Application of xanthoxylin in preparation of medicine for treating non-alcoholic fatty liver disease

Abstract

The invention provides application of xanthoxylin in preparation of a medicine for treating a non-alcoholic fatty liver disease, and belongs to the technical field of medicines. The invention aims to provide the application of the xanthoxylin in preparing the medicine for treating the non-alcoholic fatty liver disease, the xanthoxylin can inhibit EndMT and improve endothelial dysfunction, and can be used as an inhibitor of TGF betaR2 to relieve NAFLD by inhibiting the occurrence of EndMT.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technologies, and particularly to the application of psoralen in the preparation of drugs for treating non-alcoholic fatty liver disease. Background Art

[0002] Nonalcoholic fatty liver disease (NAFLD) is currently the most common type of chronic liver disease and a major global health problem. In recent years, with the change of lifestyle and the intake of high-calorie diets, the prevalence of NAFLD has gradually increased. Clinical studies have shown that patients with fatty liver can further develop into non-alcoholic steatohepatitis (NASH), and even progress to liver cirrhosis and liver cancer. By 2030, it is very likely that NAFLD will become the main cause of liver transplantation.

[0003] Vascular endothelium, as the interface between blood and other tissues of the body, is not only a physical barrier but also involved in different physiological functions such as hemostasis, metabolite transport, inflammation, thrombosis, angiogenesis, and vascular tone. Hepatic endothelium is mainly formed by liver sinusoidal endothelial cells (LSECs), which are specialized vascular cells located between the hepatic sinusoidal lumen and the hepatic sinusoidal space. Fenestrae are their most characteristic structure, and these pores can regulate the transport of macromolecules (including lipids and lipoproteins) within the sinusoids. LSECs can integrate signals from the intestine and adipose tissue and interact with other cells in the liver. Under physiological conditions, LSECs are the gatekeepers of liver homeostasis. LSECs play anti-inflammatory and anti-fibrotic roles by preventing the activation of Kupffer cells and hepatic stellate cells and regulating hepatic vascular resistance and portal vein pressure. In the NASH stage, the altered LSECs release inflammatory mediators and promote the recruitment of inflammatory cells, thus promoting liver injury and inflammation. Capillarized LSECs transform into mesenchyme and secrete fibrotic factors such as TGF-β1 and extracellular matrix proteins, as well as stimulate the activation of adjacent hepatic stellate cells, thereby promoting liver fibrosis. Endothelial-mesenchymal transformation (EndMT) is a phenotypic conversion process through which endothelial cells lose their endothelial characteristics and acquire mesenchymal characteristics, resulting in endothelial dysfunction and interstitial fibrosis. Therefore, improving LSECs is a promising approach for preventing the progression and complications of NAFLD.

[0004] Xanthotoxin is mainly present in the fruits of Zanthoxylum schinifolium Sieb. et Zucc. of the Rutaceae family and the whole plant of Rutaceae. It is commonly found in daily seasonings such as star anise and Chinese prickly ash. It has also been found in commonly used traditional Chinese medicines such as Glehnia littoralis Fr. Schmidt ex Miq., Angelica pubescens Maxim. f. biserrata Shan et Yuan, and Cnidium monnieri (L.) Cuss. It belongs to the class of furanocoumarin natural compounds and has a wide range of pharmacological activities. Modern pharmacology has proven that xanthotoxin has antioxidant effects, can induce apoptosis of gastric cancer cells, and protect myocardial cells from hypoxia-reoxygenation injury. Moreover, the latest research shows that xanthotoxin can also prevent cognitive defects and may play an antidepressant role. At the same time, it also has a strong effect of dilating coronary arteries, relieving coronary artery spasm, and antiplatelet aggregation, and is a promising drug for the treatment of coronary heart disease. Due to its strong photosensitivity, xanthotoxin is mainly used clinically in combination with psoralen and ultraviolet A therapy to treat skin diseases such as vitiligo and psoriasis.

[0005] Numerous studies have shown that most Chinese herbal medicines can treat non-alcoholic fatty liver disease (NAFLD) by slowing down the progression of NAFLD. Existing research has shown that xanthotoxin, as an active ingredient in various commonly used traditional Chinese medicines such as Cnidium monnieri (L.) Cuss., Salvia miltiorrhiza Bunge, and Angelica dahurica (Fisch. ex Hoffm.) Benth. et Hook. f. ex Franch. et Sav., has effects such as enhancing the body's immunity, treating arthritis, antioxidant, and vasodilation. However, its efficacy in liver diseases is not fully clear, especially its effect on NAFLD. NAFLD is a chronic inflammatory disease related to blood vessels. Therefore, studying the effect of xanthotoxin on the treatment of NAFLD can provide further theoretical basis for the research and development of xanthotoxin as a new drug for the treatment of NAFLD and the development and utilization of Chinese herbal medicine resources. Summary of the Invention

[0006] The purpose of the present invention is to provide the application of xanthotoxin in the preparation of drugs for the treatment of non-alcoholic fatty liver disease. Xanthotoxin can inhibit EndMT, improve endothelial dysfunction, and can be used as an inhibitor of TGFβR2 to reduce NAFLD by inhibiting the occurrence of EndMT.

[0007] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0008] The present invention provides the application of xanthotoxin in the preparation of drugs for the treatment of non-alcoholic fatty liver disease.

[0009] Preferably, the structural formula of the xanthotoxin is:

[0010]

[0011] Preferably, the xanthotoxin can inhibit EndMT.

[0012] Preferably, the xanthotoxin is an inhibitor of TGFβR2.

[0013] Preferably, the drug in the above application is a pharmaceutically common preparation prepared with xanthotoxin as the active ingredient and pharmaceutically acceptable excipients or auxiliary components.

[0014] In vivo and in vitro experiments of the present invention show that psoralen can improve endothelial dysfunction by inhibiting EndMT. Transforming growth factor-β (TGF-β) is a multifunctional cytokine of the transforming growth factor superfamily and plays an important role in regulating cell growth and differentiation. Immunofluorescence experiments confirm that psoralen inhibits the activation of the TGF-β / Smad signaling pathway in vivo and in vitro, thereby inhibiting the occurrence of EndMT. Molecular docking and surface plasmon resonance experiments confirm that psoralen can bind to TGFβR2. Therefore, we believe that psoralen can be used as an inhibitor of TGFβR2 to alleviate NAFLD by inhibiting the occurrence of EndMT. Description of the Drawings

[0015] Figure 1 Results of psoralen reducing high-fat diet-induced hepatic steatosis in mice in Example 1; (A) Representative photographs of frozen sections of mouse liver tissues stained with Oil Red O. Scale bar: 100 μm. (B) Percentage (%) of the positive area of Oil Red O-stained lipid droplets measured by Image J. (C) Content of TG in the mouse liver. (D) Content of TC in the mouse liver. (E) Content of LDL-C in the mouse liver. **P < 0.01, ***P < 0.001, ****P < 0.0001 vs Control; #P < 0.05, ##P < 0.01, P < 0.001, #P < 0.0001 vs HFD;

[0016] Figure 2 Results of psoralen reducing high-fat diet-induced non-alcoholic steatohepatitis in mice in Example 1; (A) Representative photographs of paraffin sections of mouse liver tissues stained with H&E. Scale bar: 100 μm. (B) Fatty change, ballooning degeneration of hepatocytes, lobular inflammation and NAS score in mice. (C) Detection of the contents of AST and ALT in the serum of mice. *P < 0.05, **P < 0.01, ***P < 0.001 vs Control; #P < 0.05, ##P < 0.01, P < 0.001 vs HFD;

[0017] Figure 3Results of the attenuation of high-fat diet-induced hepatic fibrosis in mice by xanthotoxin in Example 1; (A) Representative photographs of Masson staining of perivascular and interstitial regions in paraffin sections of mouse liver and the percentage of fibrotic positive area measured by Image J (%). Scale bar: 100 μm. (B) Representative photographs of Sirius red staining of perivascular and interstitial regions in paraffin sections of mouse liver and the percentage of fibrotic positive area measured by Image J (%). Scale bar: 100 μm. ****P < 0.0001 vs Control; #P < 0.05, P < 0.001, #P < 0.0001 vs HFD;

[0018] Figure 4 Results of the reversal of HFD-induced EndMT by xanthotoxin in Example 2; (A) Representative images of Western blot analysis of endothelial markers VEGFR2, CD31, VE-cadherin and mesenchymal markers Vimentin, α-SMA and FSP1 in mouse liver. (B) Quantitative analysis of the protein expression of endothelial markers VEGFR2, CD31, VE-cadherin and mesenchymal markers Vimentin, α-SMA and FSP1 in mouse liver using Image J. (C) Detection of the mRNA levels of endothelial markers VEGFR2, CD31, VE-cadherin and mesenchymal markers Vimentin, α-SMA and FSP1 in mouse liver by qRT-PCR. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs Control; #P < 0.05, ##P < 0.01, P < 0.001, #P < 0.0001 vs HFD;

[0019] Figure 5Results of Example 3: Psoralen reverses FFA-induced EndMT in HHSECs; (A) Representative images of Western blot assay for detecting endothelial markers VEGFR2, CD31, VE-cadherin and mesenchymal markers Vimentin, α-SMA, and FSP1 in HHSECs. (B) Quantification of protein expression of endothelial markers VEGFR2, CD31, VE-cadherin and mesenchymal markers Vimentin, α-SMA, and FSP1 in HHSECs using Image J. (C) Detection of mRNA levels of endothelial markers VEGFR2, CD31, VE-cadherin and mesenchymal markers Vimentin, α-SMA, and FSP1 in HHSECs using qRT-PCR. *P<0.05, **P<0.01, ***P<0.001 vs Control; #P<0.05, ##P<0.01, P<0.001, #P<0.0001 vs FFA;

[0020] Figure 6 Results of Example 4: Psoralen alleviates EndMT-induced endothelial dysfunction; (A) Detection of reactive oxygen species levels in HHSECs of each group using kits. (B) Detection of eNOS protein expression in HHSECs using Western blot. *P<0.05 vs Control; #P<0.05, ##P<0.01 vs FFA;

[0021] Figure 7 Results of Example 5: Psoralen inhibits the transduction of TGF-β / Smad signaling pathway in HHSECs; Representative images (A) and relative fluorescence intensity (B) of immunofluorescence evaluation of p-Smad2 / 3 protein expression in HHSECs of each group. Scale bar: 50 μm. ****P<0.0001 vs Control; #P<0.0001 vs FFA;

[0022] Figure 8 Results of Example 5: Psoralen inhibits the transduction of TGF-β / Smad signaling pathway in animal livers; (A) Representative images of immunofluorescence evaluation of p-Smad2 / 3 (red) protein expression in liver tissues of each group and relative fluorescence intensity. Scale bar: 50 μm. (B) Evaluation of fluorescence intensity of p-Smad2 / 3 protein expression in vascular intima of liver tissues of each group using Image J. **P<0.01, ****P<0.0001 vs Control; ##P<0.01, #P<0.0001 vs HFD;

[0023] Figure 9For the binding of xanthotoxin to TGFβR2 in Example 6; (A) Molecular docking experiments were used to detect the binding of xanthotoxin to TGFβR2. (B) Surface plasmon resonance technology experiments were used to detect the binding affinity of xanthotoxin to TGFβR2;

[0024] Figure 10 For xanthotoxin in Example 7 can inhibit the function of TGFβR2; (A) Representative pictures of the expression of p-Smad2 / 3 protein in HHSECs were detected by Western blot. (B) The expression of p-Smad2 / 3 protein was quantitatively analyzed by Image J. *P<0.05, ***P<0.001, ****P<0.0001 vs TGFβ2+XAT(5μM). Detailed implementation manners

[0025] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0026]

[0027]

[0028]

[0029]

[0030]

[0031] Example 1

[0032] 1.1 Construction of animal model

[0033] Thirty-five 6-8-week-old ApoE were selected - / -Male mice (purchased from Changzhou Cavens Biotechnology Co., Ltd.) were kept at an indoor temperature of 24 ± 2°C, with an air humidity of 55 ± 5%. They were exposed to 12 hours of light and 12 hours of darkness. The experimental animals were randomly divided into 5 groups: a control group, a model group, a low-dose psoralen treatment group (10 mg / kg), a medium-dose psoralen treatment group (20 mg / kg), and a high-dose psoralen treatment group (30 mg / kg). The control group maintained a normal diet, while the other four groups were fed a high-fat diet (specific foods included 48.3% corn, 12% wheat middlings, 17% wheat, 6% soybean meal, 4% fish meal, 4% chicken meal, 4% premix, 2% gluten, 1.1% limestone powder, 1% salad oil, 0.45% feed-grade sodium chloride, and 0.15% feed-grade magnesium oxide). While the mice in the drug treatment groups were fed a high-fat diet, psoralen (10 mg / kg, 20 mg / kg, 30 mg / kg) was prepared with 0.5% sodium carboxymethylcellulose as a solvent and administered by gavage once every 2 days for 12 consecutive weeks. During this period, the mice had free access to water and food. Before sampling, the body weights of the mice were recorded. Blood was collected by ocular phlebotomy, and the liver was quickly removed and weighed. The mouse blood was centrifuged at 3000 rpm / min at 4°C for 15 min, and the upper serum sample was separated and stored at -80°C. Part of the liver tissue was fixed in 4% paraformaldehyde for further pathological examination, and the remaining tissue was stored at -80°C in a refrigerator for further experiments.

[0034] 1.2 Preparation of frozen sections of liver tissue

[0035] Take the liver tissue fixed in 4% paraformaldehyde for more than 24 hours in the above 1.1, dehydrate it successively in the prepared 15% and 30% sucrose solutions, and then use it to make frozen sections:

[0036] (1) First, place the liver tissue in a 15% sucrose solution in a 4°C refrigerator until it sinks to the bottom after dehydration, and then transfer it to a 30% sucrose solution in a 4°C refrigerator until it sinks to the bottom after dehydration.

[0037] (2) Put the dehydrated liver tissue into a disposable embedding cassette mold, completely immerse it with OCT embedding medium, quickly place the embedding cassette on the freezing table of the cryostat for freezing, and start sectioning after the OCT turns white and hardens.

[0038] (3) Fix the embedding table on the microtome. First, trim the tissue surface roughly until it is flat, and then start sectioning. The section thickness is 8 - 10 μm. Place a clean glass slide flat above the cut tissue section to attach the tissue to the glass slide. After writing the label on the slide, store it at -20°C for later use.

[0039] 1.3 Preparation of paraffin sections of liver tissue

[0040] (1) Sample fixation: The liver tissue in the above 1.1 was placed in 4% paraformaldehyde for fixation for more than 24 hours.

[0041] (2) Tissue dehydration: Take out the liver tissue soaked in 4% paraformaldehyde and dehydrate it according to the order in Table 1.

[0042] Table 1 Dehydration sequence and time of liver tissue

[0043]

[0044]

[0045] Note: Absolute ethanol I means the liver tissue is dehydrated through absolute ethanol once; absolute ethanol II means the liver tissue is dehydrated through absolute ethanol twice; xylene I means the liver tissue is cleared through xylene once; xylene II means the liver tissue is cleared through xylene twice; paraffin I, II, and III mean the well-cleared liver tissue is infiltrated with melted paraffin at 65°C three times respectively.

[0046] (3) Paraffin embedding: Pour the melted paraffin into the embedding frame, and use heated forceps to gently place the wax-impregnated tissue flat into the embedding mold. Press the tissue embedding box, and then slowly and evenly add the melted wax to the tissue embedding box until the liquid level is flush with the mold edge. Subsequently, to accelerate the cooling of the wax block, place it on a freezing table or ice box.

[0047] (4) Paraffin tissue sectioning: Trim the excess wax blocks around each tissue block, fix the tissue on the microtome, and adjust the section thickness of the paraffin microtome to 4 μm. To present the largest tissue section, the wax block needs to be roughly trimmed. After the tissue section is trimmed completely, hold a brush in the left hand and turn the large gear with the right hand, rotating evenly. Place the cut wax slices into a water bath with clean water (temperature set at 40°C), spread the tissue in the water, and flatten the slices. Wait until the tissue is flat and has no wrinkles, then stick it on the pre-labeled adhesive-free glass slides. After sectioning, place it in a staining rack and directly bake it in an oven at 60°C for 20 min. Wait until it cools down before staining.

[0048] 1.4 Oil Red O staining

[0049] (1) Place the frozen sections of the liver tissue stored in a -20°C refrigerator in step 1.2 at room temperature for 5 - 10 min.

[0050] (2) Gently immerse the frozen sections restored to room temperature in the Oil Red O working solution and stain for 8 - 10 min (cover with a lid to avoid light).

[0051] (3) Take out the sections, wait for 3 s, and then immerse them successively in two tanks of 60% isopropanol for differentiation for 3 - 5 s.

[0052] (4) Immerse the sections successively in two tanks of pure water for washing, 10 s each time.

[0053] (5) Immerse the slices in hematoxylin staining solution to stain the cell nuclei, wash with water, blue back and then wash with water again. After slightly drying the water, add glycerol gelatin mounting medium to mount the slices.

[0054] (6) Observe the staining results under the microscope and take pictures for record.

[0055] 1.5 Triglyceride (TG) detection

[0056] (1) Take the liver tissue placed in the -80 °C refrigerator in the above 1.1, homogenize it in an ice bath with Reagent I (n-heptane: isopropanol = 1:1), then centrifuge at 8000×g and 4 °C for 10 min, and take the supernatant into a new 1.5 mL EP tube.

[0057] (2) Add Reagent II to the EP tube and mix well. Vigorously shake the EP tube for 30 s, let it stand for 3 min and then shake vigorously for 30 s again. Repeat 3 times. After the shaking is completed, let it stand at room temperature for 3 min and then take the upper layer solution into a new 1.5 mL EP tube.

[0058] (3) Add Reagent III and Reagent IV to the new EP tube in sequence and mix well. Incubate in a 65 °C water bath for 3 min, take out the EP tube and cool it at room temperature for 5 min.

[0059] (4) Then add Reagent V and Reagent VI to the EP tube in sequence and mix well. Incubate in a 65 °C water bath for 15 min, take out the EP tube and cool it at room temperature for 5 min.

[0060] (5) After cooling, transfer the liquid in the tube to a 96-well plate and measure the absorbance at 420 nm with an enzyme-linked immunosorbent assay (ELISA) reader.

[0061] 1.6 Total cholesterol (TC) detection

[0062] (1) Take the liver tissue placed in the -80 °C refrigerator in the above 1.1, homogenize it in an ice bath with isopropanol, then centrifuge at 10000×g and 4 °C for 10 min, and take the supernatant into a new 1.5 mL EP tube.

[0063] (2) Add the working solution to the EP tube and mix well. Incubate in a 37 °C water bath for 15 min and measure the absorbance at 500 nm with an ELISA reader.

[0064] 1.7 Low-density lipoprotein cholesterol (LDL-C) detection

[0065] (1) Take the liver tissue placed in the -80 °C refrigerator in the above 1.1, homogenize it in an ice bath with absolute ethanol, then centrifuge at 2500 rpm / min and 4 °C for 10 min, and take the supernatant into a new 1.5 mL EP tube.

[0066] (2) Add Reagent 1 to the EP tube and mix well. Incubate in a 37 °C water bath for 5 min, and measure the absorbance at 600 nm using a microplate reader. Record the absorbance value as A1.

[0067] (3) Subsequently, add Reagent 2 to the EP tube and mix well. Incubate in a 37 °C water bath for 5 min, and measure the absorbance at 600 nm using a microplate reader. Record the absorbance value as A2, and calculate ΔA = A2 - A1.

[0068] In the above detection, the Oil Red O staining results showed that compared with the normal diet group, a large amount of lipid accumulation occurred in the liver of the high-fat diet-fed group, while treatment with sanshool significantly reduced the liver lipid droplet area in a dose-dependent manner ( Figure 1 A, Figure 1 B). Abnormal lipid accumulation is an important factor in the pathological process of NAFLD. Hepatic steatosis is the result of impaired hepatic lipid metabolism. When the TG level in the liver is too high, lipid overload in hepatocytes will cause lipotoxicity. Analysis of the lipid levels in the mouse liver showed that TG, TC, and LDL-C, which are associated with an increased risk of NAFLD, increased after high-fat diet feeding, while treatment with sanshool significantly reduced the levels of these lipids in the liver ( Figure 1 C, Figure 1 D, Figure 1 E). The above experimental results indicate that high-fat diet-induced hepatic steatosis in mice can be improved by treatment with sanshool.

[0069] 1.8 Hematoxylin-eosin (H&E) staining

[0070] (1) Deparaffinization of paraffin sections: Immerse the sections in Environmentally Friendly Deparaffinizing Solution Ⅰ for 20 min, Environmentally Friendly Deparaffinizing Solution Ⅱ for 20 min, absolute ethanol Ⅰ for 5 min, absolute ethanol Ⅱ for 5 min, 75% alcohol for 5 min, and wash with tap water.

[0071] (2) Hematoxylin staining: Immerse the sections in hematoxylin staining solution for 3 - 5 min, wash with tap water, differentiate with differentiating solution, wash with tap water, blue with bluing solution, and rinse with running water.

[0072] (3) Eosin staining: Immerse the sections in 95% alcohol for dehydration for 1 min, and stain in eosin staining solution for 15 s.

[0073] (4) Dehydration, clearing, and mounting: Immerse the sections in absolute ethanol I for 2 min, absolute ethanol II for 2 min, absolute ethanol III for 2 min, n-butanol I for 2 min, n-butanol II for 2 min, xylene Ⅰ for 2 min, and xylene Ⅱ for 2 min for clearing, and mount with neutral gum.

[0074] (5) Microscopic examination: Observe the staining results under the microscope and take pictures for recording.

[0075] 1.9 Glutamic Oxaloacetic Transaminase (AST) Detection

[0076] (1) Take mouse serum into a new EP tube, add Reagent 1 and mix well, incubate in a 37°C water bath for 30 min.

[0077] (2) Add Reagent 2 to the EP tube and mix thoroughly, incubate in a 37°C water bath for 20 min.

[0078] (3) Subsequently, add Reagent 3 to the EP tube and mix well, incubate at room temperature for 10 min, and measure the absorbance at 505 nm with an enzyme-linked immunosorbent assay (ELISA) reader.

[0079] 1.10 Glutamic Pyruvic Transaminase (ALT) Detection

[0080] (1) Take mouse serum into a new EP tube, add Reagent 1 and mix well, incubate in a 37°C water bath for 30 min.

[0081] (2) Add Reagent 2 to the EP tube and mix thoroughly, incubate in a 37°C water bath for 20 min.

[0082] (3) Subsequently, add Reagent 3 to the EP tube and mix well, incubate at room temperature for 10 min, and measure the absorbance at 505 nm with an ELISA reader.

[0083] 1.11 Non-alcoholic Fatty Liver Activity Score

[0084] Pathologists performed a blinded histological scoring of liver stained sections. According to the Non-alcoholic Steatohepatitis - Clinical Research Network Computing (CRN) criteria, scores for steatosis, lobular inflammation, and hepatocyte ballooning were obtained from the liver stained sections. Oil Red O staining was used for steatosis scoring (0 - 3), determined by the percentage of lipid droplets in the total tissue area. H&E staining was used to evaluate lobular inflammation (0 - 3) and hepatocyte ballooning score (0 - 2), where lobular inflammation was determined by the number of inflammatory foci, and the hepatocyte ballooning score was determined by the number and size of ballooned hepatocytes. The Non-alcoholic Fatty Liver Disease (NAFLD) Activity Score (NAS) was obtained from the sum of these three scores. According to the scores of steatosis grading, lobular inflammation, and hepatocyte ballooning, NAS ≥ 5 could be used as a NASH assessment model.

[0085] Results showed that psoralen treatment restored the morphology of hepatocytes to normal, prevented the occurrence of lobular inflammation, and reduced the ballooning, lobular inflammation, and NAS scores ( Figure 2 A, Figure 2B). NAS is a method for evaluating the degree of liver cell damage through liver tissue pathology examination. Generally, the higher the NAS, the more severe the liver damage. ALT is an important indicator of liver damage. When liver cells are damaged, ALT is released into the blood, causing an increase in serum transaminase. In addition, mouse serological tests showed that a high-fat diet induced a significant increase in the levels of serum ALT and AST in mice, but these transaminase levels decreased significantly after treatment with psoralen, indicating that psoralen has a protective effect on high-fat diet-induced liver damage. Figure 2 C).

[0086] 1.12 Masson staining

[0087] (1) Dewax the paraffin sections to water: sequentially place the sections in environment-friendly dewaxing solution I for 20 min, environment-friendly dewaxing solution II for 20 min, absolute ethanol I for 5 min, absolute ethanol II for 5 min, 75% alcohol for 5 min, and wash with tap water.

[0088] (2) Immerse the sections in Masson A solution overnight and rinse with running tap water.

[0089] (3) Place the sections in a staining solution prepared by mixing Masson B solution and Masson C solution in equal proportions, immerse for 1 min, wash with tap water, differentiate with the differentiating solution for several seconds, and wash with tap water.

[0090] (4) Place the sections in Masson D solution and immerse for 6 min, then rinse with tap water.

[0091] (5) Immerse in Masson E solution for 1 min.

[0092] (6) Without washing, slightly drain and directly place in Masson F solution and stain for 2 - 30 s.

[0093] (7) Rinse and differentiate the sections with 1% acetic acid, and dehydrate with two cylinders of absolute ethanol.

[0094] (8) Clear and mount the sections: place the sections in the third cylinder of absolute ethanol for 5 min, clear with xylene for 5 min, and mount with neutral gum.

[0095] (9) Microscopic examination: observe the staining results under the microscope and take pictures for record.

[0096] 1.13 Sirius red staining

[0097] (1) Dewax the paraffin sections to water: sequentially place the sections in environment-friendly dewaxing solution I for 20 min, environment-friendly dewaxing solution II for 20 min, absolute ethanol I for 5 min, absolute ethanol II for 5 min, 75% alcohol for 5 min, and wash with tap water.

[0098] (2) Sirius red staining: The sections were stained in Sirius red staining solution for 8 min, and dehydrated with anhydrous ethanol in two or three cylinders.

[0099] (3) Sealing the slides: The sections were then placed in clean xylene for 5 min for clearing, and sealed with neutral balsam.

[0100] (4) Microscopic examination: The staining results were observed under the microscope and photographed for record.

[0101] According to the Masson and Sirius red staining results, we observed that compared with the normal group, the perivascular and interstitial collagen deposition in the high-fat diet group was significantly increased, while the collagen deposition in the mice treated with sanshools was significantly decreased ( Figure 3 ). The results of quantitative analysis of the fibrotic area were consistent. Compared with the high-fat diet group, the fibrotic area of the liver tissue in the mice treated with sanshools was significantly reduced ( Figure 3 ). Liver fibrosis is a reversible process, but if not treated in time, it will lead to the development of the disease into liver cirrhosis. Therefore, timely intervention and treatment are required. The above experimental results show that sanshools not only reduce NSAH-induced liver injury, but also help to reduce liver fibrosis.

[0102] Example 2

[0103] It is hypothesized that sanshools may reduce the pathological damage of NAFLD in mice by inhibiting EndMT. To verify this hypothesis, we evaluated the effect of sanshools on the expression of EndMT-related markers in liver tissue.

[0104] 2.1 Part of the tissues stored in the -80 °C refrigerator in Section 1.1 above were lysed in RIPA buffer containing protease inhibitors and phosphatase inhibitors. After quantification, 20 μg of protein samples were loaded on a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane. Blocked with 5% non-fat milk dissolved in PBS for 2 h, and then incubated with primary antibodies (VEGFR2, CD31, VE-cadherin, Vimentin, α-SMA, FSP1, eNOS, and p-Smad2 / 3) at 4 °C for 12 h. GAPDH (Mouse) was used as an internal reference. Incubated on a shaker at room temperature for 1 h the next day. The primary antibodies were recovered and stored at 4 °C. Washed 4 times, 5 min each time. After incubating with the secondary antibody (horseradish peroxidase-labeled goat anti-rabbit IgG (H+L)) for 2 h, the secondary antibody was recovered and stored at 4 °C. Washed 4 times, 5 min each time. Prepared the developing solution and developed.

[0105] 2.2 Real-time PCR method

[0106] (1) Sample preparation: Take about 30 mg of liver tissue and put it into a pre-cooled 1.5 mL Eppendorf tube. First, add 200 μL of RNA extraction reagent, and after grinding with a handheld grinder, add another 800 μL of RNA extraction reagent to make up to 1 mL.

[0107] (2) Liquid phase separation: Add 200 μL of chloroform, shake vigorously for 5 min, and let it stand for 5 min. Centrifuge at 12000×g at 4 °C for 15 min. After centrifugation, carefully take out the Eppendorf tube. At this time, the sample is divided into three layers (colorless upper layer, white middle layer, red lower layer), and RNA is distributed in the colorless upper aqueous phase. Carefully aspirate the upper aqueous phase into a new 1.5 mL Eppendorf tube.

[0108] (3) Precipitate RNA: Add pre-cooled isopropanol with the same volume as the supernatant, invert to mix well, and let it stand at 4 °C for 10 min. Centrifuge at 12000×g at 4 °C for 10 min, and carefully discard the supernatant.

[0109] (4) Wash RNA: Add 1 mL of 75% ethanol (prepared with RNase-free ddH 2 O), flick the bottom of the tube to suspend the precipitate, and invert several times. Let it stand at room temperature for 5 min. Centrifuge at 7500×g at 4 °C for 5 min, pour out the supernatant, and dry the precipitate in an open state at room temperature in a clean environment for 2 - 5 min. Note that it should not be over-dried, otherwise it will be difficult to dissolve the RNA.

[0110] (5) Dissolve RNA: Add an appropriate amount of RNase-free ddH 2 O to dissolve the precipitate. If necessary, gently pipette a few times. After the precipitate is completely dissolved, transfer it to a 0.2 mL Eppendorf tube and store it in a -80 °C refrigerator for later use.

[0111] (6) The extracted RNA is first treated to remove genomic DNA, then reverse transcribed into cDNA, and then Real-time PCR is performed using the primer sequences of the target genes to determine the expression status of each gene. The primer sequences of the target genes are shown in Table 1.

[0112] Table 1 Primer sequences of each target gene

[0113]

[0114]

[0115] Western blot and qRT-PCR analysis showed that in the liver tissues of mice induced by high-fat diet, the expression of endothelial markers platelet endothelial cell adhesion molecule-1 (PECAM-1 / CD31), vascular endothelial growth factor receptor 2 (VEGFR2), and vascular endothelial cadherin (VE-cadherin) was downregulated, while the expression of mesenchymal cell markers vimentin, fibroblast specific protein 1 (FSP1), and α-smooth muscle actin (α-SMA) was upregulated. However, treatment with xanthotoxin significantly inhibited high-fat diet-induced EndMT( Figure 4 ).

[0116] Example 3

[0117] To further confirm the relationship between xanthotoxin and EndMT, we conducted in vitro experiments using human hepatic sinusoidal endothelial cells (HHSECs).

[0118] 1×10 5 human hepatic sinusoidal endothelial cells (HHSECs) were placed in endothelial cell medium containing 1% endothelial cell growth factor, 5% fetal bovine serum, and 1% penicillin / streptomycin, and cultured at 37°C in 5% CO 2 2 + 95% air for 48 h. When they reached 80% - 90% confluence, they were passaged. All operations were performed in a sterile environment.

[0119] 1×DPBS and the prepared complete medium were heated in a water bath at 37°C. The original culture medium in the culture flask containing the revived cells was removed. The cells were rinsed twice with 1×DPBS to wash away the residual culture medium. After adding 0.05% trypsin solution, they were placed in an incubator at 37°C for 1 min. After observing that the cell morphology became round under the microscope, the cell solution in the culture flask was poured into a 15 mL centrifuge tube containing serum and centrifuged at 1000 rpm / min for 5 min. After centrifugation, the supernatant in the 15 mL centrifuge tube was carefully aspirated, avoiding sucking the cell pellet. The cell pellet was resuspended with complete medium and divided into two culture flasks for continued culture to obtain passaged cells.

[0120] The cells were induced with saturated fatty acids (free fatty acid, FFA; PA:OA = 1:1) for 24 h, and the treatment concentrations of xanthotoxin were 20, 40, and 80 μM. The treated HHSECs were used for subsequent experiments such as Real time-PCR (the primer sequences of the target genes are shown in Table 1), Western blot, and immunofluorescence.

[0121] Palmitic acid and oleic acid (free fatty acid, FFA) (OA:PA = 1:1) can synthesize TG, and excessive TG content can cause hepatic steatosis. Therefore, in this experiment, OA-PA (1:1) was used to stimulate HHSECs for 24 h to establish an in vitro lipid accumulation model, and the drug administration group was induced with FFA + xanthotoxin for 24 h. Consistent with the animal experiment results, Western blot and qRT-PCR results showed that in HHSECs treated with FFA, the expression of endothelial markers VEGFR2, CD31, and VE-cadherin was downregulated, while the expression of mesenchymal markers Vimentin, α-SMA, and FSP1 was upregulated. After treatment with xanthotoxin, the FFA-induced EndMT was significantly inhibited ( Figure 5 ). The above results indicate that xanthotoxin can inhibit the occurrence of EndMT, restore the characteristics of endothelial cells, and promote the normal growth of hepatic sinusoidal endothelial cells.

[0122] Example 4

[0123] EndMT is a special state in which endothelial cells transform into mesenchymal cells under the action of various stimulating factors such as high fat. In this state, both the physical barrier function and the secretory function of endothelial cells are damaged. Therefore, we detected the levels of intracellular reactive oxygen species (ROS) and endothelial nitric oxide synthase (eNOS) that maintain vascular homeostasis. The ROS level is an important signal for cell damage caused by normal physiological functions and environmental factors of cells. As Figure 6 shown in A, compared with the normal group, the expression of ROS in HHSECs was significantly increased after FFA treatment, while the ROS level was significantly decreased after treatment with xanthotoxin. In addition, Western blot experiments confirmed that the expression of eNOS was decreased after FFA treatment, but its expression level was significantly increased after treatment with xanthotoxin ( Figure 6 B).

[0124] Example 5

[0125] Since the TGF-β / Smad signaling pathway is a classical signaling pathway that induces EndMT. TGF-β exerts its function by binding to its receptor and then activating the phosphorylation of downstream Smad proteins. Therefore, we investigated whether the EndMT conversion in HHSECs induced by FFA or co-treated with FFA and sanshools was due to changes in this signaling pathway.

[0126] Tissue immunofluorescence

[0127] (1) Air-dry: The frozen sections of liver tissue taken out from the -20°C refrigerator were air-dried at room temperature for 40 min.

[0128] (2) Wash: After air-drying, the embedding medium was washed away with 1×PBS, washed 3 times, 5 min each time.

[0129] (3) Permeabilize: Permeabilize with 4% Triton-100 (diluted with 1×PBS) at room temperature for 1.5 h. Rinse with 1×PBS 3 times, 5 min each time.

[0130] (4) Block: Block with 5% BSA (diluted with 1×PBS) at room temperature for 1.5 h. Rinse with 1×PBS 3 times, 5 min each time.

[0131] (5) Incubate with primary antibody: Use a fluorescent pen to circle the tissue on the slide. Drop 50 μL of the prepared double primary antibody dilution of CD31 and p-Smad2 / 3 (CD31 concentration 1:50, p-Smad2 / 3 concentration 1:200, dilution with primary antibody dilution buffer) on each tissue, and incubate overnight at 4°C in a humidified box in the dark.

[0132] (6) Recover primary antibody: After overnight incubation with the primary antibody, recover it, and rinse with 1×PBS 3 times, 5 min each time.

[0133] (7) Incubate with secondary antibody: Drop 50 μL of the secondary antibody dilution on each tissue, which are AlexaFluor 488-conjugated goat anti-mouse IgG (H+L) and AlexaFluor 555-conjugated donkey anti-rabbit IgG (H+L). The secondary antibody concentration is 1:500. AlexaFluor 488-conjugated goat anti-mouse IgG (H+L) labels CD31, and AlexaFluor 555-conjugated donkey anti-rabbit IgG (H+L) labels p-Smad2 / 3. Incubate at room temperature for 1.5 h. Rinse with 1×PBS 3 times, 5 min each time.

[0134] (8) DAPI counterstain: Drop the anti-fluorescence quenching mounting medium containing DAPI onto the tissue, and then cover it with a coverslip.

[0135] (9) Collect images with a laser confocal microscope.

[0136] Immunofluorescence staining revealed that compared with the normal group, FFA treatment significantly increased the expression of phosphorylated Smad2 / 3 (p-Smad2 / 3), while treatment with xanthotoxin decreased its expression in HHSECs. Figure 7 A, Figure 7 B). In summary, the above experiments demonstrated that xanthotoxin could inhibit the expression of p-Smad2 / 3, thereby inhibiting the conduction of the TGF-β / Smad signaling pathway.

[0137] The liver is an organ with relatively rich blood content and well-developed blood vessels. From the above results, we have confirmed that a high-fat diet can induce EndMT in liver tissue and activate the classical TGF-β / Smad signaling pathway. Therefore, we next detected the expression of p-Smad2 / 3 in the vascular intima of mouse liver tissue. Using CD31 as the localization of the intima in the immunofluorescence staining experiment, it was found that a high-fat diet increased the expression of p-Smad2 / 3 in the intima, while xanthotoxin decreased its expression. The above results indicate that xanthotoxin can inhibit the TGF-β / Smad signaling pathway activated by a high-fat diet. Figure 8 )

[0138] Example 6

[0139] Since the activation of TGF-β signaling requires the ligand TGF-β to bind to its receptor TGFβR2 dimer, and the latter recruits the TGFβR1 dimer to form a heterotetrameric complex, we next aimed to evaluate whether psoralen plays a role in affecting the function of TGFβR2. To verify that psoralen is an inhibitor of TGFβR2, we retrieved and downloaded the three-dimensional structure model of psoralen in SDF format from the PubChem database, opened it with the Discovery Studio 2017 software package, added hydrogen and performed energy minimization (force field: CHARMm), saved it in mol2 format, and then preprocessed it with AutoDockTools1.5.7 and saved it as a pdbqt file for molecular docking. The protein structure of TGFβR2 (PDB ID: 5E92) was downloaded from the Protein DataBank (PDB), preprocessed with Discovery Studio 2017 (removing water molecules, ligands, metal ions, adding full hydrogen, etc.), and then loaded with AutoDockTools 1.5.7. The region where the original ligand was located was defined as the binding pocket, with dimensions of 56×44×58; AutoGrid4.2.5 was run to generate the receptor field file, and then AutoDock4.2.5.1 was used for molecular docking to generate 20 conformations and perform clustering analysis. The docking results were loaded with AutoDockTools 1.5.7, and considering the binding free energy and binding mode, the best binding conformation was selected and exported as a pdb format file for visualization analysis with Discovery Studio 2017.

[0140] Surface plasmon resonance experiment

[0141] (1) Immobilize the recombinant protein TGFβR2 on an AffiCoat chip and block it with a blocking solution.

[0142] (2) Inject psoralen into the chip for flow. Psoralen will interact with the TGFβR2 immobilized on the chip to form a complex.

[0143] (3) Record the change in the intensity of the reflected light over time during the flow of psoralen. When psoralen binds to TGFβR2, the amplitude of the reflected light will change.

[0144] (4) Analyze the data obtained from the experiment to calculate the change in the resonance angle and its relationship with the concentration of psoralen.

[0145] Determine the dissociation constant to judge whether the small molecule compound binds to the receptor.

[0146] The docking results showed that TGFβR2 has a pocket for binding with xanthotoxin. Among them, xanthotoxin forms hydrophobic interactions with V258, K277 and A256 of TGFβR2, forms hydrogen bonds with A256, F255, R254 and K381, and has weak electrostatic interactions with D397. The binding energy between the two is -6.98 kcal / mol. Generally, it is considered that the binding energy is less than -5 kcal / mol, indicating good binding activity between the two. Figure 9 A). Surface plasmon resonance experiments were used to evaluate the affinity and interaction kinetics between TGFβR2 and xanthotoxin in vitro. As Figure 9 shown in B, the KD value of TGFβR2-xanthotoxin was 3.61×10 -5 M, indicating a strong affinity between TGFβR2 and xanthotoxin. In summary, these results confirmed that xanthotoxin can bind to TGFβR2.

[0147] Example 7

[0148] To determine whether the binding of xanthotoxin to TGFβR2 would affect its function, and if so, whether it would promote or inhibit it? We treated cells to activate the TGF-β / Smad signaling pathway with 10 ng / mL of TGFβ2, and then detected the change in the expression of p-Smad2 / 3 protein with the increase in the concentration of xanthotoxin by Western blot. The experimental data showed that xanthotoxin would inhibit the protein expression of p-Smad2 / 3, that is to say, xanthotoxin can act as an inhibitor of TGFβR2. Figure 10 ) The above experimental results showed that xanthotoxin inhibited the TGF-β / Smad signaling pathway and the occurrence of EndMT by binding to TGFβR2, thereby improving the pathological characteristics of NAFLD.

[0149] The above is only the preferred embodiment of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Application of Zanthoxylum bungeanum toxin in the preparation of drugs for the treatment of non-alcoholic fatty liver disease.

2. The use according to claim 1, characterized in that The structural formula of the Zanthoxylum bungeanum toxin is:

3. The use according to claim 2, characterized in that The Zanthoxylum bungeanum toxin can inhibit endothelial-mesenchymal transition.

4. The use according to claim 3, characterized in that The Zanthoxylum bungeanum toxin is an inhibitor of TGFβR2.

5. The use according to claim 4, characterized in that The medicine used in the application is a commonly used pharmaceutical preparation prepared by taking Zanthoxylum bungeanum toxin as an active ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients.

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