WO2017193824A1 - 青蒿素类似物促进脂肪分解和改善糖代谢的用途、方法及组合物 - Google Patents

青蒿素类似物促进脂肪分解和改善糖代谢的用途、方法及组合物 Download PDF

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WO2017193824A1
WO2017193824A1 PCT/CN2017/082359 CN2017082359W WO2017193824A1 WO 2017193824 A1 WO2017193824 A1 WO 2017193824A1 CN 2017082359 W CN2017082359 W CN 2017082359W WO 2017193824 A1 WO2017193824 A1 WO 2017193824A1
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artemether
mice
group
fat
artemisinin
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PCT/CN2017/082359
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English (en)
French (fr)
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汤其群
党永军
路平
李希
张付闯
左建平
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复旦大学
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Priority to JP2018557930A priority Critical patent/JP2019521085A/ja
Priority to EP17795442.7A priority patent/EP3456326A4/en
Priority to US16/099,195 priority patent/US20190133997A1/en
Publication of WO2017193824A1 publication Critical patent/WO2017193824A1/zh

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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
    • A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
    • 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
    • A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
    • 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
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/541—Non-condensed thiazines containing further heterocyclic rings
    • 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
    • A61P3/04—Anorexiants; Antiobesity agents

Definitions

  • the invention belongs to the field of biomedicine, and particularly relates to the use of artemether in the preparation of a preparation for promoting lipolysis and improving sugar metabolism.
  • adipose tissue is at the core of the development and progression of obesity and plays a pivotal role in metabolic health. It is known that there are three kinds of adipose tissue, white fat, brown fat and beige fat in the human body.
  • White adipose tissue stores energy in the form of triglycerides, which consume energy through heat production.
  • Beige fat is found in white fat and has a brown fat character. In terms of phenotype, beige fat can be considered as an excessive form of white fat and brown fat.
  • browning of white fat is called browning of white fat.
  • thermogenic adipose tissue Although beige fat and brown fat have different sites and different development sources, they exert the same heat-producing function and have the same potential in terms of energy consumption. The two are called heat-producing adipose tissue. Under physiological conditions, there are two conditions that activate thermogenic adipose tissue: cold exposure and dietary intake (especially high calorie foods). During cold exposure, the heat-producing adipose tissue activates to maintain a constant body temperature; during dietary intake, the heat-producing fat activates to relieve transient energy peaks, thereby maintaining metabolic stability.
  • the heat-producing adipose tissue is the research hotspots in the field of metabolism in recent years.
  • individuals with active heat-producing adipose tissue are not prone to gain weight and are not susceptible to metabolic-related diseases such as type 2 diabetes.
  • Some drugs that improve metabolism, part of the effect is achieved by activating heat-producing adipose tissue, such as berberine and salicyl salicylic acid.
  • heat-producing adipose tissue such as berberine and salicyl salicylic acid.
  • beige adipose tissue is a heat-producing adipose tissue in human body, has the potential to consume excess energy, and can be activated under certain conditions; activates thermogenic adipose tissue, regardless of Is to enhance the function of brown fat, or to promote browning of white fat, can play a role in weight control and metabolism.
  • thermogenic adipose tissue regardless of Is to enhance the function of brown fat, or to promote browning of white fat, can play a role in weight control and metabolism.
  • UCP1 Uncoupling Protein 1
  • UCP1 is localized on the inner membrane of the mitochondria. In the case of activation, the coupling of the electron transport chain and ATP synthesis can be cut off, and the chemical energy can be directly converted into heat energy.
  • UCP1 is present in heat-producing adipose tissue. Due to this unique tissue localization, UCP1 is not only a functional molecule of thermogenic adipose tissue, but also a phenotypic marker. In addition to UCP1, PGC1 ⁇ , PRDM16 and Cytochrome C are important molecules involved in the browning process.
  • the former simulates the differentiation of adipose precursor cells into mature adipocytes, which reflects the differentiation of mesenchymal stem cells into adipocytes after orientation. process.
  • the cell model for studying the development of fat is also the Stromal Vascular Fraction (SVF), which is a primary precursor fat cell. After isolation and culture, it can differentiate into white after certain induction. Adipocytes, thereby mimicking the in vivo process of differentiation of precursor fat cells into white fat in an in vitro environment.
  • SVF Stromal Vascular Fraction
  • Artemether is an analog of artemisinin (Formula I) which is capable of killing erythrocyte endoplasmic plaques efficiently and rapidly; it is used against chloroquine falciparum malaria and The treatment of sinister malaria is markedly effective, with short-term curative effect and small side effects. It is a first-line drug for the treatment of malaria and is widely used. Recent studies have shown that artemether also has the effect of killing tumor cells and immunomodulation, showing new potential in the treatment of tumors and autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis.
  • artemisinin drugs include artesunate and dihydroartemisinin, arteether, SM905, Artemiside, Artemisone and SM934, among which dihydroartemisinin is artemisia. Secondary metabolites of ethers and other artemisinin analogues in the body.
  • the inventors of the present application intend to provide new uses of artemether and artemisinin analogues in pharmaceuticals, and in particular to the use of artemether in the preparation of a preparation for promoting lipolysis and improving glucose metabolism.
  • the object of the present invention is to provide a novel use of artemether and an artemisinin analogue in pharmaceuticals, in particular to the use of artemether in the preparation of a preparation for promoting lipolysis and improving glucose metabolism, in particular, artemether and cyanine.
  • Artemisinin analogues are useful in the preparation of browning which promotes white fat and in the preparation of reduced body weight and improved metabolic agents.
  • a first aspect of the present invention provides an artemisinin analogue represented by the following general formula (A) in the preparation of a medicament for promoting lipolysis and/or preventing or treating a metabolic-related disease use:
  • R is selected from the group consisting of: hydroxy, methoxy, ethoxy, succinic acid monoester (succinate), 2-aminoethoxy, 2R-3-tert-butylamino-2-hydroxypropoxy a thiomorpholinyl group and a 1,1-dioxythiomorpholinyl group.
  • a second aspect of the present invention provides a method for promoting lipolysis and/or preventing or treating a metabolic-related disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of the following formula (A) Artemisinin analogues:
  • R is selected from the group consisting of: hydroxy, methoxy, ethoxy, succinic acid monoester (succinate), 2-aminoethoxy, 2R-3-tert-butylamino-2-hydroxypropoxy a thiomorpholinyl group and a 1,1-dioxythiomorpholinyl group.
  • said administering a therapeutically effective amount of said artemisinin analogue is by oral or injection; more preferably, said artemisinin analogue is administered at a dose of:
  • Oral 20mg/kg ⁇ 100mg/kg; preferably 50mg/kg ⁇ 100mg/kg;
  • Injection 10 mg/kg to 50 mg/kg; preferably 20 mg/kg to 40 mg/kg.
  • a third aspect of the present invention provides an artemisinin analogue for promoting lipolysis and/or preventing or treating a metabolic-related disease, the artemisinin analogue being as shown in the following formula (A):
  • R is selected from the group consisting of: hydroxy, methoxy, ethoxy, succinic acid monoester (succinate), 2-aminoethoxy, 2R-3-tert-butylamino-2-hydroxypropoxy a thiomorpholinyl group and a 1,1-dioxythiomorpholinyl group.
  • artemisinin analogue is: artemether as shown in formula (II):
  • said promoting lipolysis comprises promoting browning of white fat.
  • the promoting lipolysis and/or preventing or treating a metabolic related disease comprises controlling body weight and improving metabolism.
  • the metabolic related disease is hyperglycemia, insulin resistance, dyslipidemia and/or fatty liver caused by obesity.
  • a fourth aspect of the present invention provides a pharmaceutical composition for promoting lipolysis and/or preventing or treating a metabolic-related disease, the pharmaceutical composition comprising: (1) artemisinin according to the third aspect of the present invention Analogs and (2) pharmaceutically acceptable carriers.
  • the oral administration dosage form of the pharmaceutical composition is selected from the group consisting of: oral suspensions, powders, and granules; and the dosage form for injection administration is selected from the group consisting of aqueous agents, oils, and suspensions.
  • the present invention also relates to artemisinin analogs other than artemether having the potential to promote browning of white fat.
  • the artemisinin structure is as shown in the formula (I), and the artemisinin analog is represented by the formula (A).
  • the artemisinin analogue of formula (A) is dihydroartemisinin, R is hydroxyl; artemether, R is ethoxy; artesunate, R is succinic acid monoester (succinate) SM934, R is 2-aminoethoxy; SM905, R is 2R-3-tert-butylamino-2-hydroxypropyloxy; artemiside, R is thiomorpholinyl; artemisinone artemisone, R is 1,1-dithiomorpholino group.
  • the artemisinin analogues of the present invention have R groups as shown in the following table:
  • artemisininin compounds have the effect of inducing browning of white fat, and by promoting the browning of white fat, thereby reducing body weight, improving metabolism, maintaining glucose homeostasis, and alleviating fatty liver. Improve the effects of diabetes-related symptoms.
  • the clinical application practice of the artemisinin drugs shows that no intolerable side effects have been found. In studies of rodent models, it has been reported that high concentrations of artemether cause anorexia, but there is no clear evidence of the dose and timing of administration. Therefore, as a clinical drug, artemether has an ideal safety.
  • Figure 1 shows that artemether renders 3T3-L1 brownish cell-like.
  • 1 shows the morphological changes of 3T3-L1 cells treated with different concentrations of ART;
  • FIG. 2 shows the mRNA levels of UCP1 after different concentrations of ART treated 3T3-L1. *p ⁇ 0.05, **p ⁇ 0.01.
  • Figures 3-5 show that artemether causes BMP4-directed C3H10T1/2 to exhibit brownish-like morphological features.
  • Figure 3 shows the cell morphology of BMP7-directed C3H10T1/2, BMP4-directed C3H10T1/2, artemether-treated BMP4-directed C3H10T1/2;
  • Figure 4 shows Western Blot detection of BMP4-directed C3H10T1/ 2, BMP4-directed C3H10T1/2 and different concentrations of artemether-treated BMP4-directed C3H10T1/2 brown fat-related genes UCP1, PGC1a, PRDM16 and CytoC protein levels;
  • Figure 5 shows artemether (15 ⁇ Mol /L) After treatment, BMP4-directed C3H10T1/2 mitochondrial MitoTracker Green staining.
  • Figures 6-7 show that artemether provides brown fat-like morphological features of SVF primary adipose precursor cells during differentiation.
  • Figure 6 shows the changes in cell morphology after treatment of SVF with different concentrations of artemether
  • Figure 7 shows the expression levels of the brown-related genes PRDM16, PGC1 ⁇ and UCP1 after treatment of SVF with different concentrations of artemether.
  • Figures 8-9 illustrate the effect of artemisinin analogues on promoting browning.
  • Figure 8 shows the cell morphology of BMP4-directed C3H10T1/2 treated with artemisinin and its analogues, artemether, artesunate and dihydroartemisinin;
  • Figure 9 shows different concentrations of dihydrocyanine The expression level of brown-related genes after treatment with BMP4-directed C3H0T1/2 by artemisinin.
  • FIG. 10 shows that artemether inhibits weight gain induced by a high fat diet.
  • 10A shows the change in body weight of a mouse subcutaneously injected with artemether under a high-fat diet condition, *p ⁇ 0.05;
  • FIG. 10B shows the body weight of a mouse subcutaneously injected with artemether under a high-fat diet. Growth, *p ⁇ 0.05.
  • FIG. 11 shows that subcutaneous injection of artemether reduced adipose tissue in mice fed high fat.
  • FIG. 11A shows the volume of brown fat of the inguinal fat, epididymal fat, and interscapular region of the mouse subcutaneously injected with artemether and the control group
  • FIG. 11B shows the subcutaneous injection of artemether.
  • the ratio of the weight of the inguinal fat, the epididymal fat, and the brown fat of the interscapular region to the mouse weight of the mice of the mice and the control group *p ⁇ 0.05
  • FIG. 11C shows the mice injected subcutaneously with artemether Morphology of brown fat cells in the inguinal fat, epididymal fat, and interscapular region of mice in the control group.
  • FIG. 12 shows that subcutaneous injection of artemether improves glucose metabolism in high fat-induced obese mice.
  • 12A shows the glucose tolerance curve of mice and control mice subcutaneously injected with artemether after eight weeks of high-fat diet feeding, *p ⁇ 0.05;
  • FIG. 12B shows that after eight weeks of high-fat diet feeding, Glucose tolerance curves of mice and control mice injected subcutaneously with artemether, *p ⁇ 0.05.
  • Figure 13 shows that artemether inhibits fatty liver induced by a high fat diet. Among them, the liver cell morphology of the mice and the control mice injected with artemether subcutaneously after eight weeks of high-fat diet feeding.
  • FIG. 14 shows the browning of subcutaneous fat in mice fed a high fat diet by subcutaneous injection of artemether.
  • FIG. 14A shows changes in body temperature of a high-fat-fed mouse in an environment of 4 ° C after administration for eight weeks.
  • Figure 14B shows the expression of UCP1 in the inguinal adipose tissue after exposure to high fat fed mice for 8 hours at 4 °C after 8 weeks of administration.
  • Figure 15 shows that intraperitoneal injection of artemether inhibits weight gain induced by a high fat diet.
  • 15A shows the change in body weight of mice injected with artemether intraperitoneally under high-fat diet conditions, *p ⁇ 0.05;
  • FIG. 15B shows the body weight of mice injected with artemether by intraperitoneal injection under high-fat diet conditions. Growth, *p ⁇ 0.05.
  • FIG. 16 shows that intraperitoneal injection of artemether significantly reduced adipose tissue in high fat fed mice.
  • FIG. 16A shows the volume of brown fat of the inguinal fat, epididymal fat, and interscapular region of the mice injected with artemether by intraperitoneal injection and the mice of the control group
  • FIG. 16B shows the intraperitoneal injection of artemether.
  • FIG. 16C shows the mice injected with artemether intraperitoneally. Morphology of brown fat cells in the inguinal fat, epididymal fat, and interscapular region of mice in the control group.
  • FIG. 17 shows that intraperitoneal injection of artemether improves glucose metabolism in high fat-induced obese mice.
  • FIG. 17A shows random blood glucose of mice and control mice intraperitoneally injected with artemether after eight weeks of high-fat diet feeding, *p ⁇ 0.05
  • FIG. 17B shows abdominal cavity after eight weeks of high-fat diet feeding Glucose tolerance test results of mice injected with artemether and control mice, *p ⁇ 0.05
  • Figure 17C shows insulin tolerance of mice and control mice injected intraperitoneally with artemether after eight weeks of high-fat diet feeding Situation, *p ⁇ 0.05.
  • Figure 18 shows the histological morphology of liver cells of mice and control mice injected intraperitoneally with artemether after eight weeks of high fat diet feeding.
  • Figure 19 shows changes in serum lipid metabolism-related indicators in mice injected with artemether after intraperitoneal injection of high-fat diet for 8 weeks, *p ⁇ 0.05, **p ⁇ 0.01.
  • Figure 20 shows the browning of subcutaneous fat in mice fed a high fat diet by intraperitoneal injection of artemether.
  • 20A shows the change in body temperature of the high-fat-fed mice in the environment of 4 ° C after 8 weeks of intraperitoneal injection of artemether, *p ⁇ 0.05;
  • FIG. 20B shows the intraperitoneal injection of artemether for eight weeks.
  • High fat-fed mice were exposed to UCP1 expression in the inguinal adipose tissue after exposure to 4 °C for 6 hr.
  • Figure 21 shows that artemether inhibits the weight gain of diabetic mouse ob/ob mice.
  • Fig. 21A shows the change in body weight of ob/ob mice by intraperitoneal injection of artemether for eight weeks;
  • Fig. 21B shows the change in body weight of ob/ob mice by intraperitoneal injection of artemether for eight weeks, *p ⁇ 0.05.
  • Fig. 22 shows that artemether improves fatty liver of diabetic mouse ob/ob, which shows the result of HE staining of liver sections after ob, ob, and eight weeks after intramuscular injection of artemether.
  • Figure 23 shows that intraperitoneal injection of artemether does not affect the growth of healthy mice.
  • Fig. 23A shows the weight of healthy mice, normal feed, intraperitoneal injection of artemether for eight weeks, mice in the administration group and control group;
  • Fig. 23B shows healthy mice, common feed, and intraperitoneal injection of artemisia Methyl ether for eight weeks, the weight gain of the mice in the drug-administered group and the control group.
  • Figure 24 shows that intraperitoneal injection of artemether does not affect glucose metabolism in healthy lean mice.
  • Fig. 24A shows a case of healthy lean mice, which was intraperitoneally injected with artemether for eight weeks, and glucose tolerance
  • Fig. 24B shows a case of healthy lean mice, which was intraperitoneally injected with artemether for eight weeks, and insulin tolerance.
  • Figure 25 shows HE staining of liver tissue sections showing intraperitoneal injection of 20 mg/kg body weight of artemether without causing liver damage in healthy lean mice, intraperitoneal injection of artemether for eight weeks.
  • Figure 26 shows that artemether does not alter the food intake of mice.
  • 26A shows the lean mice fed with conventional feed, artemether was intraperitoneally injected for 8 weeks, and the daily intake of each mouse;
  • FIG. 26B shows the obese mice fed with high fat diet, intramuscular injection of artemether. Feed consumption per mouse per day for eight weeks.
  • Example 1 Artemether induced browning of 3T3-L1 white precursor adipocytes during differentiation
  • Biotin 160 mg biotin and 80 mg calcium pantothenate are dissolved in 200 ml deionized water. Heat to dissolve. 0.22 ⁇ m filter membrane was sterilized by filtration.
  • Dexamethasone (1 mMol/L): 0.039 g of dexamethasone was dissolved in 100 ml of water and sterilized by filtration through a 0.22 ⁇ m filter.
  • Insulin (1mg/ml): 0.1g of insulin is dissolved in 100ml of water, dissolved in a few drops of hydrochloric acid, and sterilized by filtration on a 0.22 ⁇ m filter.
  • 3T3-L1 white adipose precursor cells 2.5 ⁇ 10 5 3T3-L1 white adipose precursor cells were seeded in a 3.5 cm cell culture dish, and cultured for 3 days in a maintenance medium, and the cells were proliferated to contact inhibition. 48 hours after the contact inhibition was reached, the differentiation-inducing medium was changed, and the differentiation-inducing mixture was added to induce a differentiation mixture including 0.5 mMol/L of 3-isobutyl-1-methylxanthine (Mix), 1 ⁇ Mol/L of dexamethasone. (Dex), and 1 ⁇ g/ml of insulin (Insulin).
  • the induced differentiation mixture is referred to as MDI.
  • the day of joining MDI was defined as day 0 of differentiation.
  • Artemether was stored in DMSO at a concentration of 10 mMol/L, and MDI was added on the 0th day of differentiation, and then treated with artemether (5, 10 or 15 ⁇ Mol/L), and each time the medium was changed, the same was added. The concentration of artemether until 3T3-L1 differentiated and matured.
  • Differentiated mature 3T3-L1 was induced in a 3.5 cm culture dish, and the differentiation method was as described in Method 1.4.1. After washing with PBS, fully lyse the cells with 1 ml of Trizol, mix thoroughly, add 200 ⁇ l of chloroform, shake vigorously, let stand for 15 min at room temperature, centrifuge at 4 ° C, centrifuge for 12000 g, 10 min, at this time due to chromatography The lysate is divided into three layers.
  • the corresponding cDNA was synthesized by reverse transcription using 1 ⁇ g of RNA, and the reverse transcription kit was Thermo Scientific Revert Aid First Strand cDNA Synthesis Kit.
  • the synthesis system is as follows
  • the synthesized cDNA was stored in a refrigerator at -20 °C.
  • the above system has a total of 30 ⁇ l and is divided into three multi-wells, each of which has a 10 ⁇ l.
  • the forward primer sequence for amplification of 18S is GTAACCCGTTGAACCCCATT; the reverse primer sequence is CCATCCAATCGGTAGTAGCG.
  • the forward sequence of the amplified UCP1 is AGGCTTCCAGTACCATTAGGT; the reverse primer is CTGAGTGAGGCAAAGCTGATTT.
  • the relative amount of mRNA was calculated according to the ⁇ CT method, and 18S was used as an internal reference, and the control mRNA level was set to 1, and each treatment group was compared with the control group to obtain a relative amount.
  • the morphology of the cells was observed under the microscope Olympus IX711 and recorded.
  • the 3T3-L1 of the control group exhibited typical white fat cell characteristics, the cell volume was large, the lipid droplets were large and round, and the number of lipid droplets was higher.
  • 3T3-L1 treated with Artemether (ART) showing typical brown-like characteristics, small cell volume, small and dense lipid droplets. And this change has a concentration-dependent trend. As the concentration of artemether increases, the change of 3T3-L1 adipocytes is more obvious.
  • the total RNA of 3T3-L1 was extracted and reverse transcribed into cDNA.
  • the mRNA level of UCP1 was detected by Real time qPCR. Compared with the control group, the expression of UCP1 in ART treated 3T3-L1 was significantly up-regulated and showed a concentration-dependent trend. .
  • 3T3-L1 is a classic white fat precursor cell. Artemether gives brown-like characteristics during differentiation. It not only has the morphological characteristics of brown fat, but also the expression of brown fat labeled UCP1.
  • Example 2 artemether induced C3H10T1/2 mesenchymal stem cells in the process of differentiation into adipocytes Brown fat-like features
  • dexamethasone 0.039 g was dissolved in 100 ml of water and sterilized by filtration through a 0.22 ⁇ m filter.
  • 0.1 g of insulin was dissolved in 100 ml of water, dissolved in a few drops of hydrochloric acid, and sterilized by filtration through a 0.22 ⁇ m filter.
  • Tris-HCL pH 6.8
  • SDS 2%
  • Glycerol 10%
  • NaF 10mMol/L Na3VO4 100mMol/L
  • 7.5 x 10 5 C3H10T1/2 was seeded on a 3.5 cm cell culture dish. Incubate for 3-5 days with maintenance medium. On the next day of inoculation, BMP4 (final concentration of 20 ng/ml) was added to the medium to allow the cells to proliferate to a contact inhibition state. Two days after the contact inhibition state was reached, the medium was changed to induce differentiation medium, and an induction mixture (0.5 mMol/L Mix, 1 ⁇ Mol/L Dex, 1 ng/ml insulin, 100 nMol/L indomethacin, 1 nM T3) was added.
  • BMP4 final concentration of 20 ng/ml
  • 7.5 x 10 5 C3H10T1/2 was seeded on a 3.5 cm cell culture dish. Incubate for 3-5 days with maintenance medium. On the next day of inoculation, BMP7 (final concentration of 100 ng/ml) was added to the medium to allow the cells to proliferate to a contact inhibition state. Two days after the contact inhibition state was reached, the medium was changed to induce differentiation medium, and an induction mixture (0.5 mM Mix, 1 ⁇ M Dex, 1 ng/ml insulin, 100 nM indomethacin, 1 nM T3) was added, and this day was defined as differentiation 0.
  • BMP7 final concentration of 100 ng/ml
  • the compound On the 0th day of differentiation, that is, while the differentiation-inducing mixture was added, the compound was added to the medium at a storage concentration of 10 mMol/L, and the final concentrations were 5 ⁇ Mol/L, 10 ⁇ Mol/L and 15 ⁇ Mol/L, respectively.
  • the fresh medium is replaced, the corresponding compound is supplemented, and the compound is always accompanied by cell to differentiation and maturation.
  • the differentiation method is described in 2.4.1, 300 ⁇ l lysate is added, the cells are fully lysed, the lysate is transferred to a 1.5ml centrifuge tube, heated at 100 °C for 10min, centrifuged at 13000g at 4 °C, 10min, removed For the upper layer lipid, the middle clarified lysate was transferred to a new centrifuge tube, 2.5 ⁇ l of the lysate was quantified by BCA method, 200 ⁇ l of the lysate was mixed with 50 ⁇ l of Loading Buffer, heated at 100 ° C for 10 min, and stored at -20 ° C for use. .
  • C3H0T1/2 protein 30 ⁇ g was loaded onto a 10% polyacrylamide gel, electrophoresed, electrophoresed with a layer of gel at 80 V, and subjected to electrophoresis of the separated gel at 100 V until the target strip was dispersed.
  • the membrane was transferred to a PVDF membrane in an ice bath at 100 V for 100 min. After the membrane was transferred, it was blocked for 1 hr in 5% skim milk prepared with TBST (TBS buffer + 0.5% Tween). Incubate with specific antibodies overnight at 4 °C.
  • the PVDF membrane was washed with TBST, washed at room temperature for 10 min, washed three times with the corresponding secondary antibody, at room temperature for 1 hr, washed three times with TBST, the same anti-antibiotic, co-incubated with chemiluminescent reagent, room temperature for 1 min, ImageQuant LAS4000 Mini automatic chemistry Luminometer, exposure, record results.
  • the antibody number and antibody dilution ratio are shown in the following table:
  • C3H10T1/2 is a mesenchymal stem cell with multipotential differentiation potential, differentiates into white adipose cells in the case of BMP4 orientation, and differentiates into brown fat cells in the case of BMP7 orientation.
  • artemether (15 ⁇ Mol/L) was added simultaneously.
  • BMP7-directed C3H10T1/2 has typical brown adipocyte characteristics, small cells and small lipid droplets;
  • BMP4-directed C3H10T1/2 has typical white fat cell characteristics, large cells and large lipid droplets.
  • artemether 15 ⁇ Mol/L
  • BMP7-directed C3H10T1/2 was used as a positive control.
  • BMP4-directed C3H10T1/2 was treated with different concentrations of artemether (5, 10, 15 ⁇ Mol/L), and the protein level of brown fat-related genes was detected by Western blot. Actin as an internal reference. The results showed that artemether increased the protein levels of the brown-related genes UCP1, PGC1a, PRDM16 and CytoC in BMP4-directed C3H10T1/2 cells in a concentration-dependent manner (as shown in Figure 4).
  • Example 3 artemether causes differentiation of vascular matrix components (Stromal Vascular Fraction, SVF) Brown fat cell-like features obtained during the process
  • DMEM/F12 (1:1) Up to 500ml Fetal bovine serum 10% Biotin 8mg/ml penicillin 100U/ml Streptomycin 100U/ml
  • the inguinal fat of male mice was cut, treated with collagenase at 37 ° C for 15 min, centrifuged, 250 rpm for 10 min, resuspended in F12/DMEM medium, and 2.5 ⁇ 10 5 cells were seeded in 3.5 cm culture dishes. 1 ml of polylysine was treated for 30 min.
  • induction was performed according to the adipocyte differentiation protocol as follows: with 10% fetal bovine serum, 0.5 mMol/L Mix, 1 ⁇ Mol/L Dex, 5 ⁇ g/ml Insulin 2 days after the induction of F12/DMEM of 1 ⁇ Mol/L Rosiglitazone, the culture was continued for 2 days with 10% fetal bovine serum, 5 ⁇ g/ml insulin, 1 ⁇ Mol/L Rosiglitazone, DMEM/F12, and then replaced with 10% fetal cattle every other day. Serum DMEM/F12 medium until cell differentiation to day 8.
  • the method for extracting total SVF protein is the same as 2.4.3, Western blot method and antibody number 2.4.4
  • Artemether makes brown fat-like morphological characteristics of SVF primary adipose precursor cells during differentiation
  • Example 4 in addition to artemether, other artemisinin analogues also promote browning
  • Artemisinin, artesunate and dihydroartemisinin were stored in DMSO at a concentration of 10 mMol/L.
  • the concentration shown in Figure 8 was added on day 0 of differentiation, and fresh medium was replaced every two days. The corresponding concentration of the compound until the cells are differentiated and matured.
  • the method for extracting total protein of C3H10T1/2 cells is the same as 2.4.3;
  • Dihydroartemisinin is a secondary metabolite of artemisinin and its analogues. If dihydroartemisinin promotes browning, artemisinin and its analogues promote browning. The role. After treatment of BMP4-directed C3H10T1/2 with different concentrations (1, 2.5, 5, 7.5 ⁇ Mol/L) of dihydroartemisinin, the expression levels of brown fat-related genes PGC1 ⁇ and UCP1 were detected by Western blot, and brown related genes were found. The expression level gradually increased with the concentration of dihydroartemisinin (as shown in Figure 9); the results confirmed that artemisinin analogues have the potential to promote browning of white fat.
  • Example 5 artemether inhibits weight gain induced by high fat diet
  • mice were housed in the SPF animal laboratory of the Experimental Animal Center of Fudan University. The room temperature was 21 ° C - 23 ° C, and the fat content of the high fat diet used in obese mice was 60% per 12 hr light dark cycle. When the mice were grown to 8-week age, they were randomly divided into a control group and a drug-administered group, with 8 rats in each group. At the same time as feeding with high fat diet, artemether was injected subcutaneously. Artemether was diluted in PBS at a concentration of 15 ⁇ Mol/L, injected bilaterally into the groin, 200 ⁇ l per side, and the control mice were injected with the same volume of PBS. Dosing continued for eight weeks and the body weight of the mice was recorded weekly.
  • mice injected subcutaneously with artemether were significantly lower than that of the control mice. After six weeks of administration, the difference in body weight between the two groups reached a statistical difference, as shown in Fig. 10A.
  • the weight gain of the mice in the drug-administered group was significantly slower than that in the control group, as shown in Fig. 10B.
  • Subcutaneous injection of artemether significantly inhibited the weight gain of mice under high-fat feeding conditions.
  • mice were housed in the SPF animal laboratory of the Experimental Animal Center of Fudan University. The room temperature was 21 ° C - 23 ° C, and the fat content of the high fat diet used in obese mice was 60% per 12 hr light dark cycle. When the mice were grown to 8-week age, they were randomly divided into a control group and a drug-administered group, with 8 rats in each group. At the same time as feeding with high fat diet, artemether was injected subcutaneously. Artemether was diluted in PBS at a concentration of 15 ⁇ Mol/L, injected bilaterally into the groin, 200 ⁇ l per side, and the control mice were injected with the same volume of PBS. The administration lasted for eight weeks.
  • mice were sacrificed by cervical dislocation, and the inguinal fat (Inguinal, subcutaneous fat), the fat of the epididymis (Gonadal, visceral fat) and the interscapular area of brown fat (BAT, classic brown fat) were dissected, weighed and fixed. Entrusted Shanghai Saige Biotechnology Co., Ltd. to analyze the adipose tissue histology.
  • the histological analysis process includes fixed, paraffin embedding, sectioning and HE staining.
  • adipose tissues (inguinal fat, epididymal fat and brown fat) were taken and analyzed. It was found that the volume (Fig. 11A) and weight (Fig. 11B) of the mouse adipose tissue injected subcutaneously with artemether were lower than those of the control group. Significantly small. Sectioning and staining of adipose tissue showed that the adipocytes of the mice in the administration group were significantly smaller than the control group (Fig. 11C). Artemether reduced the fat volume and weight of mice fed with high fat, and the weight loss caused by artemether was mainly caused by the decrease of adipose tissue.
  • Example 7 Subcutaneous injection of artemether to improve glucose metabolism in obese mice induced by high fat
  • mice were housed in the SPF animal laboratory of the Experimental Animal Center of Fudan University. The room temperature was 21 ° C to 23 ° C, and the light-dark cycle was every 12 hours. The fat content of the high-fat diet used in obese mice was 60%. Mouse growth At the age of 8-week, they were randomly divided into a control group and a drug-administered group, with 8 rats in each group. At the same time as feeding with high fat diet, artemether was injected subcutaneously. Artemether was dissolved in PBS at a concentration of 15 ⁇ Mol/L, bilateral groin injections, 200 ⁇ l per side, and control mice were injected with the same volume of PBS. The administration lasted for eight weeks.
  • mice were fasted for 12-16 hours, intraperitoneally injected with 2 mg/kg body weight of glucose injection, and at 0 min, 30 min, 60 min, 90 min and 120 min after injection, blood was taken from the tail and detected with OneTouch ExtraEasy blood glucose meter and OneTouch ExtraEasy blood glucose test strip. Blood sugar is recorded.
  • Insulin was injected intraperitoneally with 0.75 U/kg body weight. At 0 min, 15 min, 30 min, 45 min and 60 min after injection, blood was taken from the tail and blood glucose was measured.
  • mice with subcutaneous injection of artemether had significantly higher glucose tolerance than control mice after eight weeks of high-fat feeding (Fig. 12A), and insulin tolerance was also significantly better than control mice (Fig. 12B).
  • Fig. 12A glucose tolerance was also significantly better than control mice
  • Fig. 12B glucose metabolism and insulin sensitivity of mice injected subcutaneously with artemether were significantly better than that of the control mice after eight weeks of administration, indicating that artemether has a role in maintaining glucose homeostasis.
  • Example 8 artemether inhibits fatty liver induced by high fat diet
  • mice were housed in the SPF animal laboratory of the Experimental Animal Center of Fudan University. The room temperature was 21 ° C - 23 ° C, and the fat content of the high fat diet used in obese mice was 60% per 12 hr light dark cycle.
  • artemether was injected subcutaneously.
  • Artemether was diluted in PBS at a concentration of 15 ⁇ Mol/L, injected bilaterally into the groin, 200 ⁇ l per side, and the control mice were injected with the same volume of PBS. The administration lasted for eight weeks.
  • the mice were sacrificed by cervical dislocation, and the liver tissues of the mice were taken out and entrusted by Shanghai Saige Biotech Co., Ltd. for histological analysis of tissue fixation, paraffin embedding, sectioning and HE staining.
  • Example 9 Subcutaneous injection of artemether induced browning of subcutaneous fat in mice fed with high fat diet
  • mice were housed in the SPF animal laboratory of the Experimental Animal Center of Fudan University. The room temperature was 21 ° C - 23 ° C, and the fat content of the high fat diet used in obese mice was 60% per 12 hr light dark cycle. When the mice were grown to 8-week age, they were randomly divided into a control group and a drug-administered group, with 8 rats in each group. At the same time as feeding with high fat diet, artemether was injected subcutaneously. Artemether was dissolved in PBS at a concentration of 15 ⁇ Mol/L, bilateral groin injections, 200 ⁇ l per side, and control mice were injected with the same volume of PBS. The administration lasted for eight weeks.
  • mice were fasted, placed in a 4 ° C broth, and the mice were sacrificed at 6 months after rectal temperature.
  • the inguinal fat was taken for histological analysis. Histological analysis including tissue fixation, paraffin embedding, sectioning and UCP1 immunohistochemistry experiments commissioned by Shanghai Saige Bio Co., Ltd..
  • mice injected subcutaneously with artemether were significantly higher than that of control mice when exposed to 4 ° C (Fig. 14A).
  • Preservation of body temperature in a cold exposed environment is a physiological function undertaken by thermogenic adipose tissue. Removal of the epididymal fat of the mice for immunohistochemical examination of UCP1 showed that the subcutaneous fat of the mice in the administration group showed significant UCP1 expression (Fig. 14B).
  • the mice in the subcutaneous injection of artemether had a marked increase in body temperature retention and a marked browning of subcutaneous fat. It is indicated that artemether inhibits weight gain and improves metabolism by promoting browning of subcutaneous fat.
  • Example 10 Intraperitoneal injection of artemether inhibits weight gain induced by high fat diet
  • mice were housed in the SPF animal laboratory of the Experimental Animal Center of Fudan University. The room temperature was 21 ° C - 23 ° C, and the fat content of the high fat diet used in obese mice was 60% per 12 hr light dark cycle. When the mice were grown to 8-week age, they were randomly divided into a control group and a drug-administered group, with 8 rats in each group. Intraperitoneal injection while feeding with high fat diet. Artemether for intraperitoneal injection was stored in coconut oil at a storage concentration of 6 mg/ml (20 mMol/L), protected from light and stored at 4 °C. The mice in the administration group were injected with artemether at a dose of 20 mg/kg. The drug was administered twice a week for 8 weeks and the body weight of the mice was recorded weekly.
  • mice injected with artemether by abdominal cavity was significantly lower than that of the control mice.
  • the difference in body weight between the two groups reached a statistical difference, as shown in Fig. 15A.
  • the weight gain of the mice in the administration group was significantly slower than that in the control group, as shown in Fig. 15B.
  • Intraperitoneal injection of artemether effectively inhibited weight gain induced by a high-fat diet.
  • mice were housed in the SPF animal laboratory of the Experimental Animal Center of Fudan University. The room temperature was 21 ° C - 23 ° C, and the fat content of the high fat diet used in obese mice was 60% per 12 hr light dark cycle. When the mice were grown to 8-week age, they were randomly divided into a control group and a drug-administered group, with 8 rats in each group. Intraperitoneal injection while feeding with high fat diet. Artemether for intraperitoneal injection was stored in coconut oil at a storage concentration of 6 mg/ml (20 mMol/L), protected from light and stored at 4 °C. The mice in the administration group were injected with artemether at a dose of 20 mg/kg. It was administered twice a week for 8 weeks.
  • mice were sacrificed by cervical dislocation, and the inguinal fat (Inguinal, subcutaneous fat), the fat of the epididymis (Gonadal, visceral fat) and the interscapular area of brown fat (BAT, classic brown fat) were dissected, weighed and fixed. , commissioned by Shanghai Saige for histological analysis of adipose tissue, including fixation, paraffin embedding, sectioning and HE staining.
  • adipose tissues Three representative adipose tissues were taken for analysis, and it was found that the volume (Fig. 16A) and weight (Fig. 16B) of the adipose tissue of the mouse subcutaneously injected with artemether were significantly smaller than those of the control group. Sectioning and staining of adipose tissue showed that the fat cells of the mice in the administration group were significantly smaller than the control group (Fig. 16C). Intraperitoneal injection of artemether effectively inhibits fat accumulation caused by a high-fat diet.
  • Example 12 Intraperitoneal injection of artemether to improve glucose metabolism in obese mice induced by high fat
  • mice were housed in the SPF animal laboratory of the Experimental Animal Center of Fudan University. The room temperature was 21 ° C - 23 ° C, and the fat content of the high fat diet used in obese mice was 60% per 12 hr light dark cycle.
  • artemether was injected intraperitoneally.
  • Artemether for intraperitoneal injection was stored in coconut oil at a storage concentration of 6 mg/ml (20 mMol/L), protected from light and stored at 4 °C.
  • the mice in the administration group were injected with artemether at a dose of 20 mg/kg. It was administered twice a week for 8 weeks.
  • mice were fasted for 12-16 hr, intraperitoneal injection of 2 mg/kg body weight of glucose injection, and at 0 min, 30 min, 60 min, 90 min and 120 min after injection, blood was taken from the tail, and blood glucose was measured with OneTouch ExtraEasy blood glucose meter and OneTouch ExtraEasy blood glucose test strip. And record.
  • Insulin was injected intraperitoneally with 0.75 U/kg body weight. At 0 min, 15 min, 30 min, 45 min and 60 min after injection, blood was taken from the tail and blood glucose was measured.
  • mice were anesthetized with 10% chloral hydrate, blood was taken through the eyelids, whole blood was centrifuged, serum was taken at 4 ° C, 300 rpm, 5 min, and blood glucose calibrators Cfas and kits (Roche) were used to detect blood glucose. s level.
  • mice with intraperitoneal injection of artemether had significantly lower random blood glucose than the control group, and glucose tolerance was significantly better than that of control mice (Fig. 12A). Insulin sensitivity was also superior to control mice. 12B). Intraperitoneal injection of artemether effectively reduced blood glucose in obese mice fed high fat, significantly improved glucose metabolism and insulin sensitivity.
  • Example 13 Intraperitoneal injection of artemether effectively inhibits fatty liver development in obese mice induced by high fat diet
  • mice were housed in the SPF animal laboratory of the Experimental Animal Center of Fudan University. The room temperature was 21 ° C - 23 ° C, and the fat content of the high fat diet used in obese mice was 60% per 12 hr light dark cycle. When the mice were grown to 8-week age, they were randomly divided into a control group and a drug-administered group, with 8 rats in each group. At the same time as feeding with high fat diet, artemether was injected intraperitoneally. Artemether for intraperitoneal injection was stored in coconut oil at a storage concentration of 6 mg/ml (20 mMol/L), protected from light and stored at 4 °C. The mice in the administration group were injected with artemether at a dose of 20 mg/kg.
  • mice were sacrificed by cervical dislocation, and the liver tissue was removed.
  • the liver was subjected to histological analysis by Shanghai Saige Biotechnology Co., Ltd.
  • the histological analysis process included tissue fixation, paraffin embedding, sectioning and HE staining.
  • the liver of the control group showed obvious lipid accumulation, while the mice injected with artemether in the peritoneal cavity showed only a small amount of lipid in the liver, and the morphology of the liver cells was almost normal. 18 is shown. Intraperitoneal injection of artemether effectively inhibited the development of fatty liver induced by high-fat diet.
  • Example 14 Intraperitoneal injection of artemether inhibited elevated blood lipids caused by a high-fat diet
  • mice 14.1.1 Male C57BL/6J (4-6week) mice (Slake Laboratory Animal Co., Ltd.)
  • mice were housed in the SPF animal laboratory of the Experimental Animal Center of Fudan University. The room temperature was 21 ° C - 23 ° C, and the fat content of the high fat diet used in obese mice was 60% per 12 hr light dark cycle.
  • artemether was injected intraperitoneally.
  • Artemether for intraperitoneal injection was stored in coconut oil at a storage concentration of 6 mg/ml (20 mMol/L), protected from light and stored at 4 °C.
  • the mice in the administration group were injected with artemether at a dose of 20 mg/kg. It was administered twice a week for 8 weeks.
  • mice were anesthetized with 10% chloral hydrate, blood was taken through the eyelids, whole blood was centrifuged, 4 ° C, 300 rpm, 5 min, serum, lipid calibration standards Cfas Lipid, high density lipoprotein Cfas and low density lipoprotein Cfas, And test kits for each relevant indicator.
  • Example 15 Intraperitoneal injection of artemether induced browning of subcutaneous fat in mice fed with high fat diet
  • mice were housed in the SPF animal laboratory of the Experimental Animal Center of Fudan University. The room temperature was 21 ° C - 23 ° C, and the fat content of the high fat diet used in obese mice was 60% per 12 hr light dark cycle. When the mice were grown to 8-week age, they were randomly divided into a control group and a drug-administered group, with 8 rats in each group. At the same time as feeding with high fat diet, artemether was injected intraperitoneally. Artemether for intraperitoneal injection was stored in coconut oil at a storage concentration of 6 mg/ml (20 mMol/L), protected from light and stored at 4 °C. The mice in the administration group were injected with artemether at a dose of 20 mg/kg.
  • mice were fasted and placed in a 4 °C terrarium. The rectal temperature was measured every 2 hr. After 6 hr, the mice were sacrificed by cervical dislocation and the inguinal fat was taken for histological analysis. Histological analysis including tissue fixation, paraffin embedding, sectioning and UCP1 immunohistochemistry experiments commissioned by Shanghai Saige Bio Co., Ltd..
  • the body temperature of mice injected with artemether intraperitoneally was significantly higher than that of the control mice when exposed to 4 ° C (Fig. 19A).
  • Preservation of body temperature in a cold exposed environment is a physiological function undertaken by thermogenic adipose tissue. Immunohistochemistry of UCP1 was performed by taking out the epididymal fat of the mice, and it was revealed that the subcutaneous fat of the mice in the administration group showed significant UCP1 expression (Fig. 19B).
  • the body temperature preservation ability of the mice in the administration group was significantly enhanced, and the subcutaneous fat was significantly browned. It is indicated that the effect of artemether on inhibiting weight gain is achieved by promoting browning of subcutaneous fat.
  • mice 4.week, Nanjing University Model Animal Center
  • mice were housed in the SPF animal laboratory of the Experimental Animal Center of Fudan University, room temperature 21 ° C - 23 ° C, every 12 hr light and dark cycle. When the mice were grown to 4-week age, they were randomly divided into a control group and a drug-administered group, 4 in each group.
  • the drug-administered group was intraperitoneally injected with artemether.
  • Artemether for intraperitoneal injection was stored in coconut oil at a storage concentration of 6 mg/ml (20 mMol/L), protected from light and stored at 4 °C.
  • the administration group ob/ob mice were injected with artemether at 20 mg/kg body weight. It was administered twice a week for 8 weeks. Weighed and recorded every week.
  • the ob/ob mice of the administration group had a lower body weight than the control group, but did not reach a statistical difference (Fig. 21A). However, the weight gain of the mice in the administration group was significantly slower than that in the control group (Fig. 21B). It was shown that artemether significantly inhibited the weight gain of ob/ob mice, but the difference between the drug-administered group and the control group did not reach statistical significance at the time of administration for eight weeks. Artemether effectively inhibited the weight gain of diabetic animal model ob/ob mice.
  • mice Male ob/ob mice (4-week, Nanjing University Model Animal Center)
  • Ob/ob mice were housed in the SPF animal laboratory of the Experimental Animal Center of Fudan University, room temperature 21 ° C - 23 ° C, every 12 hr light and dark cycle. When the mice were grown to 4-week age, they were randomly divided into a control group and a drug-administered group, 4 in each group.
  • the drug-administered group was intraperitoneally injected with artemether.
  • Artemether for intraperitoneal injection was stored in coconut oil at a storage concentration of 6 mg/ml (20 mMol/L), protected from light and stored at 4 °C.
  • the administration group ob/ob mice were injected with artemether at 20 mg/kg body weight. It was administered twice a week for 8 weeks. The mice were sacrificed by cervical dislocation, and the liver tissues were removed for paraffin embedding, sectioning and HE staining. The histological analysis process was commissioned by Shanghai Saige Biotech Co., Ltd.
  • the obesity mice in the artemether-administered group showed significant relief of fatty liver (Fig. 22); artemether effectively relieved the fatty liver of the diabetic animal model ob/ob.
  • Example 18 Intraperitoneal injection of artemether does not affect the growth of healthy mice
  • mice were housed in the SPF animal laboratory of the Experimental Animal Center of Fudan University, room temperature 21 ° C - 23 ° C, every 12 hr light dark cycle. When the mice were grown to 8-week age, they were randomly divided into a control group and a drug-administered group, with 8 rats in each group.
  • the drug-administered group was intraperitoneally injected with artemether.
  • Artemether for intraperitoneal injection was stored in coconut oil at a storage concentration of 6 mg/ml (20 mMol/L), protected from light and stored at 4 °C.
  • the mice in the administration group were injected with artemether at a dose of 20 mg/kg, and the mice in the control group were injected with 100 ⁇ l of coconut oil.
  • the drug was administered twice a week for 8 weeks and the body weight of the mice was recorded weekly.
  • Example 19 Intraperitoneal injection of artemether does not affect glucose metabolism in healthy lean mice
  • mice were housed in the SPF animal laboratory of the Experimental Animal Center of Fudan University, room temperature 21 ° C - 23 ° C, every 12 hr light dark cycle. When the mice were grown to 8-week age, they were randomly divided into a control group and a drug-administered group, with 8 rats in each group.
  • Artemether for intraperitoneal injection was stored in coconut oil at a storage concentration of 6 mg/ml (20 mMol/L), protected from light and stored at 4 °C.
  • the mice in the administration group were injected with artemether at a dose of 20 mg/kg, and the mice in the control group were injected with 100 ⁇ l of coconut oil. It was administered twice a week for 8 weeks.
  • mice were fasted for 12-16 hr, intraperitoneal injection of 2 mg/kg body weight of glucose injection, and at 0 min, 30 min, 60 min, 90 min and 120 min after injection, blood was taken from the tail, and blood glucose was measured with OneTouch ExtraEasy blood glucose meter and OneTouch ExtraEasy blood glucose test strip. And record.
  • Example 20 Intraperitoneal injection of 20 mg/kg body weight of artemether in mice did not cause liver damage
  • mice were housed in the SPF animal laboratory of the Experimental Animal Center of Fudan University, room temperature 21 ° C - 23 ° C, every 12 hr light dark cycle. When the mice were grown to 8-week age, they were randomly divided into a control group and a drug-administered group, with 8 rats in each group.
  • the drug-administered group was intraperitoneally injected with artemether.
  • Artemether for intraperitoneal injection was stored in coconut oil at a storage concentration of 6 mg/ml (20 mMol/L), protected from light and stored at 4 °C.
  • the mice in the administration group were injected with artemether at a dose of 20 mg/kg, and the control group was injected with 100 ⁇ l of coconut oil. It was administered twice a week for 8 weeks.
  • the mice were sacrificed by cervical dislocation and the liver tissues were fixed. Fixation of liver tissue, paraffin embedding, sectioning and HE staining were commissioned by Sego Bio Co., Ltd
  • liver cells were normal in morphology, as shown in Figure 25; intraperitoneal injection of artemether, 20 mg/kg body weight twice a week for eight Under the conditions of the week, artemether has no liver toxicity.
  • mice were housed in the SPF animal laboratory of the Experimental Animal Center of Fudan University, room temperature 21 ° C - 23 ° C, every 12 hr light dark cycle. When the mice grew to 8-week age, they were randomly divided into four groups of 8 rats each, which were a high-fat control group, a high-fat administration group, a conventional administration group, and a conventional control group.
  • the administration group (high fat and conventional feed) was intraperitoneally injected with artemether.
  • Artemether for intraperitoneal injection was stored in coconut oil at a storage concentration of 6 mg/ml (20 mMol/L), protected from light and stored at 4 °C.
  • the mice in the administration group were injected with artemether at a dose of 20 mg/kg, and the control group was injected with 100 ⁇ l of coconut oil.
  • the feed was weighed weekly and the feed consumption of each group of mice was recorded.
  • the feed consumption of the mice in the administration group was basically the same as that in the control group, and there was no difference as shown in Fig. 26A.
  • the conventional feeding group there was no difference in the feed consumption between the mice in the drug-administered group and the control group, as shown in Fig. 26B; the intraperitoneal injection of artemether was carried out under the conditions of conventional feed and high-fat feed. Does not affect the normal eating of mice. Under the conditions of this example, artemether did not cause anorexia nervosa.

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Abstract

式(A)的青蒿素类似物在制备用于促进脂肪分解和/或预防或治疗代谢相关疾病的药物中的用途。青蒿素类似物通过促进白色脂肪棕色化的作用，起到降低体重、改善代谢，治疗肥胖引起的高血糖、胰岛素抵抗、血脂紊乱和/或脂肪肝的效果。

Description

青蒿素类似物促进脂肪分解和改善糖代谢的用途、方法及组合物 技术领域
本发明属于生物医药领域，具体涉及蒿甲醚在制备促进脂肪分解和改善糖代谢制剂中的用途。
背景技术
资料显示，肥胖症及其引起的代谢性疾病已经发展为困扰全球的流行性疾病。根据国际卫生组织(World Health Organization，WHO)2015年发布的数据，全球人口的肥胖率高达13％；而在中国，成人肥胖率为6.9％。令人堪忧的是，青少年儿童的肥胖率更高，并且呈现出激增的趋势，这意味在未来的数十年中，肥胖率会持续增长。研究显示，肥胖症会引起一系列代谢性疾病，如二型糖尿病，高血压，脂肪肝等，甚至会增加某些肿瘤发病率，这将给国家和人民带来极大的健康负担和经济负担。
研究显示，脂肪组织在肥胖的发生和进展过程处于核心位置，对于代谢健康有着举足轻重的作用。目前已知人体存在三种脂肪组织，白色脂肪，棕色脂肪和米色脂肪。白色脂肪组织以甘油三酯的形式储存能量，棕色脂肪和米色脂肪通过产热消耗能量。米色脂肪存在于白色脂肪中，具有棕色脂肪特征。从表型上看，米色脂肪可被视为白色脂肪和棕色脂肪的过度形式。白色脂肪组织中出现米色脂肪的现象被称为白色脂肪的棕色化。米色脂肪和棕色脂肪虽然所处部位不同，发育来源不同，但行使相同的产热功能，并在消耗能量方面，有同样的潜力，二者并称为产热性脂肪组织。在生理条件下，有两种激活产热性脂肪组织的情况：冷暴露和饮食摄入(尤其是高热量的食物)。在冷暴露时，产热性的脂肪组织激活，起到保持体温恒定的作用；饮食摄入时，产热脂肪激活起到缓解一过性能量高峰，从而维持代谢稳定。
产热性的脂肪组织，其分化发育、功能调节和临床应用是近几年代谢领域的研究热点。经人群调查发现，产热性脂肪组织活跃的个体，不易发胖，不易患代谢相关的疾病，如二型糖尿病等。一些改善代谢的药物，部分作用是通过激活产热性脂肪组织来实现的，如黄连素和水杨酰水杨酸等。同样，在动物模型中，无论是改变相关基因(敲除或过表达)，还是药物刺激(β肾上腺素受体激动剂等)，产热性棕色脂肪激活后，都会起到控制体重和改善代谢的作用。因此，综上所述，米色脂肪组织是人体产热性脂肪组织，具有消耗多余能量的潜力，并且能在一定条件下激活；激活产热性脂肪组织，不管 是增强棕色脂肪的功能，还是促进白色脂肪棕色化，都能起到体重控制和改善代谢目的。迄今为止，没有专门靶向产热性脂肪组织来实现改善代谢的化合物或药物发现，也没有针对这个靶点实施高通量筛选。
产热性脂肪组织之所以能将储存在脂肪酸中的化学能转化成热能释放，是因为具有一个特殊的分子，解偶联蛋白1(Uncoupling Protein 1，UCP1)。UCP1定位于线粒体的内膜上，在激活的情况下，可以切断电子传递链和ATP合成的偶联，将化学能直接转化成热能。UCP1存在于产热性的脂肪组织当中，由于这种独特的组织定位，UCP1不仅是产热性脂肪组织的功能分子，也是表型标志。除了UCP1之外，PGC1α，PRDM16和Cytochrome C是参与棕色化过程的重要分子。
研究报道，肥胖和代谢的疾病的发生是生活环境和遗传因素共同作用的结果。高脂饮食诱导的肥胖小鼠是实验室常用的研究肥胖和代谢病的发生发展机制的动物模型。ob/ob小鼠由于缺乏瘦素基因，有严重的胰岛素抵抗，自出生起就有脂肪肝，并且这些代谢障碍随着小鼠的生长逐渐加重，最终导致小鼠过早死亡。由于存在这些先天的代谢障碍，ob/ob小鼠是实验室研究糖尿病和胰岛素抵抗的常用动物模型。体外研究脂肪细胞分育的常用细胞模型有3T3-L1和C3H10T1/2，前者模拟脂肪前体细胞分化为成熟的脂肪细胞的过程，后者反映间充质干细胞经过定向后再分化成脂肪细胞的过程。除了细胞系之外，研究脂肪的发育的细胞模型还有血管基质组分(Stromal Vascular Fraction，SVF)，SVF是原代的前体脂肪细胞，经分离培养后，经过一定诱导，可分化为白色脂肪细胞，从而在体外环境模拟前体脂肪细胞向白色脂肪分化的体内过程。
现有技术公开了蒿甲醚(Artemether，ART)(式II)是青蒿素(式I)的类似物，能高效、速效地杀灭红细胞内期疟原虫；其用于抗氯喹恶性疟及凶险型疟疾的治疗，显效迅速，短期疗效好，毒副作用小，是治疗疟疾的一线药物，应用广泛。近期研究表明蒿甲醚还有杀死肿瘤细胞和免疫调节的作用，在肿瘤和自身免疫性疾病(如系统性红斑狼疮，风湿性关节炎)的治疗中，展现出新的潜力。
目前尚未发现蒿甲醚在控制体重和改善代谢的应用潜能。除了蒿甲醚之外，青蒿素类药物还包括青蒿琥酯和双氢青蒿素，蒿乙醚，SM905，Artemiside，青蒿酮Artemisone和SM934等结构，其中双氢青蒿素是蒿甲醚及其他青蒿素类似物在体内的二级代谢产物。
基于现有技术的研究基础，本申请的发明人拟提供蒿甲醚及青蒿素类似物在制药中的新用途，具体涉及蒿甲醚在制备促进脂肪分解和改善糖代谢制剂中的用途。
Figure PCTCN2017082359-appb-000001
发明内容
因此，本发明的目的在于提供蒿甲醚及青蒿素类似物在制药中的新用途，具体涉及蒿甲醚在制备促进脂肪分解和改善糖代谢制剂中的用途，尤其涉及蒿甲醚及青蒿素类似物在制备促进白色脂肪棕色化及在制备降低体重和改善代谢制剂中的用途。
为实现上述目的，本发明的第一方面提供了一种如以下通式(A)所示的青蒿素类似物在制备用于促进脂肪分解和/或预防或治疗代谢相关疾病的药物中的用途：
Figure PCTCN2017082359-appb-000002
其中，R选自：羟基、甲氧基、乙氧基、丁二酸单酯基(琥珀酸酯基)、2-氨基乙氧基、2R-3-叔丁基氨基-2-羟基丙氧基、硫代吗啉基和1,1-二氧化硫代吗啉基。
本发明第二方面提供了一种用于促进脂肪分解和/或预防或治疗代谢相关疾病的方法，所述方法包括向有需要的受试者给予治疗有效量的如以下通式(A)所示的青蒿素类似物：
Figure PCTCN2017082359-appb-000003
其中，R选自：羟基、甲氧基、乙氧基、丁二酸单酯基(琥珀酸酯基)、2-氨基乙氧基、2R-3-叔丁基氨基-2-羟基丙氧基、硫代吗啉基和1,1-二氧化硫代吗啉基。
优选地，所述给予治疗有效量所述青蒿素类似物的方式为：口服或注射方式给药；更优选地，所述青蒿素类似物的给药剂量分别为：
口服：20mg/kg～100mg/kg；优选为50mg/kg～100mg/kg；
注射：10mg/kg～50mg/kg；优选为20mg/kg～40mg/kg。
本发明第三方面提供了一种用于促进脂肪分解和/或预防或治疗代谢相关疾病的青蒿素类似物，所述青蒿素类似物如以下通式(A)所示：
Figure PCTCN2017082359-appb-000004
其中，R选自：羟基、甲氧基、乙氧基、丁二酸单酯基(琥珀酸酯基)、2-氨基乙氧基、2R-3-叔丁基氨基-2-羟基丙氧基、硫代吗啉基和1,1-二氧化硫代吗啉基。
优选地，根据前述任一方面所述的用途、青蒿素类似物或方法，所述青蒿素类似物为：如式(Ⅱ)所示的蒿甲醚：
Figure PCTCN2017082359-appb-000005
优选地，根据前述任一方面所述的所述的用途、青蒿素类似物或方法，所述的促进脂肪分解包括促进白色脂肪棕色化。
优选地，根据前述任一方面所述的用途、青蒿素类似物或方法，所述的促进脂肪分解和/或预防或治疗代谢相关疾病包括控制体重、改善代谢。
优选地，根据前述任一方面所述的用途、青蒿素类似物或方法，所述的代谢相关疾病是肥胖引起的高血糖、胰岛素抵抗、血脂紊乱和/或脂肪肝。
本发明第四方面提供了一种用于促进脂肪分解和/或预防或治疗代谢相关疾病的药物组合物，所述药物组合物包含：(1)如本发明第三方面所述的青蒿素类似物以及(2)药学上可接受的载体。
优选地，所述药物组合物的口服给药剂型选自：口服混悬剂、散剂和颗粒剂；注射给药剂型选自：水剂、油剂和混悬剂。
现结合本发明的构思，对本发明具体技术方案进一步阐述如下：
本发明所述的蒿甲醚的结构如式(II)所示。
Figure PCTCN2017082359-appb-000006
本发明还涉及除蒿甲醚之外的青蒿素类似物有促进白色脂肪棕色化的潜能。青蒿素结构如式(I)所示，青蒿素类似物如式(A)所示。
Figure PCTCN2017082359-appb-000007
式(A)所示的青蒿素类似物是，双氢青蒿素，R为羟基；蒿乙醚，R为乙氧基；青蒿琥酯，R为丁二酸单酯基(琥珀酸酯基)；SM934，R为2-氨基乙氧基；SM905，R为2R-3-叔丁基氨基-2--羟基丙氧基；artemiside，R为硫代吗啉基；青蒿酮artemisone，R为1,1-二氧化硫代吗啉基。
本发明所述的的青蒿素类似物，其R基团如下表所示：
Figure PCTCN2017082359-appb-000008
Figure PCTCN2017082359-appb-000009
本发明经体内和体外实验证实，青蒿素类化合物有诱导白色脂肪棕色化的作用，通过促进白色脂肪棕色化的作用，起到了降低体重、、改善代谢，维持糖代谢稳态、缓解脂肪肝、改善糖尿病相关症状的效果。所述的青蒿素类药物临床应用实践显示，尚未发现不可耐受的副作用。在啮齿动物模型的研究中，有报道称高浓度的蒿甲醚会引起神经性厌食，但对于给药的剂量和时间没有明确的证据。因此整体来说，作为临床药物，蒿甲醚具有理想的安全性。
附图的简要说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1图2示出了蒿甲醚使3T3-L1呈现出棕色脂肪细胞样的特征。其中，图1示出了不同浓度ART处理后的3T3-L1细胞的形态变化；图2示出了不同浓度的ART处理3T3-L1后，UCP1的mRNA水平。*p<0.05，**p<0.01。
图3-5示出了蒿甲醚使BMP4定向的C3H10T1/2出现棕色脂肪样的形态特征。其中，图3示出了BMP7定向的C3H10T1/2、BMP4定向的C3H10T1/2、蒿甲醚处理的BMP4定向的C3H10T1/2的细胞形态特征；图4示出了Western Blot检测BMP4定向的C3H10T1/2，BMP4定向的C3H10T1/2以及不同浓度的蒿甲醚处理后的BMP4定向的C3H10T1/2的棕色脂肪相关基因UCP1、PGC1a、PRDM16和CytoC的蛋白水平；图5示出了蒿甲醚(15μMol/L)处理后，BMP4定向的C3H10T1/2的线粒体MitoTracker Green染色。
图6-7示出了蒿甲醚使SVF原代脂肪前体细胞在分化过程中获得棕色脂肪样形态特征。其中，图6示出了不同浓度的蒿甲醚处理SVF后细胞形态的变化； 图7示出了不同浓度的蒿甲醚处理SVF后，棕色相关基因PRDM16、PGC1α和UCP1的表达水平。
图8-9示出了青蒿素类似物促进棕色化的作用。其中，图8示出了青蒿素及其类似物蒿甲醚、青蒿琥酯、双氢青蒿素处理BMP4定向的C3H10T1/2的细胞形态；图9示出了不同浓度的双氢青蒿素处理BMP4定向的C3H0T1/2后，棕色相关基因的表达水平。
图10示出了蒿甲醚抑制高脂饮食诱导的体重增加。其中，图10A示出了皮下注射蒿甲醚的小鼠在高脂饮食条件下的体重变化，*p<0.05；图10B示出了皮下注射蒿甲醚的小鼠在高脂饮食条件下体重增长情况，*p<0.05。
图11示出了皮下注射蒿甲醚减少了高脂饲养的小鼠的脂肪组织。其中，图11A示出了皮下注射蒿甲醚的小鼠与对照组的小鼠的腹股沟部脂肪、附睾部脂肪和肩胛间区的棕色脂肪的体积；图11B示出了皮下注射蒿甲醚的小鼠与对照组的小鼠的腹股沟部脂肪、附睾部脂肪和肩胛间区的棕色脂肪的重量与小鼠重量的比值，*p<0.05；图11C示出了皮下注射蒿甲醚的小鼠与对照组的小鼠的腹股沟部脂肪、附睾部脂肪和肩胛间区的棕色脂肪细胞的形态。
图12示出了皮下注射蒿甲醚改善高脂诱导的肥胖小鼠的糖代谢。其中，图12A示出了高脂饲料饲养八周后，皮下注射蒿甲醚的小鼠和对照小鼠的葡萄糖耐量曲线，*p<0.05；图12B示出了高脂饲料饲养八周后，皮下注射蒿甲醚的小鼠和对照小鼠的葡萄糖耐量曲线，*p<0.05。
图13示出了蒿甲醚抑制高脂饮食诱导的脂肪肝。其中，高脂饲料饲养八周后，皮下注射蒿甲醚的小鼠和对照小鼠的肝脏细胞形态。
图14示出了皮下注射蒿甲醚诱导高脂饲养的小鼠皮下脂肪出现棕色化。其中，图14A示出了给药八周后，高脂饲养的小鼠在4℃环境中的体温变化，
*p<0.05；图14B示出了给药八周后，高脂饲养的小鼠暴露在4℃6小时后，腹股沟部脂肪组织中UCP1的表达。
图15示出了腹腔注射蒿甲醚抑制高脂饮食诱导的体重增长。其中，图15A示出了腹腔注射蒿甲醚的小鼠在高脂饮食条件下的体重变化，*p<0.05；图15B示出了腹腔注射蒿甲醚的小鼠在高脂饮食条件下体重增长情况，*p<0.05。
图16示出了腹腔注射蒿甲醚显著减少了高脂饲养小鼠的脂肪组织。其中，图16A示出了腹腔注射蒿甲醚的小鼠与对照组的小鼠的腹股沟部脂肪、附睾部脂肪和肩胛间区的棕色脂肪的体积；图16B示出了腹腔注射蒿甲醚的小鼠与对照组的小鼠的腹股沟部脂肪、附睾部脂肪和肩胛间区的棕色脂肪的重量与小鼠重量的比值，*p<0.05；图16C示出了腹腔注射蒿甲醚的小鼠与对照组的小鼠的腹股沟部脂肪、附睾部脂肪和肩胛间区的棕色脂肪细胞的形态。
图17示出了腹腔注射蒿甲醚改善高脂诱导的肥胖小鼠的糖代谢。其中，图17A示出了高脂饲料饲养八周后，腹腔注射蒿甲醚的小鼠和对照小鼠的随机血糖，*p<0.05；图17B示出了高脂饲料饲养八周后，腹腔注射蒿甲醚的小鼠和对照小鼠的葡萄糖耐量实验结果，*p<0.05；图17C示出了高脂饲料饲养八周后，腹腔注射蒿甲醚的小鼠和对照小鼠的胰岛素耐量情况，*p<0.05。
图18示出了高脂饲料饲养八周后，腹腔注射蒿甲醚的小鼠和对照小鼠的肝脏细胞组织学形态。
图19示出了高脂饲料饲养八周后，腹腔注射蒿甲醚的小鼠与对照组小鼠相比，血清中脂质代谢相关指标的变化，*p<0.05，**p<0.01。
图20示出了腹腔注射蒿甲醚诱导高脂饲养的小鼠皮下脂肪出现棕色化。其中，图20A示出了腹腔注射蒿甲醚八周后，高脂饲养的小鼠在4℃环境中的体温变化，*p<0.05；图20B示出了腹腔注射蒿甲醚八周后，高脂饲养的小鼠暴露在4℃6hr后，腹股沟部脂肪组织中UCP1的表达。
图21示出了蒿甲醚抑制糖尿病小鼠ob/ob小鼠的体重增长。其中，图21A示出了ob/ob小鼠，腹腔注射蒿甲醚八周的体重变化；图21B示出了ob/ob小鼠，腹腔注射蒿甲醚八周的体重增长变化，*p<0.05。
图22示出了蒿甲醚改善糖尿病小鼠ob/ob的脂肪肝，其中显示ob/ob小鼠，蒿甲醚腹腔注射八周后，肝脏切片的HE染色结果。
图23示出了腹腔注射蒿甲醚不影响健康小鼠的生长。其中，图23A示出了健康小鼠，普通饲料喂养，腹腔注射蒿甲醚八周，给药组小鼠和对照组的体重；图23B示出了健康小鼠，普通饲料喂养，腹腔注射蒿甲醚八周，给药组小鼠和对照组的体重增量。
图24示出了腹腔注射蒿甲醚不影响健康瘦小鼠的糖代谢。其中，图24A示出了健康瘦小鼠，腹腔注射蒿甲醚八周，葡萄糖耐量的情况；图24B示出了健康瘦小鼠，腹腔注射蒿甲醚八周，胰岛素耐量的情况。
图25示出了显示小鼠腹腔注射20mg/kg体重蒿甲醚没有导致肝脏损伤，健康瘦小鼠，腹腔注射蒿甲醚八周，肝脏组织切片的HE染色。
图26示出了蒿甲醚不改变小鼠的进食量。其中，图26A示出了常规饲料饲养的瘦小鼠，蒿甲醚腹腔注射八周，每只小鼠每天的进食量；图26B示出了高脂饲料饲养的肥胖小鼠，蒿甲醚腹腔注射八周，每只小鼠每天的饲料消耗量。
实施发明的最佳方式
实施例1、蒿甲醚诱导3T3-L1白色前体脂肪细胞在分化过程中出现棕色化
1.1 材料
1.1.1 3T3-L1白色脂肪前体细胞
1.1.2 DMEM培养基(Gibco)
1.1.3 胎牛血清(Gibco)
1.1.4 小牛血清(TBD)
1.1.5 生物素(Sigma)
1.1.6 青霉素和链霉素
1.1.7 蒿甲醚(成都普菲德生物技术有限公司)
1.1.8 Trizol(Life Technology)
1.1.9 逆转录试剂盒(ThermoScientific)
1.1.10 Power SYBR Green Master Mix(ThermoScientific)
1.2 设备
1.2.1 恒温无菌孵箱Thermo Scientific Forma Series II Water Jacket
1.2.2 实时定量PCR仪Applied Biotech 7500real time PCR System
1.2.3 显微镜Olympus IX711
1.2.5 低温离心机Eppendorf Centrifuge 5427R
1.2.6 核酸定量仪器Colibri
1.3 溶液
1.3.1 培养基
3T3-L1维持培养基
DMEM 最多至500ml
小牛血清 10％
生物素 8mg/ml
青霉素 100U/ml
链霉素 100U/ml
3T3-L1诱导分化培养基
DMEM 最多至500ml
胎牛血清 10％
生物素 8mg/ml
青霉素 100U/ml
链霉素 100U/ml
1.3.2 蒿甲醚储存液溶解在DMSO中，储存浓度为10mMol/L。
1.3.3 生物素：160mg生物素和80mg泛酸钙溶于200ml去离子水中。加热溶解。0.22μm滤膜过滤除菌。
1.3.4 3-异丁基-1-甲基黄嘌呤(50mMol/L)：1.15g 3-异丁基-1-甲基黄嘌呤溶于100ml 0.2M氢氧化钾中，0.22μm滤膜过滤除菌。
1.3.5 地塞米松(1mMol/L)：0.039g地塞米松溶于100ml水中，0.22μm滤膜过滤除菌。
1.3.6 胰岛素(1mg/ml)：0.1g胰岛素溶于100ml水中,加几滴盐酸使之溶解，0.22μm滤膜过滤除菌。
1.4 方法
1.4.1 白色脂肪前体细胞3T3-L1的培养和诱导分化
在3.5cm的细胞培养皿接种2.5×105个3T3-L1白色脂肪前体细胞，用维持培养基培养3天，细胞增殖至接触抑制状态(Contact inhibition)。达到接触抑制后48小时，换诱导分化培养基，加入诱导分化混合物，诱导分化混合物包括0.5mMol/L的3-异丁基-1-甲基黄嘌呤(Mix)，1μMol/L的地塞米松(Dex)，和1μg/ml的胰岛素(Insulin)。诱导分化混合物简称MDI。加入MDI的这一天定义为分化第0天。在以上培养基中培养2天后，更换新鲜的诱导分化培养基，培养基中加入1μg/ml的胰岛素。再培养2天后，更换新鲜的诱导分化培养基，此后每隔一天，更换培养基，直至分化到第8天。
1.4.2 化合物处理
蒿甲醚储存在DMSO中，储存浓度为10mMol/L，在分化第0天加入MDI的同时，加入蒿甲醚(5，10或15μMol/L)处理，此后每次更换培养基，都加入同样浓度的蒿甲醚，直至3T3-L1分化成熟。
1.4.3 提取3T3-L1的细胞总RNA并逆转录成cDNA
在3.5cm的培养皿中诱导分化成熟的3T3-L1，分化方法参照方法1.4.1用 PBS清洗后，用1ml Trizol充分裂解细胞，彻底混匀后，加入200μl的氯仿，剧烈震荡后，室温静置15min，在4℃条件下离心，离心条件为12000g，10min，此时由于层析作用，裂解液分为三层，小心吸取上层水相溶液转移至新的离心管，加入500μl异丙醇，轻轻混匀后室温静置10分钟，4℃离心，12000g，10min，弃上清，可见管底有乳白色半透明状沉淀，用DEPC水稀释75％的乙醇清洗沉淀3遍，4℃，7500g离心，5min，弃去上清，室温晾干，60μl DEPC水溶解沉淀，用Colibri Tepertec定量后-80℃保存。
取1μg RNA进行逆转录合成相应的cDNA，逆转录试剂盒为ThermoScientific RevertAid First Strand cDNA Synthesis Kit。合成体系如下
M-MLV Reaction Buffer 4μl
10mM dNTP 2μl
RNA酶抑制剂 1μl
M-MLV逆转录酶 1μl
DEPC水 Up to 20μl
合成条件如下：
Figure PCTCN2017082359-appb-000010
合成的cDNA保存在-20℃冰箱中。
1.4.4 Real time qPCR
反应体系：
CYBR Green Master Mix 15μl
上游引物 0.5μl
下游引物 0.5μl
RNA模板 0.5μl
ddH2O 14.5μl
以上体系共30μl，分为三个复孔，每个复孔10μl。
反应条件：
Figure PCTCN2017082359-appb-000011
扩增18S的正向引物序列为GTAACCCGTTGAACCCCATT；反向引物序列为CCATCCAATCGGTAGTAGCG。
扩增UCP1的正向序列为AGGCTTCCAGTACCATTAGGT；反向引物为CTGAGTGAGGCAAAGCTGATTT。
根据ΔΔCT法计算mRNA的相对量，用18S作为内参，再将对照的mRNA水平设定为1，各个处理组与对照组对比，得出相对量。
1.4.5 数据分析
数据差异性由Student T test方法分析，p<0.05时，认为有统计学差异。
1.5 结果
1.5.1 蒿甲醚使3T3-L1呈现出棕色脂肪细胞样的特征
在显微镜Olympus IX711下观察细胞形态并记录，如图1所示，对照组(Control)的3T3-L1呈现出典型的白色脂肪细胞特征，细胞体积较大，脂滴大而圆，脂滴数量较少；经过蒿甲醚(Artemether，ART)处理后的3T3-L1，呈现出典型的棕色样特征，细胞体积较小，脂滴小而密集。并且这种变化有浓度依赖性的趋势，随着蒿甲醚的浓度增加，3T3-L1脂肪细胞的变化越明显。
1.5.2 蒿甲醚上调3T3-L1中UCP1的表达
提取3T3-L1的总RNA，逆转录成cDNA，用Real time qPCR检测UCP1的mRNA水平，与对照组相比，ART处理的3T3-L1中UCP1的表达显著上调，并呈现出浓度依赖性的趋势。
3T3-L1是经典的白色脂肪前体细胞，蒿甲醚使3T3-L1在分化过程中获得了棕色样的特征，不仅具有棕色脂肪的形态特征，也出现了棕色脂肪的标记UCP1的表达。
实施例2、蒿甲醚诱导C3H10T1/2间充质干细胞在分化成脂肪细胞的过程中出 现棕色脂肪样特征
2.1 材料
2.1.1 C3H10T1/2间充质干细胞系
2.1.2 DMEM培养基(Gibco)
2.1.3 胎牛血清(Gibco)
2.1.4 小牛血清(TBD)
2.1.5 生物素(Sigma)
2.1.6 青霉素和链霉素
2.1.7 蒿甲醚(成都普菲德生物技术有限公司)
2.1.8 BMP4(R&D)
2.1.9 BMP7(Prospec)
2.1.8 PVDF膜(Millipore Immunobilon)
2.1.9 化学发光液numECL ultra(新赛美生物科技有限公司)
2.1.10 MitoTracker Green(Life Technology)
2.1.11 其余未提及试剂均购自Sigma
2.2 设备
2.2.1 恒温无菌孵箱Thermo Scientific Forma Series II Water Jacket
2.2.2 显微镜Olympus IX711
2.2.3 电泳仪BioRad PowerPac HC
2.2.4 自动化学发光仪ImageQuant LAS4000Mini
2.3 溶液
2.3.1 培养基
C3H10T1/2维持培养基
DMEM 最多至500ml
小牛血清 10％
生物素 8mg/ml
青霉素 100U/ml
链霉素 100U/ml
C3H10T1/2诱导分化培养基
DMEM 最多至500ml
胎牛血清 10％
生物素 8mg/ml
青霉素 100U/ml
链霉素 100U/ml
2.3.2 蒿甲醚储存液溶解在DMSO中，储存浓度为10mMol/L。
2.3.3 生物素
160mg生物素和80mg泛酸钙溶于200ml去离子水中。加热溶解。0.22μm滤膜过滤除菌。
2.3.4 3-异丁基-1-甲基黄嘌呤(50mMol/L)
1.15g 3-异丁基-1-甲基黄嘌呤溶于100ml 0.2M氢氧化钾中，0.22μm滤膜过滤除菌。
2.3.5 地塞米松(1mMol/L)
0.039g地塞米松溶于100ml水中，0.22μm滤膜过滤除菌。
2.3.6 胰岛素(1mg/ml)
0.1g胰岛素溶于100ml水中,加几滴盐酸使之溶解，0.22μm滤膜过滤除菌。
2.3.7 细胞蛋白提取所用的裂解液
Tris-HCL(pH6.8) 50mMol/L
SDS 2％(w/v)
Glycerol 10％(w/v)
NaF 10mMol/L
Na3VO4 100mMol/L
2.4 方法
2.4.1 C3H10T1/2的培养和分化
将7.5×105C3H10T1/2接种在3.5cm细胞培养皿上。用维持培养基培养3-5天。接种的次日在培养基中加入BMP4(终浓度为20ng/ml)待细胞增殖至接触抑制状态。达到接触抑制状态两天后更换培养基为诱导分化培养基，同时加入诱导混合物(0.5mMol/L Mix，1μMol/L Dex，1ng/ml insulin，100nMol/L吲哚美辛，1nM T3)，这一天被定义为分化第0天，诱导2天后，换新鲜诱导分化培养基，并加入1ng/ml胰岛素和1nMol/L T3，继续培养两天后，此后每隔一天更换新鲜培养基，直至分化第8天细胞成熟。此时分化成熟的细胞为白色脂肪细胞。
将7.5×105C3H10T1/2接种在3.5cm细胞培养皿上。用维持培养基培养3-5天。接种的次日在培养基中加入BMP7(终浓度为100ng/ml)待细胞增殖至接触抑制状态。达到接触抑制状态两天后更换培养基为诱导分化培养基，同时加入诱导混合物(0.5mM Mix，1μM Dex，1ng/ml insulin，100nM吲哚美辛，1nM T3)，这一天被定义为分化第0天，诱导2天后，换新鲜诱导分化培养基，并加入1ng/ml胰岛素和1nM T3，继续培养两天后，此后每隔一天更换新鲜培养基，直至分化第8天细胞成熟。此时分化成熟的细胞为棕色脂肪细胞。
2.4.2 蒿甲醚处理
在分化第0天，即加入诱导分化混合物的同时，在培养基中加入化合物，化合物的储存浓度为10mMol/L，终浓度分别为5μMol/L，10μMol/L和15μMol/L。更换新鲜培养基时，同时补充相应的化合物，化合物一直伴随细胞至分化成熟。
2.4.3 提取细胞总蛋白
在3.5cm分化成熟的C3H10T1/2，分化方法见2.4.1，加入300μl裂解液，充分裂解细胞，将裂解液转移至1.5ml离心管中，100℃加热10min，4℃离心13000g，10min，去除上层脂质，将中层澄清的裂解液转移至新的离心管，取2.5μl裂解液用BCA法进行定量，取200μl裂解液与50μl Loading Buffer混合，100℃加热10min，保存在-20℃待用。
2.4.4 Western blot
取30μg C3H0T1/2的蛋白上样至10％的聚丙烯酰胺凝胶，进行电泳，用80V进行基层胶的电泳，100V进行分离胶的电泳，直至目的条带分散开。在冰浴中转膜至PVDF膜，转膜条件为100V，100min，转膜结束后，在用TBST(TBS缓冲液+0.5％Tween)配制的5％的脱脂牛奶中封闭1hr。与特异性抗体孵育，4℃过夜。次日，用TBST清洗PVDF膜，室温，10min，清洗三次，与对应二抗共孵，室温1hr，在用TBST清洗三次，条件同一抗，与化学发光试剂共孵，室温1min，ImageQuant LAS4000Mini自动化学发光仪，曝光，记录结果。
抗体货号和抗体稀释比例见下表：
抗体 货号 稀释比例
β-acting Proteintech HRP-60008 1：10000
PRDM16 Abcam Ab106410 1：200
PGC1α Abcam Ab54481 1：400
UCP1 Abcam Ab10983 1：200
CytoC Proteintech 10993-1-AP 1：1000
二抗 Jackson Immunology 111-035-003 1：10000
2.4.4 MitoTracker Green标记活性的线粒体
在3.5cm培养皿分化成熟后的C3H10T1/2，培养基去除，加入1ml PBS，在PBS中加入MitoTraker Green，终浓度为100ng/ml，37℃孵育10min，在荧光显微镜下观察绿色荧光，并取代表性视野拍照记录。
2.5 结果
2.5.1 蒿甲醚使BMP4定向的C3H10T1/2出现棕色脂肪样的形态特征
C3H10T1/2是具有多向分化潜能的间充质干细胞，在BMP4定向的情况下分化成白色脂肪细胞，在BMP7定向的情况下分化成棕色脂肪细胞。在BMP4定向的C3H10T1/2分化过程中，同时加入蒿甲醚(15μMol/L)处理。观察细胞形态，BMP7定向的C3H10T1/2具有典型的棕色脂肪细胞特征，细胞较小，脂滴小而多；BMP4定向的C3H10T1/2具有典型的白色脂肪细胞特征，细胞较大，脂滴大而少；在BMP4定向的C3H10T1/2分化过程中，同时加入蒿甲醚 (15μMol/L)处理，细胞出现了棕色样的特征，与BMP7定向的细胞形态类似(如图3所示)。
2.5.2 蒿甲醚使BMP4定向的C3H10T1/2细胞分化成熟后，棕色脂肪相关基因表达上调
以BMP7定向的C3H10T1/2作为阳性对照，用不同浓度的蒿甲醚(5，10，15μMol/L)处理BMP4定向的C3H10T1/2，用Western blot检测棕色脂肪相关基因的蛋白水平，其中β-actin作为内参。结果显示，蒿甲醚使BMP4定向的C3H10T1/2细胞中棕色相关基因UCP1、PGC1a、PRDM16和CytoC的蛋白水平增高，并且有浓度依赖性的趋势(如图4所示)。
2.5.3 蒿甲醚使BMP4定向的C3H10T1/2的线粒体生物合成增强
用MitoTracker Green标记BMP4定向的C3H10T1/2的线粒体，观察到蒿甲醚(15μMol/L)处理后的C3H10T1/2的线粒体生物合成明显增强(如图5所示)。
BMP4使C3H10T1/2间充质干细胞分化成具有白色脂肪特征的细胞，在蒿甲醚处理后，BMP4定向的C3H10T1/2分化成熟后，呈现棕色脂肪细胞样特征，表现在细胞形态、相关基因表达和线粒体生物合成增强三个方面。
实施例3、蒿甲醚使血管基质组分(Stromal Vascular Fraction，SVF)在分化 过程中获得棕色脂肪细胞样特征
3.1 材料
3.1.1 雄性C57BL/6J小鼠(4-6week，上海斯莱克实验动物有限公司)
3.1.2 DMEM/F12(1:1)培养基(Gibco)
3.1.3 胎牛血清(Gibco)
3.1.4 生物素(Sigma)
3.1.5 青霉素和链霉素
3.1.6 蒿甲醚(成都普菲德生物技术有限公司)
3.1.7 PVDF膜(Millipore Immunobilon)
3.1.8 化学发光试剂numECL ultra(新赛美生物科技有限公司)
3.1.10 其余未提及试剂均购自Sigma
3.2 设备
3.2.1 恒温无菌孵箱Thermo Scientific Forma Series II Water Jacket
3.2.2 显微镜Olympus IX711
3.2.3 电泳仪BioRad PowerPac HC
3.2.4 自动化学发光仪ImageQuant LAS4000Mini
3.3 溶液
3.3.1 SVF的培养基(分化和培养)
DMEM/F12(1:1) 最多至500ml
胎牛血清 10％
生物素 8mg/ml
青霉素 100U/ml
链霉素 100U/ml
3.3.2 细胞蛋白提取所用的裂解液
Tris-HCL(pH6.8) 50mMol/L
SDS 2％(w/v)
Glycerol 10％(w/v)
NaF 10mMol/L
Na3VO4 100mMol/L
3.4 方法
3.4.1 SVF的分离、培养和分化
取雄性小鼠腹股沟部脂肪剪碎，用胶原酶37℃处理15min，离心，250rpm10min，用F12/DMEM培养基重悬沉淀，取2.5×105个细胞接种在3.5cm培养皿，培养皿预先用1ml多聚赖氨酸处理30min。当细胞接触抑制两天后(定义为分化第0天)，按照脂肪细胞分化方案进行诱导，具体过程如下：用含有10％胎牛血清，0.5mMol/L Mix，1μMol/L Dex，5μg/ml Insulin，1μMol/L Rosiglitazone的F12/DMEM诱导2天后，换含有10％胎牛血清，5μg/ml insulin，1μMol/L的Rosiglitazone，DMEM/F12继续培养2天，此后每隔一天更换含10％胎牛血清的DMEM/F12培液,直至细胞分化到第8天。
3.4.2 蒿甲醚处理SVF
在分化第0天，加入不同浓度的蒿甲醚(5，10，15μMol/L)处理SVF，此后更换新鲜培养基，都加入相应浓度的蒿甲醚，直至细胞分化成熟。
3.4.3 提取SVF细胞总蛋白和Western Blot
提取SVF总蛋白的方法同2.4.3，Western blot方法以及抗体货号同2.4.4
3.5 结果
3.5.1 蒿甲醚使SVF原代脂肪前体细胞在分化过程中获得棕色脂肪样形态特征
在SVF原代前体脂肪细胞分化过程中，加入不同浓度的蒿甲醚处理细胞，直至SVF分化成熟，与标准分化的对照细胞相比，蒿甲醚处理后的细胞体积较小，具有多腔小脂滴的特征。并且这中变化有浓度依赖性的趋势(如图6所示)。
3.5.2 蒿甲醚在SVF在分化过程中上调了棕色脂肪相关基因的表达
用Western Blot检测蒿甲醚处理后SVF细胞中棕色相关基因的表达水平。发现PRDM16、PGC1α和UCP1的表达水平随着蒿甲醚浓度的升高逐渐升高。 如图7所示。SVF是原代脂肪前体细胞，在蒿甲醚的作用下，SVF在体外分化成具有棕色脂肪特征的脂肪细胞。
实施例4、除蒿甲醚之外，其它青蒿素类似物也有促进棕色化的作用
4.1 材料
4.1.1 C3H10T1/2间充质干细胞系
4.1.2 DMEM培养基(Gibco)
4.1.3 胎牛血清(Gibco)
4.1.4 小牛血清(TBD)
4.1.5 青霉素和链霉素
2.1.6 蒿甲醚，青蒿素，青蒿琥酯，双氢青蒿素(成都普菲德生物技术有限公司)
2.1.7 PVDF膜(Millipore Immunobilon)
2.1.8 化学发光第五numECL ultra(新赛美生物科技有限公司)
2.1.9 MitoTracker Green(Life Technology)
2.1.10 其余未提及试剂均购自Sigma
4.2 设备
4.2.1 恒温无菌孵箱Thermo Scientific Forma Series II Water Jacket
4.2.2 显微镜Olympus IX711
4.2.3 电泳仪BioRad PowerPac HC
4.2.4 自动化学发光仪ImageQuant LAS4000Mini
4.3 方法
4.3.1 C3H10T1/2的培养和分化
同方法2.4.1。
4.3.2 化合物处理
青蒿素、青蒿琥酯和双氢青蒿素保存在DMSO中，储存浓度为10mMol/L，在分化第0天加入图8所示的浓度，每隔两天更换新鲜培养基，都补充相应浓度的化合物，直至细胞分化成熟。
4.3.3 C3H10T1/2细胞总蛋白提取和Western blot
提取C3H10T1/2细胞总蛋白的方法同2.4.3；
Western blot方法以及抗体货号同2.4.4。
4.4 结果
与对照相比，蒿甲醚(Artemether)处理后的C3H10T1/2呈现出棕色脂肪细胞样的特征：细胞体积较小，拥有多腔小脂滴。青蒿素(Artemisinin)、青蒿琥酯(Artesunate)和双氢青蒿素(Dihydroartemisinin)，处理后的C3H10T1/2，均呈现出相似的形态特征(如图8所示)。双氢青蒿素上调了棕色相关基因的表 达。
双氢青蒿素(Dihydroartemisinin，DHA)是青蒿素及其类似物的二级代谢产物，如果双氢青蒿素有促进棕色化的作用，说明青蒿素及其类似物都有促进棕色化的作用。分别用不同浓度(1，2.5，5，7.5μMol/L)的双氢青蒿素处理BMP4定向的C3H10T1/2后，用Western blot检测棕色脂肪相关基因PGC1α和UCP1的表达水平，发现棕色相关基因的表达水平随着双氢青蒿素的浓度逐渐上升(如图9所示)；结果证实，青蒿素类似物具有促进白色脂肪棕色化的潜力。
实施例5、蒿甲醚抑制高脂饮食诱导的体重增加
5.1 材料
5.1.1 雄性C57BL/6J小鼠(4-6week)(上海斯莱克实验动物有限公司)
5.1.2 高脂饲料(Research Diets)
5.1.3 天平(赛多利斯)
5.1.4 蒿甲醚(成都普菲德生物技术有限公司)
5.2 方法
5.2.1 小鼠的饲养和给药
小鼠在复旦大学实验动物中心SPF级动物实验室饲养，室温21℃-23℃，每12hr光暗循环，诱导肥胖小鼠所用的高脂饲料脂肪含量60％。小鼠成长至8-week龄时，随机分成对照组和给药组，每组8只。在用高脂饲料喂养的同时，皮下注射蒿甲醚。蒿甲醚稀释在PBS中，浓度为15μMol/L，双侧腹股沟注射，每侧注射200μl，对照小鼠注射相同体积的PBS。给药持续八周，每周记录小鼠的体重。
5.2.2 数据分析
数据差异性由Student T test方法分析，p<0.05时，认为有统计学差异。
5.3 结果
皮下注射蒿甲醚的小鼠体重明显低于对照组小鼠，在给药六周后，两组之间的体重差异达到统计学差异，如图10A所示。给药组小鼠的体重增长明显比对照组慢，如图10B所示。皮下注射蒿甲醚显著抑制了小鼠在高脂饲养条件下的体重增长。
实施例6、皮下注射蒿甲醚减少了高脂饲养的小鼠的脂肪组织
6.1 材料
6.1.1 雄性C57BL/6J小鼠(4-6week)(上海斯莱克实验动物有限公司)
6.1.2 高脂饲料(Research Diets)
6.1.3 多聚甲醛(4％，Sigma)
6.1.4 蒿甲醚(成都普菲德生物技术有限公司)
6.2 方法
6.2.1 小鼠的饲养和给药
小鼠在复旦大学实验动物中心SPF级动物实验室饲养，室温21℃-23℃，每12hr光暗循环，诱导肥胖小鼠所用的高脂饲料脂肪含量60％。小鼠成长至8-week龄时，随机分成对照组和给药组，每组8只。在用高脂饲料喂养的同时，皮下注射蒿甲醚。蒿甲醚稀释在PBS中，浓度为15μMol/L，双侧腹股沟注射，每侧注射200μl，对照小鼠注射相同体积的PBS。给药持续八周。断颈处死小鼠，解剖取出小鼠的腹股沟部脂肪(Inguinal，皮下脂肪)，附睾部脂肪(Gonadal，内脏脂肪)和肩胛间区的棕色脂肪(BAT，经典的棕色脂肪)，称重、固定，委托上海赛戈生物技术有限公司对脂肪组织进行组织学分析，组织学分析过程包括固定、石蜡包埋、切片和HE染色等步骤。
6.2.2 数据分析
数据差异性由Student T test方法分析，p<0.05时，认为有统计学差异。
6.3 结果
取出三块具有代表性的脂肪组织(腹股沟部脂肪，附睾部脂肪和棕色脂肪)进行分析，发现皮下注射蒿甲醚的小鼠脂肪组织的体积(图11A)和重量(图11B)比对照组明显小。脂肪组织的切片和染色显示给药组小鼠的脂肪细胞明显小于对照组(图11C)。蒿甲醚降低了高脂饲养的小鼠的脂肪体积和重量，蒿甲醚引起的体重降低主要是由脂肪组织的减少引起的。
实施例7、皮下注射蒿甲醚改善高脂诱导的肥胖小鼠的糖代谢
7.1 材料
7.1.1 雄性C57BL/6J小鼠(4-6week，上海斯莱克实验动物有限公司)
7.1.2 高脂饲料(Research Diets)
7.1.3 葡萄糖注射液
7.1.4 注射用胰岛素(优泌林)
7.1.5 血糖仪(Roche OneTouch ExtraEasy)
7.1.6 血糖试纸(Roche OneTouch ExtraEasy)
7.1.8 蒿甲醚(成都普菲德生物技术有限公司)
7.2 方法
7.2.1 小鼠的饲养和给药
小鼠在复旦大学实验动物中心SPF级动物实验室饲养，室温21℃-23℃，每12小时光暗循环，诱导肥胖小鼠所用的高脂饲料脂肪含量60％。小鼠成长 至8-week龄时，随机分成对照组和给药组，每组8只。在用高脂饲料喂养的同时，皮下注射蒿甲醚。蒿甲醚溶解在PBS中，浓度为15μMol/L，双侧腹股沟注射，每侧注射200μl，对照小鼠注射相同体积的PBS。给药持续八周。
7.2.2 葡萄糖耐量实验
小鼠禁食12-16小时，腹腔注射2mg/kg体重的葡萄糖注射液，在注射后0min，30min，60min，90min和120min时，剪尾取血，用OneTouch ExtraEasy血糖仪和OneTouch ExtraEasy血糖试纸检测血糖并记录。
7.2.3 胰岛素耐量实验
腹腔注射0.75U/kg体重的胰岛素，于注射后0min，15min，30min，45min和60min时，剪尾取血，测血糖。
7.2.4 数据分析
数据差异性由Student T test方法分析，p<0.05时，认为有统计学差异。
7.3 结果
皮下注射蒿甲醚的小鼠在高脂饲养八周后，葡萄糖耐量显著优于对照小鼠(图12A)，胰岛素耐量也明显优于对照组小鼠(图12B)。在高脂饮食的饲养条件下，给药八周后，皮下注射蒿甲醚的小鼠的糖代谢和胰岛素敏感性明显比对照小鼠好，说明蒿甲醚有维持葡萄糖代谢稳态的作用。
实施例8、蒿甲醚抑制高脂饮食诱导的脂肪肝
8.1 材料
8.1.1 雄性C57BL/6J小鼠(4-6week)(上海斯莱克实验动物有限公司)
8.1.2 高脂饲料(Research Diets)
8.1.3 蒿甲醚(成都普菲德生物技术有限公司)
8.2 方法
小鼠在复旦大学实验动物中心SPF级动物实验室饲养，室温21℃-23℃，每12hr光暗循环，诱导肥胖小鼠所用的高脂饲料脂肪含量60％。小鼠成长至8-week龄时，随机分成对照组和给药组，每组8只。在用高脂饲料喂养的同时，皮下注射蒿甲醚。蒿甲醚稀释在PBS中，浓度为15μMol/L，双侧腹股沟注射，每侧注射200μl，对照小鼠注射相同体积的PBS。给药持续八周。断颈处死小鼠，取出小鼠肝脏组织，委托上海赛戈生物公司进行组织固定、石蜡包埋、切片和HE染色的组织学分析。
8.4 结果
高脂饲料饲养八周后，对照组小鼠的肝脏出现了明显的脂质堆积，而皮下注射蒿甲醚的小鼠的肝脏只出现了少量脂质，脂肪肝的情况明显比对照组小鼠改善。皮下注射蒿甲醚有效抑制了高脂饮食诱导的脂肪肝发生。
实施例9、皮下注射蒿甲醚诱导高脂饲养的小鼠皮下脂肪出现棕色化
9.1 材料
9.1.1 雄性C57BL/6J小鼠(4-6week，上海斯莱克实验动物有限公司)
9.1.2 高脂饲料(Research Diets)
9.1.3 小鼠体温仪(Physitemp BAT-12)
9.1.4 UCP1抗体(Abcam，ab10983，1:50)
9.1.5 蒿甲醚(成都普菲德生物技术有限公司)
9.2 方法
9.2.1 小鼠的饲养和给药
小鼠在复旦大学实验动物中心SPF级动物实验室饲养，室温21℃-23℃，每12hr光暗循环，诱导肥胖小鼠所用的高脂饲料脂肪含量60％。小鼠成长至8-week龄时，随机分成对照组和给药组，每组8只。在用高脂饲料喂养的同时，皮下注射蒿甲醚。蒿甲醚溶解在PBS中，浓度为15μMol/L，双侧腹股沟注射，每侧注射200μl，对照小鼠注射相同体积的PBS。给药持续八周。小鼠禁食，置于4℃饲养箱，每隔2小时直肠温度，6小时断颈处死小鼠，取腹股沟部脂肪，进行组织学分析。组织学分析包括组织固定、石蜡包埋、切片和UCP1的免疫组化实验委托上海赛戈生物有限公司代做。
9.2.2 数据分析
数据差异性由Student T test方法分析，p<0.05时，认为有统计学差异。
9.3 结果
给药八周后，当暴露在4℃环境中，皮下注射蒿甲醚的小鼠的体温明显高于对照小鼠(图14A)。在冷暴露环境中保存体温是由产热性脂肪组织承担的生理功能。取出小鼠的附睾部脂肪进行UCP1的免疫组化检测显示给药组的小鼠的皮下脂肪出现了明显的UCP1表达(图14B)。皮下给药八周后，皮下注射蒿甲醚的小鼠的体温保存能力明显增强，皮下脂肪出现明显的棕色化。说明蒿甲醚抑制体重增长和改善代谢的作用是通过促进皮下脂肪棕色化化实现的。
实施例10、腹腔注射蒿甲醚抑制高脂饮食诱导的体重增长
10.1 材料
10.1.1 雄性C57BL/6J小鼠(4-6week)
10.1.2 高脂饲料(Research Diets)
10.1.3 天平(赛多利斯)
10.1.4 蒿甲醚(武汉菲尔德生物化学有限公司)
10.1.5 椰子油(阿拉丁)
10.2 方法
10.2.1 小鼠的饲养和给药
小鼠在复旦大学实验动物中心SPF级动物实验室饲养，室温21℃-23℃，每12hr光暗循环，诱导肥胖小鼠所用的高脂饲料脂肪含量60％。小鼠成长至8-week龄时，随机分成对照组和给药组，每组8只。在用高脂饲料喂养的同时，腹腔注射。腹腔注射用的蒿甲醚保存在椰子油中，储存浓度为6mg/ml(20mMol/L),避光，4℃储存。按照20mg/kg体重给给药组小鼠注射蒿甲醚。每周给药两次，给药持续八周，每周记录小鼠体重。
10.2.2 数据分析
数据差异性由Student T test方法分析，p<0.05时，认为有统计学差异。
10.3 结果
腹腔注射蒿甲醚的小鼠体重明显低于对照组小鼠，在给药三周后，两组之间的体重差异达到统计学差异，如图15A所示。给药组小鼠的体重增长明显比对照组慢，如图15B所示。腹腔注射蒿甲醚有效抑制高脂饮食诱导的体重增长。
实施例11、腹腔注射蒿甲醚显著减少了高脂饲养小鼠的脂肪组织
11.1 材料
11.1.1 雄性C57BL/6J小鼠(4-6week，上海斯莱克实验动物有限公司)
11.1.2 高脂饲料(Research Diets)
11.1.3 多聚甲醛(4％，Sigma)
11.1.4 蒿甲醚(成都普菲德生物技术有限公司)
11.1.5 椰子油(阿拉丁)
11.2 方法
11.2.1 小鼠的饲养和给药
小鼠在复旦大学实验动物中心SPF级动物实验室饲养，室温21℃-23℃，每12hr光暗循环，诱导肥胖小鼠所用的高脂饲料脂肪含量60％。小鼠成长至8-week龄时，随机分成对照组和给药组，每组8只。在用高脂饲料喂养的同时，腹腔注射。腹腔注射用的蒿甲醚保存在椰子油中，储存浓度为6mg/ml(20mMol/L)，避光，4℃储存。按照20mg/kg体重给给药组小鼠注射蒿甲醚。每周给药两次，给药持续八周。断颈处死小鼠，解剖取出小鼠的腹股沟部脂肪(Inguinal，皮下脂肪)，附睾部脂肪(Gonadal，内脏脂肪)和肩胛间区的棕色脂肪(BAT，经典的棕色脂肪)，称重、固定，委托上海赛戈对脂肪组织进行组织学分析，包括固定、石蜡包埋、切片和HE染色。
11.2.2 数据分析
数据差异性由Student T test方法分析，p<0.05时，认为有统计学差异。
11.3 结果
取出三块具有代表性的脂肪组织进行分析，发现皮下注射蒿甲醚的小鼠脂肪组织的体积(图16A)和重量(图16B)比对照组明显小。脂肪组织的切片和染色显示给药组小鼠的脂肪细胞明显小于对照组(图16C)。腹腔注射蒿甲醚有效抑制了高脂饮食导致的脂肪堆积。
实施例12、腹腔注射蒿甲醚改善高脂诱导的肥胖小鼠的糖代谢
12.1 材料
12.1.1 雄性C57BL/6J小鼠(4-6week，斯莱克实验动物有限公司)
12.1.2 高脂饲料(Research Diets)
12.1.3 蒿甲醚(成都普菲德生物技术有限公司)
12.1.4 椰子油(阿拉丁)
12.1.5 葡萄糖注射液
12.1.6 注射用胰岛素(优泌林)
12.1.7 血糖仪(Roche OneTouch ExtraEasy)
12.1.8 血糖试纸(Roche OneTouch ExtraEasy)
12.1.9 水合氯醛(10％，Sigma)
12.1.10 全自动生化分析仪以及检测血糖的校准品Cfas和试剂盒(Roche)
12.2 方法
12.2.1 小鼠的饲养和给药
小鼠在复旦大学实验动物中心SPF级动物实验室饲养，室温21℃-23℃，每12hr光暗循环，诱导肥胖小鼠所用的高脂饲料脂肪含量60％。小鼠成长至8-week龄时，随机分成对照组和给药组，每组8只。在用高脂饲料喂养的同时，腹腔注射蒿甲醚。腹腔注射用的蒿甲醚保存在椰子油中，储存浓度为6mg/ml(20mMol/L)，避光，4℃储存。按照20mg/kg体重给给药组小鼠注射蒿甲醚。每周给药两次，给药持续八周。
12.2.2 葡萄糖耐量实验
小鼠禁食12-16hr，腹腔注射2mg/kg体重的葡萄糖注射液，在注射后0min，30min，60min，90min和120min时，剪尾取血，用OneTouch ExtraEasy血糖仪和OneTouch ExtraEasy血糖试纸检测血糖并记录。
12.2.3 胰岛素耐量实验
腹腔注射0.75U/kg体重的胰岛素，于注射后0min，15min，30min，45min和60min时，剪尾取血，测血糖。
12.2.4 随机血糖监测
用10％的水合氯醛麻醉小鼠，通过眼眶取血，全血离心，4℃，300rpm，5min，取血清，用全自动生化分析仪检测血糖的校准品Cfas和试剂盒(Roche)检测血糖的水平。
12.2.5 数据分析
数据差异性由Student T test方法分析，p<0.05时，认为有统计学差异。
12.3 结果
腹腔注射蒿甲醚的小鼠在高脂饲养八周后，随机血糖明显低于对照组，葡萄糖耐量显著优于对照小鼠(图12A)，胰岛素敏感性也显优于对照组小鼠(图12B)。腹腔注射蒿甲醚有效降低了高脂饲养的肥胖小鼠的血糖，显著改善了葡萄糖代谢和胰岛素敏感性。
实施例13、腹腔注射蒿甲醚有效抑制高脂饮食诱导的肥胖小鼠的脂肪肝发生
13.1 材料
13.1.1 雄性C57BL/6J小鼠(4-6week)(上海斯莱克实验动物有限公司)
13.1.2 高脂饲料(Research Diets)
13.1.3 蒿甲醚(成都普菲德生物技术有限公司)
13.1.4 椰子油(阿拉丁)
13.2 方法
小鼠在复旦大学实验动物中心SPF级动物实验室饲养，室温21℃-23℃，每12hr光暗循环，诱导肥胖小鼠所用的高脂饲料脂肪含量60％。小鼠成长至8-week龄时，随机分成对照组和给药组，每组8只。在用高脂饲料喂养的同时，腹腔注射蒿甲醚。腹腔注射用的蒿甲醚保存在椰子油中，储存浓度为6mg/ml(20mMol/L)，避光，4℃储存。按照20mg/kg体重给给药组小鼠注射蒿甲醚。每周给药两次，给药持续八周。断颈处死小鼠，取出肝脏组织固定，委托上海赛戈生物有限公司对肝脏进行组织学分析，组织学分析过程包括组织的固定、石蜡包埋、切片和HE染色。
13.4 结果
高脂饲料饲养八周后，对照组小鼠的肝脏出现了明显的脂质堆积，而腹腔注射蒿甲醚的小鼠的肝脏只出现了少量脂质，肝脏细胞的形态基本接近正常，如图18所示。腹腔注射蒿甲醚有效抑制了高脂饮食诱导的脂肪肝发生。
实施例14、腹腔注射蒿甲醚抑制了高脂饮食导致的血脂升高
14.1 材料
14.1.1 雄性C57BL/6J(4-6week)小鼠(斯莱克实验动物有限公司)
14.1.2 高脂饲料(Research Diets)
14.1.3 蒿甲醚(成都普菲德生物技术有限公司)
14.1.4 椰子油(阿拉丁)
14.1.5 水合氯醛(10％，Sigma)
14.1.6 全自动生化分析仪所用脂类校准标准品Cfas Lipid和其他校准品Cfas，各相关指标的检测试剂盒(Roche)
14.2 方法
14.2.1 小鼠的饲养和给药
小鼠在复旦大学实验动物中心SPF级动物实验室饲养，室温21℃-23℃，每12hr光暗循环，诱导肥胖小鼠所用的高脂饲料脂肪含量60％。小鼠成长至8-week龄时，随机分成对照组和给药组，每组8只。在用高脂饲料喂养的同时，腹腔注射蒿甲醚。腹腔注射用的蒿甲醚保存在椰子油中，储存浓度为6mg/ml(20mMol/L)，避光，4℃储存。按照20mg/kg体重给给药组小鼠注射蒿甲醚。每周给药两次，给药持续八周。
14.2.2 小鼠的血清指标检测
用10％的水合氯醛麻醉小鼠，通过眼眶取血，全血离心，4℃，300rpm，5min，取血清，脂类校准标准品Cfas Lipid，高密度脂蛋白Cfas和低密度脂蛋白Cfas，以及各相关指标的检测试剂盒。
14.2.3 数据分析
数据差异性由Student T test方法分析，p<0.05时，认为有统计学差异。
14.3 结果
在高脂饲养八周后，腹腔注射蒿甲醚的小鼠血清中的甘油三酯(Triglyceride)和高密度脂蛋白(High Density Lipoprotein，HDL)与对照组没有差别。血清胆固醇(Cholestrol)和低密度脂蛋白(Low Density Lipoprotein，LDL)与对照组相比显著下降，如图19所示。蒿甲醚显著改善了高脂饮食导致的血脂紊乱。
实施例15、腹腔注射蒿甲醚诱导高脂饲养的小鼠皮下脂肪出现棕色化
15.1 材料
15.1.1 雄性C57BL/6J小鼠(4-6week)
15.1.2 高脂饲料(Research Diets)
15.1.3 小鼠体温仪(Physitemp BAT-12)
15.1.4 UCP1特异性抗体(Abcam，ab10983，1:50)
15.1.5 蒿甲醚(成都普菲德生物技术有限公司)
15.1.6 椰子油(阿拉丁)
15.2 方法
15.2.1 小鼠的饲养和给药
小鼠在复旦大学实验动物中心SPF级动物实验室饲养，室温21℃-23℃，每12hr光暗循环，诱导肥胖小鼠所用的高脂饲料脂肪含量60％。小鼠成长至8-week龄时，随机分成对照组和给药组，每组8只。在用高脂饲料喂养的同时，腹腔注射蒿甲醚。腹腔注射用的蒿甲醚保存在椰子油中，储存浓度为6mg/ml(20mMol/L)，避光，4℃储存。按照20mg/kg体重给给药组小鼠注射蒿甲醚。每周给药两次，给药持续八周。小鼠禁食，置于4℃饲养箱，每隔2hr测其直肠温度，6hr后，断颈处死小鼠，取腹股沟部脂肪，进行组织学分析。组织学分析包括组织固定、石蜡包埋、切片和UCP1的免疫组化实验委托上海赛戈生物有限公司代做。
15.2.2 数据分析
数据差异性由Student T test方法分析，p<0.05时，认为有统计学差异。
15.3 结果
给药八周后，当暴露在4℃环境中，腹腔注射蒿甲醚的小鼠的体温明显高于对照小鼠(图19A)。在冷暴露环境中保存体温是由产热性脂肪组织承担的生理功能。取出小鼠的附睾部脂肪进行UCP1的免疫组化检测显示给药组的小鼠的皮下脂肪出现了明显的UCP1表达(图19B)。腹腔注射蒿甲醚八周后，给药组小鼠的体温保存能力明显增强，皮下脂肪出现明显的棕色化。说明蒿甲醚抑制体重增长的作用是通过促进皮下脂肪棕色化化实现的。
实施例16、蒿甲醚抑制糖尿病小鼠ob/ob小鼠的体重增长
16.1 材料
16.1.1 雄性ob/ob小鼠(4week，南京大学模式动物中心)
16.1.2 天平(赛多利斯)
16.1.3 蒿甲醚(成都普菲德生物技术有限公司)
16.1.4 椰子油(阿拉丁)
16.2 方法
16.2.1 小鼠的饲养和给药
ob/ob小鼠在复旦大学实验动物中心SPF级动物实验室饲养，室温21℃-23℃，每12hr光暗循环。小鼠成长至4-week龄时，随机分成对照组和给药组，每组4只。给药组通过腹腔注射蒿甲醚。腹腔注射用的蒿甲醚保存在椰子油中，储存浓度为6mg/ml(20mMol/L)，避光，4℃储存。按照20mg/kg体重给给药组ob/ob小鼠注射蒿甲醚。每周给药两次，给药持续八周。每周称重并记录。
16.2.3 数据分析
数据差异性由Student T test方法分析，p<0.05时，认为有统计学差异。
16.3 结果
给药组的ob/ob小鼠的体重有低于对照组的趋势，但没有达到统计学差异(图21A)。但给药组的小鼠的体重增长明显比对照组慢(图21B)。说明蒿甲醚显著抑制了ob/ob小鼠的体重增长，但给药八周时，给药组和对照组之间的差异没有达到统计学意义。蒿甲醚有效抑制了糖尿病动物模型ob/ob小鼠的体重增长。
实施例17、蒿甲醚改善糖尿病小鼠ob/ob的脂肪肝
17.1 材料
17.1.1 雄性ob/ob小鼠(4-week，南京大学模式动物中心)
17.1.2 蒿甲醚(武汉菲尔德生物化学有限公司)
17.1.3 椰子油(阿拉丁)
17.2 方法
Ob/ob小鼠在复旦大学实验动物中心SPF级动物实验室饲养，室温21℃-23℃，每12hr光暗循环。小鼠成长至4-week龄时，随机分成对照组和给药组，每组4只。给药组通过腹腔注射蒿甲醚。腹腔注射用的蒿甲醚保存在椰子油中，储存浓度为6mg/ml(20mMol/L)，避光，4℃储存。按照20mg/kg体重给给药组ob/ob小鼠注射蒿甲醚。每周给药两次，给药持续八周。小鼠断颈处死，取出肝脏组织固定进行石蜡包埋、切片和HE染色，组织学分析过程委托上海赛戈生物公司代做。
17.3 结果
与对照组相比，蒿甲醚给药组的ob/ob小鼠的脂肪肝明显缓解(图22)；蒿甲醚有效缓解了糖尿病动物模型ob/ob的脂肪肝。
实施例18、腹腔注射蒿甲醚不影响健康小鼠的生长
18.1 材料
18.1.1 雄性C57BL/6J小鼠(4-6week)
18.1.2 常规饲料(上海斯莱克实验动物有限公司)
18.1.3 天平(Satorius)
18.1.4 蒿甲醚(武汉菲尔德生物化学有限公司)
18.1.5 椰子油(阿拉丁)
18.2 方法
18.2.1 小鼠的饲养和给药
小鼠在复旦大学实验动物中心SPF级动物实验室饲养，室温21℃-23℃，每12hr光暗循环。小鼠成长至8-week龄时，随机分成对照组和给药组，每组8只。给药组通过腹腔注射蒿甲醚。腹腔注射用的蒿甲醚保存在椰子油中，储存浓度为6mg/ml(20mMol/L),避光，4℃储存。按照20mg/kg体重给给药组小鼠注射蒿甲醚，对照组小鼠注射100μl椰子油。每周给药两次，给药持续八周，每周记录小鼠体重。
18.2.2 数据分析
数据差异性由Student T test方法分析，p<0.05时，认为有统计学差异。
18.3 结果
在给药八周的时间内，给药组的小鼠的体重和对照组没有出现差异(图23A)。给药组小鼠的体重增长与对照组也没有显著差别(图23B)。腹腔注射蒿甲醚不影响健康瘦小鼠的正常生长。
实施例19、腹腔注射蒿甲醚不影响健康瘦小鼠的糖代谢
19.1 材料
19.1.1 雄性C57BL/6J小鼠(4-6week，斯莱克实验动物有限公司)
19.1.2 常规饲料(上海斯莱克实验动物有限公司)
19.1.3 葡萄糖注射液
19.1.4 注射用胰岛素(优泌林)
19.1.5 血糖仪(Roche OneTouch ExtraEasy)
19.1.6 血糖试纸(Roche OneTouch ExtraEasy)
19.2 方法
19.2.1 小鼠的饲养和给药
小鼠在复旦大学实验动物中心SPF级动物实验室饲养，室温21℃-23℃，每12hr光暗循环。小鼠成长至8-week龄时，随机分成对照组和给药组，每组8只。腹腔注射用的蒿甲醚保存在椰子油中，储存浓度为6mg/ml(20mMol/L),避光，4℃储存。按照20mg/kg体重给给药组小鼠注射蒿甲醚，对照组小鼠注射100μl椰子油。每周给药两次，给药持续八周。
19.2.2 葡萄糖耐量实验
小鼠禁食12-16hr，腹腔注射2mg/kg体重的葡萄糖注射液，在注射后0min，30min，60min，90min和120min时，剪尾取血，用OneTouch ExtraEasy血糖仪和OneTouch ExtraEasy血糖试纸检测血糖并记录。
19.2.3 胰岛素耐量实验
腹腔注射0.75U/kg体重的胰岛素，于注射后0min，15min，30min，45min 和60min时，剪尾取血，测血糖
19.2.4 数据分析
数据差异性由Student T test方法分析，p<0.05时，认为有统计学差异。
19.3 结果
健康瘦小鼠，腹腔注射蒿甲醚，持续八周后，给药组小鼠的葡萄糖耐量曲线与对照组没有差别，说明葡萄糖代谢没有受到蒿甲醚影响，如图23A所示。给药组小鼠的胰岛素耐量曲线与给药组也没有差别，如图24B所示；腹腔注射蒿甲醚不改变健康瘦小鼠的糖代谢。
实施例20、小鼠腹腔注射20mg/kg体重蒿甲醚没有导致肝脏损伤
20.1 材料
20.1.1 雄性C57BL/6J小鼠(4-6week)
20.1.2 常规饲料(上海斯莱克实验动物有限公司)
20.1.3 蒿甲醚(武汉菲尔德生物化学有限公司)
20.1.4 椰子油(阿拉丁)
20.2 方法
小鼠在复旦大学实验动物中心SPF级动物实验室饲养，室温21℃-23℃，每12hr光暗循环。小鼠成长至8-week龄时，随机分成对照组和给药组，每组8只。给药组通过腹腔注射蒿甲醚。腹腔注射用的蒿甲醚保存在椰子油中，储存浓度为6mg/ml(20mMol/L)，避光，4℃储存。按照20mg/kg体重给给药组小鼠注射蒿甲醚，对照组注射100μl椰子油。每周给药两次，给药持续八周。小鼠断颈处死，取肝脏组织固定。肝脏组织的固定、石蜡包埋、切片和HE染色委托赛戈生物有限公司代做。
20.3 结果
健康瘦小鼠在腹腔注射蒿甲醚八周后，肝脏细胞形态正常，与对照组小鼠没有差别，如图25所示；腹腔注射蒿甲醚，20mg/kg体重，每周两次，持续八周的条件下，蒿甲醚没有肝脏毒性。
实施例21、蒿甲醚不改变小鼠的进食量
21.1 材料
21.1.1 雄性C57BL/6J小鼠(4-6week，上海斯莱克实验动物有限公司)
21.1.2 常规饲料(上海斯莱克实验动物有限公司)
21.1.3 高脂饲料(Research Diets)
21.1.4 蒿甲醚(武汉菲尔德生物化学有限公司)
21.1.5 椰子油(阿拉丁)
21.2 方法
21.2.1 小鼠的饲养和给药
小鼠在复旦大学实验动物中心SPF级动物实验室饲养，室温21℃-23℃，每12hr光暗循环。小鼠成长至8-week龄时，随机分成四组，每组8只，分别是高脂对照组，高脂给药组，常规给药组和常规对照组。给药组(高脂和常规饲料)通过腹腔注射蒿甲醚。腹腔注射用的蒿甲醚保存在椰子油中，储存浓度为6mg/ml(20mMol/L)，避光，4℃储存。按照20mg/kg体重给给药组小鼠注射蒿甲醚，对照组注射100μl椰子油。每周称重饲料，记录每组小鼠的饲料的消耗量。
21.2.2 数据分析
数据差异性由Student T test方法分析，p<0.05时，认为有统计学差异。
21.3 结果
高脂饲养组，给药组小鼠的饲料消耗和对照组基本一致，没有差异，如图26A所示。常规饲养组，给药组小鼠的饲料消耗量和对照组也没有差别，如图26B所示；不管是在常规饲料饲养的条件下，还是高脂饲料饲养的条件下，腹腔注射蒿甲醚不影响小鼠的正常进食。在本实施例的条件下，蒿甲醚没有引起神经性厌食。

Claims (10)

  1. 如以下通式(A)所示的青蒿素类似物在制备用于促进脂肪分解和/或预防或治疗代谢相关疾病的药物中的用途：
    Figure PCTCN2017082359-appb-100001
    其中，R选自：羟基、甲氧基、乙氧基、丁二酸单酯基(琥珀酸酯基)、2-氨基乙氧基、2R-3-叔丁基氨基-2-羟基丙氧基、硫代吗啉基和1,1-二氧化硫代吗啉基。
  2. 一种用于促进脂肪分解和/或预防或治疗代谢相关疾病的方法，其特征在于，所述方法包括向有需要的受试者给予治疗有效量的如以下通式(A)所示的青蒿素类似物：
    Figure PCTCN2017082359-appb-100002
    其中，R选自：羟基、甲氧基、乙氧基、丁二酸单酯基(琥珀酸酯基)、2-氨基乙氧基、2R-3-叔丁基氨基-2-羟基丙氧基、硫代吗啉基和1,1-二氧化硫代吗啉基。
  3. 根据权利要求2所述的方法，其特征在于，所述给予治疗有效量所述青蒿素类似物的方式为：口服或注射方式给药；优选地，所述青蒿素类似物的给药剂量分别为：
    口服：20mg/kg～100mg/kg；优选为50mg/kg～100mg/kg；
    注射：10mg/kg～50mg/kg；优选为20mg/kg～40mg/kg。
  4. 一种用于促进脂肪分解和/或预防或治疗代谢相关疾病的青蒿素类似物，其特征在于，所述青蒿素类似物如以下通式(A)所示：
    Figure PCTCN2017082359-appb-100003
    其中，R选自：羟基、甲氧基、乙氧基、丁二酸单酯基(琥珀酸酯基)、2-氨基乙氧基、2R-3-叔丁基氨基-2-羟基丙氧基、硫代吗啉基和1,1-二氧化硫代吗啉基。
  5. 根据前述任一项权利要求所述的用途、青蒿素类似物或方法，其特征在于，所述青蒿素类似物为：如式(Ⅱ)所示的蒿甲醚：
    Figure PCTCN2017082359-appb-100004
  6. 根据权利要求1-5中任一项所述的用途、青蒿素类似物或方法，其特征在于，所述的促进脂肪分解包括促进白色脂肪棕色化。
  7. 根据权利要求1-5中任一项所述的用途、青蒿素类似物或方法，其特征在于，所述的促进脂肪分解和/或预防或治疗代谢相关疾病包括控制体重、改善代谢。
  8. 根据权利要求1-5中任一项所述的用途、青蒿素类似物或方法，其特征在于，所述的代谢相关疾病是肥胖引起的高血糖、胰岛素抵抗、血脂紊乱和/或脂肪肝。
  9. 一种用于促进脂肪分解和/或预防或治疗代谢相关疾病的药物组合物，其特征在于，所述药物组合物包含：(1)如权利要求4-5任一项所述的青蒿素类似物以及(2)药学上可接受的载体。
  10. 根据权利要求9所述的方法，其特征在于，优选地，所述药物组合物的口服给药剂型选自：口服混悬剂、散剂和颗粒剂；注射给药剂型选自：水剂、油剂和混悬剂。
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