CN113456649A - Method for relieving non-alcoholic fatty liver by activating AMPK with limonin - Google Patents
Method for relieving non-alcoholic fatty liver by activating AMPK with limonin Download PDFInfo
- Publication number
- CN113456649A CN113456649A CN202110864166.8A CN202110864166A CN113456649A CN 113456649 A CN113456649 A CN 113456649A CN 202110864166 A CN202110864166 A CN 202110864166A CN 113456649 A CN113456649 A CN 113456649A
- Authority
- CN
- China
- Prior art keywords
- limonin
- mice
- c57bl
- diet
- group
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- KBDSLGBFQAGHBE-MSGMIQHVSA-N limonin Chemical compound C=1([C@H]2[C@]3(C)CC[C@H]4[C@@]([C@@]53O[C@@H]5C(=O)O2)(C)C(=O)C[C@@H]2[C@]34COC(=O)C[C@@H]3OC2(C)C)C=COC=1 KBDSLGBFQAGHBE-MSGMIQHVSA-N 0.000 title claims abstract description 67
- VHLJDTBGULNCGF-UHFFFAOYSA-N Limonin Natural products CC1(C)OC2CC(=O)OCC23C4CCC5(C)C(CC(=O)C6OC56C4(C)C(=O)CC13)c7cocc7 VHLJDTBGULNCGF-UHFFFAOYSA-N 0.000 title claims abstract description 66
- 102100036009 5'-AMP-activated protein kinase catalytic subunit alpha-2 Human genes 0.000 title claims abstract description 37
- 101000783681 Homo sapiens 5'-AMP-activated protein kinase catalytic subunit alpha-2 Proteins 0.000 title claims abstract description 37
- 208000008338 non-alcoholic fatty liver disease Diseases 0.000 title claims abstract description 20
- 238000000034 method Methods 0.000 title claims abstract description 14
- 230000003213 activating effect Effects 0.000 title claims abstract description 13
- 241000699670 Mus sp. Species 0.000 claims abstract description 61
- 238000011740 C57BL/6 mouse Methods 0.000 claims abstract description 51
- 238000012360 testing method Methods 0.000 claims abstract description 35
- 241001465754 Metazoa Species 0.000 claims abstract description 33
- 210000004185 liver Anatomy 0.000 claims abstract description 30
- 235000009200 high fat diet Nutrition 0.000 claims abstract description 27
- 235000021590 normal diet Nutrition 0.000 claims abstract description 24
- 230000037406 food intake Effects 0.000 claims abstract description 6
- 235000012631 food intake Nutrition 0.000 claims abstract description 6
- 235000019786 weight gain Nutrition 0.000 claims abstract description 5
- 230000004584 weight gain Effects 0.000 claims abstract description 4
- 239000003814 drug Substances 0.000 claims description 19
- 230000000694 effects Effects 0.000 claims description 15
- 231100000240 steatosis hepatitis Toxicity 0.000 claims description 11
- 208000004930 Fatty Liver Diseases 0.000 claims description 10
- 206010019708 Hepatic steatosis Diseases 0.000 claims description 10
- 210000000593 adipose tissue white Anatomy 0.000 claims description 10
- 208000010706 fatty liver disease Diseases 0.000 claims description 10
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 9
- 235000021316 daily nutritional intake Nutrition 0.000 claims description 9
- 230000002503 metabolic effect Effects 0.000 claims description 8
- 238000001514 detection method Methods 0.000 claims description 5
- 201000010063 epididymitis Diseases 0.000 claims description 5
- 238000007920 subcutaneous administration Methods 0.000 claims description 5
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 4
- 230000008859 change Effects 0.000 claims description 4
- 238000013238 high-fat diet model Methods 0.000 claims description 3
- 230000004060 metabolic process Effects 0.000 claims description 3
- 230000001575 pathological effect Effects 0.000 claims description 3
- 230000035790 physiological processes and functions Effects 0.000 claims description 3
- 238000005070 sampling Methods 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 2
- 238000010998 test method Methods 0.000 claims description 2
- 235000005911 diet Nutrition 0.000 abstract description 15
- 230000037213 diet Effects 0.000 abstract description 15
- 230000037396 body weight Effects 0.000 abstract description 5
- 230000004580 weight loss Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 20
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 18
- 229940079593 drug Drugs 0.000 description 16
- 238000010186 staining Methods 0.000 description 14
- 210000002966 serum Anatomy 0.000 description 12
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 9
- 235000021314 Palmitic acid Nutrition 0.000 description 9
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 description 9
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 9
- 238000006911 enzymatic reaction Methods 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- NPGIHFRTRXVWOY-UHFFFAOYSA-N Oil red O Chemical compound Cc1ccc(C)c(c1)N=Nc1cc(C)c(cc1C)N=Nc1c(O)ccc2ccccc12 NPGIHFRTRXVWOY-UHFFFAOYSA-N 0.000 description 7
- 230000004913 activation Effects 0.000 description 7
- 238000001262 western blot Methods 0.000 description 7
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 6
- 230000003834 intracellular effect Effects 0.000 description 6
- 230000006372 lipid accumulation Effects 0.000 description 6
- 210000005228 liver tissue Anatomy 0.000 description 6
- 102100036475 Alanine aminotransferase 1 Human genes 0.000 description 5
- 108010082126 Alanine transaminase Proteins 0.000 description 5
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 5
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 5
- 108010003415 Aspartate Aminotransferases Proteins 0.000 description 5
- 102000004625 Aspartate Aminotransferases Human genes 0.000 description 5
- 241000244206 Nematoda Species 0.000 description 5
- 102000008079 Sterol Regulatory Element Binding Protein 2 Human genes 0.000 description 5
- 108010074438 Sterol Regulatory Element Binding Protein 2 Proteins 0.000 description 5
- 238000003556 assay Methods 0.000 description 5
- 210000003494 hepatocyte Anatomy 0.000 description 4
- 108020004999 messenger RNA Proteins 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 235000012000 cholesterol Nutrition 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 235000003642 hunger Nutrition 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 238000003757 reverse transcription PCR Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 230000036962 time dependent Effects 0.000 description 3
- 101710148750 5'-AMP-activated protein kinase subunit gamma Proteins 0.000 description 2
- 206010016654 Fibrosis Diseases 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 108700019535 Phosphoprotein Phosphatases Proteins 0.000 description 2
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 2
- 102000001253 Protein Kinase Human genes 0.000 description 2
- 101100373202 Rattus norvegicus Cx3cl1 gene Proteins 0.000 description 2
- 102000009822 Sterol Regulatory Element Binding Proteins Human genes 0.000 description 2
- 108010020396 Sterol Regulatory Element Binding Proteins Proteins 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 208000019425 cirrhosis of liver Diseases 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 230000004136 fatty acid synthesis Effects 0.000 description 2
- 238000003125 immunofluorescent labeling Methods 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000037356 lipid metabolism Effects 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 108060006633 protein kinase Proteins 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 230000001603 reducing effect Effects 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000007863 steatosis Effects 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 101100321927 Caenorhabditis elegans aak-2 gene Proteins 0.000 description 1
- 101150003888 FASN gene Proteins 0.000 description 1
- 238000013218 HFD mouse model Methods 0.000 description 1
- 101150053603 HMGCR gene Proteins 0.000 description 1
- 108010028554 LDL Cholesterol Proteins 0.000 description 1
- 241000341511 Nematodes Species 0.000 description 1
- 102000008078 Sterol Regulatory Element Binding Protein 1 Human genes 0.000 description 1
- 108010074436 Sterol Regulatory Element Binding Protein 1 Proteins 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N adenyl group Chemical group N1=CN=C2N=CNC2=C1N GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000007882 cirrhosis Effects 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 238000004146 energy storage Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 210000000918 epididymis Anatomy 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 230000004761 fibrosis Effects 0.000 description 1
- 229930003935 flavonoid Natural products 0.000 description 1
- 235000017173 flavonoids Nutrition 0.000 description 1
- 150000002215 flavonoids Chemical class 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 1
- 230000028974 hepatocyte apoptotic process Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 230000003908 liver function Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 206010053219 non-alcoholic steatohepatitis Diseases 0.000 description 1
- 230000007331 pathological accumulation Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 150000008442 polyphenolic compounds Chemical class 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000011158 quantitative evaluation Methods 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 230000037351 starvation Effects 0.000 description 1
- 150000003521 tetracyclic triterpenoids Chemical class 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- 239000013585 weight reducing agent Substances 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
- A61K31/585—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin containing lactone rings, e.g. oxandrolone, bufalin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
Landscapes
- Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Gastroenterology & Hepatology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The invention discloses a method for alleviating nonalcoholic fatty liver by activating AMPK with limonin, which comprises the following steps: selecting a test animal, wherein the animal is a C57BL/6 mouse; grouping of test animals, the test animals were divided into: 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; raising the test animal; and (5) testing and detecting. According to the invention, no difference in food intake amount between the control group and the limonin-treated group is found by means of experiments of feeding the C57BL/6 mice with limonin, no matter whether the mice are fed with normal calorie feed or high-fat diet. We observed that limonin-treated mice on a high-fat diet induced significant weight loss, whereas mice fed a normal diet, with or without limonin added to the diet, showed no significant difference in body weight, indicating that liver weight gain induced by high-fat diet feeding could be improved by limonin treatment.
Description
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:
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.
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;
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.
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 (5)
1. The test method for relieving the non-alcoholic fatty liver by activating AMPK with limonin is characterized by comprising 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.
2. The method of claim 1 for testing the AMPK-activated non-alcoholic fatty liver disease alleviation by limonin:
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.
3. The method of claim 1 for testing the AMPK-activated non-alcoholic fatty liver disease alleviation by limonin:
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.
4. The method of claim 1 for testing the AMPK-activated non-alcoholic fatty liver disease alleviation by limonin:
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 of 10 mice, C57BL/6, were fed NCD feed for 10 weeks and the eating weight of 10 mice in the normal control group of C57BL/6, was recorded and the mice were orally administered limonin 50mg/kg daily at week 2;
high fat diet modeling group C57BL/6 mice 10 were fed with HFD feed for 10 weeks, induced fatty liver in C57BL/6 mice, and recorded the weight of 10 mice fed with normal control group C57BL/6 mice;
high fat diet limonin treated group 10 mice C57BL/6 were fed HFD feed for 10 weeks, C57BL/6 mice were induced for fatty liver, and normal control four groups of 10 mice C57BL/6 were recorded for eating weight, and mice were orally administered limonin 50mg/kg daily at week 2.
5. The method of claim 1 for testing the AMPK-activated non-alcoholic fatty liver disease alleviation by limonin:
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;
the liver of the C57BL/6 mouse was tested, and the liver of the C57BL/6 mouse was tested for ALP level, ALT level and AST level, respectively, to compare the effect of limonic acid on the liver of the C57BL/6 mouse.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110864166.8A CN113456649A (en) | 2021-07-29 | 2021-07-29 | Method for relieving non-alcoholic fatty liver by activating AMPK with limonin |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110864166.8A CN113456649A (en) | 2021-07-29 | 2021-07-29 | Method for relieving non-alcoholic fatty liver by activating AMPK with limonin |
Publications (1)
Publication Number | Publication Date |
---|---|
CN113456649A true CN113456649A (en) | 2021-10-01 |
Family
ID=77883077
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110864166.8A Pending CN113456649A (en) | 2021-07-29 | 2021-07-29 | Method for relieving non-alcoholic fatty liver by activating AMPK with limonin |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113456649A (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115252630A (en) * | 2022-09-02 | 2022-11-01 | 深圳技术大学 | Application of obacunone in preparation of medicine for preventing, improving or treating non-alcoholic fatty liver disease |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9066965B1 (en) * | 2012-09-28 | 2015-06-30 | The United States of Americas, as represented by the Secretary of Agriculture | Purified limonin glucoside for prevention and treatment of chronic diseases |
-
2021
- 2021-07-29 CN CN202110864166.8A patent/CN113456649A/en active Pending
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9066965B1 (en) * | 2012-09-28 | 2015-06-30 | The United States of Americas, as represented by the Secretary of Agriculture | Purified limonin glucoside for prevention and treatment of chronic diseases |
Non-Patent Citations (3)
Title |
---|
DARSHAN S.KELLEY等: "Citrus limonin glucoside supplementation decreased biomarkers of liver disease and inflammation in overweight human adults", 《JOURNAL OF FUNCTIONAL FOODS》 * |
DEBASISH HALDER等: "Cyclodextrin-clathrated limonin suppresses diet-induced obesity in mice", 《JOURNAL OF FOOD BIOCHEMISTRY》 * |
MARINELLA等: "Protective effects of bergamot(citrus bergamia risso & poiteau)juice in rats fed with high-fat diet", 《PLANTA MED》 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115252630A (en) * | 2022-09-02 | 2022-11-01 | 深圳技术大学 | Application of obacunone in preparation of medicine for preventing, improving or treating non-alcoholic fatty liver disease |
CN115252630B (en) * | 2022-09-02 | 2024-02-09 | 深圳技术大学 | Application of phellodendron ketone in preparing medicine for preventing, improving or treating non-alcoholic fatty liver disease |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Cheng et al. | Diosgenin prevents high-fat diet-induced rat non-alcoholic fatty liver disease through the AMPK and LXR signaling pathways | |
Guo et al. | Beneficial effects of mangiferin on hyperlipidemia in high‐fat‐fed hamsters | |
Dyle et al. | Systems-based discovery of tomatidine as a natural small molecule inhibitor of skeletal muscle atrophy | |
Moreno et al. | Inhibitory effects of grape seed extract on lipases | |
Chung et al. | Anti-inflammatory effects of (Z)-ligustilide through suppression of mitogen-activated protein kinases and nuclear factor-κB activation pathways | |
Yang et al. | Upregulation of SIRT1-AMPK by thymoquinone in hepatic stellate cells ameliorates liver injury | |
Bezerra et al. | Caffeic acid phenethyl ester reduces the activation of the nuclear factor κB pathway by high-fat diet-induced obesity in mice | |
Choi et al. | Dieckol, a major phlorotannin in Ecklonia cava, suppresses lipid accumulation in the adipocytes of high‐fat diet‐fed zebrafish and mice: Inhibition of early adipogenesis via cell‐cycle arrest and AMPKα activation | |
Mu et al. | Betulinic acid improves nonalcoholic fatty liver disease through YY1/FAS signaling pathway | |
Kwon et al. | Acacetin enhances glucose uptake through insulin-independent GLUT4 translocation in L6 myotubes | |
Lee et al. | Alpha-lipoic acid attenuates atherosclerotic lesions and inhibits proliferation of vascular smooth muscle cells through targeting of the Ras/MEK/ERK signaling pathway | |
Choi et al. | Downregulation of fetuin-B and zinc-α2-glycoprotein is linked to impaired fatty acid metabolism in liver cells | |
Sim et al. | Long-term supplementation of esculetin ameliorates hepatosteatosis and insulin resistance partly by activating AdipoR2–AMPK pathway in diet-induced obese mice | |
Kim et al. | Methylene chloride fraction of the leaves of Thuja orientalis inhibits in vitro inflammatory biomarkers by blocking NF-κB and p38 MAPK signaling and protects mice from lethal endotoxemia | |
Lu et al. | Nrf2 induces lipocyte phenotype via a SOCS3-dependent negative feedback loop on JAK2/STAT3 signaling in hepatic stellate cells | |
Liu et al. | Quercetin oppositely regulates insulin‐mediated glucose disposal in skeletal muscle under normal and inflammatory conditions: The dual roles of AMPK activation | |
Minxuan et al. | Fisetin attenuates high fat diet-triggered hepatic lipid accumulation: A mechanism involving liver inflammation overload associated TACE/TNF-α pathway | |
Yang et al. | Silibinin improves nonalcoholic fatty liver by regulating the expression of miR‑122: An in vitro and in vivo study | |
Ho et al. | Anti-atherosclerotic action of Ger-Gen-Chyn-Lian-Tang and AMPK-dependent lipid lowering effect in hepatocytes | |
Liu et al. | Polydatin down-regulates the phosphorylation level of STAT3 and induces pyroptosis in triple-negative breast cancer mice with a high-fat diet | |
Zhang et al. | Calcium supplementation relieves high-fat diet-induced liver steatosis by reducing energy metabolism and promoting lipolysis | |
Ma et al. | Rutin promotes white adipose tissue “browning” and brown adipose tissue activation partially through the calmodulin-dependent protein kinase kinase β/AMP-activated protein kinase pathway | |
CN113456649A (en) | Method for relieving non-alcoholic fatty liver by activating AMPK with limonin | |
Bahari | Effect of Elateriospermum tapos extract as coadjuvant in ameliorating maternal obesity on female offspring at weaning | |
Chen et al. | PKM2 aggravates palmitate-induced insulin resistance in HepG2 cells via STAT3 pathway |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
RJ01 | Rejection of invention patent application after publication | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20211001 |