Application of thioquinone oxidoreductase in preparation of medicine for treating inflammatory bowel disease

Abstract

The invention discloses an application of thioquinone oxidoreductase in preparation of a medicine for treating inflammatory bowel disease, through a series of experiments such as construction of a mouse acute UC model and the like, expression of thioquinone oxidoreductase SQR in acute UC is reduced, and DSS-induced mouse acute UC symptoms can be aggravated by knockout of the thioquinone oxidoreductase SQR; the composition can promote weight loss, DAI score increase, colorectum shortening, aggravated colorectum injury, inflammatory response enhancement, intestinal barrier function reduction, intestinal epithelial cell death increase and the like. The thioquinone oxidoreductase SQR provided by the invention can be used as a potential drug target for treating UC, UC symptoms are expected to be improved by regulating and controlling expression or activity of the thioquinone oxidoreductase SQR, and a new theoretical basis and a molecular target are provided for clinical prevention and treatment of IBD.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to application of thioquinone oxidoreductase in preparing medicine for treating inflammatory bowel disease. Background Art

[0002] Inflammatory bowel disease (IBD) is a chronic, relapsing intestinal inflammatory disorder. IBD has two main clinical forms: ulcerative colitis (UC) and Crohn's disease (CD), distinguished by distinct clinical manifestations and intestinal localization. UC is more common in the colorectal area, while CD can occur anywhere in the gastrointestinal tract. The pathogenesis of IBD remains unclear and is incurable, leading to an annual increase in the number of IBD cases worldwide. It is currently believed that the pathogenesis of both UC and CD involves genetic factors, changes in the intestinal microbiome, and immune responses involving cytokines and immune cells. In recent years, it has been discovered that mitochondrial function in gastrointestinal epithelial cells plays a crucial role in maintaining intestinal health. Mitochondrial metabolism, function, and biogenesis in the intestinal epithelium influence intestinal inflammation in multiple ways. Mitochondrial dysfunction in epithelial cells leads to loss of epithelial barrier integrity, epithelial cell apoptosis, and bacterial invasion. Therefore, mitochondrial dysfunction in the intestinal epithelium is a key factor in the pathogenesis of IBD.

[0003] Sulfide quinone oxidoreductase (SQR), also known as SQRDL or SQOR, is a key enzyme involved in sulfide metabolism. In eukaryotes, SQR is located in the mitochondria and initiates the irreversible oxidation of hydrogen sulfide (H2S). Through the oxidation of H2S, SQR transfers electrons from H2S to oxidized coenzyme Q (CoQ). Electrons are then transferred to oxygen via mitochondrial complexes III and IV or to oxidized nicotinamide adenine dinucleotide (NAD+) via reverse electron transport (RET) at mitochondrial complex I. The latter pathway is believed to be required for H2S detoxification when high concentrations of H2S inhibit complex IV. In the sulfide oxidation system, SQR plays an essential role in sulfide oxidation due to its critical function of catalyzing the initial oxidation of H2S. Some studies have speculated that the mitochondria of the mouse colonic mucosa can use the thioquinone oxidoreductase SQR to oxidize sulfide produced by anaerobic metabolism of intestinal bacteria to protect the colonic mucosa.

[0004] Mitochondria are important organelles for energy production in cells. Emerging evidence suggests that alterations in mitochondrial function in intestinal epithelial cells are associated with the pathogenesis of IBD, a finding that has stimulated in-depth research into the key role mitochondria play in maintaining intestinal health. In the intestine, mitochondrial metabolism and function play a key role in immune cell activation, intestinal epithelial cell (IEC) barrier integrity, and IEC differentiation programs and stemness. It has been reported that IBD patients have decreased electron transport chain (ETC) complex activity, decreased ATP levels, accumulation of mtROS, accumulation of misfolded or unfolded proteins in the matrix, and ultrastructural changes (such as cristae dissolution) in epithelial cell mitochondria. Mitochondrial dysfunction in epithelial cells leads to loss of epithelial barrier integrity, epithelial cell apoptosis, and bacterial invasion. In addition, damaged mitochondria can release inflammasome activation signals, triggering the production of proinflammatory cytokines, thereby exacerbating the inflammatory response in the intestine.

[0005] Currently, there is a lack of application of thioquinone oxidoreductase in the preparation of drugs for treating inflammatory bowel disease. Summary of the Invention

[0006] In order to solve the problems of the prior art, the present invention aims to provide a use of thioquinone oxidoreductase in the preparation of a drug for treating inflammatory bowel disease.

[0007] To achieve the above objectives, the present invention provides the following technical solutions:

[0008] In a first aspect, the present application provides a use of a thioquinone oxidoreductase in the preparation of a drug for treating inflammatory bowel disease.

[0009] In a second aspect, the present application provides a pharmaceutical composition for treating inflammatory bowel disease.

[0010] In a third aspect, the present application provides a method for preventing or treating inflammatory bowel disease.

[0011] In a fourth aspect, the present application provides a model for screening drugs for treating inflammatory bowel disease.

[0012] In a first aspect, the present application provides a use of a thioquinone oxidoreductase in the preparation of a medicament for treating inflammatory bowel disease, wherein the inflammatory bowel disease is selected from ulcerative colitis and Crohn's disease.

[0013] Furthermore, the drug can enhance the mitochondrial function of intestinal epithelial cells, thereby alleviating the symptoms of inflammatory bowel disease, including one or more of the following: reducing intestinal epithelial cell damage; reducing the expression levels of proinflammatory cytokines and chemokines; improving intestinal barrier function; and reducing intestinal epithelial cell death.

[0014] A second aspect of the present application provides a pharmaceutical composition for treating inflammatory bowel disease, the pharmaceutical composition comprising a thioquinone oxidoreductase agonist as an active ingredient, wherein the inflammatory bowel disease is ulcerative colitis.

[0015] Furthermore, thioquinone oxidoreductase agonists can enhance the mitochondrial function of intestinal epithelial cells, including one or more of the following: increasing the activity of mitochondrial electron transport chain complexes; increasing the mitochondrial DNA copy number; increasing the ATP level of intestinal epithelial cells; inhibiting excessive mitochondrial fission and maintaining the mitochondrial dynamics homeostasis of intestinal epithelial cells; and reducing the level of reactive oxygen species in intestinal epithelial cells.

[0016] Furthermore, the thioquinone oxidoreductase agonist is selected from the following compounds: sodium sulfide NaHS, hydrogen sulfide H2S donor GYY4137 and 5-(4-hydroxyphenyl)-3H-1,2-disulfide-3-thione ADT-OH.

[0017] The third aspect of the present application provides a method for preventing or treating inflammatory bowel disease, comprising the following steps: administering a pharmaceutical composition to an individual in need thereof.

[0018] Furthermore, the individual is a patient suffering from ulcerative colitis; and the administration method includes oral administration, injection or topical administration.

[0019] The fourth aspect of the present application provides a model for screening drugs for treating inflammatory bowel disease. The model is constructed based on the role of thioquinone oxidoreductase in regulating mitochondrial function of intestinal epithelial cells, and is used to screen compounds that can regulate the activity of thioquinone oxidoreductase and thereby improve the mitochondrial function of intestinal epithelial cells.

[0020] Furthermore, the model was used to evaluate the effects of candidate compounds on intestinal epithelial cell mitochondrial function and related inflammatory responses by constructing an intestinal epithelial cell-specific thioquinone oxidoreductase knockout animal model and inducing inflammatory bowel disease.

[0021] Beneficial effects: This invention discovered and verified for the first time the important role of thioquinone oxidoreductase (thioquinone oxidoreductase SQR) in ulcerative colitis (UC), provided a new molecular target for the treatment of UC, expanded the ideas and strategies for UC treatment, and helped to develop more effective and specific therapeutic drugs.

[0022] Compared with the existing technology, the present invention has the following advantages: (1) The present invention reveals the close connection between thioquinone oxidoreductase SQR and mitochondrial dysfunction in intestinal epithelial cells, further elucidates the pathogenesis of UC, especially the changes in mitochondrial dysfunction in terms of reduced activity of mitochondrial electron transport chain complexes, decreased ATP levels, reduced mtDNA copy number, excessive ROS production, etc., providing a new theoretical basis for a deeper understanding of the pathophysiological process of UC.

[0023] (2) The present invention can improve the mitochondrial function of intestinal epithelial cells and alleviate the symptoms of UC by regulating the expression or activity of thioquinone oxidoreductase SQR, such as inhibiting inflammatory response, reducing intestinal barrier function damage, and reducing intestinal epithelial cell death. It provides a new treatment strategy and method for the clinical prevention and treatment of UC and is expected to improve the prognosis and quality of life of UC patients.

[0024] (3) The present invention constructed an intestinal epithelial cell-specific thioquinone oxidoreductase SQR knockout mouse model and an in vitro inflammatory model, and established corresponding detection methods and evaluation systems, which provided powerful tools and methods for subsequent in-depth UC-related research and helped to further explore the pathogenesis and therapeutic targets of UC. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments or descriptions of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0026] Figure 1 This is an analysis diagram of the expression of the thioquinone oxidoreductase SQR of the present invention in normal human colon tissue.

[0027] Figure 2 This is a graph showing changes in the expression level of thioquinone oxidoreductase SQR in the acute UC model of the present invention. Figure 2 A: Representative immunohistochemical results of the expression of thioquinone oxidoreductase (SQR) in the colon of WT mice. Red arrows indicate intestinal epithelial cells in the intestinal mucosa. Scale bar: 200 μm. Scale bar for the enlarged image: 100 μm. Figure 2 B: Western Blot results of colonic thioquinone oxidoreductase (SQR) in the DSS-induced acute UC model in mice, with β-tubulin as the internal reference. Figure 2 C: Band grayscale analysis results of thioquinone oxidoreductase SQR; Figure 2 D: Images of the colorectum of WT mice after 3, 5, and 7 days of DSS feeding; E: Statistical analysis of colorectal length in WT mice; F: Western blot analysis of thioquinone oxidoreductase (SQR) in the colon of mice after 3, 5, and 7 days of DSS feeding; G: Grayscale analysis of thioquinone oxidoreductase (SQR) bands. Data are expressed as mean ± standard deviation (SD) (ns: not statistically significant; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001).

[0028] Figure 3 This is a diagram of the construction of intestinal epithelial cell-specific thioquinone oxidoreductase SQR knockout mice according to the present invention. Figure 3 A: Mouse gene identification PCR electrophoresis, lane 1 identification results are thioquinone oxidoreductase SQRFL / FL mice, lanes 4 and 5 identification results are thioquinone oxidoreductase SQRCKO mice; Figure 3 B: Representative immunofluorescence staining of SQR expression in the colon of SQRFL / FL and SQRCKO mice. White arrows indicate intestinal epithelial cells in the intestinal mucosa. Scale bar: 100 μm. C: Western blot analysis of SQR expression in colorectal epithelial cells of SQRFL / FL and SQRCKO mice, using β-actin as an internal control. Data are expressed as mean ± standard deviation (SD) (ns: not statistically significant; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001).

[0029] Figure 4 Figure 2 shows the effect of the knockout of thioquinone oxidoreductase SQR on the body weight, DAI score, and colorectal length of mice with acute UC. Figure 2 shows the body weight changes of thioquinone oxidoreductase SQRFL / FL mice and thioquinone oxidoreductase SQRCKO mice in the DSS-induced acute UC model. Figure 4 A) DAI score ( Figure 4 B) and representative images of colorectum ( Figure 4 C); D: Colorectal length statistics of thioquinone oxidoreductase SQRFL / FL mice and thioquinone oxidoreductase SQRCKO mice. Data are expressed as mean ± standard deviation (SD) (ns: no statistically significant difference; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001).

[0030] Figure 5 This figure shows the effect of knockout of the thioquinone oxidoreductase SQR of the present invention on colorectal damage in acute UC mice. Figure 5 A: Representative images of H&E staining of the colorectum of thioquinone oxidoreductase SQRFL / FL mice and thioquinone oxidoreductase SQRCKO mice in the DSS-induced acute UC model. Red arrows indicate crypt damage. Scale bar: 200 μm. Scale bar: 100 μm for the enlarged image. Figure 5 B: Histological scoring of mouse colorectum. Data are expressed as mean ± standard deviation (SD) (ns: no significant statistical difference; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001).

[0031] Figure 6 The figure shows the effect of the knockout of thioquinone oxidoreductase SQR on the expression levels of proinflammatory cytokines and chemokines in acute UC mice. In the DSS-induced acute UC model, the colorectal proinflammatory cytokines IL-6, IL-1β, TNF-α ( Figure 6 A) and chemokines S100a9, Cxcl1, Ccl2 ( Figure 6 B) qRT-PCR. Data are expressed as mean ± standard deviation (SD) (ns: no statistically significant difference; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001).

[0032] Figure 7 This figure shows the effect of knockout of the thioquinone oxidoreductase SQR on the intestinal barrier function of acute UC mice. Figure 7 A is a graph of the present invention showing the intestinal permeability of thioquinone oxidoreductase SQRFL / FL mice and thioquinone oxidoreductase SQRCKO mice in the DSS-induced acute UC model by detecting the concentration of FITC-Dextran entering the peripheral blood from the colon cavity; Figure 7 B is a representative transmission electron microscopic image of tight junctions between colonic epithelial cells of thioquinone oxidoreductase SQRFL / FL mice and thioquinone oxidoreductase SQRCKO mice of the present invention, with white arrows indicating tight junctions between colonic epithelial cells; Figure 7 C- Figure 7 F: Immunofluorescence staining to detect tight junction protein Occludin in mouse colon ( Figure 7 C) and ZO-1( Figure 7 E) Representative images of expression, scale bar 200 μm, and fluorescence quantitative statistics ( Figure 7 D and Figure 7 F). Data are expressed as mean ± standard deviation (SD) (ns: no statistically significant difference; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001).

[0033] Figure 8 This figure shows the effect of knockout of the thioquinone oxidoreductase SQR of the present invention on intestinal epithelial cell death in acute UC mice. Figure 8 A: Representative images of TUNEL staining of the colon of thioquinone oxidoreductase SQRFL / FL mice and thioquinone oxidoreductase SQRCKO mice in the DSS-induced acute UC model. White arrows indicate intestinal epithelial cell death. Scale bar: 200 μm. Figure 8B: Quantitative statistics of TUNEL staining fluorescence. Data are expressed as mean ± standard deviation (SD) (ns: no significant statistical difference; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001).

[0034] Figure 9 This figure shows the effect of knockout of the thioquinone oxidoreductase SQR of the present invention on mitochondrial function in mouse intestinal epithelial cells. Figure 9 A: Representative transmission electron microscopy images of intestinal epithelial cell mitochondria from thioquinone oxidoreductase SQRFL / FL mice and thioquinone oxidoreductase SQRCKO mice in the DSS-induced acute UC model. Red arrows indicate damaged mitochondria. Scale bar: 5 μm. Scale bar for magnified images: 500 nm. Figure 9 B: Statistics of the percentage of damaged mitochondria; Figure 9 C: qRT-PCR of mitochondrial electron transport chain complex genes (including Atp5a1, Cox4i1, Uqcrc1, and Ndufab1) in mouse intestinal epithelial cells. Figure 9 D: mtDNA copy number in mouse intestinal epithelial cells; Figure 9 E: ATP levels in mouse intestinal epithelial cells; data are expressed as mean ± standard deviation (SD) (ns: no significant statistical difference; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001).

[0035] Figure 10 This is a diagram showing the effects of different concentrations of DSS on the inflammatory response of NCM460 cells. Figure 10 A: NCM460 cells were stimulated with 0, 5, 10, and 20 μg / mL DSS for 24 h, and the expressions of proinflammatory cytokines TNF-α, IL-6, and IL-1β in NCM460 cells were detected by qRT-PCR. Figure 10 B: Representative Western Blot images of thioquinone oxidoreductase SQR in NCM460 cells 72 hours after siRNA interference, with β-tubulin as an internal control; Figure 10 C: Band grayscale analysis results of thioquinone oxidoreductase SQR.

[0036] Figure 11 This figure shows the effect of knockdown of the thioquinone oxidoreductase SQR of the present invention on the mitochondrial membrane potential of NCM460 cells. Figure 11 A: After siRNA interference in NCM460 cells for 48 hours, DSS was used to stimulate NCM460 cells for 24 hours to establish an in vitro inflammation model. Representative results of mitochondrial membrane potential detection by TMRM method; Figure 11B: Quantitative analysis of mitochondrial membrane potential. Data are expressed as mean ± standard deviation (SD) (ns: no significant statistical difference; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001).

[0037] Figure 12 This figure shows the effect of knockout of the thioquinone oxidoreductase SQR of the present invention on mitochondrial dynamics in intestinal epithelial cells. Figure 12 A: After siRNA interference in NCM460 cells for 48 hours, DSS was used to stimulate NCM460 cells for 24 hours to establish an in vitro inflammatory model. Representative results of mitochondrial morphology were detected by laser confocal microscopy. Scale bar: 20 μm, scale of the magnified image: 5 μm. Figure 12 B: Representative transmission electron microscopy images of mitochondria in intestinal epithelial cells of thioquinone oxidoreductase SQRFL / FL mice and thioquinone oxidoreductase SQRCKO mice in the DSS-induced acute UC model. The red line represents the mitochondrial length measurement. Figure 12 C: Quantitative analysis of mitochondrial length in transmission electron microscopy images; Figure 12 D: Western blot images of thioquinone oxidoreductase (SQR) and DRP1 in intestinal epithelial cells from SQRFL / FL mice and SQRCKO mice in a DSS-induced acute UC model, with β-actin as an internal control. E: Grayscale analysis of DRP1 bands. FG: Representative images of immunofluorescence staining for DRP1 expression in mouse colon, scale bar 100 μm, and fluorescence quantification (G). Data are expressed as mean ± standard deviation (SD) (ns: not statistically significant; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001).

[0038] Figure 13 This figure shows the effect of knocking out the thioquinone oxidoreductase SQR of the present invention on the ROS level in intestinal epithelial cells. Figure 13 A: After siRNA interference in NCM460 cells for 48 hours, DSS stimulation of NCM460 cells for 24 hours was used to establish an in vitro inflammatory model. Representative results of DCFH probe detection of cellular ROS levels. Figure 13 B: Quantitative analysis results of ROS levels; Figure 13 C: ROS levels in intestinal epithelial cells from SQRFL / FL and SQRCKO mice expressing thioquinone oxidoreductase in the DSS-induced acute UC model. Data are expressed as mean ± standard deviation (SD) (ns: not statistically significant; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001). DETAILED DESCRIPTION

[0039] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0040] In this application, the term "and / or" describes the relationship between associated objects, indicating that three possible relationships exist. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0041] In this application, "-one or more" means one or more, and "more than one" means two or more. "The following - one or more" or similar expressions refer to any combination of these items, including any combination of single items or plural items. For example, "a, b, or c - one or more", or "a, b, and c - one or more" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0042] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution. Some or all of the steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0043] The terms used in the embodiments of the present application are for the purpose of describing specific embodiments only and are not intended to limit the present application. The singular forms "a", "an", "the" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms unless the context clearly indicates otherwise.

[0044] The weights of the relevant components mentioned in the examples of this application may not only refer to the specific content of each component, but also represent the weight ratio between the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the examples of this application, it is within the scope disclosed in the examples of this application. Specifically, the mass described in the examples of this application may be a mass unit known in the chemical industry, such as μg, mg, g, kg, etc.

[0045] The terms "first" and "second" are used solely for descriptive purposes to distinguish objects, such as substances, from one another and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the technical features being referred to. For example, a first XX could also be referred to as a second XX, and similarly, a second XX could also be referred to as a first XX, without departing from the scope of the embodiments of this application. Thus, features defined as "first" or "second" may explicitly or implicitly include one or more of such features.

[0046] In a first aspect, an embodiment of the present application provides a use of a thioquinone oxidoreductase in the preparation of a medicament for treating inflammatory bowel disease, wherein the inflammatory bowel disease is selected from ulcerative colitis and Crohn's disease.

[0047] In some embodiments, the drug can enhance the mitochondrial function of intestinal epithelial cells, thereby alleviating the symptoms of inflammatory bowel disease, including one or more of the following: reducing intestinal epithelial cell damage; reducing the expression levels of proinflammatory cytokines and chemokines; improving intestinal barrier function; and reducing intestinal epithelial cell death.

[0048] A second aspect of an embodiment of the present application provides a pharmaceutical composition for treating inflammatory bowel disease, the pharmaceutical composition comprising a thioquinone oxidoreductase agonist as an active ingredient, and the inflammatory bowel disease is ulcerative colitis.

[0049] In some embodiments, the thioquinone oxidoreductase agonist can enhance the mitochondrial function of intestinal epithelial cells, including one or more of the following: increasing the activity of mitochondrial electron transport chain complexes; increasing the mitochondrial DNA copy number; increasing the ATP level of intestinal epithelial cells; inhibiting excessive mitochondrial fission and maintaining the mitochondrial dynamics homeostasis of intestinal epithelial cells; and reducing the level of reactive oxygen species in intestinal epithelial cells.

[0050] In some embodiments, the thioquinone oxidoreductase agonist is selected from the following compounds: sodium sulfide NaHS, hydrogen sulfide H2S donor GYY4137, and 5-(4-hydroxyphenyl)-3H-1,2-disulfanyl-3-thione ADT-OH.

[0051] A third aspect of an embodiment of the present application provides a method for preventing or treating inflammatory bowel disease, comprising the following steps: administering a pharmaceutical composition to an individual in need thereof.

[0052] In some embodiments, the subject is a patient suffering from ulcerative colitis; and the administration comprises oral administration, injection, or topical administration.

[0053] The fourth aspect of the embodiments of the present application provides a model for screening drugs for treating inflammatory bowel disease. The model is constructed based on the role of thioquinone oxidoreductase in regulating the mitochondrial function of intestinal epithelial cells, and is used to screen compounds that can regulate the activity of thioquinone oxidoreductase and thereby improve the mitochondrial function of intestinal epithelial cells.

[0054] In some embodiments, the model is constructed by constructing an intestinal epithelial cell-specific thioquinone oxidoreductase knockout animal model and inducing inflammatory bowel disease to evaluate the effects of candidate compounds on intestinal epithelial cell mitochondrial function and related inflammatory responses.

[0055] Example 1

[0056] The invention relates to an application of a thioquinone oxidoreductase in the preparation of a medicine for treating inflammatory bowel disease, wherein the inflammatory bowel disease is selected from ulcerative colitis and Crohn's disease.

[0057] The drug can enhance the mitochondrial function of intestinal epithelial cells, thereby alleviating the symptoms of inflammatory bowel disease, including one or more of the following: reducing intestinal epithelial cell damage; reducing the expression levels of proinflammatory cytokines and chemokines; improving intestinal barrier function; and reducing intestinal epithelial cell death.

[0058] Example 2

[0059] The present invention provides a pharmaceutical composition for treating inflammatory bowel disease, which comprises a thioquinone oxidoreductase agonist as an active ingredient. The inflammatory bowel disease is ulcerative colitis.

[0060] Thioquinone oxidoreductase agonists can enhance the mitochondrial function of intestinal epithelial cells, including one or more of the following: increasing the activity of mitochondrial electron transport chain complexes; increasing the copy number of mitochondrial DNA; increasing the ATP level of intestinal epithelial cells; inhibiting excessive mitochondrial fission and maintaining the mitochondrial dynamics homeostasis of intestinal epithelial cells; and reducing the level of reactive oxygen species in intestinal epithelial cells.

[0061] The thioquinone oxidoreductase agonist is selected from the following compounds: sodium sulfide NaHS, hydrogen sulfide H2S donor GYY4137 and 5-(4-hydroxyphenyl)-3H-1,2-disulfanyl-3-thione ADT-OH.

[0062] Example 3

[0063] A method for preventing or treating inflammatory bowel disease of the present invention comprises the following steps: administering the pharmaceutical composition to an individual in need thereof.

[0064] The individual is a patient suffering from ulcerative colitis; the administration route includes oral administration, injection or topical administration.

[0065] Example 4

[0066] The present invention provides a model for screening drugs for treating inflammatory bowel disease. The model is constructed based on the role of thioquinone oxidoreductase in regulating the mitochondrial function of intestinal epithelial cells, and is used to screen compounds that can regulate the activity of thioquinone oxidoreductase and thereby improve the mitochondrial function of intestinal epithelial cells.

[0067] The model constructs an intestinal epithelial cell-specific thioquinone oxidoreductase knockout animal model and induces inflammatory bowel disease to evaluate the effects of candidate compounds on intestinal epithelial cell mitochondrial function and related inflammatory responses.

[0068] Example 5

[0069] 1 Experimental methods

[0070] 1.1 Construction of acute UC model in mice

[0071] Mice in the experimental group were fed 3% DSS, while mice in the control group were housed in an SPF-grade environment with free access to sterile water. Mice were weighed daily, and feces were observed. On the seventh day, mice were sacrificed by cervical dislocation. Colons were removed for imaging and length measurement. Feces were flushed with pre-chilled PBS, and the same colon location was sampled from each mouse for subsequent experiments.

[0072] 1.2 Mouse colon immunohistochemistry, immunofluorescence, H&E staining, and TUNEL staining

[0073] (1) Fixation: Place fresh colon tissue in a universal tissue fixative containing 4% paraformaldehyde for at least 24 hours.

[0074] (2) After the colon tissue was fixed, it was embedded and sectioned, and immunofluorescence, immunohistochemistry, and H&E staining experiments were performed.

[0075] (3) TUNEL detection of colon tissue was performed using the TUNEL BrightGreen Apoptosis Detection Kit according to the manufacturer's instructions.

[0076] 1.3 Western Blot Detection of SQR Protein Expression Level

[0077] (1) Take 0.1 g of colon tissue and place it in a 1.5 mL EP tube. Add 300 μL of RIPA strong lysis buffer and 2 small magnetic beads, and grind it thoroughly using a tissue homogenizer.

[0078] (2) Place the tissue homogenate on ice for 30 minutes, inverting and mixing several times to ensure thorough lysis. After 30 minutes, centrifuge the homogenate at 12,000 rpm in a refrigerated centrifuge for 10 minutes. Pipette the supernatant into a new 1.5 mL EP tube and measure the protein concentration using a BCA protein quantification kit.

[0079] (3) Add 5× Protein Loading Buffer to the supernatant at a ratio of 1:4, boil in a 100°C metal bath for 10 min, and freeze at -80°C.

[0080] (4) Electrophoresis: Load 20 μg of each protein sample and perform electrophoresis at a constant voltage of 80 V. Once the sample begins to enter the separation gel, adjust the electrophoresis voltage to 120 V.

[0081] (5) Transfer: Soak the PVDF membrane in methanol for a few seconds to activate it, and then transfer it by wet method at 300mA constant current for 1-1.5h. The specific transfer time is determined according to the molecular weight of the protein.

[0082] (6) Blocking: Transfer the membrane to a ready-to-use rapid blocking solution and complete the blocking in 5 minutes.

[0083] (7) Primary antibody incubation: Dilute the primary antibody in 5% skim milk at a ratio of 1:1000, incubate the PVDF membrane with the primary antibody at 4°C overnight, and then wash the PVDF membrane three times with 1× PBST, each time for 10 min.

[0084] (8) Secondary antibody incubation: Incubate the PVDF membrane with the secondary antibody on a shaker for 1 h, then wash the PVDF membrane three times with 1× PBST, each time for 10 min.

[0085] (9) Development: Take 1 mL each of ECL chemiluminescent substrate solution A and solution B and mix them to prepare the working solution. Use a chemiluminescent imager to detect and photograph the target protein bands.

[0086] 1.4 Extraction of intestinal epithelial cells

[0087] (1) Remove the mouse colon and remove the fat and connective tissue on the colon surface with forceps. Gently press the colon surface with a cotton swab to expel the intestinal contents, then rinse thoroughly with pre-chilled PBS buffer. Cut the colon longitudinally and cut into approximately 1 cm segments, placing them in a 50 mL centrifuge tube.

[0088] (2) Add approximately 10 mL of intestinal epithelial cell extract to the centrifuge tube containing the intestinal tissue, incubate on a shaker for 20 min, vortex for 10 s, and filter through a mesh to collect the filtrate. Repeat this step twice and combine the filtrates.

[0089] (3) Centrifuge the filtrate at 5000 rpm for 10 min, discard the supernatant, and the precipitate is the intestinal epithelial cells.

[0090] 1.5DAI Rating

[0091] The mice were scored based on weight loss, stool consistency, and fecal occult blood. The sum of the scores was divided by 3 to obtain the DAI score. The scoring details are shown in Table 1. The DAI scoring criteria are shown in Table 1:

[0092] Table 1

[0093]

[0094] 1.6 Histological scoring of mouse colon

[0095] Scores were calculated based on the severity of colon inflammation, depth of injury, and inflammatory cell infiltration in mice. The sum of the scores was the colon histological score. The specific scoring criteria for the histological scoring standard are shown in Table 2.

[0096] Table 2

[0097]

[0098] 1.7 Mouse intestinal permeability test

[0099] (1) Intestinal permeability test was performed on the day of mouse autopsy, and the mice were fasted for 4 h before oral gavage.

[0100] (2) Preparation of FITC-Dextran solution: Add 500 mg of FITC-Dextran powder to 10 mL of ddH2O and mix by inverting to fully dissolve it.

[0101] (3) Each mouse was given 200 μL of FITC-Dextran solution by gavage.

[0102] (4) After 4 hours, 1 mL of blood was collected from the eyeball, kept in the dark for 3 hours, and then centrifuged at 10,000 rpm at 4°C for 20 minutes.

[0103] (5) Prepare FITC-Dextran standard according to the instructions.

[0104] (6) The upper serum layer was aspirated into a new EP tube, and the serum was diluted 1:1 with ddH2O. 100 μL of FITC-Dextran standard and diluted serum sample were spotted into a black-bottomed 96-well plate. The fluorescence intensity at an excitation wavelength of 480 nm and an emission wavelength of 530 nm was detected by a microplate reader.

[0105] (7) Draw a standard curve and calculate the FITC-Dextran concentration.

[0106] 1.8 Transmission electron microscopy of mouse colon and rectum

[0107] (1) Sampling: Use tweezers to remove the fat and connecting tissue on the surface of the colon from fresh colon tissue. Use a cotton swab to gently press the surface of the colon to expel the colon contents. Rinse with pre-cooled PBS buffer and cut into a ring of about 2 mm in size.

[0108] (2) Fixation: Place the collected colon sample in electron microscopy fixative at room temperature in the dark for 2 h and then transfer to a 4°C refrigerator for storage.

[0109] (3) After the colon tissue was fixed, it was sent to Wuhan Sevier Biotechnology Co., Ltd. for transmission electron microscopy to observe the tight junctions of intestinal epithelial cells.

[0110] 1.9 ATP level detection

[0111] Mouse colorectal epithelial cells were extracted, and the enhanced ATP detection kit of Biyuntian Biotechnology Co., Ltd. was used according to the instructions to detect the ATP levels of intestinal epithelial cells in thioquinone oxidoreductase SQRFL / FL mice and thioquinone oxidoreductase SQRCKO mice before and after acute UC modeling.

[0112] 1.10 Detection of mitochondrial membrane potential by TRM

[0113] (1) After the in vitro inflammatory model of NCM460 cells was successfully established, the cells were washed twice with PBS and digested to prepare a cell suspension.

[0114] (2) Take an appropriate amount of TMRM probe, dilute it with serum-free DMEM at a ratio of 1:500, and place it in a 37°C incubator to preheat.

[0115] (3) Collect the cells by centrifugation at 800 rpm for 3 min, add 400 μL of TMRM probe diluent to each sample, gently pipette to resuspend the cells, and incubate in a 37°C incubator in the dark for 30 min.

[0116] (4) Collect the cells by centrifugation at 3000 rpm for 3 min at 4°C and wash the cells twice with PBS to remove excess probe.

[0117] (5) Resuspend the cells in 500 μL of PBS preheated at 37°C, and measure the mitochondrial membrane potential of NCM460 cells by flow cytometry.

[0118] 1.11 Data Analysis

[0119] Data collection and analysis were performed using Graphpad Prism 9. All data are expressed as mean ± standard deviation (SD), and differences between data were compared using a two-way ANOVA test (ns: no significant statistical difference; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

[0120] 2. Experimental Results

[0121] 2.1 Downregulation of thioquinone oxidoreductase (SQR) expression in acute UC in mice

[0122] Currently, the role of thioquinone oxidoreductase (SQR) in the progression of inflammatory bowel disease remains unclear, and there are no reports on the relationship between thioquinone oxidoreductase (SQR) and enteritis. To explore the feasibility of studying thioquinone oxidoreductase (SQR) and enteritis, the present invention conducted a database survey on the expression of thioquinone oxidoreductase (SQR) in clinical samples of normal colon tissue. According to immunohistochemical data provided by the Human Protein Atlas database, thioquinone oxidoreductase (SQR) is highly expressed in normal human colon tissue, with the highest expression level in intestinal epithelial cells ( Figure 1 ). Figure 1 This is an analysis diagram of the expression of the thioquinone oxidoreductase SQR of the present invention in normal human colon tissue.

[0123] To investigate changes in the expression of thioquinone oxidoreductase (SQR) in acute ulcerative colitis (UC), 8-week-old male WT mice were fed 3% DSS to establish an acute UC model. Immunohistochemistry revealed that SQR was highly expressed in the colonic tissue of normal mice, with the highest expression at the apex of the colonic crypts. Following DSS treatment, crypt abscesses became deformed and absent, colonic tissue was severely damaged, and SQR expression was downregulated. Figure 2 A). Western Blot results showed that the expression of thioquinone oxidoreductase SQR was significantly downregulated after DSS treatment ( Figure 2 B and Figure 2 C).

[0124] To investigate the changes in the expression of thioquinone oxidoreductase (SQR) with the aggravation of enteritis, WT male mice were given 3% DSS for 3, 5, and 7 days. The results showed that with the increase in the number of days of DSS feeding, the colorectal length gradually shortened and enteritis gradually worsened ( Figure 2 D and 2E). Western Blot results showed that as enteritis worsened, the expression of thioquinone oxidoreductase SQR gradually decreased ( Figure 2 F and 2G). Figure 2 This is a graph showing changes in the expression level of thioquinone oxidoreductase SQR in the acute UC model of the present invention. Figure 2 A: Representative immunohistochemical results of the expression of thioquinone oxidoreductase (SQR) in the colon of WT mice. Red arrows indicate intestinal epithelial cells in the intestinal mucosa. Scale bar: 200 μm. Scale bar for the enlarged image: 100 μm. Figure 2 B: Western Blot results of colonic thioquinone oxidoreductase (SQR) in the DSS-induced acute UC model in mice, with β-tubulin as the internal reference. Figure 2 C: Band grayscale analysis results of thioquinone oxidoreductase SQR; Figure 2 D: Pictures of the colorectum of WT mice after DSS feeding for 3d, 5d, and 7d; Figure 2E: Statistics of colorectal length of WT mice; Figure 2 F: Western Blot results of thioquinone oxidoreductase (SQR) in the colon of mice fed with DSS for 3, 5, and 7 days; Figure 2 G: Band grayscale analysis results of thioquinone oxidoreductase (SQR). Data are expressed as mean ± standard deviation (SD) (ns: no significant difference; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001).

[0125] 2.2 Construction of intestinal epithelial cell-specific thioquinone oxidoreductase SQR knockout mice

[0126] Thioquinone oxidoreductase SQRflox / flox mice were crossed with Villin-Cre mice and expanded for breeding. The tails of their offspring were cut and the genome was extracted. The genotypes were identified by PCR electrophoresis. The results were as follows: Figure 3 As shown in A, mice with intestinal epithelial cell-specific thioquinone oxidoreductase SQR knockout (i.e., thioquinone oxidoreductase SQRCKO mice) were successfully obtained, and the control mice were thioquinone oxidoreductase SQRFL / FL mice. Immunofluorescence staining and Western Blot results further confirmed that thioquinone oxidoreductase SQR was successfully knocked out in the intestinal epithelial cells of thioquinone oxidoreductase SQRCKO mice ( Figure 3 B and 3C). Figure 3 The invention relates to the construction of an intestinal epithelial cell-specific thioquinone oxidoreductase SQR knockout mouse. Figure 3 A: Mouse gene identification PCR electrophoresis, lane 1 identification results are thioquinone oxidoreductase SQRFL / FL mice, lanes 4 and 5 identification results are thioquinone oxidoreductase SQRCKO mice; Figure 3 B: Representative immunofluorescence staining of SQR expression in the colon of SQRFL / FL and SQRCKO mice. White arrows indicate intestinal epithelial cells in the intestinal mucosa. Scale bar: 100 μm. C: Western blot analysis of SQR expression in colorectal epithelial cells of SQRFL / FL and SQRCKO mice, using β-actin as an internal control. Data are expressed as mean ± standard deviation (SD) (ns: not statistically significant; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001).

[0127] Test Example 1

[0128] SQR knockout of thioquinone oxidoreductase promotes weight loss, increased DAI score, and colorectal shortening in DSS-induced acute UC mice

[0129] To explore the role of thioquinone oxidoreductase (SQR) in the development of enteritis, the present invention used 3% DSS to establish an acute UC model in thioquinone oxidoreductase (SQRFL / FL) and thioquinone oxidoreductase (SQRCKO) mice. By monitoring the weight of the mice, it was found that the weight of thioquinone oxidoreductase (SQRFL / FL) mice began to decrease slightly on the 5th day and began to decrease significantly on the 6th day. The weight of thioquinone oxidoreductase (SQRCKO) mice began to decrease from the 2nd day, and the weight on the 5th day was significantly lower than that of mice without DSS treatment ( Figure 4 A). By the end of the 7th day of modeling, the weight loss in SQR knockout mice was significantly greater than that in SQRFL / FL mice. Therefore, SQR knockout promotes weight loss in mice with DSS-induced acute UC.

[0130] The disease activity score results showed that after DSS treatment, the thioquinone oxidoreductase SQRFL / FL mice showed pathological reactions (i.e., weight loss, soft stools, and fecal occult blood) on the 4th day, while the thioquinone oxidoreductase SQRCKO mice showed increased DAI scores starting from the 3rd day, and on the 4th day, the DAI scores increased significantly and were higher than those of the thioquinone oxidoreductase SQRFL / FL mice ( Figure 4 B). Thus, SQR knockout promoted the elevated DAI score in DSS-induced acute UC in mice.

[0131] The colorectum of mice was taken and the length was measured. Figure 4 As shown in Figures C and 4D, there was no difference in colorectal length between SQRFL / FL mice and SQRCKO mice without DSS treatment. Following DSS treatment, the colorectum of both SQRFL / FL and SQRCKO mice shortened, with the shortening being more pronounced in SQRCKO mice. Therefore, SQR knockout promotes colorectal shortening in DSS-induced acute UC in mice.

[0132] Figure 4 Figure 2 shows the effect of the knockout of thioquinone oxidoreductase SQR on the body weight, DAI score, and colorectal length of mice with acute UC. Figure 2 shows the body weight changes of thioquinone oxidoreductase SQRFL / FL mice and thioquinone oxidoreductase SQRCKO mice in the DSS-induced acute UC model. Figure 4 A) DAI score ( Figure 4 B) and representative images of colorectum ( Figure 4C); D: Colorectal length statistics of thioquinone oxidoreductase SQRFL / FL mice and thioquinone oxidoreductase SQRCKO mice. Data are expressed as mean ± standard deviation (SD) (ns: no statistically significant difference; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001).

[0133] Test Example 2

[0134] SQR knockout promotes colorectal injury in DSS-induced acute UC in mice

[0135] H&E staining was used to observe the pathological changes of the colorectum of mice. Figure 5 As shown in A, the colorectal architecture of thioquinone oxidoreductase (SQRFL / FL) and thioquinone oxidoreductase (SQRCKO) mice, which were not treated with DSS, was intact. However, DSS treatment disrupted the epithelial structure of thioquinone oxidoreductase (SQRFL / FL) mice, with partial crypt loss and inflammatory infiltration in the colon. The severity of colonic damage in thioquinone oxidoreductase (SQRCKO) mice was significantly greater than that in thioquinone oxidoreductase (SQRFL / FL), with crypt abscesses, loss, and ulceration, and a significant increase in immune cell infiltration in the lamina propria.

[0136] The colorectal tissue of mice was scored histologically based on the severity of inflammation, depth of injury and infiltration of inflammatory cells. Figure 5 As shown in Figure B, there was no difference in histological scores between SQRFL / FL mice and SQRCKO mice without DSS treatment. However, after DSS treatment, the histological scores of SQRCKO mice were significantly higher than those of SQRFL / FL mice. Therefore, SQR knockout promotes colorectal damage in DSS-induced acute UC in mice.

[0137] Figure 5 This figure shows the effect of knockout of the thioquinone oxidoreductase SQR of the present invention on colorectal damage in acute UC mice. Figure 5 A: Representative H&E staining of the colorectum of SQRFL / FL and SQRCKO mice expressing thioquinone oxidoreductase in the DSS-induced acute UC model. Red arrows indicate crypt damage. Scale bar: 200 μm; scale bar: 100 μm for magnified images. B: Histological scoring of the colorectum of mice. Data are expressed as mean ± standard deviation (SD) (ns: not statistically significant; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001).

[0138] Test Example 3

[0139] SQR knockout promotes inflammatory response in DSS-induced acute UC in mice

[0140] Proinflammatory mediators play a key role in the pathogenesis of IBD, namely UC and CD. When enteritis occurs, significant infiltration of inflammatory-related factors such as TNF-α, IL-1β and IL-6 damages intestinal epithelial cells, thereby causing increased intestinal permeability. This pathological process reflects the important role of inflammatory factors in intestinal health and their potential impact on the development of intestinal diseases. Therefore, the present invention uses real-time fluorescence quantitative PCR to detect the levels of proinflammatory cytokines, and the results are as follows: Figure 6 As shown in A, there was no difference in the expression levels of colorectal proinflammatory cytokines between thioquinone oxidoreductase SQRFL / FL mice and thioquinone oxidoreductase SQRCKO mice without DSS treatment. After DSS treatment, the expression levels of colorectal proinflammatory cytokines TNF-α, IL-1β and IL-6 in thioquinone oxidoreductase SQRCKO mice were significantly higher than those in thioquinone oxidoreductase SQRFL / FL mice.

[0141] Studies have shown that chemokines, as key cytokines, play a key role in the development of IBD. When chemokines bind to their specific receptors, they trigger a complex biological process, namely the recruitment of a large number of Th1 and Th2 cells. This process disrupts the original immune balance in the intestine, thereby promoting the abnormal accumulation of inflammatory mediators and NF-κB in the intestine, exacerbating the inflammatory response. The present invention uses real-time fluorescence quantitative PCR to detect the expression levels of chemokines S100a9, Cxcl1 and Ccl2. The results are as follows: Figure 6 As shown in Figure B, there was no difference in the expression of colorectal chemokines between SQRFL / FL mice and SQRCKO mice without DSS treatment. However, after DSS treatment, the expression levels of S100a9, Cxcl1, and Ccl2 in the colorectal thioquinone oxidoreductase SQRCKO mice were significantly higher than those in SQRFL / FL mice. Therefore, SQR knockout promotes the inflammatory response in DSS-induced acute UC in mice.

[0142] Figure 6Figure 1 shows the effect of SQR knockout on the expression of proinflammatory cytokines and chemokines in mice with acute UC. qRT-PCR analysis of colorectal proinflammatory cytokines IL-6, IL-1β, and TNF-α (A) and chemokines S100a9, Cxcl1, and Ccl2 (B) in mice expressing SQRFL / FL and SQRCKO in the DSS-induced acute UC model. Data are expressed as mean ± standard deviation (SD) (ns: not statistically significant; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001).

[0143] Test Example 4

[0144] SQR knockout reduces intestinal barrier function in DSS-induced acute UC in mice

[0145] The intestinal barrier refers to the entire intestinal defense system. It is a complex structure that absorbs nutrients while ensuring protection against intestinal pathogens and maintaining intestinal immune balance. Dysregulation of the intestinal barrier can lead to pathological responses to tissue damage and microbiota, which is associated with a variety of chronic diseases and systemic diseases, including IBD, intestinal fibrosis and cancer. The intestinal barrier includes the intestinal epithelial barrier, intestinal flora and intestinal immune barrier. Among them, the intestinal epithelial barrier is composed of intestinal epithelial cells, which cover the surface of the intestinal mucosa and are connected together by structural substances such as zonula occludens protein-1 (ZO-1), tight junction protein (Occludin) and mucin 2 (MUC2). When intestinal epithelial cells are damaged, intestinal permeability increases, and some harmful substances (such as toxins, bacteria) and large molecules (such as proteins) pass through the intestinal wall into the body, subsequently exacerbating enteritis.

[0146] In order to evaluate the intestinal permeability of mice, the fluorescence intensity of FITC-Dextran in mouse serum was measured to detect the intestinal permeability. Figure 7As shown in A, there is no difference in the concentration of FITC-Dextran in the serum of thioquinone oxidoreductase SQRFL / FL mice and thioquinone oxidoreductase SQRCKO mice that have not been treated with DSS. The concentration of FITC-Dextran in the serum of thioquinone oxidoreductase SQRCKO mice after DSS treatment is significantly higher than that of thioquinone oxidoreductase SQRFL / FL mice. This indicates that thioquinone oxidoreductase SQR knockout increases the intestinal permeability of DSS-induced acute UC in mice. The present invention further detected the tight junctions of intestinal epithelial cells by transmission electron microscopy and found that both thioquinone oxidoreductase SQRFL / FL mice and thioquinone oxidoreductase SQRCKO mice that have not been treated with DSS showed complete tight junctions. After DSS treatment, the tight junctions of thioquinone oxidoreductase SQRFL / FL mice were relatively intact, while the tight junctions of thioquinone oxidoreductase SQRCKO mice were severely damaged ( Figure 7 B). Consistent with these results, the present invention detected the expression of tight junction proteins occludin and ZO-1 in colon tissue by immunofluorescence and found no difference in the expression of occludin and ZO-1 proteins between thioquinone oxidoreductase SQRFL / FL mice and thioquinone oxidoreductase SQRCKO mice without DSS treatment. After DSS treatment, the expression of occludin and ZO-1 proteins in both mice was reduced, and the expression levels of these two proteins in thioquinone oxidoreductase SQRCKO mice were significantly lower than those in thioquinone oxidoreductase SQRFL / FL mice ( Figure 7 C, 7D, 7E, and 7F). These results indicate that SQR knockout reduces intestinal barrier function in DSS-induced acute UC in mice.

[0147] Figure 7 This figure shows the effect of knockout of the thioquinone oxidoreductase SQR on the intestinal barrier function of acute UC mice. Figure 7 A is a graph of the present invention showing the intestinal permeability of thioquinone oxidoreductase SQRFL / FL mice and thioquinone oxidoreductase SQRCKO mice in the DSS-induced acute UC model by detecting the concentration of FITC-Dextran entering the peripheral blood from the colon cavity; Figure 7 B is a representative transmission electron microscopic image of tight junctions between colonic epithelial cells of thioquinone oxidoreductase SQRFL / FL mice and thioquinone oxidoreductase SQRCKO mice of the present invention, with white arrows indicating tight junctions between colonic epithelial cells; Figure 7 C- Figure 7 F: Immunofluorescence staining to detect tight junction protein Occludin in mouse colon ( Figure 7 C) and ZO-1( Figure 7 E) Representative images of expression, scale bar 200 μm, and fluorescence quantitative statistics ( Figure 7 D and Figure 7F). Data are expressed as mean ± standard deviation (SD) (ns: no statistically significant difference; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001).

[0148] Test Example 5

[0149] SQR knockout promotes intestinal epithelial cell death in DSS-induced acute UC in mice

[0150] The present invention detected the death of colon tissue cells by TUNEL staining, and the results were as follows: Figure 8 As shown, only minimal cell death occurred in the colons of SQRFL / FL mice and SQRCKO mice without DSS treatment, with no significant difference in cell death levels. Following DSS treatment, compared to less than 10% cell death in the colons of SQRFL / FL mice, SQRCKO mice experienced significantly increased cell death, reaching 25%, with most of the death occurring in intestinal epithelial cells. Therefore, SQR knockout promotes intestinal epithelial cell death in DSS-induced acute UC in mice. Figure 8 This figure shows the effect of knockout of the thioquinone oxidoreductase SQR of the present invention on intestinal epithelial cell death in acute UC mice. Figure 8 A: Representative images of TUNEL staining of the colon of thioquinone oxidoreductase SQRFL / FL mice and thioquinone oxidoreductase SQRCKO mice in the DSS-induced acute UC model. White arrows indicate intestinal epithelial cell death. Scale bar: 200 μm. Figure 8 B: Quantitative statistics of TUNEL staining fluorescence. Data are expressed as mean ± standard deviation (SD) (ns: no significant statistical difference; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001).

[0151] Test Example 4

[0152] SQR knockout promotes mitochondrial damage in intestinal epithelial cells

[0153] It is reported that thioquinone oxidoreductase SQR is closely related to mitochondrial homeostasis, and knockout of thioquinone oxidoreductase SQR will aggravate cisplatin-induced mitochondrial dysfunction in renal tubular cells. Therefore, the present invention observed the mitochondrial ultrastructure of intestinal epithelial cells by transmission electron microscopy. The results are as follows Figure 9As shown in Figure A, mitochondria in SQRFL / FL mice without DSS treatment showed intact mitochondrial structure with clearly visible cristae, whereas mitochondria in SQRCKO mice showed partial damage, decreased mitochondrial density, and fragmented cristae. After DSS treatment, mitochondria in intestinal epithelial cells from SQRFL / FL mice showed cristae fragmentation or loss, but more than half of the mitochondria remained relatively intact. However, after SQR knockout, mitochondrial damage, characterized by mitochondrial swelling and cristae fragmentation, was exacerbated, with the mitochondrial damage rate reaching 80%.

[0154] The activity of the mitochondrial ETC complex is inseparable from mitochondrial function. The reduction of ETC complex activity can lead to energy metabolism disorders. According to literature reports, thioquinone oxidoreductase SQR mediates H2S oxidation and participates in mitochondrial ETC electron transfer. Therefore, the present invention will explore whether the knockout of thioquinone oxidoreductase SQR will affect the expression level of ETC complex genes. The present invention used qRT-PCR to detect the expression levels of mitochondrial electron transport chain complex genes Atp5a1, Cox4i1, Uqcrc1 and Ndufab1. The results are as follows Figure 9 As shown in Figure C, compared with SQRFL / FL mice, the expression level of Atp5a1 in SQRCKO mice without DSS treatment was significantly decreased, while the expression levels of Cox4i1 and Uqcrc1 were slightly decreased, but the differences were not significant. After DSS treatment, the expression levels of Atp5a1, Cox4i1, and Ndufab1 genes in SQRCKO mice were significantly lower than those in SQRFL / FL mice. Therefore, SQR knockout reduces the expression levels of ETC complex genes in intestinal epithelial cells.

[0155] Human mitochondrial DNA (mtDNA) is a circular molecule composed of 16,568 bases, encoding ribosomal RNA (rRNA), transfer RNA (tRNA) and important components of mitochondrial ETC. The amount of mtDNA in the mitochondrial genome is called the mtDNA copy number. It is reported that a reduction in the number of mtDNA copies in cells will impair mitochondrial function and play an important role in the occurrence and development of diseases. The present invention uses real-time fluorescence quantitative PCR to detect the copy number of mtDNA in intestinal epithelial cells, and the results are as follows Figure 9As shown in Figure D, the mtDNA copy number in DSS-untreated thioquinone oxidoreductase SQRCKO mice was significantly lower than that in thioquinone oxidoreductase SQRFL / FL mice, indicating that mitochondrial function in thioquinone oxidoreductase SQRCKO mice without DSS treatment was already impaired. After DSS treatment, the mtDNA copy number in both thioquinone oxidoreductase SQRFL / FL and thioquinone oxidoreductase SQRCKO mice decreased, indicating that DSS-induced acute UC reduces mtDNA copy number in intestinal epithelial cells and impairs mitochondrial function in mouse intestinal epithelial cells. In addition, after DSS treatment, the mtDNA copy number in thioquinone oxidoreductase SQRCKO mice was lower than that in thioquinone oxidoreductase SQRFL / FL mice. These results indicate that thioquinone oxidoreductase SQR knockout reduces mtDNA copy number in intestinal epithelial cells and inhibits mitochondrial function.

[0156] Next, the present invention detected the ATP level of mouse intestinal epithelial cells. Figure 9 As shown in Figure E, the ATP level in the intestinal epithelial cells of thioquinone oxidoreductase SQRCKO mice without DSS treatment was significantly lower than that in thioquinone oxidoreductase SQRFL / FL mice, indicating that the mitochondrial energy supply of intestinal epithelial cells in thioquinone oxidoreductase SQRCKO mice is impaired under normal conditions. After DSS treatment, the ATP levels of both mice decreased, indicating that DSS-induced acute UC reduces the energy supply of intestinal epithelial cells. Moreover, the ATP level in the intestinal epithelial cells of thioquinone oxidoreductase SQRCKO mice after DSS treatment was significantly lower than that of thioquinone oxidoreductase SQRFL / FL mice, indicating that thioquinone oxidoreductase SQR knockout reduces the ATP level in intestinal epithelial cells and impairs mitochondrial function.

[0157] In summary, thioquinone oxidoreductase SQR plays an important role in mitochondrial homeostasis, and thioquinone oxidoreductase SQR knockout promotes mitochondrial damage in intestinal epithelial cells.

[0158] Figure 9 This figure shows the effect of knockout of the thioquinone oxidoreductase SQR of the present invention on mitochondrial function in mouse intestinal epithelial cells. Figure 9 A: Representative transmission electron microscopy images of intestinal epithelial cell mitochondria from thioquinone oxidoreductase SQRFL / FL mice and thioquinone oxidoreductase SQRCKO mice in the DSS-induced acute UC model. Red arrows indicate damaged mitochondria. Scale bar: 5 μm. Scale bar for magnified images: 500 nm. Figure 9 B: Statistics of the percentage of damaged mitochondria; Figure 9 C: qRT-PCR of mitochondrial electron transport chain complex genes (including Atp5a1, Cox4i1, Uqcrc1, and Ndufab1) in mouse intestinal epithelial cells. Figure 9 D: mtDNA copy number in mouse intestinal epithelial cells; Figure 9 E: ATP levels in mouse intestinal epithelial cells; data are expressed as mean ± standard deviation (SD) (ns: no significant statistical difference; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001).

[0159] Test Example 5

[0160] The inflammatory response of NCM460 cells to DSS-induced inflammation increased in a dose-dependent manner

[0161] In order to construct an in vitro inflammatory model, the present invention used different concentrations (5, 10, 20 μg / mL) of DSS to stimulate NCM460 cells for 24 h, and then used real-time fluorescence quantitative PCR to detect the expression levels of proinflammatory cytokines TNF-α, IL-6, and IL-1β. The results are as follows: Figure 10 As shown in A, with the increase of DSS concentration, the expression levels of the three key pro-inflammatory cytokines showed a clear upward trend, and this change showed a clear correlation with the dose of DSS. When the DSS concentration was 20 μg / mL, its inflammation induction effect was particularly significant. Therefore, in order to ensure the accuracy and reliability of subsequent experiments, the present invention selected 20 μg / mL as the induction concentration of DSS. At the same time, the present invention also involves the synthesis of siRNA. The present invention collected NCM460 cells 72 hours after the end of the interference, extracted cell proteins, and performed Western Blot experiments to verify the interference efficiency of the siRNA interference fragment at the NCM460 cell protein level. The results are shown in the figure. Figure 10 B, as shown in 1-10-C, the interference efficiency reached 50%, indicating that siRNA successfully interfered with the expression of the thioquinone oxidoreductase SQR gene.

[0162] Figure 10 This is a diagram showing the effects of different concentrations of DSS on the inflammatory response of NCM460 cells. Figure 10 A: NCM460 cells were stimulated with 0, 5, 10, and 20 μg / mL DSS for 24 h, and the expressions of proinflammatory cytokines TNF-α, IL-6, and IL-1β in NCM460 cells were detected by qRT-PCR. Figure 10 B: Representative Western Blot images of thioquinone oxidoreductase SQR in NCM460 cells 72 hours after siRNA interference, with β-tubulin as an internal control; Figure 10 C: Band grayscale analysis results of thioquinone oxidoreductase SQR.

[0163] Test Example 6

[0164] SQR knockdown reduces mitochondrial membrane potential in intestinal epithelial cells

[0165] Mitochondrial membrane potential is one of the important indicators for evaluating mitochondrial function. Studies have shown that increasing mitochondrial membrane potential and ATP levels can restore DSS-induced mitochondrial dysfunction in mouse intestinal epithelial cells. After establishing an in vitro inflammation model, the present invention used TMRM probe to detect the mitochondrial membrane potential of NCM460 cells. The results are as follows: Figure 11 As shown, after knockdown of the thioquinone oxidoreductase SQR, the mitochondrial membrane potential was significantly decreased compared with the control group. After DSS-induced in vitro inflammation, the mitochondrial membrane potential of both the control group and the thioquinone oxidoreductase SQR interference group decreased, and the mitochondrial membrane potential of the thioquinone oxidoreductase SQR interference group was significantly lower than that of the control group. These results indicate that knockdown of the thioquinone oxidoreductase SQR reduces the mitochondrial membrane potential of intestinal epithelial cells and exacerbates DSS-induced mitochondrial dysfunction in intestinal epithelial cells.

[0166] Figure 11 This figure shows the effect of knockdown of the thioquinone oxidoreductase SQR of the present invention on the mitochondrial membrane potential of NCM460 cells. Figure 11 A: After siRNA interference in NCM460 cells for 48 hours, DSS was used to stimulate NCM460 cells for 24 hours to establish an in vitro inflammation model. Representative results of mitochondrial membrane potential detection by TMRM method; Figure 11 B: Quantitative analysis of mitochondrial membrane potential. Data are expressed as mean ± standard deviation (SD) (ns: no significant statistical difference; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001).

[0167] Test Example 7

[0168] Thioquinone oxidoreductase (SQR) maintains mitochondrial dynamics homeostasis in intestinal epithelial cells by inhibiting excessive mitochondrial fission

[0169] Mitochondrial dynamics maintains the mitochondrial network and cellular homeostasis. Imbalances in mitochondrial dynamics are associated with the development and progression of IBD. Imbalances in mitochondrial fission affect mitochondrial function, triggering disrupted energy metabolism and exacerbating excessive ROS production, activating inflammatory signaling pathways. These chain reactions collectively exacerbate mitochondrial dysfunction, posing a serious threat to cellular health.

[0170] The present invention constructs an in vitro inflammation model by stimulating NCM460 cells with DSS, and observes the mitochondrial morphology by mitochondrial fluorescence staining. Figure 12As shown in A, mitochondria in NCM460 cells without DSS treatment exhibit interconnected filamentous and reticular structures, while punctate mitochondria increased after SQR knockdown. Mitochondrial fission in the DSS-treated NC group was significantly increased compared to untreated NCM460 cells, indicating that DSS-induced inflammation promotes mitochondrial fission in NCM460 cells. Mitochondria in the SQR knockdown group exhibited punctate distribution, with completely disrupted filamentous and reticular structures, and the degree of mitochondrial dynamics imbalance was significantly higher than in the DSS-treated NC group. Therefore, SQR knockdown promotes excessive mitochondrial fission in NCM460 cells.

[0171] In addition, the present invention observed the morphology of mouse intestinal epithelial cells mitochondria by transmission electron microscopy and measured the mitochondrial length. Figure 12 As shown in Figures B and 12C, mitochondria in intestinal epithelial cells from thioquinone oxidoreductase SQRFL / FL mice without DSS treatment exhibited both long rods and spherical shapes, with an average mitochondrial length of 0.80 μm. Intestinal epithelial cells from thioquinone oxidoreductase SQRCKO mice exhibited fewer rod-shaped mitochondria and more spherical mitochondria, with an average mitochondrial length of 0.69 μm, which is shorter than the average mitochondrial length in intestinal epithelial cells from thioquinone oxidoreductase SQRFL / FL mice. Following DSS treatment, spherical mitochondria increased in intestinal epithelial cells from thioquinone oxidoreductase SQRFL / FL mice, and the average mitochondrial length decreased to 0.71 μm, indicating that DSS-induced inflammation promotes mitochondrial fission in mouse intestinal epithelial cells. After DSS treatment, most mitochondria in intestinal epithelial cells from thioquinone oxidoreductase SQRCKO mice exhibited spherical shapes, with an average mitochondrial length decreased to 0.51 μm, significantly shorter than the average mitochondrial length in intestinal epithelial cells from thioquinone oxidoreductase SQRFL / FL mice treated with DSS. Therefore, knockout of thioquinone oxidoreductase SQR promotes excessive mitochondrial fission in intestinal epithelial cells.

[0172] DRP1 is a core protein that regulates mitochondrial fission. To further verify the above results, the present invention detected the expression level of DRP1 in intestinal epithelial cells of thioquinone oxidoreductase SQRFL / FL mice and thioquinone oxidoreductase SQRCKO mice by Western Blot method. The results are as follows: Figure 12As shown in Figures D and 12E, the expression level of DRP1 in the intestinal epithelial cells of thioquinone oxidoreductase SQRCKO mice without DSS treatment was significantly higher than that in thioquinone oxidoreductase SQRFL / FL mice, indicating that thioquinone oxidoreductase SQR knockout promoted excessive mitochondrial fission in intestinal epithelial cells. Compared with thioquinone oxidoreductase SQRFL / FL mice without DSS treatment, the expression of DRP1 in the intestinal epithelial cells of thioquinone oxidoreductase SQRFL / FL mice increased after DSS treatment, indicating that DSS-induced inflammation promotes mitochondrial fission, which is consistent with the research results of Vincent et al. After DSS treatment, the expression level of DRP1 in the intestinal epithelial cells of thioquinone oxidoreductase SQRCKO mice was further increased compared with that without DSS treatment, and was significantly higher than that of thioquinone oxidoreductase SQRFL / FL mice treated with DSS. Therefore, thioquinone oxidoreductase SQR knockout promoted excessive mitochondrial fission in intestinal epithelial cells, which is consistent with the previous research results of the present invention.

[0173] In summary, thioquinone oxidoreductase SQR inhibits excessive mitochondrial fission by regulating the level of DRP1, thereby maintaining mitochondrial dynamics homeostasis in intestinal epithelial cells.

[0174] Figure 12 This figure shows the effect of knockout of the thioquinone oxidoreductase SQR of the present invention on mitochondrial dynamics in intestinal epithelial cells. Figure 12 A: After siRNA interference in NCM460 cells for 48 hours, DSS was used to stimulate NCM460 cells for 24 hours to establish an in vitro inflammatory model. Representative results of mitochondrial morphology were detected by laser confocal microscopy. Scale bar: 20 μm, scale of the magnified image: 5 μm. Figure 12 B: Representative transmission electron microscopy images of mitochondria in intestinal epithelial cells of thioquinone oxidoreductase SQRFL / FL mice and thioquinone oxidoreductase SQRCKO mice in the DSS-induced acute UC model. The red line represents the mitochondrial length measurement. Figure 12 C: Quantitative analysis of mitochondrial length in transmission electron microscopy images; Figure 12 D: Western blot images of thioquinone oxidoreductase SQR and DRP1 in intestinal epithelial cells of thioquinone oxidoreductase SQRFL / FL mice and thioquinone oxidoreductase SQRCKO mice in the DSS-induced acute UC model, with β-actin as the internal reference; E: Grayscale analysis results of DRP1 bands. Figure 12 F- Figure 12 G: Representative images of immunofluorescence staining for DRP1 expression in mouse colon, scale bar 100 μm, and fluorescence quantitative statistics were performed (G). Data are expressed as mean ± standard deviation (SD) (ns: no significant statistical difference; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001).

[0175] Test Example 8

[0176] Thioquinone oxidoreductase (SQR) inhibits ROS production by regulating mitochondrial dynamics in intestinal epithelial cells

[0177] Mitochondria are the primary site of reactive oxygen species (ROS) production, accounting for approximately 90% of cellular ROS. Studies have shown that mitochondrial ROS are a crucial component of host defense and immunity. However, under pathological conditions, high levels of ROS can combine with iron to generate free radicals, damaging macromolecules within mitochondria and oxidizing mtDNA. Because mtDNA is located in close proximity to the ETC (the source of mitochondrial ROS), it is susceptible to oxidative damage from mitochondrial ROS. In this setting, ROS are a driving factor in the inflammatory response. Increased ROS production in the colonic mucosa of patients with active UC has been reported, and excessive ROS may trigger an imbalance in the oxidative state, leading to oxidative damage to intestinal tissue, inflammation, and apoptosis.

[0178] The present invention firstly constructed an in vitro inflammatory model by stimulating NCM460 cells with DSS for 24 h, and detected the ROS level of NCM460 cells by flow cytometry. Figure 13 As shown in A and 13B, before DSS treatment, the ROS level in the thioquinone oxidoreductase SQR interference group was significantly higher than that in the NC group. After DSS treatment, the ROS levels in both the NC and interference groups were significantly upregulated, indicating that DSS-induced inflammation promoted the increase in ROS levels in NCM460 cells. After DSS treatment, the ROS level in the thioquinone oxidoreductase SQR interference group was significantly higher than that in the NC group, indicating that knockdown of the thioquinone oxidoreductase SQR promoted the increase in ROS levels in NCM460 cells.

[0179] The present invention further detected the ROS level in intestinal epithelial cells of thioquinone oxidoreductase SQRFL / FL mice and thioquinone oxidoreductase SQRCKO mice. Figure 13 As shown in Figure C, ROS levels in intestinal epithelial cells of thioquinone oxidoreductase SQRCKO mice without DSS treatment were higher than those in thioquinone oxidoreductase SQRFL / FL mice, but the difference was not significant. After DSS treatment, ROS levels in intestinal epithelial cells of both mouse models increased, and ROS levels in intestinal epithelial cells of thioquinone oxidoreductase SQRCKO mice were significantly higher than those in thioquinone oxidoreductase SQRFL / FL mice, which is consistent with the changes in DRP1 expression. This suggests that thioquinone oxidoreductase SQR knockout leads to increased ROS levels by promoting excessive mitochondrial fission in intestinal epithelial cells. In summary, thioquinone oxidoreductase SQR inhibits excessive ROS production by regulating mitochondrial dynamics in intestinal epithelial cells, thereby maintaining normal cellular redox levels.

[0180] Figure 13This figure shows the effect of knocking out the thioquinone oxidoreductase SQR of the present invention on the ROS level in intestinal epithelial cells. Figure 13 A: After siRNA interference in NCM460 cells for 48 hours, DSS stimulation of NCM460 cells for 24 hours was used to establish an in vitro inflammatory model. Representative results of DCFH probe detection of cellular ROS levels. Figure 13 B: Quantitative analysis results of ROS levels; Figure 13 C: ROS levels in intestinal epithelial cells from SQRFL / FL and SQRCKO mice expressing thioquinone oxidoreductase in the DSS-induced acute UC model. Data are expressed as mean ± standard deviation (SD) (ns: not statistically significant; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001).

[0181] In recent years, a growing body of research suggests that mitochondrial function is crucial for maintaining intestinal health, and that mitochondrial dysfunction is associated with the pathogenesis of IBD. However, the role of thioquinone oxidoreductase (SQR), a mitochondrial inner membrane protein, in the progression of IBD has not yet been reported. An acute ulcerative colitis model was established by administering 3% DSS solution to wild-type male mice for 7 days. Western blot analysis revealed downregulation of SQR expression.

[0182] To further explore the role of thioquinone oxidoreductase (SQR) in the progression of UC, the present invention constructed intestinal epithelial cell-specific thioquinone oxidoreductase (SQR) knockout mice. The results showed that thioquinone oxidoreductase (SQR) knockout aggravated DSS-induced acute UC in mice. Specifically, compared with thioquinone oxidoreductase (SQRFL / FL) mice, thioquinone oxidoreductase (SQRCKO) mice showed faster weight loss, higher DAI scores, more significant colorectal shortening, more severe colorectal damage, and higher expression levels of proinflammatory cytokines and chemokines after DSS treatment. Intestinal barrier function plays an important role in the progression of UC. The present invention used FITC-Dextran to detect intestinal permeability and transmission electron microscopy to observe tight junctions between intestinal epithelial cells and found that thioquinone oxidoreductase (SQR) knockout reduced the intestinal barrier function of DSS-induced acute UC in mice. Subsequent immunofluorescence staining of ZO-1 and Occludin tight junction proteins further confirmed this result. TUNEL staining results showed that after DSS treatment, the level of colon cell death in mice expressing the thioquinone oxidoreductase (SQRCKO) was significantly higher than that in mice expressing the thioquinone oxidoreductase (SQRFL / FL), and was primarily concentrated in the intestinal epithelial cells. Therefore, subsequent studies in this paper will focus on intestinal epithelial cells. The results of this paper suggest that the thioquinone oxidoreductase (SQR) plays an important role in DSS-induced acute ulcer disease and is essential for ameliorating excessive inflammation in colonic epithelial cells.

[0183] Mitochondrial function is crucial for maintaining the activity of mitochondrial respiratory chain complexes, ATP synthesis, and mitochondrial membrane potential. Mitochondrial dysfunction is primarily manifested by altered mitochondrial morphology and structure, decreased ATP production, mtDNA abnormalities, excessive ROS production, and decreased mitochondrial membrane potential. Numerous studies have shown that mitochondrial dysfunction in intestinal epithelial cells contributes to the development and progression of IBD. Therefore, the present invention hypothesizes that knockout of the thioquinone oxidoreductase (SQR) leads to mitochondrial dysfunction in intestinal epithelial cells, thereby exacerbating DSS-induced acute ulcerative colitis in mice.

[0184] The present study used transmission electron microscopy to observe the mitochondrial ultrastructure of intestinal epithelial cells from mice expressing the enzyme thioquinone oxidoreductase (SQRFL / FL) and mice expressing the enzyme thioquinone oxidoreductase (SQRCKO). The study found that knockout of the enzyme thioquinone oxidoreductase (SQR) exacerbated mitochondrial damage in intestinal epithelial cells. To further validate this finding, the present study examined the expression levels of mitochondrial ETC complex genes, mtDNA copy number, and ATP levels in intestinal epithelial cells. The results showed that knockout of the enzyme thioquinone oxidoreductase (SQR) reduced ETC complex gene expression, mtDNA copy number, and ATP levels.

[0185] The present invention established an in vitro inflammation model and used a TMRM probe to measure the mitochondrial membrane potential of NCM460 cells. The results showed that knockdown of the thioquinone oxidoreductase (SQR) reduced the mitochondrial membrane potential of NCM460 cells. These results suggest that the thioquinone oxidoreductase (SQR) regulates mitochondrial function in intestinal epithelial cells through multiple effects, maintaining mitochondrial homeostasis in intestinal epithelial cells.

[0186] The role of the discovered thioquinone oxidoreductase SQR in acute ulcerative colitis, the present invention found that: thioquinone oxidoreductase SQR regulates the ROS level of intestinal epithelial cells by inhibiting excessive mitochondrial fission and promoting mitochondrial biogenesis, thereby maintaining the homeostasis of mitochondrial function in intestinal epithelial cells. When epithelial cells are stimulated by DSS, the expression of thioquinone oxidoreductase SQR is downregulated, and the expression of DRP1 is upregulated, which promotes excessive mitochondrial fission and excessive ROS production, causing oxidative stress in intestinal epithelial cells. In addition, the downregulation of thioquinone oxidoreductase SQR will lead to the downregulation of PGC1α and the mitochondrial antioxidant genes and uncoupling protein genes it regulates, and the ROS scavenging ability is weakened, which leads to a large accumulation of ROS, thereby triggering mitochondrial dysfunction and ultimately the death of intestinal epithelial cells. Targeting thioquinone oxidoreductase SQR may be a potential strategy for the future clinical treatment of IBD.

[0187] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and improvements may be made to the present invention without departing from the spirit and scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims, the description, and their equivalents.

Claims

1. Use of a thioquinone oxidoreductase in the preparation of a medicament for treating inflammatory bowel disease, characterized in that: The inflammatory bowel disease is ulcerative colitis.

2. The use according to claim 1, characterized in that: The drug can enhance the mitochondrial function of intestinal epithelial cells, thereby alleviating the symptoms of inflammatory bowel disease, including one or more of the following: reducing intestinal epithelial cell damage; reducing the expression levels of proinflammatory cytokines and chemokines; improving intestinal barrier function; and reducing intestinal epithelial cell death.

3. A pharmaceutical composition for treating inflammatory bowel disease, characterized in that: The pharmaceutical composition comprises a thioquinone oxidoreductase agonist as an active ingredient, and the inflammatory bowel disease is ulcerative colitis.

4. The pharmaceutical composition according to claim 3, wherein: The thioquinone oxidoreductase agonist can enhance the mitochondrial function of intestinal epithelial cells, including one or more of the following: Enhance the activity of mitochondrial electron transport chain complexes; Increased mitochondrial DNA copy number; Increase ATP levels in intestinal epithelial cells; Inhibit excessive mitochondrial fission and maintain mitochondrial dynamics homeostasis in intestinal epithelial cells; Reduce the level of reactive oxygen species in intestinal epithelial cells.

5. The pharmaceutical composition according to claim 1, wherein: The thioquinone oxidoreductase agonist is selected from the following compounds: sodium sulfide NaHS, hydrogen sulfide H2S donor GYY4137 and 5-(4-hydroxyphenyl)-3H-1,2-disulfide-3-thione ADT-OH.

6. A method for preventing or treating inflammatory bowel disease, characterized in that The method comprises the following steps: administering the pharmaceutical composition of claim 6 to an individual in need thereof.

7. The method according to claim 6, characterized in that: The individual is a patient suffering from ulcerative colitis; the administration method includes oral administration, injection or topical administration.

8. A model for screening drugs for treating inflammatory bowel disease, characterized in that: The model is constructed based on the role of thioquinone oxidoreductase in regulating the mitochondrial function of intestinal epithelial cells, and is used to screen compounds that can regulate the activity of thioquinone oxidoreductase and thus improve the mitochondrial function of intestinal epithelial cells.

9. The model according to claim 8, characterized in that: The model is constructed by constructing an intestinal epithelial cell-specific thioquinone oxidoreductase knockout animal model and inducing inflammatory bowel disease to evaluate the effects of candidate compounds on intestinal epithelial cell mitochondrial function and related inflammatory responses.

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