Application of chick embryo amniotic fluid in preparation of gastric ulcer medicine

Abstract

The invention belongs to the technical field of medicines, and provides a medicine capable of preventing and treating gastric ulcer. Evaluation of the acute gastric ulcer treatment effect of early chick embryo amniotic fluid finds that the early chick embryo amniotic fluid can reduce the gastric tissue ulcer area and inflammatory factor infiltration, and the protective effect of the mucosal barrier on the gastric tissue is enhanced by reducing the calcium ion content in the gastric tissue and myeloperoxidase, so that the gastric ulcer treatment effect is improved. In addition, the oxidative stress level caused by ethanol is obviously inhibited, and the level of tight junction protein of stomach tissue is up-regulated. The invention has the potential of treating gastric ulcer.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine, and in particular relates to a medicine for treating gastric ulcer, namely, application of chicken embryo amniotic fluid in preparing the medicine for treating gastric ulcer. Background Art

[0002] Gastric ulcers (GUs) are chronic peptic ulcers that develop between the cardia and pylorus. Their pathological mechanism involves erosion and necrosis of the gastric mucosal tissue, forming ulcers of varying size, shape, and depth, with clear demarcation from the surrounding tissue. Mild lesions can form scabs through autodigestion, while severe cases can lead to gastric perforation. Hydrochloric acid in gastric fluid activates pepsinogen to convert it into pepsin, which degrades protein molecules, further damaging digestive tissue and the mucosa, promoting ulcer formation. Under normal physiological conditions, the epithelial barrier, blood supply, bicarbonate, mucus secretion, prostaglandins, and growth factors all work together to maintain mucosal integrity. When harmful factors outweigh the defense mechanisms, mucosal damage occurs. Chronic alcohol consumption can cause acute and chronic gastric mucosal damage, and elevated hydrochloric acid concentrations in gastric fluid can exacerbate mucosal damage. Gastric ulcers are also a common complication in critically ill patients, with an increasing incidence in recent years. The occurrence and severity of acute gastric ulcers primarily depend on the balance between external aggressors and the body's own defenses. Previous studies have shown that oxidative stress damage can damage the natural defense system in the stomach by destroying the mucus layer of gastric tissue. It can not only enhance the sensitivity of gastric tissue to mechanical stretch by producing hydroxyl groups, but also activate redox-sensitive transcription factors and aggravate the body's inflammatory response.

[0003] Currently, the clinical treatment options for gastric ulcers are relatively limited, primarily including proton pump inhibitors (such as omeprazole and lansoprazole), H2 receptor antagonists (such as ranitidine and famotidine), anti-Helicobacter pylori antibiotics, and traditional Chinese medicine. However, clinically used gastric ulcer treatments are generally expensive, have limited efficacy, and exhibit significant side effects. Therefore, research into therapeutic agents for acute gastric ulcers and the development of more effective ulcer treatments is a topic that requires continued research.

[0004] In recent years, natural compounds, Chinese herbal medicines and ingredients extracted from natural sources have become a hot topic in therapeutic research, which provides a certain theoretical basis for the development of new drugs. The present invention aims to find an effective drug for treating gastric ulcer from natural sources. Summary of the Invention

[0005] The present invention provides application of chicken embryo amniotic fluid in preparing medicine for treating gastric ulcer.

[0006] In some embodiments, the gastric ulcer is an acute gastric ulcer.

[0007] In some embodiments, the gastric ulcer is a chronic gastric ulcer.

[0008] In some embodiments, the method for preparing the chicken embryo amniotic fluid is: incubating fertilized eggs at 37±1°C and 45-60% humidity, collecting the chicken embryo amniotic fluid incubated for 6-8 days, centrifuging the collected amniotic fluid at 10,000-14,000 r / min for 10-40 minutes, and collecting the supernatant to obtain the chicken embryo amniotic fluid.

[0009] In some embodiments, the method for preparing the chicken embryo amniotic fluid is as follows: fertilized eggs are incubated at 37±1°C and 50% humidity, the chicken embryo amniotic fluid incubated for 6 to 8 days is collected, the collected amniotic fluid is centrifuged at 12000r / min for 30 minutes, and the supernatant is collected to obtain the chicken embryo amniotic fluid.

[0010] In some embodiments, the chicken embryo amniotic fluid is collected using a sterile syringe.

[0011] In some embodiments, the chicken embryo amniotic fluid is stored at a temperature of -80°C.

[0012] In some embodiments, the drug consists of chicken embryo amniotic fluid and a pharmaceutically acceptable carrier.

[0013] In some embodiments, the drug is in the form of an external preparation, an oral preparation, or an injectable preparation.

[0014] In some embodiments, the drug is in the form of a gel, spray, aerosol, patch, tincture, paste, lotion, cream, cream, ointment, granules, capsule, pill, tablet, powder, oral solution or syrup.

[0015] Beneficial effects

[0016] Chick early amniotic fluid is a natural composition obtained from fertilized chicken embryos. As the embryonic growth environment, amniotic fluid maintains acid-base balance and provides essential growth factors for embryonic development and growth. Studies have found that chick early amniotic fluid contains nutrients, immune factors, and various growth-related factors.

[0017] Early-stage chick embryo amniotic fluid can be produced on an industrial scale. It is inexpensive, readily available, and ethically safe, making it feasible for in-depth mechanistic studies and preclinical trials. Currently, there are only a few research reports on the use of early-stage chick embryo amniotic fluid, and no approved drugs are marketed. There are also no reports on its use for acute gastric ulcers. The inventors of this patent application have studied the therapeutic effects of early-stage chick embryo amniotic fluid on acute gastric ulcers and achieved significant results.

[0018] The inventors evaluated the efficacy of early-stage chick embryo amniotic fluid in treating acute gastric ulcers. Animal experiments confirmed that early-stage chick embryo amniotic fluid can reduce the area of ​​gastric ulcers and the infiltration of inflammatory factors, enhance the protective effect of the mucosal barrier on gastric tissue by reducing calcium ion content and myeloperoxidase in gastric tissue, significantly inhibit ethanol-induced oxidative stress, and increase the level of tight junction proteins in gastric tissue. This suggests that this fluid has the potential to be used as a drug for treating gastric ulcers. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Middle A is the analysis result of the index of early chicken embryo amniotic fluid on the organs (liver, kidney, spleen) of mice with acute gastric ulcer (ns>0.05);

[0020] Figure 1 Figure B shows representative images and analysis of the effects of early chick embryo amniotic fluid treatment on gastric ulcer area in mice with acute gastric ulcers. One-way ANOVA was used to test for significant differences between groups. If the differences between groups were statistically significant, post hoc pairwise comparisons were performed using the Student-Newman-Keuls (SNK) test. The p-value was used to determine statistical significance (*P < 0.05, **P < 0.01, ***P < 0.001).

[0021] Figure 1 Middle C is a representative picture of the pathological section of gastric tissue in mice with acute gastric ulcer treated with early chick embryo amniotic fluid;

[0022] Figure 2 Figure D shows the results of an analysis of the levels of inflammatory factors TNF-α, IL-1β, and IL-6, and chemokines iNOS and COX2 in gastric tissue of mice with acute gastric ulcers using early chick embryo amniotic fluid. One-way ANOVA was used to test for significant differences between groups. If the differences between groups were statistically significant, post hoc pairwise comparisons were performed using the Student-Newman-Keuls (SNK) test. The p value was used as the criterion for statistical significance (*P < 0.05, **P < 0.01, ***P < 0.001).

[0023] Figure 2 Figure E shows the results of an analysis of calcium ion and MPO levels in gastric tissue of mice with acute gastric ulcers using early chick embryo amniotic fluid. One-way ANOVA was used to test for significant differences between groups. If the differences between groups were statistically significant, post hoc pairwise comparisons were performed using the Student-Newman-Keuls (SNK) test. The p value was used as the criterion for statistical significance (*P < 0.05, **P < 0.01, ***P < 0.001).

[0024] Figure 2 Figure F shows the analysis of SOD, MDA, and GSH levels in gastric tissue of mice with acute gastric ulcers treated with early chick embryo amniotic fluid. One-way ANOVA was used to test for significant differences between groups. If the differences between groups were statistically significant, post hoc comparisons were performed using the Student-Newman-Keuls (SNK) test. The p value was used as the criterion for statistical significance (*P < 0.05, **P < 0.01, ***P < 0.001).

[0025] Figure 3 Figure G shows representative images and analysis of the effects of early chick embryo amniotic fluid treatment on the expression of tight junction proteins ZO-1 and occcludin in small stomach tissue of patients with acute gastric ulcers. One-way ANOVA was used to test for significant differences between groups. If statistically significant, post hoc comparisons were performed using the Student-Newman-Keuls (SNK) test. Statistically significant differences were determined using p-values ​​(*P < 0.05, **P < 0.01, ***P < 0.001). DETAILED DESCRIPTION

[0026] Example 1: Study on the therapeutic effect of early chicken embryo amniotic fluid on acute gastric ulcer

[0027] (1) Experimental animals

[0028] In this study, 30 8-week-old C57BL / 6 male mice were purchased from Suzhou Sibeifu Biotechnology Co., Ltd. in Jiangsu Province. They were maintained at the SPF-grade Laboratory Animal Center of Wuxi Medical College, Jiangnan University. The experimental protocol was approved by the Animal Experimentation Ethics Committee of Wuxi Medical College, Jiangnan University. All experimental animals were housed at 22-26°C, 40-70% relative humidity, and a 12-hour light / 12-hour dark cycle. Animals had free access to food and water.

[0029] (2) Preparation of early chick embryo amniotic fluid

[0030] Fertilized chicken eggs were incubated at 37 ± 1°C and 50% humidity. Amniotic fluid was collected from 6-8 day old chick embryos using a sterile syringe. The collected amniotic fluid was centrifuged at 12,000 rpm for 30 minutes and stored at -80°C for a long term.

[0031] (III) Construction of an acute gastric ulcer mouse model and early chick embryo amniotic fluid intervention

[0032] Thirty C57BL / 6 male mice were randomly divided into five groups according to their body weight, with six mice in each group, namely: control group (10 ml / kg pure water was gavaged daily, and blood was collected from the eyeball about one hour after gavage of pure water for ten days before being killed); acute gastric ulcer group (AGC) (10 ml / kg pure water was gavaged daily, and 10 ml / kg anhydrous ethanol was gavaged once ten days later, and blood was collected from the eyeball about one hour after being killed); early chick embryo amniotic fluid low concentration group (L-ceAF) (7.5 ml / kg early chicken embryo amniotic fluid, ten days later, the last gavage of chicken embryo amniotic fluid was followed by gavage of 10 ml / kg anhydrous ethanol 30 minutes later, and the animals were killed after one hour of eye bleeding); a high concentration early chicken embryo amniotic fluid group (H-ceAF) (15 ml / kg early chicken embryo amniotic fluid was gavaged every day, ten days later, the last gavage of chicken embryo amniotic fluid was followed by gavage of 10 ml / kg anhydrous ethanol 30 minutes later, and the animals were killed after one hour of eye bleeding); a positive drug group (Omeprazole) (20 mg / kg omeprazole was gavaged every day, ten days later, the last gavage of omeprazole was followed by gavage of 10 ml / kg anhydrous ethanol 30 minutes later, and the animals were killed after one hour of eye bleeding).

[0033] (IV) Experimental results

[0034] 1. Effects of early chicken embryo amniotic fluid on organ index and ulcer area in mice with acute gastric ulcer

[0035] In toxicology experiments, liver index, kidney index and spleen index are commonly used organ indexes. These indicators can effectively reflect whether the body is contaminated by toxins and whether the immune function is affected. Therefore, they play an important role in toxicology research and are often used to evaluate the safety of test substances. In implementation cases, such as Figure 1 As shown in Part A, after mice were gavaged with different concentrations of early chicken embryo amniotic fluid and a positive drug for 10 consecutive days, the liver, kidney, and spleen indexes of the mice in each test group showed no significant differences compared with the normal group. This result indicates that a 15ml / kg dose of early chicken embryo amniotic fluid is relatively safe for mice. In addition, Figure 1 As shown in Part B, the gastric tissue morphology of the control mice was normal, with no ulcers, congestion, or edema. However, the gastric mucosal damage in the acute gastric ulcer group was severe, with extensive strip-like and patchy bleeding. The gastric ulcer area was large (14.47±2.85). Compared with the acute gastric ulcer group, the gastric ulcer area was significantly reduced in the mice treated with different concentrations of early chick embryo amniotic fluid and omeprazole (low concentration group: 10.1715±1.40; high concentration group: 9.31±1.312107719; positive drug group: 6.76±2.54), with reduced gastric mucosal damage and alleviated bleeding symptoms. In summary, early chick embryo amniotic fluid demonstrates a positive effect in inhibiting gastric ulcers.

[0036] 2. Early chicken embryo amniotic fluid relieves acute gastric ulcers caused by ethanol

[0037] The results of histopathological examination were as follows Figure 1 As shown in section C of the figure. In the gastric tissue of control mice, glands were neatly arranged and well-connected. The mucosal, submucosa, and muscular layers were structurally intact and clearly layered. No vascular dilation, congestion, or inflammatory cell infiltration was observed. However, gastric tissue sections from mice with acute gastric ulcers revealed that ethanol caused severe damage to the gastric tissue: the gastric mucosa was damaged and ruptured, with extensive epithelial cell shedding and damage even extending to the submucosal muscularis. Furthermore, excessive alcohol intake caused edema and widening of the gastric submucosa, with significant inflammatory cell infiltration and extensive bleeding, representing a typical inflammatory response. In the early chick embryo amniotic fluid group and the positive drug omeprazole group, the severity of gastric damage was alleviated. Although some epithelial loss persisted, the lesions were shallower, the number of inflammatory cells was significantly reduced, and bleeding was alleviated compared to the acute gastric ulcer group. The mucosal surface remained largely intact, with only minor epithelial shedding and superficial mucosal damage. The number of inflammatory cells and the amount of bleeding were significantly less than those in the acute gastric ulcer group. In summary, early chicken embryo amniotic fluid has the effect of alleviating ethanol-induced acute gastric ulcer.

[0038] 3. Early chicken embryo amniotic fluid improves gastric tissue inflammatory response in mice with acute gastric ulcer

[0039] Acute gastric ulcers are often accompanied by significant changes in inflammatory factors. In order to study the anti-inflammatory ability of early chicken embryo amniotic fluid, in this embodiment, the levels of pro-inflammatory factors TNF-α, IL-1β, IL-6 and chemokines iNOS and COX2 in mouse gastric tissue were measured. Figure 2 As shown in Part D, compared with the control group, the gastric secretion levels of TNF-α, IL-1β, IL-6, iNOS, and COX2 in the disease group mice were significantly increased. Compared with the disease group, the gastric secretion levels of TNF-α, IL-1β, IL-6, iNOS, and COX2 in the early chick embryo amniotic fluid group and the positive drug group were significantly decreased. This suggests that early chick embryo amniotic fluid can improve the inflammatory response in the gastric tissue of mice with acute gastric ulcers.

[0040] 4. Effects of early chicken embryo amniotic fluid on the gastric mucosal barrier in mice with acute gastric ulcer

[0041] The effects of early chicken embryo amniotic fluid on the gastric mucosal barrier of mice exposed to acute alcohol overdose were studied by measuring the calcium ion content and myeloperoxidase (MPO) activity in gastric tissue. Figure 2As shown in Part E, compared with the control group, the gastric tissue calcium ion content and MPO activity in mice with acute gastric ulcers increased. Compared with the acute gastric ulcer group, the gastric tissue calcium ion and MPO levels in the early chicken embryo amniotic fluid group were significantly reduced. These results indicate that alcohol causes a large number of inflammatory cells to invade the submucosal tissue of mouse gastric tissue, and early chicken embryo amniotic fluid can alleviate alcohol-induced damage to the gastric mucosal barrier and protect the gastric mucosa.

[0042] 5. Early chicken embryo amniotic fluid reduces oxidative stress in gastric tissue of mice with acute gastric ulcer

[0043] This case study aimed to investigate the inhibitory effect of early chicken embryo amniotic fluid on free radical damage caused by acute gastric ulcer by detecting the content of malondialdehyde (MDA) and the activities of superoxide dismutase (SOD) and glutathione (GSH) in gastric tissue. Figure 2 As shown in Section F, experimental data show that compared with the control group, SOD activity and GSH activity in the gastric tissue of mice with acute gastric ulcers decreased significantly, while MDA content increased significantly. Furthermore, compared with the disease group, SOD and GSH activities in early-stage chick embryo amniotic fluid at different concentrations were significantly increased, while MDA content was significantly decreased. These experimental results demonstrate that early-stage chick embryo amniotic fluid exhibits significant antioxidant effects in the ethanol-induced acute gastric ulcer model.

[0044] 6. Early chicken embryo amniotic fluid upregulates the expression of tight junction proteins in gastric tissue of mice with acute gastric ulcer

[0045] Gastric tight junction proteins are also one of the pathological characteristics of acute gastric ulcers. In this case, compared with the control group, the expression of gastric tight junction proteins ZO-1 and Occcludin in the acute gastric ulcer mice was significantly reduced. Compared with the disease group, the expression of gastric tight junction proteins ZO-1 and Occcludin in the different concentrations of early chicken embryo amniotic fluid and positive drug group mice was significantly upregulated ( Figure 3 The above experimental results show that in the ethanol-induced acute gastric ulcer model, early chicken embryo amniotic fluid can upregulate the expression of tight junction proteins in the gastric tissue of acute gastric ulcer mice.

[0046] Finally, it should be noted that the above embodiments are only used to illustrate rather than limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the present invention can still be modified or replaced by equivalents. Any modification or partial replacement that does not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. Application of chicken embryo amniotic fluid in the preparation of medicine for treating gastric ulcer.

2. The use according to claim 1, characterized in that The gastric ulcer is an acute gastric ulcer.

3. The use according to claim 1, characterized in that The gastric ulcer is a chronic gastric ulcer.

4. The use according to any one of claims 1 to 3, characterized in that The preparation method of the chicken embryo amniotic fluid comprises the following steps: incubating fertilized eggs at 37±1°C and 45-60% humidity, collecting the chicken embryo amniotic fluid incubated for 6-8 days, centrifuging the collected amniotic fluid at 10,000-14,000 r / min for 10-40 minutes, and collecting the supernatant to obtain the chicken embryo amniotic fluid.

5. The use according to claim 4, characterized in that The chicken embryo amniotic fluid preparation method comprises the following steps: incubating fertilized eggs at 37±1°C and 50% humidity, collecting the chicken embryo amniotic fluid incubated for 6 to 8 days, centrifuging the collected amniotic fluid at 12,000 r / min for 30 minutes, and collecting the supernatant to obtain the chicken embryo amniotic fluid.

6. The use according to any one of claims 4-5, characterized in that The chicken embryo amniotic fluid is collected using a sterile syringe.

7. The use according to any one of claims 1 to 6, characterized in that The storage temperature of the chicken embryo amniotic fluid is -80°C.

8. The use according to any one of claims 1 to 7, characterized in that The medicine consists of chicken embryo amniotic fluid and a pharmaceutically acceptable carrier.

9. The use according to any one of claims 1 to 8, characterized in that The drug exists in the form of an external preparation, an oral preparation or an injection preparation.

10. The use according to claim 9, wherein the drug is in the form of a gel, spray, aerosol, patch, tincture, paste, lotion, cream, cream, ointment, granule, capsule, pill, tablet, powder, oral liquid or syrup.

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