CN111529687B

CN111529687B - Application of GSDMD protein and target thereof in preparation of medicines for treating inflammatory bowel disease

Info

Publication number: CN111529687B
Application number: CN202010167058.0A
Authority: CN
Inventors: 杨硕; 王冰微; 马春梅; 刘雪
Original assignee: Nanjing Medical University
Current assignee: Nanjing Medical University
Priority date: 2020-03-11
Filing date: 2020-03-11
Publication date: 2022-05-27
Anticipated expiration: 2040-03-11
Also published as: CN111529687A

Abstract

The invention discloses application of GSDMD protein and a target thereof in preparing a medicament for treating inflammatory bowel disease. The invention discloses application of GSDMD protein in screening or preparing a medicament or a kit for treating intestinal diseases. The invention also discloses application of the GSDMD target cGAS protein in drug screening and application of an inhibitor thereof. The invention provides the inflammation inhibiting effect of GSDMD in enteritis, can provide treatment thought and theoretical basis for various GSDMD related inflammatory diseases, and provides a new target spot and a new thought for screening and preparing medicines for treating inflammatory bowel diseases.

Description

Application of GSDMD protein and target thereof in preparation of medicines for treating inflammatory bowel disease

Technical Field

The invention relates to application of GSDMD protein and a target thereof in preparing a medicament for treating inflammatory bowel disease.

Background

Inflammatory Bowel Disease (IBD) mainly includes both Ulcerative Colitis (UC) and Crohn's Disease (CD). UC is characterized by diffuse inflammation, mainly involving the superficial and submucosal mucosa of the colon, rectum, with recurrent attacks; CD may affect the whole or any part of the digestive tract from the oral cavity to the perianal region, and although the etiology and clinical manifestations of both are different, both are diseases mediated by the environment and genes and the immune system, and the intestinal mucosal immunity abnormality is the main pathological cause. IBD patients can have clinical symptoms such as abdominal pain, vomiting, diarrhea, rectal bleeding and the like, which brings great pain to the working and living of the patients, the disease is lack of effective radical treatment means at present, the disease is easy to recur, and long-term IBD can also induce Colorectal cancer (CRC). Epidemiological investigations have shown that IBD has become increasingly prevalent over the last three decades. Statistical data show that the number of UC cases is multiplied in recent decades, and in addition, the incidence of CD is increased year by year. Therefore, the method for deeply understanding the pathogenesis of the intestinal inflammation disease and effectively diagnosing and treating the intestinal inflammation disease is important to relieving the serious disease problem.

Recent studies have shown that immune-related diseases of the intestinal tract, such as Inflammatory Bowel Disease (IBD) and Irritable Bowel Syndrome (IBS), are closely related to the dysfunction of inflammatory bodies. Early studies found that the expression of IL-1 beta and IL-18 inflammatory factors in the serum of IBD patients is increased, suggesting that inflammasome may promote the formation of IBD, but studies of enteritis models based on knockout mice found that IL-18 or its receptor IL-18R aggravated the symptoms of DSS (dextran sulfate sodium salt) -induced enteritis. Subsequent studies have further shown that gene knock-out of NLRP6, ASC and Caspase-1 inflammasome molecules can also exacerbate DSS-induced inflammatory bowel disease symptoms. Therefore, intervention against inflammasome and its downstream molecules can effectively treat the development of inflammatory bowel disease, but no effective anti-enteritis drug against inflammasome is available at present.

Gasderm min D (cell apoptosis protein, GSDMD) is a key effector molecule found in recent two years downstream of inflammatory corpuscles, belongs to a member of a Gasdermin protein family, and also comprises GSDMA, GSDMB, GSDMC, DFNA5, DFNB59 and other molecules, and the proteins of the family have homology of about 45 percent and comprise two structural domains of Gasdermin-N and Gasdermin-C. GSDMDM pore formation has been reported to promote the release of interleukins IL-1 beta and IL-18, and excessive activation of GSDMDM has been closely correlated with the onset of sepsis. In addition, several studies have shown that GSDMD also plays an important role in the development of stroke and neuroinflammation. Research also reveals that the N-terminal oligomeric fragment of the GSDMD protein can be inserted into a bacterial membrane rich in cardiolipin to play a bactericidal role. The current research mainly focuses on GSDMD structure and basic research, and the research aiming at GSDMD in enteritis resistance and anti-inflammatory drugs aiming at the protein and the target thereof belong to the blank field. The research on the anti-enteritis mechanism of GSDMD is not clear.

Disclosure of Invention

The purpose of the invention is as follows: the invention also provides application of the GSDMD protein and a target cGAS thereof in preparing a medicament for treating inflammatory bowel disease.

The technical scheme is as follows: the invention provides application of GSDMD protein in screening or preparing medicines or kits for treating intestinal diseases.

In a second aspect, the invention provides the use of the GSDMD protein in screening or preparing a medicament or kit for treating inflammatory bowel disease.

In a preferred embodiment, the inflammatory bowel disease is ulcerative enteritis.

The third aspect of the invention provides application of the GSDMD target cGAS protein in screening medicines or kits for treating inflammatory bowel diseases.

In a fourth aspect, the invention provides the use of the cGAS inhibitor ru.521 as a medicament for the treatment of inflammatory bowel disease.

In a preferred embodiment, the inhibitor ru.521 exerts a therapeutic effect via the cGAS protein.

In a fifth aspect, the present invention provides a cGAS-STING signaling pathway inhibitor, wherein the inhibitor is GSDMD protein.

The invention provides application of the GSDMD protein in preparing a cGAS-STING signal pathway inhibitor.

In a preferred embodiment, the cGAS-STING signaling pathway is mediated by GSDMD.

Has the advantages that: (1) according to the application of GSDMD in enteritis generation and the determination of anti-inflammatory targets, the invention can provide treatment ideas and theoretical bases for various CGAS-related inflammatory diseases, and can further provide molecular targets and preparation ideas for screening and preparing medicines for treating inflammatory diseases; (2) the invention discovers that the GSDMD can play a role in inhibiting enteritis by regulating and controlling the activation of cGAS.

Drawings

FIG. 1 shows the results of the expression of immunofluorescent-labeled GSDMD in the intestinal epithelium and lamina propria, indicating that GSDMD, E-cadherin and CX3CR1 are co-localized.

FIG. 2 shows the western test result of the activation of the GSDMD protein in the intestinal tract tissue of the DSS enteritis mouse;

FIG. 3 is a DSS enteritis model study showing the protective effect of GSDMDM in the process of enteritis occurrence, wherein A is a body weight curve of a mouse, and it can be seen from the graph that the body weight of a GSDMDM knock-out mouse is gradually reduced along with the time lapse compared with a wild mouse, and B is the colon tissue length of the mouse, and it can be seen from the graph that the colon length of the GSDMDM knock-out mouse is obviously shortened compared with the wild mouse;

FIG. 4 shows the difference of characteristic molecule expression between WT mice and GSDMD knockout mice in a DSS-induced enteritis model of mice, and the results show that the GSDMD knockout mice secrete IL6, TNF and CCL2 in significantly increased amounts, but secrete IL-1 beta and IL-18 in significantly decreased amounts, compared with the WT mice;

FIG. 5 shows the possible mechanism of action of the protective effect of enteritis by GSDMD protein, and the results show that the GSDMD protein and the control group mice (GSDMD)^fl/fl) In contrast, specific myeloid cell GSDMD knockdown mice (GSDMD)^fl/flLysMcre) weight loss was significantly increased and colon length was significantly shortened, and enteritis protection by the GSDMD protein was likely exerted by immunomodulatory effects on myeloid cells;

FIG. 6 shows the RNA sequencing analysis of the signal pathways related to the GSDMD effect, and the results show that GSDMD can inhibit the activation of DNA sensing signal pathways;

fig. 7 is a result of measurement of the inflammatory inhibitory effect of GSDMD by cGAS;

FIG. 8 shows the inhibition of GSDMD-induced enteritis by RU.521;

figure 9 is the inhibition result of ru.521 on the activation of DSS-induced cGAS signaling pathway.

Detailed Description

Test materials and samples

BMDM (bone marrow-derived macrophages): the preparation method is shown in literature (Shen et al, 2019, The journal of experimental media);

DSS (dextran sulfate): TdB Labs, DB 001;

anti-GSDMDM antibody Abcam, ab 219800;

anti-caspase-11 antibodies: NOVUS, NB 120-10454;

anti-E-cadherin antibodies: BD, 612131;

anti- β -actin antibodies: sigma, A1978;

anti-phosphorylation STING antibody: affinity, AF 7416;

anti-STING antibodies: CST, 13647 s;

anti-phosphorylated IRF3 antibody: CST, 4302 s;

anti-IRF 3 antibody: CST, 4947 s;

IL-6ELISA kit: r & D Systems, DY 406;

TNF ELISA kit: r & D Systems, DY 410;

IL-1. beta. ELISA kit: r & D Systems DY 401;

IL-18ELISA kit: r & D Systems, D047-3;

CCL2 ELISA kit: r & D Systems, DY 478;

CCL5 ELISA kit: r & D Systems, DY 466;

ru.521(cGAS inhibitor): AOBIOUS, INC;

the GSDMD knock-out mouse can be purchased directly, and the GSDMD knock-out mouse used in the embodiment is given by Shaofeng academy of the Beijing institute of Life sciences;

specific myeloid lineage cell GSDMD knock-out mouse (GSDMD)^fl/flLysMcre) purchased by cantonese seiko bio ltd.

Second, test results

Example 1: GSDMD is expressed in both the intestinal epithelium and lamina propria

1.1 Experimental methods

Mice were sacrificed, colon tissue samples were collected, feces from the mouse colon were washed clean with sterile PBS, and sectioned, rolled into swiss rolls, fixed with 4% paraformaldehyde, embedded, and sectioned.

1.2 results of the experiment

The localization expression of immunofluorescent-labeled GSDMD (red and green) in mouse intestinal tract, E-cadherin (green) and CX3CR1 (red) represent intestinal epithelial and lamina propria immune cells, respectively. The results are shown in FIG. 1, showing that GSDMD, E-cadherin and CX3CR1 are co-localized.

Example 2: activation expression of GSDMD protein in intestinal tissue of DSS enteritis mouse

2.1 Experimental methods

Day zero: DSS was dissolved in sterile water to prepare a 2.5% DSS solution, and wild mice (WT) and GSDMD knockout mice were allowed to drink for 6 days.

The sixth day: the 2.5% DSS solution was changed to normal drinking water.

The ninth day: the mice were sacrificed and colonic tissue samples were collected, and the feces from the colon of the mice were rinsed clean with sterile PBS and a section of the pellet was taken and weighed. Adding protein lysate and collecting protein.

2.2 results of the experiment

The activation of GSDMD was detected in 2.1 part of protein lysates by anti-mouse GSDMD antibody, anti-mouse beta-actin antibody, and western, which showed significant increase in GSDMD expression and activation in DSS-induced colitis mice (see FIG. 2,

lanes

1 and 2 are control, and lanes 3-7 are DSS-induced).

Example 3: DSS enteritis model research shows that GSDMD has protective effect in enteritis occurrence process

3.1 Experimental methods

On the sixth day: the 2.5% DSS solution was changed to normal drinking water.

The ninth day: the mice were sacrificed, colon tissue samples were collected, feces of the mouse colon were washed clean with sterile PBS, and the colon length was measured and photographed.

3.2 results of the experiment, during the modeling period of the mice, the mice were weighed every day, and weight time curves were made, as shown in part A in FIG. 3 (black line is WT mice, red line is GSDMD knock-out mice), and the length of colon tissue of the mice is shown in part B in FIG. 3 (left in the figure is WT mice, right in the figure is GSDMD knock-out mice). The results show that after DSS modeling, the weight reduction ratio of the GSDMD deficient mice is obviously higher than that of the WT mice, and the colon length of the GSDMD deficient mice is shorter, which indicates that the enteritis is more inflammatory.

Example 4: in a DSS-induced enteritis model of mice, GSDMD knockout promotes the secretion of IL6, TNF and CCL2, but IL-1 beta and IL-18 are obviously reduced.

4.1 Experimental methods

On the sixth day: the 2.5% DSS solution was changed to normal drinking water.

The ninth day: mice were sacrificed and colonic tissue samples were collected, and the feces of the mouse colon were rinsed clean with sterile PBS and a section of the weight record was cut. The cells were cultured in a DMEM medium containing antibiotics for 12 hours at 37 ℃.

The tenth day: after colon tissue culture for 12 hours, the tissue mass supernatant was collected and centrifuged at 2500rpm at 4 ℃.

4.2 results of the experiment

Collecting the cell supernatant after 4.1 treatment, and detecting protein expressions of IL6, TNF, CCL2, IL-1 beta and IL-18 secreted by cells in the cell supernatant by an ELISA method, wherein in the figure 4, A is a protein expression result of IL6, B is a protein expression result of TNF, C is a protein expression result of CCL2, D is a protein expression result of IL-1 beta, E is a protein expression result of IL-18, the first column in the figure is a WT mouse group, and the second column is a GSDMD knocking-out mouse group. As can be seen from the results, the GSDMDM knockout mouse intestinal macrophages have significantly increased amounts of IL6, TNF and CCL2, but significantly decreased amounts of IL-1 beta and IL-18, compared with WT mice.

Example 5: the protective effect of the GSDMD protein against enteritis may be exerted by an immunomodulatory effect on myeloid lineage cells.

5.1 Experimental methods

Day zero: control mice (GSDMD)^fl/fl) And specific myeloid cell GSDMD knockdown murine GSDMD^fl/flLysMcre) and mice were scored double blindly for fecal status, coat appearance, and body posture. DSS was dissolved in sterile water to make 2.5% DSS solution, and mice were allowed to drink for 6 days.

The sixth day: the 2.5% DSS solution was changed to normal drinking water.

The ninth day: mice were sacrificed and samples were collected. The colon (including the cecum segment) was removed and photographed and the length was recorded; after the photographing is finished, removing the cecum section, longitudinally cutting the colon, and weighing and recording; different parts of the lower middle part of the colon were then harvested for protein, RNA and histopathological analysis.

Day zero to day nine: control mice (GSDMD) were weighed and recorded^fl/fl) And specific myeloid cell GSDMD knock-out mice (GSDMD)^fl/flLysMcre), double blind scoring of fecal condition, coat appearance and body posture of the mice.

5.2 results of the experiment

The test results are shown inFIG. 5, wherein A is the body weight comparison of the knock-out mice and the control group and B is the rectal length comparison of the knock-out mice and the control group, it can be seen that the mice (GSDMDM) and the control group are similar^fl/fl) In contrast, specific myeloid cell GSDMD knockdown mice (GSDMD)^fl/flLysMcre) weight loss increased significantly and colon length decreased significantly.

Example 6: RNA sequencing analysis of relevant signal pathways for GSDMD action

6.1 Experimental methods

Day zero: DSS was dissolved in sterile water to prepare a 2.5% DSS solution, and then control mice (wild type WT) and GSDMD knock-out mice (GSDMD)^-/-) The drinking is continued for 5 days.

The fifth day: the mice were sacrificed and the colons were collected, and the faeces of the colons were washed clean with PBS and the colons were cut into 1-2cm pieces. 5ml of intestinal tissue epithelium removal Solution (STEMCELL) was added thereto, and the mixture was incubated for 20 minutes at 37 ℃ on a shaker at 150 rpm. The intestinal tissue was washed three times with PBS, then minced, 5ml of digest (containing 1.5mg/ml collagenase IV and 20U/ml DNase I) was added, placed in a shaker, and incubated at 37 ℃ for 20 minutes at 150 rpm. The digested intestinal tissue was shaken vigorously, then the digest was filtered through a 74 μm cell sieve, the intestinal tissue was collected and a second digest was added to 5mL of digest. Then, the cells of the two digestions were collected by centrifugation at 1500rpm at 4 ℃. The cells were subjected to gradient centrifugation with Percoll, and the lamina propria immune cells at the interface of 40% Percoll and 80% Percoll were collected, followed by washing the cells once with PBS. FVD for single cell suspension

506, anti-CD45, anti-CD11b, anti-F4/80 antibodies, ice incubation for 25 minutes, PBS wash three times followed by intestinal macrophages on a BD FACS Aria machine (CD 45)⁺CD11b⁺F4/80⁺) And (6) sorting. Sorted macrophages were sampled for RNA whole genome sequencing.

The fifth day: the mouse is sacrificed to collect the colon of the mouse, the excrement of the colon of the mouse is washed clean by PBS, the 1-2cm colon is collected and added with cell lysate, then the homogenate is carried out, and the cell lysate is collected for western blotting analysis.

6.2 results of the experiment

See fig. 6, where a is the result of whole genome sequencing of macrophage RNA, the first column is the wild-type WT group, the second column is the wild-type WT group repeat group, the third column is the wild-type WT group repeat group, the fourth column is the GSDMD knockdown mouse group, the fifth column is the GSDMD knockdown mouse group repeat group, and the sixth column is the GSDMD knockdown mouse group repeat group. Compared with a control group, the GSDMD knock-out mouse group has obviously increased expression of inflammatory factors and genes related to DNA sensing signal paths. Cell lysates were collected and subjected to western blotting analysis using anti-mouse P-STING, P-TBK1, TBK1, P-IRF3, IRF3, GSDMD, P38, Actin antibodies. The results are shown in the B diagram in FIG. 6 (lane 1 is the wild type WT group, lane 2 is the wild type WT group, lane 3 is the wild type WT group, lane 4 is the GSDMD knock-out mouse group, lane 5 is the GSDMD knock-out mouse group, and lane 6 is the GSDMD knock-out mouse group). As can be seen from the B diagram in FIG. 6, compared with the control group wild type mouse, the activation of p-STING, p-TBK1, p-IRF3 in the GSDMD knock-out mouse group is obviously increased, that is, the activation of the DNA sensing signal pathway can be inhibited by GSDMD in the process of enteritis occurrence.

Example 7: GSDMD exerts an inflammatory inhibitory effect via cGAS

7.1 Experimental methods

The first day: the colons of wild mice (WT) and GSDMDM knockouts were collected, and the feces from the colons of mice were washed clean with PBS and the colons were cut into 1-2cm pieces. 5ml of intestinal tissue epithelium removal Solution (STEMCELL) was added thereto, and the mixture was incubated for 20 minutes at 37 ℃ on a shaker at 150 rpm. The intestinal tissue was washed three times with PBS, then minced, 5mL of digest (containing 1.5mg/mL collagenase IV and 20U/mL DNase I) was added, placed in a shaker, and incubated at 37 ℃ for 20 minutes at 150 rpm. The digested intestinal tissue was shaken vigorously, then the digest was filtered through a 74 μm cell sieve, the intestinal tissue was collected and a second digest was added to 5mL of digest. Then, the cells of the two digestions were collected by centrifugation at 1500rpm at 4 ℃. The cells were subjected to gradient centrifugation with Percoll, and the lamina propria immune cells at the interface of 40% Percoll and 80% Percoll were collected, followed by washing the cells once with PBS. Thin sheetFVD for cell suspension

506, anti-CD45, anti-CD11b, anti-F4/80 antibodies, ice incubation for 25 minutes, PBS wash three times followed by intestinal macrophages on a BD FACS Aria machine (CD 45)⁺CD11b⁺F4/80⁺) And (6) sorting. The sorted cells were dispensed into 24-well plates, each well 5X 10⁵And (4) one cell.

The next day: the supernatant was aspirated and 300. mu.L of DMEM medium was added to each well. The two intestinal macrophages are divided into three groups, six groups in total, and each group is provided with three groups of repeat.

A first group: WT mouse intestinal macrophages, negative control group.

Second group: WT mouse intestinal macrophages were transfected with 0.5. mu.g of Salmonella genomic DNA per well using Lipo2000 and incubated for eight hours.

Third group: WT mouse intestinal macrophages were incubated for three hours with the addition of a final concentration of 20. mu.g/mL cGAS inhibitor (RU.521). 0.5. mu.g of Salmonella genomic DNA was transfected with Lipo2000 and incubated for eight hours.

And a fourth group: GSDMD knockout mouse intestinal macrophages, negative control group.

And a fifth group: GSDMD knocks down mouse intestinal macrophages, transfects 0.5 μ g of salmonella genomic DNA per well with Lipo2000, and incubates for eight hours.

A sixth group: GSDMDM knockdown of murine intestinal macrophages was performed by first adding a cGAS inhibitor (RU.521) at a final concentration of 20. mu.g/mL and incubating for three hours. 0.5. mu.g of Salmonella genomic DNA was transfected with Lipo2000 and incubated for eight hours.

7.2 results of the experiment

Cell supernatants after 7.1 treatments were collected and tested for protein expression of IL6, TNF (TNF α in this example) and CCL5 secreted by cells in the cell supernatants by ELISA as shown in panel B in fig. 7, the first column was the first group of 7.1 partial treatments (WT murine negative control), the second column was the fourth group of 7.1 partial treatments (WT murine negative control), the third column was the second group of 7.1 partial treatments, the fourth column was the fourth group of 7.1 partial treatments, the fifth column was the third group described in section 7.1, and the sixth column was the sixth group of 7.1 partial treatments. Then adding TRIZol into the cells, extracting total RNA in the cells by a TRIZol method, detecting protein expression of TNF and CCL5 in the cells by a real-time quantitative PCR (qPCR) method, wherein the grouping mode is the same as the description above, and specifically comprises the following steps: the first column is the first group of 7.1 partial treatments (WT murine negative control), the second column is the fourth group of 7.1 partial treatments (WT murine negative control), the third column is the second group of 7.1 partial treatments, the fourth column is the fourth group of 7.1 partial treatments, the fifth column is the third group of 7.1 partial treatments, and the sixth column is the sixth group of 7.1 partial treatments. After salmonella genome DNA is transfected, the intestinal macrophages of GSDMD knockout mice express and secrete IL6, and the amounts of TNF and CCL5 are obviously increased compared with wild intestinal macrophages. Meanwhile, the addition of the cGAS inhibitor (RU.521) obviously inhibits the wild type and GSDMD from knocking out the expression of IL6, TNF and CCL5 in mouse intestinal macrophages, and the addition of the RU.521 leads the GSDMD to knock out the expression of IL6, TNF and CCL5 in the mouse intestinal macrophages to be consistent with the wild type.

Example 8: RU.521 can obviously inhibit enteritis induced by GSDMD deletion

8.1. The experimental method comprises the following steps:

a first group: wild mice were given 2.5% DSS solution, and then mice were allowed to drink for 6 days, with normal drinking water changed on the sixth day.

Second group: the GSDMD knock-out mice were given a 2.5% DSS solution, and then the mice were allowed to drink for 6 days, with the sixth day being changed to normal drinking water.

Third group: mice were given 10mg/kg of RU.521 intraperitoneal injections the day before the administration of the 2.5% DSS solution to the wild mice. Mice were then given 10mg/kg of ru.521 intraperitoneal injection daily before sacrifice.

And a fourth group: mice were given 10mg/kg of RU.521 intraperitoneal injections one day before administration of the GSDMD knockdown mouse 2.5% DSS solution. Mice were then given 10mg/kg of ru.521 intraperitoneal injection daily before sacrifice.

Day zero: the day of 2.5% DSS administration was day zero, four groups of mice were weighed and the mice were scored blindly for fecal status, coat appearance, and body posture. DSS was dissolved in sterile water to make 2.5% DSS solution, and mice were allowed to drink for 6 days.

On the sixth day: the 2.5% DSS solution was changed to normal drinking water.

Day zero to day nine: control mice (GSDMD) were weighed and recorded^fl/fl) And specific myeloid cell GSDMD knock-out mice (GSDMD)^fl/flLysMcre), the fecal condition, fur appearance and body posture of the mice were scored double blindly, and the third and fourth groups of mice were given daily intraperitoneal injections of 10mg/kg ru.521.

8.2. The experimental results are as follows:

the results are shown in fig. 8, the intraperitoneal injection of cGAS inhibitor (ru.521) significantly reduced the severity of enteritis in mice, and the addition of ru.521 led to the reduction of weight and colon length of the GSDMD-knocked-down mice consistent with those of wild-type mice.

Example 9: RU.521 significantly inhibited DSS-induced activation of the cGAS signaling pathway

9.1 Experimental methods:

a first group: wild mice were given a 2.5% DSS solution, and then the mice were allowed to drink for 5 days.

Second group: the GSDMD knockdown mice were given a 2.5% DSS solution, and then the mice were allowed to drink for 5 days.

Day zero: the day of 2.5% DSS administration was day zero, four groups of mice were weighed and mice were scored double blindly for stool status, coat appearance and body posture. DSS was dissolved in sterile water to make 2.5% DSS solution, and mice were allowed to drink for 5 days.

The fifth day: the mouse is sacrificed to collect colon, excrement of the colon of the mouse is washed clean by PBS, the colon of about 100mg is collected, 500ul PBS is added for homogenization, and the supernatant is collected for ELISA to detect the content of cGAMP. Collecting 1-2cm colon, adding cell protein lysate, homogenizing, collecting cell lysate, and performing western blotting analysis.

9.2 results of the experiment

The 9.1 treated homogenate supernatant was collected and the expression of cGAMP in colon tissue was measured by ELISA, as shown in panel a of fig. 9, with the first column being the first group of 9.1 partial treatments, the second column being the second group of 9.1 partial treatments, the third column being the third group of 9.1 partial treatments, and the fourth column being the fourth group of 9.1 partial treatments. As can be seen from the results of part a of fig. 9, the GSDMD knockout mice produced more cGAMP during enteritis development compared to the wild type mouse group. The 9.1 treated tissue protein lysates were collected and subjected to western blotting analysis using anti-mouse P-STING, P-TBK1, TBK1, P-IRF3, IRF3, GSDMD, P38, Actin antibodies. The results are shown in FIG. 9 for part B (lane 1 is the first group treated with part 9.1, lane 2 is the first group treated with part 9.1, lane 3 is the third group treated with part 9.1, lane 4 is the third group treated with part 9.1, lane 5 is the second group treated with part 9.1, lane 6 is the second group treated with part 9.1, lane 7 is the fourth group treated with part 9.1, and lane 8 is the fourth group treated with part 9.1. from part B of FIG. 9, the addition of cGAS inhibitor (RU.521) significantly inhibited the activation of p-STING, p-TBK1, p-IRF3 in both wild-type and GSDMD knock-out murine intestines, and the addition of RU.521 resulted in a level consistent with wild-type activation of p-STING, p-TBK1, p-IRF3 in both GSDMD-out murine intestines.

By determining the inflammation inhibition effect of the GSDMD in the enteritis, the action target of the GSDMD in the enteritis control process is researched, the treatment thought and the theoretical basis can be provided for various GSDMD-related inflammatory diseases, and a new target and a new thought are provided for screening and preparing medicines for treating inflammatory bowel diseases.

Claims

Application of GSDMD protein in preparation of medicines for treating inflammatory intestinal diseases.
2. The use according to claim 1, wherein the inflammatory bowel disease is ulcerative enteritis.