WO2020192233A1 - 穿心莲内酯在制备脂代谢异常相关疾病药物中的应用 - Google Patents

穿心莲内酯在制备脂代谢异常相关疾病药物中的应用 Download PDF

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WO2020192233A1
WO2020192233A1 PCT/CN2019/130839 CN2019130839W WO2020192233A1 WO 2020192233 A1 WO2020192233 A1 WO 2020192233A1 CN 2019130839 W CN2019130839 W CN 2019130839W WO 2020192233 A1 WO2020192233 A1 WO 2020192233A1
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andrographolide
expression
genes
liver
secretion
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管敏
张敏仪
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Shenzhen Institute of Advanced Technology of CAS
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    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics

Definitions

  • the invention belongs to the technical field of biomedicine, and specifically relates to an application of andrographolide in the preparation of drugs for diseases related to abnormal lipid metabolism.
  • Abnormal lipid metabolism refers to the abnormality of lipids and their metabolic products in plasma and other tissues and organs, often accompanied by elevated cholesterol or triglyceride (TG) levels, and can be divided into primary and secondary categories.
  • Lipid metabolism disorder is a key influencing factor of non-alcoholic fatty liver (NAFLD), atherosclerosis (AS), cardiovascular and cerebrovascular diseases and diabetes.
  • NAFLD refers to the pathological syndrome of excessive fat deposition in liver cells caused by other factors besides alcohol.
  • the incidence of NAFLD in my country has been increasing year by year and is becoming younger.
  • the detection rate of NAFLD in obese children and adolescents is reported to be as high as 23.3% to 59.6%, which is significantly higher than abroad. How to control abnormal blood lipid metabolism and stop the rapid growth of cardiovascular and cerebrovascular diseases has become a serious challenge facing my country's public health.
  • lipid-lowering drugs have become one of the hot spots in drug research in recent years.
  • the clinically used hypolipidemic drugs mainly include statins, fibrates, niacins, bile acid chelating agents, etc.
  • statins have the best therapeutic effect and can effectively reduce low-density lipoprotein cholesterol (LDL-C) and triacylglycerol (TG) in the serum.
  • LDL-C low-density lipoprotein cholesterol
  • TG triacylglycerol
  • the side effect is that large doses will produce myotoxicity and increase liver transaminases.
  • Fibrates have a significant triglyceride-lowering effect, which may cause gastrointestinal discomfort and kidney function damage.
  • niacin performs well in raising high-density lipoprotein (HDL), which may cause gastrointestinal discomfort and liver and kidney side effects.
  • the cholesterol absorption inhibitor etimibe and the bile acid chelator colesevelam mainly reduce LDL-C, but these drugs also have certain adverse reactions, such as facial or glossopharyngeal edema, and fat-soluble vitamin deficiency.
  • Andrographolide is the main active ingredient of Andrographis paniculata.
  • Andrographis paniculata is the whole plant or leaf of Andrographis (Andrographis). Also known as spring lotus and autumn willow, bliss at first sight, olive seed lotus, bitter gall grass, golden vanilla, golden ear hook, Indian grass and bitter grass. It has the effects of clearing away heat, detoxifying, reducing inflammation, reducing swelling and pain. Indications of bacterial dysentery, urinary tract infections, acute tonsillitis, enteritis, pharyngitis, pneumonia and influenza, external use can treat boils, swelling, poisoning and trauma infections.
  • Andrographis paniculata It is widely used in many countries in Asia to treat viral infections, diabetes, rheumatoid arthritis, pharyngitis and diarrhea.
  • Andrographis paniculata With the in-depth research on Andrographis paniculata, it has recently been reported that Andrographis paniculata and its analogues andrographis paniculata show good cancer treatment (Satyanarayana et al., Science 2003, 299, 363-370), and are used in the treatment or prevention of AIDS and Alzheimer’s disease.
  • Progress has been made in Haimer's disease and hepatitis.
  • andrographolide Although there are a small amount of research reports about andrographolide in regulating lipid metabolism disorders, there has been no research on the pharmacological mechanism of andrographolide for regulating lipid metabolism disorders. Report.
  • the purpose of the present invention is to overcome the above-mentioned shortcomings of the prior art and provide an application of andrographolide in the preparation of drugs for diseases related to abnormal lipid metabolism, so as to solve the problem that andrographolide is currently only used for anti-tumor and anti-viral and current There are technical problems with relatively large side effects of drugs related to lipid metabolism disorders.
  • one aspect of the present invention provides the application of andrographolide in at least one of the following aspects:
  • Another aspect of the present invention provides a drug for inhibiting lipid synthesis or reducing the formation and secretion of very low density lipoprotein.
  • the medicine includes an effective dose of andrographolide.
  • a method for interfering with the action of HNF4 ⁇ and PGC-1 ⁇ includes the step of contacting an effective dose of andrographolide or the drug used for inhibiting lipid synthesis or reducing the formation and secretion of very low density lipoproteins with cells expressing HNF4 ⁇ .
  • the method includes the step of contacting an effective dose of andrographolide or the drug used for reducing the formation and secretion of very low-density lipoprotein with cells expressing MTP, APOB and PLA2G12B.
  • a method for down-regulating the expression of genes SREBP-1, FASN and SCD-1 includes the step of contacting an effective dose of andrographolide or the drug for inhibiting lipid synthesis of the present invention with cells expressing SREBP-1, FASN, and SCD-1.
  • the andrographolide can down-regulate the expression of genes including FASN, SCD-1, and SREBP-1, and at the same time interfere with the interaction of HNF4 ⁇ and PGC-1 ⁇ , down-regulate the expression of MTP, APOB and PLA2G12B, thereby reducing lipids Synthesize and/or reduce the formation and secretion of very low-density lipoprotein.
  • using andrographolide as a regulator to reduce lipid synthesis and/or reduce the formation and secretion of very low-density lipoprotein can effectively down-regulate SREBP1c, FASN, and SCD1 Expression can reduce lipid synthesis, and can also inhibit the formation and secretion of very low-density lipoproteins.
  • interference with HNF4 ⁇ and PGC-1 ⁇ interaction and down-regulation include gene down-regulation of MTP, APOB and PLA2G12B expression.
  • the medicine used to inhibit lipid synthesis or reduce the formation and secretion of very low density lipoprotein contains an effective dose of andrographolide
  • the medicine can down-regulate the expression of lipid metabolism-related genes, thereby reducing lipid synthesis and extreme Low-density lipoprotein is formed and secreted to reduce blood lipids and lipid content in the liver, and ultimately play a role in preventing and/or treating diseases related to abnormal lipid metabolism.
  • andrographolide is an effective ingredient of natural medicine, it has good curative effect and small side effects.
  • Fig. 1 is a graph showing the influence of AP on HNF4 ⁇ -LBD activity and VLDL secretion-related gene promoter transcription activity in Example 1.
  • Fig. 1-A is a graph showing the influence of AP on HNF4 ⁇ -LBD activity
  • Fig. 1-B is a graph showing the influence of AP on HNF4 ⁇ -LBD activity
  • Figure 2-C shows the effect of AP on the transcription activity of the MTP promoter
  • Figure 3 is a graph showing the effect of different concentrations of andrographolide on lipid droplets in primary mouse hepatocytes in Example 3.
  • Figure 3-A is a graph of fluorescent staining of lipid droplets in primary hepatocytes of AP mice;
  • Figure 3-B Is a statistical histogram of intracellular lipid droplets in the control and experimental groups;
  • Figure 5 is a graph showing the effect of AP on the blood lipids of high-fat mice in the normal group, high-fat group, and high-fat drug-added group in Example 5; wherein, Figures 5-A to 5-D are CD mice, HFD mice, and The levels of triglycerides (TG), cholesterol (TC), high-density lipoprotein (HCL-C), and low-density lipoprotein (LDL-C) in the serum of HFD+AP mice.
  • Figure 5-E shows CD mice, Changes in serum triglyceride (TG) levels in HFD mice and HFD+AP mice.
  • Figure 5-F shows the detection of VLDL secretion rates in CD mice, HFD mice, and HFD+AP mice.
  • FIG. G ApoB levels in the serum of CD mice, HFD mice and HFD+AP mice.
  • Figure 6 shows the effect of AP on the liver tissue of high-fat mice in the normal group, high-fat group, and high-fat drug-added group in Example 5; among them, Figure 6-A shows CD mice, HFD mice and HFD+AP The levels of triglycerides (TG) in the liver of mice.
  • Figures 6-B to 6-C show the levels of ALT and AST in the serum of CD mice, HFD mice and HFD+AP mice.
  • Figure 6-D shows CD mice. , HFD mice and HFD+AP mice liver and oil red, HE staining; and *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001. #P ⁇ 0.05, ##P ⁇ 0.01, ###P ⁇ 0.001.
  • Andrographolide It is an active ingredient extracted from the plant Andrographolide.
  • the results of the medicinal properties and toxicity test show that in the acute toxicity test, the LD50 of andrographolide in mice is above 40g/kg. The daily maximum tolerated dose is 110.25g/kg.
  • rats or rabbits were given andrographolide 1g/kg once a day for 7 consecutive days, and no obvious toxicity was seen. Chuanhuning injection was injected intraperitoneally at 84 mg/kg once a day for 10 consecutive days without obvious toxicity.
  • Hepatocyte Nuclear Factor 4 ⁇ (HNF4 ⁇ ): It is a conservative ligand-dependent transcription factor of the nuclear hormone receptor superfamily. It is mainly expressed in the liver, kidney, and intestine.
  • PHA2G12B Secreted phospholipase A2G12B
  • PGC-1 ⁇ Coactivator-1 ⁇ (PPAR ⁇ coactivator-1 ⁇ )
  • FASN Fatty acid synthase (Fatty acid synthase)
  • SCD-1 Stearoyl Coenzyme A1
  • ACAC ⁇ Acetyl-CoA carboxylase ⁇
  • MTP microsomal triglyceride transfer protein (microsimal triglyceride transfer protein)
  • VLDL Very Low Density Lipoprotein
  • Abnormal lipid metabolism It is the abnormality of lipid and its metabolic products and amount in blood and other tissues and organs caused by congenital or acquired factors. The specific symptoms mainly include hyperlipoproteinemia.
  • the inventors of the present invention have discovered based on a large number of studies that andrographolide can down-regulate the expression of lipid metabolism-related genes to reduce blood lipids and liver fat and other related effects. Based on this, the embodiments of the present invention provide the following related aspects of andrographolide application.
  • the embodiments of the present invention provide applications of andrographolide in at least one of the following aspects:
  • the andrographolide can effectively down-regulate the expression of lipid metabolism-related genes, thereby inhibiting or reducing the lipid synthesis process, and inhibiting or reducing the formation and secretion process of very low density lipoprotein , And finally achieve the effect of reducing liver fat and blood lipids, thereby improving diseases related to abnormal lipid metabolism.
  • the andrographolide has the effect of down-regulating the expression of genes including SREBP-1, FASN, and SCD-1. Therefore, the andrographolide can be used to down-regulate the expression of genes including SREBP-1, FASN, and SCD-1, and further can be used to prepare related drugs that down-regulate the expression of genes including SREBP-1, FASN, and SCD-1 ,
  • To effectively down-regulate lipid synthesis such as inhibiting the expression of related genes in the process of liver lipid synthesis, so as to inhibit lipid synthesis, such as inhibiting liver lipid synthesis, to reduce lipid formation in related cells such as liver cells, to achieve lipid-lowering, such as reducing liver Lipid function, thereby improving the clinical effect of diseases related to abnormal lipid metabolism.
  • HNF4 ⁇ is an important transcription factor regulating liver lipid metabolism. It is a member of the nuclear receptor superfamily. HNF4 ⁇ is highly expressed in the liver, followed by kidney, small intestine, colon and pancreatic cells. Its mode of action is: HNF4 ⁇ protein contains a zinc finger structure, which binds to DNA in the form of a homodimer, and its recognition site is the same repeating AGGTCA element, which upregulates the target by recruiting transcriptional co-activators and other auxiliary proteins. Gene expression. In liver tissue, HNF4 ⁇ is only located in the nucleus and regulates the constitutive expression of a large number of genes.
  • the proteins encoded by these genes include various enzymes, transporters, other nuclear receptors, and most of the genes specifically expressed in the liver.
  • the inventors discovered in research that the andrographolide can regulate the interaction of HNF4 ⁇ and PGC-1 ⁇ , thereby down-regulating the expression of its downstream target-related genes, where down-regulating its downstream target-related genes includes MTP, APOB and PLA2G12B.
  • the andrographolide can be used as a modulator of HNF4 ⁇ , and further can be used to prepare related drugs that regulate the interaction between HNF4 ⁇ and PGC-1 ⁇ , thereby effectively regulating the interaction between HNF4 ⁇ and PGC-1 ⁇ , Down-regulate the expression of its downstream related target genes, thereby reducing the formation and secretion of very low-density lipoproteins, and ultimately reducing the effects of liver lipids and blood lipids, thereby improving the clinical effects of diseases related to abnormal lipid metabolism.
  • the andrographolide has the effect of down-regulating the expression of HNF4 ⁇ /PGC-1 ⁇ downstream related target genes including MTP, APOB, PLA2G12B and the like. Therefore, in a specific embodiment, the application of andrographolide as a regulator that down-regulates the expression of genes including MTP, APOB, and PLA2G12B, and further can be used to prepare related drugs that regulate the interaction between HNF4 ⁇ and PGC-1 ⁇ , so as to reduce The formation and secretion of very low-density lipoprotein ultimately reduces the effects of liver lipids and blood lipids, thereby improving the clinical effects of diseases related to abnormal lipid metabolism.
  • the andrographolide can effectively down-regulate the expression of lipid metabolism-related genes, thereby reducing lipid synthesis and inhibiting the formation and secretion of very low-density lipoproteins
  • the andrographolide can be used for the preparation of prevention and/ Or drugs for treating diseases related to abnormal lipid metabolism, and further used for preparing drugs for inhibiting lipid synthesis or drugs for reducing the formation and secretion of very low density lipoproteins.
  • the andrographolide can be used to prepare drugs that down-regulate the expression of genes including SREBP-1, FASN, and SCD-1, used to prepare drugs that regulate the interaction of HNF4 ⁇ and PGC-1 ⁇ , and used To prepare drugs that down-regulate the expression of genes including MTP, APOB and PLA2G12B.
  • the corresponding embodiment of the present invention also provides a drug for preventing and/or treating diseases related to abnormal lipid metabolism, and further provides a drug for inhibiting lipid synthesis or reducing the formation and secretion of very low density lipoprotein.
  • a drug that down-regulates the expression of genes including SREBP-1, FASN and SCD-1, a drug that regulates the interaction of HNF4 ⁇ and PGC-1 ⁇ , and a drug that down-regulates the expression of genes including MTP, APOB and PLA2G12B are provided.
  • the medicine includes an effective dose of andrographolide, an active ingredient.
  • the drug may also include other active ingredients that can effectively prevent and/or treat diseases related to dyslipidemia.
  • the "effective” mentioned here is a component that alone has clinical effects in preventing and/or treating diseases related to dyslipidemia. It can also be a component that can improve andrographolide to prevent and/or treat diseases related to abnormal lipid metabolism after being compounded with andrographolide.
  • the "effective dose” refers to an effective amount capable of preventing and/or treating diseases related to abnormal lipid metabolism, and refers to an amount of andrographolide sufficient to show benefits or clinical significance to an individual.
  • the effective dose of andrographolide in the drug is 50-100 mg/kg.
  • the effective dose of andrographolide is 50-100 mg/kg, which can be specifically experimental
  • the effective oral dose for mice is 50-100mg/kg.
  • the diseases related to abnormal lipid metabolism include hyperlipidemia, non-alcoholic fatty liver disease (including simple fatty liver, non-alcoholic steatohepatitis), atherosclerosis, cardiovascular and cerebrovascular diseases, and diabetes.
  • the diseases related to abnormal lipid metabolism include hyperlipidemia, non-alcoholic fatty liver disease (including simple fatty liver, non-alcoholic steatohepatitis), atherosclerosis, cardiovascular and cerebrovascular diseases, and diabetes.
  • hyperlipidemia non-alcoholic fatty liver disease (including simple fatty liver, non-alcoholic steatohepatit
  • the drug may further include a pharmaceutically acceptable carrier (excipient) component of andrographolide.
  • the carrier component of the pharmaceutically acceptable andrographolide may be a corresponding carrier of a corresponding dosage form prepared according to the drug administration mode. As long as it is a carrier that can support the andrographolide and facilitate its stability and absorption, it is within the scope of the disclosure of the present invention. Therefore, according to the choice of the carrier, the dosage form of the drug may be at least one of an oral dosage form and an injection dosage form.
  • the drug contains the above andrographolide, the drug can effectively down-regulate the expression of lipid metabolism-related genes, specifically, it can down-regulate the expression of genes including SREBP-1, FASN, and SCD-1; at the same time, it can regulate HNF4 ⁇ and PGC-1 ⁇ interacts to down-regulate the expression of its downstream target-related genes including MTP, APOB and PLA2G12B, thereby inhibiting lipid synthesis and the formation and secretion of very low-density lipoproteins, and ultimately reducing the effects of liver lipids and blood lipids. Thereby improving the clinical effect of diseases related to abnormal lipid metabolism.
  • andrographolide is an effective ingredient of natural medicine, it has good curative effect, small side effects and safety.
  • the embodiment of the present invention provides a method for interfering with the action of HNF4 ⁇ and PGC-1 ⁇ .
  • the method includes the step of contacting an effective dose of the andrographolide or the above-mentioned drug with cells expressing HNF4 ⁇ .
  • the method can effectively interfere with the contact of HNF4 ⁇ and PGC-1 ⁇ in the cell, down-regulate the expression of its downstream related target genes, thereby reducing the formation and secretion of very low-density lipoprotein, and ultimately reducing the effect of liver lipids and the effect of reducing blood lipids , Thereby improving the clinical effect of diseases related to abnormal lipid metabolism.
  • the embodiment of the present invention also provides a method for down-regulating the expression of the genes MTP, APOB and PLA2G12B.
  • the method includes the step of contacting an effective dose of andrographolide or the above-mentioned drug with cells expressing MTP, APOB, and PLA2G12B.
  • the method can effectively reduce the formation and secretion of very low-density lipoprotein, and ultimately reduce the effect of liver fat and blood lipid, thereby improving the clinical effect of diseases related to abnormal lipid metabolism.
  • the embodiment of the present invention also provides a method for down-regulating the expression of SREBP-1, FASN, and SCD-1.
  • the method includes the step of contacting an effective dose of andrographolide or the above-mentioned drug with cells expressing SREBP-1, FASN, and SCD-1.
  • the method can effectively down-regulate lipid synthesis by regulating the expression of genes related to liver lipid synthesis, thereby inhibiting or reducing lipid synthesis, such as inhibiting liver lipid synthesis, and reducing lipid formation in related cells such as liver cells to achieve lipid reduction Such as lowering liver fat, thereby improving the clinical effect of diseases related to abnormal lipid metabolism.
  • the above-mentioned cells can be, but not only, hepatocytes and liver cancer cells, but also It is related cells in other tissues, such as related cells in kidney and intestine.
  • the andrographolide in the above embodiments can be extracted from the medicinal materials of the sacred grassland according to the existing conventional methods, of course, it is also possible to design a new extraction method to extract from the medicinal materials of the sacred grassland.
  • mice 293T cells were seeded on 96 plates at a density of 2.0 ⁇ 10 4 /well, with 3 replicate wells in each group.
  • Lipofectamine3000 transfection reagent to combine the internal reference plasmid pRL-TK and the reporter plasmid pGL3-PLA2G12B (25ng) (or pGL3-MTP (25ng) and pGL3-APOB (25ng)) and The expression vectors pcDNA4-HNF4a (25ng) and pcDNA4-PGC1a (10ng) were co-transfected and transformed into cells for 6 hours and then treated with AP (10, 25, 50 ⁇ M) for 24 hours;
  • Control group refer to the experimental group, in which AP is not added
  • Blank group 293T cells.
  • mice The internal reference plasmid pRL-TK, the reporter plasmid pFR-Luc (25ng) and the expression vector pCMV-BD-HNF4a-LBD (25ng), pcDNA4-PGC1a (5ng) were co-transfected into the cells and cultured for 24 hours. Drug AP (10, 25, 50 ⁇ M) was treated for 24 hours.
  • Control group refer to the experimental group, in which AP is not added
  • Blank group 293T cells.
  • Luciferase fluorescence detection After the experimental group, control group and blank group in (1) and (2) have been cultured, aspirate the culture medium in each group, wash the cells twice with PBS, and add 50 ⁇ l of double-distilled water diluted 1 ⁇ passive lysis buffer, lyse the cells on a shaker for 30 minutes; aspirate 25 ⁇ l of cell lysate from each well, add 25 ⁇ l of luciferase substrate, use a multiwell chemiluminescence detector to detect the fluorescence intensity of the lysate, add 25 ⁇ l of 1 ⁇ Stop&Glo substrate to detect the fluorescence intensity of the internal reference phRL-TK luciferase. Calculate the ratio of luciferase fluorescence intensity to phRL-TK luciferase fluorescence intensity in each well, and perform data processing.
  • Huh-7 cells were seeded in a 6-well plate at a density of 3 ⁇ 10 5 /well, and cultured with 10% FBS high glucose DMEM. After 24 hours, the cells were treated as the control group (DMSO) and administration group (AP 10 ⁇ M). After administration treatment for 24 hours, the culture medium was discarded, washed twice with cold PBS, Trizol was added to lyse the cells, total RNA in the cells was extracted, and cDNA was prepared by reverse transcription. , The system is shown in Table 1 below:
  • Example 3 The effect of andrographolide (AP) on the secretion of lipid droplets in mouse primary hepatocytes, and the experiment to increase the level of triglyceride accumulation in primary hepatocytes
  • mice primary hepatocytes mouse liver was washed, digested, resuspended and percoll stratified to obtain primary hepatocytes. After washing three times with cold PBS, resuspend in 10% FBS (DMEM-L), count the cells with trypan blue to see the cell viability.
  • DMEM-L 10% FBS
  • OA storage concentration: 10mM
  • Control group refer to the experimental group, in which AP is not added
  • Determination of intracellular triglyceride concentration After the experimental group and the control group in (1) and (2) are cultured, the cells are lysed, and after extraction, the intracellular triglyceride and protein concentration are measured according to the kit instructions.
  • Intracellular lipid droplet fluorescence experiment 24 hours after adding the drug in the experimental group and 24 hours in the control group, the samples were collected, washed twice with PBS, fixed with 4% PVF for 15 minutes; washed 3 times with PBS, stained with BODIPY493/503 for 10 minutes, DAPI stains the nucleus for 3 minutes; after washing 3 times with PBS, place the cell slide upside down on a glass slide dripped with anti-quenching agent; finally, observe and take pictures under an Olympus upright fluorescent microscope (BX51).
  • C57BL/6J mice were randomly divided into groups (n ⁇ 6), and the andrographolide (AP) dose was 100mg/kg/day, and they were given by intragastric administration for 5 consecutive days. After execution, the liver was taken, total liver RNA was extracted, and cDNA was prepared by reverse transcription. The reverse transcription system and procedure as well as the qPCR system and procedure are as in Example 2.
  • the measured results of this embodiment are shown in Figure 4.
  • the AP drug group can inhibit the key genes SREBP-1c, FASN, and SCD-1 for lipid synthesis in mouse liver cells. At the same time, it also inhibits the expression of the key genes PLA2G12B, MTP and APOB for VLDL synthesis and secretion. It can be seen that andrographolide (AP) can regulate intracellular lipid synthesis and secretion by regulating liver lipid synthesis and VLDL secretion.
  • Example 5 Testing the effect of andrographolide (AP) on the mouse model of non-alcoholic fatty liver induced by high-fat diet
  • mice Six-week-old male C57BL/6 mice were divided into 3 groups, the normal group (CD), the high-fat group (HFD) and the high-fat andrographolide group (HFD+AP).
  • the general group was given regular feeding, and the high-fat group and the high-fat drug group were given high-fat feed.
  • the high-fat medicated group was given a high-fat diet and a daily gavage of AP with a daily dose of 50 mg/kg.
  • the control group was given a blank control solvent. After 6 weeks, the mice were sacrificed, and blood and liver samples were collected for testing.
  • AP can significantly reduce the increased activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) induced by a high-fat diet, and improve liver function.
  • AST aspartate aminotransferase
  • ALT alanine aminotransferase
  • FIG 5-E and Figure 5-F analysis of liver VLDL secretion showed that AP can reduce the increase in liver VLDL secretion induced by high-fat diet.
  • Figure 5-G and Figure 5-H the detection results of the expression of genes related to VLDL synthesis and secretion in the liver showed that AP can significantly down-regulate the expression of APOB, MTP and PLA2G12B genes in the liver of mice. It can be seen that AP inhibits VLDL synthesis and secretion by down-regulating APOB, MTP and PLA2G12B gene expression, reduces lipid output in the liver, and thereby reduces blood lipids.
  • AP can reduce lipid synthesis and secretion in liver cells by regulating lipid synthesis and VLDL secretion-related genes, thereby reducing liver fat and blood lipids, improving atherogenesis, and having a beneficial effect on non-alcoholic steatohepatitis. Efficacy.

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Abstract

提供一种穿心莲内酯作为降低脂质合成的调节剂和/或降低极低密度脂蛋白形成与分泌的调节剂和药物中的应用。进一步，含有有效剂量的穿心莲内酯的药物，作为HNF4α的调节剂、下调基因MTP、APOB和PLA2G12B的表达，同时下调基因SREBP-1、FASN、SCD-1表达的方法。所述穿心莲内酯能够下调脂代谢相关基因的表达和干扰肝脏细胞内HNF4α/PGC-1α相互作用，下调其下游靶基因的表达，因此，穿心莲内酯能够降低脂质合成和极低密度脂蛋白形成和分泌，以降低血脂和降低肝脏内脂质含量，最终起到预防和/或治疗脂代谢异常相关疾病的作用。另外，由于穿心莲内酯是天然药物的有效成分，疗效好，毒副作用小。

Description

穿心莲内酯在制备脂代谢异常相关疾病药物中的应用 技术领域
本发明属于生物医药技术领域，具体的是涉及一种穿心莲内酯在制备脂代谢异常相关疾病药物中的应用。
背景技术
脂代谢异常是指血浆及其他组织器官中脂质及其代谢产物质的异常，常伴有胆固醇或甘油三酯(TG)水平的升高，可分为原发性和继发性两类。脂代谢紊乱是非酒精性脂肪肝(NAFLD)、动脉粥样硬化(AS)、心脑血管疾病和糖尿病等发病关键影响因子。
随着社会的发展，人们的饮食呈富余化趋势，因血脂升高导致的动脉粥样硬化、冠心病等心血管脂代谢异常导致的慢性疾病率逐年升高，并呈年轻化趋势。我国成人血脂异常患病率高达18.6％，其中高胆固醇血症占2.9％，高甘油三酯血症占11.9％，低高密度脂蛋白血症占7.4％。临床统计表明，近年来心肌梗死病死亡人数中77％是由于高血脂造成的。其中，动脉粥样硬化和冠心病，是全球常见多发病，严重危害人类健康和生存质量。NAFLD是指除酒精外其他因素所致的肝细胞内脂肪过度沉积的病理综合征。近年随着肥胖及相关代谢综合征全球化趋势，NAFLD在我国发病率逐年上升且日趋低龄化，国内报道肥胖儿童和青少年中NAFLD的检出率高达23.3％～59.6％，显著高于国外。如何控制血脂代谢异常、遏止心脑血管病迅猛增长势头，已成为我国公共卫生事业面临的严重挑战。
降低血脂对于治疗脂代谢异常疾病具有重要意义，因而，降血脂药物已成为近年来药物研究的热点之一。目前临床应用的降血脂药主要有他汀类、贝特类、烟酸类、胆酸鳌合剂类等。其中他汀类药治疗效果最好，能有效地降低血清中的低密度脂蛋白胆固醇(LDL-C)和三酰甘(TG)，其副作用是大剂量服用会产生肌毒和升高肝转氨酶。贝特类降甘油三酯效果显著，可能会引起胃肠道不适和肾功能损伤。而缓释烟酸在升高高密度脂蛋白(HDL)方面表现良好，可 能引起胃肠道不适和肝肾副作用。胆固醇吸收抑制剂依替米贝和胆酸螯合剂考来维仑主要降低LDL-C，但这些药物也有一定的不良反应，如面部或舌咽部水肿，以及脂溶性维生素缺失。
用天然药物改善血脂异常具有一定效果，使用相对安全，适宜长期服用。近年来，关于中药活性成分改善血脂异常有很多报道，也筛选出了一批有效的天然产物，但大多数研究只停留在药效学研究，在治疗机制和药理作用环节方面研究较少。
穿心莲内酯(andrographolide，AP)是穿心莲的主要活性成分，穿心莲为爵床科植物穿心莲(Andrographis)的全草或叶。又名春莲秋柳、一见喜、榄核莲、苦胆草、金香草、金耳钩、印度草和苦草。有清热解毒、消炎、消肿止痛作用。主治细菌性痢疾、尿路感染、急性扁桃体炎、肠炎、咽喉炎、肺炎和流行性感冒，外用可治疗疮疖肿毒和外伤感染。在亚洲许多国家广泛用于治疗病毒感染、糖尿病、风湿关节炎、咽喉炎和腹泻。随着对穿心莲的研究不断深入，最近有报道穿心莲及其类类似物穿心莲表现出良好的癌症(Satyanarayana et al.，Science 2003，299，363-370)治疗，并在治疗或预防艾滋病、阿尔茨海默氏病和肝炎取得了进展，虽然目前发现少量有关于穿心莲内酯在调节脂代谢紊乱疾病方面的研究报道，但是至今没有看到关于穿心莲内用于调节脂代谢紊乱疾病药理作用机制的研究报道。
技术问题
本发明的目的在于克服现有技术的上述不足，提供一种穿心莲内酯在作为制备脂代谢异常相关疾病药物中的应用，以解决穿心莲内酯目前常仅局限用于抗肿瘤和抗病毒以及现有脂代谢异常相关疾病药物副作用较大的技术问题。
技术解决方案
为了实现上述发明目的，本发明的一方面，提供了穿心莲内酯在如下至少一方面的应用：
作为抑制脂质合成的调节剂；
作为降低极低密度脂蛋白形成与分泌的调节剂。
本发明的另一方面，提供了用于抑制脂质合成或降低极低密度脂蛋白形成 与分泌的药物。所述药物包括有效剂量的穿心莲内酯。
本发明的又一方面，提供了一种干扰HNF4α与PGC-1α作用的方法。所述干扰HNF4α与PGC-1α作用的方法包括将有效剂量的穿心莲内酯或本发明用于抑制脂质合成或降低极低密度脂蛋白形成与分泌的药物与具有表达HNF4α的细胞细胞接触的步骤。
同时，还提供了一种下调基因MTP、APOB和PLA2G12B表达的方法。所述方法包括将有效剂量的穿心莲内酯或本发明用于降低极低密度脂蛋白形成与分泌的药物与具有表达MTP、APOB和PLA2G12B的细胞接触的步骤。
以及，还提供了一种下调基因SREBP-1、FASN、SCD-1表达的方法。所述方法包括将有效剂量的穿心莲内酯或本发明用于抑制脂质合成的药物与具有表达SREBP-1、FASN、SCD-1的细胞接触的步骤。
与现有技术相比，本发明穿心莲内酯的应用具有以下有益效果：
经实验证明，所述穿心莲内酯能够下调包括基因FASN、SCD-1、SREBP-1表达等作用，同时干扰HNF4α与PGC-1α相互作用，下调其MTP、APOB和PLA2G12B的表达，从而降低脂质合成和/或降低极低密度脂蛋白形成与分泌，因此，将所述穿心莲内酯作为降低脂质合成和/或降低极低密度脂蛋白形成与分泌调节剂，能够有效下调SREBP1c、FASN、SCD1表达实现降低脂质合成，还能抑制极低密度脂蛋白形成和分泌，具体的如干扰HNF4α与PGC-1α相互作用和下调包括基因下调MTP、APOB和PLA2G12B表达。
有益效果
本发明用于抑制脂质合成或降低极低密度脂蛋白形成与分泌的药物由于含有有效剂量的穿心莲内酯，因此，所述药物能够下调脂代谢相关基因的表达，从而降低脂质合成和极低密度脂蛋白形成和分泌，以降低血脂和降低肝脏内脂质含量，最终起到预防和/或治疗脂代谢异常相关疾病的作用。另外，由于穿 心莲内酯是天然药物的有效成分，疗效好，毒副作用小。
附图说明
下面将结合附图及实施例对本发明作进一步说明，附图中：
图1为实施例1中AP对HNF4α-LBD活性及VLDL分泌相关基因启动子转录活性的影响图；其中，图1-A为AP对HNF4α-LBD活性的影响图，图1-B为AP对APOB启动子转录活性的影响图，图2-C为AP对MTP启动子转录活性的影响图，图2-D为AP对PLA2G12B启动子转录活性的影响图，且n＝4，与空白对照组比较，*P＜0.05，***P＜0.001；
图2为实施例2中对照组和给药组中AP为10μmol/L对Huh-7细胞脂代谢关键基因mRNA表达影响图；其中，图2-A至图2-C为AP对参与VLDL装配和分泌相关基因mRNA表达影响图；图2-D至图2-G为AP对参与调控脂肪酸和甘油三酯生物合成相关基因mRNA表达的影响图；且n＝4，与对照组比较，*P＜0.05，**P＜0.01，***P＜0.001；
图3为实施例3中不同浓度穿心莲内酯对小鼠原代肝细胞脂滴水平的影响图；其中，图3-A为AP小鼠原代肝细胞脂滴荧光染色图；图3-B为对照组和实验组中胞内脂滴荧光区域统计柱状图；图3-C为对照组和实验组中胞内甘油三酯水平图；且n＝4，与空白对照组比较，*P＜0.05，**P＜0.01，***P＜0.001；
图4为实施例4中对照组和给药组中AP为100mg/kg/天短期加药对小鼠肝脏中脂代谢相关基因表达的影响影响图；其中，图4-A至图4-C为AP对参与VLDL装配和分泌相关基因mRNA表达影响图；图4-D至图4-F为AP对参与调控脂肪酸和甘油三酯生物合成相关基因mRNA表达的影响图；且n＝6，与对照组比较，*P＜0.05，**P＜0.01；
图5为实施例5中普通组、高脂组和高脂加药组中AP对高脂小鼠血脂的 影响图；其中，图5-A至5-D为CD小鼠、HFD小鼠及HFD+AP小鼠血清中甘油三酯(TG)、胆固醇(TC)、高密度脂蛋白(HCL-C)、低密度脂蛋白(LDL-C)的水平，图5-E为CD小鼠、HFD小鼠及HFD+AP小鼠血清甘油三酯(TG)水平变化，图5-F为CD小鼠、HFD小鼠及HFD+AP小鼠VLDL分泌率的检测。G:CD小鼠、HFD小鼠及HFD+AP小鼠血清中ApoB水平，图5-H为CD小鼠、HFD小鼠及HFD+AP小鼠肝脏蛋白western blot结果。且n＝8，与空白对照组比较，*P＜0.05，**P＜0.01，***P＜0.001。#P＜0.05，##P＜0.01，###P＜0.001；
图6为实施例5中普通组、高脂组和高脂加药组中AP对高脂小鼠肝脏组织的影响图；其中，图6-A为CD小鼠、HFD小鼠及HFD+AP小鼠肝脏中甘油三酯(TG)的水平，图6-B至6-C为CD小鼠、HFD小鼠及HFD+AP小鼠血清中ALT、AST水平，图6-D为CD小鼠、HFD小鼠及HFD+AP小鼠肝脏及油红、HE染色；且*P＜0.05，**P＜0.01，***P＜0.001。#P＜0.05，##P＜0.01，###P＜0.001。
本发明的实施方式
为了使本发明要解决的技术问题、技术方案及有益效果更加清楚明白，以下结合实施例与附表，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
本文中涉及的相关专用名称的解释：
穿心莲内酯(andrographolide，AP)：是从植物穿心莲中提取得到的活性成分，药性和毒性试验结果显示，在急性毒性实验中，小鼠灌服穿心莲内酯的LD50在40g/kg以上，小鼠每日最大耐受量为110.25g/kg。亚急性毒性实验中，对大鼠或家兔灌服穿心莲内酯1g/kg，每日1次，连续7日，未见明显的毒性。穿琥宁注射液84m g/kg腹腔注射每日1次，连续10天，无明显毒性作用。
肝细胞核因子4α(HNF4α)：是核激素受体超家族的一种保守性的配体依赖性转录因子，主要表达于肝脏、肾脏、肠道。
分泌型的磷脂酶A2G12B(PLA2G12B)：是分泌型磷脂酶家族的成员之一，主要在小鼠肝脏和小肠中表达。
PGC-1α：辅助激活因子-1α(PPARγcoactivator-1α)
FASN：脂肪酸合成酶(Fatty acid synthase)
SCD-1：硬脂酰辅酶A1
ACACα：乙酰辅酶A羧化酶α
MTP：微粒体甘油三酯转运蛋白(microsimal triglyceride transfer protein)
极低密度脂蛋白(VLDL)：是由肝脏利用乳糜颗粒残粒、胆汁酸、脂肪酸、糖和蛋白质的中间代谢物与肝脏内合成的载脂蛋白组成的一种脂蛋白，在肝细胞中组装合成并携带脂类，是将肝脏脂质分泌输出到外周组织的主要途径。
脂质代谢异常：是先天性或获得性因素造成的血液及其他组织器官中脂质及其代谢产物质和量的异常，其具体病症主要包括高脂蛋白血症等。
本发明的发明人基于大量研究发现，穿心莲内酯具有下调脂代谢相关基因的表达，以降低血脂和肝脂的等相关作用，基于此，本发明实施例提供了穿心莲内酯在下文相关方面的应用。
一方面，本发明实施例提供了穿心莲内酯在如下至少一方面的应用：
(1)作为抑制脂质合成的调节剂；
(2)作为降低极低密度脂蛋白形成与分泌的调节剂。
经发明人构建的相关实验得知，所述穿心莲内酯作为活性成分，能够有效下调脂代谢相关基因的表达，从而抑制或降低脂质合成过程，抑制或降低极低 密度脂蛋白形成和分泌过程，最终达到降低肝脂和血脂的作用，从而改善脂代谢异常相关疾病。
如在一实施例中，所述穿心莲内酯具有下调包括基因SREBP-1、FASN、SCD-1表达的作用。因此，所述穿心莲内酯可以被用于下调包括基因SREBP-1、FASN、SCD-1表达的调节剂，以及进一步可以用于制备下调包括基因SREBP-1、FASN、SCD-1表达的相关药物，以有效下调脂质合成如抑制肝脏脂质合成过程中的相关基因表达，从而实现抑制脂质合成如抑制肝脏脂质合成，达到降低相关细胞如肝脏细胞脂质形成以达到降脂如降肝脂作用，从而改善脂代谢异常相关疾病的临床效果。
研究发现，HNF4α是调节肝脏脂代谢的重要转录因子。是细胞核受体超家族的成员之一，HNF4α在肝脏中高表达，接下来依次是肾、小肠、结肠和胰腺细胞。其作用模式是：HNF4α蛋白含有锌指结构，以同源二聚体的形式结合在DNA上，其识别位点是同向重复的AGGTCA元件，通过招募转录辅激活因子和其他辅助蛋白，上调靶基因的表达。在肝脏组织中，HNF4α仅定位于细胞核中，调控大量基因的组成型表达，这些基因编码的蛋白包括各种酶、转运蛋白、其他核受体以及大部分肝脏特异性表达的基因。而发明人在研究发现，所述穿心莲内酯具有调节HNF4α与PGC-1α相互作用，从而下调其下游靶相关基因表达，其中，下调其下游靶相关基因包括MTP、APOB和PLA2G12B。因此，在一实施例中，所述穿心莲内酯作可以为HNF4α调节剂的应用，以及进一步可以用于制备调节HNF4α与PGC-1α相互作用的相关药物，从而有效调节HNF4α与PGC-1α相互作用，以下调其下游相关靶基因的表达，从而降低极低密度脂蛋白形成和分泌，最终降低如肝脂作用和降低血脂的作用，从而改善脂代谢异常相关疾病的临床效果。
由于所述穿心莲内酯具有下调HNF4α/PGC-1α下游相关靶基因包括MTP、APOB和PLA2G12B等的表达的作用。因此，在具体实施例中，所述穿心莲内酯作为下调包括基因MTP、APOB和PLA2G12B表达的调节剂的应用，以及进一步可以用于制备调节HNF4α与PGC-1α相互作用的相关药物，从而实现降低极低密度脂蛋白形成和分泌，最终降低如肝脂作用和降低血脂的作用，从而改善脂代谢异常相关疾病的临床效果。
基于上文所述穿心莲内酯能够有效下调脂代谢相关基因的表达，从而降低脂质合成以及抑制极低密度脂蛋白形成和分泌过程等作用，所述穿心莲内酯可以被用于制备预防和/或治疗脂代谢异常相关疾病药物，进一步是作为制备抑制脂质合成的药物或降低极低密度脂蛋白形成与分泌的药物。具体如上文所述的，所述穿心莲内酯可以被用于制备下调包括基因SREBP-1、FASN、SCD-1表达的药物、被用于制备调节HNF4α与PGC-1α相互作用的药物、被用于制备下调包括基因MTP、APOB和PLA2G12B表达的药物。那么相应的本发明实施例还提供了一种预防和/或治疗脂代谢异常相关疾病的药物，进一步提供了一种用于抑制脂质合成或降低极低密度脂蛋白形成与分泌的药物，具体提供了一种下调包括基因SREBP-1、FASN、SCD-1表达的药物、调节HNF4α与PGC-1α相互作用的药物、下调包括基因MTP、APOB和PLA2G12B表达的药物。所述药物包括有效剂量的活性成分穿心莲内酯。另外，所述药物还可以包括能够有效预防和/或治疗脂代谢异常相关疾病的其他活性成分，此处所述的“有效”是单独预防和/或治疗脂代谢异常相关疾病有临床效果的成分，也可以是与穿心莲内酯进行复合后能够提高穿心莲内酯预防和/或治疗脂代谢异常相关疾病有临床效果的成分。所述“有效剂量”是指能够预防和/或治疗脂代谢异常相关疾病的有效量，是指足以对个体显示益处或临床意义的穿心莲内酯 的量。本领域技术人员将会理解，给药的实际量或剂量以及给药时程将取决于被预防或治疗的疾病的性质和严重性、被预防或治疗的受试者的年龄和一般状况以及给药方式等。如在一实施例中，所述穿心莲内酯在所述药物中的有效剂量为50-100mg/kg，如经动物实验，穿心莲内酯的有效剂量为50-100mg/kg，具体的可以是实验小鼠口服有效剂量为50-100mg/kg。其中，所述脂代谢异常相关疾病包括高脂血症、非酒精性脂肪肝疾病(包单纯性脂肪肝、非酒精性脂肪性肝炎)、动脉粥样硬化、心脑血管疾病和糖尿病中的至少一种。
另外，所述药物还可以进一步包括药学上可接受的穿心莲内酯的载体(辅料)成分。所述药学上可接受的穿心莲内酯的载体成分可以根据所述药物给药方式制备的相应剂型的相应载体。只要是能够负载所述穿心莲内酯，并有利于其稳定和被吸收的符合医药要求的载体均在本发明公开的范围。因此，依据所述载体的选择，所述药物的剂型可以是口服剂型、注射剂型中的至少一种。
因此，所述药物由于含有上文的穿心莲内酯，由此，所述药物能够有效下调脂代谢相关基因的表达，具体可以下调包括基因SREBP-1、FASN、SCD-1表达；同时调节HNF4α与PGC-1α相互作用，从而下调其下游靶相关基因包括MTP、APOB和PLA2G12B表达，从而抑制脂质合成和抑制极低密度脂蛋白形成和分泌过程，最终降低如肝脂作用和降低血脂的作用，从而改善脂代谢异常相关疾病的临床效果。另外，由于穿心莲内酯是天然药物的有效成分，疗效好，毒副作用小，安全。
又一方面，基于上文所述穿心莲内酯的应用和含有所述穿心莲内酯的药物，一实施例中，本发明实施例提供了一种干扰HNF4α与PGC-1α作用的方法。所述方法包括将有效剂量的所述穿心莲内酯或上文所述药物与具有表达HNF4α的细胞接触的步骤。通过所述方法能够有效干扰所述细胞中HNF4α与 PGC-1α的接触，以下调其下游相关靶基因的表达，从而降低极低密度脂蛋白形成和分泌，最终降低肝脂作用和降低血脂的作用，从而改善脂代谢异常相关疾病的临床效果。
另一实施例中，本发明实施例还提供了一种下调基因MTP、APOB和PLA2G12B表达的方法。所述方法包括将有效剂量的穿心莲内酯或上文所述药物与具有表达MTP、APOB和PLA2G12B的细胞接触的步骤。通过所述方法能够有效降低极低密度脂蛋白形成和分泌，最终降低肝脂作用和降低血脂的作用，从而改善脂代谢异常相关疾病的临床效果。
再一实施例中，本发明实施例还提供了一种下调SREBP-1、FASN、SCD-1表达的方法。所述方法包括将有效剂量的穿心莲内酯或上文所述药物与具有表达SREBP-1、FASN、SCD-1的细胞接触的步骤。通过所述方法能够有效下调脂质合成如下调肝脏脂质合成的相关基因表达，从而实现抑制或降低脂质合成如抑制肝脏脂质合成，达到降低相关细胞如肝脏细胞脂质形成以达到降脂如降肝脂作用，从而改善脂代谢异常相关疾病的临床效果。
其中，上述的细胞如具有表达HNF4α的细胞、具有表达SREBP-1、FASN、SCD-1的细胞和具有表达具有表达MTP、APOB和PLA2G12B的细胞可以但不仅仅为肝细胞、肝癌细胞，还可以是其他组织部分的相关细胞，如肾脏、肠道等中相关细胞。
其次，上文各实施例中的所述穿心莲内酯可以按照现有常规方法从甘草原药材中提取，当然也可以设计新的提取方法从甘草原药材中提取。
现结合具体实例，对穿心莲内酯用于抑制脂代谢相关基因表达的应用进行进一步详细说明。
实施例1.检测穿心莲内酯(AP)对VLDL合成和分泌关键基因启动子 活性的影响的实验：
(1)实验方法如下：
A.检测穿心莲内酯(AP)对VLDL合成和分泌关键基因启动子活性的影响：
实验组：将293T细胞以2.0×10 4/孔的密度接种在96板上，每组3个复孔。对于启动子活性检测实验，待细胞贴壁后，使用Lipofectamine3000转染试剂将内参质粒pRL-TK、报告质粒pGL3-PLA2G12B(25ng)(或pGL3-MTP(25ng)和pGL3-APOB(25ng))和表达载体pcDNA4-HNF4a(25ng)、pcDNA4-PGC1a(10ng)共转染转入细胞培养6小时后，给药AP(10、25、50μM)处理24小时；
对照组：参照实验组处理，其中，不添加AP；
空白组：293T细胞。
B.对于穿心莲内酯(AP)干扰HNF4a配体结合域(LBD)与共激活因子PGC1a相互作用的实验：
实验组：将内参质粒pRL-TK、报告质粒pFR-Luc(25ng)和表达载体pCMV-BD-HNF4a-LBD(25ng)、pcDNA4-PGC1a(5ng)共转染转入细胞培养24小时后，给药AP(10、25、50μM)处理24小时。
对照组：参照实验组处理，其中，不添加AP；
空白组：293T细胞。
(2)Luciferase荧光检测：待(1)和(2)中实验组、对照组和空白组培养完毕后，吸去各组中的培养液，PBS清洗细胞2次，加入50μl双蒸水稀释的1×passive lysis buffer，于震荡仪上震荡裂解细胞30分钟；每个孔吸出25μl细胞裂解液，加入25μl萤光素酶底物，利用多孔板化学发光检测仪检测裂解 液荧光强度，加25μl的1×Stop&Glo底物，检测内参phRL-TK萤光素酶荧光强度。计算各孔中萤光素酶萤光强度与phRL-TK萤光素酶萤光强度的比值，并进行数据处理。
(3)实验结果：本实施例测得结果如图1所示，AP通过干扰HNF4α的配体结合域与PGC-1a的相互作用，抑制了HNF4α/PGC-1a对APOB、MTP和PLA2G12B基因启动子活性的上调作用。由此可见，AP通过下调HNF4α参与的VLDL相关基因转录调控，从而参与VLDL合成和分泌的调节，而且随着AP的有效剂量浓度增加下调HNF4α参与的VLDL相关基因转录调控作用增强。
实施例2.穿心莲内酯(AP)对Huh-7细胞脂代谢关键基因的影响实验：
(1)实验方法如下：
检测穿心莲内酯(AP)在细胞水平对脂代谢关键基因的影响：将Huh-7细胞按照3×10 5/孔的密度接种在6孔板中，用10％FBS的高糖DMEM培养。24小时后，按对照组(DMSO)和给药组(AP 10μM)处理细胞。给药处理24h后弃去培养基，用冷的PBS清洗2次，加入Trizol裂解细胞，提取细胞内总RNA，反转录制备cDNA。，体系如下表1所示：
表1.反转录体系及RT-qPCR体系如下：
Figure PCTCN2019130839-appb-000001
Figure PCTCN2019130839-appb-000002
反转录PCR程序：
反应：42℃(60min)，终止反应：70℃(5min)。将反转录产物稀释20倍后，进行Real time RCR检测。
Real time PCR反应体系如下表2所示：
表2.Real time PCR反应体系
Figure PCTCN2019130839-appb-000003
Real time PCR反应程序：
预变性          94℃ 30s
PCR反应40cycles 95℃ 5s
                60℃ 15s
                72℃ 10s
溶解曲线        50℃ 30s
                50℃～95℃ 0.5℃/s
Real time PCR引物序列如下表3所示：
表3.Real time PCR引物序列
Figure PCTCN2019130839-appb-000004
Figure PCTCN2019130839-appb-000005
(2)实验结果：本实施例测得结果如图2所示。由图2可知，与对照组比较，浓度为10μM的AP可以明显抑制VLDL合成与分泌关键基因PLA2G12B、MTP和APOB以及脂合成关键基因SREBP-1c、FASN、SCD-1和ACACa表达。由此可见，AP通过抑制脂质的从头合成和VLDL分泌相关基因的表达从而调控胞内脂质合成和分泌。
实施例3.穿心莲内酯(AP)对小鼠原代肝细胞脂滴分泌的影响，提高原代肝细胞内甘油三酯积累水平实验
(1)实验方法如下：
实验组：小鼠原代肝细胞的提取：小鼠肝脏经过冲洗、消化、重悬和percoll分层后得到原代肝细胞。用冷的PBS清洗三遍后，10％FBS(DMEM-L)重悬，台盼蓝计数，看细胞活率。按1.5*105/孔铺24孔板，3.5*105/孔铺12孔板。铺板3小时后，有爬片的孔板换液；铺板12小时后，加OA(储存浓度：10mM)至培养基中，终浓度为100μM；加OA培养12小时后，加药处理，AP(10，25，50μM)。培养24小时后收细胞进行一步处理。
对照组：参照实验组处理，其中，不添加AP；
(2)检测方法：
胞内甘油三酯浓度测定：待(1)和(2)中实验组和对照组培养完毕后，裂解细胞，提取后按试剂盒说明检测胞内甘油三酯和蛋白浓度。
胞内脂滴荧光实验：对实验组加药24小时后和对照组24形式后收样，PBS清洗两遍，4％PVF固定15分钟；PBS清洗3遍，用BODIPY493/503染色10分钟，用DAPI染核3分钟；PBS清洗3遍后，把细胞爬片倒扣在滴有防淬灭剂的载玻片上；最后，在Olympus正置荧光显微镜(BX51)下观察并拍摄照片。
(3)实验结果：本实施例测得结果如图3所示。由图3-C可知，随着AP药物浓度增加，胞内甘油三酯浓度显著增加，并呈剂量依赖性，说明AP能够抑制小鼠原代肝细胞胞内甘油三酯的输出；由图3-A和图3-B所示的胞内脂滴荧光染色分析结果同样表明，AP抑制小鼠原代肝细胞胞内脂滴向胞外分泌，造成胞内脂滴积累增加。由此可见，AP可通过调控VLDL合成和分泌相关基因影响胞内脂质的输出。
实施例4.检测穿心莲内酯(AP)对于普通小鼠短期加药后，对肝脏中与脂代谢相关基因的影响
(1)实验方法如下：
C57BL/6J小鼠随机分组(n≥6)，穿心莲内酯(AP)剂量为100mg/kg/day，连续5天灌胃给药。处死后取肝脏，提取肝脏总RNA，反转录制备cDNA。反转录体系和程序以及qPCR的体系和程序按实例2。
Real time PCR引物序列如下表4所示：
表4.Real time PCR引物序列
Figure PCTCN2019130839-appb-000006
Figure PCTCN2019130839-appb-000007
(2)实验结果：本实施例测得结果如图4所示。AP加药组可以抑制小鼠肝脏细胞脂合成关键基因SREBP-1c、FASN、SCD-1。同时对VLDL合成与分泌关键基因PLA2G12B、MTP和APOB表达也具有抑制作用。由此可见，穿心莲内酯(AP)可以通过调控肝脏的脂质合成和VLDL分泌从而调节胞内脂质合成和分泌。
实施例5.检测穿心莲内酯(AP)对于高脂饲料诱导的非酒精性脂肪肝小鼠模型的影响实验
(1)实验方法如下：
将6周龄雄性C57BL/6小鼠分为3组，普通组(CD)、高脂组(HFD)和高脂穿心莲内酯加药组(HFD+AP)。普通组给予普通喂食，高脂组和高脂药组给予高脂饲料。18周后高脂加药组给予高脂饮食的同时每天灌胃剂量为50mg/kg的AP，对照组给予空白对照溶剂，6周后将小鼠处死，收集血液和肝脏等样本进行检测。
(2)实验结果：本实施例测得结果如图5和6所示。由图5-A和图5-D所示可知，血生化分析显示，与HFD组相比，AP加药组能有效降低高脂饮食导致的血浆中高甘油三酯、高胆固醇和高低密度脂蛋白。由图6-A和6-D所示，通过对肝脏甘油三酯含量、组织切片染色和肝功能检测分析，发现AP能够明显降低高脂脂饮诱导的肝脏甘油三酯含量上升，改善肝脏脂肪变性程度。同时，如图6-B和图6-C所示，AP能够明显降低高脂饮食诱导的谷草转氨酶(AST)和谷丙转氨酶(ALT)的活性上升，改善肝功能。如图5-E和图5-F所示，对肝脏VLDL分泌分析表明，AP能降低由高脂饮食诱导肝脏VLDL分泌率增加。如图5-G和图5-H所示，对肝脏中VLDL合成与分泌的相关基因表达量检测结果表明，AP能够显著下调小鼠肝脏中APOB、MTP和PLA2G12B基因的表达。由此可见，AP通过下调APOB、MTP和PLA2G12B基因表达来抑制VLDL合成和分泌，降低肝脏中脂质输出，从而降低血脂。
结合上述细胞水平研究实验结果可知，AP可以通过调控脂合成和VLDL分泌相关基因降低肝脏细胞内脂质合成和分泌，从而降低肝脂和血脂，改善动脉粥样化，对非酒精性脂肪肝炎具有疗效。
以上所述的实施例仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (9)

  1. 穿心莲内酯在如下至少一方面的应用：
    作为抑制脂质合成的调节剂；
    作为降低极低密度脂蛋白形成与分泌的调节剂。
  2. 根据权利要求1所述的应用，其特征在于：所述穿心莲内酯作为下调包括基因SREBP-1、FASN、SCD-1表达的调节剂的应用。
  3. 根据权利要求1所述的应用，其特征在于：所述穿心莲内酯作为HNF4α调节剂的应用。
  4. 根据权利要求3所述的应用，其特征在于：所述穿心莲内酯作为下调包括基因MTP、APOB和PLA2G12B表达的调节剂的应用。
  5. 一种用于抑制脂质合成或降低极低密度脂蛋白形成与分泌的药物，其特征在于：包括有效剂量的穿心莲内酯。
  6. 根据权利要求5所述的药物，其特征在于：所述药物为口服剂型、注射剂型中的至少一种。
  7. 一种干扰HNF4α与PGC-1α作用的方法，其特征在于：包括将有效剂量的穿心莲内酯或权利要求5-6任一所述的药物与具有表达HNF4α的细胞接触的步骤。
  8. 一种下调基因MTP、APOB和PLA2G12B表达的方法，其特征在于：包括将有效剂量的穿心莲内酯或权利要求5-6任一所述的药物与具有表达MTP、APOB和PLA2G12B的细胞接触的步骤。
  9. 一种下调SREBP-1、FASN、SCD-1表达的方法，其特征在于：包括将有效剂量的穿心莲内酯或权利要求5-6任一所述的药物与具有表达SREBP-1、FASN、SCD-1的细胞接触的步骤。
PCT/CN2019/130839 2019-03-28 2019-12-31 穿心莲内酯在制备脂代谢异常相关疾病药物中的应用 Ceased WO2020192233A1 (zh)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102247356A (zh) * 2011-07-12 2011-11-23 上海中医药大学 一种穿心莲内酯的应用

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Publication number Priority date Publication date Assignee Title
CN102247356A (zh) * 2011-07-12 2011-11-23 上海中医药大学 一种穿心莲内酯的应用

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