for later use.

TBS solution: Weigh 40g NaCl, 1g KCl and 15g Tris-Base powder, dissolve in double distilled water and make up to 500mL, add concentrated HCL to adjust the pH to 7.5, make 10× TBS buffer solution, and store it at room temperature for later use.

TBST solution: Measure 50mL of 10× TBS buffer solution, add 0.1% Tween-20, dilute to 500mL with double distilled water, store at room temperature for later use.

Blocking solution (5% skimmed milk powder): Weigh 1.5 g of skimmed milk powder and dissolve it in 30 mL of 1× TBST buffer solution, and prepare immediately for use.

10% AP solution: Weigh 0.1g AP and dissolve in 1mL double distilled water, store in a 4°C refrigerator for later use, and use within two weeks.

10% SDS solution: Weigh 1g of SDS powder and dissolve it in 10mL of double distilled water, shake it at room temperature and let it stand for later use.

KGF-2: Store KGF-2 powder in a refrigerator at 4°C. When used, dissolve it with cell culture medium and dispense it into 1.5mL EP tubes. Store KGF-2 dilution in a refrigerator at -20°C. Thaw at room temperature before the next use .

cell culture medium

(1) DEME cell complete medium: DMEM high glucose medium + 10% fetal bovine serum + 1X penicillin-streptomycin antibiotic concentrate.

(2) DEME cell starvation medium: DMEM high glucose medium + 0.5% fetal bovine serum + 1X penicillin-streptomycin antibiotic concentrate.

(3) DEME cell-starved heparin medium: DMEM high-glucose medium + 0.5% fetal bovine serum + 1X penicillin-streptomycin antibiotic concentrate + 100 μg/ml heparin sodium.

Statistical analysis: Graphpad Prism6.0 software was used for data processing and statistical analysis. After normality test and homogeneity of variance test, one-way analysis of variance (one-way ANOVA) was used for comparison between groups. P<0.05 means the difference is statistically significant. All experiments were repeated 3 or more times.

sample

Human scar skin fibroblasts and human normal skin fibroblasts were isolated from scar skin biopsies and normal skin biopsies from six patients with hypertrophic scars. Human studies were approved by the Ethics Committee of the School of Pharmacy, Wenzhou Medical University. All patients and controls signed consent forms approved by the local institutional review board.

Tissue embedding involved in the examples: paraffin embedding pretreatment process, stone freezing embedding pretreatment process; HE and Masson staining experiments; Western blotting; protein denaturation; SDS-PAGE electrophoresis; IHC and IF staining experiments, cell culture , cell recovery, cell subculture, cell cryopreservation, and cell counting are all routine experimental methods, and will not be repeated here.

real-time quantitative PCR

Total cellular RNA was extracted with the Total RNA Extraction Kit (Solebo) and reverse transcribed using the iScript cDNA Kit (BioRad). cDNA products were amplified in 10 μl reactions containing 5 μl of QSYBR Green SuperMix (Bio Rad) and 200 nM primers as previously described (Ray et al., 2010). To normalize template input, GAPDH transcript levels were measured for each sample. Data are expressed as fold change normalized to GAPDH.

The primers used for RT-PCR are as follows:

Col1a1, sense primer (SP): 5'-GAGGGCCAAGACGAAGACATC-3' and antisense primer (AS): 5'-CAGATCACGTCATCGCACAAC-3';

Col1A2, SP: 5'-CTCCATGGTGAGTTTGGTCTC-3' and AS: 5'-CTTCCAATAGGACCAGTAGGAC-3';

Col3a1, SP: 5'-GGAGCTGGCTACTTCTCGC-3' and AS: 5'-GGGAACATCCTCCTTCAACAG-3';

α-SMA, SP: 5'-GTGTTGCCCCTGAAGAGCAT-3' and AS: 5'-GCTGGGACATTGAAAGTCTCA-3';

KGF-2, SP: 5'-CAGTAGAAATCGGAGTTGTTGCC-3' and AS: 5'-TGAGCCATAGAGTTTCCCCTTC-3';

aFGF, SP: 5'-TTCACAGCCCTGACCGAGAA-3' and AS: 5'-CGTTGCTACAGTAGAGGAGTTTG-3';

bFGF, SP: 5'-AGAAGAGCGACCCTCACATCA-3' and AS: 5'-CGGTTAGCACACACTCCTTTG-3';

STAP-2, SP: 5'-GACCTTGGAGTGTCGGGAAAT-3' and AS: 5'-GAAGCAGGGTCAAGTCGGT-3';

GAPDH, SP: 5'-GGAGCGAGATCCCTCCAAAAT-3' and AS:

5'-GGCTGTTGTCATACTTCTCATGG-3'.

Example 1

By HE and Masson staining, it was found that the collagen fibers of the hypertrophic scar tissue were dense, and the gaps between the collagens were small.

The results of WesternBlot and RT-PCR experiments showed that α-SMA, collagen I and collagen III were highly expressed in hypertrophic scar tissue, while the expression of MMP-9, which promotes collagen degradation, was down-regulated. In addition, by examining the expressions of aFGF, bFGF and KGF-2 in hypertrophic scar tissue, the mRNA and protein expression of KGF-2 in hypertrophic scar tissue were significantly down-regulated, while the expressions of aFGF and bFGF had no significant changes.

Fibroblasts and p-STAT3 immunofluorescence colocalization test revealed that p-STAT3 was highly expressed in hypertrophic scar fibroblasts (see Figures 1, 2, 3, and 4).

Conclusion: There is excessive deposition of extracellular matrix and up-regulated expression of STAT3 phosphorylation in scar tissue. Growth factor detection revealed that the lack of KGF-2 may be related to scar formation.

Note: Compared with the normal skin group, ***p≤0.001, **p≤0.01, *p≤0.05, n=6.

Example 2

Construction of mechanical stress-induced scar model

Twenty-four 4- to 6-week-old B57CL/6 mice, clean grade (purchased from Beijing Weitong Lihua Experimental Animal Co., Ltd.). Raised in separate cages in the Phase II Animal Experiment Center of the Pilot Test Base of Wenzhou Medical University. The breeding environment is about 23±2°C at room temperature, about 60±5% relative humidity, 12 hours of alternating day and night light, fed with solid pellet mouse feed, and free to drink water. The experimental animals were repurchased and fed for 1 week to construct scar models.

After the mice were anesthetized by intraperitoneal injection of 4% chloral hydrate, the hair in the center of the back was shaved, and the scar model was prepared after local disinfection with 75% alcohol. A 1.5cm full-thickness skin shear wound was produced on the back of the mouse and sutured. After healing, a tension device was loaded on both ends of the wound, and the tension was adjusted in 3 days, and the administration time was one month.

The medicine is given every 3 days, and the treatment time is one month.

Grouped as follows:

Control group: shear injury, no tension device loaded, injected with normal saline, volume V=0.1ml;

Model group: shear injury, loaded with tension device, injected with normal saline, volume V=0.1ml;

Low-dose treatment group: shear injury, loaded tension device, injected 25μg/ml KGF-2;

High-dose treatment group: shear injury, loaded tension device, injected 50μg/ml KGF-2.

By making a mouse hypertrophic scar model induced by mechanical stress, after the shear wound healed, the upper tension device was loaded and the tension was adjusted every three days, and KGF-2 was injected subcutaneously (Figure 5).

At the end of administration, the scar marks in the suture area were observed, as shown in Figure 6, compared with the cutting injury mice without mechanical stress, the scars in the model group were obvious, and after KGF-2 treatment, the scars were significantly reduced, and there was a dose-effect relationship .

From HE and Masson staining, we can observe that the scar area of the 500 μg/kg KGF-2 administration group is significantly reduced, and the scar area is only 30% of that of the model group (Fig. 7, 8).

The results of Western Blot experiments showed that KGF-2 not only significantly reduced the expression of α-SMA, type I collagen and type III collagen, etc., but also reduced the expression of p-STAT3 in scar tissue (Figures 9-12). However, KGF-2 did not change the expression of STAT3 in skin tissue, but only affected the phosphorylation level of STAT3. Immunohistochemical staining of type Ⅰ collagen also showed that KGF-2 down-regulated the expression of collagen in mouse skin tissue in a dose-dependent manner. Down-regulation of α-SMA and p-STAT3 was also observed in mouse skin tissue immunofluorescence staining test (Figs. 13-14).

The above results indicated that KGF-2 could inhibit the formation of scar induced by mechanical stress to a certain extent.

Remarks: Compared with the normal group, ###p≤0.001, #p≤0.05; compared with the model group, ***p≤0.001, **p≤0.01, *p≤0.05, n=6.

Example 3

The cells were digested and centrifuged, then transferred to an ultra-clean bench, and the supernatant was discarded. Add 3 mL of complete medium, gently pipette to resuspend and mix. Cover the prepared cell counting plate with a cover glass, draw a certain amount of cell suspension, and drop it into the gap to avoid air bubbles. Put the cell counting plate on the inverted microscope, and record the total number of cells in the four grids according to the counting principle of counting up, not down, and left, not right. Formula: the number of cells per milliliter = (the sum of the number of cells in the four grids/4) × 10 ⁴ .

MTT: In order to detect the effect of KGF-2 on the cells, the extracted primary cells were plated on a 96-well plate, with 5000 cells per well, cultured for 24 hours and replaced with a starvation medium overnight, and the next day by three-fold concentration gradient dilution Methods: KGF-2 was applied to the cells at an initial concentration of 300 μg/ml. After acting for 24 hours, add 20ul of MTT to each well, absorb the culture medium after 4 hours, add 120ul of DMSO to each well, shake on a shaker for 5-10min, and measure the absorbance (Abs490) at 490nm on a microplate reader. The data were statistically analyzed and made statistical graphs with GraphpadPrism 6.0.

In order to further confirm the regulation of KGF-2 on the expression of extracellular matrix, primary scar fibroblasts (HSF) were extracted from clinical tissues and stimulated with a certain concentration of KGF-2.

the result shows:

The primary cell immunofluorescence identification test showed that scar fibroblasts (HSF) had high expression of α-SMA ( FIG. 15 ). In the following immunofluorescence and Western Blot experiments, the results of clinical samples and animal experiments were also confirmed: KGF-2 down-regulated α-SMA, collagen Ⅰ, collagen Ⅲ and p in scar fibroblasts (HSF) in a dose-dependent manner. - Expression of STAT3 (Figures 16-19).

Example 4

The scar mice treated with KGF-2 were collected from the normal group, model group and KGF-2 treatment group at the end of the experiment. Take an appropriate amount of tissue samples and place them in liquid nitrogen for thorough grinding. Add 4 volumes of lysis buffer and process 3 times with a high-intensity sonicator (Scientz). Cell debris was removed by centrifugation at 12,000 g for 10 min at 4°C, and the supernatant was collected, and the protein concentration was determined using the BCA kit (Beyond Biotechnology Co., Ltd.) according to the manufacturer's instructions.

Add dithiothreitol to the protein solution to make the final concentration 5mM, and reduce at 56°C for 30min. Afterwards, iodoacetamide was added to make the final concentration 11 mM, and incubated at room temperature in the dark for 15 min. Finally the urea concentration of the sample was diluted below 2M. Add trypsin at a mass ratio of 1:50 (trypsin:protein), and digest overnight at 37°C. Then trypsin was added at a mass ratio of 1:100 (trypsin:protein), and the enzymolysis was continued for 4 hours. Trypsinized peptides were desalted with StrataX C18 (Phenomenex) and then vacuum freeze-dried. After thawing, the labeling reagent was dissolved in acetonitrile, mixed with the peptide, and incubated at room temperature for 2 hours. The labeled peptide was mixed, desalted, and vacuum freeze-dried. After labeling with TMT, it was detected by liquid chromatography-mass spectrometry, and the obtained data were analyzed for differential proteins through bioinformatics software (InterProScan, KEGG Mapper, Wolfpsort, CELLO, Perl module, Blast), and cluster analysis was performed on the differential proteins , forming a transcriptome network of KGF-2-regulated genes.

The results showed that proteomic analysis was performed on mouse skin tissue, and the protein expression profile of mouse skin changed significantly before and after KGF-2 treatment ( FIG. 20 ).

Clustering analysis of cell components showed that extracellular matrix-related proteins such as fibrinogen complex in the model group were higher than those in the control group and the administration group ( FIG. 21 ). By comparing the expression of reverse protein in the administration group, and through bioinformatics analysis (Perl module), around the transcriptome network of KGF-2 regulatory genes, a network with STAP2 as the center of fibrosis and scar formation was constructed. It was further proved by co-immunoprecipitation that there is a protein-protein interaction between STAP2 and STAT3, that is, KGF-2 regulates the phosphorylation of STAT3 by down-regulating the expression of STAP-2, thereby inhibiting the formation of skin scars ( FIG. 22 ).

Example 5

siRNA transfection

STAP2 siRNA (Gimma) was at 40% confluence in NF and HSF, followed the manufacturer’s instructions, and the medium was changed 5 hours after transfection in serum-free and double-antibody-free medium, and the cells were collected after 24 hours and used for protein or total RNA extraction.

STAP2 and p-STAT3 accumulate in fibrotic skin

In order to confirm that STAP2 is an important target for scar regulation, the expression of STAP2 in normal skin (Normal Skin), hypertrophic scar (HS Skin) tissue, scar mouse skin tissue and primary scar fibroblasts was compared and analyzed.

Immunofluorescence data showed that the phosphorylation levels of STAP2 protein and STAT3 were significantly increased in the skin of HS patients compared with controls (Figs. 23-25).

The level of STAP2 protein in the scar mouse model was 6 times that of normal mice. After KGF-2 administration, the expressions of STAP2 and p-STAT3 in scarred mouse tissues were down-regulated and tended to normal. In primary scar fibroblasts, STAP2 protein levels were 1.6 times higher than in normal skin. Notably, KGF-2 downregulated STAP2 and p-STAT3 expression in a dose-dependent manner.

The above results were also confirmed by western blot experiments (Figures 26-29). The above results confirmed the presence of highly expressed STAP2 and STAT3 protein phosphorylation in scar tissue.

Remarks: Compared with the normal group, ###p≤0.001, ##p≤0.01; compared with the model group, ***p≤0.001, **p≤0.01, *p≤0.05, n=6.

Using RNA interference technology, a STAP2 knockdown cell model was constructed. The results of Western-blot and Q-RT-PCR showed that STAP2 expression was significantly down-regulated after transfection with STAP2 interference vector.

Compared with the control, STAP2 deficiency reduced the phosphorylation of STAT3 and the accumulation of type I collagen, type III collagen, and α-SMA extracellular matrix. The above results verified that STAP2 is a key target of fibrosis and scar formation.

Remarks: NC: Negative Control; KD: Positive STAP2-siRNA; compared with the control group, ***p≤0.001, **p≤0.01, *p≤0.05, n=6.

Example 6

Application of overexpression lentivirus

The overexpression lentivirus was constructed and identified by Yuanhe Biotech. At NF 60% confluency, follow the manufacturer's instructions, change the medium 5 hours after transfection in serum-free medium without double antibody, and determine the transfection efficiency under an inverted microscope. Cells were collected 24 h after administration and used for protein or total RNA extraction.

KGF-2 regulates scar formation through the interaction of STAP2 and STAT3 (STAP2 induces phosphorylation of STAT3)

The CCC-HSF-1 cell line overexpressing STAP2 was constructed. Western blot analysis showed that the expression level of STAP2 in the STAP2 overexpressed cell line was doubled (Fig. 30, 31). The data indicated that STAP2 overexpression significantly increased the expression level of p-STAT3, and the level of extracellular matrix was significantly increased.

Real-time quantitative PCR analysis showed that the mRNAs of ECM genes such as α-SMA, type I collagen, and type III collagen were significantly up-regulated in the overexpression cell lines (Figures 32-34).

Immunofluorescence results showed that the expression of p-STAT3 was significantly increased in the STAP2 overexpression cell line ( FIG. 35 ).

The results showed that: STAP2 can cause changes in the phosphorylation level of STAT3, and then affect the deposition of ECM and lead to the formation of scar. Real-time qPCR analysis also revealed that cells overexpressing STAP2 had higher expressions of collagen type I and type III and α-SMA levels.

Western-blot results showed that p-STAT3 tended to increase in overexpressed cells. Collagen levels were elevated in overexpressing cells, as were α-SMA levels, as well as fibrotic proteins (including COLⅠ, COLⅢ, and α-SMA), while MMP9 was downregulated.

To confirm that KGF-2 is involved in the regulation of scar formation through STAP2, 50 μg/ml of KGF-2 was added to STAP2 overexpressed cells.

Western-blot results showed that after 50 μg/ml KGF-2 treatment, the p-STAT3 protein level of the STAP2 overexpression cell line was reduced, and the protein levels of ECM-related factors (including COLⅠ, COLⅢ and α-SMA) were also reduced.

Real-time qPCR analysis showed that the mRNAs of COLⅠ, COLⅢ and α-SMA were down-regulated under the action of KGF-2. KGF-2 can reverse the accumulation of extracellular matrix proteins caused by STAP2 overexpression to some extent.

Example 7

Use of pathway inhibitors

In order to understand whether KGF-2 affects the cells through the MAPK pathway, the extracted primary cells were plated on a ⁶ -well plate, with 1x105 cells per well, and after 24 hours of culture, the starvation medium was replaced overnight, and the corresponding Pathway inhibitors were pretreated for one hour, and then a certain dose of KGF-2 was added to act on the cells. After 24 hours of action, the total protein was extracted, and the protein was quantitatively analyzed by Western Blot. p38 and ERK1/2 kinases jointly inhibit STAT3 signaling to STAP2 in fibroblasts

According to proteome data analysis, mitogen-activated protein kinase (mitogen-activated protein kinase, MAPK) signaling pathway is involved in KGF-2-mediated cell proliferation and cell migration.

Therefore, it was investigated whether MAPK signaling mediates overexpression of STAP2 in response to phosphorylation of STAT3. Inhibitors of the MAPK pathway were added to experiments in scar fibroblasts.

The results showed that the KGF-2 down-regulated scar effect was inhibited when the P38 pathway was inhibited. When the ERK pathway was inhibited, the expression of STAP2 was downregulated.

Furthermore, the regulatory effect of KGF-2 was enhanced in the presence of ERK pathway inhibitors due to the cascade reaction of ERK and P38 pathways.

It indicated that KGF-2 may regulate STAP2 through P38 pathway.

Each embodiment in this specification is described in a progressive manner, each embodiment focuses on the difference from other embodiments, and the same and similar parts of each embodiment can be referred to each other.

The above description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention will not be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (8)

1. The application of KGF-2 in the preparation of medicine for treating skin dysfunction. 2. The application according to claim 1, wherein the skin dysfunction is skin fibrosis and hypertrophic scar. 3. The application according to claim 2, wherein the drug down-regulates the expression of STAP2 and reduces the phosphorylation level of STAT3, thereby affecting the expression of downstream fibrosis-related proteins. 4. The use according to claim 3, wherein said protein comprises COL I, COL III and α-SMA. 5. The application according to claim 1, characterized in that KGF-2 down-regulates the expression of collagen in mouse skin tissue. 6. The application according to claim 1, wherein KGF-2 inhibits mechanical stress-induced scar formation in mice. 7. The application according to claim 1, characterized in that KGF-2 reduces the expression of extracellular matrix of primary fibroblasts. 8. A drug for inhibiting scars, characterized in that it includes KGF-2, and the dosage of KGF-2 is 250-500 μg/kg.

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