WO2017148338A1 - 木犀草素-7-二葡萄糖醛酸苷在制备抗心肌损伤或纤维化的药物中的应用 - Google Patents

木犀草素-7-二葡萄糖醛酸苷在制备抗心肌损伤或纤维化的药物中的应用 Download PDF

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WO2017148338A1
WO2017148338A1 PCT/CN2017/074842 CN2017074842W WO2017148338A1 WO 2017148338 A1 WO2017148338 A1 WO 2017148338A1 CN 2017074842 W CN2017074842 W CN 2017074842W WO 2017148338 A1 WO2017148338 A1 WO 2017148338A1
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luteolin
diglucuronide
isoproterenol
fibrosis
myocardial
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French (fr)
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张腾
朱维良
陈瑜
宁冰冰
张勇
贾成林
熊敏琪
崔金刚
王培伟
李波
徐志建
吴丹丹
李黎
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Shanghai Institute of Materia Medica of CAS
Yueyang Hospital of Integrated Traditional Chinese and Western Medicine Shanghai University of TCM
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Shanghai Institute of Materia Medica of CAS
Yueyang Hospital of Integrated Traditional Chinese and Western Medicine Shanghai University of TCM
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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

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  • the invention relates to the technical field of medicine, in particular to the application of luteolin-7-diglucuronide in the preparation of a medicament for preventing myocardial injury or fibrosis.
  • the anti-fibrotic formation mechanism of luteolin-7-diglucuronide suggests the application of its intervention in the pathogenesis of multi-system fibrosis such as liver, lung and kidney.
  • Heart disease is a general term for heart disease, including rheumatic heart disease, congenital heart disease, hypertensive heart disease, coronary heart disease, and myocarditis.
  • myocardial fibrosis CF
  • Isoproterenol is a beta agonist and is clinically used for the treatment of bronchial asthma, cardiac arrest, atrioventricular block, shock, and the like.
  • Major adverse reactions involve myocardial damage, arrhythmia, sudden death, and the like.
  • it is widely used in the establishment of animal models of myocardial injury, and is widely used in the mechanism and pharmacological research of myocardial injury.
  • Fibrosis is an important mechanism for organ dysfunction and even failure caused by various organ damage caused by various causes. It is an important pathological process that mediates irreversible changes in the morphology and function of multiple organs such as liver, kidney and lung.
  • Transforming growth factor- ⁇ 1 (TGF- ⁇ 1) is an important cytokine that promotes the formation of multi-system fibrosis. TGF- ⁇ 1 is considered to be a therapeutic target for anti-fibrosis.
  • Activated TGF- ⁇ 1 plays a role mainly by binding to its receptor, and then mediates its profibrotic effect through intracellular SMAD family proteins. SMAD-dependent TGF- ⁇ 1 signaling pathway is a classic TGF- ⁇ 1 profibrotic effect. signal path.
  • Col1a1, Col1a2, Col3a1, Col12a1, CTGF, elastin, Fibrillin1 and other genes during multi-system fibrosis is a common marker of fibrosis formation, reflecting the abnormal over-formation of extracellular matrix during fibrosis. Effective drug intervention for the mechanism of fibrosis formation is of great value in the treatment of multi-organ fibrosis.
  • Luteolin-7-O-[ ⁇ -glucuronosy (1 ⁇ 2) ⁇ -glucuronic acid] can be obtained from a variety of plant bodies Extracted, the compound is abbreviated as Luteolin-7-diglucuronide in English, and the Chinese chemical name can be abbreviated as luteolin-7-diglucuronide.
  • the chemical structural formula is as shown in Formula I:
  • Chinese patent document 201410166181.5 discloses the use of luteolin-7-diglucuronide in the preparation of a medicament for treating retinal degenerative diseases, and an effective amount of luteolin-7-glucuronide anti-photoreceptor cell death, The role of prevention and treatment of retinal degenerative diseases has been studied. However, the application of the luteolin-7-diglucuronide of the present invention in the preparation of a drug against myocardial injury and fibrosis has not been reported yet.
  • a first object of the present invention is to provide a use of luteolin-7-diglucuronide for the preparation of a medicament for preventing myocardial injury in view of the deficiencies in the prior art.
  • a second object of the present invention is to provide an application of luteolin-7-diglucuronide in the preparation of a medicament for inhibiting myocardial fibrosis.
  • a third object of the present invention is to provide a use of luteolin-7-diglucuronide for the preparation of a medicament for inhibiting the formation of multi-organ fibrosis.
  • the luteolin-7-diglucuronide inhibits isoproterenol-induced myocardial damage.
  • the luteolin-7-diglucuronide inhibits isoproterenol-induced cardiomyocyte necrosis, granulation tissue formation, inflammatory cell infiltration, and myocardial fibrosis.
  • luteolin-7-diglucuronide in the preparation of a medicament for inhibiting the formation of myocardial fibrosis.
  • the luteolin-7-diglucuronide inhibits isoproterenol-induced myocardial fibrosis formation.
  • the luteolin-7-diglucuronide inhibits isoproterenol-induced extracellular matrix including collagen gene expression.
  • luteolin-7-diglucuronide in the preparation of a medicament for inhibiting the formation of multi-organ fibrosis.
  • the luteolin-7-diglucuronide inhibits TGF- ⁇ /SMAD and extracellular matrix including collagen gene expression.
  • the luteolin-7-diglucuronide is present in the form of a pharmaceutically acceptable salt.
  • the luteolin-7-diglucuronide may be present in a prodrug form.
  • the prodrug forms include pharmaceutically acceptable forms such as esterification, acetylation, and the like.
  • the luteolin-7-diglucuronide may be used singly or in the form of a pharmaceutical composition comprising luteolin-7-diglucuronide, a pharmaceutically acceptable salt thereof Or a prodrug thereof, and at least one of a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, and vehicle.
  • the invention adopts an animal model of myocardial injury induced by isoproterenol in mice, and studies the pharmacological action and molecular mechanism of luteolin-7-diglucuronide on myocardial injury, and the research results indicate that luteolin-7- Diglucuronide has a significant inhibitory effect on isoproterenol-induced myocardial injury, mainly inhibiting myocardial necrosis, granulation tissue formation, inflammatory cell invasion, fibrosis and other pathological phenotypes.
  • Figure 1 shows the protective effect of luteolin-7-glucuronide on myocardial injury induced by isoproterenol: isoproterenol model group administered with isoproterenol (dissolved in sterile buffered phosphate)
  • the saline solution (PBS) was intraperitoneally injected at a dose of 10 mg/kg body weight, and the volume of each injection was 100 ⁇ L once daily for 5 times.
  • Luteolin-7-diglucuronide was intraperitoneally injected at the following doses: 5 mg/kg body weight, 10 mg/kg body weight, 20 mg/kg body weight, 40 mg/kg body weight. After 30 minutes, the mice received isoproterenol injection for 5 days.
  • the normal control group received 100 ⁇ L of PBS as a solvent control, respectively.
  • the mice were sacrificed 24 hours after the last injection, and the hearts were collected for paraffin embedding, sectioning and hematoxylin/eosin staining. The pathological changes of the heart were observed under light microscope.
  • the isoproterenol model group (b) showed extensive necrosis in the left ventricle, with inflammatory cell infiltration and granulation tissue formation; and low dose (5 mg/kg, 10 mg/kg).
  • the left ventricle of mice administered with luteolin-7-diglucuronide showed moderate tissue necrosis (cd); a medium dose (20 mg/kg) of luteolin-7-diglucuronide was administered.
  • the left ventricle of the mice showed mild tissue necrosis (e); no obvious tissue necrosis (f) was observed in the left ventricle of mice administered with high dose (40 mg/kg) of luteolin-7-diglucuronide.
  • Figure 2 shows the inhibition of isoproterenol-induced myocardial fibrosis by intraperitoneal injection of luteolin-7-glucuronide:
  • A The left ventricular section of mice was stained with Masson's trichrome collagen, and the results showed that it was compared with the normal control group. Compared with the isoproterenol administration group, the Masson trichrome positive area was significantly increased (*p ⁇ 0.01), and the luteolin-7-diglucuronide treatment group significantly reduced Masson III compared with the isoproterenol group. Color positive area (#p ⁇ 0.01).
  • B Representative images of left ventricular Masson trichrome staining in mice: normal control group (VC), isoproterenol group (ISO) and luteolin-7-glucuronide 40 mg/kg treatment group (LC1) .
  • Figure 3 shows that luteolin-7-diglucuronide inhibits isoproterenol-induced myocardial fibrosis at the molecular level: immunohistochemical detection indicates important molecular markers related to fibrosis formation such as ⁇ -SMA, Col1 , TGF- ⁇ 1, p-SMAD2 and Timp1 were expressed in the left ventricular myocardial injury site of mice in the isoproterenol group (a, c, e, g, i). No molecular markers were found in luteolin-7. - Expression of left ventricular myocardial tissue in mice with digluconate group (b, d, f, h, j).
  • Figure 4 shows that luteolin-7-diglucuronide inhibits isoproterenol-induced expression of extracellular matrix and inflammation-related genes involved in myocardial fibrosis: cells involved in myocardial fibrosis formation by real-time quantitative PCR The expression of the outer matrix gene was analyzed. The results showed that the expressions of Col1a1, Col1a2, Col3a1, Col12a1, CTGF, elastin, Fibrillin1, Ccl4 and Ccl12 in the isoproterenol model group were significantly up-regulated compared with the normal control group. The expression of these genes was significantly down-regulated in the -7-diglucuronide-treated group compared with the isoproterenol model group.
  • Figure 5 is a protective effect of luteolin-7-diglucuronide on myocardial injury induced by isoproterenol for 10 days: isoproterenol model group is given isoproterenol (dissolved in sterile buffered phosphate) The saline solution (PBS) was intraperitoneally injected at a dose of 10 mg/kg body weight, and the volume of each injection was 100 ⁇ L once daily for 10 times. On the fifth day of modeling, luteolin-7-diglucuronide was intraperitoneally injected at a dose of 40 mg/kg. After 30 minutes, Mice received isoproterenol injection for 6 days. The normal control group received 100 ⁇ L of PBS as a solvent control, respectively.
  • PBS saline solution
  • mice were sacrificed 24 hours after the last injection, and the hearts were collected for paraffin embedding, sectioning and hematoxylin/eosin staining. The pathological changes of the heart were observed under light microscope.
  • the isoproterenol model group (b) showed extensive necrosis in the left ventricle, with inflammatory cell infiltration and granulation tissue formation; and 40 mg/kg luteolin-7-dglycan Mild tissue necrosis (c) was seen in the left ventricle of mice administered with aldosides.
  • Figure 6 shows that luteolin-7-diglucuronide inhibits isoproterenol-induced myocardial fibrosis formation for 10 days:
  • A. Mouse left ventricular sections were stained with Masson's trichrome collagen, and the results showed that it was compared with the normal control group. Compared with the isoproterenol administration group, the Masson trichrome positive area was significantly increased (*p ⁇ 0.01), and the luteolin-7-diglucuronide treatment group significantly reduced Masson III compared with the isoproterenol group. Color positive area (#p ⁇ 0.01).
  • B Representative images of left ventricular Masson trichrome staining in mice: normal control group (VC), isoproterenol group (ISO) and luteolin-7-glucuronide 40 mg/kg treatment group (LC1) .
  • Figure 7 shows that luteolin-7-diglucuronide inhibits isoproterenol-induced myocardial fibrosis formation for 10 days:
  • A Mouse left ventricular sections were stained with Sirius red collagen, and the results showed that compared with the normal control group. In the isoproterenol-administered group, the area of Sirius red-positive was significantly increased (*p ⁇ 0.01), and the luteolin-7-diglucuronide-treated group significantly reduced Sirius red-positive compared with the isoproterenol group. Area (#p ⁇ 0.01).
  • B Representative pictures of left ventricular Sirius red collagen staining in mice: normal control group (VC), isoproterenol group (ISO) and luteolin-7-glucuronide 40 mg/kg treatment group (LC1).
  • Figure 8 is a protective effect of luteolin-7-diglucuronide on isoproterenol-induced myocardial injury: isoproterenol model group administered isoproterenol (dissolved in sterile buffered phosphate)
  • the saline solution (PBS) was intraperitoneally injected at a dose of 10 mg/kg body weight, and the volume of each injection was 100 ⁇ L once daily for 5 times.
  • Luteolin-7-diglucuronide was administered by 120 mg/kg body weight and 240 mg/kg body weight. After 30 minutes, the mice received isoproterenol injection for 5 days.
  • Normal and model control groups received 200 ⁇ L of PBS as a solvent control, respectively.
  • mice were sacrificed 24 hours after the last injection, and the hearts were collected for paraffin embedding, sectioning and hematoxylin/eosin staining. The pathological changes of the heart were observed under light microscope.
  • the isoproterenol model group (b) showed extensive necrosis in the left ventricle, with inflammatory cell infiltration and granulation tissue formation; and received LC1120 mg/kg body weight (c) and 240 mg/ The degree of myocardial tissue damage was significantly reduced in kg body weight (d).
  • Figure 9 is a graph showing the inhibitory effect of luteolin-7-diglucuronide on isoproterenol-induced myocardial fibrosis: isoproterenol model group administered with isoproterenol (dissolved in sterile buffer) Phosphate solution (PBS)) was intraperitoneally injected at a dose of 10 mg/kg body weight, each injection volume of 100 ⁇ L once daily for 5 times. Luteolin-7-diglucuronide was administered by 120 mg/kg body weight and 240 mg/kg body weight. After 30 minutes, the mice received isoproterenol injection for 5 days. Normal and model control groups received 200 ⁇ L of PBS as a solvent control, respectively.
  • PBS Phosphate solution
  • mice normal control group (VC), isoproterenol group (ISO), luteolin-7-diglucuronide 120 mg/kg treatment group (LC1L) And luteolin-7-diglucuronide 240mg/kg treatment group (LC1H).
  • VC normal control group
  • ISO isoproterenol group
  • LC1L luteolin-7-diglucuronide 120 mg/kg treatment group
  • LC1H luteolin-7-diglucuronide 240mg/kg treatment group
  • FIG 10 shows that luteolin-7-diglucuronide gavage significantly inhibits ISO-induced cardiomyocyte hypertrophy: isoproterenol model group is given isoproterenol (dissolved in sterile buffered phosphate solution ( PBS)), intraperitoneal injection at a dose of 10 mg/kg body weight, each injection volume of 100 ⁇ L, once daily for 5 times. Luteolin-7-diglucuronide was administered by 120 mg/kg body weight and 240 mg/kg body weight. After 30 minutes, the mice received isoproterenol injection for 5 days. Normal and model control groups received 200 ⁇ L of PBS as a solvent control, respectively.
  • PBS sterile buffered phosphate solution
  • mice After the last injection for 24 hours, the mice were sacrificed, the hearts were collected, paraffin-embedded, and the left ventricle sections of the mice were stained with hematoxylin/eosin. The pathological changes of the heart were observed under light microscope, and the cardiomyocytes were analyzed by Leica QWIN V3 analysis software. The cross-sectional area is measured.
  • the area of cardiomyocytes in the normal group (VC) was (215.74 ⁇ 1.61) ⁇ m 2 , compared with the normal group (VC), the myocardial cell area of the model group (ISO) increased significantly (329.52 ⁇ 11.70 ⁇ m 2 , *P ⁇ 0.05); model group (ISO), compared luteolin 7-glucuronide 120mg / kg treatment group (LC1L) (249.88 ⁇ 10.26 ⁇ m 2 , # P ⁇ 0.05), luteolin 7-glucuronide acid glycoside 240mg / kg treatment group (LC1H) (231.69 ⁇ 31.18 ⁇ m 2 , # P ⁇ 0.05) were significantly reduced. There was no significant difference between the luteolin-7-diglucuronide 120 mg/kg treatment group (LC1L) and the luteolin-7-diglucuronide 240 mg/kg treatment group (LC1H).
  • prodrug as used in the present invention means that a compound is converted into the formula (I) in vivo. The compound shown. Such transformation is affected by the hydrolysis of the prodrug in the blood or by enzymatic conversion to the parent structure in the blood or tissue.
  • the prodrug-like compound of the present invention may be an ester.
  • the ester may be used as a prodrug such as a phenyl ester, an aliphatic (C1-24) ester, an acyloxymethyl ester, a carbonate, Carbamates and amino acid esters.
  • a compound of the invention comprises a hydroxyl group, i.e., it can be acylated to give a compound in the form of a prodrug.
  • Other prodrug forms include phosphates, such as those obtained by phosphorylation of a hydroxy group on the parent.
  • salts formed by pharmaceutically acceptable non-toxic acids include, but are not limited to, mineral acid salts formed by reaction with amino groups, hydrochloride, hydrobromide, phosphate, sulfate, perchlorate, And organic acid salts such as acetates, oxalates, maleates, tartrates, citrates, succinates, malonates, or by other methods described in the literature, such as ion exchange These salts.
  • salts include adipate, alginate, ascorbate, aspartate, besylate, benzoate, disulfate, borate, butyrate, camphoric acid Salt, camphor sulfonate, cyclopentylpropionate, digluconate, lauryl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate Salt, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, Malate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, palmitate, pamoate, pectate, persulphate, 3 -Phenylpropionate
  • Salts obtained by appropriate bases include the alkali metal, alkaline earth metal, ammonium and N+(C1-4 alkyl)4 salts.
  • the pharmaceutically acceptable salts further include suitable, non-toxic ammonium, quaternary ammonium salts and amine cations formed by counter-ion ions, such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, C1- 8 sulfonate and aromatic sulfonate.
  • mice Male clean grade C57BL/6J mice, 6-8 weeks old and weighing 20-25 g, were purchased from the Chinese Academy of Sciences for regular feeding and drinking.
  • a myocardial ischemia model was established by intraperitoneal injection of isoproterenol (Sigma, USA). Isoproterenol was dissolved in sterile phosphate buffered saline (PBS) at a dose of 10 mg/kg per dose, administered in a volume of 100 ⁇ L, administered once daily at 9:00 am, and intraperitoneally.
  • PBS sterile phosphate buffered saline
  • Isoproterenol was administered 5-10 times; luteolin-7-diglucuronide (provided by Shanghai Institute of Materia Medica, Chinese Academy of Sciences, purity >98%) was dissolved in sterile phosphate buffered saline (PBS). Luteolin-7-diglucuronide was administered 30 minutes before the administration of isoproterenol, and was administered intraperitoneally and intragastrically (both daily). The doses of luteolin-7-diglucuronide administered intraperitoneally were: 5 mg/kg body weight, 10 mg/kg body weight, 20 mg/kg body weight and 40 mg/kg body weight, and the volume per administration was 100 ⁇ L.
  • PBS sterile phosphate buffered saline
  • the oral dose of luteolin-7-diglucuronide was 120 mg/kg body weight and 240 mg/kg body weight, and the volume per administration was 200 ⁇ L. Both the normal group and the model control group were given an equal volume of sterile phosphate buffer PBS for intraperitoneal injection or intragastric administration.
  • mice were sacrificed 24 hours after the end of the administration, and the hearts were collected, fixed in 4% paraformaldehyde, and then subjected to tissue embedding and sectioning. Histomorphometric analysis was performed by using a 4 ⁇ M thick paraffin section for hematoxylin-eosin staining, and the histomorphological changes of the heart were observed under light microscopy (Leica, Germany). Collagen fiber staining was observed under the light microscope after Masson's trichrome staining. Cardiac pathological changes were scored after hematoxylin-eosin staining, and the scoring criteria were as follows.
  • 1 point no myocardial necrosis, granulation tissue formation and inflammatory cell infiltration and other changes of myocardial injury
  • 2 points occasionally scattered, independent myocardial necrosis lesions
  • 3 points 30% or less myocardial damage in the subendocardial layer
  • 4 points 30%-50% extensive myocardial damage in the subendocardium
  • 5 points more than 50% of the subendocardial myocardial injury.
  • the primary antibody includes ⁇ -smooth muscle actin ( ⁇ -SMA) (Sigma, USA), collagen 1 (Col1) (Sigma, USA), tissue inhibitor of metalloproteinase 1 (Timp1) (Protein Tech, China), transforming growth factor - ⁇ 1 (TGF- ⁇ 1) (Santa Cruz, China), p-SMAD2 (Protein Tech, China).
  • Secondary antibodies were purchased from Solarbio (China), including goat anti-mouse IgG for detection of ⁇ -SMA expression, and goat anti-rabbit IgG for detection of levels of TGF- ⁇ 1, Timp1 and p-SMAD2. Immunoreactivity was finally developed using 3,3-diaminodipropylamine (DAB, Sigma, USA) and observed under light microscopy (Leica, Germany).
  • cDNA Tissue total RNA extraction and purification (Qiagen, USA) followed by reverse transcription synthesis of cDNA (Revert Aid First Strand cDNA Synthesis Kit, Termo, USA).
  • the cDNA was further analyzed by fluorescence real-time quantitative PCR using ABI Power SYBR Green PCR Master Mix (7900HT Sequence Detection System, ABI, USA).
  • Luteolin-7-diglucuronide inhibits isoproterenol-induced myocardial injury in mice:
  • Isoproterenol is a beta agonist that induces death of infarct-like cardiomyocytes.
  • ISO-induced myocardial injury animal models can mimic changes in cardiac morphology and metabolism during human myocardial infarction, and are widely used in the study of drug efficacy and mechanism of anti-myocardial injury.
  • C57/BL6J mice were used to establish an ISO-induced myocardial injury model.
  • ISO is administered intraperitoneally at a dose of 10 mg/kg once daily for 5 or 10 days.
  • luteolin-7-glucuronide As shown in Figure 1, intraperitoneal injection inhibition of luteolin-7-glucuronide (LC1) dose-dependently alleviated changes in myocardial damage such as ISO-induced cardiomyocyte degeneration, inflammatory cell infiltration, and granuloma formation. As shown in Figure 5, intraperitoneal injection of LC1 significantly reversed ISO-induced myocardial damage. Oral administration of luteolin-7-diglucuronide also exhibited significant anti-ISO-induced myocardial damage ( Figure 8 and Table 1).
  • LC1 luteolin-7-glucuronide
  • LC1L LC1 120mg/kg body weight
  • LC1H LC1 240mg/kg body weight
  • Luteolin-7-diglucuronide inhibits isoproterenol-induced myocardial fibrosis:
  • Myocardial fibrosis is defined as a significantly increased collagen volume. Therefore, collagen fibers in myocardial tissue sections were further evaluated and quantified using Masson's trichrome staining. As shown in Figure 2A, a small amount of Masson's trichrome-positive collagen fibers were found in the myocardial space of the solvent control group. The collagen fiber content of Masson's trichrome staining was significantly increased in the ISO group, and the Masson's trichrome-stained collagen fiber was observed in the high-dose LC1 treatment group. The content is significantly reduced. Representative Masson's trichrome staining pictures are shown in Figure 2B: no solvent-like connective tissue (a) is seen in the solvent-controlled myocardium.
  • Luteolin-7-diglucuronide inhibits ISO-induced cardiomyocyte hypertrophy:
  • Luteolin-7-diglucuronide inhibits the expression of molecular markers of fibrosis:
  • Col1 is the main component of the extracellular matrix, suggesting that LC1 intervention can be performed. Significant inhibition of extracellular matrix production during injury.
  • other key molecular markers of fibrosis such as TGF- ⁇ 1, p-SMAD2, which reflects the activity of TGF ⁇ signaling pathway mediating fibrosis, are significantly enhanced in ISO-induced myocardial injury sites (e, g), whereas The signal was not seen in the myocardium (f, h) of LC1-interacting mice. This result indicates that LC1 can interfere with ISO-induced myocardial fibrosis by inhibiting the activity of TGF- ⁇ 1, a key signaling pathway for fibrosis formation.
  • Timp1 an important molecule that inhibits the degradation of collagen fibers during ISO-induced fibrosis formation, is significant at myocardial injury sites. Adjust (i), and this anomaly can also be inhibited by LC1 (j), indicating that LC1 has significant intervention effects on important molecular links such as fibrosis formation and fiber degradation.

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Abstract

木犀草素-7-二葡萄糖醛酸苷在制备抗心肌损伤或纤维化形成药物中的应用。

Description

木犀草素-7-二葡萄糖醛酸苷在制备抗心肌损伤或纤维化的药物中的应用 技术领域
本发明涉及医药技术领域，具体地说，是木犀草素-7-二葡萄糖醛酸苷在制备抗心肌损伤或纤维化的药物中的应用。此外，木犀草素-7-二葡萄糖醛酸苷抗纤维化形成机制的效应提示了其干预其他系统纤维化如肝、肺、肾等多系统纤维化病理形成的应用。
背景技术
心脏病(heart disease)是心脏疾病的总称，包括风湿性心脏病、先天性心脏病、高血压性心脏病、冠心病、心肌炎等各种心脏病。
在心血管系统，当心肌因缺血、炎症、衰老等原因受损时，局部心肌细胞凋亡、心肌组织结构中胶原纤维过量积聚、胶原浓度和胶原容积分数显著增加，各型胶原比例失调以及排列絮乱，导致心肌纤维化(cardiac fibrosis,CF)发生。它是各种心血管疾病发展到一定阶段的共同病理改变，是心肌重构的主要表现之一。纤维化的心肌僵硬度增加、顺应性减少、收缩力下降、同步性降低，使得心脏功能逐步减退。临床研究表明纤维化是不良心脏预后的独立预测因子。因此有效干预心肌损伤纤维化至今仍是研究热点。
异丙肾上腺素为β受体激动剂，临床上用于支气管哮喘、心脏骤停、房室传导阻滞、休克等的治疗。其重大不良反应涉及心肌损伤、心律失常、猝死等。在实验研究中，根据其心脏毒性，多用于心肌损伤动物模型的建立，广泛用于心肌损伤的机制及药理研究。
纤维化是各种原因引起的多种器官损伤导致器官功能障碍甚至衰竭的重要机制，是介导肝、肾、肺等多个器官的形态及功能不可逆性改变的重要病理过程。转化生长因子β1(transforming growth factor-β1,TGF-β1)是促进多系统纤维化形成的重要细胞因子。TGF-β1被认为是抗器官纤维化的治疗靶点。活化的TGF-β1主要是通过与其受体结合而发挥作用，进而通过细胞内SMAD家族蛋白介导其促纤维化效应，SMAD依赖性TGF-β1信号传导通路是TGF-β1促纤维化效应的经典信号通路。Col1a1,Col1a2，Col3a1,Col12a1，CTGF，elastin，Fibrillin1等基因在多系统纤维化过程中表达上调，是纤维化形成的共同标志，反映出纤维化进程中细胞外基质的异常过度形成。针对纤维化形成机制的有效药物干预对多器官纤维化的治疗具有重要价值。
木犀草素-7-O-[β-葡萄糖醛酸基(glucuronosy)(1→2)β-葡萄糖醛酸]可从多种植物体 提取获得，该化合物英文简写为Luteolin-7-diglucuronide，中文化学名称可简写为木犀草素-7-二葡萄糖醛酸苷，化学结构式如式I所示：
Figure PCTCN2017074842-appb-000001
中国专利文献201410166181.5公开了木犀草素-7-二葡萄糖醛酸苷在制备治疗视网膜退行性病变药物中的应用，对有效量的木犀草素-7-二葡萄糖醛酸苷抗光感受器细胞死亡、防治视网膜退行性病变发生的作用进行了研究。然而，关于本发明的木犀草素-7-二葡萄糖醛酸苷在制备抗心肌损伤及纤维化的药物中的应用，目前还未见报道。
发明内容
本发明的第一个目的是针对现有技术中的不足，提供木犀草素-7-二葡萄糖醛酸苷在制备抗心肌损伤药物中的应用。
本发明的第二个目的是，提供木犀草素-7-二葡萄糖醛酸苷在制备抑制心肌纤维化形成药物中的应用。
本发明的第三个目的是，提供木犀草素-7-二葡萄糖醛酸苷在制备抑制多器官纤维化形成的药物中的应用。
为实现上述第一个目的，本发明采取的技术方案是：
木犀草素-7-二葡萄糖醛酸苷在制备抗心肌损伤药物中的应用。
所述的木犀草素-7-二葡萄糖醛酸苷抑制异丙肾上腺素诱导的心肌损伤。
所述的木犀草素-7-二葡萄糖醛酸苷抑制异丙肾上腺素诱导的心肌细胞坏死，肉芽组织形成，炎细胞侵润、心肌纤维化形成。
为实现上述第二个目的，本发明采取的技术方案是：
木犀草素-7-二葡萄糖醛酸苷在制备抑制心肌纤维化形成的药物中的应用。
所述的木犀草素-7-二葡萄糖醛酸苷抑制异丙肾上腺素诱导的心肌纤维化形成。
所述的木犀草素-7-二葡萄糖醛酸苷抑制异丙肾上腺素诱导的细胞外基质包括胶原基因表达。
为实现上述第三个目的，本发明采取的技术方案是：
木犀草素-7-二葡萄糖醛酸苷在制备抑制多器官纤维化形成的药物中的应用。
所述的木犀草素-7-二葡萄糖醛酸苷抑制TGF-β/SMAD及细胞外基质包括胶原基因表达。
所述的木犀草素-7-二葡萄糖醛酸苷以药学上可接受的盐形式存在。
所述的木犀草素-7-二葡萄糖醛酸苷可以以前药形式存在。
所述的前药形式包括酯化、乙酰化等药学上可接受的形式。
所述木犀草素-7-二葡萄糖醛酸苷可以单独使用或者以药物组合物的形式使用，所述药物组合物包含木犀草素-7-二葡萄糖醛酸苷、其药学上可接受的盐或它的前药，以及药学上的载体，赋形剂，稀释剂，辅剂和媒介物的至少一种。
本发明优点在于：
本发明采用异丙肾上腺素诱导小鼠心肌损伤的动物模型，对木犀草素-7-二葡萄糖醛酸苷干预心肌损伤的药理作用及分子机制进行了研究，研究结果表明木犀草素-7-二葡萄糖醛酸苷对异丙肾上腺素诱导的心肌损伤具有显著的抑制作用，主要表现为抑制心肌细胞坏死，肉芽组织形成，炎细胞侵润、纤维化形成等病理表型。运用荧光实时定量PCR，在分子水平揭示并进一步证实了木犀草素-7-二葡萄糖醛酸苷抗心肌纤维化形成的作用，特别是体现在木犀草素-7-二葡萄糖醛酸苷对细胞外基质包括胶原基因表达具有显著的抑制作用。因此本发明为木犀草素-7-二葡萄糖醛酸苷抗心肌损伤及心肌纤维化的效应提供了直接的实验证据。此外，木犀草素-7-二葡萄糖醛酸苷下调TGF-β/SMAD及细胞外基质基因表达的效应提示了其干预其他系统纤维化如肝、肺、肾等多系统纤维化病理形成的应用。
附图说明
附图1为木犀草素-7-二葡萄糖醛酸苷腹腔注射对异丙肾上腺素诱导的心肌损伤的保护作用：异丙肾上腺素模型组给与异丙肾上腺素(溶于灭菌的缓冲磷酸盐溶液(PBS))，按照10mg/kg体重的剂量进行腹腔内注射，每次注射体积为100μL，每日一次，共5次。木犀草素-7-二葡萄糖醛酸苷按照以下剂量腹腔内注射：5mg/kg体重、10mg/kg体重、 20mg/kg体重、40mg/kg体重。30分钟后，小鼠接受异丙肾上腺素注射，持续5天。正常对照组分别接受100μLPBS作为溶剂对照。最后一次注射24h后处死小鼠，采集心脏，进行石蜡包埋、切片及苏木素/伊红染色，光镜下观察心脏的病理形态改变。与正常对照组(a)相比较，异丙肾上腺素模型组(b)小鼠左心室呈现广泛的坏死，伴炎细胞侵润，肉芽组织形成；而低剂量(5mg/kg,10mg/kg)木犀草素-7-二葡萄糖醛酸苷给药的小鼠左心室表现为中度的组织坏死(c-d)；接受中剂量(20mg/kg)木犀草素-7-二葡萄糖醛酸苷给药的小鼠左心室表现为轻度组织坏死(e)；接受高剂量(40mg/kg)木犀草素-7-二葡萄糖醛酸苷给药的小鼠左心室未见明显组织坏死(f)。
附图2为木犀草素-7-二葡萄糖醛酸苷腹腔注射抑制异丙肾上腺素诱导的心肌纤维化形成：A.小鼠左心室切片进行Masson三色胶原染色，结果表明与正常对照组相比，异丙肾上腺素给药组Masson三色阳性的区域显著增加(*p<0.01),与异丙肾上腺素组相比，木犀草素-7-二葡萄糖醛酸苷治疗组显著减少Masson三色阳性的区域(#p<0.01)。B.小鼠左心室Masson三色胶原染色代表性图片：正常对照组(VC)，异丙肾上腺素组(ISO)及木犀草素-7-二葡萄糖醛酸苷40mg/kg治疗组(LC1)。
附图3为木犀草素-7-二葡萄糖醛酸苷在分子水平抑制异丙肾上腺素诱导的心肌纤维化形成：免疫组织化学检测表明纤维化形成相关的重要分子标志物如α-SMA，Col1，TGF-β1，p-SMAD2及Timp1在异丙肾上腺素组小鼠左心室心肌受损部位表达增强(a,c,e,g,i),未见这些分子标志物在木犀草素-7-二葡萄糖醛酸苷组小鼠左心室心肌组织的表达(b,d,f,h,j)。
附图4为木犀草素-7-二葡萄糖醛酸苷抑制异丙肾上腺素诱导的参与心肌纤维化形成细胞外基质及炎症相关基因的表达：采用荧光实时定量PCR对参与心肌纤维化形成的细胞外基质基因的表达进行分析，结果表明异丙肾上腺素模型组小鼠心脏Col1a1,Col1a2，Col3a1,Col12a1，CTGF，elastin，Fibrillin1及Ccl4，Ccl12的表达，与正常对照组相比较显著上调，木犀草素-7-二葡萄糖醛酸苷治疗组与异丙肾上腺素模型组相比，这些基因的表达呈显著的下调。
附图5为木犀草素-7-二葡萄糖醛酸苷对异丙肾上腺素诱导10天的心肌损伤的保护作用：异丙肾上腺素模型组给与异丙肾上腺素(溶于灭菌的缓冲磷酸盐溶液(PBS))，按照10mg/kg体重的剂量进行腹腔内注射，每次注射体积为100μL，每日一次，共10次。造模第5天开始，木犀草素-7-二葡萄糖醛酸苷按照40mg/kg剂量腹腔内注射，30分钟后， 小鼠接受异丙肾上腺素注射，持续6天。正常对照组分别接受100μLPBS作为溶剂对照。最后一次注射24h后处死小鼠，采集心脏，进行石蜡包埋、切片及苏木素/伊红染色，光镜下观察心脏的病理形态改变。与正常对照组(a)相比较，异丙肾上腺素模型组(b)小鼠左心室呈现广泛的坏死，伴炎细胞侵润，肉芽组织形成；而40mg/kg木犀草素-7-二葡萄糖醛酸苷给药的小鼠左心室见轻度组织坏死(c)。
附图6为木犀草素-7-二葡萄糖醛酸苷抑制异丙肾上腺素诱导10天的心肌纤维化形成：A.小鼠左心室切片进行Masson三色胶原染色，结果表明与正常对照组相比，异丙肾上腺素给药组Masson三色阳性的区域显著增加(*p<0.01),与异丙肾上腺素组相比，木犀草素-7-二葡萄糖醛酸苷治疗组显著减少Masson三色阳性的区域(#p<0.01)。B.小鼠左心室Masson三色胶原染色代表性图片：正常对照组(VC)，异丙肾上腺素组(ISO)及木犀草素-7-二葡萄糖醛酸苷40mg/kg治疗组(LC1)。
附图7为木犀草素-7-二葡萄糖醛酸苷抑制异丙肾上腺素诱导10天的心肌纤维化形成：A.小鼠左心室切片进行天狼星红胶原染色，结果表明与正常对照组相比，异丙肾上腺素给药组天狼星红阳性的区域显著增加(*p<0.01),与异丙肾上腺素组相比，木犀草素-7-二葡萄糖醛酸苷治疗组显著减少天狼星红阳性的区域(#p<0.01)。B.小鼠左心室天狼星红胶原染色代表性图片：正常对照组(VC)，异丙肾上腺素组(ISO)及木犀草素-7-二葡萄糖醛酸苷40mg/kg治疗组(LC1)。
附图8为木犀草素-7-二葡萄糖醛酸苷灌胃对异丙肾上腺素诱导的心肌损伤的保护作用：异丙肾上腺素模型组给与异丙肾上腺素(溶于灭菌的缓冲磷酸盐溶液(PBS))，按照10mg/kg体重的剂量进行腹腔内注射，每次注射体积为100μL，每日一次，共5次。木犀草素-7-二葡萄糖醛酸苷按照120mg/kg体重及240mg/kg体重灌胃。30分钟后，小鼠接受异丙肾上腺素注射，持续5天。正常与模型对照组分别接受200μLPBS作为溶剂对照。最后一次注射24h后处死小鼠，采集心脏，进行石蜡包埋、切片及苏木素/伊红染色，光镜下观察心脏的病理形态改变。与正常对照组(a)相比较，异丙肾上腺素模型组(b)小鼠左心室呈现广泛的坏死，伴炎细胞侵润，肉芽组织形成；而接受LC1120mg/kg体重(c)及240mg/kg体重(d)灌胃的小鼠心肌组织损伤程度显著减轻。
附图9为木犀草素-7-二葡萄糖醛酸苷灌胃对异丙肾上腺素诱导的心肌纤维化的抑制作用：异丙肾上腺素模型组给与异丙肾上腺素(溶于灭菌的缓冲磷酸盐溶液(PBS))，按照10mg/kg体重的剂量进行腹腔内注射，每次注射体积为100μL，每日一次，共5次。 木犀草素-7-二葡萄糖醛酸苷按照120mg/kg体重及240mg/kg体重灌胃。30分钟后，小鼠接受异丙肾上腺素注射，持续5天。正常与模型对照组分别接受200μLPBS作为溶剂对照。最后一次注射24h后处死小鼠，采集心脏，进行石蜡包埋、小鼠左心室切片，进行Masson三色胶原染色。A.Masson三色胶原染色定量结果表明与正常对照组相比，异丙肾上腺素给药组Masson三色阳性的区域显著增加(*p<0.01),与异丙肾上腺素组相比，木犀草素-7-二葡萄糖醛酸苷灌胃治疗组显著减少Masson三色阳性的区域(#p<0.01)。B.小鼠左心室Masson三色胶原染色代表性图片：正常对照组(VC)，异丙肾上腺素组(ISO),木犀草素-7-二葡萄糖醛酸苷120mg/kg治疗组(LC1L)及木犀草素-7-二葡萄糖醛酸苷240mg/kg治疗组(LC1H)。
附图10为木犀草素-7-二葡萄糖醛酸苷灌胃显著抑制ISO所诱导的心肌细胞肥大：异丙肾上腺素模型组给与异丙肾上腺素(溶于灭菌的缓冲磷酸盐溶液(PBS))，按照10mg/kg体重的剂量进行腹腔内注射，每次注射体积为100μL，每日一次，共5次。木犀草素-7-二葡萄糖醛酸苷按照120mg/kg体重及240mg/kg体重灌胃。30分钟后，小鼠接受异丙肾上腺素注射，持续5天。正常与模型对照组分别接受200μLPBS作为溶剂对照。最后一次注射24h后处死小鼠，采集心脏，进行石蜡包埋、小鼠左心室切片，进行苏木素/伊红染色，光镜下观察心脏的病理形态改变，并采用Leica QWIN V3分析软件对心肌细胞横截面积进行测算。正常组(VC)心肌细胞面积为(215.74±1.61)μm2，与正常组(VC)相比，模型组(ISO)心肌细胞面积显著增加(329.52±11.70μm2，*P<0.05)；与模型组(ISO)相比，木犀草素-7-二葡萄糖醛酸苷120mg/kg治疗组(LC1L)(249.88±10.26μm2，#P<0.05)，及木犀草素-7-二葡萄糖醛酸苷240mg/kg治疗组(LC1H)(231.69±31.18μm2，#P<0.05)均显著减少。木犀草素-7-二葡萄糖醛酸苷120mg/kg治疗组(LC1L)与木犀草素-7-二葡萄糖醛酸苷240mg/kg治疗组(LC1H)两组相比未见显著差异。
具体实施方式
下面结合具体实施方式，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明记载的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
需要说明的是，本发明所使用的术语“前药”，代表一个化合物在体内转化为式(I) 所示的化合物。这样的转化受前体药物在血液中水解或在血液或组织中经酶转化为母体结构的影响。本发明前体药物类化合物可以是酯，在现有的发明中酯可以作为前体药物的有苯酯类，脂肪族(C1-24)酯类，酰氧基甲基酯类，碳酸酯，氨基甲酸酯类和氨基酸酯类。例如本发明的化合物包含羟基，即可以将其酰化得到前体药物形式的化合物。其他的前体药物形式包括磷酸酯，如这些磷酸酯类化合物是经母体上的羟基磷酸化得到的。关于前体药物完整的讨论可以参考以下文献：T.Higuchi and V.Stella,Pro-drugs as Novel Delivery Systems,Vol.14of the A.C.S.SymposiumSeries,Edward B.Roche,ed.,Bioreversible Carriers in Drug Design,American Pharmaceutical Association and Pergamon Press,1987,J.Rautio et al,Prodrugs:Design and Clinical Applications,Nature Review Drug Discovery,2008,7,255-270,and S.J.Hecker et al,Prodrugs of Phosphates and Phosphonates,Journal of Medicinal Chemistry,2008,51,2328-2345。
本发明所使用的“药学上可接受的盐”是指化合物的有机盐和无机盐。药学上可接受的盐在所属领域是为我们所熟知的，如文献：S.M.Berge et al.,J.Pharmaceutical Sciences,66:1-19,1977所记载的。药学上可接受的无毒的酸形成的盐包括，但并不限于，与氨基基团反应形成的无机酸盐有盐酸盐，氢溴酸盐，磷酸盐，硫酸盐，高氯酸盐，和有机酸盐如乙酸盐，草酸盐，马来酸盐，酒石酸盐，柠檬酸盐，琥珀酸盐，丙二酸盐，或通过书籍文献上所记载的其他方法如离子交换法来得到这些盐。其他药学上可接受的盐包括己二酸盐，藻酸盐，抗坏血酸盐，天冬氨酸盐，苯磺酸盐，苯甲酸盐，重硫酸盐，硼酸盐，丁酸盐，樟脑酸盐，樟脑磺酸盐，环戊基丙酸盐，二葡萄糖酸盐，十二烷基硫酸盐，乙磺酸盐，甲酸盐，反丁烯二酸盐，葡庚糖酸盐，甘油磷酸盐，葡萄糖酸盐，半硫酸盐，庚酸盐，己酸盐，氢碘酸盐，2-羟基-乙磺酸盐，乳糖醛酸盐，乳酸盐，月桂酸盐，月桂基硫酸盐，苹果酸盐，丙二酸盐，甲磺酸盐，2-萘磺酸盐，烟酸盐，硝酸盐，油酸盐，棕榈酸盐，扑酸盐，果胶酸盐，过硫酸盐，3-苯基丙酸盐，苦味酸盐，特戊酸盐，丙酸盐，硬脂酸盐，硫氰酸盐，对甲苯磺酸盐，十一酸盐，戊酸盐，等等。通过适当的碱得到的盐包括碱金属，碱土金属，铵和N+(C1-4烷基)4的盐。药学上可接受的盐进一步包括适当的、无毒的铵，季铵盐和抗平衡离子形成的胺阳离子，如卤化物，氢氧化物，羧化物，硫酸化物，磷酸化物，硝酸化物，C1-8磺酸化物和芳香磺酸化物。
实施例1
在本实施方案中，我们通过病理形态学及分子生物学的研究手段，探讨并明确了木 犀草素-7-二葡萄糖醛酸苷对异丙肾上腺素诱导的小鼠心肌损伤模型的干预效应。研究结果表明木犀草素-7-二葡萄糖醛酸苷可有效地抑制异丙肾上腺素诱导的小鼠心肌细胞坏死变性，炎细胞侵润，肉芽组织形成及纤维化形成并显著抑制心肌纤维化形成过程中关键分子及基因的表达。这一研究首次揭示了木犀草素-7-二葡萄糖醛酸苷对心肌损伤的治疗干预作用及相关机制，提示木犀草素-7-二葡萄糖醛酸苷相关的药物对于心肌损伤及损伤后纤维化形成具有重要的治疗干预价值。
一、方法
1.小鼠心肌损伤动物模型及干预
6-8周龄，体重20-25g的雄性清洁级C57BL/6J小鼠购自中国科学研究院，常规摄食及饮水喂养。采用异丙肾上腺素(Sigma,USA)腹腔内注射建立心肌缺血模型。异丙肾上腺素溶解于无菌的磷酸盐缓冲液(PBS)，每次给药剂量为10mg/kg，给药体积为100μL，每日于9:00am给药一次，腹腔内注射。异丙肾上腺素给药为5-10次；木犀草素-7-二葡萄糖醛酸苷(中国科学院上海药物研究所提供，纯度＞98％)溶解于无菌的磷酸盐缓冲液(PBS)，木犀草素-7-二葡萄糖醛酸苷于异丙肾上腺素给药前30分钟给药，分腹腔注射和灌胃两个途径(均每日一次)。木犀草素-7-二葡萄糖醛酸苷腹腔注射给药剂量分别为：5mg/kg体重,10mg/kg体重,20mg/kg体重及40mg/kg体重，每次给药体积为100μL。木犀草素-7-二葡萄糖醛酸苷灌胃剂量为120mg/kg体重及240mg/kg体重，每次给药体积为200μL。正常组及模型对照组均同步给予等体积的无菌磷酸盐缓冲液PBS腹腔注射或灌胃。
2.小鼠心脏组织形态学
给药结束后24小时处死小鼠，采集心脏，固定于4％多聚甲醛后进行组织包埋及切片处理。组织形态学分析主要采用4μM厚度的石蜡切片进行苏木素-伊红染色，光镜(Leica，Germany)下观察心脏的组织形态学改变。胶原纤维染色采用Masson三色染色后光镜下进行观察。苏木素-伊红染色后对心脏病理改变进行评分，评分标准如下。1分：无心肌坏死，肉芽组织形成及炎细胞浸润等心肌损伤的改变；2分：偶见散在、独立的心肌坏死病灶；3分：心内膜下层见30％或30％以下心肌损伤；4分：心内膜下层30％-50％广泛的心肌损伤；5分：50％以上的心内膜下层心肌损伤。
3.小鼠心脏免疫组织化学检测
各组石蜡切片(厚度4μM)或冰冻切片(厚度10μM)进一步进行免疫组织化学研 究，一抗包括α-smooth muscle actin(α-SMA)(Sigma,USA),collagen 1(Col1)(Sigma,USA)，tissue inhibitor of metalloproteinase 1(Timp1)(Protein Tech,China),transforming growth factor-β1(TGF-β1)(Santa Cruz,China),p-SMAD2(Protein Tech,China)。二抗均购自Solarbio(China),包括山羊抗小鼠IgG用来检测α-SMA的表达，山羊抗兔IgG用来检测TGF-β1、Timp1及p-SMAD2的水平。免疫反应性最终采用3,3-二氨基联丙胺(DAB，Sigma，USA)显色并于光镜(Leica，Germany)下观察记录。
4.荧光实时定量PCR分析
组织总RNA抽提纯化(Qiagen,USA)后进行逆转录合成cDNA(RevertAid First Strand cDNA Synthesis Kit,Termo,USA)。cDNA进一步采用ABI Power SYBR Green PCR Master Mix进行荧光实时定量PCR的分析(7900HT Sequence Detection System,ABI,USA)。
5.统计学分析
实验结果重复至少三次，数据表达为mean±S.E.M,数据分析采用student’s t-test。p<0.05定义为统计学显著差异。
二、结果：
1.木犀草素-7-二葡萄糖醛酸苷抑制异丙肾上腺素诱导的小鼠心肌损伤：
异丙肾上腺素(isoproterenol,ISO)为一种β受体激动剂，可诱导梗死样心肌细胞死亡。ISO诱导的心肌损伤动物模型可模拟人心肌梗死过程中心脏形态及代谢的改变，在抗心肌损伤的药物疗效及机制研究中广泛采用。本研究中采用C57/BL6J小鼠建立ISO诱导的心肌损伤模型。ISO按照10mg/kg剂量腹腔内注射，每日一次，给药5或10天。如图1所示，木犀草素-7-二葡萄糖醛酸苷腹腔注射抑制(LC1)剂量依赖性地减轻ISO诱导的心肌细胞退行性改变、炎细胞侵润、肉芽肿形成等心肌损伤改变。如图5所示，LC1腹腔注射可显著逆转ISO诱导的心肌损伤。木犀草素-7-二葡萄糖醛酸苷灌胃给药亦体现出显著的抗ISO诱导心肌损伤的效应(图8及表1)。
表1 心肌损伤程度评分
Figure PCTCN2017074842-appb-000002
LC1L：LC1 120mg/kg体重灌胃；LC1H：LC1 240mg/kg体重灌胃
*与ISO组相比，P<0.05。
2.木犀草素-7-二葡萄糖醛酸苷抑制异丙肾上腺素诱导的心肌纤维化形成：
心肌纤维化定义为显著增加的胶原体积。因此，进一步采用Masson’s三色染色对心肌组织切片中的胶原纤维进行了评估并定量。如图2A所示，少量的Masson’s三色阳性的胶原纤维见于溶剂对照组的心肌空隙间，ISO组Masson’s三色染色的胶原纤维含量显著增加，而LC1高剂量治疗组Masson’s三色染色的胶原纤维含量显著降低。代表性Masson’s三色染色图片见图2B：溶剂对照心肌组织未见纤维化样结缔组织(a),大量Masson’s三色染色阳性的纤维化样结缔组织可见于ISO组的小鼠左心室，特别是损伤部位(b),而这一现象未见于LC1干预的小鼠左心室(c)。此外，如图6、7所示，LC1显著逆转ISO诱导的心肌纤维化。这些结果表明LC1对ISO诱导的心肌纤维化形成具有显著的抑制效应。木犀草素-7-二葡萄糖醛酸苷抑制异丙肾上腺素诱导的心肌纤维化形成的效应亦体现在接受灌胃给药的小鼠中(图9)。
3.木犀草素-7-二葡萄糖醛酸苷抑制ISO诱导的心肌细胞肥大：
ISO不仅引起心肌细胞坏死损伤，同时引起存活的心肌细胞肥大样改变，病理学分析表明与正常组相比，ISO组心肌细胞面积显著增加；与ISO组相比，LC1灌胃治疗显著降低心肌细胞面积，LC1两个剂量之间未见显著性差异(图10)。
4.木犀草素-7-二葡萄糖醛酸苷抑制纤维化形成分子标志物的表达：
为进一步明确LC1抗心肌纤维化的效应，本研究进一步分析了纤维化相关的关键分子标志物在各组的表达。如图3所示，在ISO诱导的心肌损伤模型中，α-SMA在心肌损伤部位的表达显著上调(a)，而这一现象未见于LC1干预的小鼠心肌(b)，提示LC1干预可显著抑制损伤过程中肌成纤维细胞的激活，从而抑制纤维化形成。在ISO诱导的心肌损伤模型组中，Col1在损伤部位的表达显著升高(c)，而LC1组则未观察到Col1的表达(d)，Col1是细胞外基质的主要成分，提示LC1干预可显著抑制损伤过程中细胞外基质的生成。此外，其他纤维化形成的关键分子标志物如反映介导纤维化形成的TGFβ信号通路活性的TGF-β1，p-SMAD2在ISO诱导的心肌损伤部位免疫反应性显著增强(e,g)，而其信号未见于LC1干预的小鼠心肌(f,h)，这一结果表明LC1可通过抑制纤维化形成的关键信号通路TGF-β1的活性而干预ISO诱导的心肌纤维化。进一步的分析表明，ISO诱导的纤维化形成过程中抑制胶原纤维降解的重要分子Timp1的表达在心肌损伤部位显著上 调(i)，而这一异常亦可被LC1所抑制(j)，表明LC1对纤维化形成及纤维降解等重要分子环节均具有显著的干预效应。
5.木犀草素-7-二葡萄糖醛酸苷抗纤维化作用的分子依据：
为进一步在分子水平揭示LC1抗心肌损伤的分子效应及机制，本研究采用荧光实时定量PCR对参与心肌纤维化形成的细胞外基质基因的表达进行分析，结果表明炎症反应基因及纤维化基因表达上调参与ISO诱导的心肌损伤，而LC1对这些重要的损伤相关的基因表达具有显著的抑制效应(图4)，这亦是其抗心肌损伤中重要分子效应及机制。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。

Claims (12)

  1. 木犀草素-7-二葡萄糖醛酸苷在制备抗心肌损伤药物中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述的木犀草素-7-二葡萄糖醛酸苷抑制异丙肾上腺素诱导的心肌损伤。
  3. 根据权利要求1所述的应用，其特征在于，所述的木犀草素-7-二葡萄糖醛酸苷抑制异丙肾上腺素诱导的心肌细胞坏死，肉芽组织形成，炎细胞侵润、心肌纤维化形成。
  4. 木犀草素-7-二葡萄糖醛酸苷在制备抑制心肌纤维化形成的药物中的应用。
  5. 根据权利要求4所述的应用，其特征在于，所述的木犀草素-7-二葡萄糖醛酸苷抑制异丙肾上腺素诱导的心肌纤维化形成。
  6. 根据权利要求4所述的应用，其特征在于，所述的木犀草素-7-二葡萄糖醛酸苷抑制异丙肾上腺素诱导的细胞外基质包括胶原基因表达。
  7. 木犀草素-7-二葡萄糖醛酸苷在制备抑制多器官纤维化形成的药物中的应用。
  8. 根据权利要求7所述的应用，其特征在于，所述的木犀草素-7-二葡萄糖醛酸苷抑制TGF-β/SMAD及细胞外基质包括胶原基因表达。
  9. 根据权利要求1-8任一所述应用，其特征在于，所述木犀草素-7-二葡萄糖醛酸苷以药学上可接受的盐形式存在。
  10. 根据权利要求1-8任一所述应用，其特征在于，所述木犀草素-7-二葡萄糖醛酸苷以前药形式存在。
  11. 根据权利要求10所述应用，其特征在于，所述木犀草素-7-二葡萄糖醛酸苷前药形式包括酯化、乙酰化等药学上可接受的形式。
  12. 根据权利要求1-8所述的应用，其特征在于，所述木犀草素-7-二葡萄糖醛酸苷可以单独使用或者以药物组合物的形式使用，所述药物组合物包含木犀草素-7-二葡萄糖醛酸苷、其药学上可接受的盐或它的前药，以及药学上的载体，赋形剂，稀释剂，辅剂和媒介物的至少一种。
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