Application of kaempferitrin in preparation of medicine for resisting non-alcoholic fatty liver

Abstract

The invention provides application of kaempferitrin in preparation of a medicine for resisting non-alcoholic fatty liver disease, and relates to the technical field of medicine. The kaempferitrin provided by the invention can reduce intracellular lipid deposition and reduce intracellular inflammatory factor level, and is related to FXR, FAS, Nrf2, TGR5 and SREBP-1c protein expression related to regulation of bile acid metabolism, lipid metabolism, oxidative stress and inflammatory reaction on a protein action mechanism, so that the kaempferitrin can well cope with the non-alcoholic fatty liver disease.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an application of kaempferol in the preparation of an anti-nonalcoholic fatty liver disease drug. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) is a spectrum of liver diseases ranging from simple steatosis to non-alcoholic fatty hepatitis (NASH), which may further develop into cirrhosis and liver cancer. Currently, the "two-hit" theory has been proposed to explain the pathogenesis of NAFLD. The first hit includes abnormal accumulation of triglycerides (TG) in the liver. If the fatty liver suffers a second hit, including activation of inflammatory mediators, reactive oxygen species, and stellate cells, it may develop into NASH. NAFLD is often associated with obesity and diabetes. Obesity is the main risk factor for the development of NAFLD. At the same time, inflammatory markers are usually elevated in patients with fatty liver. In addition to the direct impact on liver health, NAFLD is also associated with a higher risk of extrahepatic complications, including increased mortality from cardiovascular disease. At present, there is a lack of clinical therapeutic drugs for NAFLD, and this situation needs to be changed urgently. Summary of the invention

[0003] The object of the present invention is to provide an application of kaempferol in the preparation of an anti-nonalcoholic fatty liver drug.

[0004] The present invention provides an application of kaempferol in the preparation of a drug for resisting non-alcoholic fatty liver disease.

[0005] Specifically, the structural formula of kaempferol is shown below:

[0006] BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 A is a comparison of the effects of different concentrations of oleic acid on the viability of HepG2 cells;

[0008] Figure 1 B is a comparison of the effects of different concentrations of oleic acid and modeling time on the viability of HepG2 cells;

[0009] Figure 2 A is a comparison of the effects of kaempferol at different concentrations on the survival rate of HepG2 cells;

[0010] Figure 2 B is a comparison of the effects of kaempferol at different concentrations on TG content in HepG2 cells;

[0011] Figure 3 A is the oil red staining image of cells in each group;

[0012] Figure 3 B is a comparison chart of the OD values ​​of cells in each group;

[0013] Figure 4 A is a comparison of the effects of kaempferol on SOD levels in HepG2 cells;

[0014] Figure 4 B is a comparison of the effects of kaempferol on the MDA level in HepG2 cells;

[0015] Figure 5 A is a comparison chart of the effects of kaempferol on TNF-α levels in HepG2 cells;

[0016] Figure 5 B is a comparison of the effects of kaempferol on IL-6 levels in HepG2 cells;

[0017] Figure 6 This is a visual simulation diagram of the docking of kaempferol and protein;

[0018] Figure 7 This is a comparison of the effects of kaempferol on the protein levels of FXR, FAS, Nrf2, TGR5 and SREBP-1c in HepG2 cells;

[0019] Figure 8 A is a comparison chart of the weight gain rate of mice in each group;

[0020] Figure 8 B is a comparison chart of liver index changes in mice in each group;

[0021] Fig. 9 A is a comparison chart of serum total cholesterol (TC) content in the serum of mice in each group;

[0022] Fig. 9 B is a comparison chart of the total triglyceride (TG) content in the serum of mice in each group;

[0023] Fig. 9 C is a comparison chart of serum high-density lipoprotein cholesterol (HDL) content in the serum of mice in each group;

[0024] Fig. 9 D is a comparison chart of serum low-density lipoprotein cholesterol (LDL) content in the serum of mice in each group;

[0025] Fig.10 A is a comparison of AST levels in the serum of mice in each group;

[0026] Fig.10 B is a comparison of ALT levels in the serum of mice in each group;

[0027] Fig.11 A is a comparison of the superoxide dismutase (SOD) levels in the serum of mice in each group;

[0028] Fig.11 B is a comparison of glutathione (GSH) levels in the serum of mice in each group;

[0029] Fig.12 A is a comparison of TNF-α levels in the serum of mice in each group;

[0030] Fig.12 B is a comparison of IL-6 levels in the serum of mice in each group;

[0031] Fig.12 C is a comparison of IL-1β levels in the serum of mice in each group;

[0032] Fig.13 HE staining images of the liver of mice in each group;

[0033] Fig.14 Oil red O staining images of the liver of mice in each group. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Unless otherwise defined, the technical terms or scientific terms used herein should be the common meanings understood by people with general skills in the field to which the present invention belongs.

[0035] The present invention provides an application of kaempferol in the preparation of an anti-nonalcoholic fatty liver drug. Specifically, the structural formula of the kaempferol used is as follows:

[0036]

[0037] In fact, kaempferitrin can be isolated from the medicinal material of Pineapple, or a commercially available product (Kaempferitrin, kaempferol-3,7-di-O-α-L-rhamnoside, CAS: 482-38-2) can be used.

[0038] 1. Cell experiment:

[0039] HepG2 cells were pre-added to DMEM high-glucose medium (C11995500B, Thermo Fisher Scientific) containing 10% FBS (FSP500, Suzhou Yikesai Biotechnology Co., Ltd.) and 1% penicillin / streptomycin, and cultured in an incubator at 37°C and 5% CO2. The medium was changed every 48 hours until the cells reached 80% density. The cells were then digested with trypsin-EDTA digestion solution (T1300-100) for 1 min before passage.

[0040] HepG2 cells were inoculated into a 96-well plate at a density of 300,000 / mL (100 μL per well). After the cells adhered to the wall, a normal control group (oleic acid concentration 0 mmol / L) and control groups with different oleic acid concentrations (0.25 mmol / l, 0.5 mmol / L, 1 mmol / L, 2 mmol / L) were set up. After culturing for 24 hours, 10 μL CCK8 solvent was added to each well of the well plate and incubated in a 37°C incubator for 50 minutes. The OD value was detected at a wavelength of 450 nm using an enzyme reader to determine the cell survival rate. The results are shown in Figure 2. Figure 1 As shown in A.

[0041] from Figure 1 As can be seen from A, compared with the normal control group with an oleic acid concentration of 0, oleic acid concentrations of 0.25mmol / L and 0.5mmol / L had no significant effect on the survival rate of HepG2 cells, while the survival rate decreased significantly when the oleic acid concentration was greater than 0.5mmol / L.

[0042] HepG2 cells were seeded into 96-well plates at a density of 300,000 / mL (100 μL per well). After the cells adhered to the plate, a normal control group (oleic acid concentration 0 mmol / L) and control groups with different oleic acid concentrations (0.25 mmol / L, 0.5 mmol / L) were set up. After culturing for 12 h, 24 h, and 36 h, the TG content in the cells was detected. Figure 1 As shown in B.

[0043] from Figure 1 As can be seen from B, with the increase of oleic acid concentration in the three time periods, the TG value in the cells also increased, and there were significant differences compared with the normal group (P < 0.01). The cell TG content was highest when the oleic acid concentration was 0.5 mmol / L and the modeling culture was 24 hours, so it can be used as the optimal modeling concentration and the optimal modeling time.

[0044] HepG2 cells were seeded into a 96-well plate at a density of 300,000 / mL (100 μL per well). After the cells adhered to the wall, a blank control group (Control) and a kaempferol control group were set up. After culturing for 24 hours, 10 μL CCK8 solvent was added to each well of the well plate. After further incubation in a 37°C incubator, the OD value was detected at a wavelength of 450 nm using an enzyme reader to determine the cell survival rate. The results are shown in Figure 2. Figure 2 As shown in A.

[0045] from Figure 2 As can be seen from A, compared with the blank control group, kaempferol had no significant effect on cell activity at 50 μmol / L, and significantly reduced cell survival rate at concentrations above 100 μmol / L.

[0046] HepG2 cells were seeded into 96-well plates at a density of 300,000 / mL (100 μL per well). After the cells adhered to the wall, a blank control group (Control), a model group (Model), a positive drug-administered group (OCA, drug concentration 20 μmol / L), and different concentrations (25 μmol / L, 50 μmol / L, 100 μmol / L) of kaempferol experimental groups were set up. Except for the blank control group, the remaining groups were administrated and modeled (oleic acid concentration 0.5 mmol / L, modeling culture 24 h). After 24 h, the plates were collected and 150 μL of RIPA lysis buffer (R0020, Beijing Solebow Technology Co., Ltd.) was used in each well. After lysis on ice for 30 min, the TG level was detected using a TG kit (A110-1-1-1, Nanjing Jiancheng Biological Co., Ltd.). Figure 2 As shown in B.

[0047] from Figure 2 As can be seen in B, kaempferol has a significant effect on the TG content in cells at three gradient concentrations within the safe concentration range, and shows different degrees of TG-lowering effects.

[0048] HepG2 cells were seeded into 96-well plates at a density of 300,000 / mL (100 μL per well). After the cells adhered to the wall, a blank control group (Control), a model group (Model), a positive drug group (OCA, drug concentration 20 μmol / L), and a kaempferol drug group (concentration 20 μmol / L) were set up and modeled. After culturing in a 37°C incubator for 24 hours, the culture medium was removed, and the cells were rinsed with PBS 2-3 times, and then fixed with a fixative. 20-30min, wash twice with distilled water, then rinse with 60% isopropanol for 20-30s and discard, use the newly prepared Oil Red O staining solution to stain for 10-20min, then use 60% isopropanol to soak for 20-30s until the interstitium is clear, then rinse with distilled water 2-5 times, add Mayer hematoxylin staining solution to stain for 1-2min, discard the staining solution, wash 2-5 times with water, add buffer solution for 1min and discard, add distilled water to observe the Oil Red staining of each group of cells under an inverted microscope, as shown Figure 3 As shown in A; after the observation, 300 μL of 60% isopropanol was added to each well to dissolve the oil red dye on the cell surface, and the OD value of each group of cells was measured at a wavelength of 540 nm. The results are shown in Figure 3 As shown in B.

[0049] from Figure 3 As can be seen from A, compared with the Control group, the area of ​​Oil Red O staining in the cells of the Model group was significantly increased, the lipid droplets were arranged around the cells in a beaded manner, and the cell nucleus shrank significantly; compared with the Model group, the area of ​​lipid droplets stained with Oil Red O in the cells of the Kaempferia glycoside group was reduced, the cell nucleus atrophy was improved, and the cell volume was improved, which intuitively demonstrated the lipid-lowering ability of Kaempferia glycoside; Figure 3 As can be seen from B, the intracellular lipid level in the Model group was 70% higher than that in the Control group, indicating a significant modeling effect. Compared with the Model group, the intracellular lipid level in the kaempferol group was significantly reduced (P<0.001).

[0050] HepG2 cells were seeded into 96-well plates at a density of 300,000 / mL (100 μL per well). After the cells adhered to the wall, a blank control group (Control), a model group (Model), a positive drug group (OCA, drug concentration 20 μmol / L), and a kaempferol drug group (concentration 20 μmol / L) were set up and modeling was performed. After culturing in a 37°C incubator for 24 hours, the plates were collected and the levels of oxidative stress factors SOD and MDA in each group of cells were detected using a SOD kit (A001-3-2, Nanjing Jiancheng Biological Co., Ltd.) and an MDA kit (A003-1-2, Nanjing Jiancheng Biological Co., Ltd.). The results are shown as follows: Figure 4 A. Figure 4 As shown in B.

[0051] from Figure 4 As can be seen from A, the SOD content of the Model group was significantly lower than that of the normal group (P<0.01), indicating that the ability of HepG2 cells to scavenge superoxide ion free radicals decreased sharply after modeling. Compared with the Model group, the intracellular SOD content of the kaempferol group was significantly increased, increasing the SOD content of the cells in the group by 35%. Figure 4 As can be seen from B, the MDA content in the Model group was significantly higher than that in the normal group (P<0.01), indicating that the intensity of intracellular lipid peroxidation was severe after HepG2 cell modeling. Compared with the Model group, the intracellular MDA content in the kaempferol group was significantly decreased, falling to 42% of that in the model group. Therefore, it can be shown that kaempferol can significantly reduce the level of oxidative stress in the model cells.

[0052] HepG2 cells were inoculated into 96-well plates at a density of 300,000 / mL (100 μL per well). After the cells adhered to the wall, a blank control group (Control), a model group (Model), a positive drug group (OCA, drug concentration 20 μmol / L), and a kaempferol drug group (concentration 20 μmol / L) were set up and modeling was performed. After culturing in a 37°C incubator for 24 hours, the plates were harvested, the cell culture supernatant was centrifuged to remove impurities in the culture medium, and the ELISA kit was used to detect the levels of intracellular inflammatory factors TNF-α and IL-6. The results were as follows: Figure 5 A. Figure 5 As shown in B.

[0053] from Figure 5 A and Figure 5 As can be seen in B, compared with the Control group, the intracellular TNF-α and IL-6 in the Model group increased significantly (P<0.01), indicating that oleic acid modeling significantly stimulated the secretion of inflammatory factors in HepG2 cells. Compared with the Model group, kaempferol significantly reduced the intracellular IL-6 content (P<0.01), and in terms of reducing the inflammatory factor TNF-α, kaempferol also showed a significant reducing effect. In summary, kaempferol has a more prominent effect in reducing the levels of inflammatory factors TNF-α and IL-6, which are reduced to 17% and 65% of the Model group, respectively.

[0054] The PDB files of core targets (SREBP-1c, FAS, FXR, Nrf2 and TGR5) were obtained from the PDB database (https: / / www.rcsb.org / ). The target proteins were opened in AutoDock1.5.7. The kaempferol and target proteins were hydrogenated and charged. Semi-flexible docking was used for small molecule screening, that is, the protein structure remained rigid and the small molecule ligand was considered flexible. The ligand could bind in a variety of conformations in the receptor active site. The binding energy was calculated. If it was lower than -4.25 kcal / mol, -5.0 kcalmol or -7.0 kcalmol, it indicated that the ligand and receptor had certain, good or strong binding activity, respectively. The docking results are shown in Table 1. Finally, the data were visualized by PyMol software. The docking visualization of kaempferol with FXR, TGR5, Nrf2 and SREBP-1c proteins was shown in Table 1. Figure 6 A. Figure 6 B. Figure 6 C. Figure 6 As shown in D.

[0055] Table 1 Docking results of target proteins and kaempferol

[0056]

[0057]

[0058] As the binding energy is smaller, kaempferol is easier to enter the active pocket of the protein, and the hydrogen bonding and hydrophobic interactions with the amino acid residues of each protein are stronger. As can be seen from Table 1, the binding energy between kaempferol and the five target proteins is less than -5.0 kcal / mol, which indicates that kaempferol has good binding activity with the target proteins under computer simulation. Figure 6 It can be seen that kaempferol forms hydrogen bonds with SREBP-1c through DG-61 and ASN-358, and forms hydrogen bonds with Nrf2 through ASN-517, VAL-512, VAL-561, THR-560, VAL-608, and VAL-369.

[0059] HepG2 cells were inoculated into 96-well plates at a density of 300,000 / mL (100 μL per well). After the cells adhered to the wall, a blank control group (Control), a model group (Model), a positive drug-treated group (OCA, drug concentration of 20 μmol / L), and a kaempferol-treated group (concentration of 20 μmol / L) were set up and modeling was performed on all of them. After culturing in a 37°C incubator for 24 hours, the plates were collected, the culture medium was discarded, and the cells were fully lysed on ice for 30 minutes using RIPA lysis buffer (R0020, Beijing Solebow Technology Co., Ltd.), and then centrifuged at 4°C and 12,000 rpm for 10 minutes, and the supernatant was taken to quantify the proteins in each group using the BCA protein method. Electrophoresis was performed at 100V for 60min, and the membrane was transferred by sandwich method at 108V and 200mA for 90min. The membrane was blocked with 5% skim milk powder for 2h, and the primary antibody was added. The membrane was incubated at 4℃ overnight. The membrane was washed with TBST 3 times, each time for 10min. The secondary antibody (rabbit antibody 1:6000) was added. The membrane was incubated at room temperature for 1h and then washed with TBST 3 times, each time for 10min. ECL chemiluminescence reagent was added and the membrane was imaged using a fully automatic chemiluminescence imaging analyzer. The results were analyzed using Image Lab software. Figure 7 shown.

[0060] from Figure 7 It can be seen that compared with the Control group, the expression of FXR, TGR5 and Nrf2 in the Model group cells was significantly reduced, while SREBP-1c and FAS were significantly increased. The effect of kaempferol on the expression of related proteins in the model cells is summarized in Table 2. It can be seen that kaempferol has a wide range and significant effect on the expression of various proteins. Kaempferol is similar to the positive drug OCA, which can significantly increase the expression of FXR, TGR5 and Nrf2, and significantly inhibit SREBP-1c and FAS. The results of the molecular docking experiment show that the binding energy of kaempferol with the target protein is less than -7kcal / mol, indicating that kaempferol has strong binding activity with each target protein, which has been further verified in the cell experiment here.

[0061] Table 2 Effects of kaempferol on protein levels in HepG2 cells

[0062]

[0063] Note: Compared with the model group (vs. model group), "c" means P < 0.05, "d" means P < 0.01; "-" means no significant change.

[0064] 2. Animal Experimentation

[0065] When conducting animal experiments, the materials and reagents used are shown in Table 3 below:

[0066] Table 3 Animal experimental materials and reagents

[0067]

[0068]

[0069] When conducting animal experiments, the materials and reagents used are shown in Table 4 below:

[0070] Table 4 Animal experiment instruments

[0071]

[0072] 72 male C57BL / 6j mice (weight 22-24g, purchased from Hunan Slake Jingda Experimental Animal Co., Ltd., license number: SCXK (Xiang) 2019-0004) were kept at 23±3℃, humidity 50±10%, 12-hour day and night cycle, and mice were allowed to eat and drink freely. After one week of adaptive feeding, 72 C57BL / 6j male mice were randomly divided into a blank group (Control) (n=12) and a high-fat diet group (n=10). The blank group was fed with a basic diet, the high-fat group was fed with a high-fat diet D12492 (60% fat ratio) and injected with dexamethasone, and the treatment group was fed with a high-fat diet and injected with dexamethasone while being gavaged with drugs. Modeling and drug administration were carried out simultaneously for 4 weeks. At the end of the fourth week, mice in each group were randomly captured to compare the test effects, and blood was taken from the inner canthus to test serum biochemical indicators.

[0073] Weigh 5g of CMC-Na powder, add it to 1L of pure water and place it in a water bath for continuous heating and stirring. Prepare the 0.5% CMC-Na aqueous solution used for the drug. After it is completely dissolved, store it in a 4°C refrigerator and return it to room temperature before use. All drugs in each group were dissolved in 0.5% CMC-Na aqueous solution to prepare a suspension. The OCA group, Kae-H group and Kae-L group were given 10mg / kg of obeticholic acid, 50mg / kg of kaempferol and 25mg / kg of kaempferol, respectively, and dexamethasone was injected intraperitoneally at the same time. Ultrasound was performed before administration to make it fully and evenly suspended. Mice in the blank group were gavaged with 0.5% CMC-Na. The administration lasted for a total of 4 weeks.

[0074] During the feeding period, the feed intake of each group of mice was recorded every day and the general condition of the mice was observed: body shape, mental state, free movement, hair gloss and bowel movements. The mice were weighed every three days and the weight changes of each group were recorded. Figure 8As shown in A. At the end of the fourth week of drug administration, mice were fasted but not watered for 12 hours before the experiment. After blood was taken from the eyeballs and sacrificed, the whole blood was allowed to stand at room temperature for 2 hours to coagulate and stratify, and then centrifuged at 4500 rpm for 15 minutes. The supernatant was aliquoted and stored at -80°C. The livers of mice were removed and weighed wet, and the liver index of each group of mice was calculated based on the following formula. The results are shown in Figure 8 As shown in B. Feces and cecal contents were collected, and a portion of the liver was fixed in paraformaldehyde fixative and stored at room temperature.

[0075] The liver index calculation formula is: liver index = liver weight (g) / body weight (g) × 100%.

[0076] The levels of TC, TG, HDL, and LDL in the serum of each group of mice were detected using a kit. The results are as follows: Fig. 9 As shown; the levels of AST and ALT in the mouse serum of each group of mice were detected using a kit, and the results are shown Fig.10 As shown; the levels of SOD and GSH in the serum of each group of mice were detected using a kit, and the results are shown Fig.11 As shown; the levels of TNF-α, IL-6, and IL-1β in the serum of each group of mice were detected using a kit, and the results are shown Fig.12 shown.

[0077] After the livers of mice in each group were removed and made into paraffin sections, the paraffin was placed in xylene I for 20 minutes → xylene II for 20 minutes → anhydrous ethanol I for 5 minutes → anhydrous ethanol II for 5 minutes → 75% alcohol for 5 minutes for gradient dewaxing → washing with water for 3 minutes; the sections were stained with hematoxylin for 3-5 minutes, the excess dye was washed off with tap water, differentiated with 1% hydrochloric acid aqueous solution for a few seconds, rinsed with tap water, and then 0.6%-0.7% ammonia aqueous solution was returned to blue, and rinsed with running water for a few seconds; the paraffin sections were dehydrated with gradient alcohol (85% ethanol → 95% ethanol) and placed in eosin dye for 5 minutes; the paraffin sections were placed in anhydrous ethanol I for 5 minutes → anhydrous ethanol II for 5 minutes → anhydrous ethanol III for 5 minutes → n-butanol for 5 minutes → xylene I for 5 minutes → xylene II for 5 minutes to make them transparent, the sections were taken out of xylene and slightly dried, and sealed with neutral gum. A 200X microscope was used to take pictures to analyze the pathological changes of liver tissue, and the HE staining picture is shown as follows Fig.13 shown.

[0078] The liver tissues removed from each group of mice were embedded in OCT and then frozen and sectioned. The sections were stained with saturated Oil Red O solution for 8-10 minutes (protected from light and covered), differentiated with two cylinders of 60% isopropanol for 10 seconds, rinsed with distilled water, counterstained with hematoxylin for nuclei, wiped off excess moisture in the tissues, sealed with glycerol gelatin, stored in a dark environment at 4°C, and sectioned, and photographed with a 200X microscope. Fig.14 shown.

[0079] from Fig. 9 A and Fig. 9 As can be seen in B, as time goes by, the body weight growth rate and liver wet weight of mice in the model group increased significantly compared with the normal group, and the body weight growth rate and liver wet weight of mice in the OCA group and Kae group showed a significant decrease trend compared with the model group. This shows that kaempferol has a positive effect on alleviating the abnormal increase in body weight growth rate and liver wet weight of mice induced by HFD.

[0080] from Fig. 9 It can be seen that compared with the normal diet, high-fat diet intervention led to increased serum TC, TG and LDL-C levels and decreased serum HDL-C levels, indicating that long-term HFD intake disrupted lipid homeostasis. Compared with the normal group, the TG, TC and LDL in the serum of the model group mice increased significantly, and HDL decreased significantly. Compared with the model group, TG, TC and LDL in the high- and low-dose groups of kaempferol decreased, and there was statistical significance (P<0.001; P<0.01; P<0.05), and HDL increased significantly. The results showed that both high and low concentrations of kaempferol can significantly improve the abnormally expressed blood lipid indicators in the serum of model mice, and are dose-dependent.

[0081] from Fig.10 It can be seen that compared with the normal group, the ALT and AST in the serum of the model group mice increased significantly. Compared with the model group, the ALT and AST in the high- and low-dose kaempferol groups decreased, and there was statistical significance (P<0.001; P<0.01). The results showed that both high and low concentrations of kaempferol can significantly improve the abnormal liver damage indicators expressed in the serum of model mice, and in a dose-dependent manner.

[0082] from Fig.11 It can be seen that compared with the normal group, the SOD and GSH in the serum of the model group mice were significantly reduced (P<0.01; P<0.001). Compared with the model group, the SOD and GSH in the serum of the mice in the high- and low-dose groups of kaempferol increased, and both were statistically significant (P<0.01:P<0.05; P<0.001). The results showed that both high and low concentrations of kaempferol can significantly improve the abnormally expressed antioxidant indicators in the serum of NAFLD model mice, and kaempferol is dose-dependent.

[0083] from Fig.12 It can be seen that compared with the normal group, the serum TNF-α, IL-6, and IL-1β in the model group mice were significantly increased (P<0.001). Compared with the model group, the serum TNF-α, IL-6, and IL-1β in the high- and low-dose kaempferol groups were reduced, and all were statistically significant (P<0.01; P<0.05; P<0.001). The results showed that both high and low concentrations of kaempferol can significantly improve the abnormally expressed inflammatory indicators in the serum of model mice, and in a dose-dependent manner.

[0084] from Fig.13 It can be seen that the liver tissue of mice in group N (blank group) had no fatty degeneration, obvious hepatic sinusoids, neatly arranged hepatic cords, clear cell structure, cell nuclei in the center of the cell, abundant cytoplasm, and no obvious lipid deposition. Compared with group N, the liver tissue of mice in group M (model group) showed severe fatty degeneration, obvious fatty degeneration around the central vein, accompanied by inflammatory cell infiltration and hepatocyte necrosis, cell nuclei squeezed to one side, and the cytoplasm was filled with a large number of fat vacuoles with blurred boundaries, and significant lipid deposition. Compared with group M, the number of fatty degenerated cells in group Kae decreased. With the increase of Kae dose, fatty degeneration of hepatocytes was significantly alleviated, inflammatory infiltration and cell necrosis were gradually improved, and lipid deposition was significantly improved.

[0085] from Fig.14 It can be seen that the liver tissue structure of mice in group N was basically normal, with complete liver cell structure, clear outline, round nucleus, rich cytoplasm, no necrosis or degeneration; the size of the hepatic sinusoids was uniform, no expansion was observed, and the hepatic cords were arranged neatly and tightly: no inflammatory cell infiltration was observed in the liver parenchyma. Compared with group N, the liver tissue structure of mice in group M was severely abnormal, with unclear outlines, some hepatocytes were necrotic, and round lipid droplets of varying sizes were visible in the cytoplasm; inflammatory cell infiltration appeared in the liver parenchyma; compared with group M, the liver tissue structure of mice in group Kae was moderately abnormal, with a small number of hepatocytes fatty degeneration, and round lipid droplets of uniform size were visible in the cytoplasm. The above results show that kaempferol can reduce the pathological damage of liver tissue in model mice.

[0086] Although the embodiments of the present invention are described in detail above, it is obvious to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations are within the scope and spirit of the present invention as described in the claims. Moreover, the present invention described herein may have other embodiments and may be implemented or realized in a variety of ways.

Claims

1. An application of kaempferol in the preparation of an anti-nonalcoholic fatty liver disease drug.

2. The use according to claim 1, characterized in that: The structural formula of kaempferol is shown below:

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