Nitrate composition for preventing and treating non-alcoholic fatty liver disease

Abstract

The invention provides a composition for preventing and treating a non-alcoholic fatty liver disease, the composition comprises pharmaceutically acceptable nitrate, and the nitrate regulates the steady state of macrophages by up-regulating the Sialin expression level of the macrophages so as to inhibit the inflammatory response and fatty degeneration of the liver. Compared with the prior art, the nitrate composition for preventing and treating the non-alcoholic fatty liver disease has the advantages that the mode of preventing and treating the non-alcoholic fatty liver disease by dietary nitrate is defined; meanwhile, it is proved that nitrate can up-regulate the sialin expression level in macrophages and regulate the treatment mechanism of proinflammatory / anti-inflammatory homeostasis of macrophages. By absorbing nitrate components in natural vegetables, the purpose of preventing and treating the non-alcoholic fatty liver disease can be achieved economically, conveniently, safely and effectively without side effects.

Description

Technical Field

[0001] The present application relates to the field of medical technology, and in particular to a nitrate composition for preventing and treating non-alcoholic fatty liver disease. Background Art

[0002] Nitrates and nitrites are widely present in water, soil, air and plants. The main source of nitrate absorption by the human body is food, with green vegetables accounting for the main part.

[0003] In recent years, with the continuous changes in the surrounding environment, lifestyle, and dietary structure, metabolic-related diseases have become increasingly prevalent, with a trend of younger patients. Among them, non-alcoholic fatty liver disease (NAFLD) is a common metabolic disorder-related disease, one of the most common liver diseases, and has become the second leading cause of liver transplantation. If the disease progression is not well controlled, up to 25% of patients may develop cirrhosis and liver cancer, which seriously threatens human life and health. Currently, there are no effective preventive and therapeutic drugs for NAFLD in clinical practice. Summary of the Invention

[0004] In response to the above-mentioned technical limitations, the present application proposes a nitrate composition for preventing and treating non-alcoholic fatty liver disease; it overcomes the deficiencies and defects mentioned in the background technology.

[0005] To achieve the above objectives, this application adopts the following technical solutions:

[0006] The invention of the present application is to provide a composition for preventing and treating non-alcoholic fatty liver disease, comprising a pharmaceutically acceptable nitrate, which regulates macrophage homeostasis by upregulating the expression level of Sialin in macrophages, thereby inhibiting the inflammatory response and fatty degeneration of the liver.

[0007] Optionally, the above composition is a dietary supplement or a pharmaceutical composition, and the administration form is selected from oral administration, injection or local administration.

[0008] Optionally, in the above composition, the nitrate is selected from at least one of sodium nitrate, potassium nitrate, calcium nitrate or magnesium nitrate, preferably sodium nitrate.

[0009] Optionally, in the above-mentioned composition, the administration concentration of the nitrate is 0.1-10 mM, preferably 0.5-5 mM, more preferably 4 mM; the administration dosage is 0.035-3.5 mmol / kg body weight per day, preferably 0.175-1.75 mmol / kg body weight, more preferably 1.4 mmol / kg body weight; the administration method is selected as delivery through drinking water or feed.

[0010] Optionally, in the above composition, the macrophages are selected from pro-inflammatory macrophages and / or anti-inflammatory macrophages.

[0011] The second invention of this application is to provide a method for regulating macrophage homeostasis in vitro, comprising the following steps:

[0012] S1. Isolate and culture primary bone marrow-derived macrophages and culture them in vitro in culture medium. The specific method is as follows: remove the femur and tibia of the mouse and place them in a 50ml centrifuge tube containing RPMI 1640; remove the muscle tissue on the surface of the femur and tibia, and cut off the joint surface respectively; flush the bone marrow in the joint cavity with a syringe containing RPMI 1640 and place it in a sterile culture dish; filter the liquid with a 70μm filter, transfer it to a 50ml centrifuge tube, centrifuge at 2000rpm / 5min, and discard the supernatant; add 2ml of red blood cell lysis buffer to the precipitate and lyse for 3min; add 5ml of MACS buffer to terminate the lysis reaction, centrifuge at 2000rpm / 5min, discard the supernatant, resuspend the cells with MACS buffer, and add PE-labeled anti-mouse TER119 antibody, Gr-1 antibody and B220 antibody (the concentration of each antibody is 0.125μl / 10 6 cells), incubated at 4°C in the dark for 15 min; centrifuged at 2000 rpm for 5 min, discarded the supernatant, resuspended the cells in MACS buffer, and added anti-PE magnetic beads (1 μl / 10 6 Cells were incubated at 4°C in the dark for 15 min, centrifuged at 2000 rpm / 5 min, and the supernatant was discarded. The cells were resuspended in 2 ml of MACS buffer and passed through LS magnetic columns. The cells were centrifuged at 2000 rpm / 5 min, and the supernatant was discarded. The cells were resuspended in complete medium [RPMI 1640 (88%) + FBS (10%) + AA (1%) + Glu (1%)] containing GM-CSF (20 ng / ml) and transferred to a 24-well plate with a cell density of 7 × 10 cells per well. 4 cells / ml, cultured for 5 days;

[0013] S2. Adding nitrate to the culture medium at a concentration of 0.5-10 mM (preferably 2 mM);

[0014] S3. The expression level of Sialin and the changes in the ratio of pro-inflammatory / anti-inflammatory macrophages were detected. Nitrate inhibits the differentiation of pro-inflammatory macrophages by upregulating Sialin.

[0015] Optionally, the above method further includes the following steps:

[0016] S4. Validate the specific regulatory effects of nitrate on macrophage homeostasis by knocking out or overexpressing the Sialin gene. Specifically:

[0017] To knock down the gene, siRNA was designed, and the sequence was shown in SEQ ID No. 1: CAAGCCGCTGACTATTTAA;

[0018] Overexpression gene plasmid, EGFP tag.

[0019] The third invention point of the present application is to provide the use of nitrates in the preparation of a drug for preventing and treating non-alcoholic fatty liver disease, wherein the drug upregulates the expression level of Sialin in macrophages, regulates macrophage homeostasis, and inhibits hepatic inflammatory cell infiltration and fatty degeneration.

[0020] Optionally, in the above application, the drug further contains a vitamin C preparation that can be used in combination with nitrates. The vitamin C preparation increases the effective nitrate concentration in mice and thereby increases the expression level of Sialin, thereby effectively enhancing the prevention and treatment of non-alcoholic fatty liver disease.

[0021] In the combined drug, the molar ratio of sodium nitrate to vitamin C is 5:1.

[0022] Compared with the prior art, this application has the following advantages:

[0023] The nitrate composition provided in this application for preventing and treating non-alcoholic fatty liver disease (NAFLD) clarifies the role of dietary nitrate in preventing and treating NAFLD. It also demonstrates the therapeutic mechanism by which nitrate can upregulate sialin expression in macrophages and regulate the pro-inflammatory / anti-inflammatory homeostasis of macrophages. By absorbing nitrates from natural vegetables, the goal of preventing and treating NAFLD can be achieved economically, conveniently, without side effects, and safely and effectively. DETAILED DESCRIPTION

[0024] To make the purpose, technical solutions and advantages of this application more clear, this application is further described in detail below. However, it should be understood that the description herein is only used to explain this application and is not intended to limit the scope of this application.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are intended solely for the purpose of describing specific embodiments and are not intended to limit this application. The reagents and instruments used herein are commercially available, and the characterization methods involved can be found in the relevant descriptions in the prior art and will not be further elaborated herein.

[0026] In order to further understand the present application, the present application is further described in detail below in conjunction with the best embodiment.

[0027] Example 1

[0028] A composition for preventing and treating non-alcoholic fatty liver disease comprises a pharmaceutically acceptable nitrate, which upregulates the expression level of Sialin in macrophages and regulates macrophage homeostasis, thereby inhibiting inflammatory response and fatty degeneration in the liver.

[0029] The composition can be selected as a dietary supplement or a pharmaceutical composition, and the administration form can be selected as oral, injection or topical administration. The nitrate can be selected as at least one of sodium nitrate, potassium nitrate, calcium nitrate or magnesium nitrate, preferably sodium nitrate.

[0030] To verify the therapeutic effect of nitrate on non-alcoholic fatty liver disease (NAFLD), the applicant first conducted an animal model.

[0031] The MCD (methionine-choline-deficient diet) consists of 21% fat, 63% sucrose-based carbohydrates, and is deficient in choline and methionine. The MCD diet drives liver damage by impairing hepatic lipid export. Therefore, MCD mice can be used as an animal model for NAFLD.

[0032] The CDHFD (choline-deficient high-fat diet) is a dietary model that can be used to study the combined effects of obesity / metabolic syndrome. It contains 45% fat and lacks the essential nutrient choline. Choline deficiency, as the main component of low-density lipoprotein, reduces the liver's ability to secrete very low-density lipoprotein and metabolize fat through β-oxidation, resulting in lipid storage in hepatocytes in the form of triglycerides. C57 mice fed a CDHFD had a significant increase in total body fat mass and developed impaired glucose tolerance and other metabolic problems. Significant changes in transaminases were detected after feeding, and typical NAFLD histopathological features appeared. Therefore, CDHFD model mice can also be used as an animal model for NAFLD.

[0033] Nitrate was administered to the above-mentioned model mice.

[0034] The administration concentration of nitrate is 0.1-10 mM, preferably 0.5-5 mM, more preferably 4 mM; the administration dose is 0.035-3.5 mmol / kg body weight per day, preferably 0.175-1.75 mmol / kg body weight, more preferably 1.4 mmol / kg body weight; the administration method is selected as delivery through drinking water or feed.

[0035] Pharmacodynamic studies have shown that sodium nitrate (NaNO3) can significantly reduce elevated alanine aminotransferase levels in mice modeled after MCD / CDHFD. Histological staining also indicates that NaNO3 can significantly alleviate inflammatory cell infiltration and fatty degeneration in the liver of these mice, suggesting that NaNO3 can prevent and treat nonalcoholic fatty liver disease.

[0036] Further research on its therapeutic mechanism revealed that nitrate can upregulate macrophage Sialin levels to regulate macrophage homeostasis. The discovery and verification of the in vitro regulatory mechanism can supplement the nitrate pathway and play a broader role in vivo after in vitro regulatory modification. The discovery and verification of the in vitro regulatory mechanism can supplement the nitrate pathway and play a broader role in vivo after in vitro regulatory modification.

[0037] Based on the above efficacy and mechanism studies, the applicant further provides a method for regulating macrophage homeostasis in vitro, comprising the following steps:

[0038] S1. Isolate and culture primary bone marrow-derived macrophages and culture them in vitro in culture medium. The specific method is as follows: remove the femur and tibia of the mouse and place them in a 50ml centrifuge tube containing RPMI 1640; remove the muscle tissue on the surface of the femur and tibia, and cut off the joint surface respectively; flush the bone marrow in the joint cavity with a syringe containing RPMI 1640 and place it in a sterile culture dish; filter the liquid with a 70μm filter, transfer it to a 50ml centrifuge tube, centrifuge at 2000rpm / 5min, and discard the supernatant; add 2ml of red blood cell lysis buffer to the precipitate and lyse for 3min; add 5ml of MACS buffer to terminate the lysis reaction, centrifuge at 2000rpm / 5min, discard the supernatant, resuspend the cells with MACS buffer, and add PE-labeled anti-mouse TER119 antibody, Gr-1 antibody and B220 antibody (the concentration of each antibody is 0.125μl / 10 6 cells), incubated at 4°C in the dark for 15 min; centrifuged at 2000 rpm for 5 min, discarded the supernatant, resuspended the cells in MACS buffer, and added anti-PE magnetic beads (1 μl / 10 6 Cells were incubated at 4°C in the dark for 15 min, centrifuged at 2000 rpm / 5 min, and the supernatant was discarded. The cells were resuspended in 2 ml of MACS buffer and passed through LS magnetic columns. The cells were centrifuged at 2000 rpm / 5 min, and the supernatant was discarded. The cells were resuspended in complete medium [RPMI 1640 (88%) + FBS (10%) + AA (1%) + Glu (1%)] containing GM-CSF (20 ng / ml) and transferred to a 24-well plate with a cell density of 7 × 10 cells per well. 4cells / ml, cultured for 5 days;

[0039] S2. Adding nitrate to the culture medium at a concentration of 0.5-10 mM (preferably 2 mM);

[0040] S3. Detect the expression level of Sialin and the changes in the ratio of pro-inflammatory / anti-inflammatory macrophages. Nitrate inhibits the differentiation of pro-inflammatory macrophages by upregulating Sialin; or

[0041] S4. Validate the specific regulatory effects of nitrate on macrophage homeostasis by knocking out or overexpressing the Sialin gene. Specifically:

[0042] To knock down the gene, siRNA was designed, and the sequence was shown in SEQ ID No. 1: CAAGCCGCTGACTATTTAA;

[0043] Overexpression gene plasmid, EGFP tag.

[0044] To verify the therapeutic effect and therapeutic mechanism of nitrate in this application, the following studies were conducted:

[0045] In vivo studies: 8-week-old C57BL / 6 mice were fed a special diet and given nitrate in their drinking water. Liver tissue and serum were collected for histological, serological, and immunological testing to measure sialin expression levels.

[0046] In vitro studies: Primary mouse bone marrow-derived macrophages were isolated and cultured, and nitrate was added to the culture medium in advance to promote their differentiation into pro-inflammatory or anti-inflammatory cells. Cell ratios, inflammatory indicators, and sialin expression levels were measured. Cell ratios, inflammatory indicators, etc. were measured by knocking out or overexpressing sialin.

[0047] The above research fully proves that nitrates can be used to prepare drugs for the prevention and treatment of non-alcoholic fatty liver disease. The drugs upregulate the expression level of Sialin in macrophages, regulate macrophage homeostasis, and inhibit liver inflammatory cell infiltration and fatty degeneration.

[0048] In actual use, this drug may also contain vitamin C preparations that can be used in combination with nitrates. Vitamin C preparations increase the effective nitrate concentration in mice and thus achieve the purpose of increasing the expression level of Sialin, which can effectively enhance the prevention and treatment of non-alcoholic fatty liver disease.

[0049] Example 2

[0050] The preventive and therapeutic effects of sodium nitrate on NAFLD in MCD model mice:

[0051] The MCD diet drives liver damage by impairing hepatic lipid output. MCD mice can be used as an animal model for NAFLD. Sodium nitrate is administered via drinking water starting one week before modeling. Samples are collected four weeks after modeling to observe the preventive and therapeutic effects of sodium nitrate on non-alcoholic fatty liver disease.

[0052] 1.1 Experimental Animals

[0053] C57BL / 6 mice: Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[0054] 1.2 Grouping and Dosing

[0055] Starting at eight weeks old, mice were administered medication in drinking water. One week later, MCD models were established in the relevant groups, and samples were collected four weeks after modeling. The specific groups and dosages for the nitrate-based MCD nonalcoholic fatty liver disease study are shown in Table 1.

[0056] Table 1

[0057]

[0058] 1.3 Experimental methods

[0059] Drugs were administered to each group of mice starting at eight weeks of age through drinking water, with model establishment established one week later. C57BL / 6 mice and C57BL / 6 mice modeled with MCD served as controls. Four weeks after model establishment, plasma samples were collected for alanine aminotransferase (ALT) levels and nitrate concentrations, and liver tissue was collected for nitrate concentrations and stained for histological observation to analyze the extent of inflammatory cell infiltration and steatosis.

[0060] ① Mouse serum collection method

[0061] After mice were deeply anesthetized, their eyeballs were removed and blood was collected into a blood collection tube pre-filled with anticoagulant. The blood volume was recorded and the tube was centrifuged at 3500 rpm for 5 minutes to obtain the supernatant.

[0062] ② Mouse liver tissue collection method

[0063] After mice were deeply anesthetized, the abdomen was opened along the midline. Normal saline was perfused into the liver via cardiac cannulation via the aorta and hepatic artery or the portal vein. The liver was isolated and placed in normal saline. The left lobe of the liver was excised and partially fixed in 4% tissue fixative. The remaining fresh tissue sample was stored at -80°C until use.

[0064] ③ Determination of alanine amino transferase (ALT) content

[0065] ALT content was determined using the C009-2-1 kit from Nanjing Jiancheng Bioengineering Institute.

[0066] 1) Due to the upper limit of detection of the alanine aminotransferase test kit, the plasma needs to be diluted with normal saline to reach an appropriate concentration before ALT testing;

[0067] 2) Perform sample addition according to Table 2 (after each sample is drawn into the assay well, insert the pipette tip into the matrix solution), gently pipette to mix, seal, and place in a 37°C incubator for 30 minutes;

[0068] Table 2 Alanine aminotransferase sample addition operation table 1

[0069]

[0070] 3) Add samples according to Table 3 (for each sample in the control well, insert the pipette tip into the matrix solution) and gently pipette to mix.

[0071] Table 3 Alanine aminotransferase sample addition operation table 2

[0072]

[0073] 4) After completing the above steps, perform the standard curve addition procedure (Table 4). After completion, gently pipette to mix thoroughly, seal the wells, including the assay and control wells, and place in a 37°C incubator for 20 minutes.

[0074] Table 4 Alanine aminotransferase standard curve operation table

[0075]

[0076] 5) After 20 minutes, remove the plate and add 200 μl of 0.4 M sodium hydroxide to each well. Gently shake the 96-well plate horizontally to mix. Incubate at room temperature for 15 minutes and measure the OD value of each well using a microplate reader at a wavelength of 510 nm.

[0077] 6) Calculation method: With the corresponding Karman's unit as the ordinate and the absolute OD value as the abscissa, a coordinate graph fitting formula is drawn and the alanine aminotransferase level in the sample is directly calculated using the formula (absolute OD value = OD value of the assay well - OD value of the control well).

[0078] Table 5 Alanine aminotransferase reference standard curve

[0079]

[0080] ④ Determination of nitrate content

[0081] Nitrate content was determined using the total nitric oxide and nitrate / nitrite assay kit (PKGE001, R&D Systems, USA):

[0082] Preparation:

[0083] 1) Place the sample at 4°C for 2 hours, centrifuge at 14,000 rpm for 10 minutes, and aspirate the supernatant.

[0084] 2) The obtained supernatant was filtered and diluted 10-fold to obtain reaction solution 1.

[0085] 3) Preparation of reaction solution 2: Dilute reaction solution 1 10-fold with distilled water / deionized water to obtain reaction solution 2.

[0086] 4) Preparation of nitrate reductase: Reconstitute nitrate reductase with 1.0 mL of nitrate reductase stock solution, vortex vigorously, let stand at room temperature for 15 minutes, vortex again, let stand at room temperature for another 15 minutes, vortex again, and use immediately.

[0087] Use reaction solution 2 to dilute nitrate reductase to prepare nitrate reductase with a concentration of 1 / 5 of the mother solution. The steps are as follows:

[0088] a. Nitrate reductase (x wells + 2) × 5 μL;

[0089] b. Take the reaction solution 2, the volume is 4 times the volume of the solution used in step a;

[0090] c. Add the solutions from steps a and b to a clean test tube and vortex;

[0091] d. Place on ice and use within 15 minutes.

[0092] 5) NADH Reagent - Reconstitute NADH with 5.0 mL of deionized or distilled water. Let stand for 3 minutes with gentle stirring before use. Use within 15 minutes or place on ice.

[0093] 6) Preparation of nitrate standards:

[0094] Use a pipette to transfer 900 μL of reaction solution 2 into a 200 μmol / L tube. Then, prepare nitrate standards at concentrations of 100 μmol / L, 50 μmol / L, 25 μmol / L, 12.5 μmol / L, 6.25 μmol / L, and 3.12 μmol / L, respectively. Reaction solution 2 serves as a blank (0 μmol / L).

[0095] Nitrate content detection steps (follow the kit instructions):

[0096] 1) Prepare all reagents, standards, and samples according to the above preparatory steps;

[0097] 2) Add 100 μL of reaction solution 2 to the wells of the blank group;

[0098] 3) Add 100 μL of nitrate standards or samples to the remaining wells;

[0099] 4) Add 50 μL NADH to all wells;

[0100] 5) Add 50 μL of diluted nitrate reductase to all wells, mix well, and cover with tape;

[0101] 6) Incubate at 37°C for 30 min;

[0102] 7) Add 100 μL of Griess I reaction solution to all wells;

[0103] 8) Add 100 μL of Griess II reaction solution to all wells and gently tap the side of the plate to mix well.

[0104] 9) Incubate at room temperature for 10 minutes;

[0105] 10) Optical density (OD) was measured at a wavelength of 540 nm, with a wavelength correction of 690 nm;

[0106] 11) Generate a standard curve based on the measured values of the standard and calculate the nitrate content in each group of samples.

[0107] ⑤H&E staining and NAS scoring

[0108] Paraffin embedding:

[0109] 1) After obtaining liver tissue from a mouse that had undergone cardiac perfusion, the left lobe of the liver was removed and fixed in 4% formaldehyde solution for at least 24 hours.

[0110] 2) Melt paraffin wax I and II in a 70°C incubator and prepare labeled embedding cassettes;

[0111] 3) Remove the liver tissue from the 4% formaldehyde solution and cut two pieces of tissue of appropriate size, maximizing the cross-section while maintaining neat cut edges. Place the pieces in a labeled embedding cassette.

[0112] 4) Dehydrate the liver tissue using different concentrations of ethanol and xylene: Place the embedding box in 60% ethanol (≥1 hour, overnight is acceptable), 75% ethanol (1 hour), 85% ethanol (30 minutes), 95% ethanol I (15 minutes), 95% ethanol II (15 minutes), anhydrous ethanol I (10 minutes), anhydrous ethanol II (10 minutes), xylene I (15 minutes), and xylene II (15 minutes). The liquid should submerge the liver tissue and be recyclable.

[0113] 5) Use tweezers to transfer the embedding cassette to Paraffin I and incubate at 70°C for 20 minutes. Then transfer it to Paraffin II and incubate at 70°C for 20 minutes. At the same time, turn on the embedding machine and preheat and cool it.

[0114] 6) Embedding in an embedding machine;

[0115] 7) After the paraffin block has completely cooled, use a paraffin slicer to cut 4 μm paraffin sections.

[0116] Hematoxylin-eosin staining (H&E staining):

[0117] 1) Dewaxing: Place the sections in a 70°C incubator for 10 min and place them in xylene I and xylene II for 2 min each.

[0118] 2) Graded ethanol dehydration: Dehydrate the sections in absolute ethanol, 95% ethanol, 85% ethanol, and 75% ethanol for 2 minutes each. Rinse the sections with running water until they are clean.

[0119] 3) Hematoxylin staining: Use a paper scraper to scrape off the oxide film on the surface of the hematoxylin, insert the slices, and let them stand for 7 minutes. Remove the slices, rinse with running water for 2 minutes, place them in 1% hydrochloric acid alcohol for about 5 seconds (rinsing three times is enough), rinse with running water for 6 minutes, and observe the staining of the cell nuclei under a microscope.

[0120] 4) Eosin staining: Place the sections in eosin staining solution and let stand for 1 minute, then remove and rinse with running water;

[0121] 5) Place the slices in 95% ethanol I, 95% ethanol II, anhydrous ethanol I, and anhydrous ethanol II, respectively, for 3 minutes each; quickly pass through xylene I and xylene II, and then place the slices in xylene III;

[0122] 6) Remove the slices, wipe off the residual xylene on the slices with clean gauze, seal the slices with neutral resin, and dry them at room temperature (be careful to avoid the formation of small bubbles when sealing the slices).

[0123] Non-alcoholic fatty liver disease activity scores (NAS)

[0124] The activity of non-alcoholic fatty liver disease (NAFLD) in the mouse liver was semi-quantitatively assessed using three histological features: hepatic steatosis, lobular inflammation, and ballooning. The NAFLD scoring criteria are shown in Table 6:

[0125] Table 6 NAFLD Activity Score (NAS)

[0126]

[0127] ⑥ Oil red O staining and semi-quantitative analysis

[0128] 1) Storage solution: Dissolve 150 mg of Oil Red O in 50 ml of isopropanol and incubate at 37°C for 30 min. Store at 4°C.

[0129] 2) Working solution: Dilute the stock solution with double-distilled water in a ratio of 4:6. Filter the solution until it becomes clear orange before use.

[0130] 3) Calcium formaldehyde fixative: 40% formaldehyde 10 ml, CaCl2 2.0 g, ultrapure water 90 ml;

[0131] 4) After cell induction, remove the culture medium and wash twice with PBS;

[0132] 5) Formaldehyde-calcium fixation at room temperature for 15 minutes;

[0133] 6) Wash twice with PBS;

[0134] 7) Mordanting with 60% isopropyl alcohol for 1 minute;

[0135] 8) Remove isopropanol and stain with the working solution at room temperature for 30 minutes;

[0136] 9) Rinse once with 60% isopropyl alcohol;

[0137] 10) Wash twice with PBS;

[0138] 11) Under the microscope, lipid droplets are stained red;

[0139] 12) Image J pro was used to analyze the red area ratio.

[0140] Example 3

[0141] The verification results of Example 2 are as follows:

[0142] ① The alanine aminotransferase level in the MCD mice + NaNO3 group was significantly lower than that in the MCD mice + double-distilled water group. The alanine aminotransferase levels of mice in each experimental group are shown in Table 7.

[0143] Table 7 Plasma ΔLT levels (IU / L) of mice in each group

[0144]

[0145] Note: a: Compared with the mice + double distilled water group, p < 0.01, there is a very significant difference; b: Compared with the MCD mice + double distilled water group, p < 0.01, there is a very significant difference.

[0146] ②The plasma nitrate ion level in the NaNO3 group was significantly higher than that in the double-distilled water group. The plasma nitrate ion concentrations of mice in each group are shown in Table 8.

[0147] Table 8 Plasma NO3 concentration (uM) of mice in each group

[0148]

[0149] Note: a: Compared with the mice + double distilled water group, p < 0.05, there is a significant difference; b: Compared with the MCD mice + double distilled water group, p < 0.01, there is a very significant difference.

[0150] ③The nitrate ion level in the liver tissue of the NaNO3 group was significantly higher than that in the double-distilled water group. The nitrate ion concentrations in the liver tissue of mice in each group are shown in Table 9.

[0151] Table 9 NO3 concentration in liver tissue of mice in each group (μM)

[0152]

[0153] Note: a: Compared with the mice + double distilled water group, p < 0.01, there is a very significant difference; b: Compared with the MCD mice + double distilled water group, p < 0.01, there is a very significant difference.

[0154] ④ The NAS score in the MCD mice + NaNO3 group was significantly lower than that in the MCD mice + double-distilled water group. The NAS scores in the liver tissues of mice in each group are shown in Table 10.

[0155] Table 10 NAS scores of mice in each group

[0156]

[0157] Note: a: Compared with the mice + double distilled water group, p < 0.01, there is a very significant difference; b: Compared with the MCD mice + double distilled water group, p < 0.01, there is a very significant difference.

[0158] ⑤ The degree of fat accumulation in the MCD mice + NaNO3 group was significantly lower than that in the MCD mice + double-distilled water group. Semi-quantitative analysis of relative fat accumulation in liver tissue of mice in each group is shown in Table 11.

[0159] Table 11 Quantitative analysis of fatty degeneration in the liver of mice in each group

[0160]

[0161] Note: a: Compared with the mice + double distilled water group, p < 0.01, there is a very significant difference; b: Compared with the MCD mice + double distilled water group, p < 0.01, there is a very significant difference.

[0162] Example 4

[0163] The preventive and therapeutic effects of sodium nitrate on NAFLD in CDHFD model mice

[0164] Mice fed a CDHFD showed significant changes in transaminases and displayed typical NAFLD histopathological features. Therefore, CDHFD-fed mice can be used as an animal model for NAFLD. The experiment involved administering sodium nitrate via drinking water starting one week before modeling, and samples were collected 16 weeks after modeling to observe the preventive and therapeutic effects of sodium nitrate on non-alcoholic fatty liver disease.

[0165] 1.1 Experimental Animals

[0166] C57BL / 6 mice: Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[0167] 1.2 Grouping and Dosing

[0168] Starting at 8 weeks old, mice were administered medication through drinking water. One week later, the relevant groups were placed on a CDHFD model, and samples were collected 16 weeks after modeling. Specific groupings and dosages are shown in Table 12.

[0169] Table 12 Study groups and dosing of nitrates of the present invention for preventing and treating CDHFD non-alcoholic fatty liver disease

[0170]

[0171] 1.3 Experimental methods

[0172] Each group of mice was given drinking water starting at 8 weeks of age, and modeling was established 1 week later. C57BL / 6 mice and C57BL / 6 mice modeled on CDHFD served as controls. After 16 weeks of modeling, plasma samples were collected to measure alanine aminotransferase levels and nitrate concentrations. Liver tissue was collected to measure nitrate concentrations and stain for histological observation, and the degree of inflammatory cell infiltration and steatosis was analyzed (see Example 3 for experimental procedures).

[0173] Example 5

[0174] The verification results of Example 4 are as follows:

[0175] ① The alanine aminotransferase level in the CDHFD + NaNO3 group was significantly lower than that in the CDHFD + double-distilled water group. The alanine aminotransferase levels of mice in each experimental group are shown in Table 13.

[0176] Table 13 Plasma ALT levels (IU / L) of mice in each group

[0177]

[0178] Note: a: Compared with the mice + double distilled water group, p < 0.01, there is a very significant difference; b: Compared with the CHHFD mice + double distilled water group, p < 0.05, there is a significant difference.

[0179] ②The plasma nitrate ion level in the NaNO3 group was significantly higher than that in the double-distilled water group. The plasma nitrate ion concentrations of mice in each group are shown in Table 14.

[0180] Table 14 Plasma NO3 concentration (μM) of mice in each group

[0181]

[0182] Note: a: Compared with the mice + double distilled water group, p < 0.05, there is a significant difference; b: Compared with the CDHFD mice + double distilled water group, p < 0.01, there is a very significant difference.

[0183] ③The nitrate ion level in the liver tissue of the NaNO3 group was significantly higher than that in the double-distilled water group. The nitrate ion concentrations in the liver tissue of mice in each group are shown in Table 15.

[0184] Table 15 NO3 concentration in liver tissue of mice in each group (μM)

[0185]

[0186] Note: a: Compared with the double-distilled water group, p<0.05, there is a significant difference; b: Compared with the CDHFD mice I double-distilled water, p<0.01, there is a very significant difference.

[0187] ④ The NAS score of the CDHFD mice + NaNO3 group was significantly lower than that of the CDHFD + double-distilled water group. The NAS scores in the liver tissues of mice in each group are shown in Table 16.

[0188] Table 16 NAS scores of mice in each group

[0189]

[0190] Note: a: Compared with the mice + double distilled water group, p < 0.01, there is a very significant difference; b: Compared with the CDHFD mice + double distilled water, p < 0.01, there is a very significant difference.

[0191] ⑤ The degree of fat accumulation in the CDHFD mice + NaNO3 group was significantly lower than that in the CDHFD + double-distilled water group. Semi-quantitative analysis of relative fat accumulation in liver tissue of mice in each group is shown in Table 17.

[0192] Table 17 Quantitative analysis of fatty degeneration in the liver of mice in each group

[0193]

[0194] Note: a: Compared with the mice + double distilled water group, p < 0.01, there is a very significant difference; b: Compared with the CDHFD mice + double distilled water, p < 0.01, there is a very significant difference.

[0195] Example 6

[0196] Sodium nitrate upregulates sialin in the regulation of bone marrow-derived macrophages

[0197] 1.1 Cell selection

[0198] Isolate and culture primary bone marrow macrophages.

[0199] 1.2 Grouping and Dosing

[0200] After 4 days of in vitro culture, nitrate was added for another 2 days, and then differentiation factors were added to promote differentiation. Specific grouping and dosage are shown in Table 18.

[0201] Table 18 Nitrate regulation of bone marrow-derived macrophages in the present invention: Study grouping and administration

[0202]

[0203] 1.3 Experimental methods

[0204] Collect cells for flow cytometry analysis and RT-qPCR analysis (see Example 3 for experimental steps)

[0205] Example 7

[0206] The verification results of Example 6 are as follows:

[0207] ①The proportion of pro-inflammatory cells in the M1+NaNO3 group was significantly lower than that in the M1 group, and the proportion of anti-inflammatory cells in the M2+NaNO3 group was significantly lower than that in each experimental group in the M2 group. The effect of the sialin knockout group disappeared, as shown in Tables 19 and 20.

[0208] Table 19 Proportion of proinflammatory cells in each group (%)

[0209]

[0210] Note: a is significantly different from M1, P < 0.05.

[0211] Table 20 Proportion of anti-inflammatory cells in each group (%)

[0212]

[0213] Note: a Compared with M2, P < 0.05, there is a significant difference.

[0214] ②The expression level of TNFα in the M1+NaNO3 group was significantly lower than that in the M1 group, and the effect disappeared in the sialin knockout group, as shown in Tables 21 and 22.

[0215] Table 21 Relative expression levels of TNFα in each group

[0216]

[0217] Note: a is significantly different from M1, P < 0.05.

[0218] Table 22 Relative expression levels of IL10 in each group

[0219]

[0220] Note: a Compared with M2, P < 0.05, there is a significant difference.

[0221] Example 8

[0222] In order to verify the specific role of nitrate in nitrate, the applicant designed a comparative experiment, that is, adding NaCl at the same time, the purpose of which is to eliminate the influence of Na ions; the specific experimental process and results are as follows:

[0223] First, to verify the effects of non-Na ions on non-alcoholic fatty liver disease (NAFLD), the applicant conducted an animal modeling.

[0224] Similar to Example 1, the applicant established MCD and CDHFD models.

[0225] Nitrate was administered to the above-mentioned model mice.

[0226] The concentration of nitrate administered was 4 mM; the dosage was 1.4 mmol / kg body weight per day; and the administration method was selected to be delivered through drinking water.

[0227] The concentration of NaCl administered was 4 mM, and the administered dose was 1.4 mmol / kg body weight.

[0228] Pharmacodynamic studies have demonstrated that sodium nitrate (NaNO3) significantly reduces elevated alanine aminotransferase (AST) levels in mice modeled as MCD / CDHFD. Histological staining also revealed that NaNO3 significantly alleviates hepatic inflammatory cell infiltration and steatosis in these mice, suggesting that NaNO3 can prevent and treat nonalcoholic fatty liver disease. However, NaCl did not exhibit these same protective effects, suggesting that the effects of sodium nitrate are not due to the production of Na ions.

[0229] Table 23 shows the results of plasma ALT level detection in mice of each group.

[0230] Table 23

[0231] Plasma ALI levels of mice in each group (IJ / L)

[0232]

[0233] Note: a: Compared with the mice + double distilled water group, p < 0.01, there is a very significant difference; b: Compared with the MCD mice + double distilled water group, p < 0.01, there is a very significant difference.

[0234] Table 24 shows the NAS score results of mice in each group.

[0235] Table 24

[0236] NAS scores of each mouse

[0237]

[0238] Note: a: Compared with the mice + double distilled water group, p < 0.01, there is a very significant difference; b: Compared with the MCD mice + double distilled water group, p < 0.01, there is a very significant difference.

[0239] Table 25 shows the quantitative analysis results of liver steatosis in each group of mice.

[0240] Table 25

[0241] Quantitative analysis of liver steatosis in mice in each group

[0242]

[0243] Note: a: Compared with the mouse-double distilled water group, p<0.01, there is a very significant difference; b: Compared with the MCD mouse+double distilled water group, p<0.01, there is a very significant difference.

[0244] Example 9

[0245] Vitamin C used in combination with nitrates, vitamin C preparations can effectively enhance the prevention and treatment of non-alcoholic fatty liver disease by increasing the effective nitrate concentration in mice and thus increasing the expression level of Sialin.

[0246] Similar to Example 1, the applicant established MCD and CDHFD models.

[0247] The above model mice were administered nitrate (specifically sodium nitrate) combined with vitamin C.

[0248] In the combined drug, the ratio of the amount of sodium nitrate to vitamin C is 5:1, and the specific dosage is 1.7g of sodium nitrate and 0.176g of vitamin C;

[0249] The preparation method of the combination drug is specifically as follows: weigh the corresponding amount of sodium nitrate and vitamin C according to the above ratio, dissolve them, filter and sterilize them with a 0.22 μm microporous filter membrane, mix them, and divide the mixed solution into sterile vials, seal them, and freeze them at -20 to -60°C for 2 to 18 hours (the actual operation is freezing at -40°C for 10 hours), and freeze-dry them in a freeze dryer: set the temperature at -20 to 20°C for a primary sublimation of 4 to 20 hours (the actual operation is a primary sublimation of 12 hours at 0°C), and a secondary sublimation of 1 to 30 hours at 20 to 40°C (the actual operation is a secondary sublimation of 15 hours at 30°C), and take them out to obtain the combination drug composition.

[0250] The administration concentration of the combined drug was 4 mM (nitrate ion concentration), and the administration dose was 1.4 mmol / kg body weight.

[0251] Pharmacodynamic experiments have demonstrated that sodium nitrate (NaNO3) significantly reduces elevated alanine aminotransferase levels in mice modeled as MCD / CDHFD, with combined use further reducing these levels. Histological staining has shown that NaNO3 significantly alleviates hepatic inflammatory cell infiltration and fatty degeneration in MCD / CDHFD mice, with combined use achieving even greater relief. This suggests that NaNO3 can prevent and treat nonalcoholic fatty liver disease, with combined use providing a more effective treatment.

[0252] Table 26 shows the results of plasma ALT level detection in each group of mice.

[0253] Table 26

[0254] Plasma ALT levels of mice in each group (IU / L)

[0255]

[0256] Note: a: Compared with the mice + double distilled water group, p < 0.01, there is a very significant difference; b: Compared with the MCD mice + double distilled water group, p < 0.01, there is a very significant difference.

[0257] Table 27 shows the NAS score results of mice in each group.

[0258] Table 27

[0259] NAS scores of mice in each group

[0260]

[0261] Note: a: Compared with the mice + double distilled water group, p < 0.01, there is a very significant difference; b: Compared with the MCD mice + double distilled water group, p < 0.01, there is a very significant difference.

[0262] Table 28 shows the quantitative analysis results of liver steatosis in each group of mice.

[0263] Table 28

[0264] Quantitative analysis of liver steatosis in mice in each group

[0265]

[0266] Note: a: Compared with the mice + double distilled water group, p < 0.01, there is a very significant difference; b: Compared with the MCD mice + double distilled water group, p < 0.01, there is a very significant difference.

[0267] Table 29 shows the results of the NO3 concentration test in the liver plasma of each group of mice.

[0268] Table 29

[0269] NO3 concentration in liver and plasma of mice in each group (μM)

[0270]

[0271] Note: a: Compared with the mice + double distilled water group, p < 0.01, there is a very significant difference; b: Compared with the MCD mice + double distilled water group, p < 0.01, there is a very significant difference.

[0272] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent replacements or improvements made within the spirit and principles of the present application should be included in the scope of protection of the present application.

Claims

1. A composition for preventing and treating non-alcoholic fatty liver disease, characterized in that: The invention comprises pharmaceutically acceptable nitrates, which regulate macrophage homeostasis by upregulating the expression level of Sialin in macrophages, thereby inhibiting inflammatory response and fatty degeneration of the liver.

2. The composition according to claim 1, characterized in that The composition is a dietary supplement or a pharmaceutical composition, and the administration form is selected from oral administration, injection or local administration.

3. The composition according to claim 1, characterized in that The nitrate is selected from at least one of sodium nitrate, potassium nitrate, calcium nitrate or magnesium nitrate, preferably sodium nitrate.

4. The composition according to claim 1, characterized in that The administration concentration of the nitrate is 0.1-10 mM, preferably 0.5-5 mM, more preferably 4 mM; the administration dose is 0.035-3.5 mmol / kg body weight per day, preferably 0.175-1.75 mmol / kg body weight, more preferably 1.4 mmol / kg body weight; the administration method is selected as delivery through drinking water or feed.

5. The composition according to claim 1, characterized in that The macrophages are selected to be pro-inflammatory macrophages and / or anti-inflammatory macrophages.

6. A method for regulating macrophage homeostasis in vitro, characterized in that: The following steps are involved: S1. Isolate and culture primary bone marrow-derived macrophages and culture them in vitro in culture medium; S2. Add nitrate to the culture medium at a concentration of 0.5-10 mM; S3. The expression level of Sialin and the changes in the ratio of pro-inflammatory / anti-inflammatory macrophages were detected. Nitrate inhibits the differentiation of pro-inflammatory macrophages by upregulating Sialin.

7. The method according to claim 6, characterized in that The following steps are also included: S4. Validate the specific regulatory effect of nitrate on macrophage homeostasis by knocking out or overexpressing the Sialin gene.

8. Use of nitrate in the preparation of a medicament for preventing and treating non-alcoholic fatty liver disease, characterized in that: The drug regulates macrophage homeostasis by upregulating the expression level of Sialin in macrophages, thereby inhibiting hepatic inflammatory cell infiltration and fatty degeneration.

9. The use according to claim 8, characterized in that The drug also contains a vitamin C preparation that can be used in combination with nitrates. The vitamin C preparation increases the effective nitrate concentration in mice and thereby increases the expression level of Sialin, thereby effectively enhancing the prevention and treatment of non-alcoholic fatty liver disease.

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