WO2014206056A1 - 柚皮素在制备预防和/或治疗腹主动脉瘤的药物中的应用 - Google Patents

柚皮素在制备预防和/或治疗腹主动脉瘤的药物中的应用 Download PDF

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WO2014206056A1
WO2014206056A1 PCT/CN2014/000093 CN2014000093W WO2014206056A1 WO 2014206056 A1 WO2014206056 A1 WO 2014206056A1 CN 2014000093 W CN2014000093 W CN 2014000093W WO 2014206056 A1 WO2014206056 A1 WO 2014206056A1
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naringenin
mice
abdominal aortic
aortic aneurysm
abdominal
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PCT/CN2014/000093
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French (fr)
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孔炜
崔庆华
刘子懿
于芳
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北京大学
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Priority to US14/901,692 priority Critical patent/US9669005B2/en
Priority to EP14818368.4A priority patent/EP3017815B1/en
Publication of WO2014206056A1 publication Critical patent/WO2014206056A1/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
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • 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
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • naringenin in the preparation of a medicament for preventing and/or treating abdominal aortic aneurysm
  • the invention relates to the use of naringenin in the pharmaceutical field.
  • Abdominal aortic aneurysm is a highly lethal cardiovascular disease. It refers to the permanent expansion of the abdominal aorta wall. If the wall expansion is greater than 50% of the diameter of the adjacent site, or the diameter exceeds 3 cm, it can be considered to have an abdominal aortic aneurysm (Johnston KW, Rutherford RB, Tilson MD, Shah DM, Hollier L, Stanley JC. Suggested standards for reporting on Subcommittee on Reporting Standards for Arterial Aneurysms, Ad Hoc Committee on Reporting Standards, Society for Vascular Surgery and North American Chapter, International Society for Cardiovascular Surgery. J Vase Surg. 1991 ; 13(3 ): 452-458).
  • the abdominal aortic aneurysm is also called "a time bomb in the human body.”
  • the disease is common in the elderly, especially men. With the increase of age, the incidence rate is gradually increased, which can be as high as 8-10%, and the mortality rate is 50%-80%.
  • deaths from ruptured abdominal aortic aneurysms rank 15th in all causes of death and 10th in causes of death in adult males.
  • its incidence has risen three-fold rapidly in the past 30 years, but the pathogenesis and risk factors of the disease are still not well understood, so there is also a lack of effective methods for early prevention, early warning and drug prevention.
  • abdominal aortic aneurysm ie when the tumor is less than 5.5 cm in diameter, the risk of tumor rupture may be less than the risk of surgery.
  • finding a method for the treatment of abdominal aortic aneurysm has become a focus and hotspot of cardiovascular research. Studies have shown that the pathogenesis of abdominal aortic aneurysm is mainly due to oxygen free radicals in the blood vessel wall
  • ROS reactive Oxygen Species
  • MMP matrix metalloproteinase
  • statins Sudden D et al. Statins are Associated with a Reduced Infrarenal Abdominal Aortic Aneurysm Growth. Eur J Vase Endovasc Surg. 2006 32, 21 -26.
  • Vamen S et al. Randomized double-blind controlled trial of roxithromycin for prevention of abdominal aortic aneurysm expansion.
  • Br J Surg 2001 88(8): 1066-1072 adrenaline Qi block body 1 J (Gregory R et al Abdominal aortic aneurysm expansion rate:..
  • doxycycline (Baxter BT et al. Prolonged).
  • Naringenin is a natural flavonoid compound in citrus fruits such as grapefruit, grapefruit peel, orange peel and potato peel, and flavonoids in traditional Chinese medicine, such as grapefruit peel, mulberry yellow, and Chinese holly. expression.
  • naringenin can reduce blood lipids, reduce serum cholesterol, triglycerides and low-density lipoprotein (LDL-C) levels, thereby reducing the occurrence and development of atherosclerosis (Mulvihill E , Assini J, Sutherland B, DiMattia A, Khami M, Koppes J, Sawyez C, Whitman S, Huff M.
  • Naringenin Decreases Progression of Atherosclerosis by Improving Dyslipidemia. Arterioscler Thromb Vase Biol 2010, 30:742-748). It also regulates immune function and inhibits tumor growth (Lou C, Zhang F, Yang M, Zhao J, Zeng W, Fang X, Zhang Y, Zhang C, Liang W. Naringenin Decreases Invasiveness and Metastasis by Inhibiting TGF- ⁇ -Induced Epithelial to Mesenchymal Transition in Pancreatic Cancer Cells. PLoS ONE 2012;7(12). ).
  • naringenin can also treat insulin resistance and type 2 diabetes in animal models (Mulvihill EE, Allister EM, Sutherland BG, Telford DE, Sawyez CG, Edwards JY, Markle JM, Hegele RA, Huff MW. Naringenin preventing dyslipidemia, Apolipoprotein B overproduction, and hyperinsulinemia in LDL receptor-null mice with diet-induced insulin resistance. Diabetes.2009;58(10):2198-210 ) and liver disease ( Morcos PN, Brennan B, Smith PF. A grapefruit a day for patients Infected with hepatitis C Hepatology. 2008 Jun;47(6):2141 -2 ). However, the application of naringenin in the prevention and treatment of abdominal aortic aneurysm has not been reported.
  • naringenin provided by the present invention is their use in the preparation of products for the prevention and/or treatment of abdominal aortic aneurysms.
  • the product includes a drug and/or a health product.
  • the application of the naringenin may further comprise at least one of the following: 1) application in the preparation of a medicament for inhibiting rupture of the abdominal aortic elastic plate;
  • the inflammatory cells may be macrophages and/or white blood cells;
  • the inflammatory factor may be interleukin IL-6 and/or monocyte chemotactic factor MCP-1;
  • the matrix metalloproteinase may specifically be MMP-9.
  • a medicament for preventing and/or treating abdominal aortic aneurysm prepared by using naringenin as an active ingredient is also within the scope of the present invention.
  • the drug can be introduced into the body such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue by injection, jetting, nasal drops, eye drops, infiltration, absorption, physical or chemical mediated methods; or mixed or wrapped by other substances. Import the body.
  • One or more pharmaceutically acceptable carriers may also be added to the above drugs as needed.
  • the carrier includes conventional diluents, excipients, fillers, binders, humectants, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants and the like in the pharmaceutical field.
  • the medicament may be in the form of an injection, a suspension, a powder, a tablet, a granule or the like.
  • the above various dosage forms of the drug can be prepared according to a conventional method in the pharmaceutical field.
  • the present invention employs two clinically relevant abdominal aortic aneurysm disease models: Angiotensin II (1000 ng/kg/min) subcutaneously implanted to induce ApoE knockout mice; and calcium phosphate (0.5 M) smear Locally induced abdominal aortic aneurysm in C57 mice.
  • Angiotensin II 1000 ng/kg/min
  • calcium phosphate 0.5 M
  • naringenin can significantly inhibit the development of mouse abdominal aortic aneurysm induced by CaPO 4 smear and Angll buried pump, which may be used in patients with abdominal aortic aneurysm.
  • the medicine for preventing and/or treating abdominal aortic aneurysm provided by the invention has the advantages of safe and low toxicity and strong pharmacological action; the raw material source is rich, extensive and inexpensive, and can be hydrolyzed by crude naringin or monomer. Obtained, or extracted from various medicinal materials containing naringenin, or synthesized by other chemical methods; low cost, simple process, high yield; and effective curative effect, low effective dose.
  • the invention provides a new drug source for preventing, diagnosing, detecting, protecting, treating and researching abdominal aortic aneurysm diseases, and is easy to popularize and apply, and can generate huge social and economic benefits in a short period of time.
  • Figure 1 is a photograph of blood vessels in the abdominal aortic aneurysm of C57BL/6J mice treated with naringenin (NGN) and CaPO 4 smear of the inferior renal artery. Doxycycline (Dox) was used as a positive control for animal experiments.
  • NNN naringenin
  • Dox Doxycycline
  • Figure 2 shows the severity of abdominal aortic aneurysm in C57BL/6J mice treated with naringenin (NGN) by intragastric administration of CaPO 4 and smear of the inferior renal artery. Doxycycline was used as a positive control for animal experiments;
  • A is the diameter of the inferior renal artery of C57BL/6J mice and its statistics by histological staining for NaCl spray application, CaPO 4 smear, naringenin or doxycycline;
  • B is the histological staining and identification of the degree of rupture of the inferior arterial elastic plate of C57BL/6J mice by NaCl smear, CaPO 4 smear, naringenin or doxycycline.
  • Figure 3 is a naringin or doxycycline to reduce the inflammatory response of CaPO 4 coated C57BL/6J mice in the inferior renal artery;
  • A is the immunohistochemical identification of naringenin or doxycycline to reduce the infiltration of inflammatory cells (macrophages and leukocytes) in the renal blood vessel wall of C57BL/6J mice;
  • B is an immunohistochemical staining for naringenin or doxycycline to reduce the expression of inflammatory factor (interleukin IL-6/monocyte chemokine MCP-1) in the renal vascular wall of C57BL/6J mice.
  • inflammatory factor interleukin IL-6/monocyte chemokine MCP-1
  • Figure 4 shows the activity and expression of stromal metalloproteinases in the subrenal artery of C57BL/6J mice in which naringenin or doxycycline inhibits calcium phosphate application;
  • A is an in situ zymography method to verify that naringenin or doxycycline inhibits CaPO 4 -induced matrix metalloproteinase MMP activity;
  • B is a gelatin zymography method validation naringenin or doxycycline-induced inhibition of 4 CaPO MMP2 activity and MMP-9 in.
  • Figure 5 shows the effect of naringenin on the reduction of angiotensin II-induced ⁇ and rat abdominal aortic aneurysm
  • mice ⁇ The incidence of abdominal aortic aneurysm in mice ⁇ was administered by naringin.
  • Figure 6 shows the severity of angiotensin II-induced aortic aneurysm induced by angiotensin II in mice.
  • ⁇ is the histological staining to identify the maximum diameter of the renal aorta in mice with naringenin or water gavage and its statistics;
  • B For histological staining, the degree of rupture of the renal aortic elastic plate in naringenin or water-fed mice and its statistics were identified.
  • Figure 7 shows the effect of naringenin on the degree of inflammation of the renal artery wall induced by angiotensin II in ⁇ mice;
  • Intragastric administration of naringenin reduced the expression of inflammatory cytokines in the plasma of angiotensin II-induced ⁇ mice;
  • C is the naringenin to reduce the release of inflammatory factors in the blood vessel wall of mice.
  • Figure 8 shows that naringenin can reduce the activity of matrix metalloproteinase in the wall of the superior renal artery induced by angiotensin II in ⁇ mice.
  • ⁇ is an in situ zymography method to verify that naringenin inhibits angiotensin II-induced matrix metalloproteinase activity
  • B is a gelatin zymography method to verify that naringenin inhibits angiotensin II-induced MMP-2 and MMP-9 activities.
  • Figure 9 is a 12-week male C57 mouse treated with calcium phosphate for 7 days in Example 5, followed by naringenin (50 mg/kg/day) and water for 2 and 4 weeks, respectively.
  • Figure 10 is a 20-week male ApoE knockout mouse given a 28-day angiotensin II submerged pump treatment and ultrasonic examination in Example 5, and mice that have formed an aneurysm are equally divided into two groups to give naringenin or water. Feeding, and then giving 28 days of angiotensin II buried pump treatment;
  • A is a blood vessel photograph of a second angiotensin II treated mouse aneurysm
  • B is the incidence of aneurysm after a second round of angiotensin II immersion in ApoE knockout mice that have previously formed an aneurysm, relative to the number of previously established aneurysms;
  • C is a quantitative map of the maximum diameter of the abdominal aorta of mice detected by ultrasound;
  • D is the gelatin zymogram to detect the effect of naringenin on the activities of the suprarenal aorta metalloproteinases MMP-9 and MMP-2 in ApoE knockout mice; 3 in each group.
  • Example 1 Oral administration of naringenin to reduce abdominal aortic aneurysm in C57BL/6J mice with CaPO 4 smear in the inferior renal artery
  • mice Male C57BL/6J mice, 12 weeks old, were anesthetized by intraperitoneal injection of 0.1% sodium pentobarbital (injected by body weight, 6-71/g). The infrarenal abdominal aorta was isolated and the gauze (1 cm wide x 3 cm long) soaked in 0.5 M CaCl 2 solution was wrapped around the isolated sub-abdominal aorta. After 10 min, the gauze was removed and replaced with gauze soaked in PBS for 5 min. PO 4 _ 3 in CaCl 2 + PBS forms CaPO 4 crystals, which is equivalent to the application of CaPO 4 to blood vessels. The blood vessel was wrapped with gauze soaked in NaCl for 10 min, and then replaced with gauze in PBS for 5 min, as a NaCl smear control group.
  • NTN naringenin
  • Dox doxycycline
  • Doxycycline is used as a positive control.
  • mice were intragastrically administered on the first day after surgery, and the mice were given daily water, naringenin (50 mg/kg) (Sigma-Aldrich, St. Louis, MO) or doxycycline (100 mg/kg) (Sigma- Aldrich, St. Louis, MO). After 7 days of gavage, the material was taken. The mice were weighed and blood was taken from the capillary. After 6,000 rpm, after centrifugation for 10 minutes, the supernatant was taken and blood lipids were measured (the kit was purchased from Zhongshengbei Control Co., Ltd.). The mice were then irrigated with PBS. The blood vessels were separated, and four blood vessels were taken from each group at random. The result is shown in Figure 1.
  • the results showed that compared with the mice in the NaCl smear group, the blood vessels of the CaPO 4 smear had obvious bulging, and the naringin or doxycycline could reduce the incidence and extent of bulging.
  • naringenin or doxycycline can significantly increase the diameter of the subrenal blood vessels in mice (NaCl v ⁇ . CaPO 4 , 0.77 ⁇ 0.06 v ⁇ . 1.40 ⁇ 0.19 mm, R ⁇ 0.05)
  • daily injection of naringenin or doxycycline can significantly reduce the vasodilation caused by CaPO 4 application, and the effect of naringenin and doxycycline is similar (CaPO 4 + NGN ra. CaPO 4 + Dox, 1.03 ⁇ 0.18 Ra. 0.99 ⁇ 0.18 mm).
  • the data in Figure 2A is the mean ⁇ standard deviation.
  • the degree of rupture of the elastic plate is also an indicator of the severity of the abdominal aortic aneurysm.
  • Elastic board The degradation is divided into four grades. The first level refers to less than 25% degradation of the elastic sheet, the second level is 25%-50%, the third level is 50%-75%, and the fourth level is more than 75% cracking.
  • the first level refers to less than 25% degradation of the elastic sheet
  • the second level is 25%-50%
  • the third level is 50%-75%
  • the fourth level is more than 75% cracking.
  • Gomori staining to identify the rupture of the inferior abdominal aortic elastic plate in mice.
  • CaPO 4 application significantly increased the rupture of the inferior abdominal aortic elastic plate of the mouse compared with the NaCl smear, and the naringenin or doxycycline could reverse the action of CaPO 4 .
  • the data in Figure 2B is the mean ⁇ standard deviation.
  • naringenin administration did not affect the body weight and blood lipid levels of C57BL/6J mice, see Table l o
  • inflammatory cell infiltration of the vessel wall is a very common feature.
  • Mac3 is the result of immunohistochemical staining of specific antibodies against Mac3, representing macrophages
  • CD45 is the result of immunohistochemical staining of specific antibodies against CD45, representing leukocytes.
  • CaPO 4 smear can significantly increase the infiltration of macrophages and leukocytes in the outer wall of the blood vessel wall, and oral administration of naringenin or doxycycline can reduce this effect.
  • IL-6 and MCP-1 are pro-inflammatory chemokines/cytokines, mainly from the vessel wall.
  • MCP-1 mediates vascular remodeling and expansion by recruiting leukocytes and releasing more matrix metalloproteinases to mediate vascular remodeling and expansion (MacTaggart J et al. Deletion of ccr2 but not ccr5 or cxcr3 inhibits aortic aneurysm formation Surgery. 2007; 142 :284-288; Ishibashi M et al. Bone marrow-derived monocyte chemoattractant protein- 1 receptor ccr2 is critical in angiotensin ii-induced acceleration of atherosclerosis and aneurysm formation in hypercholesterolemic mice.
  • IL-6 can augment the effect of MCP-1 to aggravate the occurrence of abdominal aortic aneurysm (Tieu BC et al. An adventitial il-6/mcpl Amplification loop accelerates macrophage-mediated vascular inflammation leading to aortic dissection in mice. J Clin Invest. 2009; 1 19:3637-3651 ).
  • tissue staining to identify the expression of IL-6 and MCP-1 on the vessel wall.
  • IL-6 and MCP-1 are immunohistochemical results for anti-IL-6 and MCP-1 specific antibodies, respectively (purchased from Abeam, Cambridge, UK). The result is shown in Figure 3B. CaPO 4 smear can significantly increase the expression of IL-6 and MCP-1 in the outer membrane of the vessel wall, while naringenin or doxycycline can reduce their expression.
  • the freshly removed mouse abdominal aorta was cleared of blood vessels in the inferior part of the aorta.
  • the blood vessel was cut into a lcm ring and placed in a 24-well plate.
  • the blood vessels were cultured for 24 hours in serum-free 0.5 ml high glucose medium.
  • protein-loading buffer containing no mercaptoethanol was added and frozen at -80 °C.
  • Gelatin Sigma-Aldrich, St. Louis, MO
  • Electrophoresis was carried out at a voltage of 80 V at 4 °C.
  • the gel was placed in a 0.25% Trixon-100 solution and washed twice in 30 minutes. It was then added to the incubation solution (50 mM Tris, 0.2 M NaCl, 5 mM CaCl 2 , 0.02% sodium azide) and incubated at 37 ° C for 24-48 hours.
  • the incubated glue is placed in Coomassie Brilliant Blue for 1-2 hours at room temperature, using methanol 30%, glacial acetic acid 10% solution, methanol 20%, glacial acetic acid 10% solution and methanol 10%, glacial acetic acid 5%.
  • MMP-2 and MMP-9 are gelatinases that are capable of degrading Glue, the place where gelatin is degraded is white, and the rest is still black. Normally, MMP-2 and MMP-9 are in the form of a zymogen, and when activated, the zymogen is cleaved into an active protease, so both MMP-2 and MMP-9 are two white strips. Band, the above are all zymogen forms, the following are active metalloproteinases. MMP-2 or MMP-9, either active or in zymogen form, can degrade gelatin.
  • mice After 28 days of gavage, the material was taken. The mice were measured for blood pressure (Beijing Soft Biotechnology Co., Ltd.) using a BP-89A sphygmomanometer using a tail sleeve method and weighed. The blood was taken from the mice, the plasma was 6,000 rpm, and the supernatant was taken after centrifugation for 10 minutes to measure the blood lipid level of the mice. Naringenin reduced the levels of cholesterol, triglycerides and low-density lipoprotein in mouse plasma, consistent with previous reports, but it did not affect the blood pressure and body weight of mice, as shown in Table 2.
  • the aorta of the mouse was fixed and isolated.
  • the Angll group was an angiotensin II pump, a control group for irrigation.
  • the Angll + NGN group was an angiotensin II buried pump, and the naringenin group was administered.
  • Four blood vessels were taken from each group at random. As a result, as shown in Fig. 5A, the first two blood vessels of the Angll group had obvious bulging in the upper part of the kidney, and more than 50% of the diameter of the adjacent blood vessel formed an aneurysm.
  • naringenin-fed group only the first blood vessel had an aneurysm formation in the upper part of the kidney, and the other three did not. This indicated that naringenin can reduce the formation of abdominal aortic aneurysm induced by angiotensin II.
  • mice In the angiotensin II-embedded, water-immersed control group, 16 mice, one of which died during the first seven days of burying the pump due to rupture of the aortic dissection, and another one after burying the pump for one week, because the abdomen The aneurysm ruptured and died. Of the remaining 14 mice, 6 had abdominal aortic aneurysm on the 28th day of the buried pump, so 8 mice in the control group of angiotensin II immersed pump occurred in 8 mice. The incidence of aortic aneurysm, the aortic aneurysm, is 50%.
  • angiotensin II pump In the angiotensin II pump, one of the 15 mice in the naringenin group died due to the rupture of the aortic aneurysm, and only one of the remaining abdominal aortic aneurysms occurred 28 days after the pump was buried. Angiotensin II buried pump, only 15 of the 15 mice in the naringenin perfusion group had an aneurysm, that is, the incidence of aortic aneurysm was 13.3%. This indicates that naringenin can significantly reduce the occurrence of angiotensin II-mediated aortic aneurysm in mice. See Table 3. The statistics of the occurrence of arteriomas are shown in Figure 5B.
  • naringenin can reduce the severity of aortic aneurysm
  • the superior renal artery from the diaphragm to the right renal artery was divided into two segments, embedded in OCT and placed in liquid nitrogen. Frozen in seconds. Serial sections were made, each slice was 6 ⁇ thick, 300 ⁇ intervals, and each mouse was subjected to 16 consecutive slices.
  • the diameter of the abdominal aorta of the mouse was determined by ⁇ . ⁇ . staining, and the measurement was straight. The diameter, and the largest diameter is selected from it. The maximum diameter is compared with the diameter of the adjacent site. If it exceeds 50% of the adjacent site, the abdominal aortic aneurysm is considered to have occurred in the mouse.
  • Figure 6A shows the maximum diameter of the suprarenal abdominal aorta in surviving mice after angiotensin II immersion pump, with a population of 11 mice per group. The results showed that the maximum diameter of the abdominal aorta in mice was significantly reduced after feeding naringenin.
  • naringenin can significantly reduce the occurrence and severity of angiotensin II-mediated abdominal aortic aneurysm in mice.
  • Naringenin feeding can reduce angiotensin II-mediated vascular inflammation in mice
  • Naringenin inhibits the infiltration of inflammatory cells in the blood vessel wall
  • CD45 and Mac3 were the results of immunofluorescence staining with anti-CD45 specific antibodies and anti-Mac3 specific antibodies, respectively.
  • naringenin gavage significantly reduced the infiltration of inflammatory cells (macrophages and leukocytes) in the outer membrane as compared with the control group.
  • Naringenin reduces plasma levels of IL-6 and MCP-1
  • Naringenin reduces the expression of IL-6 and MCP-1 in the blood vessel wall
  • Example 5 Effect of naringenin on the progression and outcome of abdominal aortic aneurysm that has occurred using a mouse calcium phosphate smear model and an angiotensin II induction model to investigate whether naringenin has an established aneurysm effect.
  • angiotensin II subcutaneous pump for 28 days (1,000 ng/kg/min) to induce abdominal aortic aneurysm in 20-week male ApoE knockout mice (40 in total) and use ultrasound for in vivo detection.
  • the diameter of the superior aorta of the kidney As a standard for the occurrence of aortic aneurysm with a diameter greater than 50% of the diameter of the adjacent vessel, 28 mice developed an aneurysm after 28 days.
  • Using a similar maximum abdominal aortic diameter as the grouping criteria we divided the aortic aneurysm (28) and the non-aortic aneurysm (12) into two groups, with the average maximum abdominal aorta diameter equivalent.
  • mice One group was given naringenin and the other group was given the same amount of water. All mice continued to receive angiotensin II for 28 days. As observed in the calcium phosphate model, in mice that had not previously had an aneurysm, only one of the six mice fed naringenin developed an aneurysm after 28 days (incidence rate 16%). In the control group, 3 mice formed an aneurysm (50% incidence). In the mice that had previously formed an aneurysm, 4 of the 14 mice that were fed with water died of aneurysm rupture, and 10 of the surviving mice developed severe aneurysms (mean maximum diameter reached 3.00 ⁇ 0.58). Mm).
  • naringenin can not only inhibit the formation of abdominal aortic aneurysm, but also reverse the established aortic aneurysm to play a therapeutic role.
  • the medicine for preventing and/or treating abdominal aortic aneurysm provided by the invention has the advantages of safe and low toxicity and strong pharmacological action; the raw material source is rich, extensive and inexpensive, and can be hydrolyzed by crude naringin or monomer. Obtained, or extracted from various medicinal materials containing naringenin, or synthesized by other chemical methods; low cost, simple process, high yield; and effective curative effect, low effective dose.
  • the invention provides a new source of medicine for preventing, diagnosing, detecting, protecting, treating and studying abdominal active bronchial diseases, and is easy to popularize and apply, and can generate enormous social and economic benefits in a short period of time.

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Abstract

本发明公开了一种柚皮素的药物新用途。本发明所公开的新用途是柚皮素在制备预防和/或治疗腹主动脉瘤的产品中的应用。所述产品包括药物和/或保健品。本发明采用两种与临床相关的腹主动脉瘤疾病模型：血管紧张素II（1000ng/kg/min）皮下埋泵诱导ApoE基因敲除小鼠和磷酸钙（0.5M）涂抹腹主动脉局部诱导的C57小鼠腹主动脉瘤。实验证明，将柚皮素口服灌胃，能明显抑制CaPO4涂抹和Angll埋泵诱导的小鼠腹主动脉瘤的发生发展，可用于治疗腹主动脉瘤的病人。

Description

柚皮素在制备预防和 /或治疗腹主动脉瘤的药物中的应用 技术领域
本发明涉及柚皮素在制药领域中的应用。
背景技术
腹主动脉瘤 （Abdominal aortic aneurysm, AAA ) 是一种高致死性的 心血管疾病。 它是指腹主动脉壁的永久扩张， 如果管壁扩张大于临近部位 直径的 50%， 或者直径超过 3cm， 就可以认为患有腹主动脉瘤 （Johnston KW, Rutherford RB, Tilson MD, Shah DM, Hollier L， Stanley JC. Suggested standards for reporting on Subcommittee on Reporting Standards for Arterial Aneurysms, Ad Hoc Committee on Reporting Standards, Society for Vascular Surgery and North American Chapter, International Society for Cardiovascular Surgery. J Vase Surg. 1991 ; 13(3):452-458 ) 。 瘤体一旦破裂， 病人将因大量 失血而死亡， 因此腹主动脉瘤又被称为 "人体内的定时炸弹" 。 此病常见 于老年人， 尤其是男性， 随着年龄的增长发病率逐渐上升， 可高达 8- 10% , 致死率更是达到了 50%-80%。 在美国， 由腹主动脉瘤破裂导致的死 亡分别排在所有死亡原因的第十五位和成年男性疾病死亡原因的第十位。 而在中国， 近 30年以来， 其发病率迅速上升了 3倍， 但是关于该病的发病 机制和危险因素至今尚不十分清楚， 因此也缺乏早期预防、 早期预警和药 物防治的有效方法。 自从 1951年 DuBost首次成功地实施了腹主动脉瘤切 除术和人造血管移植术后 （DuBost C, Allary M， Oeconomos N. Resection of an aneurysm of the abdominal aorta: reestablishment of the continuity by a preserved human arterial graft, with result after five months. AM A Arch Surg. 1952 Mar;64(3):405-8 ) ， 随着技术的不断发展和改进， 血管修补术已经在 世界范围内被广泛地接受和使用， 成为了目前治疗腹主动脉瘤最为常见的 方法。 但是， 手术的缺点也很多， 譬如风险大， 费用高， 不能治本， 难以 普及。 且对于小动脉瘤 （small abdominal aortic aneurysm ) 的患者， 即瘤 体直径小于 5.5cm时， 瘤体破裂的风险可能小于手术的风险。 可是目前没 有有效的药物能够防治腹主动脉瘤， 因此寻找治疗腹主动脉瘤的防治方法 成为现在心血管研究的一个重点和热点。 研究显示腹主动脉瘤的发病机制主要是由于血管壁的氧自由基
(Reactive Oxygen Species, ROS ) 升高、 炎症细胞的浸润和炎症因子的释 放增多、 基质金属蛋白酶 （ Matrix Metalloproteinase， MMP ) 的活性升 高， 使血管壁的炎症反应和基质的降解显著性地增加， 从而导致腹主动脉 瘤的发生 ( Daugherty A, Cassis LA. Mouse models of abdominal aortic aneurysms. Arterioscler Thromb Vase Biol. 2004 Mar;24(3):429-34 ) 。 这就 提示如果使用抑制血管炎症反应或减少基质的降解的药物， 就可能减轻或 治疗腹主动脉瘤的发生发展。 目前， 尚缺乏明确的大规模临床随机对照研 究支持的药物治疗方案。 但曾有一些药物用于治疗腹主动脉瘤的小规模临 床试验， 如他汀类药物 ( Schouten D et al. Statins are Associated with a Reduced Infrarenal Abdominal Aortic Aneurysm Growth. Eur J Vase Endovasc Surg.2006 32, 21-26. ) ， 大环内酯类抗菌素 （Vammen S et al. Randomized double-blind controlled trial of roxithromycin for prevention of abdominal aortic aneurysm expansion. Br J Surg 2001 88(8): 1066—1072 ) ， 肾上腺素受体阻断齐1 J ( Gregory R et al. Abdominal aortic aneurysm expansion rate: Effect of size and beta-adrenergic blockade. J Vase Surg.1994 Apr; 19(4):727-31 ) 和强力霉素 ( Mosorin M et al. Use of doxycycline to decrease the growth rate of abdominal aortic aneurysms: a randomized, double-blind, placebo-controlled pilot study. J Vase Surg 2001 34(4):606- 610 ) 等， 都对患有腹主动脉瘤的病人有一定的治疗作用。 此外， 前列腺 素合成酶的抑制齐1 J(Walton LJ et al. Inhibition of prostaglandin E2 synthesis in abdominal aortic aneurysms: implications for smooth muscle cell viability, inflammatory processes, and the expansion of abdominal aortic aneurysms .Circulation.1999 100(1):48- 54)和抗炎药吲哚美辛 （Holmes DR et al. Indomethacin prevents elastase-induced abdominal aortic aneurysms in the rat. J Surg Res. 1996 Jun;63(l):305-9. ) 也在诱导腹主动脉瘤的动物模型 中取得了一定的效果。 这些药物大多都能降低血管的炎症反应， 或是抑制 金属蛋白酶的活性， 从而发挥作用。 但是， 上述药物均缺乏大规模随机对 照的临床研究证据支持其有效性。 有的药物在应用于临床试验时， 也产生 了较为频发的副作用， 如强力霉素 （ Baxter BT et al. Prolonged administration of doxycycline in patients with small asymptomatic abdominal aortic aneurysms: report of a prospective (Phase II) multicenter study. J Vase Surg. 2002 Jul;36(l): l- 12 ) 。 因此， 研发防治效果好， 副作用小的新药用 于治疗腹主动脉瘤， 将会具有巨大的临床应用价值。
柚皮素是一种天然的黄酮类化合物， 在葡萄柚、 柚子皮、 橘子皮和土 豆皮等橘柑类植物和中药中的黄酮类化合物中， 如胡柚皮、 桑黄、 华中冬 青等高表达。 在动物实验中， 已有报道， 柚皮素能够降低血脂、 减少血清 中胆固醇、 甘油三酯和低密度脂蛋白 （LDL-C ) 的水平， 从而减少动脉粥 样硬化的发生和发展 ( Mulvihill E， Assini J， Sutherland B， DiMattia A, Khami M， Koppes J, Sawyez C, Whitman S, Huff M. in High-Fat -Fed Low- Density Lipoprotein Receptor-Null Mice Naringenin Decreases Progression of Atherosclerosis by Improving Dyslipidemia. Arterioscler Thromb Vase Biol 2010, 30:742-748 ) 。 它还能调节免疫功能和抑制肿瘤生长 （Lou C, Zhang F， Yang M， Zhao J, Zeng W， Fang X, Zhang Y， Zhang C， Liang W. Naringenin Decreases Invasiveness and Metastasis by Inhibiting TGF-β- Induced Epithelial to Mesenchymal Transition in Pancreatic Cancer Cells. PLoS ONE 2012;7(12). ) 。 此外， 柚皮素也能在动物模型中治疗胰岛素抵 抗和 II型糖尿病 ( Mulvihill EE, Allister EM, Sutherland BG， Telford DE， Sawyez CG， Edwards JY， Markle JM， Hegele RA, Huff MW. Naringenin prevents dyslipidemia, apolipoprotein B overproduction, and hyperinsulinemia in LDL receptor-null mice with diet-induced insulin resistance. Diabetes.2009;58(10):2198-210 ) 和肝病 ( Morcos PN, Brennan B， Smith PF. A grapefruit a day for patients infected with hepatitis C Hepatology. 2008 Jun;47(6):2141 -2 ) 。 但是， 柚皮素在防治腹主动脉瘤发生方面的应用， 还 未见相关报道。
发明公开
本发明的目的是提供柚皮素的药物新应用。
本发明所提供的柚皮素的应用是它们在制备预防和 /或治疗腹主动脉瘤 的产品中的应用。 所述产品包括药物和 /或保健品。
更进一步的所述柚皮素的应用还可包括下述至少一种： 1 ) 在制备抑制腹主动脉弹力板破裂的药物中的应用；
2 ) 在制备抑制腹主动脉血管壁炎症细胞浸润的药物中的应用； 所述 炎症细胞可为巨噬细胞和 /或白细胞；
3 ) 在制备抑制血浆中 IL-6和 MCP-1的水平的药物中的应用；
4 ) 在制备抑制腹主动脉血管壁中炎症因子表达的药物中的应用； 所 述炎症因子可为白介素 IL-6和 /或单核细胞趋化因子 MCP-1；
5 ) 在制备抑制腹主动脉血管壁基质金属蛋白酶的活性与表达的药物 中的应用； 所述基质金属蛋白酶具体可为 MMP-9。
以柚皮素为活性成分制备的预防和 /或治疗腹主动脉瘤的药物也属于 本发明的保护范围。
所述药物可通过注射、 喷射、 滴鼻、 滴眼、 渗透、 吸收、 物理或化学 介导的方法导入机体如肌肉、 皮内、 皮下、 静脉、 粘膜组织； 或是被其他 物质混合或包裹后导入机体。
需要的时候， 在上述药物中还可以加入一种或多种药学上可接受的载 体。 所述载体包括药学领域常规的稀释剂、 赋形剂、 填充剂、 粘合剂、 湿 润剂、 崩解剂、 吸收促进剂、 表面活性剂、 吸附载体、 润滑剂等。
所述药物可以制成注射液、 悬浮剂、 粉剂、 片剂、 颗粒剂等多种形 式。 上述各种剂型的药物均可以按照药学领域的常规方法制备。
本发明采用两种与临床相关的腹主动脉瘤疾病模型： 血管紧张素 II (Angiotensin II) (1000ng/kg/min)皮下埋泵诱导 ApoE基因敲除小鼠； 和 磷酸钙 （0.5M) 涂抹腹主动脉局部诱导的 C57 小鼠腹主动脉瘤。 实验证 明， 将柚皮素口服灌胃， 能明显抑制 CaPO4涂抹和 Angll埋泵诱导的小鼠 腹主动脉瘤的发生发展， 可能应用于治疗腹主动脉瘤的病人。
本发明提供的预防和 /或治疗腹主动脉瘤的药品安全低毒， 药理作用较 强； 其原料来源丰富、 广泛、 价廉， 可以由柚皮苷 (Naringin)粗品或者单体 通过水解的方法得到， 或者从含柚皮素的各种药材中提取得到， 或者通过 其他化学方法合成得到； 治本成本低廉、 工艺简单、 得率高； 且疗效确 切， 有效剂量低。 本发明为预防、 诊断、 检测、 保护、 治疗和研究腹主动 脉瘤疾病提供了一种新的药物来源， 且容易推广应用， 能够在较短的时间 内产生巨大的社会效益和经济效益。 附图说明
图 1为柚皮素 (NGN)灌胃减少 CaPO4涂抹肾下动脉的 C57BL/6J小鼠 腹主动脉瘤发生的血管照片， 强力霉素 （Dox ) 作为动物实验的阳性对 照。
图 2为柚皮素 (NGN)灌胃减少 CaPO4涂抹肾下动脉的 C57BL/6J小鼠 腹主动脉瘤的严重程度， 强力霉素作为动物实验的阳性对照；
A为组织学染色鉴定 NaCl涂抹喂水、 CaPO4涂抹喂水、 柚皮素或强力 霉素的 C57BL/6J小鼠肾下动脉直径及其统计情况；
B为组织学染色鉴定 NaCl涂抹喂水、 CaPO4涂抹喂水、 柚皮素或强力 霉素的 C57BL/6J小鼠肾下动脉弹力板破裂程度及统计情况。
图 3为柚皮素或强力霉素减少 CaPO4涂抹的 C57BL/6J小鼠肾下动脉 血管的炎症反应；
A为免疫组化鉴定柚皮素或强力霉素灌胃减少 C57BL/6J小鼠肾下动 脉血管壁中炎症细胞 （巨噬细胞和白细胞） 的浸润；
B 为免疫组化鉴定柚皮素或强力霉素灌胃减少 C57BL/6J小鼠肾下动 脉血管壁中炎症因子 （白介素 IL-6/单核细胞趋化因子 MCP-1 ) 的表达。
图 4为柚皮素或强力霉素抑制磷酸钙涂抹的 C57BL/6J小鼠肾下动脉 血管壁基质金属蛋白酶的活性与表达；
A为原位酶谱方法验证柚皮素或强力霉素抑制 CaPO4诱导的基质金属 蛋白酶 MMP的活性；
B为明胶酶谱方法验证柚皮素或强力霉素抑制 CaPO4诱导的 MMP2和 MMP-9的活性。
图 5为柚皮素灌胃减少由血管紧张素 II诱导的 ΑροΚ 、鼠腹主动脉瘤 的发生；
Α为柚皮素灌胃的 ΑροΕ^小鼠腹主动脉瘤发生情况的血管照片；
Β为柚皮素灌胃的 ΑροΕ^小鼠腹主动脉瘤发病率的统计。
图 6为柚皮素灌胃减少血管紧张素 II诱导的 ΑροΕ^小鼠主动脉瘤的严 重程度；
Α为组织学染色鉴定柚皮素或水灌胃的小鼠肾上主动脉最大直径及其 统计； B 为组织学染色鉴定柚皮素或水喂养的小鼠肾上主动脉弹力板破裂程 度及其统计。
图 7为柚皮素灌胃减少血管紧张素 II诱导的 ΑροΕ^小鼠肾上动脉血管 壁的炎症程度；
Α为免疫组化鉴定柚皮素灌胃减少血管紧张素 II诱导的 ΑροΕ^小鼠小 鼠血管壁中炎症细胞的浸润；
Β为柚皮素灌胃减少血管紧张素 II诱导的 ΑροΕ^小鼠血浆中炎症因子 的表达；
C为柚皮素灌胃减少小鼠血管壁中炎症因子的释放。
图 8为柚皮素灌胃减少血管紧张素 II诱导的 ΑροΕ^小鼠肾上动脉血管 壁中基质金属蛋白酶的活性；
Α为原位酶谱方法验证柚皮素抑制血管紧张素 II诱导的基质金属蛋白 酶的活性；
B为明胶酶谱方法验证柚皮素抑制血管紧张素 II诱导的 MMP-2和 MMP-9的活性。
图 9为实施例 5中对 12周的雄性 C57小鼠利用磷酸钙涂抹处理 7天， 然后进行柚皮素 （50 mg/kg/day) 和水分别灌胃 2周和 4周， 利用 ex vivo 测量的腹主动脉图及其直径的统计图。
图 10为实施例 5中对 20周雄性 ApoE敲除小鼠给予 28天血管紧张素 I I埋泵处理并进行超声检测， 已形成动脉瘤的小鼠被平均分为两组给予柚 皮素或水喂养， 并再给予 28天血管紧张素 II埋泵处理；
A为第二轮血管紧张素 II处理后小鼠动脉瘤发生的血管照片；
B为先前已形成动脉瘤的 ApoE敲除小鼠在第二轮血管紧张素 II埋泵 后动脉瘤发生率， ％， 相对于先前已形成动脉瘤的小鼠数量；
C为超声检测的小鼠腹主动脉最大直径的定量图；
D为明胶酶谱检测柚皮素对 ApoE敲除小鼠肾上腹主动脉金属蛋白酶 MMP-9和 MMP-2活性的影响； 每组 3只。
实施发明的最佳方式
下面通过具体实施例对本发明进行说明， 但本发明并不局限于此。 下述实施例中所述实验方法， 如无特殊说明， 均为常规方法； 所述试 剂和生物材料， 如无特殊说明， 均可从商业途径获得。
实施例 1、 柚皮素灌胃减少 CaPO4涂抹肾下动脉的 C57BL/6J小鼠腹 主动脉瘤发生
1、 CaPO4涂抹小鼠肾下主动脉诱导腹主动脉瘤的模型
12周龄的雄性 C57BL/6J的小鼠， 腹腔注射 0.1 %的戊巴比妥钠 （按照 体重注射， 6-71/g ) 麻醉。 分离肾下腹主动脉， 将浸泡在 0.5 M CaCl2溶液 中的纱布 （1cm宽 x3cm长） 包裹住分离好的肾下腹主动脉。 10 min后， 将纱布取出， 换成浸泡在 PBS中的纱布包裹血管 5 min。 CaCl2 + PBS中的 PO4_3会形成 CaPO4结晶， 即相当于将血管涂抹 CaPO4。 用浸泡在 NaCl中 的纱布包裹血管 lOmin后， 换为 PBS中的纱布 5 min, 作为 NaCl涂抹对照 组。
2、 柚皮素 （NGN ) 或强力霉素 （Dox ) 灌胃的模型
强力霉素作为阳性对照药。
术后第一天开始灌胃， 每天给小鼠灌胃水、 柚皮素 （50 mg/kg ) ( Sigma-Aldrich, St. Louis, MO ) 或强力霉素 ( 100 mg/kg ) ( Sigma- Aldrich, St. Louis, MO ) 。 灌胃 7天后取材。 小鼠称重， 并用毛细管内眦取 血， 6000转， 10分钟离心后， 取上清， 测量血脂 （试剂盒购自中生北控公 司） 。 然后小鼠用 PBS 灌流冲洗。 分离血管， 每组随机各取四只血管照 相。 结果如图 1所示。 其中， NaCl为涂抹 NaCl组 （n=l l ) ， CaPO4为涂 抹 CaPO4加喂水组 （n=12 ) ， CaPO4+NGN为 CaPO4涂抹加灌胃柚皮素组 (n=l l)， CaPO4+Dox为 CaPO4涂抹加灌胃强力霉素组 (n=l 1)。 结果表明与 NaCl涂抹组的小鼠相比， CaPO4涂抹的小鼠肾下部位血管有明显的膨出， 而灌胃柚皮素或强力霉素能够减少膨出的发生率和程度。
将血管切片， H.E.染色鉴定小鼠肾下腹主动脉直径的变化情况。 结果 如图 2A所示， 与 NaCl涂抹组相比， CaPO4涂抹能够明显增加小鼠肾下血 管的直径 (NaCl v^. CaPO4, 0.77±0.06 v^. 1.40±0.19 mm, R<0.05 ) ， 但是每 天灌药柚皮素或强力霉素能够明显减少 CaPO4涂抹引起的血管扩张， 而且 柚皮素与强力霉素的作用效果相似 （CaPO4 + NGN ra. CaPO4 + Dox, 1.03±0.18 ra. 0.99±0.18 mm) 。 图 2A中的数据为平均值士标准差。
弹力板的破裂程度也是显示腹主动脉瘤严重程度的一个指标。 弹力板 的降解分为四个等级。 第一级是指弹力板少于 25%的降解， 第二级是 25%-50% , 第三级是 50%-75%， 而第四级是多于 75%的破裂。 我们用 Gomori染色鉴定小鼠肾下腹主动脉弹力板的破裂情况。 结果如图 2B所 示， 与 NaCl涂抹相比， CaPO4涂抹显著性地增加小鼠肾下腹主动脉弹力板 的破裂情况， 而灌胃柚皮素或强力霉素可以反转 CaPO4的作用。 图 2B 中 的数据为平均值±标准差。
此外， 柚皮素灌胃并不影响 C57BL/6J小鼠的体重和血脂水平， 见表 l o
表 1、 磷酸钙处理的 C57BL/6J小鼠的生化指标 组 只数 体重 （'克） 胆固醇 （毫摩） 甘油三酯 （'毫摩）
NaCl 11 21.U1.92 3.09±0.37 1.22±0.32
CaPO* 12 20.3±1.98 3.73±0.93 1.22±0.39
CaP04+ NGN 11 20.1±1.41 3.53±0.45 0. 7±0.22
CaP04+ Dox 11 21.0±1.89 3.49±0.64 1.07士 0.32 在主动脉瘤的发生中， 血管壁的炎症细胞浸润是很常见的特点。 我们 用组化染色鉴定浸润在血管壁的炎症细胞。 Mac3为抗 Mac3的特异性抗体 进行免疫组化染色的结果， 代表巨噬细胞， CD45为抗 CD45的特异性抗体 进行免疫组化染色的结果， 代表白细胞。 结果如图 3A所示。 CaPO4涂抹 可以明显增加血管壁中巨噬细胞和白细胞在外膜的浸润， 而灌胃柚皮素或 强力霉素均可以降低这一效果。
IL-6和 MCP-1 是促炎的趋化因子 /细胞因子， 主要来自于血管壁。 MCP-1 可以通过招募白细胞， 释放更多的基质金属蛋白酶来降解基质， 介 导了血管重塑和扩张 （MacTaggart J et al. Deletion of ccr2 but not ccr5 or cxcr3 inhibits aortic aneurysm formation Surgery. 2007; 142:284-288; Ishibashi M et al. Bone marrow-derived monocyte chemoattractant protein- 1 receptor ccr2 is critical in angiotensin ii-induced acceleration of atherosclerosis and aneurysm formation in hypercholesterolemic mice. Arterioscl throm vase biol. 2004;24:el74- 178 ) ， 而 IL-6可以扩大 MCP-1的 效应来加重腹主动脉瘤的发生 （Tieu BC et al. An adventitial il-6/mcpl amplification loop accelerates macrophage-mediated vascular inflammation leading to aortic dissection in mice. J Clin Invest. 2009; 1 19:3637-3651 ) 。 我 们用组化染色鉴定血管壁上 IL-6和 MCP-1的表达。 IL-6和 MCP-1分别为 抗 IL-6和 MCP-1特异性抗体的免疫组化结果 （购自 Abeam, Cambridge公 司， UK) 。 结果如图 3B所示。 CaPO4涂抹可以明显增加血管壁中 IL-6和 MCP-1在外膜的表达， 而灌胃柚皮素或强力霉素均能减少它们的表达。
腹主动脉瘤的发病机制之一就是基质金属蛋白酶的激活， 活性升高， 能显著增加基质的降解， 从而导致腹主动脉瘤的发生。 小鼠的主动脉用 PBS灌注后， 新鲜的组织直接用 OCT包埋， 放入液氮中冷冻， 然后进行连 续切片。 先用 PBS洗 3 min, 3 次。 用荧光标记的 DQ-明胶 -FITC底物 ( Sigma- Aldrich, St. Louis, MO ) ， 37°C孵育 1小时。 在正常情况下， 底物 的荧光标签是被包裹住的， 因此没有荧光， 当有活性的明胶酶降解了明胶 之后， 绿色的荧光标签便会暴露出来。 片子孵育后， 用 PBS洗 3 min, 3 次， 然后封片。 用免疫荧光共聚焦显微镜照射。 荧光越强， 意味着基质金 属蛋白酶的活性越强。 原位酶谱的结果如图 4A所示， CaPO4涂抹明显增 加基质金属蛋白酶的活性， 而灌胃柚皮素或强力霉素可以降低 CaPO4涂抹 所诱导的基质金属蛋白酶的活性。
3、 明胶酶谱
新鲜取下的小鼠腹主动脉的肾下部位血管清理淤血， 去掉脂肪和结缔 组织后， 将血管剪成 lcm的环， 放入 24孔板中。 用无血清的 0.5ml高糖培 养基培养血管 24小时。 将培养过的上清蛋白定量后， 加入不含有 -巯基 乙醇的蛋白上样缓冲液， -80°C冻存。 用水将明胶 （ Sigma-Aldrich, St. Louis, MO ) 配制成 lOmg/ml的浓度， 按照 1 : 10的比例加入蛋白电泳的 分离胶中， 使明胶的终浓度为 lmg/ml。 以 80V的电压 4°C电泳。 将胶放入 0.25%Trixon-100 的溶液中， 30分钟洗两次。 然后加入孵育液中 （50mM Tris, 0.2M NaCl, 5mM CaCl2, 0.02%叠氮钠） ， 37°C孵育 24-48小时。 孵育 好的胶放入考马斯亮蓝中室温染色 1-2 小时， 分别用甲醇 30%， 冰醋酸 10%的溶液， 甲醇 20%， 冰醋酸 10%的溶液和甲醇 10%， 冰醋酸 5%的溶 液各洗 30 分钟， 然后以 Odyssey 红外荧光扫描系统分析 （LI-COR Biosciences, Lincoln, NE)。 MMP-2和 MMP-9 都是明胶酶， 能够降解明 胶， 明胶被降解的地方为白色， 其余的仍为黑色。 通常情况下， MMP-2和 MMP-9是以酶原形式存在， 而当其被激活后， 酶原被切割成有活性的蛋白 酶， 因此 MMP-2和 MMP-9均为两条白色的条带， 上面的均为酶原形式， 下面的为有活性的金属蛋白酶。 无论是有活性的或者酶原形式的 MMP-2 或 MMP-9都能降解明胶， 结果如图 4B所示， 灌胃柚皮素或强力霉素可以 反转 CaPO4涂抹造成的 MMP-2和 MMP-9的升高。 实施例 2、 柚皮素减少由血管紧张素 II诱导的小鼠腹主动脉瘤的发 生。
首先 16周雄性的 ΑροΕ— Λ的小鼠随机分为两组， 分为对照组 (η=16)和 柚皮素组 (η=15)。 然后用 0.1%的戊巴比妥钠麻醉， 皮下埋入迷你泵 (model 2004, Alzet, Cupertino, CA) ， 泵中加入用生理盐水溶解的血管紧 张素 II ( Sigma- Aldrich, St. Louis, MO ) ， 使其能够以 lOOOng/kg/min的剂 量持续给药四周。 从术后第一天起， 每天给小鼠定点灌胃， 对照组灌水， 柚皮素组灌柚皮素 （50mg/kg ) 。 灌胃 28 天后取材。 小鼠用尾套法使用 BP-89A血压计测量血压 （北京软隆生物技术有限公司） 并称重。 小鼠内 眦取血， 血浆 6000转， 10分钟离心后取上清， 测量小鼠的血脂水平。 柚 皮素减少了小鼠血浆中胆固醇、 甘油三酯和低密度脂蛋白的水平， 和之前 的报道一致， 但是它并不影响小鼠的血压和体重， 见表 2。
表 2、 血管紧张素 II埋泵的 ApoE^敲除小鼠的生化指标 组 Angll Angll + NGN 只数 14 14
体重（克） 28.8±1.42 28.9±2.03 胆固醇 （'毫摩） 12.3士 1.36 8.46±2.45 * 甘油: Ξ酯 （毫摩） 1.66± 0.62 0.69± 0.35*
低密度脂蛋由 (毫克 /分升） 88.9± 45.7 44.1± 15.9*
高密度脂蛋白 （：毫克 /分升） 55,3±5.35 56.5±5.97 血压 （毫米汞柱） 131.0±16.20 136.6±14,27 固定并分离小鼠的主动脉。 Angll组为血管紧张素 II埋泵， 灌水的对 照组。 Angll + NGN组为血管紧张素 II埋泵， 灌柚皮素组。 每组随机各取 四只血管照相。 结果如图 5A所示， Angll组前两只血管在肾上部位均有明 显的膨出， 超过临近部位血管直径的 50%， 形成了动脉瘤。 而柚皮素喂养 组只有第一只血管在肾上部位有动脉瘤的形成， 其余三只均没有， 说明柚 皮素灌胃可以减少血管紧张素 II诱导的小鼠腹主动脉瘤的形成。
接着， 统计了所有的小鼠腹主动脉瘤的发生。 在血管紧张素 II埋泵， 灌水的对照组中， 16只小鼠， 其中有一只小鼠在埋泵的前七天内因主动脉 夹层破裂而死亡， 另有一只在埋泵一周后， 因为腹主动脉瘤的破裂而死 亡， 其余 14只小鼠中， 在埋泵第 28天有 6只产生腹主动脉瘤， 因此血管 紧张素 II埋泵的对照组 16只小鼠中共有 8只小鼠发生了主动脉瘤， 即主 动脉瘤的发病率为 50%。 而在血管紧张素 II埋泵， 灌注柚皮素组的 15只 小鼠中， 有一只因为主动脉瘤的破裂死亡， 剩下的在埋泵 28天后仅有一只 发生了腹主动脉瘤， 因此血管紧张素 II埋泵， 柚皮素灌注组的 15只小鼠 中， 只有两只发生了动脉瘤， 即主动脉瘤的发病率为 13.3%。 这说明柚皮 素灌胃能明显减少血管紧张素 II介导的小鼠主动脉瘤的发生。 见表 3。 动 脉瘤发生情况的统计情况如图 5B所示。
表 3、 由血管紧张素 II介导的灌注水 /柚皮素的 ApoE^小鼠主动脉瘤 的发生情况 组 只数 夹层 破裂 动脉瘤 无动脉瘤 ίύ 1 1 6 8
AngH+ Ci 15 Θ 1 1 13
实施例 3、 柚皮素能减少主动脉瘤的严重程度
小鼠在取材后， 主动脉被 4%的多聚甲醛灌注固定， 从膈肌到右侧的 肾动脉分支的肾上主动脉被平均分为两段， 用 OCT包埋后放入液氮中 30 秒冷冻。 进行连续切片， 每一切片 6 μπι 厚， 300 μπι 的间距， 每只老鼠都 进行连续的 16 张切片。 用 Η.Ε.染色确定小鼠腹主动脉的直径， 测量直 径， 并从中选出最大直径。 将最大直径和临近部位的直径做比较， 如果超 过临近部位的 50%， 就认为小鼠发生了腹主动脉瘤。 图 6A显示的是血管 紧张素 II埋泵后， 柚皮素或水灌注的存活的小鼠肾上腹主动脉的最大直 径， 每组均为 1 1只小鼠。 结果显示， 喂养柚皮素后小鼠腹主动脉的最大直 径明显降低。
我们用 Gomori染色确定了小鼠腹主动脉的弹力板破裂情况， 并进行 统计。 结果如图 6B所示， 喂养柚皮素后小鼠腹主动脉的弹力板破裂情况 减少， 每组检测的小鼠均为 6只。 图中数据为平均值 ±标准差。
这些都说明柚皮素灌胃能明显降低血管紧张素 II介导的小鼠腹主动脉 瘤的发生及严重程度。 实施例 4、 柚皮素喂养能降低血管紧张素 II介导的小鼠血管炎症反应
1.柚皮素抑制血管壁炎症细胞的浸润
CD45、 Mac3分别为以抗 CD45特异性抗体和抗 Mac3特异性抗体进 行免疫荧光染色的结果。 结果如图 7A所示， 与灌水的对照组相比， 柚皮 素灌胃可以显著性地减少炎症细胞 （巨噬细胞和白细胞） 在外膜的浸润。
2.柚皮素减少血浆中 IL-6和 MCP-1的水平
小鼠摘眼球取血， 血中加入 20μ1肝素， 在 6000转， 4°C离心 10分钟 后吸取上清。 取 ΙΟμΙ上清， 加入 ΙΟμΙ珠子， 在加入 ΙΟμΙ ΡΕ， 室温孵育 2 小时后， 用洗液洗两次， 再加入 200μ1洗液。 混匀后上流式进行分选。 (其中小鼠炎症因子的流式试剂盒购自 BD公司） 结果如图 7Β所示， 灌注 柚皮素后， 血浆中 IL-6 和 MCP-1 的水平被明显的降低了 （η=1 1， Ρ <0.05 ) 。 与此同时， 抑炎因子 IL-10并没有明显变化， 而 TNF-oc和 IFN-γ 也都没有变化。
3.柚皮素降低血管壁 IL-6和 MCP-1的表达
为了阐明柚皮素降低小鼠腹主动脉血管壁炎性反应的机制， 用连续切 片进行 IL-6和 MCP-1 的免疫荧光染色， 来分析它们在血管壁中的分布和 表达。 使用 confocal荧光显微镜照射， 绿光显示的是 IL-6或 MCP-1， 蓝光 显示的是细胞核。 结果如图 7C所示， 当小鼠用柚皮素灌胃后， 血管壁 IL- 6和 MCP-1的产生明显的减少了， 说明了柚皮素能够减少血管壁中 IL-6和 MCP-1的含量。
4、 柚皮素灌胃降低小鼠的金属蛋白酶的活性与表达
结果如图 8A所示， 用血管紧张素 II灌注后， 中膜和外膜的金属蛋白 酶的活性很强， 而柚皮素喂养后， 有活性的金属蛋白酶明显减少。
结果如图 8B所示， 用柚皮素喂养后， MMP-9的酶原形式显著的减 少， 也就是说柚皮素抑制了 MMP-9的合成， 但同时 MMP-9的活性形式和
MMP-2均没有明显的变化。 实施例 5、 柚皮素对已经发生的腹主动脉瘤进展和转归中的作用 分别利用小鼠磷酸钙涂抹模型和血管紧张素 II诱导模型来探究柚皮素 对于已形成的动脉瘤是否有作用。
在磷酸钙模型中， 我们在 12周龄的雄性 C57小鼠 （共 36只） 肾下腹 主动脉处涂抹磷酸钙 7天后形成动脉瘤。 之后， 将 36只小鼠随机分为 2 组， 一组每天给予柚皮素 （50 mg/kg/day) 灌胃， 另一组给予同等量的水 灌胃。 分别灌胃 2周 （每组 8只） 和 4周 （每组 10只） 。 利用 ex vivo的 测量方法 （结果如图 9所示） ， 我们发现柚皮素和对照组相比， 可以显著 抑制动脉瘤发展进程和减小肾下腹主动脉的平均直径 (2周， 0.884+0.33 ra. 1·488±0·42 mm, P<0.05; 4周， 0·9935±0·5 vs.l.718±0.64 mm, Ρ<0·05)。
此外， 我们利用血管紧张素 II皮下埋泵 28天 （1,000 ng/kg/min) 的方 法， 在 20周雄性 ApoE敲除小鼠 （共 40只） 上诱发腹主动脉瘤并利用超 声在体检测肾上腹主动脉直径。 以直径大于临近血管直径的 50%作为发生 主动脉瘤的标准， 28天后有 28只小鼠罹患动脉瘤。 以相似的最大腹主动 脉直径作为分组标准， 我们分别将发生主动脉瘤 （28只） 和未发生主动脉 瘤 （12只） 的小鼠平均分为两组， 平均最大腹主动脉直径相当， 一组给予 柚皮素灌胃， 另一组给予同量的水灌胃。 全部的小鼠继续给予血管紧张素 II埋泵 28天。 与在磷酸钙模型中观察到的结果相同， 在先前未发生动脉瘤 的小鼠中， 喂养柚皮素的 6只小鼠中只有 1只在 28天后形成了动脉瘤 （发 生率 16%) ， 而对照组中有 3只小鼠形成了动脉瘤 （发生率 50%) 。 在先 前已形成动脉瘤的小鼠中， 喂养水的 14只小鼠中有 4只死于动脉瘤破裂， 另外 10只存活小鼠都发生了严重的动脉瘤 （平均最大直径达到 3.00±0.58 mm) 。 与此相比， 在第二轮的血管紧张素 II刺激后， 柚皮素喂养组小鼠 全部正常存活。 在这 14只小鼠中， 只在 5只小鼠中观察到动脉瘤， 其余小 鼠的动脉瘤都被翻转， 血管最大直径降低到 1.62±0.45 mm。 此外， 通过利 用明胶酶谱检测金属蛋白酶活性， 我们发现柚皮素可以显著抑制金属蛋白 酶 MMP-9的活性。
综上所述， 柚皮素不仅可以抑制腹主动脉瘤的形成， 而且可以逆转已 形成的主动脉瘤从而起到治疗作用。
工业应用
本发明提供的预防和 /或治疗腹主动脉瘤的药品安全低毒， 药理作用较 强； 其原料来源丰富、 广泛、 价廉， 可以由柚皮苷 (Naringin)粗品或者单体 通过水解的方法得到， 或者从含柚皮素的各种药材中提取得到， 或者通过 其他化学方法合成得到； 治本成本低廉、 工艺简单、 得率高； 且疗效确 切， 有效剂量低。 本发明为预防、 诊断、 检测、 保护、 治疗和研究腹主动 脉瘤疾病提供了一种新的药物来源， 且容易推广应用， 能够在较短的时间 内产生巨大的社会效益和经济效益。

Claims

权利要求
1、 柚皮素在制备预防和 /或治疗腹主动脉瘤的产品中的应用。
2、 根据权利要求 1 所述的应用， 其特征在于： 所述产品为药物和 /或 保健品。
3、 柚皮素在制备下述任一产品中的应用：
1 ) 抑制腹主动脉弹力板破裂的药物；
2 ) 抑制腹主动脉血管壁炎症细胞浸润的药物；
3 ) 抑制血浆中白介素 IL-6 和 /或单核细胞趋化因子 MCP-1 的水平的 药物；
4) 抑制腹主动脉血管壁中炎症因子表达的药物；
5 ) 抑制腹主动脉血管壁基质金属蛋白酶的活性与表达的药物。
4、 根据权利要求 3 所述的应用， 其特征在于： 所述炎症因子为白介 素 IL-6和 /或单核细胞趋化因子 MCP_1。
5、 根据权利要求 3 所述的应用， 其特征在于： 所述炎症细胞为巨噬 细胞和 /或白细胞。
6、 根据权利要求 3 所述的应用， 其特征在于： 所述基质金属蛋白酶 为 MMP-9。
7、 一种预防和 /或治疗腹主动脉瘤的产品， 其活性成分为柚皮素。
8、 根据权利要求 Ί 所述的产品， 其特征在于： 所述产品为药物和 /或 保健品。
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