WO2021098826A1 - 芍药内酯苷或芍药苷用于预防和/或治疗肾性贫血的用途 - Google Patents

芍药内酯苷或芍药苷用于预防和/或治疗肾性贫血的用途 Download PDF

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WO2021098826A1
WO2021098826A1 PCT/CN2020/130437 CN2020130437W WO2021098826A1 WO 2021098826 A1 WO2021098826 A1 WO 2021098826A1 CN 2020130437 W CN2020130437 W CN 2020130437W WO 2021098826 A1 WO2021098826 A1 WO 2021098826A1
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paeoniflorin
anemia
renal
composition containing
epo
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PCT/CN2020/130437
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French (fr)
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张作光
田晖
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张作光
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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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics

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  • the invention belongs to the field of medicine and relates to the use of paeoniflorin or paeoniflorin or a composition containing paeoniflorin or paeoniflorin for preventing and/or treating renal anemia. More specifically, the present invention relates to the use of paeoniflorin, paeoniflorin or a composition containing paeoniflorin or paeoniflorin to prevent and/or treat renal anemia by up-regulating erythropoietin (EPO).
  • EPO erythropoietin
  • the present invention also provides the use of paeoniflorin, paeoniflorin or a composition containing paeoniflorin or paeoniflorin in the preparation of medicines, health products, food nutrients, and food additives for the prevention and/or treatment of renal anemia use.
  • Renal anemia is one of the main complications of chronic kidney disease (CKD) in the compensatory phase of renal dysfunction.
  • the cause of renal anemia is the reduction of erythropoietin (EPO) production, because the production of erythropoietin in the kidney is destroyed.
  • EPO erythropoietin
  • patients with renal anemia mostly relied on supplementation of exogenous erythropoietin for treatment.
  • CKD chronic kidney disease
  • Patients with renal anemia are more difficult to correct than conventional anemia, and the patients are severely fatigued and have poor quality of life. Whether it is dialysis or non-dialysis-dependent chronic kidney disease patients, the morbidity and mortality of renal anemia are very high.
  • Chronic kidney disease can occur at any age and is more common in the elderly. At present, there are more than 100 million patients with chronic nephritis in China, of which more than 1 million patients are end-stage and require dialysis or kidney transplantation. 98.2% of dialysis patients have anemia, and 52.1% of non-dialysis patients have renal anemia. Treatment drugs Mainly iron and erythropoietin, the method of administration is injection.
  • EPO erythropoietin
  • the main indication of erythropoietin (EPO) is anemia caused by renal failure. It can improve the patient's hematocrit and hemoglobin level.
  • the use of erythropoietin to treat renal failure anemia has a significant effect, even without the need for blood transfusion.
  • the reticulocyte count begins to increase; within 2 to 6 weeks, the hematocrit and hemoglobin level begin to increase.
  • intravenous or subcutaneous injection of erythropoietin 50-150IU/kg three times a week can maintain the hematocrit at about 35%.
  • Erythropoietin is also effective for the treatment of anemia in primary bone marrow disorders and certain secondary anemias, including aplastic anemia, myeloproliferative and myelodysplastic diseases, multiple myeloma, and chronic inflammation, AIDS and Anemia caused by tumors. Especially if the patient's serum erythropoietin level is too low compared to the degree of anemia, such as less than 100IU/L, treatment with erythropoietin may be more effective.
  • rosarestat a drug for the treatment of renal anemia jointly developed by Fabojin and AstraZeneca, as the world's first innovative drug, was listed in 2018 through the CFDA priority review and approval process.
  • the drug inhibits the activity of HIF2a prolyl hydroxylase in the hypoxic pathway, reduces the degradation of HIF2a, continuously activates the HIF2a hypoxic pathway, promotes endogenous EPO production, and enhances iron utilization, thereby improving anemia in patients with chronic kidney disease .
  • the drug DDO-3055 tablets developed by Jiangsu Hengrui Pharmaceutical Co., Ltd. for the treatment of renal anemia has obtained the CFDA "Clinical Trial Notice". Hengrui stated in the announcement that it will carry out clinical trials of the drug in the near future, and it is intended to be used Treatment of anemia caused by chronic kidney disease (including dialysis and non-dialysis).
  • the new drugs currently under development for the treatment of renal anemia include HEC53856 capsules from Dongyang Sunshine and AND017 capsules from Hangzhou Andao Pharmaceutical.
  • EPO endogenous erythropoietin
  • the purpose of the present invention is to provide a new use of paeoniflorin or albiflorin or a composition containing paeoniflorin or paeoniflorin in view of the deficiencies of the prior art.
  • paeoniflorin, paeoniflorin, or a composition containing paeoniflorin or paeoniflorin can regulate the endocrine of erythropoietin (EPO), which can be used to prevent and/or treat renal disease. anemia.
  • EPO erythropoietin
  • the invention also provides the use of the paeoniflorin or paeoniflorin for preparing medicines, health products or food nutrients for the prevention and/or treatment of renal anemia.
  • Albiflorin is a monoterpene compound with a molecular formula of C 23 H 28 O 11 and a molecular weight of 480.46.
  • the molecular structure is shown in the following formula. It is a natural active substance derived from the Ranunculaceae plant Paeonia lactiflora ( Paeonia lactiflora Pall) or Sichuan red peony (Paeonia veitchii Lynch) root, peony (P. suffrsticosa Andrz) root.
  • the present invention provides paeoniflorin, paeoniflorin or a composition containing paeoniflorin in the preparation of medicines, health products, food nutrients or food additives for increasing endogenous erythropoietin (EPO) In the use.
  • EPO erythropoietin
  • the composition containing paeoniflorin or paeoniflorin is an extract containing paeoniflorin or paeoniflorin, more preferably a white peony extract containing paeoniflorin or paeoniflorin.
  • the present invention provides the preparation of paeoniflorin, paeoniflorin or a composition containing paeoniflorin or paeoniflorin for the prevention and/or treatment of primary anemia, secondary anemia or renal anemia Use in medicines, health products, food nutrients or food additives.
  • the primary anemia is anemia of primary bone marrow disease
  • the secondary anemia is aplastic anemia, or secondary anemia caused by myeloproliferative and myelodysplastic diseases, multiple myeloma, chronic inflammation, AIDS or tumors.
  • the renal anemia is renal anemia complicated by diabetes.
  • the composition containing paeoniflorin or paeoniflorin is an extract containing paeoniflorin or paeoniflorin, more preferably a white peony extract containing paeoniflorin or paeoniflorin.
  • the present invention provides that paeoniflorin, paeoniflorin or a composition containing paeoniflorin or paeoniflorin are prepared for the prevention and/or treatment of primary erythropoietin (EPO) by up-regulating endogenous erythropoietin (EPO).
  • EPO primary erythropoietin
  • EPO endogenous erythropoietin
  • the primary anemia is anemia of primary bone marrow disease.
  • the secondary anemia is aplastic anemia, or secondary anemia caused by myeloproliferative and myelodysplastic diseases, multiple myeloma, chronic inflammation, AIDS, or tumors.
  • the renal anemia is renal anemia complicated by diabetes.
  • the composition containing paeoniflorin or paeoniflorin is an extract of paeoniflorin or paeoniflorin, more preferably a white peony extract containing paeoniflorin or paeoniflorin.
  • paeoniflorin, paeoniflorin or a composition containing paeoniflorin or paeoniflorin can activate the PI-3K pathway by activating adenosine A1 and A2 receptors in the hypoxic environment of chronic nephritis Enhance the stability of hypoxia inducible factor (HIF) and promote the expression of EPO.
  • hypoxia inducible factor HIF
  • the physiological role of hypoxia inducible factor (HIF) not only increases the expression of EPO, but also increases the expression of EPO receptors and proteins that promote iron absorption and circulation.
  • the present invention also provides a method for increasing endogenous erythropoietin (EPO), the method comprising administering to a subject in need thereof an effective amount of paeoniflorin, paeoniflorin or containing paeoniflorin A combination of ester glycosides or paeoniflorin.
  • EPO endogenous erythropoietin
  • the composition containing paeoniflorin or paeoniflorin is an extract of paeoniflorin or paeoniflorin, more preferably a white peony extract containing paeoniflorin or paeoniflorin.
  • the present invention provides a method for preventing and/or treating primary anemia, secondary anemia or renal anemia, the method comprising administering an effective amount of paeoniflorin to a subject in need thereof Glucoside, paeoniflorin or a composition containing paeoniflorin or paeoniflorin.
  • the primary anemia is anemia of primary bone marrow disease.
  • the secondary anemia is aplastic anemia, or secondary anemia caused by myeloproliferative and myelodysplastic diseases, multiple myeloma, chronic inflammation, AIDS or tumors.
  • the renal anemia is renal anemia complicated by diabetes.
  • the composition containing paeoniflorin or paeoniflorin is an extract of paeoniflorin or paeoniflorin, more preferably a white peony extract containing paeoniflorin or paeoniflorin.
  • the present invention provides a method for preventing and/or treating primary anemia, secondary anemia or renal anemia by up-regulating endogenous erythropoietin (EPO), the method comprising administering
  • EPO endogenous erythropoietin
  • the subject in need thereof has an effective amount of paeoniflorin, paeoniflorin or a composition containing paeoniflorin or paeoniflorin.
  • the primary anemia is anemia of primary bone marrow disease.
  • the secondary anemia is aplastic anemia, or secondary anemia caused by myeloproliferative and myelodysplastic diseases, multiple myeloma, chronic inflammation, AIDS or tumors.
  • the renal anemia is renal anemia complicated by diabetes.
  • the composition containing paeoniflorin or paeoniflorin is an extract of paeoniflorin or paeoniflorin, more preferably a white peony extract containing paeoniflorin or paeoniflorin.
  • the inventors of the present invention have discovered through a series of research results: 1.
  • the main cause of renal anemia is the reduction of erythropoietin (EPO) production: chronic nephritis continues to develop, and residual renal function decreases.
  • EPO erythropoietin
  • the residual kidney cannot respond adequately to hypoxic stimulation caused by anemia.
  • Paeoniflorin or paeoniflorin and their compositions containing paeoniflorin or paeoniflorin can prevent and treat renal anemia by increasing the expression of endogenous EPO; 3.
  • Paeoniflorin or paeoniflorin and its contents are adenosine A1 and A2 receptor agonists, which can activate the adenosine A1 and A2 receptors in the hypoxic environment of chronic nephritis to promote the expression of EPO.
  • the present invention has the following advantages:
  • the paeoniflorin or paeoniflorin of the present invention is derived from the white peony medicinal material with the same medicine and food. It is a naturally-occurring monomer drug and has higher safety and patient compliance than other compound drugs in the prior art. .
  • Figure 1 shows the results of agarose gel electrophoresis of RT-PCR products of EPO mRNA of mouse bone marrow, where M: labeled group, A: normal group, B: model group, and C: paeoniflorin group;
  • Figure 2 shows the results of agarose gel electrophoresis of RT-PCR products of mouse bone marrow G-CSF mRNA, where M: labeled group A: normal group B: model group C: paeoniflorin group;
  • Figure 3 shows the effect of different doses of paeoniflorin on EPO gene expression in HepG2 cells under hypoxic conditions, where 1: control group, 2: paeoniflorin 0.02 ⁇ mol ⁇ L -1 group, and 3: paeoniflorin Ester glycoside 0.2 ⁇ mol ⁇ L -1 group, 4: Paeoniflorin 2 ⁇ mol ⁇ L -1 group, 5: Paeoniflorin 20 ⁇ mol ⁇ L -1 group;
  • Figure 4 shows the effects of DPCPX and LY294002 on the promotion of EPO gene expression by paeoniflorin, in which 1: control group, 2: paeoniflorin 2 ⁇ mol ⁇ L -1 group, 3: DPCPX 10 ⁇ mol ⁇ L -1 group, 4: DPCPX 10 ⁇ mol ⁇ L -1 + paeoniflorin 2 ⁇ mol ⁇ L -1 group, 5: LY294002 30 ⁇ mol ⁇ L -1 group, 6: LY294002 30 ⁇ mol ⁇ L -1 + paeoniflorin 20 ⁇ mol ⁇ L -1 group;
  • Figure 5 shows the effects of SCH58261 and CPA on the promotion of EPO gene expression by paeoniflorin, in which 1: control group, 2: paeoniflorin 2 ⁇ mol ⁇ L -1 group, 3: SCH58261 0.2 ⁇ mol ⁇ L -1 group, 4 : SCH58261 0.2 ⁇ mol ⁇ L -1 + paeoniflorin 2 ⁇ mol ⁇ L -1 group, 5: CPA 10 ⁇ mol ⁇ L -1 group, 6: CPA 10 ⁇ mol ⁇ L -1 group + paeoniflorin 20 ⁇ mol ⁇ L -1 group ;
  • Figure 6 shows the results of different doses of paeoniflorin (0.02, 0.2, 2, 20 ⁇ mol ⁇ L-1) after 36 hours of normal oxygen culture to inhibit EPO gene expression in HepG 2 cells, in which 1: control group, 2: Paeoniflorin 0.2 ⁇ mol ⁇ L -1 group, 3: Paeoniflorin 2 ⁇ mol ⁇ L -1 group, 4: Paeoniflorin 20 ⁇ mol ⁇ L -1 group;
  • Figure 7 shows the effect of different doses of paeoniflorin on the expression of EPO protein in HepG2 cells under hypoxic conditions, where 1: control group, 2: paeoniflorin 0.02 ⁇ mol ⁇ L -1 group, and 3: paeoniflorin Ester glycoside 0.2 ⁇ mol ⁇ L -1 group, 4: Paeoniflorin 2 ⁇ mol ⁇ L -1 group, 5: Paeoniflorin 20 ⁇ mol ⁇ L -1 group;
  • Figure 8 shows the results of different doses (0.02, 0.2, 2 , 20 ⁇ mol ⁇ L -1) of paeoniflorin inhibiting the expression of EPO protein in HepG 2 cells after 36 hours of normoxia culture, in which 1: control group, 2: peony Lactone 0.02 ⁇ mol ⁇ L -1 group, 3: Paeoniflorin 0.2 ⁇ mol ⁇ L -1 group, 4: Paeoniflorin 2 ⁇ mol ⁇ L -1 group, 5: Paeoniflorin 20 ⁇ mol ⁇ L -1 group ;
  • Figure 9 shows the effect of hypoxia on the expression of adenosine A2 receptor, where 1: normoxia condition group, 2: hypoxia 8 hours group, 3: hypoxia 16 hours group, 4: hypoxia 24 hours group.
  • the Applicant discovered through the effect of the expression of EPO gene in bone marrow of radiation-induced anemia mice, and found that paeoniflorin and extracts containing paeoniflorin have hematopoietic effects, can promote the expression of bone marrow EPO gene, and treat renal anemia.
  • Example 1 Effect of paeoniflorin on EPO and G-CSF genes in bone marrow of radiation-induced anemia mice Elevating effect
  • Paeoniflorin glycosides were provided by Beijing Onar Bioengineering Technology Co., Ltd. According to the human dosage, it is determined that the daily dosage of mouse paeoniflorin is 14mg.kg-1. Prepare the paeoniflorin solution with water, the concentration is 1.4mg.ml-1.
  • AWV reverse transcriptase AMV/rfl 5 ⁇ reaction buffer, Taq DNA synthetase, RNaSe inhibitor, etc. were purchased from Promega, and TRIZOL, Oligo dT, dNTP, etc. were purchased from Gibco.
  • EPO, IL-3 and L-glutamine were purchased from Sigma, methyl cellulose was purchased from Whatman, IL-11 was purchased from Chongqing Duotai products, G-CSF was a product of Kylin Zhushi Company of Japan, DU640 nucleic acid protein UV The analyzer was purchased from Beckman, the Gel Doc 1000 gel imager was a Bio-RAD product, the DNA synthesizer was a product of PE, and the Sysmex-820 automatic blood cell counter was purchased from Japan.
  • mice C57BL/6J mice, female, 60 mice, 6-8 weeks old, weight 20 ⁇ 2g, purchased from Beijing Weitonglihua Experimental Animal Center. According to the experimental plan, they were randomly divided into three groups: normal control group, model control group, and paeoniflorin group. Each group of 20.
  • mice After the mice were bred routinely for several days to adapt to the environment, the model control group and paeoniflorin were irradiated with 60 CO gamma rays once, with an irradiation dose of 3.5 Gy and a dose rate of 1.60 Gy.min -1 , to make a blood deficiency model. After irradiation, mice were given intragastric administration immediately. The paeoniflorin group was given 0.2ml each time, once a day; the normal control group and the model control group were given the same amount of normal saline for 7 days.
  • mice Take 10 mice from each group. On the 3rd, 5th, 7th, 10th, and 13th days after modeling, 20 ⁇ l of blood was collected from the tail vein to detect peripheral blood leukocytes.
  • CFU-GM granulocyte-macrophage colony forming unit
  • BFU-E red blood cell colony forming unit
  • CFU-mix mixed colony forming unit
  • RT-PCR Reverse transcription-polymerase chain reaction
  • mice from each group were taken, sacrificed by cervical dislocation, and femoral bone marrow was taken. Wash the bone marrow with RNase-free phosphate buffer, add 1ml TRIZOL reagent, place at room temperature for 5 minutes, add 200 ⁇ l of chloroform, shake vigorously for 15sec, place at room temperature for 3 minutes, 4°C,
  • Reverse transcription Take total RNA 2.5 ⁇ g, 1 ⁇ g ⁇ ml -1 oligo dT2.5 ⁇ l, make up the volume to 12.5 ⁇ l with DEPC treated water, 70°C water bath for 60min, ice bath for 5min, add to the reaction system (including 5 ⁇ AMV/Tfl Reaction buffer 10 ⁇ l, 25mmol.L -1 MgCl 2 10 ⁇ l, 10mmol1.L -1 dNTP 2.5 ⁇ l, RNaSe inhibitor 1.25 ⁇ l, AMV 2.5 ⁇ l with DEPC-treated water to make up the volume to 37.5 ⁇ l), put at room temperature for 5min, 42°C water bath 62min, water bath at 70°C for 15min.
  • the reaction system including 5 ⁇ AMV/Tfl Reaction buffer 10 ⁇ l, 25mmol.L -1 MgCl 2 10 ⁇ l, 10mmol1.L -1 dNTP 2.5 ⁇ l, RNaSe inhibitor 1.25 ⁇ l, AMV 2.5 ⁇ l with DEPC-treated water to make up the volume to 3
  • Polymerase chain reaction 20 ⁇ l reaction system, containing 1 ⁇ l of reverse transcription product, PCR-mix 15 ⁇ l, Taq DNA synthetase 0.2 ⁇ l, shaking and mixing, reaction on the machine, using ⁇ -actin as internal reference.
  • PCR parameters 94°C for 5 minutes; denaturation at 94°C for 30 seconds, renaturation at 55°C for 30 seconds, and extension at 72°C for 30 seconds; 30 cycles at 72°C for 5 minutes.
  • Epo primer sequence upstream primer SEQ ID NO:1
  • G-CSF primer sequence upstream primer SEQ ID NO: 3
  • ⁇ -actin primer sequence upstream primer SEQ ID NO: 5
  • the length of the amplified product is 178bp.
  • Agarose gel electrophoresis take 5 ⁇ l PCR product, electrophoresis on a 1.5% agarose gel at 150V for 20min, and stain with ethidium bromide.
  • the agarose gel was scanned and photographed, and the image was analyzed. Peripheral blood white blood cell count and hematopoietic progenitor cell colony count were expressed as mean ⁇ standard deviation, and t test was used for statistical analysis.
  • Example 2 Paeoniflorin promotes EPO under hypoxic conditions by activating adenosine A1 and A2a receptors gene expression
  • HepG2 cells were purchased from Shanghai Jining Industrial Co., Ltd., DMEM medium, pancreatin and TRzol were purchased from Invitrogen, fetal bovine serum was purchased from Hangzhou Sijiqing, PI-3kinase pathway inhibitor (LY294002), adenosine A1 receptor antagonist Drugs, adenosine A1 receptor agonist and adenosine A2a receptor antagonist were all purchased from Sigma, paeoniflorin was provided by Beijing Onar Bioengineering Technology Co., Ltd., HotStartDNA polymerase was purchased from Tiangen, AMV Transcriptase was purchased from TaKaRa company, EPO antibody was purchased from R&D company, and Tubulin was purchased from SantaCruz subpackage product of Zhongshan Jinqiao Company. The secondary antibody was purchased from Zhongshan Jinqiao Company.
  • the hypoxic incubator is the product of Thermo Forma
  • the Western blot electrophoresis and transfer instrument is the product of Bio-Rad
  • the PCR machine is the product of PERK N ELMER.
  • SEQ ID NO: 8 5'-ATGGTAGGTGCGAAAACAGG-3' amplified length 300bp;
  • Downstream primer SEQ ID NO: 10 5′-CAGCAGCTCCTGAACCCTAG-3
  • Amplification length is 607bp
  • the PCR products were subjected to agarose gel electrophoresis, and the results were analyzed by Gel-Pro software.
  • the ratio of the integrated optical density of the target band to the integrated optical density of ⁇ -actin was used as the relative expression of EPO gene, and the calculation was repeated 3 times for each experimental group.
  • DPCPX and LY294002 can inhibit the effect of paeoniflorin on EPO gene
  • SCH58261 can significantly inhibit the promotion of EPO gene expression by paeoniflorin.
  • CPA has an inhibitory effect on EPO expression at 8h, and has no effect on EPO expression at 16 and 24h, as shown in Figure 5.
  • Paeoniflorin (0.02, 0.2, 2 , 20 ⁇ mol ⁇ L -1) inhibits HepG2 after 36 hours of normal oxygen culture
  • the EPO gene expression in the cell is shown in Figure 6.
  • Hypoxic culture can promote the expression of adenosine A2 receptor in HepG2 cells after 8, 16, and 24 hours, as shown in Figure 9.
  • Paeoniflorin can promote the expression of EPO gene under hypoxic conditions by activating adenosine A1 and A2 receptors and treat renal anemia.
  • Example 3 Study on the affinity of paeoniflorin and paeoniflorin to adenosine A1 and A2 receptors
  • High-speed refrigerated centrifuge H1ACH1 20PR-5.
  • UV-250 ultraviolet spectrophotometer Shimadzu Corporation, Japan.
  • Multi-head suction filter Shaoxing Instrument and Equipment Company.
  • Type 49 glass fiber filter membrane Shanghai Yuguang Purification Material United Company.
  • the labeling ligand used in the experiment was a product of PE company.
  • the non-labeled ligand used in the experiment is Sigma
  • Methyllycaconitine MAA
  • PEI Polyethyleneimine
  • BSA Bovine Serum Albumin
  • PMSF and protease inhibitors
  • the scintillation fluid is a product of PE company.
  • Folin-phenol reagent is a product of Warwick Keyi Company.
  • Tris-HCl buffer 50mM Tris-HCl Buffer, 1mM EDTA, 5mM MgCl2, 1mM PMSF, 0.1%.NaN3, 3 ⁇ g/ml protease inhibitor, pH7.4
  • Other reagents are of analytical grade.
  • test tube Place the test tube in the reaction conditions at 25°C. 100 ⁇ g of receptor protein extracted from rat hippocampus or other brain regions was sequentially added to all test tubes. Add 50 ⁇ l (10-4M) of the corresponding non-labeled ligand to the non-specific binding tube, the final concentration is 10uM, and react for 30 minutes in advance. 30 ⁇ l of test drugs paeoniflorin and paeoniflorin were added to the test tube in sequence. Add 40 ⁇ l of [3H]-labeled compound to all test tubes, and the final concentration of labeled ligand is as shown in the table above.
  • Tris-HCl buffer 50mM Tris-HCl, 1mM EDTA, 5mM MgCl2, 0.1mM PMSF, 0.1% NaN3, pH7.4 to make up the volume of all reaction tubes to 300 ⁇ l. React at 25°C for 1 hour.
  • Tris-HCl buffer 50mM Tris-HCl Buffer, 1mM EDTA, 5mM MgCl2, 1mM PMSF, 0.1%.NaN3, 3 ⁇ g/ml Protease inhibitor, pH7.4 wash, 2ml each time, 3 times in total, drain the filter paper and take out the filter paper and dry it, put it in a scintillation bottle, add 1ml scintillation fluid, and measure the radioactivity intensity with a liquid scintillation counter.
  • Tris-HCl buffer 50mM Tris-HCl Buffer, 1mM EDTA, 5mM MgCl2, 1mM PMSF, 0.1%.NaN3, 3 ⁇ g/ml Protease inhibitor, pH7.4 wash, 2ml each time, 3 times in total, drain the filter paper and take out the filter paper and dry it, put it in a scintillation bottle, add 1ml scintillation fluid, and measure the radioactivity intensity with a liquid scintillation
  • paeoniflorin and paeoniflorin have significant affinity for adenosine A1 and A2 receptors.

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Abstract

芍药内酯苷、芍药苷或含有芍药内酯苷或芍药苷的组合物在制备用于增加内源性促红细胞生成素的药品、保健品、食品营养剂或食品添加剂中的用途；芍药内酯苷、芍药苷或含有芍药内酯苷或芍药苷的组合物在制备用于通过上调内源性促红细胞生成素预防和/或治疗原发性贫血、继发性贫血或肾性贫血的药品、保健品、食品营养剂或食品添加剂中的用途。芍药内酯苷、芍药苷源于药食同源的白芍药材，是天然存在的单体药物，与现有技术的其他化合物药物相比，具有更高的安全性和患者依从性。

Description

芍药内酯苷或芍药苷用于预防和/或治疗肾性贫血的用途 技术领域
本发明属于医药领域，涉及芍药内酯苷或芍药苷或含有芍药内酯苷或芍药苷的组合物用于预防和/或治疗肾性贫血的用途。更具体地，本发明涉及芍药内脂苷、芍药苷或含有芍药内酯苷或芍药苷的组合物通过上调促红细胞生成素(EPO)预防和/或治疗肾性贫血的用途。本发明还提供了芍药内脂苷、芍药苷或含有芍药内酯苷或芍药苷的组合物在制备用于预防和/或治疗肾性贫血的药品、保健品、食品营养剂、食品添加剂中的用途。
背景技术
肾性贫血是慢性肾脏病(CKD)肾功能失调代偿期主要并发症之一。肾性贫血产生的原因，是促红细胞生成素(EPO)生成的减少，因为促红细胞生成素在肾脏的生成遭到破坏，以往肾性贫血病人多是依靠补充外源性红细胞生成素进行治疗。随着慢性肾脏病(CKD)的进展，肾性贫血的发病率和严重程度逐渐增加，肾性贫血患者较常规贫血更难以纠正，患者乏力严重，生活质量低下。无论是透析还是非透析依赖性慢性肾病患者，肾性贫血的发病率和死亡率都非常高。慢性肾病可发病于任何年龄，在老年中更为常见。目前在中国慢性肾炎患者超过1亿，其中超过100万患者是终末期，需要接受透析或肾移植治疗，98.2％的透析患者合并贫血症，52.1％的非透析患者合并有肾性贫血，治疗药物主要是铁剂和促红细胞生成素，给药方式均为注射。
促红细胞生成素(EPO)的主要适应症是肾衰引起的贫血，它可以改善病人的红细胞压积和血红蛋白水平，使用促红细胞生成素治疗肾衰贫血，疗效显著，甚至可以不需要再输血。通常，使用外源性促红细胞生成素后10天内，网织红细胞计数开始增加；2～6周内，红细胞压积和血红蛋白水平开始上升。对大多数病人来说，静脉或皮下注射红细胞生成素50-150IU/kg，每周三次，可使红细胞压积维持在35％左右。如治疗无效，多因并发缺铁，需要同时服用铁剂。有些病人还需要同时补充叶酸。促红细胞生成素对治疗原发性骨髓疾患的贫血和某些继发性贫血也有效，包括再生障碍性贫血、骨髓增生性和骨髓发育不良性疾病、多发性骨髓瘤、以及慢性炎症、艾滋病和肿瘤引起的贫血。特别是如果病人血清促红细胞生 成素水平同贫血程度相比显得过低，例如低于100IU/L，用促红细胞生成素治疗可能更有效。目前，肾性贫血的病人主要依靠注射外源性促红细胞生成素进行治疗。这种给予外源性红细胞生成素进行治疗的方法有以下缺陷：1.给药方式为注射，一定要在医院进行治疗，患者用药不方便；2.有可能引起血压升高或形成血栓；3.外源性促红细胞生成素是生物制剂，用药费用比较高。
针对肾性贫血的发病原因和外源性促红细胞生成素治疗的缺陷，各国药企都在研发促进内源性促红细胞生成素生成的药物，用于肾性贫血的治疗。其中，珐博进和阿斯利康联合研制的治疗肾性贫血药物罗沙司他，作为全球首创新药，已于2018年通过CFDA优先审评审批程序上市。该药通过抑制低氧通路中HIF2a脯氨酰羟化酶的活性，减少HIF2a的降解，使HIF2a低氧通路持续活化，促进内源性EPO生成，增强铁利用率，从而改善慢性肾脏病患者贫血。江苏恒瑞医药股份有限公司研发的用于治疗肾性贫血的药品DDO-3055片，已获得CFDA《临床试验通知书》，恒瑞在公告中称将于近期开展该药品临床试验，拟用于治疗慢性肾病所致贫血(包括透析和非透析)。据网上披露，目前在研的治疗肾性贫血新药包括东阳光的HEC53856胶囊、杭州安道药业的AND017胶囊。
通过提升内源性促红细胞生成素(EPO)治疗慢性肾炎贫血药物是临床急需的药物，其市场容量很大，此前在罗沙司他上市时，科睿唯安就曾测算该产品在2022年的销售额将达20亿美元，而Evalute Pharma分析师更是测算该品的销售额可达58-81亿美元。
因此，针对肾性贫血的治疗药物存在巨大的市场和临床需求，而且发现具有治疗EPO相关疾病且具有很好的生物利用度和患者依从性的药物是具有挑战性的工作。
发明内容
本发明的目的是针对现有技术的不足，提供一种芍药内酯苷或芍药苷(Albiflorin)或含有芍药内酯苷或芍药苷的组合物的新用途。具体地，本发明人发现，芍药内脂苷、芍药苷或含有芍药内酯苷或芍药苷的组合物能够调节促红细胞生成素(EPO)的内分泌，从而可以用于预防和/或治疗肾性贫血。本发明还提供了所述芍药内酯苷或芍药苷用于制备预防和/或治疗肾性贫血的药品、保健品或食品营养剂的用途。
本发明的目的是通过以下技术方案实现的：
芍药内酯苷(Albiflorin)为单萜类化合物，其分子式为C 23H 28O 11，分子量为480.46，分子结构如下式所示，是一种天然活性物质，来源于毛莨科植物白芍(Paeonia lactiflora Pall)或川赤芍(Paeonia veitchii Lynch)的根、牡丹(P.suffrsticosa Andrz)的根。
Figure PCTCN2020130437-appb-000001
一方面，本发明提供了芍药内酯苷、芍药苷或含有芍药内酯苷的组合物在制备用于增加内源性促红细胞生成素(EPO)的药品、保健品、食品营养剂或食品添加剂中的用途。
优选地，所述含有芍药内酯苷或芍药苷的组合物为含有芍药内脂苷或芍药苷的提取物，更优选地为含有芍药内酯苷或芍药苷的白芍提取物。
另一方面，本发明提供了芍药内酯苷、芍药苷或含有芍药内酯苷或芍药苷的组合物在制备用于预防和/或治疗原发性贫血、继发性贫血或肾性贫血的药品、保健品、食品营养剂或食品添加剂中的用途。
根据本发明所述的用途，所述原发性贫血为原发性骨髓疾患的贫血；
根据本发明所述的用途，所述继发性贫血为再生障碍性贫血，或者骨髓增生性和骨髓发育不良性疾病、多发性骨髓瘤、慢性炎症、艾滋病或肿瘤引起的继发性贫血。
优选地，所述肾性贫血为糖尿病并发的肾性贫血。
优选地，所述含有芍药内酯苷或芍药苷的组合物为含有芍药内脂苷或芍药苷的提取物，更优选地为含有芍药内酯苷或芍药苷的白芍提取物。
另一方面，本发明提供了芍药内酯苷、芍药苷或含有芍药内酯苷或芍药苷的组合物在制备用于通过上调内源性促红细胞生成素(EPO)预防和/或治疗原发性贫血、继发性贫血或肾性贫血的药品、保健品、食品营养剂或食品添加剂中的用途。
根据本发明所述的用途，所述原发性贫血为原发性骨髓疾患的贫血。
根据本发明所述的用途，所述继发性贫血为再生障碍性贫血，或者骨髓增生性和骨髓发育不良性疾病、多发性骨髓瘤、慢性炎症、艾滋病或肿瘤引 起的继发性贫血。
优选地，所述肾性贫血为糖尿病并发的肾性贫血。
优选地，所述含有芍药内酯苷或芍药苷的组合物为含有芍药内脂苷或芍药苷提取物，更优选地为含有芍药内酯苷或芍药苷的白芍提取物。
本发明的发明人发现，芍药内脂苷、芍药苷或含有芍药内酯苷或芍药苷的组合物可以在慢性肾炎的低氧环境中通过激活腺苷A1、A2受体，激活PI-3K通路增强低氧诱导因子(HIF)的稳定性，促进EPO的表达。低氧诱导因子(HIF)的生理作用，不仅使EPO表达增加，也能使EPO受体以及促进铁吸收和循环的蛋白表达增加。
另一方面，本发明还提供了一种增加内源性促红细胞生成素(EPO)的方法，所述方法包括给予需要其的受试者有效量的芍药内脂苷、芍药苷或含有芍药内酯苷或芍药苷的组合物。
优选地，所述含有芍药内酯苷或芍药苷的组合物为含有芍药内脂苷或芍药苷提取物，更优选地为含有芍药内酯苷或芍药苷的白芍提取物。
另一方面，本发明提供了一种用于预防和/或治疗原发性贫血、继发性贫血或肾性贫血的方法，所述方法包括给予需要其的受试者有效量的芍药内脂苷、芍药苷或含有芍药内酯苷或芍药苷的组合物。
根据本发明所述的用途，所述原发性贫血为原发性骨髓疾患的贫血。
根据本发明所述的用途，所述继发性贫血为再生障碍性贫血，或者骨髓增生性和骨髓发育不良性疾病、多发性骨髓瘤、慢性炎症、艾滋病或肿瘤引起的继发性贫血。
优选地，所述肾性贫血为糖尿病并发的肾性贫血。
优选地，所述含有芍药内酯苷或芍药苷的组合物为含有芍药内脂苷或芍药苷提取物，更优选地为含有芍药内酯苷或芍药苷的白芍提取物。
再另一方面，本发明提供了一种芍通过上调内源性促红细胞生成素(EPO)预防和/或治疗原发性贫血、继发性贫血或肾性贫血的方法，所述方法包括给予需要其的受试者有效量的芍药内脂苷、芍药苷或含有芍药内酯苷或芍药苷的组合物。
根据本发明所述的用途，所述原发性贫血为原发性骨髓疾患的贫血。
根据本发明所述的用途，所述继发性贫血为再生障碍性贫血，或者骨髓增生性和骨髓发育不良性疾病、多发性骨髓瘤、慢性炎症、艾滋病或肿瘤引起的继发性贫血。
优选地，所述肾性贫血为糖尿病并发的肾性贫血。
优选地，所述含有芍药内酯苷或芍药苷的组合物为含有芍药内脂苷或芍药苷提取物，更优选地为含有芍药内酯苷或芍药苷的白芍提取物。
本发明的发明人通过一系列的研究成果发现：1、肾性贫血产生的主要原因是促红细胞生成素(EPO)生成的减少：慢性肾炎不断发展，残余肾功能下降，一方面促红细胞生成素(EPO)减少，另一方面残余肾无法对贫血引起的缺氧刺激产生足够的应答反应。2、芍药内酯苷或芍药苷及其含有芍药内酯苷或芍药苷的组合物，可以通过增加内源性EPO的表达，防治肾性贫血；3、芍药内酯苷或芍药苷及其含有芍药内酯苷或芍药苷的提取物，是腺苷A1、A2受体激动剂，可以在慢性肾炎的低氧环境中通过激活腺苷A1、A2受体，促进EPO的表达。
与现有技术相比，本发明具有以下优点：
本发明的芍药内酯苷或芍药苷源于药食同源的白芍药材，是天然存在的单体药物，与现有技术的其他化合物药物相比，具有更高的安全性和患者依从性。
附图说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1示出小鼠骨髓的EPO mRNA的RT-PCR产物琼脂糖凝胶电泳结果，其中M:标记组，A：正常组，B：模型组，C：芍药内酯苷组；
图2示出小鼠骨髓G-CSF mRNA的RT-PCR产物琼脂糖凝胶电泳结果，其中M:标记组A：正常组B：模型组C：芍药内酯苷组；
图3示出不同剂量的芍药内酯苷在低氧条件下对HepG2细胞中EPO基因表达的影响，其中1：对照组，2：芍药内酯苷0.02μmol·L -1组，3：芍药内酯苷0.2μmol·L -1组，4：芍药内酯苷2μmol·L -1组，5：芍药内酯苷20μmol·L -1组；
图4示出DPCPX和LY294002对芍药内酯苷促进EPO基因表达的影响，其中1：对照组，2：芍药内酯苷2μmol·L -1组，3：DPCPX 10μmol·L -1组，4：DPCPX 10μmol·L -1+芍药内酯苷2μmol·L -1组，5：LY294002 30μmol·L -1组，6：LY294002 30μmol·L -1+芍药内酯苷20μmol·L -1组；
图5示出SCH58261和CPA对芍药内酯苷促进EPO基因表达的影响，其中1：对照组，2：芍药内酯苷2μmol·L -1组，3：SCH58261 0.2μmol·L -1组，4：SCH58261 0.2μmol·L -1+芍药内酯苷2μmol·L -1组，5：CPA 10μmol·L -1组，6：CPA 10μmol·L -1组+芍药内酯苷20μmol·L -1组；
图6示出不同剂量的芍药内酯苷(0.02，0.2，2，20μmol·L-1)在常氧培养36 h后抑制HepG 2细胞中EPO基因表达的结果，其中1：对照组，2：芍药内酯苷0.2μmol·L -1组，3：芍药内酯苷2μmol·L -1组，4：芍药内酯苷20μmol·L -1组；
图7示出不同剂量的芍药内酯苷在低氧条件下对HepG2细胞中EPO蛋白表达的影响，其中1：对照组，2：芍药内酯苷0.02μmol·L -1组，3：芍药内酯苷0.2μmol·L -1组，4：芍药内酯苷2μmol·L -1组，5：芍药内酯苷20μmol·L -1组；
图8示出不同剂量(0.02、0.2、2、20μmol·L -1)的芍药内酯苷在常氧培养36h后抑制HepG 2细胞中EPO蛋白表达的结果，其中1：对照组，2：芍药内酯苷0.02μmol·L -1组，3：芍药内酯苷0.2μmol·L -1组，4：芍药内酯苷2μmol·L -1组，5：芍药内酯苷20μmol·L -1组；
图9示出为低氧对腺苷A2受体表达的影响，其中1：常氧条件组，2：低氧8小时组，3：低氧16小时组，4：低氧24小时组。
具体实施方式
下面结合具体实施案例，进一步阐释本发明。但下述实施案例仅限于说明本发明，而不是用于限制本发明的范围。
申请人通过对放射线致贫血小鼠骨髓EPO基因表达的影响，发现芍药内酯苷及含有芍药内酯苷的提取物，具有生血作用，能够促进骨髓EPO基因的表达，治疗肾性贫血。
实施例1：芍药内酯苷对放射造模贫血小鼠骨髓EPO和G-CSF基因的 升高作用
1.1药物
芍药内酯苷由北京欧纳尔生物工程技术有限公司提供。按照人用药剂量折合，确定小鼠芍药内酯苷每天用量14mg.kg-1。用水配置芍药内酯苷药液，浓度为1.4mg.ml-1。
1.2主要试剂与仪器
AWV逆转录酶、AMV/rfl 5×反应缓冲液、Taq DNA合成酶、RNaSe抑制剂等购自Promega公司，TRIZOL、Oligo dT、dNTP等购自Gibco公司，
EPO、IL-3及L-谷氨酰胺购自Sigma公司，甲基纤维素购自Whatman公司，IL-11购自重庆多泰产品，G-CSF为日本麒麟株氏会社产品，DU640核酸蛋白紫外分析仪购自Beckman公司，Gel Doc 1000凝胶成像仪为 BIo-RAD产品，DNA合成仪为PE公司产品，Sysmex-820自动血细胞计数仪购自日本。
1.3动物
C57BL/6J小鼠，雌性，60只，6～8周龄，体重20±2g，购自北京维通利华实验动物中心。按照实验方案随机分为三组：正常对照组、模型对照组、芍药内酯苷组。每组20只。
2方法
2.1动物造模及给药
小鼠常规饲养数天适应环境后，模型对照组和芍药内酯苷采用 60COγ射线全身1次照射，照射剂量3.5Gy，剂量率1.60Gy.min -1，制成血虚证模型。照射后小鼠立即灌胃给药，芍药内酯苷组每次0.2ml，每日1次；正常对照组和模型对照组灌以等量生理盐水，连续灌胃7d。
2.2外周血象检测
每组分别取10只小鼠，于造模后第3，5，7，10，13天，分别由尾静脉取血20μl，检测外周血白细胞。
2.3造血祖细胞培养
造模后第7天，每组分别取4只小鼠，颈椎脱臼处死，无菌取骨髓，进行集落培养，观察并计数粒细胞一巨噬细胞集落形成单位(CFU-GM)、爆裂型红细胞集落形成单位(BFU-E)、红细胞集落形成单位(CFU-E)、混合集落形成单位(CFU-mix)。
2.4逆转录-聚合酶链反应(RT-PCR)
2.4.1骨髓细胞总RNA提取
最后一次灌胃24h后，每组分别取6只小鼠，颈椎脱臼处死，取股骨骨髓。用无RNA酶的磷酸盐缓冲液洗涤骨髓，加入1ml TRIZOL试剂，室温放置5min，加入200μl氯仿，剧烈振摇15sec，室温放置3min，4℃、
11 990r·min -1、离心15min，取上层水相，加入500μl异丙醇，室温放置10min，4℃、11 990r·min -1、离心10min，弃上清，沉淀用75％乙醇洗涤，干燥后用DEPC处理水20μl溶解，测定RNA纯度和浓度。
2.4.2骨髓EpO和GCSF RT-PCR
逆转录：取总RNA 2.5μg，1μg·ml -1oligo dT2.5μl，用DEPC处理水补足体积至12.5μl，70℃水浴60min，冰浴放置5min，加至反应体系(含5×AMV/Tfl反应缓冲液10μl，25mmol.L -1MgCl 210μl，10mmol1.L -1dNTP 2.5μl，RNaSe抑制剂1.25μl，AMV 2.5μl用DEPC处理水补足体积至37.5μl)，室温 放置5min，42℃水浴62min，70℃水浴15min。
聚合酶链反应：20μl反应体系，含逆转录产物1μl，PCR-mix 15μl，Taq DNA合成酶0.2μl，振荡混匀，上机反应，以β-actin为内参照。PCR参数：94℃5min；94℃变性30s、55℃复性30s、72℃延伸30s共30个循环；72℃5min。
Epo引物序列：上游引物SEQ ID NO:1
5’AAAATGTCAC-GATGGGTTG3’，
下游引物SEQ ID NO:2 5’GAGTGTTCG-GAGTGTAGC 3’，扩增产物长度332bp。
G-CSF引物序列：上游引物SEQ ID NO:3
5’AGGGAAGGAGATGGTAA-AT 3’，
下游引物SEQ ID NO:4 5’TGGAGGGTGAGGGTGGAT3’，扩增产物长度428bp。
β-actin引物序列：上游引物SEQ ID NO:5
5’ATCGTGCGTGACATCAAAGA 3’，
下游引物SEQ ID NO:6 5’AGAAGGAAGGCTGGAAAAGA 3’，
扩增产物长度178bp。
琼脂糖凝胶电泳，取5μl PCR产物，在1.5％的琼脂糖凝胶上150V电泳20min，溴化乙锭染色。
2.5凝胶扫描分析及统计处理
琼脂糖凝胶扫描拍照，图像分析。外周血白细胞计数和造血祖细胞集落数以均数±标准差表示，统计分析采用t检验。
3.结果
3.1芍药内酯苷对贫血小鼠外周血白细胞数量的影响
造模后第3，5，7，10，13天对模型组及芍药内酯苷组小鼠外周血白细胞计数，结果见表1。
表1芍药内酯苷对贫血小鼠外周白细胞数量的影响
Figure PCTCN2020130437-appb-000002
Figure PCTCN2020130437-appb-000003
与模型组比较*P<0.05，**P<0.01，***P<0.001
由表1可见，与正常组相比，造模后小鼠外周血白细胞计数显著降低(P<0.001)。造模后第5，10，13天芍药内酯苷组与模型组无显著差别；造模后第3，7天与模型组相比，芍药内酯苷组小鼠外周血白细胞计数显著升高(P<0.05)。
3.2芍药内酯苷对贫血小鼠骨髓造血祖细胞增殖的影响
各组小鼠骨髓CFU-GM、CFU-E、BFU-E、CFU-miX集落数见表2。
表2芍药内酯苷对小鼠造血祖细胞增殖的影响
Figure PCTCN2020130437-appb-000004
Figure PCTCN2020130437-appb-000005
与模型组比较**P<0.01，***P<0.001
由表2可见，与正常组相比，造模后小鼠骨髓CFU-GM、CFU-E、BFU-E、CFU-mix集落数显著降低(P<0.001)，芍药内酯苷可显著升高贫血小鼠骨髓各种造血祖细胞集落数(P<0.01，P<0.001)。
3.3芍药内酯苷对贫血小鼠骨髓Epo基因表达的影响
各组小鼠骨髓Epo mRNA的RT-PCR产物琼脂糖凝胶电泳结果表明，与正常组相比，模型组骨髓Epo mR-NA表达减少，芍药内酯苷组则使其回升，见图1。
3.4芍药内酯苷对贫血小鼠骨髓G-CSF基因表达的影响
各组小鼠骨髓G-CSF mRNA的RT-PCR产物琼脂糖凝胶电泳结果表明，与正常组相比，模型组骨髓G-CSF表达上调，芍药内酯苷组则进一步上调，见图2。
实施例2：芍药内酯苷通过激活腺苷A1、A2a受体促进低氧条件下EPO 基因表达
1材料与方法
1.1药品与试剂
HepG2细胞购自上海纪宁实业有限公司，DMEM培养基、胰酶和TRzol 购自Invitrogen公司，胎牛血清购自杭州四季青公司，PI-3kinase通路抑制剂(LY294002)、腺苷A1受体拮抗剂、腺苷A1受体激动剂和腺苷A2a受体拮抗剂均购自Sigma公司，芍药内酯苷由北京欧纳尔生物工程技术有限公司提供，HotStartDNA聚合酶购自天根公司，AMV反转录酶购自TaKaRa公司，EPO抗体购自R&D公司，Tubulin购自中杉金桥公司的SantaCruz分装产品。二抗购自中杉金桥公司。
1.2仪器
低氧培养箱为Thermo Forma公司产品，Western blot电泳及转膜仪为Bio-Rad公司产品,PCR仪为PERK N ELMER公司产品。
1.3方法
1.3.1 HepG2细胞培养及处理
使用10％的胎牛血清的DMEM培养液，37℃下置于5％的CO 2培养箱中，待细胞长至80％去掉培养液，加入含药的无血清DMEM培养基，置于普通培养箱培养12h后放入低氧培养箱3％O 2，分别于8，16，24h后取出。
实验分组:
1.按不同浓度分别加入0、0.02、0.2、2、20μmol·L -1芍药内酯苷；
2.分别加入0、2μmol·L -1芍药内酯苷、10μmol·L -1DPCPX、10μmol·L -1DPCPX+2μmol·L -1芍药内酯苷、30μmol·L -1LY294002、30μmol·L -1LY294002+2μmol·L -1芍药内酯苷；
3.分别加入0、2μmol·L -1芍药内酯苷、0.2μmol·L -1SCH58261、0.2mol·L -1SCH58261+2μmol·L -1芍药内酯苷、10μmol·L -1CPA、10μmol·L -1CPA+2μmol·L -1芍药内酯苷。
1.3.2 RT-PCR方法研究EPO基因的变化
从培养箱中取出细胞倒掉上清，加入TRzol，按说明书的方法提取总RNA，用紫外分光光度计测总RNA浓度及纯度。按照TaKaRa公司AMV反转录酶说明书进行反转录反应。得到反转录产物后按天根公司Hot Start DNA聚合酶说明书进行PCR扩增目的基因。根据Gen-Bank公布的EPO、腺苷A2受体和β-actin全长mRNA序列，利用生物软件Primer5.0设计引物:
EPO上游引物
SEQ ID NO:7 5′-CTCCCTCACCAACATTGCTT-3′，
下游引物
SEQ ID NO:8 5′-ATGGTAGGTGCGAAAACAGG-3′扩增长度300bp；
腺苷A2a受体上游引物
SEQ ID NO:9 5′-CTTGGGTTCTGAGGAAGCAG-3′，
下游引物SEQ ID NO:10 5′-CAGCAGCTCCTGAACCCTAG-3
′扩增长度253bp；
内参β-actin的引物上游引物SEQ ID NO:11
5′-CATCCTGCGTCTGGACCTGGCTGGCCGGGA-3′
下游引物SEQ ID NO:12
5′-CTAGAAGCATTTGCGGTGGACGATGGAGGGGC-3′
扩增长度607bp；
PCR产物进行琼脂糖凝胶电泳，结果用Gel-Pro软件分析测定目的条带的积分光密度与β-actin的积分光密度的比值作为EPO基因的相对表达量,每个实验组重复3次计算统计量。
1.3.3 Western blot鉴定细胞EPO表达情况
将细胞从培养箱中取出后上清倒掉，用PBS冲洗两次，按每2×10 6个细胞加10ml·L -1裂解液(150mmol·L -1NaCl，0.01Triton100，5mmol·L -1EDTA，0.01去氧胆酸，0.001SDS，NaF50mmol·L -1,100mmol·L -1Tris，pH＝8.0)，如果提取样品后要检测EPO的表达还应加入0.3倍裂解液体积的胰酶，冰上裂解1h后在4℃12000×g离心15min,收集上清，用Bradford′s法测定蛋白浓度后，在样品中加入上样缓冲液，并在99℃水浴中加热10min变性后分装保存于-20℃。
依据蛋白浓度测定结果，按每孔15μg上样后进行聚丙烯酰胺凝胶电泳，然后转膜。转膜后封闭1h，按1∶1 000浓度一抗4℃孵育过夜。用辣根过氧化物酶标记的羊抗兔和羊抗鼠抗体1∶3 000，孵育1h，洗膜后显影。
1.4.统计学方法
所有数据均采用x±s表示，control组与芍药内酯苷组、芍药内酯苷组与DPCPX组、芍药内酯苷组与LY294002组之间用成组t检验，用SPSS13.0
进行统计学处理。
2.结果
2.1芍药内酯苷在低氧条件下对HepG2细胞中EPO基因表达的影响
在8、16、24h，2μmol·L -1浓度的芍药内酯苷对EPO表达与对照组
相比有促进作用，24h处0.02，0.2，2，20μmol·L -1对EPO表达有促进作用，其中2μmol·L -1浓度差异有显著性P<0.05，见图3。
2.2 DPCPX和LY294002对芍药内酯苷促进EPO基因表达的影响
在8、16、24h，DPCPX和LY294002能抑制芍药内酯苷对EPO基因
表达的促进作用，差异有显著性，P<0.05，见图4。
2.3 SCH58261和CPA对芍药内酯苷促进EPO基因表达的影响
在8、16、24hSCH58261能明显抑制芍药内酯苷对EPO基因表达的促
进作用(P<0.05),CPA在8h处对EPO表达有抑制作用，在16和24h处对EPO表达没有影响，见图5。
2.4芍药内酯苷(0.02，0.2，2，20μmol·L -1)在常氧培养36h后抑制HepG2
细胞中EPO基因表达，见图6。
2.5芍药内酯苷在低氧条件下对HepG2细胞中EPO蛋白表达的影响
低氧培养12h后,0.02、0.2、20μmol·L -1浓度的芍药内酯苷对EPO表达抑制作用，而2μmol·L -1的芍药内酯苷尽管对EPO表达有一定抑制但较其它3个浓度抑制作用较小。低氧培养24h后，上述浓度芍药内酯苷对其EPO蛋白的表达没有影响，见图7。
2.6不同剂量(0.02，0.2，2，20μmol·L-1)的芍药内酯苷在常氧培养36h后抑制HepG2细胞中EPO蛋白表达，见图8。
2.7低氧对腺苷A2受体表达的影响
低氧培养8、16、24h后能促进HepG2细胞中腺苷A2受体的表达，见图9。
结论：芍药内酯苷可以通过激活腺苷A1、A2受体促进低氧条件下EPO基因的表达，治疗肾性贫血。
实施例3：芍药内酯苷、芍药苷对腺苷A1、A2受体亲和力的研究
1.实验材料和试剂
1.1实验材料：
从大鼠的海马以及稳定转染的细胞系中提取的各种不同受体膜蛋白。
高速冷冻离心机，H1ACH1 20PR-5。
超速冷冻离心机，H1ACH1 SCP85H。
匀浆器，ULTRA-TURRAXT25。
UV-250紫外分光光度计，日本岛津公司。
多头抽滤器，绍兴仪器设备公司。
49型玻璃纤维滤膜，上海羽光净化材料联合公司。
LS6500型液闪计数仪，Beckman公司。
培养皿，12孔板，96孔板，Corning公司
1.2试剂
试验中所用标记配体为PE公司产品。试验中所用非标记配体为Sigma
公司产品。Methyllycaconitine(MLA)、Polyethyleneimine(PEI)、牛血清白蛋白(BSA)、PMSF、蛋白酶抑制剂为Sigma公司产品。闪烁液为PE公司产品。Folin-酚试剂为华威科仪公司产品。Tris-HCl缓冲液(50mM Tris-HCl Buffer，1mM EDTA，5mM MgCl2，1mM PMSF，0.1％.NaN3，3μg/ml蛋白酶抑制剂，pH7.4)。其他试剂均为分析纯。
2.实验方法
芍药苷(10uM)和芍药内酯苷(10uM)与表中所示受体和放射性配体的
结合的竞争抑制试验
在25℃的反应条件中摆放试管。所有试管中依次加入从大鼠海马或者其他脑区中提取的100μg受体蛋白量。非特异结合管中加入50μl(10-4M)的相对应的非标记性配体,终浓度为10uM，预先反应30min。测试管中依次加入30μl受试药物芍药苷和芍药内酯苷。全部试管依次加入40μl[3H]-标记性化合物，标记配体终浓度为如上表所示。以Tris-HCl缓冲液(50mM Tris-HCl，1mM EDTA，5mM MgCl2，0.1mM PMSF，0.1％NaN3，pH7.4)补足所有反应管体积为300μl。在25℃反应1小时。然后点样于49型玻璃纤维滤纸上，经负压抽滤，再用冰冷的Tris-HCl缓冲液(50mM Tris-HCl Buffer，1mM EDTA，5mM MgCl2，1mM PMSF，0.1％.NaN3，3μg/ml蛋白酶抑制剂，pH7.4)洗涤，每次2ml，共3次，抽干滤纸滤纸取出烘干后，放在闪烁瓶中，加1ml闪烁液，用液闪计数器测定放射性强度。
根据如下公式测出I％：
Figure PCTCN2020130437-appb-000006
3.实验结果
芍药苷和芍药内酯苷对上述几种受体配体的竞争抑制力如下表所示：
Figure PCTCN2020130437-appb-000007
由以上结果可以看出：芍药内酯苷和芍药苷对腺苷A1和A2受体具有显著亲和力。
尽管以上已经对本发明作了详细描述，但是本领域技术人员理解，在不偏离本发明的精神和范围的前提下可以对本发明进行各种修改和改变。本发明的权利范围并不限于上文所作的详细描述，所述修改和改变应归属于权利要求书的范围。

Claims (6)

  1. 芍药内酯苷、芍药苷或含有芍药内酯苷或芍药苷的组合物在制备用于增加内源性促红细胞生成素(EPO)的药品、保健品、食品营养剂或食品添加剂中的用途；
    优选地，所述含有芍药内酯苷或芍药苷的组合物为含有芍药内脂苷或芍药苷提取物，更优选地为含有芍药内酯苷或芍药苷的白芍提取物。
  2. 芍药内酯苷、芍药苷或含有芍药内酯苷或芍药苷的组合物在制备用于预防和/或治疗原发性贫血、继发性贫血或肾性贫血的药品、保健品、食品营养剂或食品添加剂中的用途；
    优选地，所述原发性贫血为原发性骨髓疾患的贫血；
    优选地，所述继发性贫血为再生障碍性贫血，或者骨髓增生性和骨髓发育不良性疾病、多发性骨髓瘤、慢性炎症、艾滋病或肿瘤引起的继发性贫血；
    优选地，所述肾性贫血为糖尿病并发的肾性贫血；
    优选地，所述含有芍药内酯苷或芍药苷的组合物为含有芍药内脂苷或芍药苷的提取物，更优选地为含有芍药内酯苷或芍药苷的白芍提取物。
  3. 芍药内酯苷、芍药苷或含有芍药内酯苷或芍药苷的组合物在制备用于通过上调内源性促红细胞生成素(EPO)预防和/或治疗原发性贫血、继发性贫血或肾性贫血的药品、保健品、食品营养剂或食品添加剂中的用途；
    优选地，所述原发性贫血为原发性骨髓疾患的贫血；
    优选地，所述继发性贫血为再生障碍性贫血，或者骨髓增生性和骨髓发育不良性疾病、多发性骨髓瘤、慢性炎症、艾滋病或肿瘤引起的继发性贫血；
    优选地，所述肾性贫血为糖尿病并发的肾性贫血；
    优选地，所述含有芍药内酯苷或芍药苷的组合物为含有芍药内脂苷或芍药苷提取物，更优选地为含有芍药内酯苷或芍药苷的白芍提取物。
  4. 一种增加内源性促红细胞生成素(EPO)的方法，所述方法包括给予需要其的受试者有效量的芍药内酯苷、芍药苷或含有芍药内酯苷或芍药苷的组合物；
    优选地，所述含有芍药内酯苷或芍药苷的组合物为含有芍药内脂苷或芍药苷提取物，更优选地为含有芍药内酯苷或芍药苷的白芍提取物。
  5. 一种用于预防和/或治疗原发性贫血、继发性贫血或肾性贫血的方法，所述方法包括给予需要其的受试者有效量的芍药内酯苷、芍药苷或含有芍药内酯苷或芍药苷的组合物；
    优选地，所述原发性贫血为原发性骨髓疾患的贫血；
    优选地，所述继发性贫血为再生障碍性贫血，或者骨髓增生性和骨髓发育不良性疾病、多发性骨髓瘤、慢性炎症、艾滋病或肿瘤引起的继发性贫血；
    优选地，所述肾性贫血为糖尿病并发的肾性贫血；
    优选地，所述含有芍药内酯苷或芍药苷的组合物为含有芍药内脂苷或芍药苷提取物，更优选地为含有芍药内酯苷或芍药苷的白芍提取物。
  6. 一种通过上调内源性促红细胞生成素(EPO)预防和/或治疗原发性贫血、继发性贫血或肾性贫血的方法，所述方法包括给予需要其的受试者有效量的芍药内酯苷、芍药苷或含有芍药内酯苷或芍药苷的组合物；
    优选地，所述原发性贫血为原发性骨髓疾患的贫血；
    优选地，所述继发性贫血为再生障碍性贫血，或者骨髓增生性和骨髓发育不良性疾病、多发性骨髓瘤、慢性炎症、艾滋病或肿瘤引起的继发性贫血；
    优选地，所述肾性贫血为糖尿病并发的肾性贫血；
    优选地，所述含有芍药内酯苷或芍药苷的组合物为含有芍药内脂苷或芍药苷提取物，更优选地为含有芍药内酯苷或芍药苷的白芍提取物。
PCT/CN2020/130437 2019-11-22 2020-11-20 芍药内酯苷或芍药苷用于预防和/或治疗肾性贫血的用途 WO2021098826A1 (zh)

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