CN102018702B - New application of ginkgolide B - Google Patents

Abstract

The invention discloses application of ginkgolide B to preparation of medicament for preventing and/or treating autosomal dominant polycystic kidney disease. A madin-darby canine kidney (MDCK) vesicle model is used for screening to find that the ginkgolide B inhibits formation and growth of vesicles. An experimental result shows that: the ginkgolide B has an obvious inhibiting effect on the formation and growth of MDCK vesicles and the effect of the ginkgolide B is in dose response relationship; the ginkgolide B has no cytotoxicity to MDCK cells, so that the vesicle inhibiting effect of the ginkgolide B is independent of the cytotoxicity; the ginkgolide B does not obviously induce MDCK cell apoptosis, so that the vesicle inhibiting effect of the ginkgolide B is independent of cell apoptosis promotion of the ginkgolide B; the ginkgolide B can promote the MDCK cells or vesicles to form tubular structures; and the effect is in dose response relationship; and the ginkgolide B has an inhibiting effect on the growth of the embryonic kidney vesicles. The ginkgolide B provides experimental data for development of a specific medicament for preventing and/or treating autosomal dominant polycystic kidney disease.

Description

A kind of purposes of ginkalide B

Technical field

The present invention relates to a kind of purposes of ginkalide B.

Background technology

One, the chemical feature of ginkalide B and pharmacological action

1, the general characteristic of ginkalide B

Bilobalide (ginkgolides) is from Semen Ginkgo, to extract a kind of terpene lactone compounds that obtains, and comprises ginkalide A, B, C, J, K, L and M, and bilobalide is platelet activating factor (platelet activating factor; PAF) inhibitor, wherein, ginkalide B (ginkgolideB; GB; The chemical structural formula of ginkalide B is as shown in Figure 1) be the strongest a kind of (van Beek TA.Ginkgolides and bilobalide:their physical, chromatographic and spectroscopic properties.Bioorg Med Chem, 2005 of pharmacologically active in the bilobalide; 13 (17): 5001-12.Vogensen SB; Stromgaard K, Shindou H, et al.Preparation of7-substituted ginkgolide derivatives:potent platelet activating factor (PAF) receptor antagonists.J Med Chem; 2003,46 (4): 601-8.).Research shows; Ginkalide B has effect (Xia SH such as antiinflammatory, antioxidation, antiallergic, neuroprotective; Fang DC.Pharmacological action and mechanisms of ginkgolide B.Chin Med J, 2007,120 (10): 922-8.).

2, the antiinflammatory action of ginkalide B

Many discovering, ginkalide B has antiinflammatory action, and body inflammatory reaction and inflammatory factor such as TNF-α, IL-1, IL-6, IL-8 etc. are closely related.Wherein, IL-8 is topmost inflammatory factor, and neutrophilic granulocyte is had obvious chemotactic and functional activation effect.Discover (Mao YJ; Wang L, Wang WJ.Effect of ginkgolide B on the function of rat aorta smooth cells and U937 cells stimulated by oxLDL.Yao Xue Xue Bao, 2006; 41 (1): 36-40.); OX-LDL (oxidized ldl) can induce U937 emiocytosis inflammation chemotactic factor such as MCP-1, IL-8 etc., and ginkalide B can suppress this process, and its mechanism maybe be relevant with the inhibition paf receptor.In addition; The expression that 4-hydroxyl nonenyl aldehyde can stimulate bronchial epithelial cell IL-8; Ginkalide B can reduce IL-8 (Yu L through suppressing ERK 1-AP 1 signal path; Xiong LH; Ran PX.The mechanism of inhibition by ginkgolide B on interleukin-8 production induced by4-hydroxynonenal in bronchial epithelial cells.zhonghua Jie He He Hu Xi Za Zhi, 2008,31 (5): 372-7.).

TNF-α is a kind of monokine, is mainly produced by mononuclear cell and macrophage, and LPS (lipopolysaccharide) is stronger stimulant.Ginkalide B can suppress the inductive mouse peritoneum macrophage of LPS TNF-α and produce; And can suppress the inductive rat pleura of LPS (lipopolysaccharide) polymorphonuclear leukocyte NF-KB and activate (Nie ZG; Peng SY; Wang WJ.Effects of ginkgolide B on lipopolysaccharide-induced TNFalpha productionin mouse peritoneal macrophages and NF-kappaB activation in rat pleuralpolymorphonuclear leukocytes.Acta Pharmacol Sin (Chin); 2004,39 (6): 415-8.).Ginkalide B can restraining chronic inflammation property granuloma model mice serum, IL-1 β and TNF express in the U937 cell; And angiogenesis (the Ou-Yang XY of remarkable restraining chronic inflammation property granuloma model mice; Wang WJ, LiaoWH, et al.Inhibitory effect of ginkgolide B on angiogenesis in chronic inflammation.Yao Xue Xue Bao; 2005,40 (4): 311-5.).

In addition; (Peng SY, Zhang FY, Ou-Yang XY are also found in research; Et al.Effect of ginkgolide Bon the platelet-activating factor induced changes of chemotaxis and cytoskeleton ofmacrophages.Acta Pharmacol Sin (Chin); 2006,41 (2): 156-60.), at Ca ²⁺Under the condition that exists, ginkalide B can suppress F-actin (filamentous actin) polymerization in the inductive muroid peritoneal macrophages of PAF, thereby suppresses the chemotactic of peritoneal macrophages, brings into play its antiinflammatory action.Ginkalide B can be induced a series of signal variation among the PMN (human polymorphonuclear leukocyte); Like tyrosine phosphorylation, Choline phosphatase activation, calcium transient, p38 activation etc., possibly defend relevant (Lenoir M, Muntaner O with PMN; Pedruzzi E; Et al.Ginkgolide B stimulates signaling events in neutrophils and primes defense activities.Biochem Biophys Res Commun, 2005,335 (4): 1149-54.).

3, the antioxidation of ginkalide B

In the neurocyte model of oxidative, the ginkalide B of variable concentrations all can improve H ₂O ₂Injured nerve cells survival rate (Wu XM; Chen HS; Jing SY; Et al.Effects of ginkgolides against injury ofneurocytes induced by H2O2 and expression of immediate early genes.Chin J ClinPhamacol Ther, 2003,8 (4): 401-4.).In alcohol induced Hep G2 cellular oxidation damage; The ginkalide B of low concentration (5-25 μ M) can suppress the generation of intracellular reactive oxygen and suppress apoptosis; But the ginkalide B effect of high concentration (50-100 μ M) is opposite; Prompting should be selected proper dosage (Chan WH for use; HsuuwYD.Dosage effects of ginkgolide B on ethanol-induced cell death in human hepatomaG2cells.Ann N Y Acad Sci, 2007,1095:388-98.).

4, the anti-allergic effects of ginkalide B

Ginkalide B and antioxidant ASX (carotenoid astaxanthin) coupling can suppressor T cell activate; Be superior to cetirizine (CTZ) and azelastine (AZE) coupling; Prompting can be used for asthma new drug development (MahmoudFF; Haines DD, Abul HT, et al.In vitro effects of astaxanthin combined with ginkgolideB on T lymphocyte activation in peripheral blood mononuclear cells from asthmaticsubjects.J Pharmacol Sci; 2004,94 (2): 129-36.).

5, the vascular protection effect of ginkalide B

Bilobalide all has protective effect (Xu JP to rat transience and permanent focal middle cerebral artery occlusion model and mice imperfection cerebral ischemic model; Sun LS; Yang XM.Protective effects of ginkgolideon cerebral ischemia-reperfusion injury in rats.Chin J Integr Tradit West Med IntensiveCrit Care, 2003,10 (1): 31-3.Wu XF; Wang QJ; Lou FC.Protective effect of ginkgolideson rat focal brain ischemia.J China Pharm Univ, 2001,32 (2): 141-5.).Ginkalide B can suppress calcium increase in the VSMC by selectivity; And non-selective vasopressin effect (the Wang Y that alleviates of ability; Yu YQ; Xu JG.Inhibition of intracellular calcium elevation and blunting of vasopressorresponse due to serotonin by ginkgolide B.Planta Med, 2002,68 (6): 501-4.).Left heart function and the Bcl-2 that ginkalide B also can increase the ischemia-reperfusion rat expresses, reduces and block area and lactic acid dehydrogenase release; Thereby performance cardioprotection (Hao Y; Sun Y; Xu C; Et al.Improvement of ContractileFunction in Isolated Cardiomyocytes From Ischemia-Reperfusion Rats by Ginkgolide BPretreatment.J Cardiovasc Pharmacol, 2009, doi:10.1097/FJC.0b013e3181a91410.).But discover (Jihye KIM; Qun LI; Cindy X FANG, et al.Paradoxical effects of ginkgolide Bon cardiomyocyte contractile function in normal and high-glucose environments.ActaPharmacologica Sinica, 2006; 27 (5): 536-42.), the ginkalide B effect of variable concentrations is different.In high sugar (25.5mmol/L glucose) culture environment, myocardium sarcoplasmic reticulum Ca ²⁺-ATP expression of enzymes significantly increases, and 0.5-1.0 μ g/mL ginkalide B can reduce myocardium sarcoplasmic reticulum Ca ²⁺-ATP expression of enzymes.In low sugar (5.5mmol/L glucose) culture environment, 2.0 μ g/mL ginkalide B can increase myocardium sarcoplasmic reticulum Ca-ATP expression of enzymes.In the sugared culture environment of height, the ginkalide B of two kinds of concentration all can suppress Na ⁺-Ca ²⁺Exchanger (NCX) is expressed.

6, the neuroprotective of ginkalide B

Beta-amyloid polypeptide 1-can activate the calcium channel that high voltage relies on through the AC-cAMP-PKA signal path; The calcium increase induces calcium overload to cause neurotoxicity in making; Ginkalide B can suppress the inductive calcium overload of beta-amyloid polypeptide 1-, performance neuroprotective (Chen L, Liu CJ; Tang M; Et al.Action of beta-amyloidpeptide (1-40) on I (HVA) and its modulation by ginkgolide B.Acta Physiol Sin (Chin), 2006,58 (1): 14-20.).Bilobalide also can produce anti-amnesia (Lee TF to the depression effect of cholinergic transmission through alleviating the amyloid-beta related peptides; Chen CF; Wang LC.Effect of ginkgolides onbeta-amyloid-suppressed acetylocholine release from rat hippocampal slices.PhytotherRes; 2004,18 (7): 556-60.).Discover that ginkalide B can increase, raise calbindin D28K mRNA, protection 6-hydroxyl DOPA (6-hydroxydopamine through suppressing intracellular Ca2+; 6-OHDA) PC 12 cells (the Meng H of effect; Li C, Feng L, et al.Effects of Ginkgolide B on 6-OHDA-inducedapoptosis and calcium over load in cultured PC 12.Int J Dev Neurosci; 2007,25 (8): 509-14.).Express increase at local cerebral ischemia spider cell glial fibrillary acidic protein (GFAP); Ginkalide B can be brought into play neuroprotective (Zhang JG, Li SQ, Wng SR through suppressing the GFAP expression; Et al.The effect of ginkgolide B on astrocyte GFAP expression after the focal cerebralischemia.Sichuan Da Xue Xue Bao Yi Xue Ban; 2007,38 (2): 284-6,301.).At the rat hippocampus teleneuron, ginkalide B can activate PKA, through acting on voltage-dependent N, P/Q type Ca ²⁺Passage promotes Ca ²⁺Get into, thereby promote glutamic acid to discharge.Prove that simultaneously ginkalide B can promote Ca ²⁺Carrier mediated glutamic acid discharges, and the prompting ginkalide B can directly act on Ca ²⁺The secretory in downstream (Wang SJ; ChenHH.Ginkgolide B; A constituent of Ginkgo biloba; Facilitates glutamate exocytosis fromrat hippocampal nerve terminals.Eur J Pharmacol, 2005,514 (2-3): 141-9.).

NO is a body endogenous active medium, also is a kind of endogenous neurotransmitter, and NO has dual function in many pathology and physiological process.Discover (Xu Y; Tao YX.Involvement ofthe NMDAreceptor/nitric oxide signal pathway in platelet-activating factor-induced neurotoxicity.Neuroreport.2004; 15 (2): 263-6.); Ginkalide B suppresses long term exposure NO synthase activity in the neuron of PAF, reduces the neurotoxicity of excessive N O.But bilobalide also time-dose-dependent inhibition LPS, IFN-α, TNF-α, IL-1 and inductive astrocytoma of IL-2 and SK-N-SH HNB in NO produce; Thereby suppress NO neurotoxic effect (Zhao HW; Li XY.Ginkgolide A, B, and huperzine Ainhibit nitric oxide-induced neurotoxicity.Int Immunopharmacol; 2002,2 (11): 1551-6.).Neurotransmitter L-Glu can cause the rat cerebellum neuron mortality of cultivating; Give neurotoxicity (the Chandrasekaran K that EGb761 or bilobalide can obviously reduce Glu in advance; Mehrabian Z; Spinnewyn B, et al.Neuroprotective effects of bilobalide, a component of Ginkgo biloba extract (EGb 761) in global brain ischemia and in excitotoxicity-induced neuronal death.Pharmacopsychiatry; 2003,36 (Suppl 1): S89-94.).

Discover (Bate C; Salmona M; Williams A.Ginkgolide B inhibits the neurotoxicityofprions or amyloid-betal-42.J Neuroinflammation; 2004,1 (1): 4.), PAF antagonist ginkalide B also possibly be used for alzheimer ' Gadamer disease or prion disease treatment.The neural ball of NSC (NSCs) and single cell clone can be induced to differentiate into neuron (Jin GH by ginkalide B; Huang Z; Tan XF; Et al.Effects of Ginkgolide on the development of NOS and AChE positive neurons in theembryonic basal forebrain.Cell Biol Int, 2006,30 (6): 500-4.).Ginkalide B can promote NSC (NSCs) to be divided into the male astrocyte of GFAP (Yoshida H; Imaizumi T; Tanji K; Et al.Platelet-activating factor enhances the expression of nerve growth factor in normalhuman astrocytes under hypoxia.Brain Res Mol Brain Res, 2005,133 (1): 95-101.).

7, ginkalide B genotoxic potential effect

Ginkalide B only has some faint side effect; Comprise (Guinot P.Clinical experience with platelet-activating factor antagonists.Past, present and nearfuture.Clin Rev Allergy 1994 such as singultus, headache, drowsiness, weakness; 12:397-417.Brown SL; Jala VR, Raghuwanshi SK, et al.Activation and regulation of platelet-activating factor receptor:role of G (i) and G (q) inreceptor-mediated chemotactic; Cytotoxic; And cross-regulatory signals.J Immunol, 2006,177 (5): 3242-9.).Ginkalide B is external to suppress embryonic stem cell proliferation and blastocyst is grown; Cause fetal development damage (Chan WH.Ginkgolide B induces apoptosis and developmental injury inmouse embryonic stem cells and blastocysts.Hum Reprod in the body; 2006,21 (11): 2985-95.).

Two, autosomal dominant inheritance, AD MCKD pathogenesis and therapeutic advance

Autosomal dominant inheritance, AD MCKD (autosomal dominant polycystic kidney disease; ADPKD) be a kind of common monogenic inheritance disease; Sickness rate abroad accounts for the 4th of China's end stage renal failure cause of disease up to 1/1000-1/400.At present, found that three genes are relevant with the ADPKD morbidity, wherein PKD1 gene (polycystic kidney disease 1) is positioned chromosome 16p13.3, and the sudden change of this gene accounts for 85% of ADPKD; PKD2 gene mapping is in 4q21-23, and the sudden change of this gene accounts for 15% of ADPKD; Several familys, still no-fix are only found in the sudden change of PKD3 gene.The ADPKD pathological changes is a principal character with multiple the carrying out property of two kidneys vesicle, causes change in renal function, and cause renal failure whole latter stage, often with organ cystic lesion and cardio cerebrovascular affections such as liver, spleen, pancreases.At present, remove and use renal transplantation and dialysis treatment end-stage renal failure, still do not have generation and growth that specific short stops vesicle.Therefore, the course of disease that delays ADPKD morbidity even reverse ADPKD is this hot research fields both at home and abroad.

The albumen of PKD1 gene and PKD2 gene code be called respectively many capsules albumen 1 (polycystin-1, PC1) with many capsules albumen 2 (polycystin-2, PC2).Wherein, PC1 is the renal cells membrane receptor, and main Subcellular Localization is in renal epithelial cell cilium, tight connecting portion, adhesion connection, desmosome, talin etc.; PC2 has the effect of calcium channel, and main Subcellular Localization is in cilium, endoplasmic reticulum, centrosome etc.At renal cells cilium place, PC1 and PC2 interaction composition function complex, but PC1 perception renal tubules intracavity liquid and passes to PC2 with signal to the mechanical stimulus of cilium, and PC2 mediates Ca ²⁺Interior stream, Ca ²⁺Interior stream further causes endocytoplasmic reticulum Ca ²⁺Release, keep Ca in the born of the same parents ²⁺Concentration is to normal level.Ca ²⁺In the normal cell of level, Ca ²⁺Can be through suppressing the generation that adenyl cyclase 6 suppress cAMP, simultaneously can also activating phosphatase diesterase (PDE) and degraded cAMP, therefore, Ca in the cell ²⁺The concentration of cAMP in can the negative regulation cell.When PKD1 gene or PKD2 gene are undergone mutation, Ca in the cell ²⁺Concentration reduces, Ca ²⁺The effect of negative regulation cAMP weakens, and cAMP concentration increases in the cell.CAMP activated protein kinase A (PKA) in the cell that increases, PKA can activate the Ras/Raf/MEK/ERK signal path and promote the epithelial propagation of vesicle; PKA can also activate cystic fibrosis transmembrance regulator (CFTR) and promote chloride ion secretion to blister cavities, improves osmotic pressure in the vesicle, causes that sodium and water Secondary cases transport in vesicle and promote the secretion of capsule liquid, finally causes the pathological change of polycystic kidney.Other there are some researches show; PC1 can act on protein tyrosine kinase (JAK2) in the presence of PC2; Raise the p21waf expression of gene through signal transduction and activating transcription factor 1 (STAT1); P21 cyclin-dependent kinase capable of inhibiting cell (CDK) and the G1 phase is prolonged, thus cell proliferation suppressed ^[51]PC1 can also act on JAK2 in the presence of PC2, promote the renal tubules differentiation of hepatocyte growth factor (HGF) mediation through signal transduction and activating transcription factor 3 (STAT3).When PKD1 gene or PKD2 gene were undergone mutation, the p21waf expression of gene reduced, and cell proliferation increases, and renal tubules differentiation simultaneously reduces, and promotes the generation of polycystic kidney.PC1 can be through forming the activity that complex suppresses mTOR with tuberous sclerosis (TSC2) gene and mTOR, thereby regulate the mTOR signal path.In ADPKD; The disappearance of PC1 can cause the abnormal activation of mTOR signal path, makes cell hyperproliferation (Bhunia AK, Piontek K; Boletta A; Et al.PKD1induces p21 (waf1) and regulation of the cell cycle via direct activation of the JAK-STATsignaling pathway in a process requiring PKD2.Cell, 2002,109 (2): 157-68.).In normal renal cells, PC1 participates in β-chain of rings, and plain (β-catenin) forms the process of complex with E-calcium adhesion molecule (E-cadherin), keep the cell connection; PC1 can also raise the degraded of the active β-catenin of promotion of glycogen synthase kinase 3 β (GSK3 β); Thereby reduced levels (the MostovKE.mTOR is out of control in polycystic kidney disease.Proc Natl Acad Sci USA of β in the maintenance Cytoplasm-catenin concentration; 2006,103 (14): 5247-8.).In ADPKD; The disappearance of PC1 can make cell connect β in damage, the kytoplasm-catenin concentration and raise; While Wnt signal path abnormal activation; Wnt blocks β-catenin degraded through the formation of Dishevelled albumen antagonism β-catenin degraded complex (APC/Axin) also can make β-catenin in Cytoplasm, assemble, and promotes cell hyperproliferation through transcribing into nuclear.Therefore according to the pathogenesis of polycystic kidney, can screen the medicine (see figure 2) of treatment ADPKD through suppressing methods such as vesicle epithelial cell proliferation and the secretion of capsule liquid.

At present, the research of treatment ADPKD still is in the exploratory stage, and therapeutic strategy mainly is to concentrate on cell proliferation, suppress the secretion of capsule liquid, regulate Ca in the born of the same parents according to its pathogenesis ²⁺Concentration with alleviate aspect such as secondary affection.Set forth from these several aspects below:

1, cell proliferation

The epithelial propagation of ADPKD vesicle possibly relate to multiple somatomedin; Like epidermal growth factor (epidermal growth factor; EGF) can stimulate the epithelial propagation of vesicle, its mechanism is closely related with the tyrosine kinase activity of EGF-R ELISA Erb B/EGFR (epidermal growth factor receptor), Src, MEK mitogen-activated protein kinase kinase (mitogen-activated protein kinase kinase) etc.Therefore, tyrosine kinase inhibitor has very big potentiality in the treatment of ADPKD.Discover Erb B like Wilson etc. ₂Inhibitor can recover the normal configuration of polycystic kidney vesicle cell and improve its function (BocaM; D ' Amato L; Distefano G; Et al.Polycystin-1 induces cell migration by regulatingphosphatidylinositol 3-kinase-dependent cytoskeletal rearrangements andGSK3beta-dependent cell cell mechanical adhesion.Mol Biol Cell, 2007,18 (10): 4050-61.).Other discovers; The Src inhibitor can be repaired abnormal epithelial cell (the Wilson SJ of polycystic kidney kidney; Amsler K, Hyink DP, et al.Inhibition of HER-2 (neu/ErbB2) restores normal functionand structure to polycystic kidney disease (PKD) epithelia.Biochim Biophys Acta; 2006,1762 (7): 647-55.).Mek inhibitor can suppress the vesicle cell proliferation of pcy polycystic kidney mice; Delay depleted (the Sweeney WE Jr of its kidney merit; Von Vigier RO, Frost P, Avner ED.Src inhibitionameliorates polycystic kidney disease.J Am Soc Nephrol; 2008,19 (7): 1331-41.).Other thunderous handkerchief mycin (Omori S; Hida M, Fujita H, et al.Extracellular signal-regulated kinaseinhibition slows disease progression in mice with polycystic kidney disease.J Am SocNephrol; 2006; 17 (6): 1604-14.), cyclin dependant kinases inhibitor (Berthier CC, Wahl PR, Le Hir M; Et al.Sirolimus ameliorates the enhanced expression ofmetalloproteinases in a rat model of autosomal dominant polycystic kidney disease.Nephrol Dial Transplant; 2008,23 (3): 880-9.), caspase-3 inhibitor (Bukanov NO, Smith LA; Klinger KW; Et al.Long-lasting arrest of murine polycystic kidney diseasewith CDK inhibitor roscovitine.Nature, 2006,444 (7121): 949-52.) waiting also can be through influencing the effect that cell proliferation plays treatment ADPKD.

2, suppress the secretion of capsule liquid

(cystic fibrosis transmembrane conductanceregulator is the activated ATP gate property of an a kind of cAMP chloride channel CFTR) to cystic fibrosis transmembrance regulator, expresses at the renal epithelial cell teleblem.In the ADPKD pathogenesis, but activated adenyl cyclases such as vasopressin are activated the interior cAMP concentration increase of born of the same parents, PKA; PKA can activate the CFTR that is positioned at vesicle epithelial cell teleblem makes chloride ion secretion to blister cavities, improves osmotic pressure in the vesicle, cause that sodium and water Secondary cases transport in vesicle, and in vesicle, accumulate, and accumulating of capsule liquid has caused vesicle epithelial cell compensatory hypertrophy.Therefore, suppress CFTR and can treat ADPKD effectively.Many researchs show that the CFTR overexpression plays important effect in the polycystic kidney morbidity.MDCK (mdck cell) PKD model, external embryo kidney model and PKD mouse model confirm that all the CFTR inhibitor significantly suppresses vesicle formation and growth (Tao Y in vitro and in vivo; Kim J; Faubel S; Et al.Caspase inhibition reduces tubular apoptosis and proliferation and slows diseaseprogression in polycystic kidney disease.PNAS, 2005,102 (19): 6954-9.).Other are like vasopressin V ₂Receptor (V ₂R) antagonist (Yang B, Sonawane ND, Zhao D; Et al.Small-moleculeCFTR inhibitors slow cyst growth in polycystic kidney disease.J Am Soc Nephrol; 2008,19 (7): 1300-10.), SSA (Wang X, Wu Y; Ward C J; Et al.Vasopressindirectly regulates cyst growth in polycystic kidney disease.J.Am Soc Nephrol, 2008,19 (1): 102-8.) wait also and can be used for treating ADPKD through regulating the secretion of capsule liquid.

3, regulate Ca in the born of the same parents ²⁺Concentration

In the ADPKD morbidity, PKD1 gene or PKD2 gene are undergone mutation, Ca in the cell ²⁺Concentration reduces, Ca in the low-level cell ²⁺Through influencing in the cell and cell differentiation growth, signal transduction pathway that gene expression is relevant with the epicyte protein function controlling, cause that vesicle forms, vesicle epithelial cell abnormality proliferation and vesicle liquid excessive secretion, cause the polycystic kidney pathological change then.Can use Ca ²⁺Channel agonist, Ca ²⁺Carrier (Ruggenenti P, Remuzzi A, Ondei P; Et al.Safety and efficacy of long-acting somatostatin treatment inautosomal-dominant polycystic kidney disease.Kidney Int; 2005,68 (1): 206-16.), PDE agonist (Yamaguchi T, Hempson SJ; Reif GA; Et al.Calcium restores a normalproliferation phenotype in human polycystic kidney disease epithelial cells.J Am SocNephrol, 2006,17 (1): 178-87.) wait through raising Ca in the born of the same parents ²⁺Concentration is treated ADPKD.In addition, Crews group finds that Triptolide can be through Ca in the cell that promotes the PC2 mediation ²⁺Release and promote the expression (P21 is the mortifier of CDK) of P21 thus performance suppresses the effect of cell proliferation; This process does not rely on the expression of PC1; The prompting Triptolide can be used for treating ADPKD (Cheng J; Grande JP.Cyclic nucleotidephosphodiesterase (PDE) inhibitors:novel therapeutic agents for progressive renal disease.Exp Biol Med, 2007,232 (1): 38-51.).

4, alleviate secondary affection

The growth of ADPKD vesicle and the reconstruction of tissue need the degraded and the reorganization of substrate; Because metalloprotein endonuclease capable matrix degradation increases vesicle easily, and makes surrounding tissue reconstruct in tissue; Therefore can use inhibitors of metalloproteinase to treat ADPKD (Leuenroth SJ; Okuhara D, Shotwell JD, et al.Triptolide is a traditional Chinese medicine-derived inhibitor of polycystic kidney disease.PNAS; 2007,104 (11): 4389-94.).Some hypoglycemic medicines also can be alleviated ADPKD symptom (Nakamura T, Ushiyama C, Suzuki S through regulating matrix build-up; Et al.Elevation of serum levels of metalloproteinase-1; Tissue inhibitor of metalloproteinase-1 and type IV collagen, and plasma levels of metalloproteinase-9 in polycystic kidney disease.Am J Nephrol, 2000; 20 (1): 32-6.Muto S; Aiba A, Saito Y, et al.Pioglitazone improves the phenotype and molecular defects of a targeted Pkd1 mutant.Hum Mol Genet; 2002,11 (15): 1731-42.).The ADPKD pathogenic process often is accompanied by reconstructed tissue and blood vessel hyperplasia; And VEGF (VEGF) is main short angiogenesis factor; Discover that Application V EGF inhibitor can suppress formation and growth (Yuan ZZ, the Xu CG of Han:SPRD rat (Cy/+) vesicle; Gao CF; Mei CL.Effect of lovastatin on proliferation and extracellular matrix secretion in cyst-lining epithelial cells of autosomal dominant polycystic kidney disease.Acad J Sec Mil Med Univ, 2006,27 (1): 111-2.).The formation of ADPKD patient's scrotum bubble can activate renin angiotensin aldosterone system (renin-angiotensin-aldosterone system RAAS), makes sclerosis of blood vessels and causes hypertension, left ventricular hypertrophy with enlarging.Zoopery confirms; Use angiotensin converting enzyme inhibitor (angiotensin-converting enzyme inhibitors, ACEI) or angiotensin receptor blocker (angiotensin receptor blocker, ARB) controlling blood pressure possibly delay the polycystic kidney renal failure; But some clinical researches do not confirm its effect (Tao Y; Kim J, Yin Y, et al.VEGF receptor inhibition slows the progression of polycystic kidney disease.Kidney Int; 2007,72 (11): 1358-66.).

According to pathogenesis and the therapeutic strategy of ADPKD, the screening vesicle forms and growth inhibitor, and the novel drugs of research and development treatment ADPKD is present this hot research fields.

Summary of the invention

A kind of purposes that the purpose of this invention is to provide ginkalide B.

The purposes of ginkalide B provided by the present invention is specially:

1) with the ginkalide B is the medicine that prevents and/or treats the autosomal dominant inheritance, AD MCKD of active component;

2) ginkalide B prevents and/or treats the application in the medicine of autosomal dominant inheritance, AD MCKD in preparation;

3) with the ginkalide B be the inhibition vesicle formation of active component and/or the medicine of growth;

4) application of ginkalide B in the medicine of preparation inhibition vesicle formation and/or growth.

In the such use, said cell is MDCK or mice embryonic nephrocyte.

The above-mentioned medicine that prevents and/or treats the autosomal dominant inheritance, AD MCKD can import body through injection or oral mode.

The said amount of drug that prevents and/or treats the autosomal dominant inheritance, AD MCKD is generally: 20-100mg/ people/sky (injection) or 200-1000mg/ people/sky (oral).

The present invention uses MDCK vesicle model discrimination and is inhibited that vesicle forms and the ginkalide B of growth, confirms the toxicity of this chemical compound and to the influence of normal cell Growth and Differentiation through mtt assay; Generate of the influence of experimentation ginkalide B with the MDCK tubule to the renal epithelial cell differentiation; Confirm pharmacologically active in the whole kidney of ginkalide B through external embryo kidney model; The specific medicament that prevents and/or treats the autosomal dominant inheritance, AD MCKD for exploitation provides experimental data.Experimental result shows that the MDCK vesicle is formed ginkalide B and growth has the obvious suppression effect, and its effect is dose-effect relationship; Ginkalide B explains that to the effect of mdck cell no cytotoxicity effect and its cytotoxicity of ginkalide B inhibition vesicle is irrelevant; The not obvious induction MDCK apoptosis of ginkalide B confirms that effect and its promotion apoptosis of ginkalide B inhibition vesicle is irrelevant; Ginkalide B can promote mdck cell or vesicle to form the tubule spline structure, and effect is dose-effect relationship; And ginkalide B is also inhibited to the growth of embryo kidney vesicle.

Description of drawings

Fig. 1 is the chemical structural formula of ginkalide B

Fig. 2 is the pathogenesis of ADPKD

Fig. 3 is MDCK vesicle and cell colony

Fig. 4 is the inhibitory action that ginkalide B forms the MDCK vesicle

Fig. 5 is the inhibitory action of ginkalide B to the vesicle growth

Fig. 6 is the inhibitory action curve of ginkalide B to the vesicle growth

Fig. 7 is the reversible inhibition curve of ginkalide B to the vesicle growth

Fig. 8 detects the cytotoxicity of ginkalide B to mdck cell for mtt assay

Fig. 9 is that ginkalide B is to mdck cell apoptosis induced effect picture

Figure 10 is that ginkalide B is to the effect of mdck cell apoptosis induced

Figure 11 is the facilitation that ginkalide B generates the MDCK tubule

Figure 12 is ginkalide B forms the tubule spline structure to vesicle a facilitation

Figure 13 is ginkalide B forms the tubule spline structure to vesicle a facilitation picture

Figure 14 is the regulating action of ginkalide B to the mdck cell signal transduction pathway

Figure 15 is mice embryonic scrotum bubble model sketch map

Figure 16 is the forming process of mice embryonic scrotum bubble

Figure 17 is the inhibitory action of ginkalide B to the growth of embryo kidney vesicle

Figure 18 suppresses the dosage effect of mice embryonic scrotum steep raising exhibition for ginkalide B

The specific embodiment

Experimental technique described in the following embodiment like no specified otherwise, is conventional method; Said reagent and biomaterial like no specified otherwise, all can obtain from commercial sources.

The inhibitory action that embodiment 1, ginkalide B form and grow the MDCK vesicle

One, ginkalide B can suppress the formation of vesicle, and effect is dose-effect relationship

1, vesicle forms and suppresses experiment

MDCK (MDCK) is stimulated by cAMP when in three dimensional matrix glue, cultivating and forms vesicle, and sustainable growth.Forskolin is the activator of adenyl cyclase; Adenyl cyclase can mediate the generation of cAMP; Therefore forskolin can promote the MDCK vesicle to form and growth; Its vesicle characteristic is similar with the characteristic of polycystic kidney vesicle, is the best external model of screening and assessment compounds for treating polycystic kidney pharmacologically active.

Mdck cell (available from U.S. ATCC company, catalogue No CCL-34) is incubated at respectively contains 10 μ M forskolin (Buddhist SCH, available from Sigma company, article No. F6886) and concentration is respectively 0M, 12.5 * 10 ^-8M, 5 * 10 ^-7M and 2 * 10 ^-6The ginkalide B of M is (available from Sigma company; Catalogue No G6910) three dimensional matrix glue (Purecol Collagen; Available from Inamed Biomaterials Fremont company, article No. 5409) in, about 400 cells in every hole; Cultivate after 4-5 days, in the hole of only containing 10 μ M forskolin, form the vesicle model of mdck cell.Each dosage repeats 3 holes.The colony of counting sacculus rotundus (diameter is greater than 50 μ m) and non-vesicle cell when cultivating the 6th day calculates the percentage rate that vesicle accounts for total cell colony (vesicle and non-vesicle colony).Three repetitions are established in experiment, and the result shows that the percentage rate that vesicle accounts for total cell colony is 33.82 ± 0.93%.The form of vesicle and non-vesicle cell is as shown in Figure 3.Wherein, control representes to add in this hole the culture fluid that does not contain forskolin, and forskolin representes to add in this hole the culture fluid that contains 10 μ M forskolin.Ginkalide B is as shown in Figure 4 to the inhibitory action that the MDCK vesicle forms.As can be seen from Figure 4; Ginkalide B can obviously reduce the inductive MDCK vesicle of forskolin number; And inhibitory action is dose-effect relationship (black part among the figure), but ginkalide B does not influence total colony number (comprising vesicle and non-vesicle colony, the summation of white and black part among the figure).Above presentation of results, ginkalide B form the MDCK vesicle has the obvious suppression effect, but does not have cytotoxicity.

2, vesicle growth inhibited experiment

Mdck cell is incubated in the three dimensional matrix glue that contains 10 μ M forskolin, cultivates and to form the sacculus rotundus that diameter is 50-100 μ m after 4-5 days; And then the adding final concentration is respectively 0M, 1.25 * 10 in Tissue Culture Plate ^-7M, 5 * 10 ^-7M and 2 * 10 ^-6The ginkalide B of M continues to cultivate.Renew the bright culture fluid that contains ginkalide B and forskolin every day, each vesicle of tracking shot and measure the vesicle diameter and suppress the effect that vesicle is grown to estimate ginkalide B was observed 8-12 days altogether every other day.Each dosage repeats 3 holes, and 10 above vesicles of every hole counting are made the vesicle growth curve.Three repetitions are established in experiment, and ginkalide B is as shown in Figure 5 to the inhibitory action of vesicle growth.Wherein, First row's expression used the culture fluid that only contains forskolin to cultivate in 4-12 days; Second row's expression used the culture fluid that contains ginkalide B and forskolin to cultivate in 4-12 days; The 3rd row's expression was only cultivated with the culture fluid that contains forskolin with the culture fluid cultivation that contains ginkalide B and forskolin in 4-7 days in 8-12 days.Ginkalide B is as shown in Figure 6 to the inhibitory action curve of vesicle growth.The result shows; Mdck cell is cultivated in the three dimensional matrix glue that contains 10 μ M forskolin can form sacculus rotundus after 4 days; And vesicle is progressivity growth (seeing Fig. 5 first row's cell), and as can be seen from Figure 6, ginkalide B can obviously suppress the vesicle growth.

Two, ginkalide B can suppress the growth of vesicle reversiblely, and effect is dose-effect relationship

Mdck cell is incubated in the three dimensional matrix glue that contains 10 μ M forskolin, cultivates and to form circular vesicle after 4 days; In Tissue Culture Plate, adding final concentration since the 4th day is 1.25 * 10 ^-7The ginkalide B of M continues to cultivate, and renews the bright culture fluid that contains ginkalide B and forskolin every day; During by the 8th day, in Tissue Culture Plate, only add the forskolin that final concentration is 10 μ M, and do not add ginkalide B, until the 12nd day.Measure the vesicle diameter every day, three repetitions are established in experiment, and the situation of ginkalide B reversibility inhibition vesicle growth is as shown in Figure 7.Wherein, the medicine ball curve representation adds forskolin and the ginkalide B that final concentration is 10 μ M simultaneously, and it is the forskolin of 10 μ M and do not add ginkalide B that the hollow ball curve representation only adds final concentration.The result shows, remove ginkalide B after, vesicle can recover growth, explain that ginkalide B does not damage the vesicle epithelial cell, shows that the inhibitory action that ginkalide B is grown to vesicle is reversible.

The influence of the cytotoxicity of embodiment 2, ginkalide B, cell growth and differentiation

1, confirms the cytotoxicity of ginkalide B through mtt assay

In 96 well culture plates, every hole contains 4 * 10 with the mdck cell suspension inoculation of logarithmic (log) phase ³Individual cell; Every hole gives 200 μ l mdck cell culture fluid (by the DMEM culture medium (available from American I nvitrogen company; Catalogue No 12100-046) and the F12 culture medium (available from American I nvitrogen company; Catalogue No 21700-075) equal-volume mixes), place 37 ℃ 5%CO ₂Cultivated 24 hours in the incubator.In Tissue Culture Plate, add final concentration then and be respectively 1 * 10 ^-4M, 1 * 10 ^-5M, 1 * 10 ^-6M and 1 * 10 ^-7The ginkalide B 20 μ l of M continue to cultivate 24 hours.Remove supernatant, add 200 μ l mdck cell culture fluid and 20 μ l concentration are the MTT solution of 5mg/ml, continue to cultivate 3 hours.Remove supernatant, every hole adds 150 μ l dimethyl sulfoxide, puts 100 rev/mins of vibration 10min on the shaking table, and crystal is fully dissolved.ELIASA detects each hole OD value (the detection wavelength is 492nm), and zeroing hole (culture medium, MTT and dimethyl sulfoxide) and control wells (dissolve medium of the ginkalide B of cell, same concentrations, culture medium, MTT and dimethyl sulfoxide) are set, 5 multiple holes of every settings.Calculate suppression ratio according to the following equation: suppression ratio=[(control wells-zeroing hole)-(dosing holes-zeroing hole)]/(control wells-zeroing hole) * 100%.Three repetitions are established in experiment, and the MTT result of cell is as shown in Figure 8 behind the adding ginkalide B.Wherein, control representes with the not culture fluid cultivation of bilobalide-containing B.The result shows 1 * 10 ^-5The ginkalide B of the following concentration of M explains that to the effect of mdck cell no cytotoxicity effect and its cytotoxicity of ginkalide B inhibition vesicle is irrelevant.

2, Tunel test kit (available from Roche company, article No. 11 684 795 910) is confirmed the influence of ginkalide B pair cell apoptosis

Mdck cell is incubated in the culture fluid of being made up of isopyknic DMEM culture medium and F12 culture medium, and when cell density reaches 30-50%, adding concentration respectively is 1.25 * 10 ^-7M, 5 * 10 ^-7M, 2 * 10 ^-6The ginkalide B of M continues to cultivate 48 hours; Remove culture fluid, with the PBS washed cell once, then under 15-25 ℃ condition, using volumn concentration is 4% paraformaldehyde (fixative) fixed cell 60 minutes.Reuse PBS washing once adds the sodium citrate agent (changing liquid thoroughly) of the 0.1% quality percentage composition that contains 0.1% volumn concentration Triton X-100, and (2-8 ℃) hatched 2 minutes on ice.Removing liquid, experimental group, the positive controls that ginkalide B is handled add 50 μ l TUNEL respectively and detect liquid, and negative control group adds 50 μ l fluorescent labeling liquid (Label solution), and in a humid environment, 37 ℃ of lucifuges were hatched 60 minutes.With PBS washing 3 times, observe down, count, take a picture with fluorescence microscope after anti-fluorescent quenching mounting liquid (available from Beyotime, the article No. P0126) mounting.Under optical microscope, the nucleus of apoptotic cell is dyed pale brown color, and the visible cell nuclear morphology is broken point-like.Under fluorescence microscope, select the excitation wavelength of 450-500nm and the emission wavelength of 515-565nm, apoptotic cell shows green fluorescence.5 high power fields of picked at random, the apoptosis cell of each group (experimental group, positive controls and negative control group) of statistics is also done statistical analysis.Three repetitions are established in experiment, and ginkalide B is to the effect of mdck cell apoptosis induced such as Fig. 9 and shown in Figure 10.Among Fig. 9 and Figure 10, control representes the apoptosis situation of negative control group cell, and gentamycin representes the apoptosis situation of positive controls cell, and ginkalide B representes to add the apoptosis situation of the experimental group cell of ginkalide B.The result shows, the not obvious induction MDCK apoptosis of ginkalide B confirms that effect and its promotion apoptosis of ginkalide B inhibition vesicle is irrelevant.

3, MDCK tubule generation experiment confirm ginkalide B can promote MDCK to form the tubule spline structure, and effect is dose-effect relationship

Mdck cell is being contained 1.25 * 10 respectively ^-7M, 5 * 10 ^-7M, 2 * 10 ^-6The ginkalide B of M and 3T3 fibroblast culture fluid (use the DMEM culture fluid that contains 10% hyclone (available from American I nvitrogen company; Catalogue No 12100-046; Cultivated the 3T3 fibroblast 3; The gained culture fluid is 3T3 fibroblast culture fluid) three dimensional matrix glue in cultivated 15 days; Renew bright culture fluid that contains ginkalide B and 3T3 fibroblast culture fluid and photograph every day, 10 above tubules are followed the tracks of in every hole, count through tubule and estimate the influence of ginkalide B to the mdck cell differentiation.Three repetitions are established in experiment, and ginkalide B is shown in figure 11 to the facilitation that the MDCK tubule generates.Wherein, control representes to cultivate mdck cell 15 days with the 3T3 fibroblast culture fluid of bilobalide-containing B not.The result shows that ginkalide B can promote mdck cell to form the tubule spline structure, and effect is dose-effect relationship.

4, MDCK tubule generation experiment confirm ginkalide B can promote vesicle to form the tubule spline structure, and effect is dose-effect relationship

Mdck cell is incubated in the three dimensional matrix glue that contains 10 μ M forskolin, cultivates and to form the sacculus rotundus that diameter is 50-100 μ m after 4 days; Stop to add forskolin, change into and contain 1.25 * 10 respectively ^-7M, 5 * 10 ^-7M, 2 * 10 ^-6The HGF of M ginkalide B or 3T3 fibroblast culture fluid continue to cultivate, and renew bright HGF that contains ginkalide B or 3T3 fibroblast culture fluid every day.The MDCK vesicle sprouts in 3T3 fibroblast culture fluid, synapse, one-tenth rope with become pipe, be similar to kidney epithelial cell tubule generative process.After 15 days, add up the length of formed tubule number and tubule.Three repetition are established in experiment, ginkalide B to the facilitation of vesicle formation tubule spline structure shown in Figure 12 and 13.Among Figure 12, control representes to cultivate with the 3T3 fibroblast culture fluid of bilobalide-containing B not 4 days MDCK vesicle 15 days.The result shows that ginkalide B can promote vesicle to form the tubule spline structure, and effect is dose-effect relationship.

5, the action target spot of ginkalide B and mechanism

Utilize the Western engram technology, detect the influence of ginkalide B vesicle epithelial cell signal transduction pathway.Concrete experimentation is following:

Mdck cell is cultured to 70% cell density, changes serum-free medium and cultivated 24 hours, add the culture fluid that contains 10 μ M forskolin then respectively, give ginkalide B simultaneously respectively, make its final concentration in culture medium be respectively 0M, 1.25 * 10 ^-7M, 5 * 10 ^-7M and 2 * 10 ^-6M, the mdck cell of cultivating with the culture fluid that does not contain forskolin is simultaneously organized as control, and above-mentioned each group all stimulates 240min.

Extract the above-mentioned total protein of respectively organizing cell, make protein quantification with the Bradford method.SDS-PAGE electrophoresis, commentaries on classics film add corresponding one anti-(p-ERK, sc7383, ERK2; Sc-153, B-raf; Sc-166, Raf-1, sc-227 and β-actin, sc47778; Above-mentioned each antibody is all available from U.S. Santa Cruz Biotechnology company) and two anti-(available from U.S. Amersham company, catalogue No NA934VS).With ECL PLUS (available from U.S. GE Healthcare company, catalogue No ALSZR004-096412) chemiluminescence, development, photographic fixing.Find the influence of ginkalide B through detecting B-raf, Raf-1 and p-ERK protein level to the Ras-B-Raf-MEK-ERK signal path.In ADPKD, B-raf expresses to be increased, the Raf-1 expression decreased, and ginkalide B can be reduced B-raf, raises Raf-1, and can suppress the p-ERK phosphorylation, and the result is shown in figure 14.

Embodiment 3, confirm the inhibitory action of ginkalide B to embryo kidney vesicle growth through external embryo kidney model

Carry out male and female with the cage copulation with 6 week ICR mices in age (available from Department Of Medicine, Peking University's Experimental Animal Center) according to 1: 1 quantity the 1st day afternoon; Whether observe female Mus the 2nd day morning has cloudy bolt; If the female Mus of cloudy bolt pool expression conceived half day arranged; To not have the mice of cloudy bolt to divide cage earlier, mate afternoon again, observed in 1st again; The pregnant female Mus is continued to feed 13 days separately, got embryo kidney and cultivate in the 13rd day with the transwell plate.

Get above-mentioned 13.5 days mice embryonic kidney at the 8-Br-cAMP of 100 μ m (available from Sigma company; Article No. B-1381) effect down; In nephridial tissue, form scrotum bubble multiple, the growth of carrying out property, can be used as and estimate the external whole organ model that ginkalide B prevents and/or treats ADPKD.Simultaneously with the mice embryonic kidney handled without 8-Br-cAMP as contrast.The sketch map of embryo kidney vesicle model is shown in figure 15.The vesicle forming process of mice embryonic kidney is shown in figure 16.Wherein, control representes the embryo kidney that 8-Br-cAMP of no use handles, and 8-Br-cAMP representes the embryo kidney that the 8-Br-cAMP with 100 μ m handled.

It is 1.25 * 10 that the embryo kidney of above-mentioned formation vesicle is used concentration respectively ^-7M, 5 * 10 ^-7M, 2 * 10 ^-6The ginkalide B of M is handled, and three repetition are established in experiment, and the inhibitory action that ginkalide B is grown to the embryo kidney vesicle is shown in Figure 17 and 18.Among Figure 17, control representes not the embryo kidney handled with 8-Br-cAMP and ginkalide B; 8-Br-cAMP representes only to handle with 8-Br-cAMP, and the embryo kidney of not handling with ginkalide B; Ginkalide B 1.25 * 10 ^-7, ginkalide B 5 * 10 ^-7, ginkalide B 2 * 10 ^-6Expression is simultaneously with the embryo kidney of the ginkalide B of 8-Br-cAMP and respective concentration processing respectively.

The result shows, the vesicle that ginkalide B can suppress in the inductive 13.5 days mice embryonic nephridial tissue of 8-Br-cAMP forms, and inhibitory action strengthens with the increase of ginkalide B concentration.

Claims (2)

1. ginkalide B prevents and/or treats the application in the medicine of autosomal dominant inheritance, AD MCKD in preparation.

2. the application of ginkalide B in the medicine of preparation inhibition cell formation vesicle and/or the growth of inhibition vesicle; Said cell is MDCK or mice embryonic nephrocyte.

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