CN114748484B

CN114748484B - Application of ursodeoxycholic acid in preparation of medicine for preventing and treating colibacillosis

Info

Publication number: CN114748484B
Application number: CN202111460070.1A
Authority: CN
Inventors: 曹志军; 何至远; 马玉林
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2021-12-02
Filing date: 2021-12-02
Publication date: 2024-04-09
Anticipated expiration: 2041-12-02
Also published as: CN114748484A

Abstract

The invention provides application of ursodeoxycholic acid in preparing a medicine for preventing and treating colibacillosis. The invention proves that ursodeoxycholic acid can promote the production of short-chain fatty acid by inhibiting inflammatory reaction in the colibacillosis of cows through in vitro cell tests and mouse tests, thereby playing a role in protecting the colibacillosis of cows. The ursodeoxycholic acid has a good protection effect on colonitis caused by colibacillosis of cows, and lays a theoretical foundation for preventing and treating the colibacillosis of the cows by adopting the ursodeoxycholic acid.

Description

Application of ursodeoxycholic acid in preparation of medicine for preventing and treating colibacillosis

Technical Field

The invention relates to the field of biological medicine, in particular to application of ursodeoxycholic acid or conjugated bile acid or pharmaceutically acceptable salt thereof in preparation of medicines for preventing and treating colibacillosis.

Background

Diarrhea of cows is a disease caused by various factors, and causes of the diarrhea mainly comprise pathogenic microorganisms such as escherichia coli, rotavirus, coronavirus, cryptosporidium and the like, and external factors such as nutrition, environment, feeding management and the like, wherein the incidence rate and death rate of diarrhea caused by Diarrhea Escherichia Coli (DEC) are high, and the infection of the diarrhea is very common in cows and seriously endangers the development of dairy cow breeding industry. After the herd is infected with DEC, the herd is mainly manifested by clinical symptoms such as diarrhea, toxemia, colonitis and the like, and can cause secondary infection, thereby causing growth retardation and dysplasia. Highly pathogenic plasmids or virulence genes carried by bacteria are often capable of encoding specific adhesins, toxins, siderophores, etc., leading to an imbalance in the collective immune system, and in particular, inflammatory responses caused by DEC infection are key factors in colon injury. Since pathogenesis is not clear, no effective preventive and therapeutic measures are clinically available at present. Therefore, the search for more effective and safe control measures against colibacillosis in cows has been a research hotspot in this field.

Animal bile is a traditional Chinese medicine, is a bitter viscous dark alkaline liquid, mainly derived from poultry, pigs, cows, snakes and bear gall, and bile acid is the main component of bile, and the physiological function of bile is mainly mediated by bile acid. The bile acid can be used for detoxication, liver protection, gallbladder function promotion, blood pressure reduction, bacteriostasis, inflammation diminishing, immunity adjustment and the like when being used as a medicine, and is widely applied to the clinical treatment of Chinese medicine at home and abroad.

Ursodeoxycholic acid (Ursodeoxycholic Acid, UDCA) is chemically named 3 alpha, 7 beta-dihydroxy-5 beta-cholestan-24-acid, which is epimeric with chenodeoxycholic acid, and is distinguished by the hydroxy configuration at the 7-position. UDCA is a natural secondary bile acid, widely found in human and animal bile, and has a clinically important role, and has been approved by the FDA. The traditional Chinese medicine composition is mainly used for treating primary cholangitis and primary biliary cirrhosis and dissolving cholesterol calculus clinically at present, has an immunoregulatory effect, and is an important medicine for clinically protecting liver and resisting inflammation.

The clinical colibacillosis is a digestive system disease seriously harming the health of cows, and the pathogenic mechanism is not clear, so that the clinical treatment is mainly symptomatic treatment, including antibiotics, nutrition support, probiotics or probiotic additives and the like. If the traditional antibiotic medicines are adopted for clinical treatment, the problems of multiple drug resistance, drug residues and the like are easy to occur, the treatment effect of the medicines is affected, and especially bla is carried _CTX-M DEC strains that produce genetically ultra-broad spectrum beta-lactamase (ESBL) have been widely prevalent worldwide. Therefore, development of a safe and efficient antibiotic substitute or method for preventing and treating colibacillosis of cows and improving intestinal health of cows has become a technical problem to be solved.

The current research shows that the occurrence and the development of the colonitis and the colibacillosis of the dairy cows are closely related. Cattle with colibacillosis often have severe colonic inflammatory reactions, indicating that colonic inflammatory reactions play an important role in the occurrence and development of colibacillosis in cows.

Disclosure of Invention

The invention aims to provide application of ursodeoxycholic acid in preparing a medicine for preventing and treating colibacillosis.

According to the pharmaceutical properties of the UDCA and the pathogenesis characteristics of the colibacillosis of the dairy cows, namely the UDCA relieves colon inflammatory reaction, the invention verifies that the UDCA possibly plays an important role in relieving the colibacillosis of the dairy cows, and analyzes the action mechanism of the UDCA by taking the colibacillosis of the dairy cows as a main study object on the basis of fully understanding the related characteristics of the known UDCA and the colibacillosis of the dairy cows and evaluating the safety and feasibility of experimental design. According to the invention, a new accurate and efficient treatment measure is explored through a cell model and a mouse model, the intervention effect of ursodeoxycholic acid on the colibacillosis model of the dairy cows is estimated, and the important effect of ursodeoxycholic acid in treating the colibacillosis of the dairy cows is further verified.

In order to achieve the aim of the invention, in a first aspect, the invention provides application of ursodeoxycholic acid or conjugated bile acid or pharmaceutically acceptable salt thereof in preparing medicines for preventing and treating colibacillosis.

Preferably, the E.coli is a CTX-M type ESBL-DEC strain.

In a second aspect, the invention provides the use of ursodeoxycholic acid or a conjugated bile acid or a pharmaceutically acceptable salt thereof in the preparation of a diarrheal E.coli bacteriostat.

Preferably, the E.coli is a CTX-M type ESBL-DEC strain.

In a third aspect, the present invention provides the use of ursodeoxycholic acid or its conjugated bile acid or its pharmaceutically acceptable salt in the manufacture of a medicament for the prevention and treatment of a disease caused by diarrheal E.coli infection.

The diseases include colitis and diarrhea.

Preferably, the E.coli is a CTX-M type ESBL-DEC strain.

In a fourth aspect, the present invention provides the use of ursodeoxycholic acid or a conjugated bile acid or a pharmaceutically acceptable salt thereof in the manufacture of a formulation for reducing the inflammatory response of the colon induced by a diarrheal escherichia coli infection.

The colon inflammatory response comprises the increase of IL-1 beta, IL-6 and TNF-alpha expression.

Preferably, the E.coli is a CTX-M type ESBL-DEC strain.

In a fifth aspect, the present invention provides the use of ursodeoxycholic acid or a conjugated bile acid or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for maintaining the weight of a diarrheal escherichia coli infected mouse and improving the survival rate of the mouse.

According to the results of non-targeted metabonomics research of calf feces in the early stage, the invention discovers that the ursodeoxycholic acid content in the intestinal tract of healthy calves is obviously higher than that of diarrhea calves, and based on the difference, the research of prevention and treatment of the ursodeoxycholic acid for colic diarrhea is further developed. The diarrhea Escherichia coli (Diarrheagenic Escherichia coli) -Escherichia coli 1587 is isolated from calves clinically suffering from Escherichia coli diarrhea, and can cause acute diarrhea of cows. The strain is preserved in China general microbiological culture Collection center (CGMCC) with the address of 1 st Xila No. 3 in the Korean area of Beijing, and the preservation number of CGMCC No.23694, the preservation date of 2021, 10 months and 29 days.

The construction method of the diarrhea escherichia coli infected mice comprises the following steps: preparing a bacterial suspension of diarrhea escherichia coli, and injecting the bacterial suspension into a mouse by intraperitoneal injection.

In the method, the mouse is CD-1 mouse.

In the foregoing method, the mice are postnatal 35-42 days (preferably 42 days) of female mice.

The concentration of diarrhea E.coli in the bacterial suspension is 5×10 ⁷ ～1×10 ⁸ CFU/mL, formulated with PBS solution.

Further, the method comprises: the mice are injected with 1mL of bacterial suspension in the abdominal cavity, can eat freely, drink enough water, and are kept for 18-24 h (preferably 24 h) under the condition of ventilation and drying.

In a sixth aspect, the invention provides the use of ursodeoxycholic acid or a conjugated bile acid or a pharmaceutically acceptable salt thereof for promoting the production of short chain fatty acids (e.g. acetic acid, butyric acid) in the intestine of an animal.

In a seventh aspect, the invention provides the use of ursodeoxycholic acid or a conjugated bile acid or a pharmaceutically acceptable salt thereof for the preparation of a formulation for modulating colon development in an animal, said modulation being up-regulation.

In an eighth aspect, the present invention provides the use of ursodeoxycholic acid or a conjugated bile acid or a pharmaceutically acceptable salt thereof for the treatment or prevention of a disease caused by a diarrhea-predominant E.coli infection.

In a ninth aspect, the present invention provides the use of ursodeoxycholic acid or a conjugated bile acid or a pharmaceutically acceptable salt thereof for reducing the inflammatory response of the colon induced by a diarrheal escherichia coli infection.

In a tenth aspect, the present invention provides the use of ursodeoxycholic acid or a conjugated bile acid or a pharmaceutically acceptable salt thereof for maintaining the weight of diarrhea-predominant E.coli infected mice and improving survival in mice.

In an eleventh aspect, the present invention provides the use of ursodeoxycholic acid or a conjugated bile acid or a pharmaceutically acceptable salt thereof for protecting normal development of the colon in an animal.

In a twelfth aspect, the present invention provides the use of ursodeoxycholic acid or its conjugated bile acid and pharmaceutically acceptable salt thereof for the preparation of a medicament for preventing or treating colibacillosis in cows.

Further, the ursodeoxycholic acid or the conjugated bile acid thereof and the pharmaceutically acceptable salt have an effect of reducing inflammatory reaction of colon.

Further, the ursodeoxycholic acid or its conjugated bile acid and pharmaceutically acceptable salt can reduce the expression of interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha).

Further, the ursodeoxycholic acid or the conjugated bile acid and the pharmaceutically acceptable salt thereof can protect the normal growth and development of colon.

Further, the ursodeoxycholic acid or its conjugated bile acid and pharmaceutically acceptable salt can increase the production of short chain fatty acids in colon contents.

In the invention, the ursodeoxycholic acid pharmaceutically acceptable ester can be at least one selected from glycoursodeoxycholic acid, tauroursodeoxycholic acid, ursodeoxycholic acid, alkali metal salt or ammonium salt of glycoursodeoxycholic acid, alkali metal salt or ammonium salt of tauroursodeoxycholic acid and the like; the sodium salt of tauroursodeoxycholic acid is preferred.

The invention proves that UDCA can maintain the tight connection between cells by inhibiting the expression of interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha), thereby playing a role in protecting the colibacillosis of cows. The invention provides a new application of UDCA, which has a good protection effect on colon inflammatory reaction caused by cow colibacillosis, interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) are obviously reduced under the action of UDCA, and UDCA intervention can maintain the weight of a cow colibacillosis mouse model, protect the growth and development of colon, increase the production of short chain fatty acid in colon, improve the survival rate of the cow colibacillosis model mouse, and lay a theoretical foundation for preventing and treating cow (calf) colibacillosis by ursodeoxycholic acid.

Drawings

FIG. 1 is a schematic in vitro culture of E.coli in a preferred embodiment of the invention.

FIG. 2 is a photograph showing a culture of human colon cancer epithelial cells (Caco-2 cells) according to a preferred embodiment of the present invention.

FIG. 3 is a graph showing the effect of MEM+DMSO, MEM+UDCA, lipopolysaccharide+DMSO, lipopolysaccharide+UDCA groups on the mRNA levels of Caco-2 cell pro-inflammatory factors in a preferred embodiment of the invention.

FIG. 4A is a schematic representation of phosphorylated IκB (p-IκB) and IκB in the NF- κB signaling pathways of four sets of Caco-2 cells of FIG. 2, and the expression of cell-tight junctions.

FIG. 4B is a graph showing the quantitative analysis of the ratio of the relative expression amounts of phosphorylated IκB (p-IκB) and IκB. Wherein, the abscissa represents four groups of MEM+DMSO, MEM+UDCA, lipopolysaccharide+DMSO, lipopolysaccharide+UDCA, and the ordinate represents the ratio of p-IκB to IκB relative expression.

FIG. 4C is a graph showing the quantitative analysis of the ratio of the relative expression amounts of cell-closely coupled protein occludin and intracellular reference protein beta-actin. Wherein, the abscissa represents four groups of MEM+DMSO, MEM+UDCA, lipopolysaccharide+DMSO, lipopolysaccharide+UDCA, and the ordinate represents the ratio of the relative expression amounts of occludin and beta-actin.

FIG. 5 is a schematic diagram showing the modeling process of a cow colibacillosis model mouse according to the preferred embodiment of the present invention;

FIG. 6 is a graph showing survival rates of mice infected with DEC and K12 bacteria in accordance with the preferred embodiment of the present invention.

FIG. 7 is a schematic diagram showing hematoxylin & eosin staining (H & E) of colon tissue after mice infected with DEC in accordance with a preferred embodiment of the present invention.

FIG. 8 is a schematic representation of colonic bacterial load in mice infected with DEC in accordance with a preferred embodiment of the present invention.

FIG. 9A is a graph showing the change in body weight of mice in the PBS, placebo and UDCA groups infected with DEC in a preferred embodiment of the invention.

FIG. 9B is a graph showing survival rates of mice from groups PBS, placebo and UDCA infected with DEC in a preferred embodiment of the invention.

FIG. 10 is a schematic representation of proinflammatory factor mRNA expression after infection with DEC in three groups of mice of FIG. 9. Wherein the abscissa represents three groups of PBS, placebo and UDCA, and the ordinate represents the relative expression amount of cytokine mRNA.

FIG. 11 is a schematic representation of the isolation of phosphorylated IκB (p-IκB) and IκB in the NF- κB signaling pathways of colon tissue of the three groups of mice of FIG. 9, and the expression of the tight junction proteins.

Fig. 12 is a schematic diagram of colon length for the three groups of mice of fig. 9. Wherein the abscissa represents three groupings of PBS, placebo and UDCA, respectively, and the ordinate represents the colon length of the mice.

Fig. 13 is a graphical representation of the results of histologically observed and pathologically scored colon intestinal wall of the three groups of mice of fig. 9. Wherein pathology pictures are three groups of PBS, placebo and UDCA, respectively, and right side results are colon pathology scores of mice.

FIG. 14 is a schematic representation of the isolation of short chain fatty acid production in the colon of the three groups of mice in FIG. 9. Wherein the abscissa represents three groups of PBS, placebo and UDCA, and the ordinate represents the content of short chain fatty acids of colon contents of mice, acetic acid, propionic acid and butyric acid, respectively.

In the figures, the differences between the different treatment groups are statistically significant, P <0.05, P <0.01, and P <0.001.

Detailed Description

The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and all raw materials used are commercially available.

Ursodeoxycholic acid (Ursodeoxycholic Acid) used in the following examples was purchased from Allatin Corp., china and has the formula C ₂₄ H ₄₀ O ₄ The molecular weight is 392.57g/moL, and the purity is more than or equal to 99 percent. The product is dissolved in DMSO to prepare a mother solution of 15g/L, and the mother solution is diluted to corresponding action concentration in proportion for in vitro experiments.

LPS (Lipopolysaccharide) extraction kit was purchased from Sigma Co. The LPS component of the DEC strain is extracted by using an LPS extraction kit, and is dissolved in a Modified Eagle's Medium (MEM) to prepare a mother solution with the concentration of 1mg/mL, and the mother solution is diluted to the corresponding action concentration in proportion for experiments.

EXAMPLE 1 isolation and purification of DEC Strain

The DEC strain, escherichia coli 1587 (collection number CGMCC No. 23694), was isolated from calves clinically suffering from colibacillosis, and was capable of causing acute diarrhea in cows. The strain was identified as diarrhea E.coli (Diarrheagenic Escherichia coli) by 16S rDNA sequencing, and the serotype was O101: H9 based on the sequencing results as well as microbiological and biochemical characteristics. The strain has cell adhesion.

EXAMPLE 2 ursodeoxycholic acid inhibits proliferation of DEC Strain in vitro

DEC strain in logarithmic growth phase is inoculated into LB liquid culture medium containing ampicillin resistance according to the ratio of 1:1000, and the strain is inoculated into the bacterial culture solution (initial concentration OD600 of bacterial solution in experimental group _nm =0.2), UDCA (0.03 g/L, 0.3 g/L) was added, equal volumes of DMSO were added to the control group, bacterial cultures were collected at each of 0, 4, 8, 12, 24 hours after inoculation, and absorbance values of the bacterial solutions at 600nm were detected by spectrophotometry, respectively, as shown in fig. 1, proliferation of DEC strain was significantly inhibited after UDCA addition, and as the addition amount of UDCA was increased, the antibacterial phenomenon was increasingly apparent.

Example 3 ursodeoxycholic acid inhibits Caco-2 cell inflammatory response

Caco-2 cells were grown in MEM medium supplemented with 10% fetal bovine serum, according to 2X 10 ⁶ cell/well was inoculated into 6-well cell culture plates, 37 ℃,5% co ₂ Culturing in a cell culture box for 12h to enable the cells to be completely adhered. After the cells were attached, the stock culture was aspirated, 1mL of MEM complete medium containing UDCA (0.15 g/L) was added to the experimental group, 1mL of MEM complete medium containing an equal amount of ethanol solvent was added to the control group, the culture was continued for 24 hours, the stock culture was aspirated, the cells (10. Mu.g/mL) were treated with Lipopolysaccharide (LPS), and an untreated group was established. Harvesting cells 6h after treatment, wherein a part of cells are used for extracting cellular RNA, and detecting mRNA expression levels of interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) in the cells by a real-time fluorescence quantitative PCR method; and adding cell lysate into the other part of cells, harvesting cell proteins, and detecting the expression level of cell tight junction proteins and the activation condition of NF- κB signal paths by using an immunoblotting method. FIG. 2 is a photograph of a Caco-2 cell culture, wherein A in FIG. 2 is a Caco-2 control cell, B in FIG. 1 is Lipopolysaccharide (LPS) induced Caco-2 cell, and C in FIG. 2 shows that the intervention of UDCA can maintain the normal state of the cell. The results of the in vitro experiments are shown in FIG. 3 and FIG. 4A to FIG. 4C. FIG. 3 is a graph showing the effect of UDCA on expression of Lipopolysaccharide (LPS) -treated Caco-2 cell pro-inflammatory factor mRNA. The results show that UDCA intervention can reduce the expression of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) in Caco-2 cells, and has no obvious effect on the expression of interleukin-1 beta (IL-1 beta).

FIG. 4A is the effect of UDCA on phosphorylated IκB (p-IκB) and IκB proteins in Lipopolysaccharide (LPS) -treated Caco-2 cell NF- κB signaling pathways, as well as on cell-tight junction protein expression. As a result, UDCA intervention can inhibit the intracellular NF- κB signaling pathway to some extent, maintaining tight junctions between cells

FIGS. 4B and 4C are graphs showing the quantitative ratios of the amounts of phosphorylated IκB (p-IκB) and IκB expressed and the amounts of occludin expressed, respectively.

EXAMPLE 4 ursodeoxycholic acid inhibits the colon inflammatory response in mice

1. Construction of mouse infection model

In this example, the modeling procedure shown in FIG. 5 was used, and 6 week old groups of CD-1 mice without Specific Pathogen (SPF) were kept in separate cages under constant temperature and air drying conditions, and kept continuously for one week with free feeding and drinking water, and then the mice were infected by intraperitoneal injection with DEC strain (1X 10) ⁸ CFU), while the reference strain of e.coli K12 was established to infect the control group and PBS control group, 6 mice per group, and survival rate of mice was counted within one week after infection.

CD-1 mice were infected with sublethal dose of DEC strain (5X 10) ⁷ CFU), setting PBS blank control mice, dissecting dead mice and control mice after infection, collecting tissue samples, fixing the tissue with 10% formaldehyde solution, preparing paraffin sections, and adopting hematoxylin&Eosin staining method (H)&E) Pathological changes in the corresponding tissues are detected and pathological scoring is performed (double-blind method). Next, three doses of DEC strain (1X 10) ⁷ CFU，1×10 ⁸ CFU and 1×10 ⁹ CFU) infected CD-1 mice. Mice were harvested on day 7 post infection, sacrificed by cervical induction, the general morphology of the internal organs of the mice was observed, tissues or organs were harvested, colonic tissue pieces were ground to allow adequate release of internal bacteria, diluted at a 10-fold ratio, and dilutions of different dilutions were plated on mecKai solid medium to determine bacterial load in different organs at two time points. The test results are shown in FIGS. 6 to 8.

As shown in fig. 6, DEC had a significantly higher mortality than the K12 strain (p=0.0138). The survival rate of the DEC group was 16.67% and the PBS group was 100%.

As shown in FIG. 7, the three groups of mice were examined by sectioning, and the PBS control group and the K12 strain-infected control group were found to have normal colon color morphology. DEC infected mice have colonic wall edema, poor intestinal elasticity and fragile. Microscopic examination revealed that the intestinal glands in the colon of the DEC-infected group were severely destroyed, that part of the tissue necrosis disappeared, and that the intestinal wall of the K12 strain-infected group had developed a degree of edema.

As shown in FIG. 8, the bacteria load on the colon tissue tended to rise with increasing infection dose, indicating that the bacteria were well colonic.

2. Evaluation of ursodeoxycholic acid on mouse infection model

Under the condition of constant temperature ventilation and drying, 4 weeks old same group of SPF grade CD-1 mice are grouped and fed into independent squirrel cages, 6-8 mice are fed with UDCA (500 mM) in each group, a placebo (edible oil) group and a PBS control group are simultaneously established, and sublethal dose (5×10) is infected by intraperitoneal injection after 1 week of continuous feeding ⁷ CFU) DEC strain, mice were tested for weight change and survival within 1 week after infection. The results are shown in fig. 9A and 9B.

As shown in fig. 9A, UDCA can maintain body weight in DEC-infected mice, UDCA-interfered groups decreased in body weight drop and were statistically different from placebo group (p < 0.05). As shown in fig. 9B, UDCA was able to increase survival in mice, with UDCA-interfered groups being increased compared to placebo groups. The survival rates of the PBS control group and the UDCA intervention group were 100% and the placebo group was 85.12%.

3. Identification of ursodeoxycholic acid for inhibiting colon inflammatory response in mice

Harvesting the mice on day 7 after infection, killing the mice by cervical guide, isolating the colon, and using a part of colon tissue for extracting cellular RNA, and detecting mRNA expression levels of interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) in the cells by a real-time fluorescent quantitative PCR method; and adding cell lysate into the other part of colon tissue, harvesting cell proteins, and detecting the expression level of cell tight junction proteins and the activation condition of NF- κB signaling channels by using an immunoblotting method. The experimental results are shown in fig. 10 to 11.

As shown in fig. 10, the expression of proinflammatory factors was significantly higher in the intestinal tissue of placebo group than in PBS group, while UDCA intervention group was able to significantly reduce the expression of interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor- α (TNF- α).

As shown in FIG. 11, the ratio of phosphorylated IκB (p-IκB) to the relative expression of IκB proteins in the intestinal tissue of mice in the placebo group was higher than that in the PBS group, while the UDCA-intervention group significantly reduced the ratio of phosphorylated IκB (p-IκB) to the relative expression of IκB proteins. Meanwhile, the expression of the cell tight junction protein after DEC infection is in a decreasing trend, and the UDCA intervention can obviously maintain the cell tight junction.

Meanwhile, the colon of the mice was collected after harvesting, the length of the colon was measured, the above tissues were fixed with 10% formaldehyde and apparent pathology data of the UDCA treated group, placebo group and PBS control group were analyzed by hematoxylin & eosin staining (H & E). The experimental results are shown in fig. 12 to 13.

As shown in fig. 12, DEC infection significantly inhibited colonic growth and development, and UDCA dry prognosis was able to maintain colonic normal growth (p < 0.0001).

As shown in fig. 13, after the end of the experiment, the three groups of mice were examined by dissection, and it was found that the organs of the PBS control group mice were normal. DEC infected mice have colonic wall edema, poor intestinal elasticity and fragile. Microscopic examination revealed that the intestinal glands in the colon of the DEC-infected group were severely destroyed and that the necrosis of part of the tissue structure disappeared. The UDCA intervention group has slight edema of colon intestinal wall, better elasticity, complete colon gland and epithelium and no inflammatory cell infiltration. Pathology scoring: including 0, 1, 2, 3, 4, 5 minutes. From this figure, UDCA intervention was able to significantly reduce pathology scores after DEC infection with statistical differences (p=0.0007).

4. Influence of ursodeoxycholic acid on short chain fatty acid production in colon

On day 7 post infection, colon contents of UDCA treated, placebo and PBS control mice were collected and assayed for short chain fatty acid (acetic acid, propionic acid, butyric acid, etc.) content by gas chromatography. The experimental results are shown in fig. 14. The expression level of short chain fatty acids in colon contents after DEC infection is significantly reduced, and the expression level of acetic acid and butyric acid after DEC infection can be significantly increased (p < 0.001) through UDCA intervention.

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims

1. Application of ursodeoxycholic acid or pharmaceutically acceptable salt thereof in preparing medicine for preventing and treating colibacillosis is provided.

2. Application of ursodeoxycholic acid or pharmaceutically acceptable salt thereof in preparing diarrhea escherichia coli bacteriostat.

3. The application of ursodeoxycholic acid or its pharmaceutically acceptable salt in preparing medicine for preventing and treating colitis caused by diarrhea colibacillus infection.

4. Use of ursodeoxycholic acid or its pharmaceutically acceptable salt in preparing medicine for preventing and treating diarrhea caused by diarrhea colibacillus infection.

5. Use of ursodeoxycholic acid or a pharmaceutically acceptable salt thereof for the preparation of a formulation for alleviating the inflammatory reaction of the colon induced by a diarrheal escherichia coli infection.

6. The use according to claim 5, wherein the inflammatory response of the colon comprises an increase in the expression of IL-1 β, IL-6, TNF- α.

7. Use of ursodeoxycholic acid or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for maintaining the weight and improving the survival rate of a mouse infected with diarrheal escherichia coli.

8. The use according to any one of claims 1 to 7, wherein the escherichia coli is a CTX-M ESBL-DEC strain.