Drugs used to prevent or treat enteritis and intestinal cancer

Abstract

The present invention provides a drug for preventing or treating enteritis and intestinal cancer. Specifically, the present invention relates to the use of amniotic fluid in the manufacture of a drug for preventing or treating enteritis and intestinal cancer; the use of amniotic fluid in the manufacture of a drug for preventing colon cancer in a subject; and the use of amniotic fluid in the manufacture of a drug for activating Nrf2 and inhibiting NF-κB to reduce inflammation and oxidative damage caused by the destruction of the intestinal mucosal barrier in a subject. The amniotic fluid is derived from an egg with an embryonic age of 5 to 12 days, or from an egg of a bird other than a chicken whose developmental stage corresponds to the developmental stage in which the egg with the embryonic age is placed, or from a rodent embryo with an embryonic age of 8 to 14 days, or from an embryo of a non-human mammal other than a rodent whose developmental stage corresponds to the developmental stage of a rodent with an embryonic age of 8 to 14 days.

Description

[Technical field]

[0001] The present invention relates to a drug for use in the prevention or treatment of enteritis and intestinal cancer. [Background technology]

[0002] Enteritis is an inflammation of the intestine caused by bacteria, viruses, fungi, parasites or unknown causes, including small intestine and colitis. Clinical symptoms mainly include abdominal pain, diarrhea, watery stool or mucopurulent bloody stool. Enteritis can be divided into two types, acute enteritis and chronic enteritis, according to the duration of illness. The duration of illness of chronic enteritis is generally more than 2 months, and the most common clinical conditions are chronic bacterial dysentery, chronic amebic dysentery, schistosomiasis, nonspecific ulcerative colitis and regional enteritis.

[0003] Ulcerative colitis (UC) is a chronic, relapsing inflammatory disease affecting the colon and rectum, characterized by weight loss (BW), diarrhea, colonic / rectal inflammation, bloody stools and ulcers. Currently, the pathogenesis of UC is not yet fully clear. However, it is believed that environmental and genetic factors, oxidative stress, colonic inflammation, gut microbiome imbalance, and mucosal immune response dysfunction may all be related to the progression of the disease. The colonic mucus layer forms an important barrier between the external environment and the host's internal environment and can regulate the interaction between gut microbiome and immunity. Currently, UC treatments, such as corticosteroids, thiopurines, and aminosalicylates, are relatively ineffective and often cause serious adverse events. Therefore, there is a need to develop more effective and less toxic treatments.

[0004] Cellular oxidative stress is associated with a series of signal transduction and can cause the occurrence and progression of inflammatory diseases. Nuclear transcription factor Nrf 2 (nuclear erythroid 2-related factor 2) is a major transcription factor that can bind to genes containing antioxidant response elements (AREs) and further activate antioxidant-related genes such as NAD(P)H quinone oxidoreductase 1 (NQO 1) and hemoglobin oxygenase 1 (HO-1). It has been reported that Nrf 2 can control the inflammatory response of intestinal cancer associated with UC and colitis. In another study, Nrf 2 activators were used to alleviate dextran sulfate sodium (DSS)-induced chronic and acute colitis. The Nrf 2 signaling pathway can activate multiple signaling pathways and play an important role in mediating inflammation and oxidative stress. Nrf 2 also exerts a certain anti-inflammatory effect by suppressing the production of inflammatory cytokines IL-1β, IL-6 and TNF-α. Overexpression of inflammatory cytokines is considered to be a distinctive feature of UC inflammation induced by reactive oxygen species (ROS) and the nuclear factor-κB (NFκB) pathway. Overall, simultaneously targeting the Nrf2 and NFκB pathways is highly effective in treating and preventing UC. Summary of the Invention

[0005] A first aspect of the present invention provides an application of amniotic fluid in the manufacture of a medicament for preventing or treating enteritis and intestinal cancer in a subject, wherein the amniotic fluid is derived from an egg having an embryonic age of 5-12 days, preferably an egg having an embryonic age of 6-11 days, more preferably an egg having an embryonic age of 7-9 days, more preferably an egg having an embryonic age of 7-8 days, from an egg of a bird other than a chicken that corresponds to the developmental stage at which the egg of said embryonic age is placed, from a rodent having an embryonic age of 8-14 days, or from an embryo of a non-human mammal other than a rodent that corresponds to the developmental stage of a rodent having an embryonic age of 8-14 days.

[0006] In one or more embodiments, enteritis includes, but is not limited to, enteritis and colitis.

[0007] In one or more embodiments, the enteritis is chronic enteritis, including chronic bacterial dysentery, chronic amebic dysentery, schistosomiasis, nonspecific ulcerative colitis, and regional enteritis.

[0008] In one or more embodiments, the enteritis is inflammatory bowel disease, including, but not limited to, ulcerative colitis and Crohn's disease.

[0009] A second aspect of the present invention provides an application of amniotic fluid in the manufacture of a medicament for preventing or treating enteritis and intestinal cancer in a subject, wherein the amniotic fluid is derived from an egg having an embryonic age of 5-12 days, preferably an egg having an embryonic age of 6-11 days, more preferably an egg having an embryonic age of 7-9 days, more preferably an egg having an embryonic age of 7-8 days, from an egg of a bird other than a chicken that corresponds to the developmental stage at which the egg of said embryonic age is placed, from a rodent having an embryonic age of 8-14 days, or from an embryo of a non-human mammal other than a rodent that corresponds to the developmental stage of a rodent having an embryonic age of 8-14 days.

[0010] A third aspect of the present invention provides amniotic fluid, including but not limited to, drug applications for alleviating inflammation and oxidative damage caused by Nrf2 activation and NF-κB inhibition, and intestinal mucosal barrier destruction in a subject, wherein the amniotic fluid is derived from an egg having an embryonic age of 5-12 days, preferably an egg having an embryonic age of 6-11 days, more preferably an egg having an embryonic age of 7-9 days, more preferably an egg having an embryonic age of 7-8 days, from an egg of a bird other than a chicken having a developmental stage corresponding to the developmental stage in which the egg of said embryonic age was placed, from a rodent having an embryonic age of 8-14 days, or from an embryo of a non-human mammal other than a rodent having a developmental stage corresponding to the developmental stage of a rodent having an embryonic age of 8-14 days. [Brief description of the drawings]

[0011] [Figure 1-1]Figure 1: Mice were administered 3% DSS in sterile water for 7 days and normal water for 7 days. Changes in relevant parameters during this process: (A) chicken early amniotic fluid (ceAF) improved the morphological symptoms of DSS-induced acute colitis in mice, resulting in improved (B) mouse body weight, (C) colon length, (D) spleen weight, and (E) disease activity index (DAI). Data are presented as mean ± SD; *P<0.05, **P<0.01, ***P<0.001. [Figure 1-2] Same as above. [Figure 1-3] Same as above. [Diagram 2] Figure 2: ceAF has a therapeutic effect by improving colonic structure and goblet cell abundance in DSS-induced colitis mice, and there is no obvious morphological damage or abnormality in the major organs of the mice. (A) H&E stained images and (C) histological scores of representative slices. Scale: 200 μm. (B) Representative images of PAS stained. Scale: 100 μm. [Figure 3-1] Figure 3: ceAF exhibits anti-inflammatory and antioxidant activity in DSS-induced colitis by regulating DSS-induced inflammatory responses and colonic barrier dysfunction. (A) Western blot to evaluate the expression of NFκB p65, pIκB, IL6, Nrf2, HO-1, ZO-1 and Occludin in colonic tissues using GAPDH as an internal reference. (B) Relative expression intensity of NFκB p65, pIκB, IL6, Nrf2, HO-1, ZO-1 and Occludin after normalization with GAPDH. The mRNA expression levels of (C) TNF-α, (D) IL-6, (E) IL-1β, (F) Nrf2, (G) HO-1, (H) Occludin and (I) ZO-1 genes were measured by real-time PCR in colonic tissues. Laser confocal images of (J) CD68 and (K) iNOS labeled M1 macrophages. Scale: 500 μm. Data are presented as mean ± SD; *P<0.05, **P<0.01, ***P<0.001, n=10. [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Figure 4-1] Figure 4: ceAF alleviated oxidative stress and inflammation in mice with DSS-induced colitis. Chemical chromatography detected the levels of (A) SOD, (B) GSH-Px, (C) MPO, and (D) MDA in colonic tissues. ELISA method measured the levels of inflammatory cytokines (E) IL-6 and (F) TNF-α in serum. (G, H) Immunostaining of colonic tissue slices. All representative images were captured using an inverted fluorescence microscope. Data are presented as mean ± SD. *P<0.05, **P<0.01, and ***P<0.001; ns indicated no significant difference (n=10). [Figure 4-2] Same as above. [Diagram 5] Figure 5: ceAF suppresses inflammation and oxidative stress in LPS-stimulated RAW264.7 cells by activating Nrf2 and suppressing the NFκB pathway. (A) RAW264.7 cell viability after progressive ceAF treatment at different concentrations. (B) Western blot analysis of ceAF-related proteins in the NFκB and Nrf2 signaling pathways. GAPDH was used as an internal reference to detect the expression of TLR4, NFκB p65, pIκB, IL6, TNF-α, Keap1, Nrf2, and HO-1. (C) Quantitative analysis of the Western blot bands shown in (B) by optical densitometry. Data are mean ± SD; *P<0.05, **P<0.01, ***P<0.001. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (as in the examples) can be combined with each other to form a preferred solution to the problem.

[0013] The present inventors have discovered that amniotic fluid from non-human animals can activate Nrf2 and suppress NF-κB to reduce inflammation and oxidative damage caused by intestinal mucosal barrier destruction, and thus can be used to treat or prevent intestinal inflammation and intestinal cancer associated with intestinal inflammation.

[0014] In the present invention, the amniotic fluid can be derived from an egg of a hawk and a non-human mammal. An egg of a hawk refers to an egg of a bird. Preferably, the bird is a poultry such as a chicken, a duck, or a goose. In the present invention, preferably, an avian egg having an embryonic age of 5 to 20 days, more preferably, an avian egg having an embryonic age of 6 to 15 days is used. It should be understood that the appropriate embryonic age may vary depending on the poultry egg. For example, when eggs are used, preferably, an egg having an embryonic age of 5 to 12 days, more preferably, an egg having an embryonic age of 6 to 11 days, more preferably, an egg having an embryonic age of 7 to 9 days, more preferably, an egg having an embryonic age of 7 to 8 days is used. When eggs of other birds are used, eggs corresponding to the developmental stage in which the eggs of the above embryonic ages are placed can be used. For example, when duck eggs are used, duck eggs having an embryonic age of 8 to 10 days, particularly 8 to 9 days, may be the best.

[0015] Amniotic fluid from bird eggs can be obtained by traditional methods. For example, the blunt end of an egg of the relevant embryonic age is struck to break the skin, and the skin is peeled to make a hole of about 2 cm in diameter. Then, the membrane and vitelline membrane are carefully peeled off using tweezers, taking care not to damage the amniotic membrane. The amniotic membrane and associated tissues encasing the embryo are poured from the skin into a Petri dish, and the amniotic membrane is pierced with a syringe until the amniotic membrane is tightly attached to the embryo to collect the amniotic fluid, thereby obtaining the amniotic fluid of the present invention.

[0016] In the present invention, the amniotic fluid may be derived from a non-human mammal, particularly a rodent such as a mouse. Other non-human mammals may be common livestock such as cows, sheep, dogs, cats, pigs, etc. In some embodiments, the amniotic fluid is derived from a rodent embryo having an embryonic age of 8-14 days or from a non-human mammalian embryo corresponding to a rodent developmental stage having an embryonic age of 8-14 days. The amniotic fluid may be obtained by traditional methods. For example, the abdominal cavity of a mouse having a pregnancy of 8-14 days is incised with surgical scissors, the uterus is carefully removed and incised, and the amniotic membrane is pierced with a syringe to collect the amniotic fluid until the amniotic membrane adheres to the embryo, thereby obtaining the amniotic fluid for use in the present invention.

[0017] It should be understood that, if necessary, the amniotic fluid is centrifuged to separate impurities that it may contain in order to obtain amniotic fluid as pure as possible. The supernatant obtained after centrifugation is the amniotic fluid used in the present invention. All steps for obtaining amniotic fluid must be performed under sterile conditions, and the "amniotic fluid" described in this text refers to "pure" amniotic fluid, i.e., amniotic fluid separated from an avian egg or a non-human mammalian embryo, free of other components inside the avian egg or the non-human mammalian embryo, and uncontaminated by external substances. Pure amniotic fluid can be stored in a refrigerator at -60°C or below and can be used after thawing.

[0018] The amniotic fluid described herein can be used as an active ingredient in a drug in a subject in need of internal administration, for example, an effective amount of amniotic fluid or a drug composition containing amniotic fluid described herein can be provided to a subject in need thereof.

[0019] In this text, the subject can be an animal, such as a mammal, particularly a human.

[0020] Enteritis includes enteritis and colitis based on the site of onset. Enteritis can be divided into two types, acute enteritis and chronic enteritis, based on the course. Common clinical chronic enteritis includes chronic bacterial dysentery, chronic amebic dysentery, schistosomiasis, nonspecific ulcerative colitis and regional enteritis. Enteritis is an inflammatory bowel disease (IBD). IBD is a special chronic intestinal inflammatory disease that includes ulcerative colitis (UC) and Crohn's disease (CD). UC is a chronic nonspecific, noninfectious, inflammatory bowel disease that mainly affects the colonic mucosa and submucosa. The characteristic symptoms are continuous, diffuse distribution, continuous mucosal ulcers in the rectum and colon, starting from the rectum and extending to various degrees, and at its longest, can extend to the cecum. UC is a common type of inflammatory bowel disease (IBD) and is associated with the development of colon cancer. UC is mainly associated with immune abnormalities and genetic mutations, and infection, food, lifestyle, environmental factors, and emotions are essential inducing factors.

[0021] According to research, chronic ulcerative colitis may be associated with colon cancer. The cause of colon cancer may be related to chronic inflammatory stimulation of colon mucosa, and it is generally recognized that cancer develops through an inflammatory polyp stage in the process of increased inflammation. Therefore, in some embodiments, the present invention also relates to preventing colon cancer associated with chronic inflammatory stimulation of colon mucosa by using the non-human animal amniotic fluid or pharmaceutical composition described herein. The colon cancer is associated with chronic inflammatory stimulation of colon mucosa.

[0022] In a particularly preferred embodiment of the invention, amniotic fluid, in particular poultry egg amniotic fluid as described herein, more preferably egg amniotic fluid, is used for the treatment and prevention of UC and intestinal cancer associated with chronic inflammatory stimulation of the colonic mucosa.

[0023] In some embodiments, the amniotic fluid used in the present invention is egg amniotic fluid, particularly for avian species as described herein, and more preferably egg amniotic fluid is used for one or more of the following purposes: (1) For use in slowing or preventing colonic shortening in a subject; (2) To inhibit splenomegaly in a subject; (3) Use in mitigating the progression of colitis; (4) Alleviating symptoms of acute enteritis (e.g., acute UC) in a subject (e.g., colonic mucosal damage, loss of tissue architecture, epithelial erosion, glandular fluid loss, inflammatory cell infiltration, etc.); (5) use of increasing goblet cell abundance and improving goblet cell morphology in a subject to produce more mucus; (6) Use of downregulating the expression of NF-κB p65, PIκB, IL-6 and TNF-α in a subject, and upregulating the expression of Occludin, ZO-1, Nrf2 and HO-1 in a subject. Preferably, the subject is a colitis patient.

[0024] In some embodiments, the present invention relates to the use of amniotic fluid, particularly amniotic fluid from avian eggs as described herein, and more preferably amniotic fluid from eggs, to produce a formulation for one or more of the following uses: (1) For use in slowing or preventing colonic shortening in a subject; (2) To inhibit splenomegaly in a subject; (3) Use in mitigating the progression of colitis; (4) use to alleviate symptoms of acute enteritis (e.g., acute UC) in a subject (e.g., colonic mucosal damage, loss of tissue architecture, epithelial erosion, reduced number of glands, inflammatory cell infiltration, etc.); (5) increasing the abundance of goblet cells and improving goblet cell morphology in a subject to produce more mucus; and (6) Use for downregulating NF-κB p65, PIκB, IL-6 and TNF-α expression in a subject, and upregulating Occludin, ZO-1, Nrf2 and HO-1 expression in a subject.

[0025] Thus, the present invention provides a method for reducing the severity of colitis, treating and preventing colitis, or preventing colon cancer associated with chronic inflammatory stimulation of the colon mucosa, comprising administering an effective amount of the amniotic fluid of the present invention or a pharmaceutical composition comprising the amniotic fluid to a subject in need thereof. The present invention also provides an application of amniotic fluid for use in the manufacture of a medicament for treating or preventing colitis in a subject, preventing colon cancer associated with chronic inflammatory stimulation of the colon mucosa, or suppressing activation of Nrf2 and NF-κB, or reducing inflammation and oxidative damage caused by intestinal mucosal barrier destruction in a subject, and the amniotic fluid or pharmaceutical composition thereof described herein is used for treating or preventing colitis, preventing colon cancer associated with chronic inflammatory stimulation of the colon mucosa, or suppressing activation of Nrf 2b and NF-κB, thereby reducing inflammation and oxidative damage caused by intestinal mucosal barrier destruction.

[0026] In some embodiments, the present invention provides a method comprising: (1) slowing or preventing colon shortening in a subject; and / or (2) inhibiting splenomegaly in a subject; and / or (3) alleviating the progression of colitis; and / or (4) alleviating symptoms of acute colitis (e.g., acute UC) in a subject (e.g., colonic mucosal damage, tissue architecture loss, epithelial erosion, reduced glandular quantity, inflammatory cell infiltration, etc.); and / or (5) increasing goblet cell abundance and improving goblet cell morphology to produce more mucus in a subject; and / or (6) downregulating expression of NF-κB p65, PIκB, IL-6, and TNF-α in a subject, and upregulating expression of Occludin, ZO-1, Nrf2, and HO-1 in a subject, comprising administering to a subject in need thereof an effective amount of amniotic fluid or a pharmaceutical composition containing said amniotic fluid according to the present invention. Preferably, the subject is a colitis patient.

[0027] As used herein, an effective amount refers to a dosage that can treat, prevent, reduce and / or alleviate a disease or condition in a subject. The effective amount for treatment can be determined based on factors such as the patient's age, sex, disease and severity, and other physical conditions of the patient. As used herein, subject or patient or subject generally refers to mammals, particularly humans. As used herein, treatment and prevention have the meanings well known in the art.

[0028] The amniotic fluid described herein may be used directly or in the methods and uses described herein and administered to a subject in need thereof. The administration method may be parenteral, such as intravenous administration. In some embodiments, the therapeutically effective amniotic fluid may be mixed equally with saline for injection, water for injection, or glucose injection, and administered by intravenous injection or the like. Preferably, the administered pharmaceutical composition may contain 5-40% (v / v) or 10%-35% (v / v) amniotic fluid, and preferably 15-30% (v / v) amniotic fluid.

[0029] The pharmaceutical compositions containing amniotic fluid described herein generally include pharma- ceutically acceptable auxiliary materials. As used herein, "pharmaceutically acceptable auxiliary materials" refers to, but is not limited to, pharmacologically and / or physiologically compatible carriers and / or excipients. The carriers and / or excipients include antibiotics, humectants, pH control agents, surfactants, carbohydrates, adjuvants, antioxidants, chelating agents, ionic strength enhancers, preservatives, carriers, lubricants, sweeteners, dyes / colorants, flavor enhancers, wetting agents, dispersants, suspending agents, stabilizers, isotonicity agents, solvents, or emulsifiers. In some embodiments, the pharma-ceutically acceptable auxiliary materials may include one or more inactive ingredients, including but not limited to stabilizers, preservatives, additives, adjuvants, enteric solvents, or other inactive ingredients suitable for administration with the medicinal compounds. The dosage and frequency of administration may be determined by a physician depending on the particular condition, age, and sex of the patient. In general, a therapeutically effective amount in the treatment of a particular disease refers to an amount sufficient to improve or alleviate symptoms associated with the disease. Such doses can be administered in a single dose or according to an effective treatment plan. The dose of the drug is generally administered to improve the symptoms of the disease, although it may cure the disease completely. Repeated administration is generally required to improve the desired symptoms. For example, the administered dose is generally 1-200 mL / time, and can be administered daily or weekly. In some implementations, the frequency of drug administration is several times a day, twice a day, once every 2, 3, 4, 5 or 6 days, once every half month or once a month.

[0030] Another pharmaceutical composition is provided herein. The pharmaceutical composition is the amniotic fluid described herein, particularly amniotic fluid contained in an avian egg, more preferably amniotic fluid from an egg with an embryonic age of 5-12 days, more preferably 6-11 days, more preferably 6-9 days, more preferably 7-8 days. The pharmaceutical composition may be amniotic fluid stored frozen at -60°C or below or a lyophilized reagent such as lyophilized amniotic fluid. The pharmaceutical composition may also include other pharmaceutically acceptable carriers or excipients, such as saline for injection, water for injection, or glucose injection. Preferably, the pharmaceutical composition contains 5-40% (v / v) or 10-35% amniotic fluid, preferably 15-30%.

[0031] The present invention will be described below with specific examples. It should be understood that such examples are merely illustrative and are not intended to limit the scope of the present invention. The methods, reagents and equipment used in the examples are common methods, reagents and equipment in the art unless otherwise specified.

[0032] Materials and Methods ceAF manufacturing Fertilized eggs were incubated at 38 ± 1°C and 50% humidity, and collected from the eggs 6–8 days later. Samples were centrifuged at 2500 g for 20 min, and the supernatant was filtered through a 0.22 μm sterilizer and stored at −80°C.

[0033] cell culture The experimental cells were RAW264.7 mouse macrophage cell line obtained from the Cell Bank of the Chinese Academy of Sciences. The cells were cultured in DMEM high glucose medium containing 10% fetal bovine serum (Gibco, USA), penicillin 100U / ml, and streptomycin 100mg / mL (Gibco, USA) at 37°C in a 5% CO2 incubator. Nrf2 inhibitor ML385 was purchased from Selleckchem. Lipopolysaccharide (LPS) was purchased from Sigma. The cells were cultured at 5 × 10 5The cells were seeded into 6-well plates at a density of 100 / mL, ML385 dissolved in dimethylsorboxal (25 μM) was added, and the cells were incubated at 37°C for 12 hours. Then, 2 μg LPS (1 μg / mL) and 10% CEAF were added, and the cells were incubated at 37°C for 12 hours.

[0034] Cell viability detection Cell viability detection was performed in 96-well plates using the CCK-8 method. After starvation for 12 hours to synchronize the cell cycle, the cells were treated with 0, 2%, 4%, 6%, 8% and 10% ceAF for 24 hours, respectively, and then washed three times with phosphate-buffered saline (PBS). Then, incomplete medium (100 μL) was mixed with CCK-8 solution (10 μL). The enzyme-linked immunosorbent analyzer selected 450 nm wavelength to determine the absorbance value.

[0035] Experimental animals and interventions C57BL / 6J mice (female, 7-8 weeks old) were purchased from the Model Animal Research Center of Nanjing University. The mice were kept in a specific pathogen-free (SPF) environment with an environmental temperature of 22 ± 1°C, relative humidity of 50 ± 1%, and a light / dark cycle of 12 / 12 h. They were randomly divided into five groups (n = 10). (1) Control group: Eat food ad libitum, drink water, and receive distilled water via tube feeding. (2) Colitis group (DSS, dextran sulfate sodium): 3% DSS solution was used instead of distilled water for tube feeding. (3) Low-dose ceAF treatment group (DSS + 5% ceAF): Patients were asked to drink a 3% DSS solution and receive 5% ceAF via tube feeding. (4) High-dose ceAF treatment group (DSS+ceAF): Patients were administered a 3% DSS solution and 10% ceAF via tube feeding. (5) High-dose ceAF group (distilled water + 10% ceAF): Eat food and drink water freely, and receive 10% ceAF via tube feeding.

[0036] Experimental colitis was induced in mice by administering 3% DSS (w / v) in drinking water for 7 consecutive days. For treatment, mice received ceAF treatment via gastric intubation for 12 days from the same day that they received DSS. Mice in the normal control group only received sterile water. The behavior, body weight, food intake, and fecal condition of the mice were measured or observed daily. After 2 weeks, the mice were sacrificed and samples were collected and stored at -80°C. The colon was fluorescently stained.

[0037] Assessment of body weight, Disease Activity Index (DAI) score, naked eye ulcer score, and colon length During the experiment, body weight change, stool characteristics, and fecal occult blood were recorded, and the Disease Activity Index (DAI) was calculated and scored. The DAI score was calculated by recording the degree of weight loss, stool consistency, and fecal blood content to evaluate the severity of the disease. DAI score is DAI = (body weight loss score + stool characteristics score + fecal blood score) / 3. Weight loss was classified into 0 (0 points), 1-5% (1 point), 5-10% (2 points), 10-20% (3 points), and >20% (4 points). Stool consistency was classified into normal particles (0 points), loose stool (1 point), semi-formed stool (2 points), liquid stool (3 points), and diarrhea (4 points). Bleeding type was classified into no blood (0 points), trace (1 point), slight occult blood (2 points), obvious occult blood (3 points), and excessive bleeding (4 points). The scores of all sub-items were then added together and the total score was divided by 3 to obtain the DAI score (0–4 points). The colon was scored 0–5 points under a stereomicroscope for obvious damage considering the area of ​​inflammation and the presence or absence of ulcers, according to the scoring criteria proposed by a previously described maturation scoring method. The following criteria were used to evaluate naked eye ulcers: 0 point: no ulcers, no inflammation; 1 point: no ulcers, local congestion; 2 point: ulcers without congestion; 3 point: ulcers and inflammation only in one area; 4 point: ulcers and inflammation in two or more areas; 5 point: ulcers extending beyond 2 cm. Mice were sacrificed by isolating the cervical vertebrae and the colon was cut from the cecum to the anus to measure its length.

[0038] Hematoxylin-eosin (HE) and periodic acid-seep (PAS) staining For histopathological evaluation, distal colon segments were fixed in 10% formalin, embedded in paraffin, and stained with HE. The severity of DSS-induced tissue damage was classified by a histological scoring system as follows: no tissue damage (0 points), 1-25% (1 point), 26-50% (2 points), 51-75% (3 points), and 76-100% (4 points). Tissue damage was classified as mucosa (1 point), mucosa and submucosa (2 points), and submucosa excess (3 points). The extent of crypt damage was as follows: 1 / 3 of the base damaged (1 point), 2 / 3 of the base damaged (2 points), only the epithelial surface intact (3 points), and total crypt and epithelial loss (4 points). The degree of inflammation was classified as mild (1 point), moderate (2 points), and severe (3 points). At the same time, PAS staining was performed to measure mucus-secreting cells.

[0039] Western blot analysis Colon tissue was extracted using RIPA lysis solution and centrifuged (5000×g, 10 min) to obtain proteins. Protein concentration was detected by BCA protein assay kit. The same amount of protein (30 μg) was separated by 10% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane and blocked with 5% BSA buffer for 90 min. After overnight incubation with primary antibodies (1:1000 dilution) of TLR4, pIR4, IL6, NF64 p65, TNF-α, Keap1, ZO-1, Occludin, HO-1 and Nrf2 at 4°C, the membranes were rinsed three times for 6 min with TBS-T (pH 7.4) and then incubated with HRP-labeled anti-chlorine secondary antibody (1:40000 dilution) for 90 min. After rinsing three times with TBS-T for 6 min, all membranes were visualized with immobilon western chemiluminescence HRP substrate. The expression level of each protein was normalized to the internal reference GAPDH.

[0040] Assessment of cytokine levels and antioxidant activity TNF-α and IL-6 levels in serum were detected using commercial ELISA kits, while total protein content was assessed using BCA protein assay kit after colon was excised and homogenized at 4°C. Chromatography was used to measure the activities of GSH-Px, MDA, MPO and SOD in colon tissue.

[0041] RNA extraction and mRNA expression analysis Total RNA was extracted according to the instructions of the FastPure Cell / Tissue Total RNA Isolation Kit. cDNA synthesis of RNA samples (500 ng) was performed using PrimeScript real-time premix to prepare cDNA. Samples were subjected to real-time PCR analysis on a real-time fluorescent quantitative PCR system, QuantStudio 5, using Applied Biosystems Power SYBR Green PCR Master Mix reagent. The relative expression of each target gene was normalized using GAPDH. Table 1 shows the primer sequences (SEQ ID NO: 1-16) of the target genes. The threshold cycle (CT) value and relative mRNA concentration (E=2 -ΔΔCt ) was measured.

[0042] [Table 1]

[0043] Immunofluorescence (IF) detection Colon tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Tissue slides were delaminated with xylene, polarized in decreasing alcohol concentrations, and washed with deionized water. After blocking with bovine serum albumin (1%) for 1 h, slides were incubated overnight with primary antibodies (4°C). They were then stained using secondary antibodies labeled with Alexa Flour 488 or 594. After secondary antibody incubation, DAPI solution was added to stain cell nuclei, and the entire procedure was performed under light-proof conditions. Images were captured using a Leica DMIRE2 laser confocal scanning microscope.

[0044] statistical analysis All test results were statistically analyzed by GraphPad Prism v6.0, and data were expressed as mean ± standard deviation. Two-way analysis and least significant difference method were used to compare differences between groups. Significance levels of P value were set at <0.05, <0.01, and <0.001.

[0045] result 1. The alleviating effect of ceAF on the symptoms of DSS-induced colitis in mice To evaluate the basic protective effect of ceAF on DSS-induced colitis mice, we treated mice as described above to establish a colitis model (Fig. 1, A). Weight loss, stool concentration, and bleeding are all important symptoms of DSS-induced colitis. We observed weight loss, DAI, colon length, and intestinal damage. Five days after the start of DSS feeding, the body weight of the mice was significantly decreased, whereas ceAF alleviated this trend (Fig. 1, B). Colon length is another reproducible indirect indicator of colitis severity. The colon length of DSS-containing mice was significantly shorter compared to the control group. Surprisingly, ceAF supplementation significantly reversed the colon shortening of mice exposed to DSS (Fig. 1, C). DSS was also observed to increase spleen along with inflammatory changes. However, ceAF treatment could suppress DSS-induced splenomegaly (Fig. 1, D). Continuous use of ceAF could improve stool characteristics, occult blood, and even purulent blood-like stool changes in mice in the colitis group. DAI scores (an index of colonic inflammation severity) were increased in mice exposed to DSS compared to mice receiving only water, and administration of ceAF significantly improved these symptoms (Figure 1E). Overall, these observations indicate that ceAF intake can effectively alleviate the pathogenesis of colitis, and that 10% ceAF has a better therapeutic effect than 5% ceAF.

[0046] 2. ceAF ameliorates histological parameters of DSS-induced colitis in mice We further confirmed the effect of ceAF on DSS-induced colitis through histopathological analysis. The acute phase of ulcerative colitis showed many histological features, including mucosal erosion, crypt shortening, edema, and inflammatory cell infiltration in the mucosa and lamina propria. Data from H&E stained slices showed that the morphological structures of colonic tissues were significantly different. Compared with the control group, mice in the DSS group developed colonic mucosal damage, tissue structure damage, severe epithelial erosion, reduced glandular quantity, and obvious inflammatory cell infiltration. In contrast, mice treated with 5% or 10% ceAF showed reduced acute UC inflammation symptoms (Figure 2, A). Administration of ceAF could also reduce the histological scores of DSS-induced colitis (Figure 2, C), indicating that ceAF had a substantial protective effect on intestinal damage in mice induced by inflammation.

[0047] PAS staining was used to evaluate the abundance and integrity of goblet cells in the colonic mucus layer, which is the main barrier that protects the intestine. Compared with the control group, DSS significantly reduced the abundance of goblet cells in the colitis group mice, and there was no mucus layer at the inflammatory site (Figure 2, B). Surprisingly, treating the colitis mice with ceAF greatly increased the abundance and morphological improvement of goblet cells, and healthier goblet cells mean more mucus production (Figure 2, B). Such recovery may be attributed to the preservation of epithelial goblet cells that produce various mucins, which are the main cause of reducing tissue damage. It was also shown that a higher concentration of ceAF has a better therapeutic effect.

[0048] 3. Oral administration of ceAF reversed DSS-induced inflammatory responses and colonic barrier dysfunction. The Nrf2 pathway plays an important role in the cellular defense system, reducing the risk of intestinal inflammation and oxidative stress by regulating various detoxification enzymes and antioxidant proteins, including HO-1, SOD, and GSH-Px. HO-1 is the rate-limiting enzyme in heme catabolism and has an intrinsic defense mechanism. Activation of Nrf2 positively regulates HO-1 transcription, which is important for reducing the risk of gastrointestinal inflammation and oxidative stress. NF-κB is a redox-sensitive transcription factor that is important in inflammation, innate immunity, and maintaining tissue integrity, and regulates the expression of various inflammatory factors, including IL-6, IL-1β, tumor necrosis factor-α, epoxidase-2, and chemokines. These cytokines ultimately induce local inflammation and immune dysfunction, and these changes induce a positive feedback cycle to induce inflammation and intestinal mucosal damage.

[0049] To investigate the possible mechanism of the therapeutic action, we used Western blot and real-time fluorescent quantitative PCR methods to detect the protein and mRNA expression levels of cytokines in mouse intestinal tissues after ceAF administration. Compared with the control group, the expression of NF-κB p65, PIκB, IL-6, and TNF-α was significantly increased, whereas the expression of Nrf2 and HO-1 was significantly decreased in mice with DSS-induced colitis (Figure 3, A and B). Interestingly, after ceAF treatment, the expression of such typical inflammatory and oxidative stress indicators was significantly downregulated (Figure 3, A and B). Consistent with the protein expression, ceAF treatment significantly suppressed these typical indicators at the mRNA level (Figure 3, C-G). Compared with the control group, the DSS-treated group showed a significant decrease in the expression of ZO1 and occludin proteins, indicating the disruption of tight junction (TJ) structure. In contrast, ceAF treatment completely reversed the expression levels of these proteins (Figure 3, A, H, and I). In conclusion, the protective efficacy of 10% ceAF was superior to that of 5% ceAF. M1 macrophages in colonic tissues treated with 10% ceAF were detected by immunofluorescence staining. The results showed that 10% ceAF treatment significantly reduced the number of M1 macrophages (Figure 3, J and K).

[0050] 4. ceAF regulates enzymes and inflammatory cytokines involved in oxidative stress responses in DSS-induced experimental colitis. Colitis (UC) is characterized by the persistent expression of various inflammatory cytokines, such as IL-1β, IL6, and TNF-α, and the accumulation of oxidative stress. The use of antioxidant enzymes can effectively combat inflammation-related diseases by targeting GSH-Px, MDA, MPO, and SOD.

[0051] Compared with the control group, DSS significantly decreased SOD and GSH-Px activities in colonic tissues (Figure 4, A and B). In contrast, MDA and MPO activities were activated after DSS treatment, but gavage by intubation could significantly reverse the DSS-mediated changes of 10% ceAF (Figure 4, C and D). Also, serum levels of IL-6 and TNF-α were significantly upregulated in colitic mice compared with control mice. However, this upregulation was significantly suppressed by 10% ceAF (Figure 4, E and F). The results of immunofluorescence (IF) analysis (Figure 4, G and H) are consistent with the results of Western blot mentioned above (Figure 3, A). Our study indicates that ceAF can directly suppress the upregulation of infectious cytokines and oxidative stress by activating Nrf2 and suppressing the NF-κB signaling pathway.

[0052] 5. ceAF attenuates inflammation and oxidative stress in lipopolysaccharide (LPS)-stimulated RAW264.7 cells through the NF-κB and NRF2 signaling pathways. Nrf2 deficiency can exacerbate intestinal inflammation in animal models of various diseases such as emphysema, pleurisy, and sepsis, indicating that it plays an important role in mediating inflammation and oxidative stress. When evaluating the efficacy of anti-inflammatory and antioxidant agents on target cells, biocompatibility is an important consideration. Therefore, the in vitro cytotoxicity of ceAF was measured by measuring the viability of treated cells.

[0053] Our findings: ceAF shows better biocompatibility when the concentration is less than 10% (Figure 5, A). However, when the ceAF concentration is more than 40%, the cell viability is significantly decreased (Figure 5, A). Therefore, we studied the subsequent protein expression related to inflammation and oxidative stress in RAW264.7 cells stimulated by lipopolysaccharides with or without 10% ceAF. Lipopolysaccharides significantly upregulated the expression levels of TLR4, PIκB, NFκB p65, IL6, TNF-α, and Keap1, and decreased the expression levels of Nrf2 and HO1 in RAW 264.7 cells (Figure 5, B and C), indicating that lipopolysaccharides significantly activate such signal pathways related to inflammation and oxidative stress. Interestingly, the expression of these proteins was significantly reversed after treatment with 10% ceAF. To further confirm the potential mechanism of the antagonistic effect of cEAF on toxin-stimulated RAW 264.7 cells, we performed experiments using an Nrf2 inhibitor (ML385). We found that ML385 effectively inhibited Nrf2 activity, whereas the effect of cEAF on inflammation and oxidative stress in toxin-stimulated RAW 264.7 cells could be counteracted by pretreatment with ML385. Thus, we demonstrate that ceAF can directly inhibit the upregulation of inflammatory cytokines and oxidative stress by activating Nrf2 and suppressing NF-κB.

[0054] conclusion Taken together, our data indicate that ceAF can reduce the severity of DSS-induced colitis in terms of both macroscopic and histological parameters, and ceAF regulates the Nrf2 and NF-κB signaling pathways, thereby attenuating inflammation and oxidative damage due to intestinal mucosal barrier disruption.

Claims

1. An application characterized by utilizing amniotic fluid derived from eggs with an embryonic age of 5 to 12 days, derived from eggs of birds other than chickens whose developmental stage corresponds to the developmental stage at which the eggs of the embryonic age were laid, derived from rodents whose embryonic age is 8 to 14 days, or derived from embryos of non-human mammals other than rodents whose developmental stage corresponds to the developmental stage of rodents whose embryonic age is 8 to 14 days, in the manufacture of a drug for preventing enteritis in a subject.

2. 2. The application according to claim 1, characterized in that the amniotic fluid is derived from eggs with an embryonic age of 6 to 11 days.

3. 2. The application according to claim 1, characterized in that the amniotic fluid is utilized which is derived from eggs whose embryonic age is between 7 and 9 days, more preferably from eggs whose embryonic age is between 7 and 8 days.

4. The application according to any one of claims 1 to 3, characterized in that the enteritis is enteritis and colitis.

5. 4. The application according to any one of claims 1 to 3, characterized in that the enteritis is a chronic enteritis, including chronic bacterial dysentery, chronic amebic dysentery, schistosomiasis, nonspecific ulcerative colitis and regional enteritis.

6. The application according to any one of claims 1 to 3, characterized in that the intestinal inflammation is an inflammatory bowel disease, including ulcerative colitis and Crohn's disease.

7. An application characterized by utilizing amniotic fluid derived from eggs with an embryonic age of 5 to 12 days, derived from eggs of birds other than chickens whose developmental stage corresponds to the developmental stage at which the eggs of the embryonic age are laid, derived from rodents whose embryonic age is 8 to 14 days, or derived from embryos of non-human mammals other than rodents whose developmental stage corresponds to the developmental stage of rodents whose embryonic age is 8 to 14 days, in the manufacture of a drug for preventing intestinal cancer in a subject.

8. 8. The application according to claim 7, characterized in that the amniotic fluid is utilized which is derived from eggs which are 6 to 11 days old, preferably from eggs which have an embryonic age of 7 to 9 days, more preferably from eggs which have an embryonic age of 7 to 8 days.

9. An application characterized by utilizing amniotic fluid derived from eggs with an embryonic age of 5 to 12 days, from eggs of birds other than chickens whose developmental stage corresponds to the developmental stage at which the eggs of said embryonic age were laid, from rodents whose embryonic age is 8 to 14 days, or from embryos of non-human mammals other than rodents whose developmental stage corresponds to the developmental stage of rodents whose embryonic age is 8 to 14 days, in the manufacture of a drug that activates Nrf2 and inhibits NF-κB to reduce inflammation and oxidative damage caused by barrier destruction of the intestinal mucosa in a subject.

10. 10. The application according to claim 9, characterized in that amniotic fluid is used which is derived from eggs whose embryonic age is between 6 and 11 days, preferably from eggs whose embryonic age is between 7 and 9 days, more preferably from eggs whose embryonic age is between 7 and 8 days.

11. (1) slowing or preventing colon shortening in a subject; (2) inhibiting splenomegaly in a subject; (3) mitigating the progression of colitis; (4) alleviating symptoms of acute enteritis (e.g., acute UC) in a subject (e.g., colonic mucosal damage, loss of tissue structure, epithelial erosion, loss of glandular fluid, inflammatory cell infiltration, etc.); (5) increasing goblet cell abundance and improving goblet cell morphology to produce more mucus in a subject; and (6) inhibiting NF-κB in a subject. The present invention relates to an application for down-regulating the expression of p65, PIκB, IL-6, and TNF-α, and up-regulating the expression of Occludin, ZO-1, Nrf2, and HO-1 in a subject, wherein amniotic fluid is used to prepare one or more pharmaceutical compositions, and the amniotic fluid is derived from an egg having an embryonic age of 5 to 12 days, from an egg of a bird other than a chicken whose developmental stage corresponds to the developmental stage at which the egg of the embryonic age was laid, from a rodent embryo having an embryonic age of 8 to 14 days, or from an embryo of a non-human mammal other than a rodent whose developmental stage corresponds to the developmental stage of a rodent having an embryonic age of 8 to 14 days.

12. 12. The application according to claim 11, characterized in that the amniotic fluid is derived from eggs with an embryonic age of 6 to 11 days.

13. 13. The application according to claim 12, characterized in that the amniotic fluid is derived from eggs that are 7 to 9 days of embryonic age, more preferably from eggs that are 7 to 8 days of embryonic age.

14. The use according to any one of claims 11 to 13, characterized in that the symptoms of acute enteritis are selected from one or more of colonic mucosal damage, tissue structure loss, epithelial erosion, glandular count loss, and inflammatory cell infiltration.

15. The application according to any one of claims 11 to 13, characterized in that the subject is a patient with enteritis.

Images