Medicine for treating cholestatic liver injury and application

Abstract

According to the medicine for treating cholestatic liver injury and application, stevioside in the medicine provides an effective traditional Chinese medicine monomer component for treating cholestatic diseases, and the stevioside reduces the levels of bile acid TBA, total bilirubin TBIL and direct bilirubin DBIL in serum of cholestatic mice induced by BDL; the inflammatory response of BDL-induced cholestasis mice is improved; the effect of improving liver in-situ inflammation infiltration is achieved; new clinical indications of the stevioside are expanded, and the stevioside has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology and relates to a medicine for treating cholestatic liver injury and its application. Background Art

[0002] Cholestatic Liver Injury (CLI) is a pathological condition in which bile flow is obstructed due to genetic defects, mechanical obstruction of the bile duct, toxins or immune system disorders, causing bile components to accumulate in the hepatobiliary system and cause damage.

[0003] Depending on the site of obstruction, cholestasis can be categorized as extrahepatic cholestasis (caused by stones, tumors, biliary atresia, and primary sclerosing cholangitis) or intrahepatic cholestasis (caused by drugs, genetic defects, infections, and primary biliary cholangitis). Characteristic phenotypes include jaundice, dark urine, and clay-like stools, and long-term skin pruritus and hyperpigmentation. In terms of blood biochemistry, cholestasis elevates serum levels of total bile acid (TBA), direct bilirubin (DBIL), and total bilirubin (TBIL), accompanied by a strong inflammatory response. The accumulated toxic bile acids, acting as potent pro-inflammatory molecules, activate receptors on immune cells such as hepatocytes, bile duct cells, and Kupffer cells, inducing the release of pro-inflammatory cytokines and the infiltration of inflammatory cells such as neutrophils, macrophages, and lymphocytes. As the disease progresses, a progressive fibrotic response is triggered, manifested by bile duct cell proliferation, matrix remodeling, and inflammatory cell infiltration. During this process, activated bile duct cells and inflammatory cells secrete key profibrotic factors, inducing the activation of hepatic stellate cells (HSCs) into myofibroblasts and promoting the deposition of large amounts of extracellular matrix components, leading to the gradual progression of peribiliary fibrosis to bridging fibrosis and ultimately to biliary cirrhosis. Therefore, bile acid homeostasis imbalance, inflammatory response, and fibrosis are key to the development of CLI.

[0004] Stevioside (Ste) is a natural sweetener extracted from the leaves of the Stevia rebaudiana plant (Asteraceae). It belongs to the tetracyclic diterpenoid family. Studies have shown that stevioside is highly safe and non-toxic, leading to its widespread use as a sweetener in the food industry. Furthermore, stevioside exhibits multiple pharmacological activities, including anti-inflammatory, antioxidant, anti-tumor, and hypotensive and hypoglycemic properties. Currently, stevioside is used in the treatment of conditions such as hypertension and diabetes, but its use in cholestatic diseases has not been reported. Summary of the Invention

[0005] Based on the above objectives, the present invention provides a medicine for treating cholestatic liver injury and its application.

[0006] To achieve the above object, the present invention adopts the following technical solutions: The present application provides a medicine for treating cholestatic liver injury, which comprises stevioside.

[0007] In the present application, the dosage of stevioside is 90 mg / kg.

[0008] The present invention relates to the use of a drug for treating cholestatic liver injury in the preparation of a drug for repairing cholestatic liver injury.

[0009] The present invention has the following beneficial effects: (1) The stevioside in this application reduces the levels of bile acid TBA, total bilirubin TBIL, and direct bilirubin DBIL in the serum of mice with BDL-induced cholestasis; (2) Stevioside improves the inflammatory response in BDL-induced cholestasis mice; (3) The improving effect of stevioside on in situ inflammatory infiltration of the liver; (4) This drug for treating cholestatic liver injury provides an effective monomer component of traditional Chinese medicine for the treatment of cholestatic diseases, expands the new clinical indications of stevioside, and shows good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The results of the detection of TBA, TBIL, and DBIL levels in the serum of mice in each group one week after BDL modeling; Figure 2 The results of HE staining of liver tissue and statistical diagram of injury area of ​​mice in each group one week after BDL modeling; Figure 3 The results of immunofluorescence staining of CK19, a bile duct epithelial marker, in the livers of mice in each group 1 week after BDL modeling and the statistical results are shown; Figure 4 The immunofluorescence staining results and statistical results of the inflammatory factor marker CD45 in the liver of mice in each group are shown; Figure 5 The immunofluorescence staining results and statistical results of the liver inflammatory factor marker F4 / 80 in each group of mice are shown. DETAILED DESCRIPTION

[0011] The technical solution of the present invention is further explained and illustrated by means of specific embodiments below.

[0012] The stevioside used in the examples of this application has a CAS number of 57817-89-7, which is a commercially available product and has the molecular formula: HOOHOOHOOOHOHOCH2CH3H3CHHOOHHOHOHOHOOHHO.

[0013] Example 1: Establishment of mouse cholestasis disease model and drug administration strategy Eight-week-old, SPF-grade C57BL / 6J male mice weighing 18-22 g were fed a standard chow diet. After one week of acclimation, 15 mice were randomly divided into three groups: a control group (abbreviated as Sham), a cholestasis model group (abbreviated as BDL + Vehicle), and a stevioside-treated group (abbreviated as BDL + Ste). The Sham control group received normal saline orally for three days, underwent sham surgery on the fourth day, and received normal saline six times after surgery. Samples were collected on the eighth day. The cholestasis model group received normal saline orally for three days, underwent common bile duct ligation on the fourth day, and received normal saline six times after surgery. Samples were collected on the eighth day. The stevioside-treated group received stevioside orally for three days, underwent common bile duct ligation on the fourth day, and received stevioside six times after surgery. Samples were collected on the eighth day. All mice were anesthetized with 1% sodium pentobarbital intraperitoneally after treatment, and body weights were recorded. Blood was collected from the eyeballs of each mouse group and placed into 1.5 mL centrifuge tubes. After standing at room temperature for 30 minutes, the tubes were centrifuged at 15,000 rpm for 15 minutes at 4°C. The supernatant was collected and stored at -80°C. Liver tissue was weighed and fixed in 4% paraformaldehyde. Following fixation, the tissues were dehydrated in 30% sucrose solution or various concentrations of ethanol. Frozen and paraffin sections were prepared for subsequent immunofluorescence and histochemistry experiments.

[0014] Example 2: Evaluation of serum biochemical indicators and liver inflammation in mice The serum liver function of mice in each group was evaluated using Nanjing Jiancheng kit. Figure 1 , where the evaluation indicators are TBIL, DBIL and TBA.

[0015] The steps for evaluating serum biochemical indicators of mice using serum liver function indicators TBIL and DBIL include: 1) Add the corresponding reagents to the 96-well culture plate in the following order: add 7 μL of double-distilled water to the blank well, add 7 μL of standard solution to the standard well, and add 7 μL of the sample to the measurement well; 2) Add 200 μL of reagent 1 from the kit to all three wells; 3) After adding the sample, gently shake to mix and incubate at 37°C for 5 minutes. Immediately measure the absorbance of each well at 450 nm using a microplate reader and record it as A1.

[0016] 4) After measuring A1, add 50 μL of Reagent 2 from the kit to each of the three wells. Gently shake the wells again to mix thoroughly, and incubate at 37°C for another 5 minutes. After incubation, measure the absorbance of each well at 450 nm using a microplate reader and record this as A2.

[0017] 5) Calculate the absorbance change of each well: △A=A1-A2.

[0018] 6) The calculation formula is total bilirubin / direct bilirubin content (μmol / L) = [(△A 测定 -△A 空白 ) / (△A 标准 -△A 空白 )]*C 标准 .

[0019] The steps for evaluating serum biochemical indicators of mice using TBA include: 1) Add the corresponding reagents to the 96-well culture plate in sequence: add 10 μL of double-distilled water to the blank well, add 10 μL of standard solution to the standard well, and add 10 μL of the test sample to the measurement well; 2) Add 720 μL of Reagent 1 from the kit to each of the three wells. Gently shake to mix thoroughly and incubate at 37°C for 5 minutes. After incubation, add 240 μL of Reagent 2 from the kit to each of the three wells. Gently shake to mix thoroughly and incubate at 37°C for another 1 minute. After incubation, measure the absorbance of each well at 405 nm using a microplate reader and record it as A0. Continue incubating at 37°C for 3 minutes and record it as A1.

[0020] 3) Calculate the absorbance change of each well: △A = A1-A0.

[0021] 4) The calculation formula is: total bile acid content (μmol / L) = [(△A 测定 -△A 空白 ) / (△A 标准 -△A 空白 )]*C 标准 .

[0022] By the attached Figure 1 As shown, compared with the control group, the serum levels of TBIL, DBIL, and TBA in mice in the cholestasis model group were significantly increased, indicating that the cholestasis injury model was successfully established. Compared with the cholestasis model group, the serum levels of TBIL, DBIL, and TBA in mice in the stevioside treatment group were significantly decreased, indicating that stevioside can alleviate liver function in mice with BDL-induced cholestasis.

[0023] Example 3: HE staining and CK19 immunofluorescence staining of bile duct epithelial marker In the present embodiment, HE staining and CK19 immunofluorescence staining, a marker of bile duct epithelium, were used to evaluate the infiltration of liver tissue inflammation. Figure 2 、 3 The HE staining steps are as follows: 1) Embed the tissue blocks in paraffin and then slice them. Bake the liver tissue sections in an oven at 55°C for 1 hour before dewaxing.

[0024] 2) Dewaxing of liver tissue sections: a. Soak in xylene I for 5 min; b. Soak in xylene II for 5 min; c. Soak in 100% ethanol I for 5 min; d. Soak in 95% ethanol for 5 min; e. Soak in 80% ethanol for 5 min; f. Soak in 70% ethanol for 5 min; h. Wash with distilled water for 5 min.

[0025] 3) Stain with hematoxylin for 20 seconds and rinse with running water 2-3 times.

[0026] 4) Differentiate in differentiation solution for 1 minute, then rinse 2-3 times with running water to remove excess dye.

[0027] 5) Anti-blue for 1 minute, rinse with running water 2-3 times, and the cell nucleus will appear blue.

[0028] 6) Stain with eosin solution for 3 minutes, rinse with running water 2-3 times, and the cytoplasm will appear red.

[0029] 7) Dehydration: a. Soak in 95% ethanol I for 3 min; b. Soak in 95% ethanol II for 3 min; c. Soak in 100% ethanol I for 5 min; d. Soak in 100% ethanol II for 5 min; e. Soak in xylene I for 5 min; f. Soak in xylene II for 5 min.

[0030] 8) After sealing with neutral resin, observe under microscope to obtain the attached Figure 2 .

[0031] The steps for immunofluorescence staining of bile duct epithelial marker CK19 are as follows: 1) Fixation: Fix liver tissue in 4% paraformaldehyde for 1 hour. After fixation, wash three times with phosphate buffered saline (PBS) and dehydrate in 30% sucrose solution for 12 hours. Finally, transfer the tissue to an embedding box and embed in OCT (optimal cutting temperature compound).

[0032] 2) Sectioning: Cut the liver tissue into 10 μm thick sections.

[0033] 3) Staining: After the sections have dried, scrape off any excess OCT and apply a waterproof pen to prevent subsequent staining. Soak the sections in phosphate-buffered saline for 15 minutes to facilitate OCT removal. Cover the sections with the primary antibody CK19 (1:500) diluted in PBST buffer (1X PBS + 0.2% Triton X-100) and incubate overnight at 4°C in a light-proof, humidified chamber. The next day, rinse the sections three times in phosphate-buffered saline (PBS) for 5 minutes each. Incubate the sections with a donkey anti-rabbit secondary antibody (1:1000 dilution) for 30 minutes at room temperature in the dark. After incubation, rinse thoroughly in phosphate-buffered saline (PBS) three times for 5 minutes each to remove any unbound secondary antibody. Finally, mount the sections with anti-fluorescence quenching mounting medium.

[0034] 4) Perform immunofluorescence detection on the seal to obtain the attached Figure 3 .

[0035] By the attached Figure 2 、 3 It can be seen that compared with the control group, the liver tissue of mice in the BDL-induced cholestasis model group had obvious fibrous connective tissue generation, disordered arrangement, and inflammatory cell infiltration; compared with the cholestasis model group, the inflammatory cell infiltration, fibrous connective tissue, and bile duct reaction in the liver of mice in the stevioside treatment group were reduced, indicating that stevioside can improve BDL-induced cholestasis disease.

[0036] Example 4: Immunofluorescence staining Inflammation accelerates the process of fibrosis by activating macrophages. CD45 and F4 / 80 are specific markers of mouse macrophage populations and can therefore be used as indicators of inflammation in cholestasis models. In the examples of this application, immunofluorescence staining was used to detect the expression of mouse inflammatory proteins CD45 and F4 / 80. The specific steps are as follows: 1) Fixation: Fix liver tissue in 4% paraformaldehyde for 1 hour. After fixation, wash three times with phosphate buffered saline (PBS) and dehydrate in 30% sucrose solution for 12 hours. Finally, transfer the tissue to an embedding box and embed in OCT (optimal cutting temperature compound).

[0037] 2) Sectioning: Cut the liver tissue into 10 μm thick sections.

[0038] 3) Staining: After the sections have dried, scrape off any excess OCT and apply a waterproof pen to prevent subsequent staining. Soak the sections in phosphate-buffered saline for 15 minutes to facilitate OCT removal. Cover the sections with primary antibodies CD45 (1:500) and F4 / 80 (1:500) diluted in PBST buffer (1X PBS + 0.2% Triton X-100) and incubate overnight at 4°C in a light-proof, humidified chamber. The next day, rinse the sections three times in phosphate-buffered saline (PBS) for 5 minutes each. Incubate the sections with donkey anti-rabbit secondary antibody (1:1000 dilution) for 30 minutes at room temperature in the dark. After incubation, rinse thoroughly in phosphate-buffered saline (PBS) three times for 5 minutes each to remove any unbound secondary antibody. Finally, mount the sections with anti-fluorescence quenching mounting medium.

[0039] 4) Perform immunofluorescence detection on the seal to obtain the attached Figure 4 、 5 .

[0040] By the attached Figure 4 、 5 It can be seen that compared with the cholestasis model group, the protein expression levels of CD45 and F4 / 80 in the liver of mice in the stevioside treatment group were significantly reduced, indicating that stevioside can inhibit the inflammatory response of the liver tissue of BDL-induced cholestasis model mice.

[0041] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. A drug for treating cholestatic liver injury, characterized in that: Includes stevioside.

2. The drug for treating cholestatic liver injury according to claim 1, characterized in that The dosage of the stevioside is 90 mg / kg.

3. Use of the drug for treating cholestatic liver injury according to claim 1 or 2 in the preparation of a drug for treating cholestatic liver injury.

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