Application of mononuclear cell inhibitor in preparation of medicine for treating biliary atresia

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of a mononuclear cell inhibitor in preparation of a medicine for treating biliary atresia. The invention provides application of a mononuclear cell inhibitor in preparation of a medicine for treating biliary atresia. A mouse is used as an experimental subject, and an Anti-Ly6C antibody is used for eliminating Ly6C + monocytes in the mouse to treat biliary atresia. Experimental results prove that the Anti-Ly6C antibody can significantly improve symptoms of biliary atresia mice, provides a new potential drug target and a treatment strategy for treatment of biliary atresia, and has an important clinical application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of a monocyte inhibitor in the preparation of a drug for treating biliary atresia. Background Art

[0002] Biliary atresia (BA) is a severe bile duct disease mediated by extrahepatic bile duct obstruction, usually occurring in the neonatal period. It is characterized by inflammation and fibrosis of the bile ducts, resulting in obstruction of bile flow and liver damage, causing pathological jaundice and liver failure, with a high fatality rate. The causative factors are complex and not yet clear. The Kasai operation (portoenterostomy) is currently the preferred method for treating biliary atresia, but most children with BA still develop cirrhosis due to progressive intrahepatic bile duct inflammatory damage and finally die of liver failure within 2 years.

[0003] Clinical findings have shown that the progression of biliary atresia is closely related to progressive inflammatory damage of the bile ducts. Repeated bile duct inflammatory damage easily leads to irreversible bile duct obstruction, ultimately resulting in cirrhosis and liver failure. However, the specific triggering factors are currently unclear, and the treatment methods are limited. Most children ultimately need liver transplantation. Therefore, it is urgent to clarify the pathological mechanism of the occurrence of bile duct inflammatory damage in biliary atresia and urgently need new strategies for specific prevention and treatment of bile duct inflammatory damage. The latest research shows that the bile duct inflammatory damage in biliary atresia is related to multiple factors, including genetic susceptibility, viral infections (such as cytomegalovirus, rotavirus), and autoimmune responses. Genome-wide association studies (GWAS) on patients with biliary atresia have identified several risk genes that are involved in regulating processes such as immune responses, bile duct development, and repair. Some studies have found that in biliary atresia diseases induced by viral infections, the Notch signaling pathway in bile duct epithelial cells is significantly activated, exacerbating bile duct inflammatory damage. Inhibitors targeting the Notch signaling pathway, anti-inflammatory drugs (such as corticosteroids), and T cell regulators have shown certain efficacy in animal models. Although corticosteroids can relieve bile duct damage through broad-spectrum anti-inflammatory effects, their long-term use easily leads to serious side effects such as immunosuppression and metabolic disorders. T cell regulators (such as cyclosporine) can inhibit adaptive immune responses but lack specificity for monocyte-mediated innate inflammatory responses and have limited efficacy.

[0004] In addition, immune-mediated inflammatory responses play a central role in the development of bile duct damage and fibrosis. Studies have found that around the bile ducts with liver fibrosis and liver damage, there is infiltration of immune cells (such as macrophages and T cells), and these immune cells exacerbate bile duct inflammatory damage and fibrosis by releasing inflammatory factors and cytokines. Although certain progress has been made in the study of bile duct inflammatory damage in biliary atresia, its exact mechanism is still not fully understood, and there are no effective intervention targets.

[0005] Therefore, it is necessary to develop new drugs for biliary atresia and related mechanism targets. SUMMARY OF THE INVENTION

[0006] The purpose of the present invention is to provide the use of monocyte inhibitors in the preparation of drugs for treating biliary atresia.

[0007] In order to achieve the above object of the present invention, the technical solution adopted by the present invention is:

[0008] The present invention provides the use of monocyte inhibitors in the preparation of drugs for treating biliary atresia

[0009] In some embodiments of the present invention, the subjects of the drug include humans and mice.

[0010] In some embodiments of the present invention, when the subject of the drug is a human, the monocyte is CD14 + CD16 - positive monocytes.

[0011] In some embodiments of the present invention, when the subject of the drug is a mouse, the monocyte is Ly6C + positive monocytes.

[0012] In some embodiments of the present invention, the monocyte inhibitor includes substances that inhibit the number and / or activity of monocytes.

[0013] In some embodiments of the present invention, the substances include proteins and nucleic acid molecules.

[0014] In some embodiments of the present invention, the proteins include specific antibodies against CD14 and / or specific antibodies against Ly6C.

[0015] In some embodiments of the present invention, the specific antibody against Ly6C is purchased from BioXCell, clone number Mouts-1. Those skilled in the art can expect that Ly6C antibodies from other companies or other clone types can also achieve the present technical solution. The specific procurement source of the Ly6C antibody does not constitute a limitation of the present invention.

[0016] In some embodiments of the present invention, the nucleic acid molecules include microRNA, siRNA, shRNA, dsRNA, sgRNA and / or antisense oligonucleotides; the nucleic acid molecules can inhibit or knock out the expression of CD14 and / or Ly6C.

[0017] In some embodiments of the present invention, the drug includes pharmaceutically acceptable excipients.

[0018] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, antiadhesives, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, clathrates, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, carriers.

[0019] The above-mentioned pharmaceutically acceptable excipients are generally recognized for this purpose and serve as inactive ingredients of the medicament. Compilations of pharmaceutically acceptable excipients can be found in "Handbook of Pharmaceutical Excipients" (2nd Edition, edited by A. Wade and P. J. Weller; published by the American Pharmaceutical Association, Washington and The Pharmaceutical Press, London, 1994); "List of Medicinal Excipients in the Pharmacopoeia of the People's Republic of China", and other reference books.

[0020] In some embodiments of the present invention, the dosage forms of the drug include gastrointestinal dosage forms or parenteral dosage forms.

[0021] In some embodiments of the present invention, the gastrointestinal dosage forms include at least one of powders, tablets, granules, capsules, sustained-release agents, solutions, dry suspensions, effervescent tablets, emulsions, suspensions, syrups, drops, chewable tablets;

[0022] In some embodiments of the present invention, the parenteral dosage forms include at least one of injectable dosage forms, respiratory dosage forms, skin dosage forms, mucosal dosage forms, cavity dosage forms.

[0023] In some embodiments of the present invention, the drug further includes any one or more other active ingredients.

[0024] In some embodiments of the present invention, the other active ingredients include conventional drugs for treating biliary atresia, including but not limited to at least one of ursodeoxycholic acid, phenobarbital, obeticholic acid, fat-soluble vitamins, methylprednisolone, amoxicillin.

[0025] In the preliminary pre-experiment, the inventors found through single-cell sequencing data analysis that CD14 in the liver tissue of children with biliary atresia + CD16 -The number of monocytes increases, and their pro-inflammatory function is enhanced, and they are significantly positively correlated with liver function injury indicators. Based on the differential expression of surface markers, human monocytes are divided into CD14 + CD16 - , CD14 - CD16 + and CD14 + CD16 + three subsets. CD14 + CD16 - monocytes are classical monocytes, the most abundant monocyte subset in the blood, with efficient phagocytic ability and high affinity for pathogen components (such as lipopolysaccharide LPS). When the human body is infected with pathogens such as bacteria and viruses, these cells activate the immune response and secrete inflammatory factors TNF-α, IL-1, etc. by recognizing specific molecules such as LPS on the surface of pathogens, attracting more immune cells to the infection site and promoting the inflammatory response.

[0026] Therefore, in order to explore the role of CD14 + CD16 - monocytes in biliary atresia, the present invention further conducts relevant experiments on mice. Due to the differences between mouse and human monocytes, the present invention selects mouse Ly6C + CD16 - monocytes corresponding to human pro-inflammatory CD14 + monocytes as the experimental subjects. The corresponding relationship between mouse Ly6C + monocytes and human pro-inflammatory CD14 + CD16 - monocytes has been proven by relevant literature. Including but not limited to: the literature (2010. Nomenclature of monocytes and dendritic cells in blood. Prepublished online July 13, 2010; doi:10.1182 / blood-2010-02-258558) points out that human classical monocytes highly express CD14 and do not express CD16 (CD14 + CD16 - ); mouse classical monocytes highly express Ly6C and lowly express CD43 (Ly6C + CD43 -)。Both are the main monocyte subsets in their respective species and are similar in terms of function, phenotype, etc. During infection or inflammation, the quantity and function of both will change accordingly, and both have certain immunomodulatory effects; the literature (2010. Comparison of gene expression profiles between human and mouse monocyte subsets. Prepublished online as Blood First Edition paper, November 12, 2009; DOI 10.1182 / blood-200907-235028.) conducted a comparative analysis of the gene expression profiles of human and mouse monocyte subsets and found that human CD14 + CD16 - classical monocytes and mouse Ly6C + classical monocytes have similar expression profiles and are similar in aspects such as being able to quickly migrate to the inflammatory site to play a role in the early stage of the inflammatory response. Research results in terms of gene expression patterns, cell surface protein expression, and functional characteristics indicate that mouse Ly6C + classical monocytes and human CD14 + CD16 - classical monocytes are similar, and there is a strong corresponding relationship between the two.

[0027] The beneficial effects of the present invention are as follows:

[0028] The present invention provides the application of a monocyte inhibitor in the preparation of a drug for treating biliary atresia. Using mice as experimental subjects, Anti-Ly6C antibody is used to clear Ly6C + monocytes in mice to treat biliary atresia. The experimental results prove that Anti-Ly6C antibody can significantly improve the symptoms of mice with biliary atresia, providing a new potential drug target and treatment strategy for the treatment of biliary atresia, and having important clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The following further describes the present invention in conjunction with the drawings and embodiments, wherein:

[0030] Figure 1 is the flowchart for constructing a mouse model.

[0031] Figure 2 is the appearance result on the 12th day of the mouse experiment, where ① RRV + PBS; ② RRV + Isotype; ③ RRV + Anti-Ly6C; ④ Vehicle.

[0032] Figure 3Results of the change in the body weight of mice over the experimental days.

[0033] Figure 4 Results of the change in the survival rate of mice over the experimental days.

[0034] Figure 5 Results of the change in the jaundice rate of mice over the experimental days.

[0035] Figure 6 Results of the appearance of the liver and bile ducts of mice.

[0036] Figure 7 Results of the pathological sections of mouse liver tissue.

[0037] Figure 8 Results of HE pathological scoring.

[0038] Figure 9 Results of the detection of mouse liver function, where: A is alanine aminotransferase (ALT); B is aspartate aminotransferase (AST); C is total bilirubin (TBIL); D is direct bilirubin (DBIL); E is alkaline phosphatase (ALP); F is gamma-glutamyl transferase (GGT); G is total bile acids (TBA). Detailed implementation manners

[0039] The concept and technical effects of the present invention will be clearly and completely described below in combination with embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0040] Example 1 Construction of a mouse model

[0041] 1. Experimental materials

[0042] Experimental animals: BALB / c WT neonatal mice within 24 hours after birth.

[0043] Main reagents and antibodies:

[0044] (1) Rhesus rotavirus (RRV) MMU18006, titer 1.5×10 6PFU.

[0045] (2) Anti-Ly6C antibody (BioXCell, clone number Mouts-1).

[0046] (3) Isotype control antibody (Isotype, BioXCell, clone number 2A3).

[0047] (4) PBS buffer.

[0048] (5) Fluorescent contrast agent FITC-dextran (ThermoFisher, catalog number D1823).

[0049] 2. Experimental grouping

[0050] 1) RRV + PBS group;

[0051] 2) RRV + Isotype group;

[0052] 3) RRV + Anti-Ly6C group;

[0053] 4) Vehicle group.

[0054] 3. Experimental procedures

[0055] S1) BALB / c WT neonatal mice within 24 hours after birth were randomly divided into 4 groups: 1) RRV + PBS group; 2) RRV + Isotype group; 3) RRV + Anti-Ly6C group; 4) Vehicle group. Each group contained 12 neonatal mice (n = 12), and the experiment was repeated 3 times. The data were expressed as mean ± standard deviation, and one-way analysis of variance (ANOVA) was used for inter-group comparison. P < 0.05 was considered statistically significant. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Guangzhou Medical University.

[0056] S2) Neonatal mice in the RRV + Anti-Ly6C group were intraperitoneally injected with 40 μg of Anti-Ly6C antibody (BioXCell, clone number Mouts-1, diluted with PBS to a total intraperitoneal injection volume of 20 μL). Neonatal mice in the RRV + Isotype group were intraperitoneally injected with 40 μg of isotype control antibody (Isotype) (BioXCell, clone number 2A3, diluted with PBS to a total intraperitoneal injection volume of 20 μL). The remaining 2 groups were intraperitoneally injected with 20 μL of solvent control PBS.

[0057] S3) 4 hours after the above operations, the first to third groups were intraperitoneally injected with 20 μL of rhesus rotavirus (RRV) (titer 1.5×10 6 PFU) to establish a BA mouse animal model, and the fourth group was injected with 20 μL of solvent control PBS.

[0058] S4) Perform the experimental operation described in S2) once every two days until the detection on the 12th day. Monitor the status of the mice every day, record the changes in body weight, survival, and the occurrence of jaundice. On the 12th day, detect the mouse model to obtain data on liver tissue inflammation changes, liver function data, and extrahepatic bile duct obstruction changes.

[0059] 4. Observation indicators and their detection methods

[0060] 1) Observation of general condition of mice: Monitor the mental state, activity, diet, and water intake of the mice every day, and record the changes in body weight, survival, and the occurrence of jaundice.

[0061] 2) Mouse dissection and sample collection: On the 12th day, euthanize and dissect the mice, observe the appearance of the liver and bile ducts, and perform fluorescence angiography of the extrahepatic bile ducts using a fluorescent contrast agent.

[0062] 3) Histopathological examination of liver tissue: Fix, embed, and section the liver tissue, and perform HE staining. Observe the pathological changes of the liver tissue under a microscope, including the degree of periductal inflammatory infiltration, etc., and perform HE pathological scoring. The severity of liver injury is evaluated according to the following criteria: necrosis, inflammatory infiltration, ballooning degeneration, and disruption of hepatic cord structure, using a semi-quantitative scoring system (0 - 3 points, where 0 = none, 1 = mild, 2 = moderate, 3 = severe). When the observed inflammation is between two defined levels, an intermediate increment of 0.5 is used. Randomly select three high-power fields (HPFs) for each tissue section using a Leica microscope for scoring, and the scoring is independently completed by two blinded pathologists to ensure result consistency and reduce subjective bias.

[0063] 4) Detection of liver function indicators: Use a biochemical analyzer to detect alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), direct bilirubin (DBIL), alkaline phosphatase (ALP), γ-glutamyl transferase (GGT), and total bile acid (TBA) in the serum of mice.

[0064] 5. Results of model construction

[0065] The flow chart of model construction is as Figure 1 shown. A biliary atresia mouse model was successfully constructed by RRV infection. Compared with the Vehicle group, the mice in the RRV infection group (RRV + PBS group, RRV + Isotype group) showed obvious symptoms such as weight loss, jaundice, and reduced survival rate, indicating the successful construction of the model. Observe and detect the changes in various indicators of the RRV + Anti-Ly6C group.

[0066] Example 2: Effect of Anti-Ly6C antibody on the general appearance of mice

[0067] On the 12th day of the experiment, the general appearance was observed, and the results are as Figure 2 shown.

[0068] Compared with the RRV+PBS group and the RRV+Isotype group, the general appearance of the mice in the RRV+Anti-Ly6C group was significantly improved. The mice in the RRV+PBS group and the RRV+Isotype group showed obvious symptoms such as emaciation, messy hair, and jaundice, while the appearance of the mice in the RRV+Anti-Ly6C group was relatively normal, with a milder degree of weight loss and alleviated jaundice symptoms.

[0069] Example 3: Effect of Anti-Ly6C antibody on the body weight of mice.

[0070] During the experiment, the body weight changes were recorded daily until the 21st day, and the results are as Figure 3 shown.

[0071] The results showed that during the experiment, the body weights of the mice in the RRV+PBS group and the RRV+Isotype group continued to decrease, while the body weight of the mice in the RRV+Anti-Ly6C group decreased more slowly. On the 12th day, the body weight of the mice in the RRV+Anti-Ly6C group was significantly higher than that of the RRV+PBS group and the RRV+Isotype group (P<0.05), indicating that the Anti-Ly6C antibody could significantly restore the body weight of the biliary atresia mice.

[0072] Example 4: Effect of Anti-Ly6C antibody on the survival rate of mice

[0073] The results are as Figure 4 shown. All the mice in the Vehicle group survived, while the survival rates of the mice in the RRV+PBS group and the RRV+Isotype group gradually decreased. In contrast, the survival rate of the mice in the RRV+Anti-Ly6C group was significantly increased. On the 20th day, the survival rate of the mice in the RRV+Anti-Ly6C group remained at a relatively high level (P<0.05), indicating that the Anti-Ly6C antibody could effectively improve the survival rate of the biliary atresia mice.

[0074] Example 5: Effect of Anti-Ly6C antibody on the jaundice rate of mice

[0075] The results are as Figure 5As shown, the jaundice rates of the RRV+PBS group and the RRV+Isotype group of mice were relatively high and gradually increased over time. The jaundice rate of the RRV+Anti-Ly6C group of mice was significantly lower than that of the RRV+PBS group and the RRV+Isotype group (P<0.05), indicating that the Anti-Ly6C antibody could significantly reduce the jaundice rate in mice with biliary atresia. The jaundice rate was calculated according to the following criteria: The sclera and skin were observed daily for yellow staining, and a yellow staining area > 30% was determined as positive (referring to the Neonatal Jaundice scoring criteria).

[0076] Example 6 Effect of Anti-Ly6C Antibody on the Appearance of Mouse Liver and Bile Ducts

[0077] The results were as Figure 6 shown. The livers of the mice in the RRV+PBS group and the RRV+Isotype group were significantly yellowish in color, with a hardened texture, and obvious obstructive phenomena occurred in the extrahepatic bile ducts. The appearance color of the livers of the mice in the RRV+Anti-Ly6C group was similar to that of the Vehicle group, and the obstructive condition of the extrahepatic bile ducts was significantly improved visually. Subsequently, 2% FITC-dextran was slowly injected into the gallbladder for fluorescence imaging of the extrahepatic bile ducts, and the patency rate of the bile ducts was observed through a small animal in vivo imaging system. The results showed that the fluorescence signal of the extrahepatic bile ducts in the RRV+Anti-Ly6C group of mice was approximately intact, indicating that the patency of the extrahepatic bile ducts was significantly improved.

[0078] Example 7 Effect of Anti-Ly6C Antibody on the Appearance of Mouse Liver and Bile Ducts

[0079] The results were as Figure 7 shown. In the liver tissues of the mice in the RRV+PBS group and the RRV+Isotype group, obvious inflammatory infiltration around the bile ducts occurred, the bile duct structure was damaged, and the hepatocytes showed varying degrees of degeneration and necrosis. In the liver tissues of the mice in the RRV+Anti-Ly6C group, the inflammatory infiltration around the bile ducts was significantly reduced, the bile duct structure was relatively intact, and the degree of hepatocyte damage was also significantly reduced. The results of HE pathological scoring showed( Figure 8 ) that the HE pathological score of the mice in the RRV+Anti-Ly6C group was significantly lower than that of the RRV+PBS group and the RRV+Isotype group (P<0.05), further demonstrating the inhibitory effect of the Anti-Ly6C antibody on liver tissue inflammation in mice with biliary atresia.

[0080] Example 8 Effect of Anti-Ly6C Antibody on Changes in Mouse Liver Function Indexes

[0081] The results were as Figure 9As shown, the recovery of liver function is one of the main evaluation indicators for the improvement of biliary atresia symptoms. Liver function tests showed that compared with the solvent control group (Vehicle), the levels of serum ALT, AST, TBIL, DBIL, ALP, GGT, and TBA in the RRV group and the RRV + isotype control group of mice were significantly increased, indicating severe liver function damage. However, after treatment with Anti-Ly6C antibody, all seven liver function indicators shown in the figure were significantly improved.

Claims

1. Application of monocyte inhibitors in the preparation of drugs for the treatment of biliary atresia.

2. The use according to claim 1, characterized in that: The subjects of the drug include humans and mice.

3. The application according to claim 2 is characterized in that: When the subject of the drug is human, the monocytes are CD14 + CD16 - Classical monocytes.

4. The use according to claim 2, characterized in that: When the subject of the drug is a mouse, the monocytes are Ly6C + Classical monocytes.

5. The use according to claim 1, characterized in that: The monocyte inhibitors include substances that inhibit the number and / or activity of monocytes.

6. The use according to claim 1, characterized in that: The substances include antibodies that specifically bind to CD14 or Ly6C, and nucleic acid molecules that target CD14 or Ly6C genes.

7. The use according to claim 1, characterized in that: The drug includes pharmaceutically acceptable excipients.

8. The use according to claim 7, characterized in that: The pharmaceutically acceptable excipients include at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, and carriers.

9. The use according to claim 8, characterized in that: The dosage form of the drug includes a dosage form for gastrointestinal administration or a dosage form for parenteral administration.

10. The use according to claim 7, characterized in that: The medicament may further comprise any one or more other active ingredients.

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