New application of laminarin

Abstract

The invention discloses an application of Laminarin in preparation of a medicine for treating cholestatic liver diseases. The invention reveals that the Laminarin can simultaneously inhibit the expression of inflammatory factors and promote liver regeneration for the first time, and fills the blank of the prior art.

Description

Technical Field

[0001] The present invention relates to the technical field of the treatment of cholestatic liver diseases, and particularly relates to the use of laminarin in the preparation of a drug for treating cholestatic liver diseases. Background Art

[0002] In recent years, studies on the pathophysiological mechanism of cholestasis have shown that bile acids mediate inflammatory responses by stimulating cytokines, thereby causing cholestatic liver diseases.

[0003] Currently, the treatment of cholestatic liver diseases is limited, such as obeticholic acid (OCA) and ursodeoxycholic acid (UDCA), but they all have various drawbacks, such as obvious side effects. Therefore, there is an urgent need to develop a new treatment method and new candidate drugs. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art to some extent. To this end, the present invention provides the use of laminarin in the preparation of a drug for treating cholestatic liver diseases.

[0005] According to one aspect of the present invention, there is provided the use of laminarin in the preparation of a drug for treating cholestatic liver diseases.

[0006] Preferably, the drug is an oral preparation or a non-oral preparation.

[0007] Preferably, the oral preparation is one or more of capsules, tablets, oral liquids, granules, pills, powders, pills or plasters.

[0008] Preferably, the non-oral preparation is one or more of injections, creams, patches, ointments or sprays.

[0009] According to another aspect of the present invention, there is provided a method for promoting the expression of Lifr in BDL cholangiospheres, using laminarin to treat the BDL cholangiospheres.

[0010] The present invention provides the use of laminarin in the preparation of a drug for treating cholestatic liver diseases. Brief Description of the Drawings

[0011] The drawings described herein are used to provide a further understanding of the present invention, and constitute a part of this application. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0012] Figure 1 Shows the chemical structure and experimental process of laminarin provided according to the embodiments of the present invention, as well as the liver morphology and histological staining results;

[0013] Figure 2 Shows the effect of laminarin on the body weight of bile duct ligated mice and the results of serum biochemical tests provided according to the embodiments of the present invention;

[0014] Figure 3 Shows the results of laminarin improving differential metabolites provided according to the embodiments of the present invention;

[0015] Figure 4 shows the inhibitory effect of laminarin on Dectin-1, the results of inflammation and oxidative stress related indicators, and the protein and mRNA expression levels of liver fibrosis related markers in liver tissue provided according to the embodiments of the present invention;

[0016] Figure 5 Shows the results of immunofluorescence staining of liver tissue sections and the results of bulkRNA-seq provided according to the embodiments of the present invention;

[0017] Figure 6 Shows the results of the effect of laminarin on bile duct organoids, the observation results and mRNA results of the action of Lifr inhibitor EC359 on bile duct organoids in the BDL + laminarin group. Detailed implementation manners

[0018] The following listed embodiments are to enable those skilled in the art to understand the present invention more clearly. It should be noted that the following embodiments do not limit the scope of protection required by the present invention and are only illustrative embodiments. The raw materials, reagents or devices mentioned in the following embodiments can be obtained from commercial channels or by known existing methods without special instructions.

[0019] In this invention, laminarin: manufacturer (Sigma-Aldrich); product number L9634; dissolve it in PBS when using. α-SMA (alpha smooth muscle actin): manufacturer (Cell Signaling); product number (48938S); dilution ratio (1:200). IL-6 (Interleukin-6): manufacturer (Abcam); product number (AB290735); dilution ratio (1:50). Desmin: manufacturer (Proteintech); product number (16520-1-AP); dilution ratio (1:400). Alb (Albumin): manufacturer (Proteintech); product number (66051-1-Ig); dilution ratio (1:100). Dectin-1: manufacturer (Invitrogen); product number (PA5-34382); dilution ratio (1:50).

[0020] The detection platform used for serum biochemical detection: automatic biochemical analyzer (manufacturer: Shenzhen Rayto Life Science Co., Ltd.; model: Chemray 240).

[0021] Steps for H&E staining: First, fix, embed and cut the tissue samples into thin slices, then dewax; next, stain the cell nuclei with hematoxylin and perform differentiation and blueing, then stain the cytoplasm and matrix with eosin; subsequently, dehydrate with ethanol and clear with xylene; finally, mount the slices with a mounting medium and dry to complete the preparation for microscopic observation. Ensure that the tissue structure and cell morphology are clearly visible throughout the process.

[0022] Steps for Sirius red staining: First, fix, embed and cut the tissue samples into thin slices, then dewax; next, immerse the slices in Sirius red staining solution to stain the collagen fibers red or orange; subsequently, use an acidic differentiating solution to remove the excess dye to make the collagen fibers clearly visible; finally, perform counterstaining, dehydration, clearing and mounting as needed for microscopic observation and analysis of the distribution and density of collagen fibers. The whole process can specifically detect and quantify the collagen fibers in the tissue.

[0023] Steps for Masson staining: First, fix, embed and cut the tissue samples into thin slices, then dewax; next, stain the cell nuclei with hematoxylin and then counterstain with phosphotungstic acid hematoxylin to stain the collagen fibers blue; subsequently, use an acidic differentiating solution to remove the excess dye, and then stain the cytoplasm and matrix with eosin; finally, perform dehydration, clearing and mounting for microscopic observation. This method can clearly distinguish collagen fibers, muscle and cell components and is commonly used in histological and pathological research.

[0024] RNA-seq Library Construction Method: ① RNA Extraction: Total RNA was extracted from the organoid samples. ② Library Preparation for mRNA Transcriptome Sequencing: 1 μg of total RNA from each sample was used as the input material for mRNA library preparation. ③ Sequencing After the library passed the quality inspection, the library preparation was sequenced on the NovaSeq 6000 platform (CHI BIOTECH CO., LTD), generating 150-bp strand-specific paired-end reads. ④ Data Analysis: Quality Control: First, the raw data (raw reads) in FASTQ format were processed using an internally written Perl script. At the same time, the Q20, Q30, and GC content of the clean data were calculated. All subsequent analyses were based on the high-quality clean data. Then, the reference genome and gene model annotation files were directly downloaded from the genome website. Finally, featureCounts (v2.0.3) was used to count the number of reads mapped to each gene. Then, TPM (Transcripts Per Million mapped reads per Kilobase of exon model) was calculated. ⑤ Differential Expression Analysis: Before performing differential gene expression analysis, for each sequencing library, the read counts were adjusted using the edgeR package with a scaling normalization factor. Differential expression analysis between the two conditions was performed using the edgeR R package (3.40.2). The P-values were adjusted using the Benjamini & Hochberg method. A corrected P-value of 0.05 was set as the threshold for significantly differential expression.

[0025] Sham Surgery Procedure: ① Anesthesia: 6 - 8-week-old Balb / c mice were anesthetized by intraperitoneal injection of 100 - 200 μl / 10 g of 2.5% tribromoethanol before surgery. ② Disinfection: After shaving the abdominal hair of the mice, the mice were fixed in the supine position on the operating table, and the abdominal skin was disinfected with povidone-iodine. ③ Incision: A small incision was made in the midline of the mouse abdomen, usually about 1 - 2 cm in length. Incision of Skin and Muscle: The skin and muscle were carefully incised with a scalpel and scissors to expose the abdominal cavity. ④ Tissue Separation: The liver and surrounding tissues were gently separated with a fine cotton swab to find the common bile duct. The common bile duct is located between the liver and the duodenum and is usually a slender tubular structure. The common bile duct was carefully dissected free with fine forceps without ligation. Then, the organs were restored to their original positions with a cotton swab. ⑤ Incision Closure: The muscle and skin were sutured layer by layer with 5-0 needle suture. The surgical area was disinfected again with povidone-iodine. ⑥ Body Temperature Recovery: The mice were placed on a heating blanket to recover body temperature.

[0026] Bileduct ligation (BDL) surgical procedure: ① Anesthesia: 2.5% tribromoethanol 100-200μl / 10g intraperitoneal injection anesthetize 6-8 week old Balb / c mice before surgery. ② Disinfection: After preparing the skin of the mouse abdomen, fix the mouse in a supine position on the operating table and disinfect the abdominal skin with iodine. ③ Incision: Make a small incision in the midline of the mouse abdomen, usually about 1-2 cm in length. Cut the skin and muscle: Use a scalpel and scissors to carefully cut the skin and muscle to expose the abdominal cavity. ④ Separate tissue: Use a fine cotton swab to gently separate the liver and surrounding tissues and find the common bile duct. The common bile duct is located between the liver and the duodenum and is usually a long and thin tubular structure. Use fine forceps to free the common bile duct. ⑤ Place ligatures: Use fine thread (6-0 suture) to place a ligature at the proximal and distal ends of the common bile duct. Ligate the distal end first, then the proximal end. Do not cut the bile duct. ⑥ Close the incision: Use 5-0 sutures to suture the muscles and skin layer by layer. Disinfect the surgical area with iodine again. ⑦ Body temperature resuscitation: Place the mouse on a heating blanket to rewarm.

[0027] Model success criteria: One week after surgery, mice showed symptoms such as erect hair, yellow ears, yellow abdomen and yellow urine. Four weeks after surgery, serum liver function indicators such as alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBIL) were significantly higher than the normal range. Histological staining of mouse liver showed that large areas of necrosis and varying degrees of collagen fiber deposition occurred in the liver of mice that underwent BDL.

[0028] Immunofluorescence steps: ① Sample preparation: Obtain tissue samples, fix them, and use fixatives such as paraformaldehyde to maintain the stability of tissue structure and antigens. ② Sectioning: Dehydrate, make them transparent, dip them in wax, and embed them after fixing them. Then use a slicer to cut them into thin slices and attach them to a glass slide. ③ Dewaxing and hydration: Remove the paraffin on the slices and hydrate them to restore the tissue slices to a near-natural state. ④ Antigen repair: Restore the immune reactivity of antigens in the tissue by heating. ⑤ Blocking: Treat the slices with blocking solutions such as BSA to reduce nonspecific binding. ⑥ Primary antibody incubation: Add a primary antibody against the target protein and allow it to bind to the antigen in the tissue slice. ⑦ Washing: Remove unbound primary antibodies. ⑧ Secondary antibody incubation: Add a secondary antibody labeled with a fluorescent dye to bind to the primary antibody. ⑨ Wash again: Remove unbound secondary antibodies. ⑩ DAPI incubation, sealing and observation: DAPI incubation for 10 minutes. Use sealing agents to seal the slices to prevent fluorescence quenching. The sections were observed using a confocal microscope, and the fluorescence signals were recorded and analyzed.

[0029] Example 1: Mouse Experiment

[0030] In this invention, laminarin (150 mg / kg / day) was intraperitoneally injected into normal mice continuously for 18 days, and no hepatotoxicity was found in the mice, indicating that laminarin is safe and non-toxic. The detected values of serum liver function indexes in the Sham+Laminarin group of mice, such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL), etc., were all within the normal range. Moreover, the histopathological results of liver H&E staining, Sirius red staining, and Masson staining, etc., were no different from those of the Sham group.

[0031] The liver disease is cholestatic liver disease induced by bile duct ligation (BDL). To evaluate the effect of laminarin on cholestatic liver disease, Balb / c mice aged 6 - 8 weeks (regardless of gender) were selected for the experiment. They were randomly divided into a control group (Sham and Sham+Laminarin groups), a model group (BDL group), and a treatment group (BDL+Laminarin group). Among them, the mice in the BDL+Laminarin group and the Sham+Laminarin group were intraperitoneally injected with laminarin (150 mg / kg / d) continuously for 18 days; the mice in the Sham group and the BDL group were intraperitoneally injected with an equal volume of phosphate buffered saline (PBS).

[0032] On the 27th day, blood was collected from the eyeballs of the mice to obtain serum, and then liver tissues were taken for H&E staining, Sirius red staining, etc. The chemical structure and experimental process of laminarin are shown in Figure 1 Figures a and 1b respectively. The liver morphology and histological staining results confirmed (Figures 1c and 1d) that compared with the control treatment, administration of laminarin for 18 days significantly reduced the fibrotic and necrotic areas in bile duct ligated mice.

[0033] To further study the effect of laminarin on the body weight of bile duct ligated mice, the body weight was first statistically analyzed ( Figure 2 Figure a), and the results indicated that laminarin could significantly reduce the decrease in body weight of mice induced by BDL. Then, serum biochemical tests were performed to evaluate liver function. The experimental results ( Figure 2b) It shows that laminarin can significantly reduce the levels of serum ALT (alanine aminotransferase), AST (aspartate aminotransferase) and TBIL (total bilirubin), and at the same time keep DBIL (direct bilirubin), ALB (albumin) and γ-GT (γ-glutamyl transferase) within the normal range. The normal value range of ALT in mice is 10.06 - 96.47 U / L. The normal value range of AST in mice is 36.31 - 235.48 U / L. The normal value range of TBIL in mice is 6.09 - 53.06 μmol / L. The normal value range of DBIL in mice is 0.45 - 33.89 μmol / L. The normal value range of ALB in mice is 21.22 - 39.15 g / L. The normal value range of γ-GT in mice is 0 - 7.78 U / L.

[0034] Metabolites are the end products of the interaction between genes, proteins and the environment, directly reflecting the real-time physiological or pathological state of an organism. Metabolomics reveals the metabolic state and its dynamic changes of a biological system by comprehensively analyzing small molecule metabolites (such as sugars, lipids, amino acids, nucleotides, etc.) in an organism. The results of metabolomics in the negative ion mode of serum suggest that laminarin can significantly improve the content of differential metabolites such as Octadecanoic acid ( Figure 3 )(p-value < 0.05). It is worth noting that the VIP (Variable Importance in Projection) values of these differential metabolites are all greater than 1, indicating that laminarin can improve the changes of important metabolites in mice caused by common bile duct ligation.

[0035] Laminarin has an inhibitory effect on Dectin-1 ( Figure 4a ). To further study the mechanism of the liver protection of laminarin in bile duct ligated mice, ELISA was used to detect the indexes related to inflammation and oxidative stress ( Figure 4b ). The results suggest that laminarin can significantly reduce the expression of inflammatory factors (the expression of IL-6, IL-1β and TNF-α) in the liver of mice, and reduce the level of MDA in the liver, indicating the improvement effect of laminarin on liver inflammation and oxidative stress in the common bile duct ligated mouse model. Immunofluorescence and RNA-seq were used to detect the protein and mRNA expression of liver fibrosis related markers (α-SMA, Desmin and Fn1) in liver tissues ( Figure 4c ). The results suggest that laminarin can significantly improve the fibrotic state of the common bile duct ligated mouse model.

[0036] Alb is a marker of hepatocytes. MKi-67 is a non-histone protein present in the nuclei of proliferating cells and is the "gold standard" marker for evaluating cell proliferation activity. Upp2 regulates proliferation, differentiation, and regeneration by controlling the balance of pyrimidine metabolism. Hes1 plays a key role in stem cell maintenance, cell differentiation, and development. Pcna is proliferating cell nuclear antigen, which is related to DNA replication. Lifr plays a key role in liver regeneration, stem cell maintenance, inflammation regulation, and metabolic balance. Combining the results of bulk RNA-seq of liver tissues ( Figure 5 ), laminarin promoted the expression of hepatocyte markers (Alb), proliferation and / or differentiation markers (Mki67, Upp2, Hes1, Lifr, and Pcna). The above results suggest that laminarin promotes hepatocyte regeneration.

[0037] In summary, discovering effective natural ingredients for cholestatic liver disease and liver regeneration induced by common bile duct ligation may be a promising research direction for the prevention and treatment of cholestatic liver disease. The results of this invention confirmed that the candidate compound Dectin-1 inhibitor laminarin, as a potential new preparation for treating cholestatic liver disease, alleviated liver inflammation, oxidative stress, fibrosis, and necrosis in a mouse model of cholestatic liver disease induced by common bile duct ligation, improved liver function, and revealed multiple mechanisms of liver protection and promotion of liver regeneration through bulk RNA-seq studies. These findings provide a scientific basis for the possible clinical use of laminarin in the prevention or treatment of cholestatic liver disease.

[0038] Example 2: In Vitro Experiment

[0039] Extraction and culture of biliary organoids: Cells expressing EpCAM + were mixed with 50 μL of Matrigel and seeded in a 4-well plate. After the Matrigel solidified, 500 μL of biliary organoid growth medium was added. The medium was changed every 2 / 3 days. Laminarin could promote the growth of biliary organoids (by seeding single cells mixed with 50 μL of Matrigel in plates) ( Figure 6 a).

[0040] qPCR procedure for bile duct organoids: ① Cell culture and treatment: Cultivate bile duct organoids according to the experimental purpose and perform corresponding treatments, such as drug treatment, etc. ② Organoid lysis and nucleic acid extraction: After dissolving Matrigel, use an appropriate lysis buffer to lyse the organoid cells, and then extract DNA or RNA by column purification. For RNA detection, it is also necessary to pay attention to avoiding RNase contamination. ③ Reverse transcription (when detecting RNA): Use reverse transcriptase to reverse transcribe the extracted RNA into cDNA. ④ Design primers and probes: Design specific primers and probes according to the sequence of the target gene. Primers are used for PCR amplification, and probes are used to detect the amplification products. ⑤ qPCR reaction setup: Mix components such as cDNA (or DNA), primers, probes, and Taq enzyme in a certain proportion and add them to a PCR reaction tube or plate. ⑥ qPCR amplification and detection: Place the set-up PCR reaction tube or plate into a real-time fluorescence quantitative PCR instrument and perform amplification according to the preset program. During the amplification process, the PCR instrument will detect the change in fluorescence signal in real time, thereby calculating the number of starting templates. ⑦ Data analysis: According to the data provided by the qPCR instrument, use appropriate software for data analysis to calculate the expression level of the target gene or the number of starting templates.

[0041]

[0042]

[0043] Recent studies have shown that Lifr is an important factor related to liver regeneration (PMID: 39147934, PMID: 36993315). Interestingly, in in vitro experiments, compared with the bile duct organoids in the BDL + laminarin group without adding EC359 (a Lifr inhibitor, MCE, HY - 120142), the bile duct organoids in the BDL + laminarin group with added EC359 were slightly fewer in number and slightly smaller in volume (after mixing 9000 single cells / well of the same number with 50 μL of Matrigel and then seeding into plates) ( Figure 6 b). The qPCR results showed that laminarin could upregulate the expression of Lifr in BDL bile duct organoids, and when the Lifr inhibitor EC359 (1 μM, 48 h) was added to the culture medium of the organoids in the BDL + laminarin group, the expressions of Lifr, Ki67, and Krt19 in the bile duct organoids were downregulated ( Figure 6 b). The above results suggest that laminarin can promote the regeneration of bile duct cells through Lifr. At the in vitro experimental level, to promote the expression of Lifr in BDL bile duct organoids, laminarin can be used to treat BDL bile duct organoids.

[0044] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of laminarin in the preparation of a drug for treating cholestatic liver disease.

2. The use according to claim 1, wherein The drug is an oral preparation or a non-oral preparation.

3. The use according to claim 2, characterized in that, The oral preparation is one or more of capsules, tablets, oral liquids, granules, pills, powders, pills or plasters.

4. The use according to claim 2, characterized in that, The non-oral preparation is one or more of injections, creams, patches, ointments or sprays.

5. A method for promoting the expression of Lifr in BDL bile duct organoids, characterized in that, Treat the BDL bile duct organoids with laminarin.

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