Use of 2,6-dimethoxy-1,4-benzoquinone for the preparation of a medicament for the treatment of colitis and / or peritonitis

Abstract

The application relates to application of 2,6-dimethoxy-1,4-benzoquinone in preparation of a drug for treating colitis and / or peritonitis. The application provides a new choice for treatment of colitis and / or peritonitis, solves the problem of obvious side effects of the existing drugs for treating ulcerative colitis, solves the problem of drug resistance caused by the existing drugs for treating peritonitis, and provides a safer, more efficient and more potential drug for treatment of ulcerative colitis and acute peritonitis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to the use of 2,6-dimethoxy-1,4-benzoquinone in the preparation of a drug for treating colitis and / or peritonitis. BACKGROUND

[0002] Ulcerative colitis (UC) is a common and difficult-to-cure chronic nonspecific inflammatory bowel disease (IBD) in clinic, which has the characteristics of continuity and diffuse distribution, mainly involving the colonic mucosa and submucosa. The typical clinical manifestations of the disease include recurrent mucopurulent stool, diarrhea, tenesmus, abdominal pain, and other symptoms. Some patients may also have fever, weight loss and other systemic symptoms. UC has complex and diverse clinical manifestations, is difficult to diagnose, has alternating remission and active periods, and is prone to recurrence throughout life, which seriously affects the quality of life of patients.

[0003] In terms of pathogenesis, current research suggests that UC is the result of the combined effects of genetic susceptibility, environmental factors, intestinal flora imbalance, immune regulation abnormalities, and intestinal epithelial barrier dysfunction. Genome-wide association studies (GWAS) have identified more than 200 UC-related susceptibility gene loci, such as IL-23R and ATG16L1. These genes are involved in host-microbe interactions and immune regulation processes. Environmental triggers can alter the composition and metabolic products of intestinal flora, and disrupt intestinal mucosal immune tolerance. In the pathological process, activated innate immune cells (such as neutrophils and macrophages) and adaptive immune cells (such as Th1 and Th17 cells) massively infiltrate the intestinal mucosal lamina propria, release pro-inflammatory factors (TNF-α, IL-6, IL-17, etc.), and insufficient secretion of anti-inflammatory factors (IL-10, TGF-β), leading to uncontrolled inflammatory response. This imbalance in the immune microenvironment further exacerbates the abnormal expression of intestinal epithelial tight junction proteins (occludin and claudin-2), forming a vicious cycle of "inflammation-barrier damage-bacterial translocation".

[0004] Clinical studies have shown that multiple inflammation-related signaling pathways are abnormally activated in the intestinal mucosa of UC patients, including key pathways such as NF-κB and MAPK. Overactivation of these pathways leads to a significant increase in the secretion of pro-inflammatory cytokines such as IL-1β, IL-18, and others. These cytokines promote the release of more inflammatory mediators through a complex network, forming a positive feedback loop. It is particularly noteworthy that inflammation-mediated intestinal epithelial cell death, including apoptosis and necrosis, can lead to a decrease in the number of goblet cells and a thinning of the mucus layer, ultimately resulting in the destruction of both the physical and chemical barriers of the intestinal mucosa. Animal experiments have confirmed that regulating these inflammatory pathways can significantly reduce the pathological damage of experimental colitis, providing important evidence for the development of new treatment strategies.

[0005] Acute peritonitis is a common clinical acute inflammatory disease of the abdominal cavity, and its typical features include abdominal vascular dilation, increased permeability, accumulation of inflammatory exudate, and accompanying severe abdominal pain, rebound pain, and systemic inflammatory response syndrome (SIRS). In experimental studies, alum (potassium aluminum sulfate) is widely used as a classic inorganic salt adjuvant to establish an acute peritonitis model due to its stable crystal structure and clear immune stimulating properties. After intraperitoneal injection of alum, the positively charged aluminum salt crystals can be taken up by peritoneal macrophages, dendritic cells, and other innate immune cells through phagocytosis. This process can cause changes in lysosomal membrane permeability, leading to potassium ion efflux and inducing mitochondrial dysfunction, resulting in a burst of reactive oxygen species (ROS). These cellular stress responses can activate multiple inflammatory signaling pathways, and these inflammatory mediators can infiltrate the abdominal cavity through immune cells such as neutrophils and monocytes, activate TLR4 / MyD88 signaling pathways, and ultimately form an "inflammatory factor storm" leading to typical acute peritonitis pathological changes.

[0006] UC is one of the common inflammatory bowel diseases in China, and its pathogenesis is complex, involving multiple factors such as genetics, environment, and immunity. Acute peritonitis is a severe abdominal inflammation caused by infection, chemical stimulation, or organ perforation, and both conditions exhibit excessive inflammatory responses.

[0007] Current clinical treatment of UC mainly relies on different treatment methods such as aminosalicylates, immunosuppressants, and biological agents. These methods can control disease progression to some extent, but they generally have significant side effects such as abdominal pain, diarrhea, and fever, which severely affect patient treatment adherence and quality of life. Similarly, the treatment of acute peritonitis mainly uses a combination of drug therapy and surgical intervention, but the widespread use of antibiotics can easily lead to drug resistance problems and lack of specific treatment methods. Therefore, it is particularly important to develop a new drug that can treat colitis or peritonitis with low side effects and high efficiency. SUMMARY

[0008] Therefore, the application aims to provide an application of 2,6-dimethoxy-1,4-benzoquinone in the preparation of a drug for treating colitis and / or peritonitis, to provide a new option for the treatment of colitis and / or peritonitis, to solve the problem of obvious side effects of the existing drugs for treating ulcerative colitis, and to solve the problem of drug resistance caused by the existing drugs for treating peritonitis, so as to provide a safer, more efficient and more potential drug for treating ulcerative colitis and acute peritonitis.

[0009] In order to achieve the above-mentioned purpose, the technical scheme adopted by the application is as follows:

[0010] 2,6-dimethoxy-1,4-benzoquinone in the preparation of a drug for treating colitis.

[0011] Among them, the natural chemical substance 2,6-dimethoxy-1,4-benzoquinone (2,6-dimethoxy-1,4-benzoquinone, DMQ) is the main bioactive component in fermented wheat germ extract, exists in various plants, and can regulate various biological processes such as electron transfer activity and oxidative phosphorylation. DMQ can increase skeletal muscle mass and performance by regulating the AKT / mTOR signaling pathway and enhancing mitochondrial function, and may be helpful for the treatment and prevention of skeletal muscle atrophy. DMQ has significant anti-tumor activity, has a preventive effect on TPA-induced skin cancer, has a significant inhibitory effect on 4- (methyl nitrosamine) -1- (3-pyridyl) -1-butanone (NNK) induced lung tumors in mice, and can also inhibit the growth of gastric cancer cells and induce apoptosis of gastric cancer cells. DMQ can reduce the expression of mTOR signaling pathway Ki-67, phosphorylated mTOR and phosphorylated p70S6K in gastric cancer cells, and is a new type of mTOR protein kinase inhibitor. In addition, DMQ also plays an important role in fat formation inhibition, and can significantly reduce the expression of various fat formation transcription factors, including peroxisome proliferator-activated receptor-γ (PPARs-γ) and CCAAT / enhancer binding protein α (CEBPα) as well as adipocyte protein 2 (aP2) and fatty acid synthase. It is reported that DMQ has good anti-inflammatory activity and can inhibit LPS-induced NO production in a dose-dependent manner, but the role of DMQ in ulcerative colitis (Ulcerative Colitis, UC) and acute peritonitis has not been reported. Therefore, the present inventors found that DMQ has a significant therapeutic effect on ulcerative colitis and acute peritonitis by using a dextran sulfate sodium salt (DSS) induced C57BL / 6J mouse model of ulcerative colitis and an alum induced acute peritonitis model, thereby providing a new way for the clinical treatment of UC and acute peritonitis.

[0012] Due to the problem that existing drugs for treating ulcerative colitis generally have obvious side effects, and the problem that existing drugs for treating peritonitis are prone to cause drug resistance. The limitations of these drugs in clinical treatment promote the development of natural anti-inflammatory ingredients. The present inventors found in long-term experimental research that 2, 6-dimethoxy-1, 4-benzoquinone can effectively control the inflammatory cascade reaction by regulating the release of key inflammatory factors such as IL-1β, IL-18, etc., and reduce the secondary damage of inflammation storm to the tissue. And 2, 6-dimethoxy-1, 4-benzoquinone as a natural chemical substance, while ensuring the efficacy, significantly reduces the risk of adverse reactions caused by traditional drug treatment, effectively reduces the side effects, solves the problem that existing drugs for treating ulcerative colitis generally have obvious side effects, provides a new choice for the treatment of inflammatory diseases, thereby solving the problem that existing drugs for treating peritonitis are prone to cause drug resistance. Traditional Chinese medicine and its natural active ingredients (extracts) are China's traditional advantage resources, which are widely used in the prevention and treatment of various chronic diseases due to their advantages of multi-target, high safety coefficient, etc. Chinese herbal medicine natural products have multiple effects such as regulating immune cell differentiation, reducing inflammatory response and oxidative stress damage, regulating intestinal flora, and restoring intestinal mucosal barrier function, which have significant advantages in reducing adverse reactions and preventing recurrence. Thus, a safer and more efficient treatment is provided for patients with ulcerative colitis and acute peritonitis.

[0013] Preferably, the amount of 2, 6-dimethoxy-1, 4-benzoquinone is 20 mg / kg.

[0014] Experimental research shows that the 2, 6-dimethoxy-1, 4-benzoquinone of the present application has a comparable effect of improving colitis at a low dose (20 mg / kg) compared with myricetin (40 mg / kg), bilirubin (30 mg / kg) and naringin (300 mg / kg). Thus, it is proved that DMQ may have higher bioavailability and stronger anti-inflammatory activity, which provides important experimental basis for the development of new anti-colitis drugs.

[0015] Preferably, the drug for treating colitis is made by adding pharmaceutical excipients with 2, 6-dimethoxy-1, 4-benzoquinone as the active ingredient.

[0016] Preferably, the dosage form of the drug is at least one of tablets, capsules, granules, powders, patches, suspensions, syrups, oral liquids, injections and suppositories.

[0017] Preferably, the colitis is ulcerative colitis.

[0018] Preferably, the ulcerative colitis is dextran sulfate sodium salt (DSS) induced ulcerative colitis.

[0019] Preferably, the 2,6-dimethoxy-1,4-benzoquinone is used to reduce the secretion level of IL-1β in colon tissue.

[0020] Preferably, the 2,6-dimethoxy-1,4-benzoquinone is used to reduce the secretion level of IL-17A in mesenteric lymph nodes and spleen. + CD4 + T cell ratio.

[0021] The present application also provides the use of 2,6-dimethoxy-1,4-benzoquinone in the preparation of a drug for treating peritonitis.

[0022] Preferably, the 2,6-dimethoxy-1,4-benzoquinone is used in an amount of 20 mg / kg.

[0023] Experimental studies have found that the 2,6-dimethoxy-1,4-benzoquinone of the present application has comparable or even better therapeutic effect at a low dose (20 mg / kg) compared to myricetin (40 mg / kg), bilirubin (30 mg / kg) and naringin (300 mg / kg). Thus, it is proved that DMQ may have higher bioavailability and stronger anti-inflammatory activity, which provides an important clue for the development of new anti-peritonitis drugs.

[0024] Preferably, the drug for treating peritonitis takes the 2,6-dimethoxy-1,4-benzoquinone as an active ingredient and is prepared by adding pharmaceutical excipients.

[0025] Preferably, the drug is at least one of a tablet, a capsule, a granule, a powder, a patch, a suspension, a syrup, an oral solution, an injection and a suppository.

[0026] Preferably, the peritonitis is acute peritonitis.

[0027] Preferably, the peritonitis is aluminum potassium sulfate (Alum) induced acute peritonitis.

[0028] In experimental studies, alum (potassium aluminum sulfate) is widely used to establish acute peritonitis model as a classic inorganic salt adjuvant due to its stable crystal structure and clear immune stimulating properties. After intraperitoneal injection of alum, the positively charged aluminum salt crystals can be taken up by peritoneal macrophages, dendritic cells and other innate immune cells through phagocytosis. This process can cause changes in lysosomal membrane permeability, leading to potassium ion efflux and inducing mitochondrial dysfunction, resulting in reactive oxygen species (ROS) burst. These cellular stress responses can activate multiple inflammatory signaling pathways, induce inflammatory cell death, and release a large amount of damage-associated molecular patterns (DAMPs). These inflammatory mediators infiltrate the peritoneal cavity through immune cells such as neutrophils and monocytes, activate TLR4 / MyD88 signaling pathways, and ultimately form an 'inflammatory factor storm', leading to typical acute peritonitis pathological changes.

[0029] Preferably, the 2,6-dimethoxy-1,4-benzoquinone is used to inhibit the secretion of IL-1β in the eyeball blood and peritoneal fluid.

[0030] Preferably, the 2,6-dimethoxy-1,4-benzoquinone is used to reduce the proportion and number of Ly6G + CD11b + neutrophils in the peritoneal lavage fluid.

[0031] Advantages of the present application:

[0032] The application of the 2,6-dimethoxy-1,4-benzoquinone in the preparation of a drug for treating colitis has the following significant beneficial effects compared with the prior art: 1) significantly reducing side effects and improving drug safety. The existing drugs for treating ulcerative colitis (such as 5-aminosalicylic acid, glucocorticoids or immunosuppressants) are often accompanied by obvious side effects such as gastrointestinal irritation, liver and kidney function damage, and immune system suppression. 2,6-dimethoxy-1,4-benzoquinone can selectively inhibit the inflammatory response of colonic mucosa through its unique anti-inflammatory and antioxidant mechanism, reduce the release of pro-inflammatory factors (such as IL-1β), and at the same time avoid non-specific damage to normal tissue cells, thereby significantly reducing gastrointestinal discomfort, infection risk and organ toxicity, especially for patients who need long-term medication. 2) effectively overcoming drug resistance and improving treatment potential. Traditional antibiotic or anti-inflammatory drugs are prone to drug resistance when treating peritonitis due to pathogen mutation or compensatory activation of inflammatory pathways, affecting efficacy. 2,6-dimethoxy-1,4-benzoquinone can synergistically inhibit the inflammatory cascade through multi-target action (such as regulating the NF-κB and NLRP3 inflammasome pathways and scavenging reactive oxygen free radicals), and its non-antibiotic properties can avoid directly inducing microbial drug resistance, providing a more sustainable treatment for recurrent or drug-resistant peritonitis. 3) high efficiency, 2,6-dimethoxy-1,4-benzoquinone shows significant anti-inflammatory effect in in vitro and in vivo experiments. It can effectively inhibit the secretion of inflammatory cytokines, significantly reduce the secretion level of IL-1β in serum and peritoneal fluid, and thus rapidly relieve inflammatory symptoms and accelerate the healing process of ulcerative colitis and acute peritonitis, improving treatment efficiency. 4) dual indications expand, broad clinical application prospect, 2,6-dimethoxy-1,4-benzoquinone shows excellent efficacy in ulcerative colitis and acute peritonitis models: ulcerative colitis: significantly reduces colonic tissue edema, ulcer and crypt structure damage, and reduces disease activity index (DAI); acute peritonitis: rapidly inhibits inflammatory mediators in peritoneal exudate, reduces bacterial translocation and peritoneal adhesion. 5) natural source advantage, mature synthesis process. 2,6-dimethoxy-1,4-benzoquinone can be extracted from plants (such as clove and salvia miltiorrhiza) or obtained by chemical synthesis, and the raw material is easy to obtain and the structure is clear, which is convenient for industrialized production. Its stable physicochemical properties (such as moderate lipid solubility) are conducive to the preparation of oral preparations (tablets, capsules) or topical administration preparations (enema, peritoneal perfusion), meeting different clinical needs. Therefore, the 2,6-dimethoxy-1,4-benzoquinone provided by the application has high efficiency, safety and broad-spectrum anti-inflammatory properties, and provides a breakthrough solution for the treatment of ulcerative colitis and acute peritonitis, which has significant clinical value and social and economic benefits. In the field of biomedical materials technology, it has popularization and application value. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1Figures of the results of the changes in the body weight and disease activity index (DAI) of mice in each group over time;

[0034] Figure 2 Figures of the results of the colon length, spleen weight, HE staining and histological score of mice in each group;

[0035] Figure 3 Figures of the results of flow cytometry detection of IL-17A + CD4 + T cells and IFN-γ + CD8 + T cells;

[0036] Figure 4 Figures of the results of Western blot detection of tight junction proteins (Occludin, ZO-1);

[0037] Figure 5 Figures of the results of the effect of DMQ on the activation of NLRP3 inflammasome in the intestinal tissues of mice;

[0038] Figure 6 Figures of the results of flow cytometry and ELISA detection of DMQ in relieving peritonitis in mice. DETAILED DESCRIPTION

[0039] The present application will be described in detail with reference to preferred embodiments. Other advantages and effects of the present application can be easily understood by those skilled in the art from the contents disclosed in the present specification. The present application can also be implemented or applied in other different specific embodiments, and various modifications or changes can be made to the details in the present specification based on different views and applications without departing from the spirit of the present application. It should be understood that the preferred embodiments are only for illustrating the present application, but not for limiting the protection scope of the present application.

[0040] Experimental reagents and materials

[0041] 2,6-dimethoxy-1,4-benzoquinone belongs to a natural plant extraction small molecule compound, which is the main active ingredient of fermented wheat germ extract, and its abbreviation is DMQ, the molecular formula is C8H8O4, and the molecular structure formula is as shown in the following formula:

[0042] Table 1: Mice used in the experiment

[0043]

[0044] Table 2: Consumables involved in the experiment

[0045]

[0046] Table 3: Antibody information

[0047]

[0048] Table 4: Experimental reagents involved

[0049]

[0050] Example 1

[0051] DSS-induced ulcerative colitis experiment

[0052] 1. Animals and grouping: SPF level 8-week-old male C57BL / 6J mice with a body weight of 24-26 g were randomly divided into three groups, and each mouse was numbered: a blank control group (Control group, n=6), a DSS-induced colitis model group (DSS group, n=6), and a DSS-induced colitis model group treated with DMQ (DMQ+DSS group, n=6).

[0053] 2. Model establishment and treatment: The blank control group was continuously provided with normal drinking water. The DSS-induced colitis model group was provided with 3% DSS drinking water for 6 consecutive days, and new 3% DSS drinking water was replaced every two days to ensure drug efficacy. On the 7th day, normal high-pressure tap water was replaced, and sterile PBS was injected intraperitoneally for 10 consecutive days. The DSS-induced colitis model group treated with DMQ was injected intraperitoneally with DMQ (20 mg / kg) for 10 consecutive days, and 3% DSS drinking water was provided for the first 6 days, and normal high-pressure tap water was replaced on the 7th day.

[0054] 3. Observation and recording: The body weight of the mice was monitored and recorded at fixed times every day. The disease activity of the mice was observed, including the following indicators: loose stool, bloody stool, diarrhea, hair standing, rectal prolapse, etc.

[0055] 4. Experimental results and sample collection: On the 11th day of the experiment, the mice were sacrificed. The mesenteric lymph nodes and spleen were extracted, and flow cytometry was used to analyze the changes in Th and Tc cell subsets. The colon length of the mice was measured and photographed. Part of the colon was selected for HE staining and histopathological scoring. The colon tissue samples were stored at low temperature, the samples were ground, and the protein was extracted. ELISA was used to detect the secretion of inflammatory factors IL-1β and TNF-α in the supernatant, and Western blot was used to detect the expression of tight junction proteins and NLRP3 inflammasome-related component proteins.

[0056] Detection analysis

[0057] Analysis of body weight changes and disease activity index (DAI) of mice in each group

[0058] DAI (disease activity index) is determined by the following five aspects: loose stool (+1 point): stool is normal and granular, and loose stool represents soft stool; diarrhea (1 point): water-like stool appears; blood in stool detection (+1 point): blood in the anus or stool is directly observed; rectal prolapse (+1 point): the part of the rectum close to the anus is prolapsed from the anus, and swollen and red tissue can be seen at the anus; hair erection (+1 point): normal mouse hair is smooth, dense and lustrous, and when the disease occurs, the hair is disheveled, dull and may appear "erect hair" phenomenon.

[0059] The results are shown in Figure 1 .

[0060] Among them, Figure 1 A is the change of mouse body weight, Figure 1 B is the change of mouse disease activity index.

[0061] From the analysis in Figure 1 , it can be seen that the DMQ intervention group significantly improves the weight loss of mice, and the disease activity index (DAI) is also significantly reduced, thereby proving that through the DMQ intervention in Example 1 of the present application, the weight loss of mice caused by DSS is effectively alleviated and the progress of disease activity is delayed.

[0062] 2) Analysis of colon length, spleen weight, HE staining and histological score of mice in each group

[0063] The specific operation steps of HE staining are as follows: the colon tissue of the mouse is taken out, and the tissue is fixed, paraffin-embedded and paraffin sectioned, and then conventional hematoxylin-eosin staining is carried out (mainly through melting wax, transparency, hydration, staining nucleus and cytoplasm, dehydration, transparency and gum sealing), and the colon inflammatory cell infiltration and mucosal damage are observed under a microscope, and the colon histopathology score is carried out.

[0064] The histological score method: the score range of this scale is 0-4, 0 represents no inflammation; 1 represents a small amount of inflammatory cell infiltration in the lamina propria; 2 indicates that single cell infiltration leads to crypt structure separation, accompanied by mild mucosal hyperplasia; 3 shows a large number of inflammatory cell infiltration, leading to mucosal structure disorder, reduction of goblet cells and obvious mucosal hyperplasia; 4 represents crypt abscess and ulcer formation. The higher the score, the more severe the degree of intestinal inflammation. The results are shown in Figure 2 .

[0065] Among them, Figure 2 A and 2B show the change of colon length in each group, Figure 2 C shows the change of spleen weight, Figure 2 D and 2E show HE staining and histological score.

[0066] FromFigure 2 It can be seen that, compared with the DSS model group, the colon length of the DMQ intervention group of mice was significantly restored, and the spleen weight was significantly increased. The HE staining results showed that after DMQ intervention, the degree of inflammatory infiltration of the colon tissue of the mice was significantly reduced, and the intestinal mucosa damage was improved. The histological score showed that the inflammation score of the DMQ treatment group of mice was significantly reduced. Through the intervention of DMQ at a dose of 20mg / kg, the intestinal inflammation and related pathological characteristics of the DSS model mice were effectively alleviated, thereby proving that the intestinal inflammatory response of the DSS model mice was effectively alleviated by the DMQ intervention in Example 1 of the present application.

[0067] 3) Flow cytometry was used to detect Th17 cells and CD8 + T cells secreting Th1 type cytokine IFN-γ in the mesenteric lymph nodes and spleen tissues of mice.

[0068] The specific operation steps are: cells are separated and counted from the mesenteric lymph nodes and spleen tissues, and are transferred to EP tubes. After being stimulated by cytokines, blocked, labeled by external standard antibodies, fixed and broken by cells, and labeled by internal standard antibodies, the cells are resuspended using PBS, filtered into flow tubes, detected using a flow cytometer, and the changes in Th cell and Tc cell subgroups are analyzed by FlowJo software. The results are shown in Figure 3 .

[0069] Among them, Figure 3 A shows the representative IL-17A + CD4 + T cell flow cytometry graphs and CD4 + T cell IL-17A + cell percentage statistics of each group in the mesenteric lymph nodes of mice. Figure 3 B shows the representative IL-17A + CD4 + T cell flow cytometry graphs and CD4 + T cell IL-17A + cell percentage statistics of each group in the spleen of mice. Figure 3 C shows the representative IFN-γ + CD8 + T cell flow cytometry graphs and CD8 + T cell IFN-γ + cell percentage statistics of each group in the mesenteric lymph nodes of mice. Figure 3 D shows the representative IFN-γ + CD8 + T cell flow cytometry graphs and CD8 + T cell IFN-γ +The percentage of cells.

[0070] From Figure 3 analysis, Figure 3 A and Figure 3 B show that DMQ intervention significantly reduces the proportion of Th17 cells in mesenteric lymph nodes and spleen T cells, Figure 3 C and Figure 4 D results show that DMQ can significantly reduce the proportion of IFN-γ + CD8 + T cells in the mesenteric lymph nodes and spleen of mice, thereby proving that the proportion of Th17 cells and CD8 + T cells secreting IFN-γ is significantly reduced by DMQ intervention in Example 1 of the present application.

[0071] 4) Western blot detection of tight junction proteins (Occludin, ZO-1).

[0072] The specific operation steps are: extract the protein of mouse colon tissue, perform SDS-PAGE electrophoresis separation, transfer to PVDF membrane, block, then incubate primary antibody (Occludin, ZO-1 and β-actin) and HRP labeled secondary antibody, and finally develop using hypersensitive luminescent liquid. The gray value of the target band is analyzed by Image J software, and the relative expression level of the target protein is calculated. The results are shown in Figure 4 .

[0073] Among them, Figure 4 A, Figure 4 B and Figure 4 C show the protein expression levels of Occludin and ZO-1 in colon tissue.

[0074] From Figure 5 analysis, compared with the DSS model group, the expression levels of tight junction proteins Occludin and ZO-1 in the colon tissue of the DMQ intervention group of mice were significantly up-regulated, thereby proving that the damage to the intestinal barrier structure of the DSS model mice is effectively alleviated by DMQ intervention in Example 1 of the present application.

[0075] 5) Evaluate the effect of DMQ on NLRP3 inflammasome activation

[0076] Western blot detection of NLRP3 inflammasome activation related protein (Caspase-1, i.e. p20) expression

[0077] Specific operation steps are: extract mouse colon tissue protein, perform SDS-PAGE electrophoresis separation, transfer to PVDF membrane, block, then incubate primary antibody (p20 and β-actin) and HRP labeled secondary antibody in turn, and finally develop color using hypersensitive luminescent liquid. The gray value of the target band is analyzed by Image J software, and the relative expression level of the target protein is calculated.

[0078] ELISA was used to detect the secretion of inflammatory factors (IL-1β, TNF-α).

[0079] Specific operation steps are: attach the capture antibody to the solid carrier; the next day, after blocking buffer treatment, add the sample to be tested, incubate with the detection antibody, combine with horseradish peroxidase (HRP), and after substrate TMB reaction, use the stop solution to stop and measure the OD value within 5 min to analyze the secretion levels of IL-1β and TNF-α. The results are shown in Figure 5 .

[0080] wherein, Figure 5 A shows the protein expression levels of p20 and Pro-caspase1 (Pro-casp1) in colon tissue, Figure 5 B shows the secretion amount of IL-1β in colon tissue homogenate, Figure 5 C shows the secretion amount of TNF-α in colon tissue.

[0081] From the analysis in Figure 6 , it can be known that after DMQ (20 mg / kg) intervention, the expression of caspase-1 activated fragment p20 in intestinal inflammatory cells is significantly inhibited, but the protein level of Pro-caspase-1 is not affected. ELISA results show that after DMQ (20 mg / kg) intervention, the secretion of IL-1β in intestinal cells is significantly reduced, while the production of TNF-α is not significantly affected, thereby proving that the NLRP3 inflammasome activation in the intestine of DSS model mice is effectively inhibited by DMQ intervention in Example 1 of the present application.

[0082] In summary, the ulcerative colitis model induced by 3% DSS can cause typical inflammatory symptoms in mice, including weight loss, blood in stool and diarrhea, and other intestinal function disorder manifestations. The colon inflammation symptoms of DSS-induced mice can be significantly improved by DMQ intervention, which specifically manifests as: alleviating weight loss, reducing disease activity index score, improving the appearance characteristics of colon atrophy and whitening, inhibiting colon length shortening, and reducing pathological changes such as splenomegaly. In terms of immune regulation, it is observed that DMQ can significantly reduce the proportion of Th17 cell subgroups in mesenteric lymph nodes and spleen, while reducing CD8 +The number of IFN-γ positive cells in T cells. IFN-γ, as an important pro-inflammatory cytokine, not only can activate inflammatory cells, but also can affect the intestinal barrier function and increase the intestinal permeability, thereby aggravating the inflammatory response. In terms of intestinal barrier protection, the experimental results show that through DMQ intervention, the expression level of tight junction proteins ZO-1 and Occludin can be effectively inhibited, which indicates that DMQ has a positive effect on maintaining the integrity of the intestinal barrier. At the same time, DMQ treatment significantly reduces the secretion level of IL-1β in colon tissue, showing good anti-inflammatory effect. It is worth noting that compared with known anti-inflammatory compounds such as apigenin (100 mg / kg), artemisinin (150 mg / kg) and L-fucose (250 mg / kg), DMQ shows comparable improvement in colitis effect at a lower dose (20 mg / kg), showing its potential therapeutic advantage. These findings provide important experimental evidence for the development of new anti-colitis drugs.

[0083] Example 2

[0084] Alum-induced acute peritonitis experiment

[0085] 1. Animals and grouping: SPF level 8-week-old male C57BL / 6J mice with similar body weight were selected and randomly divided into the following three groups, and each mouse was numbered: blank control group (Control group, n=6), Alum-induced acute peritonitis model group (Alum group, n=6), and DMQ-treated Alum-induced acute peritonitis model group (DMQ + Alum group, n=6).

[0086] 2. Model establishment and treatment: the blank control group was not treated; the DMQ intervention group was intraperitoneally injected with DMQ (20 mg / kg), and 1 h later, Alum (1 mg / each) was intraperitoneally injected, while the acute peritonitis model group was intraperitoneally injected with Alum (1 mg / each).

[0087] 3. Experimental results and sample collection: after 12 hours, the eyeball was taken to collect blood, and the mice were sacrificed by cervical dislocation, and the peritoneal fluid was collected. Flow cytometry was used to detect the proportion of neutrophils in the peritoneal lavage fluid, and ELISA was used to detect the secretion levels of inflammatory factors in the eyeball blood and peritoneal fluid.

[0088] Detection analysis

[0089] 1) Flow cytometry was used to detect the proportion of neutrophils in the peritoneal lavage fluid.

[0090] 2) ELISA was used to detect the secretion levels of inflammatory factors (IL-1β, TNF-α) in the eyeball blood and peritoneal fluid.

[0091] The specific operation steps are: the capture antibody is attached to the solid phase carrier; the sample to be detected is added after blocking buffer treatment the next day, and the detection antibody is incubated; horseradish peroxidase (HRP) is combined; after the substrate TMB is reacted, the termination liquid is terminated and the OD value is measured within 5 minutes, and the secretion levels of IL-1β and TNF-α are analyzed. The results are shown in Figure 6

[0092] Among them, Figure 6 A shows the percentage of CD11b + Ly6G + neutrophils in the abdominal cavity fluid of mice in each group, Figure 6 B shows the percentage of CD11b + Ly6G + neutrophils in the abdominal cavity fluid of mice in each group, Figure 6 C shows the number of CD11b + Ly6G + neutrophils in the abdominal cavity fluid of mice in each group, Figure 6 D shows the secretion level of IL-1β in the eyeball blood of mice in each group, Figure 6 E shows the secretion level of IL-1β in the abdominal cavity fluid of mice in each group, Figure 6 F shows the secretion level of TNF-α in the eyeball blood of mice in each group, Figure 6 G shows the secretion level of TNF-α in the abdominal cavity fluid of mice in each group.

[0093] From the analysis, ​ it can be seen that compared with the WT group, the proportion and number of neutrophils in the Alum group are significantly increased, and after the intervention of DMQ, the proportion and number of CD11b + Ly6G + neutrophils are obviously reduced. The ELISA results show that DMQ can obviously reduce the secretion of IL-1β in the eyeball blood and the abdominal cavity fluid, but has no great effect on TNF-α, thereby proving that through the intervention of DMQ in Example 2, the acute peritonitis induced by Alum in mice is effectively relieved.

[0094] From the above, it can be seen that the acute peritonitis model of mice is induced by intraperitoneal injection of Alum (1 mg per mouse), and the treatment effect of DMQ is evaluated by multiple dimensions. Flow cytometry analysis shows that the proportion and number of Ly6G + CD11b + ​The proportion and absolute number of neutrophils were significantly reduced. At the same time, ELISA results showed that DMQ can significantly inhibit the release of inflammatory factors (IL-1β) in the eye ball blood and abdominal lavage fluid. These data fully demonstrate that DMQ can effectively alleviate the acute peritonitis induced by alum. In terms of dose effect, DMQ shows a significant advantage compared with known anti-inflammatory compounds: myricetin (40 mg / kg), bilirubin (30 mg / kg) and naringin (300 mg / kg) can all improve the symptoms of peritonitis, but DMQ shows comparable or even better therapeutic effect at a lower dose (20 mg / kg). This finding suggests that DMQ may have higher bioavailability and stronger anti-inflammatory activity, providing an important clue for the development of new anti-peritonitis drugs.

[0095] The above research results provide important clues for further exploring the mechanism of DMQ in treating colitis and peritonitis, and open up potential pathways for the development of new anti-inflammatory drugs. It is particularly noteworthy that DMQ exhibits better therapeutic effect than a variety of positive control drugs at a dose of 20 mg / kg, suggesting that it has unique pharmacodynamic characteristics and good development prospects. These findings provide new research directions for the treatment strategies of inflammatory diseases, and lay an experimental foundation for the clinical application of DMQ. It has promotional application value in the field of medical technology.

[0096] The above examples are only preferred embodiments for fully illustrating the present application, and the protection scope of the present application is not limited thereto. Any equivalent replacement or transformation made by those skilled in the art on the basis of the present application is within the protection scope of the present application.

Claims

1. Use of 2,6-dimethoxy-1,4-benzoquinone in the preparation of a drug for treating ulcerative colitis.

2. Use according to claim 1, characterized in that, The amount of 2,6-dimethoxy-1,4-benzoquinone is 20 mg / kg; The drug for treating colitis takes 2,6-dimethoxy-1,4-benzoquinone as an active ingredient and is prepared by adding pharmaceutical excipients; And / or, the dosage form of the drug is at least one of tablets, capsules, granules, powders, patches, suspensions, oral liquids, injections and suppositories.

3. Use according to claim 1, characterized in that, The ulcerative colitis is sodium dextran sulfate (DSS) induced ulcerative colitis.

4. Use according to claim 1, characterized in that, The 2,6-dimethoxy-1,4-benzoquinone is used to reduce the secretion level of IL-1β in the colon tissue; and / or the 2,6-dimethoxy-1,4-benzoquinone is used to reduce IL-17A in the mesenteric lymph nodes and spleen + CD4 + T cell ratio.

5. Use of 2,6-dimethoxy-1,4-benzoquinone in the preparation of a drug for treating acute peritonitis.

6. Use according to claim 5, characterized in that, The amount of 2,6-dimethoxy-1,4-benzoquinone is 20 mg / kg; And / or, the drug for treating peritonitis takes 2,6-dimethoxy-1,4-benzoquinone as an active ingredient and is prepared by adding pharmaceutical excipients; And / or, the dosage form of the drug is at least one of tablets, capsules, granules, powders, patches, suspensions, oral liquids, injections and suppositories.

7. The use according to claim 1, characterized in that, The peritonitis is aluminum potassium sulfate (Alum) induced acute peritonitis.

8. The use according to claim 1, characterized in that, The 2,6-dimethoxy-1,4-benzoquinone is used to inhibit the secretion of IL-1β in the eyeball blood and peritoneal fluid; and / or the 2,6-dimethoxy-1,4-benzoquinone is used to reduce Ly6G + CD11b + the proportion and number of neutrophils.

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