CN113456649A

CN113456649A - Method for relieving non-alcoholic fatty liver by activating AMPK with limonin

Info

Publication number: CN113456649A
Application number: CN202110864166.8A
Authority: CN
Inventors: 张峰; 王思为; 陈杭飞; 蓝天; 郑芳; 徐礼锋
Original assignee: Quzhou Peoples Hospital
Current assignee: Quzhou Peoples Hospital
Priority date: 2021-07-29
Filing date: 2021-07-29
Publication date: 2021-10-01

Abstract

The invention discloses a method for activating AMPK by limonoid to relieve non-alcoholic fatty liver disease, comprising the following steps: selecting test animals, the animals are C57BL/6 mice; Normal diet limonin-treated group, high-fat diet modeling group, high-fat diet limonin-treated group; feeding of experimental animals; experimental detection. The present invention finds that there is no difference in food intake between the control group and the limonin-treated group regardless of whether they are fed a normal-calorie diet or a high-fat diet through a test method of feeding C57BL/6 mice with limonin. We observed that limonin-treated mice on a high-fat diet caused significant weight loss, whereas mice fed a normal diet, with or without limonin, showed no significant difference in body weight, suggesting that Liver weight gain induced by high-fat diet feeding can be ameliorated by limonoid treatment.

Description

Method for relieving non-alcoholic fatty liver by activating AMPK with limonin

Technical Field

The invention relates to the field of non-alcoholic fatty liver disease, in particular to a method for activating AMPK to relieve non-alcoholic fatty liver disease by limonin.

Background

Non-alcoholic fatty liver disease, also known as metabolic-related fatty liver disease, is a disease characterized by pathological accumulation of triglycerides and other lipids in hepatocytes, which can progress to non-alcoholic steatohepatitis and liver fibrosis, the latter ultimately leading to cirrhosis and hepatocellular carcinoma. Although there are many small molecule chemical drugs for non-alcoholic fatty liver disease in clinical trials, there are very limited treatment options for non-alcoholic fatty liver disease in clinical practice.

AMPK is a key metabolic regulator for sensing energy state, controlling energy consumption and storage in cells, and the activation of AMPK is shown to have a therapeutic effect on non-alcoholic fatty liver diseases, and AMPK is activated through the combination of AMP and ADP, and the activation state of AMPK is inhibited by ATP. Liver-specific AMPK knockouts can cause liver lipid accumulation, steatosis, inflammation, fibrosis, and hepatocyte apoptosis. There are many studies that have shown that synthetic polyphenols can activate liver AMPK and counteract hepatic steatosis by inhibiting the activity of Sterol Regulatory Element Binding Protein (SREBP). Our previous studies indicate that flavonoids can improve mouse liver steatosis by activating AMPK.

Limonin is a tetracyclic triterpenoid, and is a secondary metabolite with high biological activity in plants. Many traditional Chinese medicines and fruits are rich in limonin, which has been recognized as one of the most beneficial and effective ingredients in medicinal foods. In recent years, many pharmacological studies have found that limonin has various biological activities, including anti-tumor, anti-inflammatory, anti-oxidant, and liver-protecting activities. Limonin can reduce low density lipoprotein cholesterol in HepG2 cells and regulate the expression of genes related to lipid metabolism in mice. However, the influence of limonin on liver lipid metabolism and the mechanism thereof are still unknown, so that a method for activating AMPK to relieve non-alcoholic fatty liver by limonin is designed.

Disclosure of Invention

The invention aims to provide a method for activating AMPK to relieve nonalcoholic fatty liver by limonin, so as to solve the problems in the background technology.

In order to achieve the purpose, the invention provides the following technical scheme: a test method for relieving nonalcoholic fatty liver by activating AMPK with limonin and an application thereof comprise the following steps:

the method comprises the following steps: selecting a test animal, wherein the animal is a C57BL/6 mouse;

step two: grouping of test animals, the test animals were randomly divided into 4 groups: a normal diet control group, a normal diet limonin treatment group, a high-fat diet building group and a high-fat diet limonin treatment group;

step three: feeding test animals and treating with medicine, feeding test animals with normal diet NCD feed or high fat diet HFD feed, and optionally adding limonin;

step four: and (3) test detection, namely sampling test animals, detecting physiological and metabolic parameters of the animals, comparing the physiological and metabolic parameters, and comparing the influence of the limonin on the pathological physiological state of the liver of the mouse.

Preferably, in step one, the animals are selected from a plurality of C57BL/6 mice, the test C57BL/6 mice are 8 weeks old, the mice are weighed and the data are recorded, NCD feed, HFD feed and limonin are prepared, and the test devices are sterilized before use.

Preferably, in step two, 10 mice in the normal diet control group C57 BL/6;

10 mice in the normal diet limonin-treated group C57 BL/6;

10 mice were made on high fat diet model C57 BL/6;

high fat diet limonin treated group 10 mice, C57 BL/6.

Preferably, in step three, 10 mice in the normal diet control group C57BL/6 were fed with NCD feed for 10 weeks, and daily food intake was recorded;

the normal diet limonin treated group C57BL/6 mice were fed NCD feed for 10 weeks with daily food intake recorded and at week 2 mice were orally administered limonin 50mg/kg daily;

feeding 10 mice with HFD feed for 10 weeks in a high fat diet modeling group C57BL/6, inducing fatty liver in C57BL/6 mice, and recording daily food intake;

high fat diet limonin treated group 10 mice C57BL/6 were fed HFD feed for 10 weeks, induced fatty liver in C57BL/6 mice, daily food intake was recorded, and mice were orally administered limonin 50mg/kg daily at week 2.

Preferably, in step four, the change of animal index is detected, and the probability comparison is carried out on the food intake, the weight curve, the weight gain, the weight of epididymal white adipose tissues and subcutaneous white adipose tissues and the liver weight index of the C57BL/6 mouse, so as to compare the influence of the citric acid on the whole metabolism of the C57BL/6 mouse;

h & E staining of liver tissue of C57BL/6 mice, serum alkaline phosphatase ALP level, serum alanine aminotransferase ALT level, serum aspartate aminotransferase AST level of C57BL/6 mice were examined to compare the effect of limonin on liver of C57BL/6 mice.

The invention has the technical effects and advantages that:

according to the invention, through the test mode that the mice fed with the C57BL/6 limonin are fed with the normal calorie feed or the high-fat diet, the food intake amount between the control group and the limonin treated group is not different, and the mice fed with the normal feed have the advantages that the limonin treated high-fat diet can cause obvious weight reduction, while the mice fed with the normal feed have no obvious weight difference with or without the limonin added in the feed, so that the increase of the liver weight induced by the high-fat diet feeding can be improved through the limonin treatment.

Drawings

FIG. 1 is a schematic diagram of the chemical structure of limonin (Lim) of the present invention.

FIG. 2 is a schematic representation of the HFD-induced fatty liver and Lim dosing regimen of the present invention.

FIG. 3 is data of food intake for mice fed with different diets and treated with drugs according to the invention.

FIG. 4 is a graph of body weight of mice fed with different diets and treated with drugs according to the invention.

Figure 5 is a graph of body weight gain data for mice fed different diets and drug treated according to the invention.

FIG. 6 shows the white adipose tissue weight data of epididymis of mice fed with different diets and treated with different drugs according to the invention.

FIG. 7 shows subcutaneous white adipose tissue weight data of mice fed with different diets and treated with drugs according to the invention.

FIG. 8 is data of liver weights of mice fed with different diets and treated with drugs according to the invention.

FIG. 9 is data of serum alkaline phosphatase levels of mice fed with different diets and treated with drugs according to the invention.

FIG. 10 is a graph showing serum alanine aminotransferase levels for various diet-fed and drug-treated mice in accordance with the present invention.

FIG. 11 is a graph showing serum aspartate transaminase level data for various diet-fed and drug-treated mice of the present invention.

FIG. 12 is a representative image of H & E staining of livers of mice fed with different diets and treated with drugs according to the invention.

FIG. 13 shows the H & E staining histological characteristics NAS quantitative evaluation data of liver sections of mice fed with different diets and treated with drugs according to the invention.

FIG. 14 is data of total liver cholesterol (TC) levels for various diet-fed and drug-treated mice of the present invention.

FIG. 15 is data of total hepatic Triglyceride (TG) levels in mice fed different diets and drug treated according to the invention.

FIG. 16 is a representative image of oil-red O staining of liver tissue sections of mice fed with different diets and treated with drugs according to the invention.

FIG. 17 is a statistical data of the oil red O staining quantification of the present invention.

FIG. 18 shows data on the expression of fatty acid synthesis genes in liver tissues of mice according to the present invention.

FIG. 19 shows data on the expression of cholesterol synthesis genes in liver tissues of mice according to the present invention.

FIG. 20 is a graph showing the results of Western blot analysis of the protein levels of phospho-AMPK, phospho-ACC and ACC in liver tissues of mice fed with various foods and treated with drugs according to the present invention.

FIG. 21 is a quantitative statistical representation of the results of FIG. 20 according to the present invention.

Figure 22Lim data for dose and time dependent increase of AMPK activity in AML12 cells.

Fig. 23 is a quantitative statistical data of Lim activating AMPK in a dose-dependent manner in fig. 22 of the present invention.

FIG. 24 is a graph showing the quantitative statistics of Lim activating AMPK in a time-dependent manner in FIG. 22 according to the present invention

FIG. 25 is a graph showing representative results of protein level measurements of phospho-AMPK and phospho-ACC after treatment with Palmitic Acid (PA) and/or Lim of the liver cell line AML12 of the present invention.

FIG. 26 is a quantitative statistical representation of the results of FIG. 25 in accordance with the present invention.

Fig. 27 is a representative image of oil red O staining to counteract the Lim lipid accumulation reducing effect in AML12 cells using the AMPK inhibitor Comp C of the present invention.

FIG. 28 is a graph showing intracellular TG levels measured by a colorimetric enzymatic method according to the present invention.

FIG. 29 is a graph showing intracellular TC levels measured by a colorimetric enzymatic method according to the present invention.

Fig. 30 is a representative image of oil red O staining according to the present invention using an inactivated AMPK expression plasmid to inhibit AMPK activity in AML12 cells, thereby counteracting the lipid accumulation reducing effect of Lim.

FIG. 31 shows the intracellular TG levels measured by the colorimetric enzymatic method according to the present invention.

FIG. 32 is a graph showing intracellular TC levels measured by a colorimetric enzymatic method according to the present invention.

Fig. 33 is a representative image of oil red O staining of the effect of Lim treatment of the invention on nematode lipid accumulation.

The diagram is a schematic diagram of the lipid-lowering effect of AMPK-DN on elimination of Lim.

FIG. 34 is a graph showing TC levels in nematodes assayed by the colorimetric enzymatic method of the present invention.

FIG. 35 is a graph showing TG levels in nematodes measured by a colorimetric enzymatic method according to the present invention.

FIG. 36 shows the result of Western blotting of protein kinase and phosphatase abundance or activation in upstream of AMPK in Lim-treated HepG2 cells of the present invention.

FIG. 37 is a representative result of the detection of AMPK activation at different times of Lim processing after transferring the HepG2 cell of the present invention into wild-type or R531G mutated AMPK γ 2.

FIG. 38 is a quantitative statistical data of the results of FIG. 37 according to the present invention.

FIG. 39 is a graph showing the change in the ADP to ATP ratio in HepG2 cells after treatment with different concentrations of Lim according to the invention.

FIG. 40 is a representative image of SREBP1c immunofluorescent staining in AML cells of the present invention.

FIG. 41 is a representative image of SREBP2 immunofluorescent staining in AML cells of the present invention.

FIG. 42 is a quantitative statistical result of fluorescence intensity of SREBP1 and SREBP2 of the present invention in nucleus versus cytoplasm.

FIG. 43 shows the results of RT-PCR assay of the mRNA expression levels of Fasn, Acc1 genes in AML cells according to the present invention.

FIG. 44 shows the results of RT-PCR assay of the present invention for the expression level of mRNA of Hmgcs and Hmgcr genes in AML cells.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention provides a method for activating AMPK to relieve nonalcoholic fatty liver by limonin, which comprises the following steps:

In the first step, a plurality of C57BL/6 mice are selected, the age of the C57BL/6 mice is 8 weeks, the mice are weighed and recorded, NCD feed, HFD feed and limonin are prepared, and test devices are subjected to sterile treatment before use.

In the second step, 10 mice in the control group C57BL/6 of normal diet are treated;

10 mice in the normal diet limonin-treated group C57 BL/6;

10 mice were made on high fat diet model C57 BL/6;

high fat diet limonin treated group 10 mice, C57 BL/6.

In step three, 10 mice in the normal diet control group C57BL/6 were fed with NCD feed for 10 weeks, and daily food intake was recorded;

In the fourth step, the change of the animal index is detected, and the probability comparison is carried out on the food intake, the weight curve, the weight gain, the weight of the epididymal white adipose tissues, the weight of subcutaneous white adipose tissues and the liver weight index of the C57BL/6 mouse, so as to compare the influence of the citric acid on the whole metabolism of the C57BL/6 mouse;

h & E staining of liver tissue of C57BL/6 mice, measurement of serum alkaline phosphatase ALP level, serum alanine aminotransferase ALT level, and serum aspartate aminotransferase AST level of C57BL/6 mice were performed to compare the effect of limonic acid on liver of C57BL/6 mice.

Fig. 1 to 8 show that limonin inhibits HFD-induced increases in body weight, fat weight and liver weight in mice, fig. 1 shows the chemical structure of Lim, whose molecular weight is 470.53, fig. 2 shows the administration of HFD-induced fatty liver and Lim, C57BL/6 mice were fed with both NCD and HFD feeds for 10 weeks, respectively, to induce fatty liver in mice, fig. 3 shows the intake of food, fig. 4 shows the body weight profile, fig. 5 shows the weight increase, fig. 6 and 7 show the weight of epididymal white adipose tissue and subcutaneous white adipose tissue, fig. 8 shows the liver weight, and the data are expressed as mean ± SD (n ═ 8).

Fig. 9-13 are plots of Lim effect on liver function in HFD mice, fig. 9 is serum alkaline phosphatase (ALP) levels, fig. 10 is serum alanine Aminotransferase (ALT) levels, fig. 11 is serum aspartate Aminotransferase (AST) levels, fig. 12 is a representative image of H & E staining of various groups of livers, fig. 13 is 3 histological feature NAS quantification of H & E stained liver sections, NAFLD activity score, steatosis 0-3, inflammation 0-3, hepatocyte ballooning 0-2.

Fig. 14-19 are images of Lim inhibiting high fat diet induced liver lipid accumulation in mice, fig. 14 is liver TC levels, fig. 15 is liver TG levels, and fig. 16 is a representative image of oil red O staining of liver sections of each group. The scale bar is 300 μm, fig. 17 is the quantitative statistics of the oil red O stained area, and fig. 18 and 19 are the detection of the mRNA levels of the genes involved in fatty acid synthesis and cholesterol synthesis by real-time PCR, respectively.

FIGS. 20-26 show that Lim is effective in activating AMPK both in vivo and in vitro. FIG. 20 is a Western blotting assay of the protein abundance of phospho-AMPK and phospho-ACC in the liver of HFD-induced mice. FIG. 22 is a graph of Western blotting assays for Lim increasing AMPK activity in a dose and time dependent manner in AML12 cells. FIG. 25 shows AML12 cells after 24 hours of starvation in serum-free DMEM medium and treated with DMSO solution, 0.4mM PA + 50. mu.M Lim or 0.4mM PA + 100. mu.M Lim for 16 hours, respectively. Western blot detects changes in phospho-AMPK and phospho-ACC. Fig. 21, fig. 23 and 24, and fig. 26 are the quantitative statistical results of fig. 20, fig. 22, and fig. 25, respectively.

Figures 27-32AMPK activation is required for Lim-mediated reduction in hepatocyte lipid accumulation. After starving AML12 cells for 24 hours in serum-free DMEM, they were treated with DMSO solution, 0.4mM PA +100 μ M Lim or 0.4mM PA +100 μ M Lim +1 μ M Comp C, respectively, for 16 hours, fig. 27 is a representative image of oil red O cell staining, scale bar 300 μ M, fig. 28 and 29 are the intracellular TG and TC levels determined by the colorimetric enzymatic method. FIG. 30, FIG. 31 and FIG. 32 AML12 expressing inactive AMPK plasmid (AMPK-DN) was treated with PA and Lim, and intracellular TG, TC were measured by oil-Red-O staining and colorimetric enzymatic methods, and AMPK-DN was found to counteract the lipid-lowering effect of Lim.

FIGS. 33-35 are graphs demonstrating that AMPK is required for reduction of Lim-induced fat deposition by knockout C.elegans. Nematodes of N2 (wild type) and aak-2(AMPK knock-out) were treated with Lim (100 μ M) for 7 days, fig. 33 is a representative image of nematode oil red O staining at a scale bar of 300 μ M, and fig. 34 and 35 are colorimetric enzymatic determinations of the TC and TG levels of the nematodes.

FIGS. 36-39 show that the activation of AMPK by Lim is adenine nucleotide dependent. The HepG2 cells were treated with different concentrations of Lim for 4 hours, fig. 36 shows that changes in abundances or activities of protein kinases and phosphatases upstream of AMPK were detected by Western blotting, fig. 37, 38 and 39 show that HepG2 cells expressing wild-type (WT) or R531G (RG) AMPK γ 2 plasmid were treated with Lim, and Western blotting detected AMPK activation and ACC phosphorylation.

FIGS. 40-44 show that Lim inhibits the transcriptional activity of SREBP1c and SREBP2 in hepatocytes by AMPK. After starving AML12 cells for 24 hours in serum-free DMEM, they were treated with DMSO solution, 0.4mM PA +100 μ M Lim or 0.4mM PA +100 μ M Lim +1 μ M Comp C for 16 hours, respectively, fig. 40 and 41 are representative pictures of SREBP1C and SREBP2 immunofluorescent stained cells, scale bar 300 μ M, fig. 42 is quantitative statistical data of fluorescence intensity of SREBP1C and SREBP2 in the nucleus versus fluorescence intensity of the cytoplasm, fig. 43 and 44 are RT-PCR assay for fas, Acc1, hcr, hcgcs gene mRNA expression levels.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments or portions thereof without departing from the spirit and scope of the invention.

Claims

1. the test method that limonin activates AMPK to relieve non-alcoholic fatty liver, is characterized in that, comprises the following steps:

Step 1: Selection of test animals, C57BL/6 mice were selected as animals;

Step 2: Grouping of test animals, the test animals were randomly divided into 4 groups: normal diet control group, normal diet limonin treatment group, high-fat diet modeling group, high-fat diet limonin treatment group;

Step 3: Feeding and drug treatment of experimental animals, feeding the experimental animals with NCD feed or high-fat diet HFD feed, with or without limonin;

Step 4: Test detection, sampling the test animals, detecting and comparing the physiological and metabolic parameters of the animals, and comparing the effect of limonin on the pathophysiological state of the mouse liver.

2. the test method that limonin according to claim 1 activates AMPK to alleviate non-alcoholic fatty liver disease is characterized in that:

In step 1, the animal selects several C57BL/6 mice, and the age of the test C57BL/6 mice is 8 weeks old, and weighs them to record data, prepare NCD feed, HFD feed and limonin, and use the test equipment. Aseptic processing is required before.

3. the test method that limonin according to claim 1 activates AMPK to relieve non-alcoholic fatty liver, is characterized in that:

In step 2, there were 10 C57BL/6 mice in the normal diet control group;

10 C57BL/6 mice in the normal diet limonin-treated group;

10 C57BL/6 mice in the high-fat diet model group;

There were 10 C57BL/6 mice in the high-fat diet limonin-treated group.

4. the test method that limonin according to claim 1 activates AMPK to relieve non-alcoholic fatty liver, is characterized in that:

In step 3, 10 C57BL/6 mice in the normal diet control group were fed NCD feed for 10 weeks, and the daily food intake was recorded;

The 10 C57BL/6 mice in the normal diet limonin-treated group were fed NCD diet for 10 weeks, and the feeding weight of the 10 C57BL/6 mice in the normal control group 2 was recorded, and the mice were orally administered limonin every day in the second week. 50mg/kg;

10 C57BL/6 mice in the high-fat diet model group were fed with HFD diet for 10 weeks to induce fatty liver in C57BL/6 mice, and the feeding weight of 10 C57BL/6 mice in three groups of normal control groups was recorded;

10 C57BL/6 mice in the high-fat diet limonin-treated group were fed with HFD for 10 weeks to induce fatty liver in C57BL/6 mice, and the food weight of 10 C57BL/6 mice in the four normal control groups was recorded. For 2 weeks, mice were orally administered limonoid 50 mg/kg daily.

5. the test method that limonin according to claim 1 activates AMPK to relieve non-alcoholic fatty liver, is characterized in that:

In step 4, the change of animal index was detected, and the food intake, body weight curve, body weight gain, epididymal white adipose tissue and subcutaneous white adipose tissue weight, and liver weight index of C57BL/6 mice were compared with probability, so as to compare lemon bitterness. The effect of acid on the overall metabolism of C57BL/6 mice;

The liver of C57BL/6 mice was detected, the serum alkaline phosphatase ALP level, the serum alanine aminotransferase ALT level, and the serum aspartate aminotransferase AST level were detected in the liver of C57BL/6 mice, so as to compare the citric acid Effects on the liver of C57BL/6 mice.