CN102018689A - New application of curcumin - Google Patents

Abstract

The invention discloses the application of curcumin in the preparation of medicines for preventing and/or treating autosomal dominant polycystic kidney disease. The present invention uses the MDCK vesicle model to screen and obtain curcumin that inhibits vesicle formation and growth, and the experimental results show that curcumin has a significant inhibitory effect on MDCK vesicle formation and growth, and its effect is in a dose-effect relationship; curcumin has an effect on MDCK The cells have no cytotoxic effect, indicating that the effect of curcumin on inhibiting vesicles has nothing to do with its cytotoxicity; curcumin does not significantly induce apoptosis in MDCK cells, confirming that the effect of curcumin on inhibiting vesicles has nothing to do with its promotion of apoptosis; curcumin can promote MDCK cell apoptosis. Or vesicles form tubule-like structures, and the effect is in a dose-response relationship; and curcumin also has an inhibitory effect on the growth of embryonic kidney vesicles. Curcumin may be developed as a specific drug for the prevention and/or treatment of autosomal dominant polycystic kidney disease.

Description

A kind of new purposes of curcumin

Technical field

The present invention relates to a kind of new purposes of curcumin.

Background technology

One, the characteristic of curcumin and pharmacological action

1, the general characteristic of curcumin

(curcumin Cur) is a kind of natural polyphenol compounds (its chemical constitution as shown in Figure 1) that extracts to curcumin from rhizome such as Zingiberaceae curcuma Rhizoma Curcumae Longae, Rhizoma Curcumae, Radix Curcumae.Curcumin is soluble in organic solvents such as methanol, ethanol, alkali, acetic acid, acetone, dimethyl sulfoxide, and water-soluble hardly, molecular formula is C ₂₁H ₂₀O ₆, molecular weight is 368.38, its main chain is unsaturated aliphatic and aromatic group.Curcumin has multiple pharmacological effect such as antioxidation, fibrosis, antitumor, antidepressant, antiinflammatory, antiviral, antithrombotic, angiogenesis inhibitor and immunomodulating, and toxicity is very low.But the blood drug level of oral curcumin is low, and bioavailability is not high.

2, the antioxidation of curcumin

In recent years, many curcumins that studies confirm that can raise the various antioxidation factors, reduce malonaldehyde (MDA) generation, and the performance antioxidation.In various oxidative stresss, because enzymatic oxidation and antioxidation dysequilibrium cause tending to the former infringement, act on lipid generation peroxidization as free radical, end-product is MDA, and the latter can cause the cross-linked polymeric of life macromolecules such as protein, nucleic acid, and has cytotoxicity.The antioxidation factor has: (catalase CAT), glutathion peroxidase (GSH-Px), reduced glutathion (GSH) etc., has very important physiological action to catalase.Glutathion peroxidase (GSH-Px) is a kind of important peroxide breakdown enzyme, can become GSSG by catalysis GSH, makes peroxide be reduced into hydroxy compounds, and promotes H ₂O ₂Decomposition, the performance antioxidation.Catalase equally can catalysis H ₂O ₂Decomposition, thereby the protection cell membrane 26S Proteasome Structure and Function.

In the inductive male Swiss mice Liver and kidney lipid peroxidation experiment of aflatoxin, giving curcumin (50mg/kg) can raise the antioxidation factor of enzyme and non-enzyme on the 45th and bring into play antioxidation (Verma R J, Mathuria N.Curcumin ameliorates aflatoxin-induced lipid-peroxidation in liver andkidney of mice.Acta Pol Pharm, 2008,65 (2): 195-202.), and can alleviate Liver and kidney DNA, RNA, proteinic infringement (Mathuria N, Verma R J.Ameliorative effect of curcumin onaflatoxin-induced toxicity in DNA, RNA and protein in liver and kidney of mice.ActaPol Pharm, 2007,64 (6): 497-502.).Curcumin can also alleviate oxidative damage (the Tirkey N of the inductive rat kidney of cyclosporin, Kaur G, Vij G, et al.Curcumin, a diferuloylmethane, attenuatescyclosporine-induced renal dysfunction and oxidative stress in rat kidneys.BMCPharmacol, 2005,5:15.).Give curcumin in advance and also can alleviate the oxidative stress damage.As giving 2 weeks of curcumin (200mg/kg) in advance, can improve the inductive male rat kidney of gentamycin oxidative damage (Farombi E O, Ekor M.Curcumin attenuates gentamicin-induced renal oxidative damage in rats.FoodChem Toxicol, 2006,44 (9): 1443-8.).Give curcumin (50mg/kg) 3 days in advance, can alleviate the inductive male Wistar rat kidney of FeNTA lipid peroxidation injury (Eybl V, Kotyzova D, Cerna P, et al.Effect of melatonin, curcumin, quercetin, and resveratrol on acute ferricnitrilotriacetate (Fe-NTA)-induced renal oxidative damage in rats.Hum Exp Toxicol, 2008,27 (4): 347-53.), inductive male mice lipid peroxidation injury (the Eybl V of Caddy (Cleary), Kotyzova D, Koutensky J, et al.Comparative study of natural antioxidants-curcumin, resveratrol andmelatonin-in cadmium-induced oxidative damage in mice.Toxicology, 2006,225 (2-3): 150-6.).Give curcumin (200mg/kg) in advance and can pass through to raise glutathion peroxidase (GSH-Px) antioxidation on the 7th, and alleviate effect (the Bayrak O of rat kidney ischemical reperfusion injury, Uz E, BayrakR, et al.Curcumin protects against ischemia/reperfusion injury in rat kidneys.World JUrol, 2008,26 (3): 285-91.).

3, the anti-fibrosis effect of curcumin

Curcumin has the effect of fibrosis, as can be used for the fibrosis of organs such as anti-liver, lung, kidney, pancreas.Its mechanism of action mainly produces, increases the vigor of metalloproteases and regulate various signal factors relevant with inhibition fibroblasts proliferation, minimizing extracellular matrix (ECM).

Curcumin can be induced peroxisome proliferation body activated receptor r (PPAR r) gene expression, activate PPARr, the activation and the propagation that suppress hepatic stellate cell, thereby make degree of hepatic fibrosis reduce (Xu J, Fu Y, Chen A.Activation of peroxisome proliferator-activated receptor-gamma contributes to theinhibitory effects of curcumin on rat hepatic stellate cell growth.Am J PhysiolGastrointest Liver Physiol, 2003,285 (1): G20-30.), and can also induce apoptosis (the Tourkina E of scleroderma lung fibroblast (SLF), Gooz P, Oates J C, etal.Curcumin-induced apoptosis inscleroderma lung fibroblasts:role of protein kinase cepsilon.Am J Respir Cell Mol Biol, 2004,31 (1): 28-35.).Curcumin (50mg/kg) can reduce SD rat nephrotoxic serum nephritis model mesonephric glomerulus collagen protein IV, fibronectin content, performance anti-fibrosis effect (Bao H Y, Chen R H, Huang SM, et al.[Effect of curcumin on extracellular matrix accumulation in the glomeruli innephrotoxic sera nephritis rats] .Zhong Xi Yi Jie He Xue Bao, 2004,2 (1): 30-2.).Matrix metalloproteinase (matrix metallo-proteinase, the effect that MMPs) have degraded ECM composition, slows down fibrosis lesion.Curcumin can be expressed by suppressing matrix metallo-proteinase inhibitor (TIMPs), improve local host metalloproteases (MMPs) activity and effect (the Jiang Y of performance fibrosis, Li Z S, Jiang F S, et al.Effects of different ingredients of zedoary on gene expression of HSC-T6 cells.World JGastroenterol, 2005,11 (43): 6780-6.).Curcumin can suppress lung fibroblast α-smooth muscle filamentous actin (expression (Hu Y of α-SMA) of the beta induced In vitro culture of TGF-, Peng J, Feng D, etal.Role ofextracellular signal-regulated kinase, p38 kinase, and activator protein-1 in transforminggrowth factor-betal-induced alpha smooth muscle actin expression in human fetal lungfibroblasts in vitro.Lung, 2006,184 (1): 33-42.).In the beta induced kidney fibroblast experiment of In vitro culture TGF-, curcumin can be reduced TGF-β II receptor, reduces the phosphorylation of Smad, suppress the c-jun vigor, reduce generation (the Gaedeke J of ECM, Noble N A, Border W A, et al.Curcumin blocks multiplesites of the TGF-beta signaling cascade in renal cells.Kidney Int, 2004,66 (1): 112-20.).In rat unilateral ureteral occlusion (UUO) model, curcumin can be reduced short fiber factor performance anti-fibrosis effect, and suppress nuclear Factor-Kappa B (NF-κ B) signal pathway (the Kuwabara N that may play an important role, Tamada S, Iwai T, etal.Attenuation of renal fibrosis by curcumin in rat obstructive nephropathy.Urology, 2006,67 (2): 440-6.).In the glomerulonephritis rat model, curcumin can be by raising heme oxidase-1 (heme oxygenase 1, HO-1) bring into play anti-fibrosis effect, and be dose dependent, reach maximum inhibitory action (Gaedeke J during 50-100mg/kg, Noble N A, Border W A, et al.Curcumin blocksfibrosis in anti-Thy 1 glomerulonephritis through up-regulation of heme oxygenase 1.Kidney Int, 2005,68 (5): 2042-9.).

In recent years, still there is many disputes (Egan M E in the effect of the anti-cystic fibrosis of relevant curcumin, Pearson M, Weiner S A, et al.Curcumin, a major constituent of turmeric, corrects cystic fibrosisdefects.Science, 2004,304 (5670): 600-2.Dragomir A, Bjorstad J, Hjelte L, et al.Curcumin does not stimulate cAMP-mediated chloride transport in cystic fibrosis airwayepithelial cells.Biochem Biophys Res Commun, 2004,322 (2): 447-51.), discover recently, although curcumin can not strengthen the expression of cystic fibrosis transmembrance regulator (deltaF508-CFTR), but can be by downward modulation calcium net matter albumen (calreticulin, CRT) improve function (the Harada K of hamster ovary cell deltaF508-CFTR, Okiyoneda T, Hashimoto Y, etal.Curcumin enhances cystic fibrosistransmembrane regulator expression by down-regulating calreticulin.Biochem BiophysRes Commun, 2007,353 (2): 351-6.).

4, the antitumor action of curcumin

The antitumor action of curcumin mainly suppresses tumor cell proliferation, inducing apoptosis of tumour cell by a series of signal approach, and curcumin also has effects such as the neoplasm metastasis of inhibition, angiogenesis inhibitor, raising immunity simultaneously.

Curcumin can make cell division arrest G2 and G2/S phase, thereby suppresses the propagation of breast cancer cell.Holy (Holy J M.Curcumin disrupts mitotic spindle structure and induces micronucleationin MCF-7breast cancer cells.Mutat Res, 2002,518 (1): 71-84.) discover that curcumin blocking-up cell is relevant with the dyskaryosis assembling in the G2/M phase.(Squires M S such as Squires, Hudson E A, HowellsL, etal.Relevance of mitogen activated protein kinase (MAPK) andphosphotidylinositol-3-kinase/protein kinase B (PI3K/PKB) pathways to induction ofapoptosis by curcumin in breast cells.Biochem Pharmacol, 2003,65 (3): 361-76.) confirm that curcumin is by influencing the propagation that the S/G2/M cell cycle suppresses HBL100 and MDA-MB-468 human breast cancer cell.Choudhuri (Choudhuri T, Pal S, Agwarwal M L, etal.Curcumin inducesapoptosis in human breast cancer cells through p53-dependent Bax induction.FEBS Lett, 2002,512 (1-3): 334-40.) etc. the discovery curcumin can be induced the apoptosis of MCF-7 breast cancer cell, and its mechanism is with to raise wild type p53 relevant with the Bax expression.

Curcumin can be induced the apoptosis of human lung carcinoma cell, its mechanism and relevant (the Radhakrishna Pillai G of expression that reduces p53, bcl-2, bcl-X, Bak and caspase, Srivastava A S, Hassanein T I, etal.nductionof apoptosis in human lung cancer cells by curcumin.Cancer Lett, 2004,208 (2): 163-70.).

Curcumin can be induced K562 leukaemia's apoptosis, it may mechanism be: suppress the expression of glutathione transferase GSTP-1, the expression of blocking-up tumor necrosis factor TNF-alpha, suppressing Buddhist ripple inductive transcription factor activated protein-1 of ester (AP-1) and NF-κ B combines with the promoter of GSTP-1 gene in conjunction with the transcription factor that forms, suppress the inductive GSTP-1 genetic transcription of TNF-α activity (Duvoix A, Morceau F, Delhalle S, etal.Induction of apoptosis by curcumin:mediation by glutathione S-transferase P1-1inhibition.Biochem Pharmacol, 2003,66 (8): 1475-83.).

Discover (Sindhwani P, Hampton J A, Baig M M, etal.Curcumin preventsintravesical tumor implantation of the MBT-2 tumor cell line in C3H mice.J Urol, 2001,166 (4): 1498-501.) curcumin and people UMUC and Mus MBT-2 transitional cell bladder carcinoma cell line are hatched altogether, can suppress the growth of two kinds of cells, and can observe the existence of apoptotic cell, prompting has significant inhibitory effect to the growth and the transplanting of transitional cell bladder carcinoma cell line.Other discovers (Tong Q S, Zheng L D, Lu P, et al.Apoptosis-inducingeffects of curcumin derivatives in human bladder cancer cells.Anticancer Drugs, 2006,17 (3): 279-87.) curcumin can raise apoptotic proteins enzyme-3 (caspase-3) in the bladder cancer EJ cell and the apoptosis of promotion EJ cell, but does not influence normal renal cells.

Curcumin can stop the mispairing reparation of rectum cancer cell DNA, and bring into play antitumaous effect (Chauhan D P by activity that suppresses cytochrome P 450 enzymes and the activity that improves the glutathion invertase, etal.Chemotherapeutic potential of curcumin for colorectal cancer.Curr Pharm Des, 2002,8 (19): 1695-706.).

Curcumin also can suppress the growth of hepatocarcinoma QGY cell, its tumour inhibiting rate has dose dependent and time dependence, flow cytometry analysis confirms that cell stops at the S phase, electron microscopic observation finds that cell has degeneration, necrosis, apoptosis (Li Hongyuan, the car skill, Deng. curcumin is to the influence of human liver cancer cell propagation and apoptosis. Chinese hepatopathy magazine, 2002,10 (6): 449-451.).

5, the antidepressant effect of curcumin

In recent years, discover (Xu Y, Ku B S, Yao HY, etal.Antidepressant effects of curcuminin the forced swim test and olfactory bulbectomy models of depression in rats.PharmacolBiochem Behav.2005,82 (1): 200-6.), curcumin has certain antidepressant effect in experiment of rat forced swimming and bilateral olfactory bulb injury experiment.Discover that subsequently curcumin can repair the neuronic damage of chronic stress rat depression model hippocampus, can raise five hydroxytryptamine 1A (5-HT simultaneously _1A) receptor and Brain Derived Neurotrophic Factor (BDNF) level, thereby performance antidepressant effect (Xu Y, Ku B, Cui L, et al.Curcuminreverses impaired hippocampal neurogenesis and increases serotonin receptor 1A mRNAand brain-derived neurotrophic factor expression in chronically stressed rats.Brain Res, 2007,1162:9-18.).Discover that further the curcumin antidepressant mediates by serotonergic system, and confirm at least and 5-HT _1A/1BReceptor and 5-HT _2CReceptor related (Wang R, Xu Y, Wu H L, etal.Theantidepressant effects of curcumin in the forced swimming test involve 5-HT1 and5-HT2 receptors.Eur J Pharmacol, 2008,578 (1): 43-50.).

6, the antiinflammatory action of curcumin

Inflammatory factors such as tumor necrosis factor (TNF-α), interleukin have important effect in inflammation generation evolution.Curcumin can be by suppressing the rat kidney inflammation infringement that TNF-α improves cisplatin induction, and be dose dependent (Kuhad A, Pilkhwal S, Sharma S, et al.Effect of curcumin oninflammation and oxidative stress in cisplatin-induced experimental nephrotoxicity.JAgric Food Chem, 2007,55 (25): 10150-5.).Wistar rat acute pancreatitis model latter stage, curcumin can be reduced TNF-α and interleukin-6 (IL-6) and performance antiinflammatory action (Gulcubuk A, Altunatmaz K, Sonmez K, et al.Effects of curcumin on tumour necrosis factor-alpha and interleukin-6in the late phase of experimental acute pancreatitis.J Vet Med A Physiol Pathol ClinMed, 2006,53 (1): 49-54).Discover recently, curcumin can reduce TNF-α and induce the IL-1 β of HaCaT cell and IL-6 to produce, but do not influence IL-8 (Cho J W, Lee K S, Kim C W.Curcuminattenuates the expression of IL-l beta, IL-6, and TNF-alpha as well as cyclin E inTNF-alpha-treated HaCaT cells; NF-kappaB and MAPKs as potential upstream targets.Int J Mol Med, 2007,19 (3): 469-74.).

7, the antivirus action of curcumin

Curcumin has active (the Araujo C C of resisting HIV (HIV), Leon L L Biologicalactivities of Curcuma longa L.Mem Inst Oswaldo Cruz, 2001,96 (5): 723-8.), its mechanism is main by inhibition long terminal repeat, the relevant enzyme (reverse transcriptase, protease and HIV-1 intergrase) of virus replication and the activity of cytokine, and brings into play anti-HIV effect.Curcumin also can reach effect (the Hergenhahn M that suppresses Epstein-Barr virus by suppressing the BZLF1 gene transcription, Soto U, Weninger A, etal.Thechemopreventive compound curcumin is an efficient inhibitor of Epstein-Barr virusBZLF1 transcription in Raji DR-LUC cells.Mol Carcinog, 2002,33 (3): 137-45.).

8, the anti thrombotic action of curcumin

(Srivastava R is found in research in 1985, Dikshit M, Srimal R C, etal.Anti-thromboticeffect of curcumin.Thromb Res, 1985,40 (3): 413-7.), curcumin all can suppress the inductive platelet aggregation of collagen and epinephrine in vitro and in vivo.In recent years, studies confirm that (Chen H W, Kuo H T, Chai CY, et al.Pretreatment of curcumin attenuates coagulopathy and renal injury inLPS-induced endotoxemia.J Endotoxin Res, 13 (1): 15-23.), intraperitoneal gives curcumin and can improve the infringement of lipopolysaccharide-induced rat disseminated inravascular coagulation (DIC) in advance, its mechanism is: curcumin can reduce the consumption of platelet, plasma fibrinogen, and can reduce the renal glomerulus fibrin deposition.

9, the blood vessel formation against function of curcumin

Studies have shown that (Mohan R, Sivak J, Ashton P, et al.Curcuminoids inhibit the angiogenicresponse stimulated by fibroblast growth factor-2, including expression of matrixmetalloproteinase gelatinase B.J Bio Chem, 2000,275 (14): 10405-12.), curcumin part and oral administration all have the effect of angiogenesis inhibitor.Curcumin can by suppress the active generation that suppresses blood vessel of MMP (Li Jianming, poplar peace. bisdemethoxycurcumin blood vessel formation against function molecules Mechanism Study. Chinese clinical rehabilitation, 2004,8 (27): 5830-1.).Curcumin can also suppress the cattle endothelial cell migration of Ox blood serum and the promotion of tumor cell conditioned medium, thus angiogenesis inhibitor (Gao Chengxian, Ding Zhishan, Liang Bingbing, Deng. curcumin is to the experimentation of angiogenesis influence. Chinese crude drug, 2003,26 (7): 499-502.).Curcumin can suppress vegf protein and mRNA among the human liver cancer cell BEL-7402 expression (Sun Jun, Wang Heling, Li Yan. curcumin is to the influence of human liver cancer cell medium vessels endothelial growth factor expression. China's digestion magazine, 2006,26 (12): 843-4.).

10, the immunoregulation effect of curcumin

Studies confirm that (Varalakshmi Ch, Ali A M, Pardhasaradhi, B V, et al.Immunomodulatory effects of curcumin:in-vivo.Int Immunopharmacol, 2008,8 (5): 688-700.), secular lumbar injection curcumin does not weaken the immunologic function of natural killer cell, macrophage, T cell, can promote the propagation of T cell on the contrary, be a kind of immunomodulator safely and effectively therefore.Curcumin can suppress the activation and the hypertrophy of T lymphocyte, bone-marrow-derived lymphocyte and macrophage, and the secretion that suppresses production of antibodies and lymphokine, mechanism is with the expression of reducing CD28, CD80 and raise relevant (the Sharma S of CTL related antigen, Chopra K, Kulkarni S K, et al.Resveratrol and curcuminsuppress immune response through CD28/CTLA-4and CD80 co-stimulatory pathway.Clin Exp immunol, 2007,147 (1): 155-63.).Curcumin also can suppress lymphocytic propagation (the Ranjan D that makes a variation, Chen C, Johnston T D, et al.Curcumin inhibits mitogen stimulatedlymphocyte proliferation, NFkappaB activation, and IL-2 signaling.J Surg Res, 2004,121 (2): 171-7.), suppress mitogen ConA, PHA, propagation (the Gururajan M of the inductive people's splenocyte of PMA, Dasu T, Shahidain S, et al.Spleen tyrosine kinase (Syk), a novel target ofcurcumin, is required for B lymphoma growth.J Immunol, 2007,178 (1): 111-21.), and can influence the immunologic function of normal natural killer cell (NK cell), increase cytotoxicity (the YadavV S of NK cell, Mishra K P, Singh D P, etal.Immunomodulatory effects of curcumin.Immunopharmacol Immunotoxicol.2005,27 (3): 485-97.).Curcumin can suppress the tumor necrosis factor (TNF-2) of lipopolysaccharide or the inductive macrophage of mitogen, mononuclear cell, endotheliocyte and medullary cell and express (Kim G Y, Kim K H, Lee S H, etal.Curcumin inhibits immunostimulatory functionof dendritic cells:MAPKs and translocation of NF-kappa B as potential targets.JImmunol, 2005,174 (12): 8116-24.).

11, the effect of curcumin treatment diabetes

Curcumin can improve the renal function of the inductive male SD diabetes rat of streptozotocin; its mechanism is: suppress the expression of heat shock protein-27 and map kinase p38; dephosphorization acid and the acetylation (TikooK of inhibition of histone H3; Meena R L; Kabra D G; et al.Change in post-translational modifications of histoneH3; heat-shock protein-27 and MAP kinase p38 expression by curcumin instreptozotocin-induced type I diabetic nephropathy.Br J Pharmacol.2008,153 (6): 1225-31.).

12, the effect for reducing blood fat of curcumin

Curcumin can be by increasing various antioxidases such as Epoxide hydrolase, glutathione s-transferase, the activity of glutathion peroxidase etc., the metabolism that changes fatty acid is drained LDL as increasing gallbladder, suppress the picked-up of spleen to LDL, and reduction hyperlipidemia model rat fat level, play blood fat reducing and study of anti-atherogenic effect (Asai, A, Miyazawa T.Dietary curcuminoids prevent high fat diet-induced lipidaccumulation in rat liver and epididymal adipose tissue.J Nutr, 2001,131 (11): 2932-5.).

13, curcumin genotoxic potential effect

The blood drug level of curcumin is low, and bioavailability is not high, and oral Rhizoma Curcumae Longae have 40%～75% without changing by gastrointestinal tract, and absorbed curcumin is metabolism in small intestinal mucosa and liver mainly.Acute and chronic animal experiment confirms its mouse oral clothes Rhizoma Curcumae Longae ethanol extraction (ETE), does not see significant mortality difference; The mice that inferior chronic body weight is handled is compared with matched group and is not seen that tangible body weight changes, and the weight of internal organs such as the heart, lung obviously changes, and the RBC in the blood, WBC level significantly reduce; Do not find significant sperm teratogenesis toxicity.

In sum, have many pharmacotoxicological effects, have not yet to see any itself and multicystic kidney disease relevant report with treatment takes place though have been found that curcumin.

Two, autosomal dominant inheritance, AD multicystic kidney disease pathogenesis and therapeutic advance

Autosomal dominant inheritance, AD multicystic kidney disease (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 the research focus in this field both at home and abroad.

The albumen of PKD1 gene and PKD2 gene code be called many capsules albumen 1 (polycystin-1, PC1) and 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 by 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 by signal transduction and activating transcription factor 1 (STAT1), p21 cyclin-dependent kinase capable of inhibiting cell (CDK) and the G1 phase is prolonged, thereby suppress cell proliferation (Bhunia AK, Piontek K, Boletta A, et al.PKD1 induces p21 (waf1) and regulation of thecell cycle via direct activation of the JAK-STAT signaling pathway in a process requiringPKD2.Cell, 2002,109 (2): 157-68.).PC1 can also act on JAK2 in the presence of PC2, promote the renal tubules differentiation of hepatocyte growth factor (HGF) mediation by 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 by 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, make cell hyperproliferation (MostovKE.mTOR is out of control in polycystic kidney disease.Proc Natl Acad Sci USA, 2006,103 (14): 5247-8.).In normal renal cells, PC1 participates in β-chain of rings, and plain (β-catenin) and E-calcium adhesion molecule (E-cadherin) form the process of complex, 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 Boca M of β in the maintenance Cytoplasm-catenin concentration, D ' Amato L, Distefano G, et al.Polycystin-1induces cellmigration by regulating phosphatidylinositol 3-kinase-dependent cytoskeletalrearrangements and GSK3beta-dependent cell cell mechanical adhesion.Mol Biol Cell, 2007,18 (10): 4050-61.).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 by the formation of Dishevelled albumen antagonism β-catenin degraded complex (APC/Axin) also can make β-catenin assemble in Cytoplasm, 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 by 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 and alleviate aspect such as secondary affection.Set forth from these several aspects below:

1, cell proliferation

The epithelial propagation of ADPKD vesicle may relate to multiple somatomedin, as 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 as Wilson etc. ₂Inhibitor can recover the normal configuration of polycystic kidney vesicle cell and improve its function (Wilson SJ, Amsler K, Hyink DP, et al.Inhibition of HER-2 (neu/ErbB2) restores normalfunction and structure to polycystic kidney disease (PKD) epithelia.Biochim BiophysActa, 2006,1762 (7): 647-55.).Other discovers, the Src inhibitor can be repaired abnormal epithelial cell (the Sweeney WE Jr of polycystic kidney kidney, von Vigier RO, Frost P, Avner ED.Src inhibitionameliorates polycystic kidney disease.J Am Soc Nephrol, 2008,19 (7): 1331-41.).Mek inhibitor can suppress the vesicle cell proliferation of pcy polycystic kidney mice, delay its kidney merit depletion (Omori S, Hida M, Fujita H, et al.Extracellular signal-regulated kinase inhibition slows disease progressionin mice with polycystic kidney disease.J Am Soc Nephrol, 2006,17 (6): 1604-14.).Other thunderous handkerchief mycin (Berthier CC, Wahl PR, Le Hir M, et al.Sirolimus ameliorates the enhancedexpression of metalloproteinases in a rat model of autosomal dominant polycystic kidneydisease.Nephrol Dial Transplant, 2008,23 (3): 880-9.), cyclin dependant kinases inhibitor (Bukanov NO, Smith LA, Klinger KW, et al.Long-lasting arrest of murinepolycystic kidney disease with CDK inhibitor roscovitine.Nature, 2006,444 (7121): 949-52.), caspase-3 inhibitor (Tao Y, Kim J, Faubel S, et al.Caspase inhibitionreduces tubular apoptosis and proliferation and slows disease progression in polycystickidney disease.PNAS, 2005,102 (19): 6954-9.) waiting also can be by 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 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 accumulate in vesicle, and accumulating of capsule liquid has caused vesicle epithelial cell compensatory hypertrophy.Therefore, suppress CFTR and can treat ADPKD effectively.Many studies show that, the CFTR overexpression plays important effect in the polycystic kidney morbidity.Madin-Darby canine kidney(cell line) (mdck cell) PKD model, external embryo kidney model and PKD mouse model confirm that all the CFTR inhibitor significantly suppresses vesicle formation and growth (Yang B in vitro and in vivo, Sonawane ND, Zhao D, etal.Small-molecule CFTR inhibitors slow cyst growth in polycystic kidney disease.J AmSoc Nephrol, 2008,19 (7): 1300-10.).Other are as vasopressin V ₂Receptor (V ₂R) antagonist (WangX, Wu Y, Ward C J, et al.Vasopressin directly regulates cyst growth in polycystic kidneydisease.J.Am Soc Nephrol, 2008,19 (1): 102-8.), somatostatin analogue (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.) wait also and can be used for treating ADPKD by 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⁺By 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 (Yamaguchi T, Hempson SJ, Reif GA, et al.Calcium restores a normal proliferation phenotype in humanpolycystic kidney disease epithelial cells.J Am Soc Nephrol, 2006,17 (1): 178-87.), PDE agonist (Cheng J, Grande JP.Cyclic nucleotide phosphodiesterase (PDE) inhibitors:novel therapeutic agents for progressive renal disease.Exp Biol Med, 2007,232 (1): 38-51.) wait by raising Ca in the born of the same parents ²⁺Concentration is treated ADPKD.In addition, Crews group finds that Triptolide can be by 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 (Leuenroth SJ, Okuhara D, Shotwell JD, et al.Triptolide is atraditional Chinese medicine-derived inhibitor of polycystic kidney disease.PNAS, 2007,104 (11): 4389-94.).

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, vesicle is increased in tissue easily, and make surrounding tissue reconstruct, therefore can use inhibitors of metalloproteinase to treat ADPKD (Nakamura T, Ushiyama C, Suzuki S, et al.Elevation of serumlevels of metalloproteinase-1, tissue inhibitor of metalloproteinase-1 and type IVcollagen, and plasma levels of metalloproteinase-9 in polycystic kidney disease.Am JNephrol, 2000,20 (1): 32-6.).Some hypoglycemic medicines also can be alleviated ADPKD symptom (Muto S by regulating matrix build-up, Aiba A, Saito Y, et al.Pioglitazone improves the phenotype and moleculardefects of a targeted Pkd1 mutant.Hum Mol Genet, 2002,11 (15): 1731-42.Yuan ZZ, XuCG, Gao CF, Mei CL.Effect of lovastatin on proliferation and extracellular matrixsecretion in cyst-lining epithelial cells of autosomal dominant polycystic kidney disease.Acad J Sec Mil Med Univ, 2006,27 (1): 111-2.).The ADPKD pathogenic process often is accompanied by reconstructed tissue and blood vessel hyperplasia, and VEGF (VEGF) is main short angiogenesis factor, discover, use formation and growth (Tao Y that the VEGF inhibitor can suppress Han:SPRD rat (Cy/+) vesicle, KimJ, Yin Y, et al.VEGF receptor inhibition slows the progression of polycystic kidneydisease.Kidney Int, 2007,72 (11): 1358-66.).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 may delay the polycystic kidney renal failure, but some clinical researches do not confirm its effect (Van Dijk MA, Breuning MH, Duiser R, et al.No effect ofenalapril on progression in autosomal dominant polycystic kidney disease.Nephrol DialTransplant, 2003,18:2314-20.).

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 the research focus in present this field.

Summary of the invention

A kind of new purposes that the purpose of this invention is to provide curcumin.

The new purposes of curcumin provided by the present invention is specially:

1) with the curcumin is the medicine that prevents and/or treats the autosomal dominant inheritance, AD multicystic kidney disease of active component;

2) curcumin prevents and/or treats application in the medicine of autosomal dominant inheritance, AD multicystic kidney disease in preparation;

3) with the curcumin be the inhibition cell formation vesicle of active component and/or the medicine that suppresses the vesicle growth;

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

In the above-mentioned new purposes, described cell is Madin-Darby canine kidney(cell line) or mice embryonic nephrocyte.

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

The described amount of drug that prevents and/or treats the autosomal dominant inheritance, AD multicystic kidney disease is generally: 0.1-3mg/kg body weight (injection) or be lower than 1.2g/kg body weight (oral).

The present invention uses MDCK vesicle model discrimination and is inhibited that vesicle forms and the curcumin of growth, determines the toxicity of this chemical compound and to the influence of normal cell Growth and Differentiation by mtt assay; Generate of the influence of experimentation curcumin with the MDCK tubule to the renal epithelial cell differentiation; Determine pharmacologically active in the kidney of curcumin by external embryo kidney model.Experimental result shows that the MDCK vesicle is formed curcumin and growth has the obvious suppression effect, and its effect is dose-effect relationship; In the dosage range that detects, curcumin illustrates that to the effect of mdck cell no cytotoxicity effect and its cytotoxicity of curcumin inhibition vesicle is irrelevant; Curcumin is suppressing that vesicle generates and the dosage level of growth induction MDCK apoptosis not, confirms that the effect that curcumin suppresses vesicle has nothing to do with its promotion apoptosis; Curcumin can promote mdck cell or vesicle to form the tubule spline structure, and effect is dose-effect relationship; And curcumin is also inhibited to the growth of embryo kidney vesicle.

Description of drawings

Fig. 1 is the chemical structural formula of curcumin

Fig. 2 is the pathogenesis of ADPKD

Fig. 3 is MDCK vesicle and cell colony

Fig. 4 is the inhibitory action that curcumin forms vesicle

Fig. 5 is the inhibitory action of curcumin to the vesicle growth

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

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

Fig. 8 detects the cytotoxicity of curcumin to mdck cell for MTT

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

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

Figure 11 is the facilitation that curcumin generates the MDCK tubule

Figure 12 is curcumin forms the tubule spline structure to vesicle a facilitation picture

Figure 13 is curcumin forms the tubule spline structure to vesicle a facilitation

Figure 14 is mice embryonic scrotum bubble model sketch map

Figure 15 is the forming process of mice embryonic scrotum bubble

Figure 16 is the inhibitory action of curcumin to the growth of embryo kidney vesicle

Figure 17 is the inhibiting dosage effect of curcumin to the growth of embryo kidney vesicle

Figure 18 is the effect of curcumin to the mdck cell signal transduction pathway

The specific embodiment

Experimental technique described in the following embodiment if no special instructions, is conventional method; Described reagent and biomaterial if no special instructions, all can obtain from commercial channels.

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

1, curcumin can suppress the formation of vesicle

Madin-Darby canine kidney(cell line) (MDCK) is stimulated by cAMP when cultivating in three dimensional matrix glue 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 to 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 (the Buddhist SCH is available from U.S. Sigma company, catalogue No F6886) and concentration is respectively 0M, 4 * 10 ^-7M, 2 * 10 ^-6M and 1 * 10 ^-5The curcumin of M is (available from U.S. Fluka company, catalogue No 28260) three dimensional matrix glue (Purecol Collagen, available from Inamed Biomaterials Fremont company, catalogue No 5409) in, about 400 cells in every hole, cultivate after 4-5 days, in the culture hole that contains 10 μ Mforskolin, form the vesicle of mdck cell.Each dosage is established 3 parallel 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 percentage rate that vesicle accounts for total cell colony is 33.82 ± 0.93%.The form of vesicle and non-vesicle cell as shown in Figure 3.Wherein, control represents to add in this hole the culture fluid that does not contain forskolin, and forskolin represents to add in this hole the culture fluid that contains 10 μ M forskolin.The inhibitory action that curcumin forms the MDCK vesicle as shown in Figure 4.As can be seen from Figure 4, curcumin can obviously reduce the inductive MDCK vesicle of forskolin number, and inhibitory action is dose-effect relationship (black part among the figure), but curcumin 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, curcumin form the MDCK vesicle has the obvious suppression effect, but does not have cytotoxicity.

2, curcumin can suppress the growth of vesicle

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, 4 * 10 in Tissue Culture Plate ^-7M, 2 * 10 ^-6M and 1 * 10 ^-5The curcumin of M continues to cultivate.Renew the bright culture fluid that contains curcumin and forskolin every day, each vesicle of tracking shot and measure the vesicle diameter to estimate the effect that curcumin suppresses the vesicle growth 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 repetition are established in experiment, and the inhibitory action that curcumin is grown to vesicle as shown in Figure 5.Wherein, first row's expression used the culture fluid that contains forskolin to cultivate in 4-12 days, second row's expression used the culture fluid that contains curcumin 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 curcumin and forskolin in 4-7 days in 8-12 days.Curcumin to the inhibitory action curve of vesicle growth as shown in Figure 6.The result shows that mdck cell is cultivated can form sacculus rotundus after 4 days in the three dimensional matrix glue that contains 10 μ M forskolin, and vesicle is progressivity growth (seeing Fig. 5 first row's photo), and as can be seen from Figure 6, curcumin can obviously suppress the vesicle growth.

3, curcumin can reversibly suppress the growth of vesicle

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; Adding final concentration since the 4th day in Tissue Culture Plate is 4 * 10 ^-7The curcumin of M continues to cultivate, and renews the bright culture fluid that contains curcumin 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 curcumin, until the 12nd day.Measure the vesicle diameter every day, three repetitions are established in experiment, and the situation of curcumin reversibility inhibition vesicle growth as shown in Figure 7.Wherein, the medicine ball curve representation adds forskolin and the curcumin that final concentration is 10 μ M simultaneously, and it is the forskolin of 10 μ M and do not add curcumin that the hollow ball curve representation only adds final concentration.The result shows, remove curcumin after, vesicle can recover growth, illustrate that curcumin does not damage the vesicle epithelial cell, shows that the inhibitory action that curcumin is grown to vesicle is reversible.

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

1, determines the cytotoxicity of curcumin by 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 curcumin of M continues 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.Microplate reader 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 curcumin of cell, same concentrations, culture medium, MTT and dimethyl sulfoxide) are set, and sets 5 multiple holes for every group.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 as shown in Figure 8 behind the adding curcumin.Wherein, control represents to cultivate with the culture fluid that does not contain curcumin.The result shows 1 * 10 ^-5The curcumin of the following concentration of M illustrates that to the effect of mdck cell no cytotoxicity effect and its cytotoxicity of curcumin inhibition vesicle is irrelevant.

2, Tunel test kit (available from U.S. Roche company, catalogue No 11684795910) is determined the influence of curcumin pair cell apoptosis

Adding concentration when mdck cell is cultured to cell density 30-50% respectively is 4 * 10 ^-7M, 2 * 10 ^-6M, 1 * 10 ^-5The curcumin of M continues to cultivate 48 hours; Removing culture fluid, with the PBS washed cell once, then under 15-25 ℃ condition, is 4% paraformaldehyde (fixative) fixed cell 60 minutes with volumn concentration.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 curcumin is handled add 50 μ l TUNEL respectively and detect liquid, and negative control group adds 50 μ l fluorescent labeling liquid, 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 catalogue 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 curcumin 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 represents the apoptosis situation of negative control group cell, and gentamycin represents the apoptosis situation of positive controls cell, and curcumin represents to add the apoptosis situation of the experimental group cell of curcumin.The result shows, the not obvious induction MDCK apoptosis of curcumin confirms that effect and its promotion apoptosis of curcumin inhibition vesicle is irrelevant.

3, MDCK tubule generation experiment confirm curcumin can promote MDCK to form the tubule spline structure

Mdck cell is being contained 4 * 10 respectively ^-7M, 2 * 10 ^-6M, 1 * 10 ^-5The curcumin of M and 3T3 fibroblast culture fluid are (with 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 curcumin and 3T3 fibroblast culture fluid and photograph every day, 10 above tubules are followed the tracks of in every hole, count by tubule and estimate the influence of curcumin to the mdck cell differentiation.Three repetition are established in experiment, and curcumin to the facilitation of MDCK tubule generation as shown in figure 11.Wherein, control represents to cultivate mdck cell 15 days with the 3T3 fibroblast culture fluid that does not contain curcumin.The result shows that curcumin can promote mdck cell to form the tubule spline structure.

4, MDCK vesicle tubule generation experiment confirm curcumin can promote vesicle to form the tubule spline structure

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 4 * 10 respectively ^-7M, 2 * 10 ^-6M, 1 * 10 ^-5The 3T3 fibroblast culture fluid of M curcumin continues to cultivate, and renews the bright 3T3 fibroblast culture fluid that contains curcumin 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 repetitions are established in experiment, and curcumin forms the facilitation of tubule spline structure shown in Figure 12 and 13 to vesicle.Among Figure 13, control represents to cultivate with the 3T3 fibroblast culture fluid that does not contain curcumin 4 days MDCK vesicle 15 days.The result shows that curcumin can promote vesicle to form the tubule spline structure.

Embodiment 3, determine the inhibitory action of curcumin to embryo kidney vesicle growth by 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 cloudy bolt, if the female Mus of cloudy bolt pool expression conceived half day arranged, to there be 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 U.S. Sigma company, catalogue 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 curcumin prevents and/or treats ADPKD.The sketch map of embryo kidney vesicle model as shown in figure 14.The vesicle forming process of mice embryonic kidney as shown in figure 15.Wherein, control represents the embryo kidney that 8-Br-cAMP of no use handles, and 8-Br-cAMP represents the embryo kidney that the 8-Br-cAMP with 100 μ M handled.

With the embryo kidney of above-mentioned formation vesicle is 4 * 10 with concentration respectively ^-7M, 2 * 10 ^-6M, 1 * 10 ^-5The curcumin of M is handled, and three repetition are established in experiment, and the inhibitory action that curcumin is grown to the embryo kidney vesicle is shown in Figure 16 and 17.Among Figure 16, control represents not use the embryo kidney of 8-Br-cAMP and curcumin processing; 8-Br-cAMP represents only to handle with 8-Br-cAMP, and the embryo kidney of not handling with curcumin; Curcumin 4 * 10 ^-7, curcumin 2 * 10 ^-6, curcumin 1 * 10 ^-5Represent to handle the embryo kidney of the curcumin processing of back reuse variable concentrations respectively with 8-Br-cAMP.

The result shows that the vesicle that curcumin can suppress in the inductive 13.5 days mice embryonic nephridial tissue of 8-Br-cAMP forms, and effect is dose-effect relationship.

The action target spot of embodiment 4, curcumin and mechanism

Utilize the Western engram technology, detect the influence of curcumin vesicle epithelial cell signal transduction pathway.Concrete experimentation is as follows:

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, give curcumin simultaneously respectively, make its final concentration in culture medium be respectively 0M, 4 * 10 ^-7M, 2 * 10 ^-6M and 1 * 10 ^-5M, the mdck cell of cultivating with the culture fluid that does not contain forskolin is organized as control simultaneously, and the above-mentioned cell of respectively organizing 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. GEHealthcare company, catalogue No ALSZR004-096412) chemiluminescence, development, photographic fixing.Find the influence of curcumin by 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 curcumin can be reduced B-raf, raises Raf-1, and can suppress the p-ERK phosphorylation, and the result as shown in figure 18.

Claims (5)

1. A drug for preventing and/or treating autosomal dominant polycystic kidney disease with curcumin as an active ingredient. 2. The application of curcumin in the preparation of medicines for preventing and/or treating autosomal dominant polycystic kidney disease. 3. A drug that uses curcumin as an active ingredient to inhibit cell formation of vesicles and/or inhibit vesicle growth. 4. The application of curcumin in the preparation of a drug that inhibits cells from forming vesicles and/or inhibits vesicle growth. 5. The application according to claim 4, characterized in that: the cells are canine kidney cells or mouse embryonic kidney cells.

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