CN114177262A

CN114177262A - Application of zucchini in inhibiting gastric mucositis caused by helicobacter pylori infection

Info

Publication number: CN114177262A
Application number: CN202111572763.XA
Authority: CN
Inventors: 石岩岩; 格桑罗布; 丁士刚; 詹思延; 李渊; 李默; 李楠; 宁静; 周凤; 白玛; 洛桑顿珠; 尼玛曲珍; 洛桑次仁
Original assignee: Tibet Ganlu Tibetan Medicine Co ltd
Current assignee: Tibet Ganlu Tibetan Medicine Co ltd
Priority date: 2021-12-21
Filing date: 2021-12-21
Publication date: 2022-03-15

Abstract

The invention relates to an application of zuodaxisi in inhibiting gastric mucositis caused by helicobacter pylori infection, belonging to the technical field of biological medicines. The invention is proved by animal in vivo experiments that: the Zuozhudaxi can reverse gastric mucosa injury caused by helicobacter pylori infection to a certain extent, and reduce gastric mucosa inflammation caused by the helicobacter pylori infection. Therefore, the zucchini has important clinical application value in the aspect of inhibiting inflammation caused by helicobacter pylori infection.

Description

Application of zucchini in inhibiting gastric mucositis caused by helicobacter pylori infection

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of zuodaxixi in inhibiting gastric mucositis caused by helicobacter pylori infection.

Background

Helicobacter pylori (Hp) is a gram-negative bacterium parasitic on gastric mucosa, is a common bacterium in chronic digestive system diseases, can infect stomach to cause gastric ulcer, can be degraded into gastric cancer in severe cases, and seriously threatens the health and life of patients. Helicobacter pylori is closely related to peptic ulcer, digestive system tumor and lymphatic tissue lymphoma related to digestive tract mucosa, and is listed as a class I carcinogen of gastric cancer by the world health organization. In China, the average Hp infection rate is about 58.07%.

With the development of Chinese medicine, researchers of traditional Chinese medicine find that helicobacter pylori can be effectively treated by using the traditional Chinese medicine. The clinical research of the Tibetan medicine shows that the Tibetan medicine has the effective rate of up to 90 percent when used for treating the Powa wood cloth (gastric ulcer) by taking the Zuozhu Daxi orally every day, treats the long-term numbness cloth (duodenal ulcer), has the healing rate of 81 percent and has the Hp clearance rate of 15 percent. Zuozhuda was originally recorded in "Wan Schli" of medicine, has the effects of soothing liver, invigorating stomach, clearing heat, healing ulcer and reducing swelling, and is used for treating lingering and unhealed diseases of wood cloth, gastric upset, food poisoning, old internal medicine diseases and the like.

Currently, the anti-helicobacter pylori infection-induced gastric mucositis and its mechanism are unclear.

Disclosure of Invention

The application aims to solve the technical problem and provides application of zucchini in inhibiting gastric mucositis caused by helicobacter pylori infection.

The application is realized by the following technical scheme:

application of zucchini in inhibiting gastric mucositis caused by helicobacter pylori infection is provided.

Further, the use includes reversing gastric mucosal damage caused by helicobacter pylori infection and reducing gastric mucositis caused by helicobacter pylori infection.

Preferably, the helicobacter pylori is a standard strain.

Further, the pathological degree of the gastric mucositis is related to the expression amount of inflammatory factors.

Preferably, the inflammatory factors include TNF- α, IL-1 α, IL-6, IL-8, PGE2, NOD 1.

Further, the assay for TNF- α, IL-1 α, IL-8 and NOD1 was real-time PCR.

Further, the detection method of IL-1. alpha., IL-6 and PGE2 was ELISA method.

Application of zucchini in preparing medicine for inhibiting gastric mucositis caused by helicobacter pylori infection is provided.

Preferably, the dose of the zucchini is 0.083g/kg or 0.166 g/kg.

Furthermore, the medicine also comprises pharmaceutically acceptable auxiliary materials.

Compared with the prior art, the method has the following beneficial effects:

in animal experiments, HE pathological staining of mouse gastric mucosal tissues shows that the gastric mucosa is damaged after Hp infection, and the gastric mucosa damage caused by Hp infection can be reversed to a certain extent by Darcy sitting beads. Compared with the Hp infection group, the mRNA levels of IL-8 and NOD1 are obviously reduced after the dacarbazine is added, the ELISA result shows that IL-1 alpha, IL-6 and PGE2 are obviously reduced in the drug-added group, and the dacarbazine can reduce the gastric mucositis caused by Hp infection to a certain extent.

Drawings

The accompanying drawings, which are included to provide a further understanding of the embodiments of the invention and are incorporated in and constitute a part of this application, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention.

In fig. 1, A is an animal experiment environment diagram of the invention, B is an animal experiment control system interface diagram, and C is a feeding box and a padding schematic diagram;

in FIG. 2, A is a schematic diagram of mouse feeding and B is a schematic diagram of mouse gavage;

in FIG. 3, A is a mouse anatomy diagram, B is a mouse stomach diagram, and C is a mouse blood drawing diagram;

in FIG. 4, A is a schematic diagram of Hp immunohistochemical staining of gastric mucosal tissue of mice in a negative control group in a pre-test, and B is a schematic diagram of Hp immunohistochemical staining of gastric mucosal tissue of mice in a Hp infection group in the pre-test;

FIG. 5A is a schematic diagram of Real-time PCR detection of IL-1 alpha of gastric mucosal tissue of mice in each group in a preliminary test, B is a schematic diagram of Real-time PCR detection of IL-8 of gastric mucosal tissue of mice in each group in a preliminary test, C is a schematic diagram of Real-time PCR detection of TNF-alpha of gastric mucosal tissue of mice in each group in a preliminary test, and D is a schematic diagram of Real-time PCR detection of NOD1 of gastric mucosal tissue of mice in each group in a preliminary test;

FIG. 6 is a graph showing the ROS levels in the serum of groups of mice in a pre-test;

FIG. 7A is a schematic diagram of ELISA kit detection of IL-1. alpha. in each mouse serum group in the preliminary test, B is a schematic diagram of ELISA kit detection of IL-6 in each mouse serum group in the preliminary test, and C is a schematic diagram of ELISA kit detection of PGE2 in each mouse serum group in the preliminary test;

in FIG. 8, A is a schematic diagram of Hp immunohistochemical staining of gastric mucosal tissue of mice in a negative control group in a formal test, B is a schematic diagram of Hp immunohistochemical staining of gastric mucosal tissue of mice in a Hp infection group in a formal test, C is a schematic diagram of Hp immunohistochemical staining of gastric mucosal tissue of mice in a drug treatment group (administration for 7 days, dosage of 0.083g/kg) after Hp infection in a formal test, D is a schematic diagram of Hp immunohistochemical staining of gastric mucosal tissue of mice in a drug treatment group (administration for 7 days, dosage of 0.166g/kg) after Hp infection in a formal test, and E is a schematic diagram of Hp immunohistochemical staining of gastric mucosal tissue of mice in a drug treatment group (administration for 14 days, dosage of 0.166g/kg) after Hp infection in a formal test;

FIG. 9 is a schematic diagram of pathological staining of HE in gastric mucosal tissue of mice in a negative control group in a formal test, B is a schematic diagram of pathological staining of HE in gastric mucosal tissue of mice in a Hp infection group in the formal test, C is a schematic diagram of pathological staining of HE in gastric mucosal tissue of mice in a drug treatment group (with a dose of 0.083g/kg) after Hp infection in the formal test (with a dose of 7 days and a dose of 0.166g/kg), D is a schematic diagram of pathological staining of HE in gastric mucosal tissue of mice in a drug treatment group (with a dose of 0.166g/kg) after Hp infection in the formal test, and E is a schematic diagram of pathological staining of HE in gastric mucosal tissue of mice in a drug treatment group (with a dose of 14 days and a dose of 0.166g/kg) after Hp infection in the formal test;

FIG. 10A is a schematic diagram of Real-time PCR detection of mouse gastric mucosal tissue IL-1 alpha in each group in the official test, B is a schematic diagram of Real-time PCR detection of mouse gastric mucosal tissue IL-8 in each group in the official test, C is a schematic diagram of Real-time PCR detection of mouse gastric mucosal tissue TNF-alpha in each group in the official test, and D is a schematic diagram of Real-time PCR detection of mouse gastric mucosal tissue NOD1 in each group in the official test;

FIG. 11 is a graph showing the ROS levels in serum of various groups of mice in a formal test;

in FIG. 12, A is a schematic diagram of ELISA kit detection of mouse serum IL-1. alpha. in each group in the main test, B is a schematic diagram of ELISA kit detection of mouse serum IL-6 in each group in the main test, and C is a schematic diagram of ELISA kit detection of mouse serum PGE2 in each group in the main test.

Detailed Description

In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments. It is to be understood that the described embodiments are only a few embodiments of the present invention, and not all embodiments. The components of embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.

Hp is parasitic bacteria of the stomach wall of a human, is not easy to reproduce in animals, and a simple and efficient Hp infection animal model capable of simulating the human infection pathological process is a commonly used infection model for research. The animal species that can be used to establish the model are various, and for an Hp infection animal model, the animal selection must satisfy the following conditions: the animal source is sufficient, and the operation is economic and simple; secondly, no homological bacteria are planted and propagated in the gastrointestinal tract; the histopathological characteristics of human infection Hp can be fully simulated; the infection duration is long enough, and long-term or long-term observation is easy to carry out; pure animals are easy to control various treatment factors; and sixthly, the model has strong repeatability and is convenient for international comparison and communication. Mice are the most commonly used rodents for pharmacodynamic testing at present. Because the animal and the human have similar anatomical physiology and metabolic characteristics, and have the advantages of clear genetic background, small volume, small consumed dosage, stable reaction to the drug, small individual difference, easy reproduction and feeding and the like, the animal and the human are the recognized animal types at home and abroad for pharmacodynamic experiments at present. The construction of helicobacter pylori infection animal models is mature, and is a common animal model for researching helicobacter pylori infection and related gastritis. Therefore, the invention adopts the mouse to carry out modern pharmacological experiments, clarifies the function and mechanism of the Tibetan medicine zuozhidaxixi for resisting the helicobacter pylori, forms a new method, a new technology and a new scheme for treating the helicobacter pylori related diseases by the Tibetan medicine, and lays a foundation for expanding the clinical application of the Tibetan medicine. The animal experiment adopted by the invention is approved by review of the Beijing university biomedical ethics committee experimental animal welfare ethics division with the approval number LA 2019343.

Example A preliminary test

1. Test materials

(1) Test subjects: the SPF grade C57BL/6 mice 36 were male, 6 weeks old, and the production license number SCXK (Jing) 2017-. Animal experimental environment as shown in fig. 1, a constant temperature and humidity spf (specific pathogen free) grade animal laboratory was used. The mice adopt CP-3 type mouse rearing cages, the padding is corncob padding, and the mice fed with SPF mice maintain the feed and drink experimental animal drinking water (figure 2A).

TABLE 1 animal weight information Table

Purchasing animal weight and age	Animal weight and age at the time of administration
		16g to 20g (about 6 weeks old)	27g to 32g (about 12 weeks old)

(2) Strain: the International Standard helicobacter pylori (Hp) SS1 strain is available in the laboratory of the third Hospital, Beijing university.

(3) Test article and solvent: finished pills (1 g/pill) of the zuzhudaxi are prepared, the dosage of the zuzhudaxi is 0.166g/kg, and the pill is set according to a common treatment cycle of a helicobacter pylori eradication treatment scheme, and the pill is administered for 7 days and 14 days; the solvent is sterile PBS buffer solution.

TABLE 2 information sheet for sample preparation

Dosage to be administered	Weighing (g)	Volume of preparation (mL)	Concentration (g dry extract powder/mL)
				0.166g/kg	1680mg	168mL	10mg/mL

(4) Instruments and reagents

TABLE 3 Main Instrument information Table

TABLE 4 Main reagent information Table

2. Test method

(1) Culture of helicobacter pylori strains

The international standard helicobacter pylori (Hp) SS1 strain can be recovered directly and used for experiments. The recovery method comprises the following steps: the frozen strains were cultured in Columbia selective solid medium at 37 ℃ in a microaerophilic environment (10% CO)₂，5％O₂，85％N₂) Recovering, culturing for 48-72 h, and collecting bacteria. The liquid culture method comprises the following steps: after recoveryAnd (3) collecting bacteria, adding the bacteria into the high-pressure sterilized Brookfield culture broth, and shaking the bacteria in a constant temperature shaking table at the temperature of 37 ℃ and the rpm of 110 for 24-48 h under a microaerophilic environment. The specific time of the culture depends on the size of the inoculum size.

(2) Helicobacter pylori infection animal model

SPF grade C57 mice were kept in 12 hour light and 12 hour dark room environment. Recovering and culturing Hp, extracting sterile physiological saline with sterile syringe to wash Hp culture plate, collecting Hp, adjusting the amount of suspension to 1 × 10 by turbidimetry⁸CFU/mL. The method comprises the steps of removing foreign bacteria in the stomach of a mouse by using antibiotics, pretreating, and then feeding each mouse with 0.5mL of intragastric Hp bacterial suspension for 1 time every other day for 5 times. Fasting and water deprivation were performed for 24h before each gavage, and food was fed 4h after gavage, followed by normal feeding (fig. 2B).

(3) Test grouping

Group 1-negative control group (12);

group 2-Hp infection group (12);

group 5-Hp infection + drug treatment group (6), bead-on-bead was administered for 7 days (bead-on-bead dose: 0.166g/kg, dissolved in PBS);

group 6-Hp infection + drug treatment group (6), Cyperus rotundus was administered for 14 days (Cyperus rotundus dose: 0.166g/kg, dissolved in PBS).

(4) Grouping animal treatment situations

1) Group 1: the water is freely fed.

2) Group 2, group 5, group 6: intragastric administration of Hp, 3X 10⁸CFU/mL, 0.5 mL/mouse, 1 time every other day, 5 times in total. Fasting and water prohibition are carried out for 24h before each intragastric administration, and food is fed after intragastric administration for 4h, and then normal feeding is carried out.

3) Animals were normally kept for 5 weeks. At 5 weeks after the last gavage, the first batch of mice was sacrificed. The success of the infection was demonstrated.

First batch sacrifice: negative control group (6); hp-infected group (6).

4) And in the drug treatment group, the drugs are infused into the stomach 1 time a day for 7-14 days continuously. And (4) after the gavage, water is forbidden within 30 minutes, and then the fish is normally bred. After the last administration, the patient is fasted and water is not forbidden for 24 hours. The second batch of mice was sacrificed.

Second batch sacrifice: negative control group (6); hp-infected group (6); hp infection + drug treatment groups (6 each).

(5) Pre-testing groups of pathological assays

As shown in FIG. 3, the stomach was taken for gross observation, the pathological type was clarified by H & E staining, and the Hp infection was clarified by WS staining. The curative effect of Tibetan medicine treatment on Hp-related gastric mucosa diseases and the curative effect on Hp eradication are discussed. Collecting mouse gastric mucosa tissue, grinding Hp infected animal gastric mucosa tissue in liquid nitrogen environment, extracting tissue total RNA by Trizol method to perform real-time PCR, and measuring molecular expression related to inflammation and oxidative stress in gastric tissue, including inflammatory factors TNF-alpha, IL-1 alpha, IL-6, IL-8, etc. Mouse serum was collected and tested for ROS levels and IL-1 α, IL-6, PGE2, etc. by ELISA.

3. Test results

(1) Hp immunohistochemical staining

As shown in fig. 4, the immunohistochemical staining of Hp in mouse gastric mucosal tissue showed: the successful construction of the Hp-infected C57BL/6 mouse model (10X) (NC: negative control, negative control; HP: H.pylori-infected group) indicated that the Hp-infected mouse animal model had been successfully constructed.

(2) Real-time PCR detection of inflammatory factors of gastric mucosal tissue

As shown in FIG. 5, IL-1. alpha. was different between 1/6 and 2/6 groups, and IL-1. alpha. was decreased but was lower than that in the normal 1 group after the drug was added. No difference was found between 1/2 groups.

IL-8 differed between group 1/group 2, with a marked increase following Hp infection. Compared with the Hp infection group, IL-8 has a reduction trend after the drug is added, but the difference has no statistical significance.

TNF- α was distinct in groups 1/2, and was markedly elevated after Hp infection. But the trend of reduction after adding the medicine is not seen.

NOD1 differed between group 1/group 2, with a marked increase following Hp infection. NOD1 decreased in the 7-day medicated group.

(3) Serum Reactive Oxygen Species (ROS) detection kit for detecting ROS level in serum

As shown in FIG. 6, there was a slight trend of increase after Hp infection compared between group 1/2, but the difference was not statistically significant. ROS were reduced in the 14-day dosing group (6) compared to the 2 group.

(4) ELISA kit for detecting inflammatory factors in serum

As shown in fig. 7, IL-1 α was higher in group 2 than group 1, but the difference was not statistically significant (P ═ 0.057). The 5 and 6 groups were lower than the 2 group, and the difference was statistically significant.

IL-6 was elevated in group 2 compared to group 1, but the difference was not statistically significant. The 5 and 6 groups were lower than the 2 group, and the difference was statistically significant.

PGE2 was higher in group 2 than group 1, but the difference was not statistically significant (P ═ 0.093). The 5 and 6 groups were lower than the 2 group, and the difference was statistically significant.

Example II official test

1. Test materials

(1) Test subjects: 30 SPF grade C57BL/6 mice, male, 6 weeks old, production license number SCXK (Jing) 2017-. The experimental environment and the feeding condition of the animals are the same as those in the first embodiment.

TABLE 5 animal weight information Table

Purchasing animal weight and age	Animal weight and age at the time of administration
		16g to 20g (about 6 weeks old)	35g to 40g (about 19 weeks old)

(2) Strain: the same as the first embodiment.

(3) Test article and solvent: finished pills (1 g/pill) of the zuzhudaxi are prepared, the application dosage of the zuzhudaxi is 0.083g/kg and 0.166g/kg, and the treatment period and the solvent are the same as those of the first embodiment.

TABLE 6 information table for preparing test sample

Dosage to be administered	Weighing (g)	Volume of preparation (mL)	Concentration (g dry extract powder/mL)
				0.083g/kg	152.72mg	23mL	6.64mg/mL
0.166g/kg	863.2mg	65mL	13.28mg/mL

(4) Instruments and reagents: the same as the first embodiment.

2. Test method

(1) Culturing helicobacter pylori strains: the same as the first embodiment.

(2) Modeling of helicobacter pylori infection animal model: the same as the first embodiment.

(3) Test grouping

Group 1-negative control group (6);

group 2-Hp infection group (6);

6 groups-Hp infection + drug treatment group (6), Cyperus rotundus was administered for 7 days (Cyperus rotundus dose: 0.083g/kg, dissolved in PBS);

7 groups-Hp infection + drug treatment group (6), Cyperus rotundus was administered for 7 days (Cyperus rotundus administration dose: 0.166g/kg, dissolved in PBS);

group 8-Hp infection + drug treatment group (6), 14 days of Cyclamen Darcy administration (Cyclamen Darcy administration dose: 0.166g/kg, dissolved in PBS).

(4) Grouping animal treatment situations

1) Group 1: the water is freely fed.

Group

2, 6, 7 and 8: intragastric administration of Hp, 3X 10⁸CFU/mL, 0.5 mL/mouse, 1 time every other day, 5 times in total. Fasting and water prohibition are carried out for 24h before each intragastric administration, and food is fed after intragastric administration for 4h, and then normal feeding is carried out.

2) Animals were normally kept for 12 weeks. At 12 weeks after the last gavage, the first batch of mice was sacrificed. The success of the infection was demonstrated.

First batch sacrifice: negative control group (3); hp-infected group (3).

3) And in the drug treatment group, the drugs are infused into the stomach 1 time a day for 7-14 days continuously. And (4) after the gavage, water is forbidden within 30 minutes, and then the fish is normally bred. After the last administration, the patient is fasted and water is not forbidden for 24 hours. The second batch of mice was sacrificed.

Second batch sacrifice: negative control group (3); hp-infected group (3); hp infection + drug treatment groups (6 each).

(5) Pre-testing each group of pathological examination: the same as the first embodiment.

3. Test results

(1) Hp immunohistochemical staining

As shown in fig. 8, the immunohistochemical staining of Hp in mouse gastric mucosal tissue showed: the Hp-infected C57BL/6 mouse model was successfully constructed (10X), indicating that the Hp-infected mouse model was successfully constructed.

(2) Pathological histological examination of gastric mucosa

As shown in fig. 9, HE pathological staining of mouse gastric mucosal tissue revealed: after Hp infection, gastric mucosal injury caused by Hp infection can be reversed to a certain extent by Darcy sitting on beads (10X).

(3) Real-time PCR detection of inflammatory factors of gastric mucosal tissue

As shown in fig. 10, IL-1 α was significantly increased in group 2 compared to group 1, with P ═ 0.008; there was no difference between 6/7/8 groups compared to 2 groups (8 groups had a decreasing trend).

Compared with the group 1, the IL-8 is obviously increased in the group 2, and the P is 0.008; the 8 groups were decreased compared to the 2 groups, the difference was statistically significant, and P ═ 0.032.

TNF- α was not distinguished between groups 1/2. No reduction trend is seen after the medicine is added.

NOD1 tended to increase in group 2 compared to group 1, but the difference was not statistically significant. The NOD1 tended to decrease compared to the group 2 with the 6/8 dosing group, but the difference was not statistically significant.

(4) Serum Reactive Oxygen Species (ROS) detection kit for detecting ROS level in serum

As shown in FIG. 11, there was a tendency for the Hp infection to increase in comparison between group 1 and group 2, and the difference was not statistically significant. The drug adding group has no reduction trend.

(5) ELISA kit for detecting inflammatory factors in serum

As shown in FIG. 12, IL-1. alpha. was significantly higher in group 2 than in group 1, P < 0.001, and significantly decreased in group 6/group 7/group 8 compared to group 2, with statistical significance of the difference.

IL-6 was significantly higher in group 2 than in group 1, P0.001, and the difference was significantly lower in group 7/8 compared to group 2, which was statistically significant.

PGE2 was significantly higher in group 2 than in group 1, P was < 0.001, and the difference was significantly lower in group 6/7/8 compared to group 2, with statistical significance.

In summary, the results of two animal experiments and statistical analysis of the data show that: compared with the simple Hp infection, the inflammation degree of the gastric mucosa is reduced after the Zuozhudaxi treats the Hp infection, the Zuozhudaxi has obvious inhibition effect on the expression of inflammatory factor mRNA in the gastric mucosa tissue when the Hp infection is in short time (7 days animal infection), and the Zuozhudaxi has obvious inhibition effect on the expression of the inflammatory factor in the serum when the Hp infection is in long time (14 days animal infection). The Tibetan medicine ZUZHUDAXIE is prompted to effectively control the inflammatory reaction caused by Hp.

The above embodiments are provided to explain the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above embodiments are merely exemplary embodiments of the present invention and are not intended to limit the scope of the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. Application of zucchini in inhibiting gastric mucositis caused by helicobacter pylori infection is provided.

2. Use according to claim 1, characterized in that: the applications include reversing gastric mucosal damage caused by helicobacter pylori infection and reducing gastric mucositis caused by helicobacter pylori infection.

3. Use according to claim 1 or 2, characterized in that: the helicobacter pylori is a standard strain.

4. Use according to claim 2, characterized in that: the pathological degree of the gastric mucositis is related to the expression amount of inflammatory factors.

5. Use according to claim 4, characterized in that: the inflammatory factors include TNF-alpha, IL-1 alpha, IL-6, IL-8, PGE2, NOD 1.

6. Use according to claim 5, characterized in that: the assay for TNF- α, IL-1 α, IL-8 and NOD1 was real-time PCR.

7. Use according to claim 5, characterized in that: the detection method for IL-1 alpha, IL-6 and PGE2 was ELISA method.

8. Application of zucchini in preparing medicine for inhibiting gastric mucositis caused by helicobacter pylori infection is provided.

9. Use according to claim 8, characterized in that: the dose of the zucchini is 0.083g/kg or 0.166 g/kg.

10. Use according to claim 9, characterized in that: the medicine also comprises pharmaceutically acceptable auxiliary materials.