阅读说明：本技术 牛磺熊脱氧胆酸在治疗新生儿坏死性小肠结肠炎中的应用 (Application of tauroursodeoxycholic acid in treatment of neonatal necrotizing enterocolitis ) 是由 付东 李朋 于 2021-01-27 设计创作，主要内容包括：本发明提供了牛磺熊脱氧胆酸(TUDCA)或其药学上可接受盐在制备预防或治疗新生儿坏死性小肠结肠炎的药物中的新用途,TUDCA对新生儿坏死性小肠结肠炎(NEC)引起的内质网应激起到很好的保护作用,CHOP和BiP在TUDCA作用下显著降低,TUDCA干预可维持NEC模型新生小鼠的体重,降低小肠上皮细胞凋亡,提高NEC模型新生小鼠的存活率。本发明通过细胞和凋亡水平实验,证实TUDCA在NEC中可以通过抑制内质网应激、降低细胞凋亡比例,从而起到对NEC的保护作用,这为采用TUDCA进行新生儿坏死性小肠结肠炎的防治奠定了理论基础。(The invention provides a new application of tauroursodeoxycholic acid (TUDCA) or pharmaceutically acceptable salts thereof in preparing a medicine for preventing or treating neonatal necrotizing enterocolitis, the TUDCA plays a good role in protecting endoplasmic reticulum stress caused by neonatal Necrotizing Enterocolitis (NEC), CHOP and BiP are remarkably reduced under the action of the TUDCA, and TUDCA intervention can maintain the weight of a neonatal mouse of an NEC model, reduce the apoptosis of epithelial cells of the small intestine and improve the survival rate of the neonatal mouse of the NEC model. The invention proves that TUDCA can play a role in protecting NEC by inhibiting endoplasmic reticulum stress and reducing apoptosis ratio in NEC through cell and apoptosis level experiments, which lays a theoretical foundation for preventing and treating necrotizing enterocolitis of newborn by TUDCA.)

1. Application of tauroursodeoxycholic acid or pharmaceutically acceptable salt thereof in preparing a medicament for preventing or treating neonatal necrotizing enterocolitis.

2. The use according to claim 1, wherein the tauroursodeoxycholic acid or the pharmaceutically acceptable salt thereof has an effect of alleviating endoplasmic reticulum stress response.

3. The use according to claim 2, wherein tauroursodeoxycholic acid or a pharmaceutically acceptable salt thereof is capable of reducing the expression of the endoplasmic reticulum stress markers CHOP and BiP.

4. The use of claim 1, wherein the tauroursodeoxycholic acid or the pharmaceutically acceptable salt thereof is capable of reducing the expression of the cleared caspase3 protein.

5. The use of claim 1, wherein the tauroursodeoxycholic acid or the pharmaceutically acceptable salt thereof is capable of reducing apoptosis of small intestine epithelial cells.

6. The use of claim 1, wherein the pharmaceutically acceptable salt of tauroursodeoxycholic acid is at least one of an alkali metal salt and an ammonium salt of tauroursodeoxycholic acid.

7. The use of claim 1, wherein the medicament comprises pharmaceutically acceptable excipients, and the dosage form comprises tablets, oils, granules and powders.

Technical Field

The invention relates to the technical field of medicines, in particular to application of tauroursodeoxycholic acid in treating necrotizing enterocolitis of a newborn.

Background

Necrotizing Enterocolitis Neonatorum (NEC) is one of the most severe and common diseases in the neonatal period and is the leading cause of death of premature infants from gastrointestinal diseases. At present, in China very low-weight births, the incidence rate of necrotizing enterocolitis of the newborn is 4.53 percent, and the related mortality rate of necrotizing enterocolitis of the newborn is up to 41.7 percent. The development and progression of necrotizing enterocolitis in the newborn involves complex pathophysiological processes of immaturity, abnormal colonization of bacteria and excessive immune response in the intestine of premature infants. Because the exact etiology and pathogenesis are unclear, no effective prevention and treatment measures are still available in clinic at present, and severe complications such as intestinal stenosis, short-bowel syndrome, neurodevelopmental retardation and the like often appear after the operation treatment. Therefore, the search for more effective and safe prevention and treatment measures for children with necrotizing enterocolitis of newborn is always a hot spot in the research field.

Tauroursodeoxycholic acid (TUDCA) has a chemical name of 3 α, 7 β dihydroxycholanyl-N-taurine, and is a conjugated bile acid formed by the shrinkage between the shuttle group of ursodeoxycholic acid (UDCA) and the amino group of taurine. TUDCA is a conjugated natural bile acid widely present in human and animal bile, and acts as a chaperone involved in protein folding to alleviate endoplasmic reticulum stress. At present, the traditional Chinese medicine composition is mainly used for treating diseases such as cholestatic liver disease, gall-stone and the like clinically. The structural formula and synthetic route of TUDCA are shown below:

at present, clinical NEC is a digestive system disease seriously harming the health of newborn, and has a limited clinical treatment method due to unknown exact etiology, and mainly treats the symptoms, including antibiotics, probiotics, nutrition support, surgical intervention and the like. In pediatrics, the study investment of the pediatric diseases is much less and less compared with that of adults in the whole clinical field, so that the understanding of the pediatric diseases is much slower, and the drug development of the pediatric diseases is far away and untimely. Certain side effects may be caused if clinical NEC intervention is performed with traditional drugs, and therefore, it is imperative to find a safe and effective drug or method for the prevention and treatment of necrotizing enterocolitis.

The preventive and therapeutic effects of tauroursodeoxycholic acid (TUDCA) in neonatal diseases are not clear. There are studies that indicate Endoplasmic Reticulum Stress (ER Stress) is an important pathogenic factor for NEC initiation. NEC infants tend to be associated with high levels of ER stress, and NEC occurs well in preterm infants, which are also found by comparison to be significantly elevated compared to term infants. Thereby further demonstrating the important role of ER stress in the generation and development of NEC. The anti-ER stress characteristic of TUDCA provides a certain theoretical basis for the research of the drug action of TUDCA in NEC.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention verifies that TUDCA possibly plays an important regulation and mitigation role in NEC according to the pharmaceutical properties of TUDCA and the pathogenesis characteristics of NEC, namely the endoplasmic reticulum stress mitigation role of TUDCA and the high endoplasmic reticulum stress level in NEC to promote the generation and development of intestinal inflammation, fully understands the related characteristics of known TUDCA and NEC, evaluates the feasibility and the safety of experimental design, and explores the role and the mechanism of TUDCA by taking NEC as a research object. The invention further verifies the application of the tauroursodeoxycholic acid in treating the neonatal necrotizing enterocolitis by establishing a neonatal necrotizing enterocolitis mouse model to find a new and more effective treatment means and evaluating the intervention effect of the tauroursodeoxycholic acid on the neonatal necrotizing enterocolitis mouse model.

In order to achieve the purpose, the invention adopts the technical scheme that:

the invention provides application of tauroursodeoxycholic acid or pharmaceutically acceptable salts thereof in preparing a medicament for preventing or treating neonatal necrotizing enterocolitis.

Further, the tauroursodeoxycholic acid or the pharmaceutically acceptable salt thereof has an effect of alleviating endoplasmic reticulum stress response.

Further, the tauroursodeoxycholic acid or the pharmaceutically acceptable salt thereof can reduce the expression of endoplasmic reticulum stress markers CHOP and BiP.

Further, the tauroursodeoxycholic acid or the pharmaceutically acceptable salt thereof can reduce the expression of the cleared caspase3(CC3) protein.

Further, the tauroursodeoxycholic acid or the pharmaceutically acceptable salt thereof can reduce apoptosis of epithelial cells of the small intestine.

Further, the pharmaceutically acceptable salt of tauroursodeoxycholic acid is at least one of alkali metal salt and ammonium salt of tauroursodeoxycholic acid. More preferably tauroursodeoxycholic acid sodium salt.

Further, the medicine contains medically acceptable auxiliary materials, and the dosage forms of the medicine comprise tablets, oil solutions, granules and powder, and more preferably water-soluble powder.

By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:

the invention provides a new application of TUDCA, which has a good protection effect on endoplasmic reticulum stress caused by NEC, CHOP and BiP are obviously reduced under the action of TUDCA, and the intervention of TUDCA can maintain the weight of a new mouse of the NEC model, reduce the apoptosis of epithelial cells of small intestine and improve the survival rate of the new mouse of the NEC model. The invention proves that TUDCA can play a role in protecting NEC by inhibiting endoplasmic reticulum stress and reducing apoptosis ratio in NEC through cell and apoptosis level experiments, which lays an effective theoretical basis for preventing and treating necrotizing enterocolitis of newborn by TUDCA.

Drawings

FIG. 1 is a schematic diagram of the modeling process of a neonatal mouse model of NEC in one embodiment of the present invention; wherein, A is a nitrogen bottle required by molding, and the nitrogen concentration is 99.99 percent; b is an anoxic bottle required by molding; c is milk powder fed by a mouse formula, and 1.9F required by feeding is placed in a central venous catheter of a human through a peripheral vein; d is the operation of feeding; e is the state of mouse hypoxia after nitrogen is introduced; f is the operation of cold stimulation of mice in a refrigerator at 4 ℃.

FIG. 2 is a schematic illustration of the intestinal wall dissected after the experiment of the normal CONTROL group (CONTROL), the neonatal necrotizing enterocolitis model group (NEC), and the neonatal necrotizing enterocolitis model TUDCA intervention group (TUDCA + NEC) in accordance with one embodiment of the present invention;

FIG. 3 is a graphical representation of the results of histomicroscopic observation of the intestinal wall and pathological scoring of the intestinal wall of FIG. 2;

FIG. 4A is a graph showing the body weight of mice in the CONTROL, NEC, TUDCA + NEC groups according to one embodiment of the present invention;

FIG. 4B is a graph showing survival rates of mice in the CONTROL, NEC, TUDCA + NEC groups according to one embodiment of the present invention;

FIG. 5A is a graph showing the expression of endoplasmic reticulum stress markers CHOP and BiP in intestinal tissue of mice in the CONTROL, NEC, TUDCA + NEC groups according to one embodiment of the present invention;

FIG. 5B is a graph showing the change in expression levels of CHOP and BiP with increasing physiological age;

FIG. 6 is a graph showing the results of immunofluorescence staining of terminal ileal tissue BiP and Tunel in mice of the CONTROL, NEC, TUDCA + NEC groups according to an embodiment of the present invention;

FIG. 7 is a graph showing the results of flow-type apoptosis assays for isolating epithelial cells from the terminal ileal tissue of the three groups of mice described in FIG. 6;

FIG. 8 is a photograph showing the culture of rat small intestine crypt epithelial cells (IEC-6 cells) in an example of the present invention;

FIG. 9 is a graph showing the results of detecting the effect of TUDCA on the expression of the IEC-6 endoplasmic reticulum stress and apoptosis-related proteins CHOP, BiP and CC3 treated with LPS lps in accordance with one embodiment of the present invention;

FIG. 10 is a graph showing the results of in vitro Tunel apoptosis assay of IEC-6 cells in accordance with one embodiment of the present invention;

FIG. 11 is a diagram illustrating the results of an in vitro flow apoptosis assay for IEC-6 cells in an embodiment of the present invention.

Detailed Description

The invention relates to application of tauroursodeoxycholic acid or pharmaceutically acceptable salts thereof in preparing a medicament for preventing or treating neonatal necrotizing enterocolitis.

The following description of the embodiments of the present invention will be made with reference to the accompanying drawings. The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby.

Example 1

In this example, a necrotic enterocolitis model of 5-day-old C57BL/6 newborn mice was prepared by a modeling process shown in FIG. 1 using artificial feeding in combination with hypoxia and cold stimulation. The experiment was divided into three groups: a normal control group (10), a neonatal necrotizing enterocolitis model group (15), and a neonatal necrotizing enterocolitis model TUDCA intervention group (20).

The specific experimental process is as follows:

artificially feeding mice: adopting mouse milk substitute such as petag brand milk powder, total energy of 840kJ/kg/d, feeding amount of 50 μ l/g, and feeding at interval of 4 h.

Hypoxia and cold stimulation: hypoxic cold stimulation was controlled at 1h after meal. Placing in an oxygen-deficient bottle containing 100% nitrogen gas for 60 s, immediately placing in a refrigerator at 4 deg.C for 10 min, and performing cold stimulation for 2 times every 24h, wherein the molding process is 96 h. After the model is established, the mouse is closely observed for food intake, defecation, abdominal condition, activity reaction and the like. Newborn mice were weighed daily before the experiment and after the model building. Mice were sacrificed on the appearance of overt clinical symptoms (severe abdominal distension, bloody stool and cyanosis) or at 96h cervical dislocation after molding, the general morphology of the intestinal tracts of mice was observed, intestinal tract specimens were collected for histological sectioning, HE staining, pathological observation, histological scoring and pathological scoring (double blind method).

TUDCA intervention: TUDCA was administered at a dose of 500mg/kg by intraperitoneal injection of TUDCA using a 1ml syringe. Injecting 0.5h before anoxia-induced cold stimulation, 1 time every 24h, and 4 times in molding process.

Preparation of TUDCA: 50mg of TUDCA was dissolved in 1ml of physiological saline and injected with about 10ul of the solution per g of mice.

Grouping experiments: the 45 mice were randomly divided into three groups:

normal control group: the female mice were fed with 10 mice. (CONTROL)

NEC model set: 15 animals were fed under combined anaerobic and cold stimulation. (NEC)

NEC model TUDCA intervention group: TUDCA was injected intraperitoneally (30 min before cold-hypoxic stimulation, 1 time daily at a dose of 500mg/kg, 20). (TUDCA + NEC)

Collecting the intestinal canal. The intestinal mass changes, growth and development, and weight changes were compared among the three groups. The experimental results are shown in fig. 2 to 7.

As shown in FIG. 2, after the experiment, the mice of the above three experimental groups were dissected, and it was found that the normal control group had red and elastic intestinal wall and appeared yellow. The lesion intestinal wall edema, congestion and necrosis of the NEC model group are red-black and yellow-red, and the lesion of the ileocecal part is the most serious; the intestinal tract is poor in elasticity and fragile. Pathological changes similar to those of the clinical NEC infant intestinal tract appear under the mirror: congestion and edema of mucosa and submucosa with different degrees, sloughing and necrosis of partial villi, disorder of gland arrangement, thinning or breaking of muscle layer, infiltration of inflammatory cells, and disappearance of intestinal villi with necrosis of the whole layer in severe cases. The NEC model TUDCA pre-treated intestinal wall has mild edema and good elasticity, and the gland part disappears but the epithelium is complete and inflammatory cell infiltration is not obvious through microscopic observation.

Fig. 3 is intestinal histomicroscopic observation and pathology scoring (HE staining, 200 ×). Wherein, A.0 is divided; b.1, dividing; c.2 min; d.3, dividing; e.4 points. From this figure, TUDCA was able to reduce NEC pathology score with statistical differences (P < 0.05), and normal control group pathology scores were: 0.30 ± 0.466, NEC group: 2.80 ± 1.11, NEC model TUDCA intervention group: 1.78 ± 0.97 (F of fig. 3).

As shown in fig. 4A, TUDCA can maintain the body weight of new mice in NEC model, the NEC model TUDCA dried group has reduced and statistically different body weight reduction compared to NEC group (P < 0.05), and the body weight after 96h in normal control group is: 5.99 ± 0.25, NEC model set: 2.98 ± 0.25, NEC model TUDCA intervention group: 3.89 +/-0.22. As shown in fig. 4B, TUDCA was able to increase survival, and NEC model TUDCA intervention group had statistically different (P < 0.05) than NEC group. The survival rate of the normal control group after 96h is 100%, and the NEC group is as follows: 46.67%, NEC model TUDCA intervention group: 66.67 percent.

As shown in FIG. 5A, the expression of CHOP and BiP, markers of endoplasmic reticulum stress in intestinal tissues of mice in the NEC model group, is significantly higher than that of the normal control group, TUDCA has a good protective effect on the endoplasmic reticulum stress caused by NEC, and CHOP and BiP are significantly reduced under the effect of TUDCA. As shown in fig. 5B, CHOP and BiP expression levels tended to decrease with increasing physiological age. The stress level of the endoplasmic reticulum of premature infants is significantly higher than that of term infants.

FIG. 6 shows three groups of mice with ileal end tissue BiP and Tunel immunofluorescent staining (200X). BiP expression was significantly higher in NEC model group than in normal control group, and BiP protein expression was decreased after TUDCA-dried (part A, B, C of fig. 6). The BiP protein is expressed in the cytoplasm of the small intestine epithelium, the NEC model group BiP is obviously stained in an inflammation area, and the staining intensity and range of the intervention group are obviously lower than those of the inflammation group. Apoptotic cell counts were very low in the normal control and TUDCA groups as observed by Tunel immunofluorescence staining, and positive apoptotic cells were detected at the villus axis and crypts in the NEC model group (portion D, E, F of fig. 6).

FIG. 7 shows the flow-type apoptosis assay performed by isolating epithelial cells of the small intestine of the terminal ileum tissue of three groups of mice. Apoptosis of small intestine epithelial cells in the NEC model group is obviously increased, and apoptosis of small intestine epithelial cells after TUDCA stem prediction is obviously reduced. (. P < 0.05 compared to normal control).

Example 2

In this example, an in vitro IEC-6 cell line experiment was performed to further verify the efficacy of TUDCA.

IEC-6 was grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 1X 10⁶cells/mL were plated in 6-well cell culture plates at 37 ℃ with 5% CO₂The cells were fully attached to the wall by 12h of culture. Lps and TUDCA were prepared using physiological saline. IEC-6 cells were treated with lps (50. mu.g/ml) for 6h, co-treated with TUDCA at final concentrations of 50. mu.M, 100. mu.M, 200. mu.M, after which the cells were harvested for subsequent studies. FIG. 8 is a photograph of an IEC-6 cell culture in which lps induces an increase in IEC-6 cell death, as shown in section B of FIG. 8, and intervention with TUDCA improves IEC-6 death and promotes cell survival, as shown in section C of FIG. 8. The results of the in vitro experiments are shown in FIGS. 9 to 11. FIG. 9 is a graph showing the effect of TUDCA on lps-treated IEC-6 cell in vitro assays. TUDCA (50. mu.M, 100. mu.M, 200. mu.M) intervention reduced the IEC-6 cell BiP, in IEC-6 cells after 6h of lps (50ng/ml) treatment,Expression of CHOP and CC3 proteins.

FIG. 10 shows in vitro Tunel experiments with IEC-6 cells. TUDCA (50. mu.M, 100. mu.M, 200. mu.M) intervention reduced apoptosis in IEC-6 cells.

FIG. 11 shows in vitro flow assay of IEC-6 cells for apoptosis. C. D, E TUDCA (50. mu.M, 100. mu.M, 200. mu.M) intervention reduced IEC-6 cell apoptosis. (comparing with normal control group, P is less than 0.05)

From the above examples, it is shown that TUDCA can protect NEC by inhibiting endoplasmic reticulum stress and reducing apoptosis ratio in NEC through cell and apoptosis level experiments.

The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.