WO2019091397A1 - Use of azido phlorizin in preparation of medicament for treating non-alcoholic fatty liver - Google Patents

Abstract

The present invention discloses a use of azido phlorizin (4-Az) as a key branching enzyme GGPPS inhibitor in a mevalonate pathway, especially in the preparation of a medicament for treating non-alcoholic fatty liver. Through a series of experiments, the present invention has proved that 4-Az can cause ubiquitination-proteasome pathway-dependent degradation of GGPPS protein, has an inhibitory effect on GGPPS protein expression, and can be used as an inhibitor of GGPPS protein expression. More specifically, the azido phlorizin of the present invention can reduce lipid accumulation and fatty degeneration in the liver, and can reduce levels of alanine aminotransferase and aspartate aminotransferase in the blood, can actively protect liver function, and can be used for the preparation of a medicament for treating non-alcoholic fatty liver.

Description

Use of azido phloridin in the preparation of a medicament for treating nonalcoholic fatty liver Technical field

The invention belongs to the field of medicine, and particularly relates to the use of azidoin for the preparation of a medicament for treating non-alcoholic fatty liver. Azidopidin can reduce lipid accumulation in the liver and has a significant effect on the treatment of nonalcoholic fatty liver.

Background technique

Nonalcoholic fatty liver disease refers to excessive deposition of fat in liver cells caused by factors other than alcohol. The development process generally includes simple fatty degeneration of the liver, steatohepatitis, liver fibrosis, etc., and severe cirrhosis. , liver failure and even liver cancer. The prevalence of the disease continues to increase globally, in parallel with the increase in obesity, and is increasingly becoming a serious public health problem.

Nonalcoholic fatty liver disease is the result of multiple factors such as genetic-environment-metabolism-stress, and is closely related to metabolic syndrome such as insulin resistance, hyperglycemia and hyperlipidemia. At present, many international guidelines on early nonalcoholic fatty liver disease treatment strategies focus on lifestyle changes, as well as prevention of accompanying metabolic and cardiovascular complications. However, in the later stages of development, drug treatment for the liver must be used to prevent the progression of liver disease and reduce or prevent liver cirrhosis and its complications.

The mevalonate pathway (MVA pathway) is an important pathway in the cellular metabolic pathway. In the mevalonate pathway, acetyl-CoA is first produced by 3-hydroxy-3-methylglutaryl coenzyme A synthetase to form 3-hydroxy-3-methylglutaryl coenzyme A, followed by 3- Mevalonate is produced by the action of hydroxy-3-methylglutaryl coenzyme A reductase. The product of the pathway can be regarded as an activated isoprene unit, which is a synthetic precursor of biomolecules such as steroids and terpenoids. Among them, metabolic intermediates such as farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) are also important substrates for the synthesis of cholesterol, terpenoids and terpenoids. , occupy an important position in a variety of physiological processes. The key branching enzyme in the mevalonate pathway, Geranylgeranyl Diphosphate Synthase (GGPPS), catalyzes the formation of geranylgeranyl pyrophosphate by farnesyl pyrophosphate, which mediates the protein isoprenic acid. The balance of olefination affects the development of non-alcoholic fatty liver and type 2 diabetes by affecting the regulation of protein prenylation in the liver. Studies have shown that GGPPS is involved in the formation of insulin resistance under high insulin status by regulating the relative activation of MAPK/PI3K/AKT signaling pathway; GGPPS-regulated protein prenylation modification promotes fatty acid-induced muscle insulin resistance; GGPPS The proportion of FPP/GGPP regulated in vivo is involved in the regulation of lipid metabolism in the liver. In non-alcoholic fatty liver disease, GGPPS can participate in the regulation of obesity-induced liver fat degeneration by regulating the proportion of FPP/GGPP in vivo. Therefore, knocking out GGPPS gene can effectively alleviate the formation of nonalcoholic fatty liver, so it is proposed to use GGPPS. Screening for effective treatment of nonalcoholic fatty liver for the target.

Liver lipid accumulation was effectively inhibited in mice with non-alcoholic fatty liver induced by high-fat treatment with GGPPS competitive binding inhibitor DGBP (Digeranyl Bisphosphonate). Due to the large toxic side effects of DGBP, its disease control for nonalcoholic fatty liver disease is limited.

Azidopidin is an analog of phenolic compounds found in apples, apple bark and leaves. Phlorizin is extracted from apple, apple bark and leaves, and is a phenolic substance in apple trees. 4-Azidophlorizin (4-Az) is a derivative of phloridin with the following chemical structure:

Azidoin has been reported as a kidney-expressed sodium-dependent glucose transporter 2 (SGLT-2) inhibitor, which inhibits renal reabsorption of glucose, but no studies have shown Whether 4-Az has the effect of promoting degradation of the GGPPS protein. Azidotin can directly target GGPPS, trigger GGPPS degradation through the ubiquitination-proteasome pathway, thereby blocking the transmission of multiple signaling pathways involved in the guanosine triphosphate family (GTPase), and inhibiting the liver. The role of lipid accumulation, but the use of azidopidin in the treatment of nonalcoholic fatty liver disease has not been reported so far.

Summary of the invention

In view of the above, an object of the present invention is to provide a use of azidopidin in the field of medicine, and in particular to its use in the preparation of a medicament for treating nonalcoholic fatty liver.

To achieve the above objectives, the inventors established a non-alcoholic fatty liver mouse model by high-fat induction, and observed the effects of azidoin on the body weight, liver tissue lipid metabolism and accumulation, and liver in nonalcoholic fatty liver mice. The impact of the function.

The medicament of the present invention may comprise dimethyl sulfoxide, azidoin and a pharmaceutically acceptable carrier, which may be administered intraperitoneally, and the dose may be 1-10 mg. /kg. The administration mode of the drug may also be subcutaneous, intradermal, intraarterial, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional, intracranial injection or infusion, oral, topical, rectal , nasal, buccal, vaginal, sublingual, intradermal, mucosal, tracheal, urethra administration, by inhalation aerosol, implant accumulation and acupuncture.

The examples of the present invention examined changes in body weight and liver weight and liver lipid accumulation in mice injected with non-alcoholic fatty liver after azigal glucosinolate injection. The results show that the present invention has the following beneficial effects:

(1) The azidoin glucoside of the present invention can effectively reduce lipid accumulation and fatty degeneration in the liver, and can lower the levels of alanine aminotransferase and aspartate aminotransferase in blood, and has activity for protecting liver function, and thus can be used for The preparation of drugs for treating nonalcoholic fatty liver has potential and good application prospects in the field of nonalcoholic fatty liver treatment.

(2) The present invention not only provides a high level of targeted drugs for the development of new drugs for nonalcoholic fatty liver, but also provides a new idea for individualized diagnosis and precise medical treatment of metabolic diseases caused by protein isoprene imbalance. .

To further confirm the effect and beneficial effects of azidoin in the treatment of nonalcoholic fatty liver disease, the inventors compared the effects of azidoin and phloridin on lipid metabolism in hepatocytes at the cellular level. effect. The results showed that the effect of azidoin on hepatic lipid metabolism was far more significant than phloridin.

BRIEF DESCRIPTION OF THE DRAWINGS:

Figure 1 shows the expression levels of GGPPS under different conditions. Among them, both A and B maps show that the expression of GGPPS is decreased in TSC2-null cells under 4-Az treatment; C is 4-Az causing reversal of RhoA protein prenylation; Figure D is 4-Az does not cause GGPPS Changes in transcript levels; E map shows that the proteasome inhibitor MG132 is able to reverse the decrease in GGPPS expression caused by 4-Az; F is a graph showing that the autophagosome-lysosomal inhibitor CQ does not reverse the decrease in GGPPS expression caused by 4-Az.

Figure 2 shows the inhibition of GGPPS by 4-Az. Among them, Panel A shows that 4-Az promotes GGPPS ubiquitination accumulation, thereby causing degradation. Panels B to E are micro thermophoresis screening systems showing binding of 4-Az to GGPPS recombinant protein.

Figure 3 is a graph showing changes in body weight and major lipid metabolism tissue weight in nonalcoholic fatty liver mice after continuous administration of different concentrations of azidoin for 14 days. Among them, the graph A shows the change in body weight of the mouse within 14 days (administered and monitored every other day); the graph B shows the ratio of liver weight to body weight of the mouse after the last administration on the 14th day; The ratio of epididymal fat weight to body weight in mice after the last dose of day.

Fig. 4 is a graph showing changes in lipid content in the liver of mice with nonalcoholic fatty liver after 14 days of continuous administration of different concentrations of azidoin. Among them, the picture A is the CT scan of the liver of the mouse after the last administration on the 14th day; the picture B is the liver tissue density of the mouse obtained by the 40keV tube voltage scan after the last administration on the 14th day; Mouse liver lipid content after the last dose of 14 days.

Fig. 5 is a photomicrograph of liver paraffin sections and HE staining of nonalcoholic fatty liver mice after continuous administration of different concentrations of azidoin for 14 days. Among them, the A picture is the light micrograph of the liver of the mouse after the last administration on the 14th day, and the B picture is the liver triglyceride content of the mouse after the last administration on the 14th day.

Fig. 6 is a graph showing changes in the contents of alanine aminotransferase and aspartate aminotransferase in the blood of mice with nonalcoholic fatty liver after 14 days of continuous administration of different concentrations of azidoin. Among them, Figure A shows the content of alanine aminotransferase in the blood of mice after the last administration on the 14th day; B is the content of aspartate aminotransferase in the blood of mice after the last administration on the 14th day.

"ns" in Figures 3 to 6 indicate no significant difference; "*" indicates a significant difference between the vehicle group and the 4-Az 1 mg/kg group, p < 0.05; "**" indicates the vehicle group and 4- There was a significant difference between the Az 1 mg/kg group, p<0.001; “#” indicates a significant difference between the 4-Az 1 mg/kg group and the 4-Az 10 mg/kg group, p<0.05; “##” indicates 4 There was a significant difference between the -Az 1 mg/kg group and the 4-Az 10 mg/kg group, p < 0.001.

Detailed ways

The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The following examples are merely illustrative of the invention and are not intended to limit the scope of the invention. The experimental method in which no specific conditions are specified in the examples is usually carried out according to the conditions described in the conventional conditions, for example, "Molecular Cloning: A Laboratory Manual", or according to the conditions recommended by the manufacturer.

Inhibition of GGPPS protein expression by azidopidin

General materials and methods

MATERIALS: The recombinant plasmid pET28a and its expression vector were constructed by the company. The target gene was inserted between the NcoI and BamI restriction sites, and cloned into the GGPPS-EGFP-His gene to facilitate the green fluorescence excitation of the exogenous protein GGPPS. E. coli BL21 was purchased and stored by the company. TSC2-null cells are a gift from the American laboratory. A plasmid extraction kit, a gel recovery kit, and a PCR product purification kit were purchased from Axygen Corporation. CaCl ₂ , LB medium, glycerol, inducer IPTG, nickel (Ni) column equilibration buffer, washing buffer and elution buffer reagents Tris, NaCl, imidazole, purchased from American sigma reagent company and China Solitaire Reagent company. Common organic reagents such as methanol, ethanol, propanol, isopropanol, chloroform, paraformaldehyde, xylene, formaldehyde, ammonia, etc., were purchased from Nanjing Chemical Reagent Company. DMSO, HEPES, TEMED, TWEEN-20, NP40, hydrogen peroxide, etc. were purchased from Shanghai Shenggong Company. Type IV collagenase, trypsin was purchased from Gibco. Protease inhibitors cocktail, BSA, SDS, GGPP, FPP, STA-21, etc. were purchased from Sigma. GGPPS, Ub antibody was purchased from Santa Cruz. PVDF membranes were purchased from Roche. Ni-IDA Resin, GST-Resin, chromatography column, Ni column affinity filler, purchased from Jiangsu Zibo Biotechnology Co., Ltd. The ultracentrifuge was purchased from Beckman. The cryogenic centrifuge was purchased from Kubota. The Western Blot system was purchased from Tianneng.

Method 1. Culture of TSC2-null cells

TSC2-null was grown in DMEM containing 10% calf serum, 90% high glucose DMEM + 10% FBS medium, 0.25% trypsin, PBS, DMSO. Place in a 5% CO2, 95% air, 37 ° incubator. Pay attention to the change of PH value in the medium, change the liquid regularly, and repeat the subculture or cryopreservation when the cell density reaches 70%-80%.

Method 2, siRNA cell transfection

Transfection was carried out by culturing TSC2-null cells (60 mm culture dishes) until the cell fusion rate was 60% or more. Lightly mix 10 ul of siRNA with 500 ul of OPTI medium in a sterile tube according to Invitrogen's LipofectamineTM 2000 Transfection Instructions. 10 ul of LipofectamineTM 2000 was diluted in 500 ul of OPTI medium, allowed to stand at room temperature for 5 min, and then the above two solutions were gently mixed, so that the liposome lipo was wrapped in the plasmid and incubated at room temperature for 20 min. Plasmid transfection is then performed. After 48 hours of transfection, cells were collected for sampling.

Method 3, extraction of cellular proteins

When the cells were cultured for the required time, the culture solution was discarded, and the monolayer cells were washed twice with pre-PBS (pH 7.4), and then the cells were lysed by adding an appropriate amount of a protease-containing cell lysate, and the lysate was heated at 100 ° C for 10 minutes. The cell debris was removed by centrifugation at 13,000 rpm, and the supernatant was taken and stored at -20 ° C or -70 ° C.

Method 4, RNA extraction refers to Real time PCR

After the monolayer adherent cells are cultured for a predetermined period of time, the culture solution is discarded. The monolayer cells (35 mm dish) were washed with PBS, 1 mL of Trizol Reagent was added, and immediately pipetted to thoroughly mix. After the cells were detached, the lysate was transferred to a centrifuge tube and allowed to stand at room temperature for 5 minutes. 0.2 mL of chloroform was added, and the mixture was thoroughly mixed, allowed to stand at room temperature for 15 minutes, and centrifuged at 4 ° C for 12,000 g for 10 minutes. The supernatant was carefully aspirated, an equal volume of isopropanol was added, and the mixture was allowed to stand at room temperature for 10 minutes, and centrifuged at 4 ° C for 12,000 g for 10 minutes. The supernatant was discarded, and the precipitate was washed twice with 75% ethanol and naturally dried at room temperature. It was dissolved in RNase-free water and frozen in a -80 ° C refrigerator. Primers were designed and the target fragment was amplified by PCR using the reverse transcription product as a template. The relative amount of mRNA was calculated from the CT value. Using 18S as the internal reference quantification, the expression levels of other time points relative to the pre-induction were calculated.

Western blotting

An electrophoresis gel was prepared and subjected to SDS-PAGE. The electrophoresis time is generally 4 to 5 hours. First, 80V pre-electrophoresis is performed, and the sample is sent to the separation gel to change the voltage to 120V. Electrophoresis until the bromophenol blue has just run out can terminate the electrophoresis and transfer the film. Stacked in the order of filter paper-gel-PVDF film-filter paper, the gel is close to the negative electrode, and the film faces the positive electrode, and the bubbles between the layers are removed. Immunoblotting reactions were performed with the corresponding antibodies. The first antibody dilution and reaction conditions were as follows: α-Tubulin (1:1000), GGPPS (1:200), Rap1A (1:200), RhoA (1:200) 4 °C Incubate overnight for 12-16 hours, wash the membrane 3 times with PBS for 10 min each time, incubate HRP-labeled fluorescent secondary antibody for 2 hours at room temperature, and wash the membrane 6 times with PBS for 5 min each time. After washing, it is chemiluminescence, and exposure and development are retained.

Prokaryotic expression and purification

The BL-21 strain containing the recombinant plasmid of the target gene was cultured on a large scale, and the expressed target protein was labeled with His, the loading speed was 2 mL/min, and the penetrating sample was collected for subsequent sample preparation to determine the gel. Whether the target protein is hung on the column; after the sample is finished, use the Ni column buffer buffer buffer W to clean the non-protein bound to the Ni column, elute the buffer buffer B with Ni column, 3 mL/min, elute with the Ni column. Binding the target protein, observe the change of the absorption peak at 280 nm, collect the eluted protein at the sample outlet at the appropriate time, inject the collected eluted protein into the dialysis card, and place the dialysis card in PBS buffer containing 10% glycerol. After standing at 4 ° C for 12 h, 1 L of PBS (without glycerol) was prepared, and a dialysis card was placed therein, and placed at 4 ° C for 12 h. The prepared prokaryotic purified protein GGPPS-EGFP-His was partially protected from light at 4 °C for subsequent experiments, and partially frozen at -80 °C for long-term storage.

Micro thermophoresis experiment identification combination

Microscale Thermophoresis (MST) is a fluorescent colorimetric quantitative technique that has been applied to biological analysis in recent years based on physical principles. The change in molecular hydration shell, electrical or size can be monitored by measuring the change in motion of the molecule under a microtemperature gradient. Using this technology, the interaction between multiple biomolecules, such as liposomes and ribonic acid, can be quickly monitored from ions or fragments.

The prepared prokaryotic purified protein GGPPS-EGFP-His was used as a macromolecular ligand, and the small molecule substrate FPP of GGPPS was used as a positive detecting substance for the GGPPS binding screening model. At the same time, 4-Az was used as the compound to be detected. Prior to the test, the mixture was stirred by centrifugation (5 minutes, 13,000 rpm, 4 ° C). Tween 20 (0.01% - 0.1%) was used as a cleaning agent to remove the original polymerization. The fluorescence value of the GGPPS fusion fluorescent protein at 20% LED POWER was measured and controlled to be in the range of 200 to 1000. The concentration of the fluorescent binding protein was unchanged, and the label-free binding ligand was diluted with a dilution buffer of 1:1 into 16 concentrations of the titration solution. The fluorescent binding protein was incubated with the label-free binding ligand in a 1:1 ratio and loaded into a capillary tube for micro thermophoresis.

Example 1. Effect of 4-Az on endogenously highly expressed GGPPS protein levels.

TSC2-null cells were cultured to a density of 50%. At this time, the cells were changed, and blanks, drug solvent negative control (DMSO), GGPP transferase inhibitor (GGTI), GGPPS small interfering RNA were used for different treatment groups. siGGPPS), and concentration gradient of 4-Az drug (0.5 uM, 5 uM, 50 uM). After 48 h treatment, cells were harvested and protein levels were detected. It was found that the concentration of GGPPS was down-regulated as the concentration gradient increased. The TSC2-null protein was collected at 0 hours, 12 hours, 24 hours, and 48 hours, respectively, and the expression level of GGPPS was detected. It was found that the protein level of GGPPS showed a time-dependent decrease after drug treatment. The above results show that 4-Az can significantly reduce endogenous GGPPS protein levels.

Example 2. Effect of 4-Az on endogenously highly expressed GGPPS transcript levels.

To further verify that the effect of 4-Az on GGPPS expression levels is regulated by transcriptional, translational or post-translational levels, we received RNA from different treatment groups for RNA extraction and reverse transcription of cDNA for Real time PCR. The mRNA expression of GGPPS was detected. The results showed that the mRNA level of GGPPS in the siGGPPS treatment group was significantly decreased, while the drug treatment group 4-Az did not cause an effect on GGPPS mRNA.

Example 3, 4-Az promotes ubiquitination degradation of GGPPS

After treatment with 4-Az, the ubiquitination-proteasome inhibitor MG132 and the lysosomal-autophagosome fusion inhibitor CQ were treated respectively. When the two degradation pathways were destroyed, only the treatment of MG132 prevented the expression of GGPPS. decline. And as the concentration of MG132 increases, the expression of GGPPS rises. We therefore concluded that the ubiquitination-proteasome inhibitor MG132 was able to reverse the decrease in GGPPS expression caused by 4-Az. 293T co-transfected with GGPPS and Ub plasmids were divided into four groups: negative control group (DMSO), drug treatment group (4-Az, 10 uM), proteasome inhibition group (DMSO + MG132), proteasome and drug combination Group (4-Az+MG132). MG132 prevents degradation by preventing the target protein labeled by ubiquitinated small molecules from entering the proteasome. When 4-Az and MG132 simultaneously treated the cells, 4-Az promoted the ubiquitination of GGPPS, and was blocked by MG132 when it had not entered the proteasome, and the ubiquitination accumulated GGPPS increased. Therefore, we can conclude that 4-Az can promote ubiquitination of GGPPS protein.

Embodiment 4, 4-Az can directly combine the mode of action of GGPPS

The concentration of the GGPPS-EGFP-His fluorescent expression fusion protein obtained by the purification was kept constant, and the concentration of the FPP concentration of the GGPPS positive control substrate used for each titration was changed. The thermophoretic motion without GGPPS is different from the motion of the substrate FPP which has been combined with GGPPS. When the time is t=30 seconds, the fluorescence signal of GGPPS is normalized and processed at different FPP concentrations to form Kd. The fitted binding curve. The titrant was replaced with the naturally active small molecule to be identified, and fluorescence detection showed direct binding of 4-Az to GGPPS compared to control EGFP. It can be seen that 4-Az promotes GGPPS degradation by directly binding to GGPPS to promote the ubiquitination of GGPPS.

Therapeutic effect of azidoin on nonalcoholic fatty liver disease

General materials and methods

Material

Four-week-old mice (C57BL/6J) were purchased from the Institute of Model Animals, Nanjing University. The mouse feed was purchased from Jiangsu Synergy Bioengineering Co., Ltd. High fat feed was purchased from Research Diets, New Brunswick, NJ. Azidopidin is purchased from Shanghai Taosu Biochemical Co., Ltd. The liver tissue triglyceride test kit was purchased from Pratt Bioengineering Co., Ltd. The alanine aminotransferase and aspartate aminotransferase assay kits were purchased from KGI Biotechnology Development Co., Ltd.

2. Preparation of reagents

Preparation of azidopidin preparation: Dissolve azid phlorizin powder in Dimethyl Sulphoxide (DMSO), dissolved in two dose groups of 1 mg/kg and 10 mg/kg. The concentration was 0.15 g/L and 1.5 g/L.

The placebo solution was the solvent DMSO of the azidopidin preparation.

3. Method

Measurement of liver tissue triglyceride: Take about 50 mg of the right lobe of mouse liver tissue and record the exact weight. The PBS was washed with water and placed in 1 ml of lysate and mechanically homogenized. The lysed tissue homogenate was divided into two parts. A portion of 500 ul was placed in a 70 ° C metal bath, heated for 10 min, and centrifuged at 2,000 x rpm for 5 min. Take 1-10 ul for tissue TG kit detection. An additional 500 ul was used to detect protein concentration by the BCA method.

Paraffin section preparation: Take the right amount of liver tissue, fix it with 4% paraformaldehyde for 24-48h, dehydrate the ethanol step by step, transparent xylene, dipped in wax, embedded in paraffin, sliced, and the thickness is 5μm.

HE staining of paraffin sections: The sections were immersed in xylene for 10 min and 5 min for dewaxing, respectively, and then water was added to the gradient ethanol. After 4 min of hematoxylin staining, the steamed water was slightly washed. It was blanched with 0.25% NH ₃ ·H ₂ O for 30 s and then washed with water. After dehydration with a gradient of ethanol to 95% ethanol, 0.5% eosin was stained for 2-3 s, then dehydrated in 100% ethanol, and soaked in xylene for 2 min. Finally, the film is sealed with a neutral gum.

Detection of alanine aminotransferase and aspartate aminotransferase levels: After anesthesia in mice, blood was taken through the orbital vein and placed at room temperature for 30 min until the serum was precipitated and centrifuged. 10 ul of serum was taken for detection of alanine aminotransferase and aspartate aminotransferase kits.

Example 1. Establishing a non-alcoholic fatty liver mouse model

Non-alcoholic fatty liver model rats were induced by food-borne induction. High-fat diets (containing 60% fat) were fed to 4-week-old mice in the "General Materials and Methods" for 8 weeks, and 8 weeks later, they were successfully induced to produce fatty liver.

Example 2 Effect of azidozin on body weight and major lipid metabolism in non-alcoholic fatty liver mice

A total of 18 non-alcoholic fatty liver mice in Example 1 were randomly assigned to 3 groups, 6 in each group, and were set to "4-Az 1" group, "4-Az 10" group, and control group. The above three groups of mice were intraperitoneally injected with an equal volume of 1 mg/kg dose of azid phagemidin preparation, 10 mg/kg dose of azid phagemidin preparation and dimethyl sulfoxide (as a placebo). The injection was given every other day for 14 consecutive days, and the changes in body weight of the mice were simultaneously monitored.

As shown in Figure A and Figure B, the body weight, liver weight and epididymal fat of mice injected with azidopistin were significantly lower than those of the control group, indicating azide. The phloridin can significantly reduce the body weight of non-alcoholic fatty liver mice.

Example 3 Effect of azidopidogrel on lipid accumulation in liver of mice with nonalcoholic fatty liver disease

After continuous administration for 14 days as in Example 2, each group of mice was sacrificed and the relevant physiological indexes were examined. The liver tissues of each group of mice were subjected to CT scanning, and the liver tissues of each group of mice were subjected to triglyceride level detection and paraffin section HE staining according to the procedure described in "General Materials and Methods".

As shown in the A-C diagram of Fig. 2, the liver tissue density of the mice injected with the azidopidin preparation was significantly higher than that of the control mice, and the lipid content was significantly lowered. As shown in Fig. 3, Panel A shows that non-alcoholic fatty liver mice administered with an azido phlorizin preparation have significant remission of hepatic macrovesicular steatosis compared with the control group, while B shows The triglyceride content was significantly reduced, indicating that azidoin can reduce the lipid-induced hepatic lipid degeneration.

Example 4 Effect of azidoin on the levels of alanine aminotransferase and aspartate aminotransferase in blood of mice After 14 days of continuous administration in Example 2, the levels of alanine aminotransferase and aspartate aminotransferase in the blood of mice were measured. As shown in Fig. 4, the results of A and B show that the mice injected with azidopidin preparation have significantly lower levels of alanine aminotransferase and aspartate aminotransferase in the blood compared with the control mice. Azidoin spectrase relieves hepatotoxicity caused by high-fat feeding and has activity to protect liver function.

Example 5: Differences in lipid metabolism in hepatocytes at the cellular level by azidoin and phloridin 1. Construction of hepatic steatosis model

This model is a cellular model that mimics the cellular environment of nonalcoholic fatty liver with lipid-denatured hepatocytes. Primary hepatocytes from 8 week old mice were isolated and plated to approximately 80% of culture dishes. The bovine serum albumin (BSA), which is a free fatty acid and a free fatty acid, was used as a negative control to stimulate the cells. Hepatocytes were cultured in a mixture of free fatty acids (monounsaturated fatty acids: saturated fatty acids = 2:1). As the concentration of free fatty acids increased and the time prolonged, the results of oil red staining and triglyceride detection showed that the accumulation of lipids was deepened.

2. Azido spectrinin (4-Az) significantly reduced triglyceride (TG) content in hepatocyte lipid degeneration model

On the basis of the hepatocyte lipid degeneration model, three groups were internally set as DMSO group, 4-Az drug treatment group (10 μM, 20 μM, 50 μM) and phlorizin drug treatment group (100 μM). The results of the cytology triglyceride kit showed that in the BSA group, the TG content of the cells decreased when the drug treatment concentration reached 50uM, and the triglyceride group was 4 times more than the negative control BSA group after the free fatty acid treatment. Significantly elevated, on the basis of this, it was found that the triglyceride content had been significantly decreased and the TG reduction rate was 65% under the treatment of the lowest concentration of 10 uM 4-Az. Under the treatment of 100uM phloridin, the content of triglyceride did not decrease significantly, and the TG reduction rate was only 83%, as shown in Table 1. The results indicate that the effect of azidoin on hepatic lipid metabolism is far more significant than phlorizin.

Table 1

The above embodiments are only used to illustrate the technical solutions of the present invention, and the present invention is not limited thereto. Although the present invention is described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or substituted. The spirit and scope of the present invention should be construed as being included in the scope of the appended claims.

Claims (6)

The use of azidated lignin as a GGPPS inhibitor. Use of azidated lignin in the preparation of a medicament for treating a disease associated with GGPPS inhibition. The use according to claim 1, wherein the disease is nonalcoholic fatty liver. The use according to claim 2 or 3, characterized in that the medicament comprises dimethyl sulfoxide, azidopidin and a pharmaceutically acceptable carrier. The use according to claim 2, wherein the drug administration mode comprises intraperitoneal injection, subcutaneous, intradermal, intraarterial, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, and focal Internal, intracranial injection or infusion, oral, topical, rectal, nasal, buccal, vaginal, sublingual, intradermal, mucosal, tracheal, urethral administration, by inhalation aerosol, implant accumulation and acupuncture medicine. The use according to claim 3, characterized in that the medicament is administered at a dose of from 1 to 10 mg/kg.

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