阅读说明：本技术 Fxyd3蛋白和基因在炎症性肠道疾病的诊断和治疗中的应用 (Application of FXYD 3protein and gene in diagnosis and treatment of inflammatory bowel disease ) 是由 来利华 王青青 宋寅敬 薛越 杨文娟 于 2021-06-16 设计创作，主要内容包括：本发明公开了FXYD3蛋白和基因在炎症性肠道疾病的诊断和治疗中的应用。本发明不仅发现了FXYD3基因主要表达在结肠杯状细胞,且其在炎症性肠病患者的炎症组织中表达显著下降,该发现为临床诊断炎症性肠病及判断易感性提供了重要的依据和生物标志物。进一步研究发现在肠道上皮细胞特异性敲除FXYD3的小鼠中,结肠杯状细胞数量和mucin 2的分泌明显受到抑制,且炎症性肠病的疾病程度更严重。细胞因子IL-22,IL-10可促进肠上皮细胞中FXYD3的表达,FXYD3通过正向调控STAT3信号活化从而促进杯状细胞分化和黏蛋白的分泌,增强肠道黏膜屏障,该发现预示着FXYD3可作为临床治疗炎症性肠病的新靶点。(The invention discloses application of FXYD 3protein and gene in diagnosis and treatment of inflammatory intestinal diseases. The invention not only discovers that the FXYD3 gene is mainly expressed in colon goblet cells, but also remarkably reduces the expression of the FXYD3 gene in inflammatory tissues of patients with inflammatory bowel diseases, and the discovery provides important basis and biomarkers for clinical diagnosis of inflammatory bowel diseases and judgment of susceptibility. Further studies found that in mice with intestinal epithelial cell-specific knockdown of FXYD3, colonic goblet cell numbers and mucin2 secretion were significantly inhibited and the disease severity of inflammatory bowel disease was more severe. The cell factors IL-22 and IL-10 can promote the expression of FXYD3 in intestinal epithelial cells, and FXYD3 can promote goblet cell differentiation and secretion of mucin and enhance intestinal mucosal barrier by positively regulating STAT3 signal activation, so the discovery indicates that the FXYD3 can be used as a new target point for clinically treating inflammatory bowel diseases.)

1. Application of human FXYD3 gene mRNA as a target point in preparing a kit for diagnosing inflammatory bowel diseases.

2. A kit for diagnosing inflammatory bowel disease, comprising:

(1) a reverse transcription reagent for reverse transcribing mRNA into cDNA,

(2) a primer for PCR amplification of a cDNA sequence of the human FXYD3 gene,

(3) a positive control containing an amplified fragment obtained by PCR amplification of human FXYD3 gene cDNA using the primers in (2) at a known concentration.

3. The kit of claim 2, further comprising a fluorescent dye for real-time fluorescent quantitative PCR detection.

4. Application of human FXYD 3protein as a target point in preparing a kit for diagnosing inflammatory bowel diseases.

5. A kit for diagnosing inflammatory bowel disease, comprising:

(1) an antibody that specifically binds to human FXYD 3protein,

(2) when the antibody of (1) as a primary antibody binds to the human FXYD 3protein, it can bind to a secondary antibody of the primary antibody, which is an antibody with a detection marker.

6. The kit according to claim 5, wherein the detection marker is peroxidase, phosphatase or luciferase, and the kit further comprises a chemiluminescent compound, a chromogenic substrate or a fluorescent dye as a reaction target for the detection marker.

7. A medicament for treating inflammatory bowel disease, characterized by the effective ingredient being at least one of:

(1) a recombinant expression vector capable of exogenously expressing FXYD3 gene;

(2) a compound capable of increasing the expression of the endogenous FXYD3 gene.

8. The pharmaceutical composition of claim 7, wherein the compound of (2) is cytokine IL-22 or IL-10.

Technical Field

The invention relates to a clinical diagnosis marker and a treatment target of diseases, in particular to the effect of an ion channel regulatory protein FXYD3 in diagnosis and treatment of inflammatory bowel diseases.

Background

Inflammatory Bowel Disease (IBD), which results from a disturbed intestinal homeostasis, is a chronic non-specific Inflammatory bowel Disease that is immune-mediated and susceptible to relapse, including Crohn's Disease (CD) and Ulcerative Colitis (UC). The crowd with the IBD at the peak of morbidity is young and strong years of 20-40 years old, has the characteristics of easy relapse and delayed non-healing, and seriously influences the life quality of patients, so that the method has important significance for the deep research of the pathogenesis of IBD, and is expected to provide a new target point for the clinical treatment of IBD.

The mucosal barrier of the intestinal tract is closely related to the occurrence of inflammatory bowel diseases, mucin secreted by goblet cells in intestinal epithelium, water, inorganic salt and the like jointly form mucus covering the surface of epithelial cells, so that a strong barrier between the mucosal epithelial cells and intestinal contents is formed, and the epithelial cells are prevented from being damaged by various physical, chemical and biological factors. The loss of mucus can lead to the development of inflammatory bowel disease. However, the mechanism of how mucus production and mucosal barrier integrity are finely regulated is not clear, and further research is needed to elucidate the relationship between the mucus production and the inflammatory bowel disease, so as to provide a potential target for clinical treatment.

FXYD (FXYD domain-ligation transport regulator) family molecules are specific accessory subunits of Na, K-ATPase, each family of which is encoded by genes of 6 to 9 small exons, the proteins are about 60 to 160 amino acids in size, the common structure is a core structure consisting of a single transmembrane segment and 35 amino acid residues around it, and the core structure is mainly responsible for maintaining the balance of Na ions and K ions concentration on the cell membrane (Lindzen M., Gottschalk K.E., Fuzesi M., Garty H. & Karlish S.J. structural interactions FX. and Na +, K + -ATPase: alpha/beta/YD cosmetic residues and cross-linking. the Journal of biological chemistry 281, 5947). FXYD3, also known as Mat-8, differs in structure from type I membrane proteins of other members of the family in that FXYD3 has a double transmembrane-like domain (Crambert G., Li C., Claeys D. & Geering K. FXYD3(Mat-8), a new regulator of Na, K-ATPase. molecular biology of the cell 16, 2363-. FXYD3 is mainly expressed in epithelial tissues such as gastrointestinal tract, skin, lung and bladder, and studies have shown that FXYD3 has a regulatory role in the development of tumors (Zhu z.l., Zhao z.r., Zhang y.s., Yang y.h., Wang z.m., Cui d.s., Wang m.w., Kleeff j., Kayed h., Yang B.Y. Sun x.f.expression and signaliciency of FXYD-3protein in gastrointestinal inflammatory bowel disease 28,63-69 (2010)), but the role and specific mechanism of FXYD3 in colon homeostasis and inflammatory bowel disease are not clear.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides the application of FXYD 3protein and gene in the diagnosis and treatment of inflammatory intestinal diseases.

The sequence of the human FXYD3 gene is shown as SEQ ID No.1, and the amino acid sequence of the human FXYD 3protein is shown as SEQ ID No. 2.

The invention firstly provides application of human FXYD3 gene mRNA as a target point in preparation of a kit for diagnosing inflammatory bowel diseases.

The present invention also provides a kit for diagnosing inflammatory bowel disease, comprising:

(1) a reverse transcription reagent for reverse transcribing mRNA into cDNA,

(2) a primer for PCR amplification of a cDNA sequence of the human FXYD3 gene,

(3) a positive control containing an amplified fragment obtained by PCR amplification of human FXYD3 gene cDNA using the primers in (2) at a known concentration.

The kit also comprises a fluorescent dye for real-time fluorescent quantitative PCR detection.

The invention also provides application of the human FXYD 3protein as a target point in preparation of a kit for diagnosing inflammatory bowel diseases.

The present invention also provides a kit for diagnosing inflammatory bowel disease, comprising:

(1) an antibody that specifically binds to human FXYD 3protein,

(2) when the antibody of (1) as a primary antibody binds to the human FXYD 3protein, it can bind to a secondary antibody of the primary antibody, which is an antibody with a detection marker.

Preferably, the kit comprises a detection marker which is peroxidase, phosphatase or luciferase, and a chemiluminescent compound, a chromogenic substrate or a fluorescent dye which is a reaction object of the detection marker.

The invention also provides a medicament for treating inflammatory bowel diseases, which comprises at least one of the following active ingredients:

(1) a recombinant expression vector capable of exogenously expressing FXYD3 gene;

(2) a compound capable of increasing the expression of the endogenous FXYD3 gene.

The medicine is characterized in that the compound in (2) is cytokine IL-22 or IL-10.

The invention not only discovers that the FXYD3 gene is mainly expressed in colon goblet cells, but also remarkably reduces the expression of the FXYD3 gene in inflammatory tissues of patients with inflammatory bowel diseases, and the discovery provides important basis and biomarkers for clinical diagnosis of inflammatory bowel diseases and judgment of susceptibility. Further studies found that in mice with intestinal epithelial cell-specific knockdown of FXYD3, colonic goblet cell numbers and mucin2 secretion were significantly inhibited and the disease severity of inflammatory bowel disease was more severe. The cell factors IL-22 and IL-10 can promote the expression of FXYD3 in intestinal epithelial cells, and FXYD3 can promote goblet cell differentiation and secretion of mucin and enhance intestinal mucosal barrier by positively regulating STAT3 signal activation, so the discovery indicates that the FXYD3 can be used as a new target point for clinically treating inflammatory bowel diseases.

Drawings

Figure 1 shows that FXYD3 was found to be expressed predominantly in colon goblet cells, a: human healthy colon tissue FXYD3 immunohistochemical staining (scale bar: 50 μm); b: human healthy colon tissue mucin2 and FXYD3 are subjected to immunofluorescence staining, mucin2 is green fluorescence, FXYD3 is red fluorescence, cell nucleus is blue fluorescence, and the green fluorescence and the red fluorescence are highly overlapped.

Fig. 2 shows that expression of FXYD3 in colonic epithelial cells in inflammatory bowel disease is significantly reduced, a: comparing the results of FXYD3 immunohistochemical staining in colon tissue of healthy and crohn's patients, scale bar: 100 μm; b: FXYD3 immunohistochemical detection of colon tissue after modelling of control and 2% DSS mice, scale bar: 50 μm; c: detecting the expression conditions of FXYD3 in colon epithelial cells of a control group and a 2% DSS mouse model mouse by qRT-PCR; d: the Western Blot experiment detects the expression of FXYD3 in colon epithelial cells of a control group and a 2% DSS mouse model group mouse.

FIG. 3 shows that the colon epithelium specificity knockdown FXYD3 transgenic mouse FXYD3^IEC-KOThe secretory function of goblet cells is impaired and intestinal mucus layer is reduced. A: PAS-AB staining detects goblet cell number, scale bar: counting the number of goblet cells and the thickness of mucus layer in colon at 50 μm; b: EUB338 in situ hybridization to detect thickness of mucus layer in intestinal lumen, scale: 50 μm. C: transmission electron microscopy detected changes in colon tissue following FXYD3 colon epithelial cell knockdown. D: and detecting the intestinal luminal surface change after the FXYD3 colon epithelial cell is knocked out by a scanning electron microscope.

FIG. 4 shows the finding of FXYD3^IEC-KOThe susceptibility of the mice to inflammatory bowel disease is increased. A: recording the weight change of mice after the mice are infected by Citrobacter rodentium (C.R.), measuring the colon length of each group of mice seven days later, and observing the damage condition of colon tissues by HE staining; b: feeding mice with 2% DSS to establish an enteritis model, recording the weight change of the mice,after nine days, the length of the colon was measured in each group of mice, and the destruction of the colon tissue was observed by HE staining.

Figure 5 is a graph showing that FXYD3 inhibits activation of STAT3 signaling in cells that are deficient in intestinal epithelial cells. A: WT and FXYD3^IEC-KOSequencing a mouse colon epithelial cell transcriptome, and obtaining a gene expression change volcanic chart; b: analyzing the KEGG signal pathway change difference; c: differences in expression of STAT3 signal-related genes; d: separation of WT and FXYD3^IEC-KOMouse colonic epithelial cells were tested for STAT3 signaling activation by Western blot analysis after treatment with IL-22, IL-10, and IL-6.

Figure 6 is a finding that FXYD3 promotes STAT3 and JAK1 binding. The protein co-immunoprecipitation method detects the binding change of JAK1 and FXYD3 after normal and silent FXYD3 expression in LS174T cells.

FIG. 7 shows that the cytokine IL-22, IL-10, was found to promote the expression of FXYD3 in LS174T cells.

Detailed Description

Example 1

Human colon tissue samples (from Shaoyifu hospital affiliated at Zhejiang university) were fixed in 10% formalin, paraffin-embedded for one week, and sectioned. Paraffin sections were placed in a 67 ℃ oven, baked for 2 hours, deparaffinized to water, and rinsed three times with PBS at pH7.4 for 3 minutes (3X 3') each. Adding a certain amount of citrate buffer solution with the pH value of 6.0 into a microwave box, heating the citrate buffer solution to boiling by microwave, placing the dewaxed and hydrated tissue slices on a high-temperature-resistant plastic slice frame, placing the tissue slices into the boiling buffer solution, carrying out medium-grade microwave treatment for 10 minutes, taking out the flowing water of the microwave box, naturally cooling, taking out the slide from the buffer solution, firstly washing the slide twice by using distilled water, and then washing the slide by using PBS (phosphate buffer solution) for 2 multiplied by 3'. 1 drop of 3% H2O2 was added to each section and incubated at room temperature for 10 minutes to block the activity of endogenous peroxidase. PBS washed 3 × 3'. The PBS was removed, and 1 drop of anti-human FXYD3(abcom ab205534) (corresponding dilution factor) was added to each section, followed by incubation at room temperature for 2 hours. PBS washed 3 × 5'. The PBS was removed and 1 drop of polymer enhancer was added to each section and incubated at room temperature for 20 minutes. PBS washed 3 × 3'. PBS was removed and 1 drop of enzyme-labeled anti-rabbit polymer was added to each section and incubated at room temperature for 30 minutes. PBS washed 3 × 5'. PBS was removed and 1 drop of freshly prepared DAB solution (diaminobenzidine) was added to each section and observed under the microscope for 5 minutes.

Hematoxylin counterstaining, 0.1% HCl differentiation, tap water washing, bluing, gradient alcohol dehydration and drying of the slices, xylene transparence, neutral gum sealing, air drying and microscopic observation, and FIG. 1A shows that FXYD3 is mainly expressed in colon goblet cells.

Human colon tissue samples were frozen and sectioned at a thickness of 20-30 μm. The desired tissue sections were selected, placed in PBS and rinsed 3 times 5 min/time on a shaker. The tissue sections were placed in blocking solution (0.01M PBS, 0.3% Triton X-100, 3% goat serum, in principle, sera homologous to the second antibody species were selected as much as possible, and sera identical to the tissue source could not be used for the cut note) and were gently shaken in a shaker at room temperature, permeabilized, and blocked for 1 h. FXYD3/MUC2 primary antibody was incubated overnight at 4 ℃. Tissue sections were placed in PBS and rinsed 4 times 10 min/time on a shaker to wash away non-specifically bound antibodies.

Adding the secondary antibody, and incubating for 2h at room temperature in a dark place in a shaking table. Tissue sections were placed in PBS and rinsed 3 times 10 min/time on a shaking table away from light. And sealing the anti-quenching sealing agent, drying in the dark, and observing by a confocal microscope. The results in FIG. 1B show that green fluorescent mucin2 is highly coincident with red fluorescent FXYD3, indicating that FXYD3 is predominantly localized to mucin2 positive cells, i.e., gut goblet cells.

Example 2

We performed immunohistochemical staining with FXYD3 using healthy humans and crohn's patients and colonic tissue from ulcerative colitis as in example 1, and the results in figure 2A found that expression was significantly reduced in FXYD3 in patient samples. Likewise, the results in figure 2B show that FXYD3 expression was also significantly reduced in colonic goblet cells in a DSS-induced murine enteritis model.

qRT-PCR: the method for separating the intestinal epithelial cells comprises the following steps: preparing an intestinal epithelium separating medium: PBS buffer plus MTT 1mM, EDTA.2Na 5mM and 5% FBS and streptomycin. After the mice are sacrificed, colon tissues are taken, the intestinal contents are flushed out by precooled PBS, the colon is cut longitudinally into 5mm, and the cut colon is put into the intestinal epithelium separating medium and is kept at 37 ℃ for 45 minutes, and the table is rotated by 150 revolutions per minute. The cells were then filtered through a vortex cell screen (200 mesh) and collected as colonic epithelial cells.

The Trizol method performs total RNA extraction according to the instructions of the reagents, and reverse transcription into cDNA according to the procedures of the Toyobo reverse transcription kit (Toyobo, Japan).

Reverse transcription system:

diluting the cDNA by 10 times with RNase-free water, and then carrying out real-time PCR by using a reaction system as follows:

the primer sequence is as follows:

mouse FXYD3 upstream primer: TACAGCATGTCCTACTCGCAG the flow of the air in the air conditioner,

mouse FXYD3 downstream primer: GAGGAAGAGGTAACCACAGGG, respectively;

human FXYD3 upstream primer: AGGTTGGCGGGCTCATC the flow of the air in the air conditioner,

human FXYD3 downstream primer: CATTTGCATTTTGCACTCATGAC are provided.

The Real-time reaction conditions are as follows:

50 ℃ for 2 min; 95 deg.C for 10 min; 95 ℃, 15sec, 60 ℃, 35sec, 40 cycles.

And (3) after the sample is added, sealing the sample by using a sealing plate membrane, centrifuging for several seconds, performing amplification reaction by using a CFX-Touch fluorescent quantitative PCR (polymerase chain reaction) instrument, wherein the result of a graph in FIG. 2C shows that the expression of FXYD3 in the intestinal epithelial cells of the DSS-induced inflammatory bowel disease mouse is obviously reduced compared with that of a normal mouse.

Western blot experiment: mouse colonic epithelial cells were isolated, centrifuged at 500g for 5min at 300-. After SDS electrophoresis, the membrane was transferred to PVDF membrane, which was immersed in a blocking solution (5% skim milk powder) and blocked on a shaker at room temperature for 1 hour. anti-FXYD 3(Santa Cruz 67245) primary antibody was added and incubated overnight at 4 ℃. After washing off the primary antibody, adding the secondary antibody and incubating for 1h at normal temperature. After washing off the excess secondary antibody, the substrate Supersonignal TM West Pico PLUS Chemicalcent (Thermo) was added for color development, and FIG. 2D results show a significant decrease in FXYD 3protein expression in colonic epithelial cells of mice with inflammatory bowel disease.

Example 3

Constructing a transgenic mouse with a colon epithelial cell specificity knockout FXYD3 through a Cre/loxp recombination system: namely FXYD3^fl/flThe mice (carrying loxp sequences) are bred by crossing with transgenic mice carrying Pvillin-cre, and Pvillin is identified⁺FXYD3 ^f/fMouse as FXYD3^IECKO laboratory mice. At the same time, the same fossa-born Pvillin will be used^-FXYD3^fl/flMice served as WT control groups. Wherein, FXYD3^fl/flBoth mice and Pvillin-cre strain mice were C57BL/6 background, purchased from Nanjing university-Nanjing model animal center.

Alizarin blue glycogen staining (AB-PAS): after paraffin-embedded sections of mouse intestinal tissues, dewaxing was carried out to water: sequentially placing the slices into xylene I20 min-xylene II 20 min-absolute ethyl alcohol I5 min-absolute ethyl alcohol II 5 min-75% alcohol 5min, and washing with tap water. Alisin blue staining: the slices are dyed in an Alisin blue dye solution for 5min, and washed with tap water for 2 min; periodic acid dyeing: slicing into periodic acid dye solution for 15min, washing with tap water, and washing with distilled water twice; and (3) performing snow dyeing: placing the slices in a Xuefu dye solution, dyeing for 30min in a dark place, and washing for 5min with running water; hematoxylin staining: and (3) dyeing the slices in hematoxylin dyeing solution for 3-5min, washing with tap water, differentiating the differentiation solution, washing with tap water, returning blue to the blue solution, and washing with running water. Dewatering and sealing: and sequentially adding absolute ethyl alcohol I for 5min, absolute ethyl alcohol II for 5min, absolute ethyl alcohol III for 5min, xylene I for 5min and xylene II for 5min, performing transparency, and sealing with neutral gum. Microscopic examination and image acquisition and analysis. FIG. 3A results show that FXYD3^IEC-KOThe number of goblet cells and the thickness of the mucus layer in colon tissue of mice were significantly reduced.

EUB338 in situ hybridization staining: after paraffin-embedded sections of mouse intestinal tissues, the slides were immersed in xylene at room temperature for 10 min. Repeating twice, each time with fresh xylene; dehydrating the glass slide in 100% ethanol at room temperature for 5min, and extracting with 85% ethanol3min, 70% ethanol for three minutes; the slides were immersed in 1 XPBS containing 0.1% DEPC for 10min and repeated once. And (3) hybridization: add 200. mu.l 3% BSA on the slide, incubate 2h at 37 ℃; diluting the FISH probe by using a hybridization buffer until the final concentration is about 0.1-0.4 mu M, carrying out denaturation at 84 ℃ for 5min, then placing 20-50 mu l of the FISH probe at 37 ℃ for 3min before a glass slide, and covering a cover glass and sealing the cover glass by Rubber cement; hybridizing in a hybridization chamber with the pre-temperature of 38-42 ℃ and incubating overnight; and (3) eluting after hybridization: carefully remove the mounting gel, treat the slide in wash solution eluent at room temperature for 15min to allow the cover slip to fall off (until the cover slip is separated from the slide), repeat once; washing in wash solution eluent for 15min, and repeating once; soaking the glass slide in 75% ethanol for 2min, 100% ethanol for 2min, and air drying for 20 min; counterstaining was performed with the addition of 20. mu.l of DAPI-antipade solution under dark conditions. Cover the glass, incubate for 10min, and then observe the staining under a fluorescent microscope. FIG. 3B results show FXYD3^IEC-KOThe mucus thickness in the intestinal lumen of mice was significantly thinner than in WT mice.

Transmission electron microscopy: a2 mm long colon tissue 1cm from the anus was fixed with 2.5% glutaraldehyde solution for 2 hours. The samples were washed three times with 1ml 0.1M PBS buffer after four degrees overnight, 15 minutes each time. After washing was completed, fixation with 50-100. mu.l of 1% osmic acid was performed for 1 hour. After fixation, rinsing with water and three washes in 10 minutes. After washing, the cells were fixed/stained with 100. mu.l of 2% uranium acetate for 30 minutes. And then dehydrating: 50%, 70%, 90% ethanol each for 15 minutes, 100% ethanol for 20 minutes, and 100% acetone for 20 minutes twice. Embedding medium + pure acetone (1:1) at room temperature for 2 hours, embedding medium + pure acetone (3:1) overnight. Pure embedding medium was exchanged and polymerization was embedded in the correct orientation at 37 ℃. The polymerized ultrathin sections (Leica UC7) were stained on a transmission electron microscope TECNAI instrument for observation. FIG. 3C results show FXYD3^IEC-KOMucin in mouse colon goblet cells cannot break through cell membrane secretion into the intestinal lumen.

Scanning electron microscope: a colon tissue with the length of 2mm at the position 1cm away from the anus of the colon is taken and longitudinally cut, a sealing film is flattened and fixed by 2.5 percent glutaraldehyde solution for 2 hours, and then the mixture is kept overnight at four degrees. After fixation was completed, the cells were washed three times with 1ml of 0.1M PBS buffer for fifteen minutes each. PBS was removed and used as 1Ososmic acid solution fixed samples for 1.5 hours. Samples were washed three times for fifteen minutes in PBS buffer. Then 50%, 70%, 90% ethanol 15 minutes each, 100% ethanol 20 minutes two times. And drying the sample at the near point, coating the sample, and observing by using an electron microscope. The results in FIG. 3D show FXYD3^IEC-KOThere are many "bubbles" on the luminal surface of mouse intestine, which appear grape-like and are distributed in the intestinal epithelial tissue, while the luminal surface of WT mouse goblet cells has many mucins that have been secreted. These results indicate that knockdown of FXYD3 from colonic goblet cells results in impaired secretory function of the goblet cells.

Example 4

Citrobacter murine infection: 8-week male WT and FXYD3^IEC-KOThe mouse is fed with antibiotics 7 days before Citrobacter infection (streptomycin 1mg/ml, metronidazole 1mg/ml, vancomycin 0.5mg/ml, neomycin 1mg/ml), and sterilized water for one day, and then fed with 2 × 10 antibiotics⁹Per 200. mu.l of the mixture was used for intragastric administration. The weight change of each group of mice is counted. Seven days later, the mice were sacrificed, the colon length was measured, and the colon tissue was HE stained. The results in FIG. 4A show FXYD3^IEC-KOThe weight loss of the mice is obviously higher than that of the WT. Mice were sacrificed at the seventh day of infection, and colon length of each group of mice was counted to find FXYD3^IEC-KOThe colon length of the mice is significantly shorter than that of the WT mice. The colon tissue HE staining shows that FXYD3^IEC-KOThe destruction of intestinal tissue in mice was more severe. These results indicate that the absence of FXYD3 in intestinal epithelial cells exacerbates intestinal inflammation caused by citrobacter murine infection.

Mouse inflammatory bowel disease model: 8-week male WT and FXYD3^IEC-KOThe mice were fed with 2% DSS water for 7 days to observe the condition of the mice, and the weight change of each group of mice was counted. Mice were sacrificed after another two days of DSS-free water feeding, the mouse colon was dissected out, colon length was measured, and colon tissue was HE stained. FIG. 4B results show FXYD3^IEC-KOThe mice lost weight significantly more than the WT group. Mice were sacrificed on day nine, the colon removed and FXYD3 found^IEC-KOThe length of the colon of the mouse is obviously shorter than that of the WT, colon tissue H&E staining showed FXYD3^IEC-KOThe colon destruction in mice was more severe. These results indicate that the absence of FXYD3 in intestinal epithelial cells promotes DSS-induced inflammatory bowel disease.

Example 5

Separation of unmolded WT and FXYD3^IEC-KOMouse colon epithelial cells, the separation method is the same as that of example 2, transcriptome sequencing is carried out, the expression of differential genes is analyzed in figure 5A, the KEGG signal path (pathway) related to the differential genes is analyzed in figure 5B, and the analysis result of figure 5C shows that the STAT3 signal path related genes are FXYD3^IEC-KOExpression was decreased in mouse-derived intestinal epithelial cells, indicating that FXYD3 is able to modulate STAT3 signaling.

Separation of unmolded WT and FXYD3^IEC-KOMouse colonic epithelial cells were isolated as described in example 2, Western blot assay after IL-22(10ng/ml), IL-10(50ng/ml) and IL-6(50ng/ml) treatment to detect signal activation. Western blotting was carried out in the same manner as in example 2. FIG. 5D results show FXYD3^IEC-KOThe STAT3 signal activation degree of the mouse intestinal epithelial cells under the stimulation of IL-22, IL-6 and IL-10 is obviously lower than that of the intestinal epithelial cells from WT mice, which shows that the FXYD3 can promote STAT3 signal activation

Example 6

The protein co-immunoprecipitation method detects the interaction of STAT3 and FXYD3 and the interaction of JAK1 and FXYD3 in LS174T cells.

The protein co-immunoprecipitation method comprises the following steps: after cells are stimulated, NP40 protein lysate is used for cracking for 20-30min on ice, 12000g is carried out, centrifugation is carried out for 15min at 4 ℃, protein supernatant is used for measuring the protein concentration of the cell total lysate by a BCA method, 200 mu g of protein sample is taken and is evenly shaken at 4 ℃ and combined with a target antibody which is well coupled with Flag-M2 magnetic beads or agarose beads overnight, the sample which forms the magnetic bead protein compound is washed three times by an IP washing solution at room temperature, 15min is carried out each time, 1x protein loading buffer solution is added, and the mixture is boiled for 15min at 100 ℃ to carry out an immune imprinting experiment. The results in figure 6 show that FXYD3 is able to bind JAK1 and STAT 3. Knockdown of FXYD3 in LS174T cells resulted in reduced binding of JAK1 and STAT3, indicating that FXYD3 can promote binding of JAK1-STAT3 to promote signaling.

Example 7

LS174T cells were cultured and treated with IL-22(10ng/ml) or IL-10(50ng/ml) for 24, 48h, after which the cells were harvested and lysed. The Western blotting experiment was performed in the same manner as in example 5 to detect the change in the expression of FXYD 3protein in the cells. The results in FIG. 7 show that cytokines IL-22 and IL-10 are able to promote expression of FXYD3 in intestinal epithelial cells.

Sequence listing

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