WO2019104936A1 - Composition containing butyric acid compound and use thereof - Google Patents

Abstract

Disclosed are a composition containing a butyric acid compound and the use of same for preventing and treating biliary atresia and its complications, i.e. cholestatic liver fibrosis. The composition containing a butyric acid compound comprises a butyric acid compound at a concentration of 5 to 70 mM, as well as a nutrition preparation or a pharmaceutical excipient. The intervention method that usually acts on the intestines is used to prevent and treat biliary atresia and its complications, i.e. cholestatic liver fibrosis. The butyric acid compound can be directly used in or added to a nutrition preparation during pregnancy and for newborn individuals when no symptoms are present, thereby fundamentally preventing and treating the occurrence and development of biliary atresia.

Description

Composition containing butyric acid compound and application thereof Technical field

The invention relates to the technical field of food medicine, in particular to a composition containing a butyric acid compound and the use thereof for preventing and treating biliary atresia and complications thereof and cholestasis liver fibrosis.

Background technique

Liver fibrosis refers to the massive deposition of extracellular matrix components in the liver. It is an inevitable stage in the development of various chronic liver diseases into liver cirrhosis. Because liver fibrosis is a substantial lesion of the liver, it is generally considered difficult to reverse in the medical field. In addition to liver transplantation, there are no other effective prevention measures. There are many types of liver fibrosis, and viral hepatitis, alcoholic hepatitis, nonalcoholic fatty liver, and cholestasis can cause liver fibrosis. Different types of liver fibrosis, the etiology and pathogenesis are different, and the control measures are completely different.

Cholestatic liver fibrosis caused by cholestatic disease is a major clinical difficulty. There are no safe and effective prevention measures. As the disease progresses, the cholestatic disease will gradually develop into chronic liver fibrosis, cirrhosis and liver failure, and eventually liver transplantation is required. Cholestatic diseases can be divided into extrahepatic cholestasis and intrahepatic cholestasis. Extrahepatic cholestasis refers to cholestasis caused by mechanical obstruction of the extrahepatic bile duct system; intrahepatic cholestasis refers to a series of pathological and clinical manifestations caused by diffuse obstruction of small bile ducts in the liver. Common cholestatic diseases include cholestasis associated with intestinal failure, biliary atresia, primary biliary cirrhosis, and primary sclerosing cholangitis.

Among them, biliary atresia (BA) is a cholestatic disease that endangers the life of infants and young children. It is characterized by progressive obstruction of intrahepatic and extrahepatic bile ducts, with an incidence rate of 1/5000-1/8000. Most children with BA usually do not have obvious symptoms within a few days after birth. With the obstruction of the intrahepatic and extrahepatic bile ducts, they gradually show obvious jaundice and terracotta color stools. Hepatic jejunostomy is the treatment of choice for biliary atresia. However, even if the bile duct patency is successfully restored by hepaticojejunostomy, progressive bile duct obstruction still occurs after normal bile drainage, leading to rapid development of liver fibrosis to cirrhosis. If liver transplantation is not performed, the long-term survival rate of BA is less than 30%, which is the leading cause of liver transplantation in children. The etiology and pathogenesis of biliary atresia are still unclear and are a major clinical problem. Because the early diagnosis of biliary atresia is very difficult, it is difficult to distinguish with neonatal cholestasis, infant hepatitis syndrome and viral hepatitis in the neonatal period. It usually takes a long time for the child to be diagnosed, which has led to the development of severe fiber at the time of diagnosis. Even cirrhosis. On the other hand, due to the lack of effective prevention and treatment methods, even if there is no effective treatment for the diagnosis of biliary atresia, liver transplantation is still needed.

Therefore, the prevention and treatment of biliary atresia is a difficult problem to be solved clinically. However, the incidence of biliary atresia is neonatal. At present, there is no effective method for early diagnosis of biliary atresia. How to prevent biliary atresia safely and effectively in neonates without obvious symptoms? How to safely and effectively inhibit progressive liver fibrosis in children who have been diagnosed with biliary atresia? These problems are all technical problems that currently exist. On the other hand, cholestatic liver fibrosis, which is complicated by biliary atresia, progresses very rapidly, and usually develops into cirrhosis within a few months, which is also a clinically difficult point. There are many types of liver fibrosis, and viral hepatitis, alcoholic hepatitis, nonalcoholic fatty liver, and cholestasis can cause liver fibrosis. Different types of liver fibrosis, the etiology and pathogenesis are different, and the control measures are completely different. At present, research on liver fibrosis has achieved some results at home and abroad. The drugs commonly used in clinical treatment of liver fibrosis mainly include interferon, colchicine, interleukin-10, etc., but these drugs have limited therapeutic effects and large side effects. For cholestatic liver fibrosis, there is currently no safe and effective prevention and treatment method.

Butyric acid is a natural component in mammalian milk and is one of the metabolites of the intestinal flora in the body. Butyric acid, as a kind of short-chain fatty acid, plays an important role in maintaining the normal physiological function of the intestine. However, the role of butyric acid in the prevention and treatment of biliary atresia has not been reported. On the other hand, studies have confirmed that butyric acid has protective effects on some intestinal diseases such as ulcerative colitis and inflammatory bowel disease, but the effect of butyric acid is not the same in cholestatic diseases.

The study found that in animal models of cholestatic disease caused by bile duct ligation, the liver's ability to metabolize fatty acids decreased, especially short-chain fatty acids such as butyric acid could not be catabolized, resulting in elevated concentrations of short-chain fatty acids, which may aggravate cholestasis. Reichen J, ect.Mechanisms of impaired hepatic fatty acid metabolism in rats with long-term bile duct ligation. Hepatology. 1994 May; 19(5): 1272-81)

S, ect. Reversibility of hepatic mitochondrial damage in rats with long-term cholestasis. J Hepatol. 1998 Jun; 28(6): 1000-7). On the other hand, Notch signaling pathway and cholangiocarcinogenesis play an important role in progressive cholestatic liver fibrosis. Zhang X et al. found that butyric acid promotes cholecytic liver fibrosis by promoting Notch signaling pathway and differentiation of liver progenitor cells into biliary epithelial cells (Zhang X, et a1. Inhibition of notch signaling pathway preventing cholestatic liver fibrosis By decreasing the differentiation of hepatic progenitor cells in to cholangiocytes.Lab Invest.2016 Mar;96(3):350-60). Therefore, based on these findings, short-chain fatty acids such as butyric acid are considered to be important factors in promoting cholestatic liver fibrosis.

Summary of the invention

The technical problem to be solved by the present invention is to provide a composition containing a butyric acid compound and an application thereof against the deficiencies of the prior art.

The present invention provides a composition that can be used to prevent and treat biliary atresia and its complications. In particular, it provides its role in the prevention and treatment of biliary atresia and its complications of liver fibrosis.

The present invention also provides a composition that can be used to prevent and treat cholestatic liver fibrosis. It is particularly provided for its role in the prevention and treatment of cholestatic liver fibrosis.

The innovative use of butyric acid compounds in the invention can effectively prevent the occurrence of biliary atresia after intervention of pregnant mice. As a safe and effective prevention and treatment method, butyric acid compounds can be directly used or added to nutritional preparations for pregnant and newborn individuals without obvious symptoms, thereby fundamentally preventing the occurrence and development of biliary atresia.

The innovative intervention of the butyric acid compound of the invention is very effective in preventing and treating cholestatic liver fibrosis and solving the clinical problem.

Complications of biliary atresia Liver fibrosis is a cholestatic liver fibrosis, and prevention is also very difficult. The usual anti-inflammatory drugs can alleviate the progression of liver fibrosis even if they can alleviate the liver inflammatory response. The role of drugs for bile acid synthesis in cholestatic liver fibrosis is also very limited. Because of bile duct obstruction and other intrahepatic bile duct obstruction such as biliary atresia, the degree of cholestasis in the liver is very serious, and the concentration of bile acid is usually ten or more times higher than the normal value. Some drugs that inhibit bile acid synthesis, even if they reduce bile acid synthesis, have bile acid concentrations in the liver that are dozens of times higher than normal. And studies have confirmed that the degree of liver fibrosis is not related to bile acid levels in cholestatic diseases such as biliary atresia, thus inhibiting the role of bile acid synthesis in preventing and treating biliary atresia, especially cholestatic liver fibrosis. Very limited.

The study found that in animal models of cholestatic disease, the liver's ability to metabolize fatty acids decreased, resulting in elevated concentrations of short-chain fatty acids may aggravate cholestatic liver disease (Reichen J, ect.Mechanisms of impaired hepatic fatty acid metabolism in rats with long -term bile duct ligation.Hepatology.1994 May;19(5):1272-81)(

S, ect. Reversibility of hepatic mitochondrial damage in rats with long-term cholestasis. J Hepatol. 1998 Jun; 28(6): 1000-7). However, we have carefully analyzed that there is no direct evidence in these studies that the role of butyric acid in progressive cholestatic liver fibrosis. On the other hand, Zhang X et al. found that sodium butyrate plays a role in promoting cholestatic liver fibrosis by promoting Notch signaling pathway and differentiation of liver progenitor cells into biliary epithelial cells (Zhang X, et a1.Inhibition of notch signaling pathway Prevention cholestatic liver fibrosis by decreasing the differentiation of hepatic progenitor cells in to cholangiocytes.Lab Invest.2016 Mar;96(3):350-60). However, this study only confirmed the effect of sodium butyrate by in vitro experiments, and its effect in vivo is not necessarily the case.

The innovative use of butyric acid compounds in the animal model of cholestatic liver fibrosis interferes with and overcomes the prior art prejudice, and is very effective in preventing and treating biliary atresia and complications of liver fibrosis, especially effective. The prevention and treatment of cholestatic liver fibrosis overcomes the prior art bias and solves the clinical problem.

Studies have confirmed that the degree of liver fibrosis of cholestatic diseases such as biliary atresia is not associated with bile acid levels, inflammatory cell infiltration, ALT, AST and other indicators. The present invention finds for the first time that the proportion of Th1 type cells in the liver of the bile duct ligation model is positively correlated with the degree of liver fibrosis, and Th1 type cells play an important role in promoting cholestatic liver fibrosis. After intervention or treatment with butyric acid compounds, the proportion of Th1 type cells decreased significantly. Butyric acid compounds play an important role in the prevention and treatment of biliary atresia with progressive liver fibrosis and cholestasis of liver fibrosis by specifically inhibiting the proportion of liver Th1 type cells.

The object of the invention is achieved by the following technical solutions:

In a first aspect, the present invention provides the use of a butyric acid compound for the preparation of a composition for the prevention and treatment of biliary atresia and complications thereof.

In a second aspect, the present invention provides the use of a butyric acid compound for the preparation of a composition for preventing and treating cholestatic liver fibrosis.

In a third aspect, the present invention provides a composition containing a butyric acid compound, comprising a butyric acid compound, and a nutritional preparation or a pharmaceutical auxiliary; the concentration of the butyric acid compound is 5 to 70 mM (mmol/L).

Preferably, the concentration of the butyric acid compound is 15 to 45 mM.

Preferably, the effective dose of the butyric acid compound as a drug is 0.5-7 mmol/kg/d.

More preferably, the effective dose of the butyric acid compound as a drug is 1.5 to 4.5 mmol/kg/d.

Preferably, the butyric acid compound is at least one selected from the group consisting of sodium butyrate, potassium butyrate, calcium butyrate, magnesium butyrate, butyric acid, glyceryl butyrate, and isoamyl butyrate.

Preferably, the butyric acid compound is at least one selected from the group consisting of sodium butyrate, glyceryl butyrate, and butyric acid.

Preferably, the nutritional preparation is selected from at least one of a conventional formula, a special medical use formula, a parenteral nutrition preparation, and an enteral nutrition preparation.

Preferably, the conventional formula comprises at least one of infant formula, gestational and lactating formula; the special medical formula comprises preterm/low birth weight infant formula, lactose-free formula or low lactose formula , milk protein partially hydrolyzed formula, milk protein deep hydrolysis formula or amino acid formula, fortifiers or nutritional supplements for breast milk and infant formula, for food intolerance, allergy, special formula for liver disease, disease or function At least one of the barrier formulas; the parenteral nutrition preparation comprising at least one of a fat emulsion injection, an all-in-one nutrient solution, and an intravenous injection; the enteral nutrition preparation comprising an amino acid enteral nutrition preparation At least one of a short peptide type enteral nutrition preparation, a whole protein type enteral nutrition preparation, and a component type enteral nutrition preparation.

In a fourth aspect, the present invention provides a method for preparing a composition containing a butyric acid compound, characterized in that the preparation method comprises: adding a butyric acid compound to the nutritional preparation or the pharmaceutical auxiliary in proportion, Mix evenly, just.

Food for Special Medical Purpose (FSMP) is a formula that is specially formulated to meet the special needs of people with limited food intake, digestive dysfunction, metabolic disorders or specific disease states. Such products must be consumed alone or in combination with other foods under the direction of a doctor or clinical nutritionist. Special medical use formulas are special dietary foods. When the target population cannot eat ordinary meals or can not meet their nutritional needs with daily diet, special medical use formulas can be used as a nutritional supplement, which plays an important nutritional support role in treatment, rehabilitation and maintenance of body functions.

Preferably, the pharmaceutical excipient comprises a pharmaceutically acceptable carrier or excipient such as lactose hydrate, microcrystalline cellulose, mannitol, sodium citrate, calcium phosphate, glycine, starch; disintegrating agents such as cross-linked polycondensation Wortone, copovidone, sodium starch glycolate, croscarmellose sodium and specific complex silicates; binders such as polyvinylpyrrolidone, hydroxypropyl methylcellulose (HPMC), hydroxypropyl fibers (HPC), sucrose, gelatin and gum arabic.

Preferably, when the butyric acid compound is butyric acid, the butyric acid compound is added in the form of a coating or a coating.

It should be noted that the application form of the butyric acid compound in the present invention is not important, and the butyric acid-containing compound is added to the formula milk powder suitable for infants and pregnant women, the formula for special medical use, and other enteral and parenteral nutrition. It can be used in the preparation or the pharmaceutical auxiliary, and the effect of preventing and treating biliary atresia and its complications can be achieved as long as the effective amount is applied.

As an alternative to the formulation of the formula, the butyric acid compounds of the present invention can be applied as a supplement rather than integrated into the formula. For example, the butyric acid compound can be ingested in the form of a pill, a tablet, a capsule, a caplet, a powder, a liquid or a gel. For example, butyric acid compounds can be taken in combination with other nutritional supplements such as breast milk supplements.

Compared with the prior art, the present invention has the following beneficial effects:

1. The present invention innovatively uses an intervention method usually applied to the intestine to prevent and treat biliary atresia. After the intervention of butyric acid compounds in pregnant mice, it is very effective in preventing the occurrence of biliary atresia. As a safe and effective prevention and treatment method, butyric acid compounds can be directly used or added to nutritional preparations for pregnant and newborn individuals without any symptoms, thereby fundamentally preventing the occurrence of biliary atresia.

2. Current research suggests that in animal models of cholestatic disease, the liver's ability to metabolize fatty acids decreases, resulting in elevated concentrations of short-chain fatty acids that may aggravate cholestatic liver disease; and sodium butyrate promotes Notch signaling pathways and liver ancestors The cells differentiate into bile duct epithelial cells, thereby playing a role in promoting cholestatic liver fibrosis. The invention overcomes the prior art prejudice and uses the butyric acid compound to intervene and treat cholestatic liver fibrosis. The results show that the model group shows severe liver fibrosis with a degree of fibrosis of grade 3, while the intervention group and the treatment group have no obvious. Liver Fibrosis. Therefore, the present invention solves the technical problem of prevention and treatment of liver fibrosis in biliary atresia complications, and provides a safe, economical and effective prevention and treatment method.

3, studies have confirmed that the degree of liver fibrosis of cholestatic diseases such as biliary atresia is not related to bile acid levels, inflammatory cell infiltration, ALT, AST and other indicators. The present invention finds for the first time that in the liver of the bile duct ligation model, the proportion of Th1 type cells is positively correlated with the degree of liver fibrosis, and Th1 type cells play an important role in the process of progressive liver fibrosis. After intervention or treatment with butyric acid compounds, the proportion of Th1 type cells decreased significantly. Butyric acid compounds play an important role in the prevention and treatment of biliary atresia with progressive liver fibrosis by specifically inhibiting the proportion of liver Th1 type cells. 4. Because the metabolism of butyric acid in the body is very fast, in order to better exert the effect of butyric acid, we add the butyric acid compound to the nutrient preparation suitable for a specific population, so that the butyric acid slowly enters the body with the nutrient preparation. Digestion and absorption, play a longer role.

Therefore, the addition of nutrient preparations and drugs of butyric acid compounds is a safe, effective and feasible method for preventing and treating biliary atresia and its complications of liver fibrosis, especially cholestasis of liver fibrosis.

DRAWINGS

Other features, objects, and advantages of the present invention will become apparent from the Detailed Description of Description

Figure 1 shows liver HE and Masson staining of the bile duct ligation (BDL) model; Figure 1a shows liver HE staining in the BDL model group; Figure 1b shows liver Masson staining in the BDL model group; Figure 1c shows BDL + sodium butyrate intervention (30 mM) liver HE staining. Figure 1d is BDL + sodium butyrate intervention (30 mM) liver Masson staining; Figure 1e is BDL + sodium butyrate treatment (45 mM) liver HE staining; Figure 1f is BDL + sodium butyrate treatment (45 mM) liver Masson staining; Figure 1g is BDL + Sodium butyrate treatment (70 mM) liver HE staining; Figure 1h is BDL + sodium butyrate treatment (70 mM) liver Masson staining;

Figure 2 shows the proportion of Th1 cells in liver lymphocytes by bile duct ligation (BDL) model. Figure 2a shows the proportion of liver Th1 cells in the BDL model group (black circle); Figure 2b shows the BDL + sodium butyrate intervention (30 mM) liver. Th1 type cell ratio (black circle); Figure 2c shows BDL + sodium butyrate treatment (45 mM) liver Th1 type cell ratio (black circle); Figure 2d is BDL + sodium butyrate treatment (70 mM) liver Th1 type cell ratio (black circle) Figure 2e shows the ratio of liver Th1 type cells in the BDL model group and each sodium butyrate intervention group; Fig. 2f shows the ratio of liver Th1 type cells in the BDL model group and each sodium butyrate treatment group.

Detailed ways

The invention will now be described in detail in connection with specific embodiments. The following examples are intended to further understand the invention, but are not intended to limit the invention in any way. It should be noted that a number of changes and modifications may be made by those skilled in the art without departing from the inventive concept. These are all within the scope of protection of the present invention.

Example 1

The present embodiment provides an infant formula containing coated butyric acid and a preparation method thereof, wherein the food grade coated butyric acid is added to the infant formula and uniformly mixed. The prepared formula had a concentration of coated butyric acid of 5 mmol/L.

Example 2

The present embodiment provides a maternal formula containing sodium butyrate and a preparation method thereof, wherein the food grade sodium butyrate is added to the maternal formula and mixed uniformly, that is, obtained. The concentration of sodium butyrate in the prepared formula was 15 mmol/L.

Example 3

The present embodiment provides a preterm infant formula containing butyric acid glyceride and a preparation method thereof, wherein the food grade butyric acid glyceride is added to the formula of premature infant formula, and the mixture is uniformly obtained. The concentration of butyric acid glyceride in the prepared formula was 20 mmol/L.

Example 4

The present embodiment provides a fat emulsion injection containing sodium butyrate and a preparation method thereof, wherein the injection grade sodium butyrate is added to a fat emulsion injection, and the mixture is uniformly obtained. The concentration of sodium butyrate in the prepared fat emulsion injection was 30 mmol/L.

Example 5

The present embodiment provides an all-in-one nutrient solution containing sodium butyrate and a preparation method thereof, wherein the injection grade sodium butyrate is added to the all-in-one nutrient solution, and the mixture is uniformly obtained. The concentration of sodium butyrate in the prepared all-in-one nutrient solution was 45 mmol/L.

Example 6

The present embodiment provides a short peptide type enteral nutrition preparation containing glyceryl butyrate and a preparation method thereof, wherein the food grade butyric acid glyceride is added to a short peptide type enteral nutrition preparation, and the mixture is uniformly mixed. That is. The concentration of the butyric acid glyceride in the prepared short peptide type enteral nutrition preparation was 55 mmol/L.

Example 7

The present embodiment provides a special disease formula containing sodium butyrate and a preparation method thereof, which comprises: adding food grade sodium butyrate to a special disease formula food, and uniformly mixing, that is, obtaining. The concentration of sodium butyrate in the prepared special disease formula was 70 mmol/L.

Example 8

This embodiment provides a pharmaceutical composition comprising a butyric acid compound and a pharmaceutical adjuvant.

The butyric acid compound is at least one selected from the group consisting of sodium butyrate, potassium butyrate, calcium butyrate, magnesium butyrate, butyric acid, glyceryl butyrate, and isoamyl butyrate.

The effective dose of the butyric acid compound is 0.5 to 7 mmol/kg/d.

Also included in the pharmaceutical compositions are other drugs compatible with the butyric acid compound and pharmaceutically acceptable carriers and/or adjuvants.

The pharmaceutical composition is in any pharmaceutically acceptable dosage form.

The dosage form is a powder, an injection, a capsule, a tablet or an oral solution.

When the butyric acid compound is butyric acid, the butyric acid compound is added to the drug in the form of a coating or a coating.

The butyric acid-containing composition prepared in the above examples can effectively prevent and treat biliary atresia and complications of liver fibrosis. The optimal effective dose for prevention is 15-30 mmol/L or 1.5-3 mmol/kg/d; the optimal effective dose for treatment is 30-45 mmol/L or 3-4.5 mmol/kg/d.

Animal test results verification:

1. Verification of animal models of biliary atresia induced by rotavirus infection in newborn mice

1.1 Experimental animals and grouping

SPF-class 7-day pregnant BALB/C pregnant mice were purchased from Shanghai Xipuer-Beikai Experimental Animal Co., Ltd. Raised at the Animal Experimental Center of Xinhua Hospital affiliated to Shanghai Jiaotong University School of Medicine, divided into model group, sodium butyrate intervention group and sodium butyrate treatment group:

Biliary atresia model: Within 24 hours after birth, pregnant rats were injected intraperitoneally with rotavirus to perform biliary atresia modeling. Newborn rats were breastfed; 14 days after model establishment, peripheral blood, bile duct, liver and intestinal tissue were taken. Carry out testing;

Sodium butyrate intervention group (45 mM): Sodium butyrate was added to water to feed pregnant mice for 7 days until production. In the 24 hours after delivery, pregnant rats were injected intraperitoneally with newborn mice for biliary atresia. Newborn rats were used. Breastfeeding; 14 days after modeling, peripheral blood, bile duct, liver, and intestinal tissue were taken for testing;

Sodium butyrate treatment group (45 mM): Within 24 hours after birth, pregnant rats were injected intraperitoneally with newborn mice for biliary atresia, and sodium butyrate was added to the water to feed the mother rats. The newborn rats were breastfed; After 14 days, peripheral blood, bile duct, liver and intestinal tissue were taken for testing.

1.2 results

Biliary atresia model: the patency of extrahepatic bile duct was detected. The results showed that the extrahepatic bile duct obstruction of the model mice was accompanied by severe cholestasis, abnormal bile duct hyperplasia and hepatic inflammatory infiltration.

Sodium butyrate intervention group (45 mM): The patency of extrahepatic bile duct was detected. It was found that the extrahepatic bile duct of mice in the sodium butyrate intervention group was patency, and the liver inflammation was infiltrated, and no obvious cholestasis and bile duct hyperplasia were observed.

Sodium butyrate treatment group (45 mM): The patency of extrahepatic bile duct was detected. It was found that the extrahepatic bile duct of the sodium butyrate treatment group was still unobstructed, and liver inflammatory cells were infiltrated, and no obvious cholestasis and bile duct hyperplasia were observed.

Therefore, our innovative use of butyric acid compounds to interfere with pregnant rats is very effective in preventing the occurrence and development of biliary atresia in neonatal rats, which is of great significance for the prevention and treatment of clinical biliary atresia.

2. The role of extrahepatic bile duct ligation model in the diagnosis of hepatic fibrosis and cholestasis of liver fibrosis in biliary atresia

2.1 experimental animals

Clean-grade three-week-old Sprague-Dawley (SD) rats, male or female, weighing approximately 50 g, were purchased from Shanghai Xipuer-Beikai Experimental Animal Co., Ltd. Raised at the Animal Experimental Center of Xinhua Hospital affiliated to Shanghai Jiao Tong University School of Medicine.

2.2 Processing methods

Biliary tube ligation (BDL) model group: rats were fed with water and normal feed. After 7 days, the common bile duct of the rats was double-ligated and cut from the middle; no administration was performed, and the 14th day was fasted overnight, and blood was taken for blood biochemistry. Liver and intestinal tissue were taken for subsequent experiments. Tissue sections were fixed with neutral formalin and H-E staining, Masson and immunohistochemistry were performed.

BDL+ sodium butyrate intervention group: sodium butyrate was added to water to feed the rats. The concentrations of sodium butyrate were 5 mM, 15 mM, 30 mM, 45 mM, respectively, and the doses were 0.5 mmol/kg/d and 1.5 mmol/kg/d, respectively. 3 mmol/kg/d, 4.5 mmol/kg/d. After 7 days of feeding, the rat common bile duct was double-ligated and cut from the middle. Fasting overnight on the 14th day after surgery, blood was taken to measure blood biochemistry. Liver and intestinal tissue were taken for subsequent experiments. Tissue sections were fixed with neutral formalin and H-E staining, Masson and immunohistochemistry were performed.

BDL+ coated butyric acid intervention group: rats were fed with butyric acid in water, the concentration of butyric acid was 5 mM, 15 mM, 30 mM, 45 mM, respectively, the dosage was 0.5 mmol/kg/d, 1.5 mmol/kg/d, 3 mmol. /kg/d, 4.5 mmol/kg/d. After 7 days of feeding, the rat common bile duct was double-ligated and cut from the middle. Fasting overnight on the 14th day after surgery, blood was taken to measure blood biochemistry. Liver and intestinal tissue were taken for subsequent experiments. Tissue sections were fixed with neutral formalin and H-E staining, Masson and immunohistochemistry were performed.

BDL + butyric acid glyceride intervention group: glycerol butyrate was added to water to feed the rats, the concentration of butyric acid glyceride was 5 mM, 15 mM, 30 mM, 45 mM, respectively, the dosage was 0.5 mmol/kg/d, 1.5 mmol/kg. /d, 3 mmol/kg/d, 4.5 mmol/kg/d. After 7 days of feeding, the rat common bile duct was double-ligated and cut from the middle. Fasting overnight on the 14th day after surgery, blood was taken to measure blood biochemistry. Liver and intestinal tissue were taken for subsequent experiments. Tissue sections were fixed with neutral formalin and H-E staining, Masson and immunohistochemistry were performed.

BDL+ sodium butyrate treatment group: The rat common bile duct was double-ligated and cut from the middle. From the 7th day after the model establishment, sodium butyrate was added to the water to feed the rats, and the administration was continued until the 14th day. The sodium butyrate concentrations were 15 mM, 30 mM, 45 mM, 70 mM, respectively, and the doses were 1.5 mmol/kg. /d, 3 mmol/kg/d, 4.5 mmol/kg/d, 7 mmol/kg/d. On the 14th day, fasting overnight, taking blood and measuring blood biochemistry. Liver and intestinal tissue were taken for subsequent experiments. Tissue sections were fixed with neutral formalin and H-E staining, Masson and immunohistochemistry were performed.

BDL+ Clostridium butyricum intervention group: Adding Clostridium butyricum to water-fed rats, the number of viable bacteria of Clostridium butyricum was 1×10 ⁷ -4×10 ⁷ CFU/mL, and the dosage was 1×10 ⁹ - 4 × 10 ⁹ CFU / kg / d. After 7 days of feeding, the rat common bile duct was double-ligated and cut from the middle. Fasting overnight on the 14th day after surgery, blood was taken to measure blood biochemistry. Liver and intestinal tissue were taken for subsequent experiments. Tissue sections were fixed with neutral formalin and examined by HE staining, Masson and immunohistochemistry.

BDL+ Clostridium butyricum treatment group: The rat common bile duct was double-ligated and cut from the middle. From the 7th day after modeling, the rats were fed with water to the rats, and the rats were continuously administered until the 14th day. The viable count of Clostridium butyricum was 5×10 ⁷ -9×10 ⁷ CFU/mL. The dose is 5 x 10 ⁹ -9 x 10 ⁹ CFU/kg/d. On the 14th day, fasting overnight, taking blood and measuring blood biochemistry. Liver and intestinal tissue were taken for subsequent experiments. Tissue sections were fixed with neutral formalin and examined by HE staining, Masson and immunohistochemistry.

2.3 Results

Biliary tube ligation (BDL) model group: Animal liver function indexes ALT, AST, GGT, TBA, TBIL were significantly increased, liver color was yellow, surface was not smooth, texture was hard; liver tissue was severe liver fibrosis, fibrosis The degree reaches level 3 (Fig. 1a, Fig. 1b). The proportion of Th1 type cells in the progenitor fibrosis process in the BDL model group was significantly increased, with a mean value of 21.3% (Fig. 2a, Fig. 2e).

BDL+ sodium butyrate intervention group:

Sodium butyrate intervention group (5mM): liver function index decreased slightly, liver color was slightly yellow, pathological staining showed moderate liver fibrosis, liver fibrosis level 2, compared with BDL group, liver Th1 type cell ratio Declining, the average is 12.7% (Figure 2e);

Sodium butyrate intervention group (15 mM): liver function index was significantly reduced, liver color was red, surface was smooth, soft texture, pathological staining showed mild liver fibrosis, liver fibrosis level 1, compared with BDL group, liver The proportion of Th1 cells decreased significantly, with a mean of 6.9% (Fig. 2e);

Sodium butyrate intervention group (30 mM): liver function index was significantly reduced, liver color was bright red, surface was smooth, soft texture, pathological staining showed no obvious fibrosis in the liver (Fig. 1c, Fig. 1d); and flow cytometry results showed The proportion of liver Th1 cells in the process of profibrosis was significantly decreased and close to normal, with a mean of 1.8% (Fig. 2b, Fig. 2e);

Sodium butyrate intervention group (45 mM): liver function index was significantly reduced, liver color was bright red, surface was smooth and soft, and pathological staining showed mild liver fibrosis, liver fibrosis level 1 and liver Th1 type cell ratio decreased. 2.4% (Figure 2e).

The results of the coated butyrate intervention group and the butyrate intervention group were consistent with the results of the sodium butyrate intervention group.

BDL + sodium butyrate treatment group:

Sodium butyrate treatment group (15 mM): liver function index decreased, liver color was slightly yellow, liver fibrosis level 2, compared with BDL group, the proportion of liver Th1 type cells decreased, the average value was 15.7% (Fig. 2f);

Sodium butyrate treatment group (30 mM): The liver function index was significantly reduced, the liver color was red, the texture was soft, the liver was mild liver fibrosis, and the degree of fibrosis was grade 1. Compared with the BDL group, the ratio of liver Th1 type cells Significant decline, with an average of 7.6% (Figure 2f);

Sodium butyrate treatment group (45 mM): liver function indicators were significantly reduced, liver color was bright red, smooth surface, soft texture, liver fibrosis was not observed in the liver (Fig. 1e, Fig. 1f); liver Th1 compared with BDL group The proportion of type cells decreased significantly, and the mean decreased to 4.8% (Fig. 2c, Fig. 2f);

Sodium butyrate treatment group (70 mM): liver function indicators were significantly reduced, liver color was bright red, smooth surface, soft texture, liver fibrosis, liver fibrosis level 1 (Fig. 1g, Fig. 1h), liver Th1 type The cell ratio dropped to 5.5% (Fig. 2d, Fig. 2f).

BDL+ Clostridium butyricum group:

The results of the intervention group of Clostridium butyricum showed that the degree of liver fibrosis and the proportion of Th1 type cells were not significantly decreased after intervention with 1×10 ⁷ -4×10 ⁷ CFU/mL live Clostridium butyricum. The degree of fibrosis was Level 2-3;

The results of the treatment group of Clostridium butyricum showed that 5×10 ⁷ -9×10 ⁷ CFU/mL live bacteria of Clostridium butyricum were used for treatment. The degree of liver fibrosis and the proportion of Th1 type cells did not decrease significantly, and the degree of fibrosis remained. It is level 3.

Therefore, the intervention of butyric acid compounds not only effectively prevent the occurrence of biliary atresia, but also has a very good effect on the prevention and treatment of liver fibrosis and cholestasis fibrosis in biliary atresia complications; especially when the sodium butyrate concentration reaches 30 mM After -45 mM, the control effect is particularly remarkable. The BDL model showed severe liver fibrosis with a grade of fibrosis of grade 3, while no significant liver fibrosis was seen in the BDL intervention group and the treatment group. Therefore, it is indicated that butyric acid compounds can be used for the prevention and treatment of biliary atresia and its complications of liver fibrosis. However, the effect of Clostridium butyricum is not obvious, and the current health department stipulates that only three probiotics, Lactobacillus, Bifidobacterium and Streptococcus, can be safely added to foods. Only Bifidobacteria and Lactobacillus can be safely applied. Infant formula. The effect of Clostridium butyricum as an active microorganism in the body is unclear and may present a safety hazard.

In summary, butyric acid compounds can effectively prevent biliary atresia and its complications, cholestasis of liver fibrosis. Butyric acid is a short-chain fatty acid present in mammalian milk, and from the results of animal tests, butyric acid compounds are administered within this concentration range, and no damage or side effects are caused to the test animals, indicating that they are administered. High security. Butyric acid compounds have great application prospects in the prevention and treatment of biliary atresia and its complications, and cholestasis of liver fibrosis.

The specific embodiments of the present invention have been described above. It is to be understood that the invention is not limited to the specific embodiments described above, and various changes or modifications may be made by those skilled in the art without departing from the scope of the invention. The features of the embodiments and the embodiments of the present application may be combined with each other arbitrarily without conflict.

Claims (11)

1. A use of a butyric acid compound for the preparation of a composition for preventing and treating biliary atresia and complications thereof.
2. A use of a butyric acid compound for the preparation of a composition for preventing and treating cholestatic liver fibrosis.
3. A composition containing a butyric acid compound, comprising a butyric acid compound and a nutritional preparation or a pharmaceutical auxiliary; the concentration of the butyric acid compound is 5 to 70 mM.
4. The butyric acid-containing compound-containing composition according to claim 3, wherein the butyric acid compound has a concentration of 15 to 45 mM.
5. The butyric acid-containing compound-containing composition according to claim 3, wherein the effective dose of the butyric acid compound as a drug is 0.5 to 7 mmol/kg/d.
6. The butyric acid-containing compound-containing composition according to claim 5, wherein the effective dose of the butyric acid compound as a drug is 1.5 to 4.5 mmol/kg/d.
7. The butyric acid-containing compound according to any one of claims 1 to 6, wherein the butyric acid compound is selected from the group consisting of sodium butyrate, potassium butyrate, calcium butyrate, magnesium butyrate, and butyl. At least one of acid, glyceryl butyrate, and isoamyl butyrate.
8. The butyric acid-containing compound-containing composition according to claim 7, wherein the butyric acid-based compound is at least one selected from the group consisting of sodium butyrate, glyceryl butyrate, and butyric acid.
9. The butyric acid-containing compound-containing composition according to claim 3, wherein the nutritional preparation is at least one selected from the group consisting of a conventional formula, a special medical use formula, a parenteral nutrition preparation, and an enteral nutrition preparation. .
10. The butyrate-containing composition according to claim 9, wherein the conventional formula comprises at least one of infant formula, pregnancy and lactating formula; and the special medical use formula comprises Premature/low birth weight infant formula, lactose-free or low-lactose formula, milk protein partially hydrolyzed formula, milk protein deep hydrolysis formula or amino acid formula, fortified or nutritional supplements for breast and infant formulas, suitable for use in At least one of a food intolerance, an allergy, a special formula for liver disease, a disease or a dysfunctional formula; the parenteral nutrition preparation includes a fat emulsion injection, an all-in-one nutrient solution, and an intravenous solution At least one; the enteral nutrition preparation comprises at least one of an amino acid type enteral nutrition preparation, a short peptide type enteral nutrition preparation, a whole protein type enteral nutrition preparation, and a component type enteral nutrition preparation.
11. A method for preparing a butyric acid-containing compound according to claim 3, wherein the preparation method comprises: adding a butyric acid compound to the nutrient preparation or the pharmaceutical excipient in proportion, and uniformly mixing , you can.

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