CN107335051A - A kind of pharmaceutical composition for protecting chronic heart failure renal function - Google Patents

Abstract

The invention discloses a kind of pharmaceutical composition for protecting chronic heart failure renal function, the pharmaceutical composition is by antisterone and relaxain in mass ratio 15000：1 mixing composition.Present invention determine that the Renoprotective Effect of joint antisterone and relaxain in chronic heart failure; result of study shows in the model that myocardial fibrosis occurs; the scheme for combining significantly improves heart function, myocardial hypertrophy, fibrosis, protects kidney, and effect is better than single therapy.When Cardiorenal syndrome (Cardio renalsyndrome, CRS) is that the infringement of heart or kidney to another organ can not be compensatory, reciprocal causation, vicious circle, the final common infringement and exhaustion for accelerating heart and renal function are formed.With kidney damage during chronic heart failure, therefore, it is proposed by the present invention use antisterone and pharmaceutical composition that relaxain mixes for chronic heart failure during protection renal provide a kind of new approaches.

Description

A kind of pharmaceutical composition for protecting chronic heart failure renal function

Technical field

The present invention relates to medicine, in particular to a kind of pharmaceutical composition for protecting chronic heart failure renal function.

Background technology

Chronic heart failure (Chronic cardiac failure, CHF) be many angiocardiopathies such as myocardial infarction, Cardiomyopathy, the terminal stage of hypertensive heart disease occurrence and development, and progressively fibrosis of the generation of heart failure then with heart has Close.Myocardial fibrosis refers to the collagenous fibres excess accumulation in the normal organization of cardiac muscle, in heart tissue collagen concentration and Collagen volume fraction dramatically increases or collagen component changes.Collagenous fibres decline the compliance of ventricle wall, and stiffness index increases Add, diastolic filling is limited；Fibrosis coronarius so that tube wall thickening, luminal stenosis, elasticity reduce, cardiac muscle cell's blood For reducing；The contractile function of myocardium cell necrosis and cicatrization infringement cardiac muscle.In addition, the electrical conduction of cardiac muscular tissue is abnormal, lead The generation of proarrhythmia.Chronic heart failure is the terminal stage of most of angiocardiopathy development, is current whole world model Jeopardize one of subject matter of health in enclosing, its drug research application prospect is extensive.

Cardiorenal syndrome (Cardio-renalsyndrome, CRS) is that the infringement of heart or kidney to another organ can not When compensatory, reciprocal causation, vicious circle, the final common infringement and exhaustion for accelerating heart and renal function are formed.1 type heart kidney is comprehensive The characteristics of simulator sickness (CRS1) be heart function acute exacerbation patient's secondary acute kidney injury (Acute kidney injury, ) and renal insufficiency AKI.The direct or indirect effect of heart failure can cause acute injury of kidney and dysfunction.II types are slow Property chronic renal insufficiency caused by cardiac insufficiency.Kidney function damage can increase patient in short term and the death rate at a specified future date, and this Kind, which changes to exist with the fibrosis of heart, kidney, maintains close ties with.The acute and chronic damage of heart kidney organ can cause immunocyte The expression increase of inner cell signal protein, promotes inflammatory cell infiltration, and activated fibroblast promotes I, type III collagen deposition, Extracellular matrix is ultimately resulted in increase and heart and the irreversible fibrosis of kidney.Therefore, Cardiorenal syndrome Mechanism Study and open The medicine for sending out anti-fibrosis effective is the difficult point and focus of current heart failure and renal failure research field.

Circulation and local renin-angiotensin-aldosterone system take part in the response of myocardial fibrosis.Renovascular is high Blood pressure animal model, that is, the side arteria renalis is ligatured, circulation RAS systems can be caused significantly to raise, fiber occurs in Ventricular Change.And the partial ligation arteria renalis, with lower abdominal aorta, this hypertension is with the characteristics of pressure load increase, not with obvious RAS systems The activation of system, then early stage fiberization is not observed.This explanation RAS system plays important in myocardial fibrosis generating process Effect.In RAS systems, Angiotensin II is different to myocardial fibrosis mechanism of action from aldosterone, myocardial fibrosis reaction Just start to occur after aldosterone is transfused 3 weeks, and be transfused after Angiotensin II only needs 3-4 days and obvious cardiac muscle fibre occur Change.Speculate that effect of the aldosterone to myocardial fibrosis is not direct.Further research is found, systemic vascular Angiotensin Converting Enzyme II liters Height, it is whether exogenous (through injecting pathway) or endogenic (reaction to renal ischaemia), then go out with early stage myocardial necrosis Existing reparative fibrosis is relevant.And relatively late reparative fibrosis occurs in aldosterone application, itself does not directly result in myocardium thin Born of the same parents' necrosis.In addition, after subcutaneous rat infusion aldosterone and salt raise individual month, it is high caused by renovascular hypertension and aldosterone infusion In blood pressure model, aldosterone receptor antagonist-spirolactone still has the function that anti-fibrosis in the case where not reducing blood pressure. This explanation Angiotensin II causes the mechanism of action of reparative fibrosis (essence is myocardium cell necrosis) different from aldosterone. They also likely to be present difference to fibrocollagenous metabolic effect.

In recent years, in the research of heart failure mechanism the effect of renin-angiotensin-aldosterone system (RAAS) by extensive Approval, intervenes and achieves success by the treatment of the RAAS systems of excessive activation during heart failure occurrence and development.It is but enough Suppression of the converting enzyme inhibitor to aldosterone and temporary transient, up to 40% patient goes out in antiangiotensin II treatments Existing aldosterone Escape.Aldosterone stimulates the protein synthesizes, and its bone collagen increased is myocardial fibrosis and Cardiovascular Remodeling Key factor.Aldosterone Escape reduces the therapeutic effect of heart failure, and always dying of illness for heart failure can be further reduced using its antagonist Rate.Therefore, aldosterone antagonist treatment plays an important roll in heart failure treatment.

Antisterone has similar chemical constitution with aldosterone, and both rise competing at the cortex position of distal convoluted tubule and concetrated pipe The effect of striving, it is that direct antagonism occurs in the level of the mineralcorticoid receptor of cell membrane, so as to which antagonism aldosterone is made With.Research finds that antisterone has function of resisting myocardial fibrillation, can reduce the patients with heart failure death rate, be it is current clinical most Conventional aldosterone receptor antagonist.

Multinomial research has proven to oxidative stress of the aldosterone in kidney trouble, important in inflammatory reaction and fibrotic processes Property.In the first month of chronic renal disease early stage patient's spironolactone, by tightly monitoring renal function and electricity Xie Zhi, find using spirolactone it is safe.In chronic heart failure rat model, antisterone reduces kidney cell infiltration, Fibrosis is reduced, protects kidney.

In addition, antisterone is cheap, there is to protect potassium diuresis, clinical practice is extensively and often and other drugs Use in conjunction.

Relaxain (relaxin), when nineteen twenty-six Frederick Hisaw is studying the change of gestation pelvic girdle first It was found that the obvious relaxation of Cooper's ligament can be caused, so gain the name.Clearly at present, relaxain not only has in During Pregnancy Important function, and blood vessel structure to non-pregnant animal and function etc. can also produce material impact.Research finds that relaxain can The synthesis of stimulation nitric oxide and cAMP, assemble with expansion blood vessel, degraded collagen, regulation fluid balance and platelet Series of biologic function.

In the atrium and ventricular muscles of patients with congestive heart failure is suffered from, the sustainable high expression of Endogenous relaxin.Slowly Property aorta arch constriction the loose left room of mouse in the expression of relaxain add 3-5 times.In isoproterenol induction In myocardial ischemia in rats damage model, the relaxain in cardiac muscle and blood plasma is horizontal and the mRNA expression of myocardial relaxation element is obvious Rise.The relaxain mRNA and egg of spontaneous hypertensive rat (spontaneously hypertensive rat, SHR) heart Plain boiled water averagely significantly raises.Extensive research has been carried out in effect of the relaxain in cardiovascular system, and it has to atrium sinistrum Positivity frequency conversion and the effect of variable force, expansion blood vessel reduce blood pressure, and protect and adjust myocardial hypertrophy, participate in the reparation after miocardial infarction With regeneration etc. effect.Research, which has proven to relaxain, can reduce a variety of cardiomyopathy models (such as relaxation gene defect；Heart is overexpressed Beta 2 adrenoreceptor and hypertension) left ventricular fibrosis.Recently, relaxain is also proved to mitigate on heavy dose of isopropyl kidney Cardiac fibrosis in the myocardial ischemic injury model of parathyrine induction.These researchs have shown that relaxain is a kind of effective anti-fibre The dimensionization factor.

Protective effect of the relaxain to kidney is in different experiments model validation.Mouse causes kidney after knocking out relaxation gene Dirty loose, dysfunction and fibrosis, exogenous loose extract for treating improve glomerulus and renal tubule fibrosis.Relaxain improves old Age Progression of Glomerulosclerosis, reduce renal dysfunction.Relaxain induces in kidney papillary necrosis model, Angiotensin II Anti-fibrosis effect is played in hypertension model and glomerular basement membrane disease model, in addition, relaxain also has increase kidney Blood flow, improve the effect of glomerular filtration rate(GFR.

The early-stage Study of this research group confirms the function of resisting myocardial fibrillation and protection renal effect of relaxain, and demonstrate,proves Its bright anti-fibrosis effect is in dose dependent.Relaxain is expected to the treatment for being used for human heart fibrosis disease, and together When protect kidney.Relaxain is expensive at present, need to consider treated with combined medication, reduces relaxain dosage.Therefore, antisterone And relaxain combined treatment provides a kind of new approaches for the protection renal that chronic heart failure is treated.

Recently, endothelium-myofibroblast conversion or endothelium-mesenchyma conversion (Endothelial-to- Mesenchymal transition, EndMT) have been found that fibrosis can be promoted.It is similar with endothelium transformation of myofibroblasts, During EndMT, endothelial cell gradually loses its own feature, and cellular morphology is changed into elongated spindle-type, and gradually obtains The phenotypic features of the mesenchymal cells such as propagation, migration and synthesis collagen.It is related to many specific biochemical factors during EndMT Change, mainly include endothelial cell marker such as platelet-endothelial cell adhesion molecule (Platelet endothelial cell Adhesion molecule-1, CD31), blood vessel endothelium calcium mucin (vascular endothelial cadherin, VE- Cadaerin), the expression of vWF ELISA (vonWillibrandfactor, vWF) is lowered, and interstitial cell mark is such as α-smooth muscle actin (α-smooth muscle actin, α-SMA), vimentin (Vimentin) and fibroblast The up-regulated expression of specific proteins (Fibroblast specific protein1, FSP1), passes through these cell sign things Change susceptible of proof EndMT presence.And transforming growth factor-β (Transcription grower factor- β, TGF-β) has Have and acted on caused by promotion and induction EndMT phenomenons.In pulmonary fibrosis, liver fibrosis, comea fibrousization, Colon Fibrosis, wound In a variety of diseases such as healing, cardiac fibrosis and kidney fibrosis, find EndMT to be present and take part in fibrosis to send out. Zeisberg etc. confirms that EndMT can cause renal fibrosis first, and they have been found that in three kinds of kidney fibrosis models, has quite A part of myofibroblast can express endothelial cell marker CD31 (also known as Platelet endothelial cell adhesion molecule-1) simultaneously And mesenchymal cell markers thing α-SMA, fibroblast-like cell specific albumen -1.Lee etc. was also confirmed that in diabetes renal fibrosis early stage EndMT be present and the generation of myofibroblast can be promoted.Therefore, EndMT is provided newly for the Mechanism Study of Cardiorenal syndrome Direction.

The content of the invention

It is an object of the invention to provide protection renal during chronic heart failure a kind of protection chronic heart failure renal function Pharmaceutical composition.Antisterone has heart, Renoprotective Effect, and cheap, and clinical practice is extensive.Relaxain equally has There are heart, Renoprotective Effect, its anti-fibrosis effect is in dose dependent, and relaxain is expensive at present, need to consider medicine Therapeutic alliance, reduce relaxain dosage.The pharmaceutical composition of protection chronic heart failure renal function provided by the invention, by antisterone With relaxain in mass ratio 15000：1 mixing composition.

The therapeutic effect of pharmaceutical composition of the present invention is better than single therapy, and the model of myocardial fibrosis is occurring for the present invention In, antisterone has function of resisting myocardial fibrillation with relaxain use in conjunction.

The beneficial effects of the invention are as follows：Pharmaceutical composition of the present invention is by antisterone and relaxain in mass ratio 15000：1 Mixing composition, it is the conjunctive use of the relaxain of antisterone and low dose of scope containing effective dose.The pharmaceutical composition With heart function protective effect, Renoprotective Effect, renal function is protected by suppressing EndMT mechanism, is Cardiorenal syndrome Treatment provides new thinking.

Brief description of the drawings

The pharmaceutical composition that Fig. 1, antisterone and relaxain combine raises LVSP ,+dp/ in Chronic heart failure model Dtmax ,-dp/dtmax, reduce LVEDP, and effect is better than single therapy；Experiment packet：Blank group (Control)；Model group (Iso)；Antisterone group (Iso+SP)；Relaxation group group (Iso+RLX)；Therapeutic alliance group (Iso+SP+RLX).

The pharmaceutical composition that Fig. 2, antisterone and relaxain combine reduces left ventricle matter in Chronic heart failure model Volume index (LVWI), right ventricle performance figure (RVWI) and kidney performance figure (KWI) (P<0.05), effect is better than single therapy (P<0.05)。

The pharmaceutical composition that Fig. 3, antisterone and relaxain combine reduces cardiac muscular tissue in Chronic heart failure model Interstitial proliferation, myocardium arrangement disorder, cardiac muscle cell's plumpness, vacuolization, cell infiltration (Fig. 3 A), it is small to reduce renal tissue kidney Pipe epithelial cell vacuolar degeneration, fibroplasia and cell infiltration (Fig. 3 B), effect are better than single therapy.

The pharmaceutical composition that Fig. 4, antisterone and relaxain combine reduces cardiac muscular tissue in Chronic heart failure model Blue cellulose tissue area (Fig. 4 B, D) (P in middle blue cellulose tissue area (Fig. 4 A, C) and renal tissue<0.01) heart, is reduced Collagen I and collagen in collagen I and collagen III contents (Fig. 4 E) and renal tissue in muscular tissue III contents (Fig. 4 F) (P<0.01).Combined treatment effect is better than single therapy (P<0.05).

It is small that the pharmaceutical composition that Fig. 5, antisterone and relaxain combine increases kidney blood in Chronic heart failure model Plate-endothelial cell adhesion molecule fluorescence signal (Platelet endothelial cell adhesion molecule-1, CD31), α-smooth muscle actin (α-smooth muscle actin, α-SMA) fluorescence signal (Fig. 5 A, B) (P is reduced< 0.01), increase CD31 protein expressions, reduce α-SMA and TGF-β protein expression (Fig. 5 C, D and E) (P<0.01) medicine, is prompted It is existing that composition suppresses kidney endothelial-mesenchyma conversion (Endothelial-to-mesenchymal transition, EndMT) As effect is better than single therapy (P<0.01).

Embodiment

With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are unlimited In this.

The pharmaceutical composition that the present invention determines antisterone by experiment and relaxain combines is in chronic heart failure Renoprotective Effect.

First, the pharmaceutical composition protection heart function that antisterone and relaxain combine, effect are better than single therapy.

(1) present invention establishes chronic heart failure cardiac fibrosis animal model using isoprel.

(2) animal packet：The present invention chooses after 50 male SD rat adaptability are fed one week and is randomly divided into 5 groups：Control Group, model group, antisterone group, relaxain group and therapeutic alliance group.

(3) each group cardiac function is evaluated：Rat gives right carotid intubation record analysis LVSP, LVEDP ,+dp/ in the present invention Dtmax and-dp/dtmax.As a result show：Chronic heart failure model group LVSP ,+dp/dtmax ,-dp/dtmax are reduced, LVEDP liters It is high.Pharmaceutical composition improves influence of the isoprel to heart function, and effect is better than single therapy (Fig. 1).

(4) left and right ventricles performance figure is measured：Rats underwent left ventricular mass index (LVWI) in the present invention, right ventricle quality Index (RVWI) measures.As a result show：Chronic heart failure model group LVWI, RVWI raises.Pharmaceutical composition improves isoproterenol Influence of the element to left and right ventricles performance figure, effect are better than single therapy (Fig. 2A, B).

(5) each group myocardiac histology is observed：Rabbit Myocardium row HE dyeing of the present invention.As a result show：Chronic heart failure model Group is shown in extensive interstitial proliferation, myocardium arrangement disorder, cardiac muscle cell's plumpness, vacuolization, cell infiltration.Pharmaceutical composition changes Myocardiac histology caused by kind isoprel changes, and effect is better than single therapy (Fig. 3 A).

(6) each group left ventricular fibrosis is evaluated：

Row Masson ' trichome, which are dyed, in the present invention determines left ventricular fibrosis area under light microscopic.As a result show：The chronic heart Failure model group is shown in a large amount of blue cellulose tissues.Pharmaceutical composition reduces left ventricular fibrosis area, and effect is better than single therapy (figure 4A,C)。

Collagen I and collagen III assays in Zhong Hang cardiac muscular tissues of the present invention.As a result show：The chronic heart Collagen I and the rise of collagen III contents in failure model group cardiac muscular tissue.Pharmaceutical composition reduces Chronic heart failure model Collagen I and collagen III contents in Rabbit Myocardium, effect are better than single therapy (Fig. 4 E).

2nd, the pharmaceutical composition protection renal function that antisterone and relaxain combine, effect are better than single therapy.

(1) kidney performance figure is measured:Take double kidneys to weigh in this experiment, calculate kidney performance figure (KWI, mg/g).Knot Fruit shows：Chronic heart failure model group kidney performance figure raises.Pharmaceutical composition reduces the kidney quality in Chronic heart failure model Index variation, effect are better than single therapy (Fig. 2 C).

(2) each group renal histology is observed：Row HE decoration methods observation renal histology changes in this experiment.As a result show： Chronic heart failure model group renal cells vacuolar degeneration, fibroplasia and cell infiltration.Pharmaceutical composition reduces chronic Renal histology change in Heart Failure Model, effect are better than single therapy (Fig. 3 B).

(3) each group renal fibrosis is evaluated：

Renal fibrosis area is determined under Masson ' trichome dyeing light microscopics.As a result show：Chronic heart failure model group kidney A large amount of blue cellulose tissues are seen in dirty.Pharmaceutical composition reduces the renal fibrosis area in Chronic heart failure model, and effect is excellent In single therapy (Fig. 4 B, D).

Collagen I and collagen III contents in ELISA method measure renal tissue.As a result show：Chronic heart failure mould Collagen I and the rise of collagen III contents in type group renal tissue.Pharmaceutical composition reduces kidney in Chronic heart failure model Collagen I and collagen III contents in dirty tissue, effect are better than single therapy (Fig. 4 F).

3rd, the pharmaceutical composition that antisterone and relaxain combine suppresses kidney EndMT in Chronic heart failure model Phenomenon, effect are better than single therapy.

(1) fluorescence immunoassay observation each group renal tissue CD31, α-SMA expression：α in immuno-fluorescence assay endothelial cell- SMA (fibroid labelled protein) and CD31 (endothelial lining labelled protein) expression are to observe EndMT phenomenons.As a result show：It is chronic CD31 expression is reduced in Heart Failure Model group renal tissue, α-SMA increases, prompts EndMT phenomenons be present.Pharmaceutical composition improves slow EndMT phenomenons in property Heart Failure Model, effect are better than single therapy (Fig. 5 A, B).

(2) expression of the detection of Western blot methods each group renal tissue TGF-β, CD31, α-SMA.As a result show：It is chronic CD31 expression is reduced in Heart Failure Model group renal tissue, TGF-β, α-SMA increases, prompts EndMT phenomenons be present.Pharmaceutical composition Improve the EndMT phenomenons in Chronic heart failure model, effect is better than single therapy (figure C, D and E).

It is implemented as follows：

1) experimental animal and experimental situation condition.

Healthy male SD rat (about 6 weeks) 50, body weight 200g-220g, standard rodent animal feed is fed.It is real Test environment comply fully with international animal protection association as defined in standard, reach toy life necessary to fundamental space requirement.

2) Animal Model：Rat chronic Heart Failure Model is established using isoprel method.

3) animal packet：Cleaning grade Wistar male rats 50, weight (220 ± 10) g, are randomly divided into 5 groups.Model Group：Rat back hypodermic injection isoprel, 5mg/kg/d, totally 7 days.Control group gives the physiological saline of equal parts.An Ti Relax and lead to group：Rat oral gavage be administered 30mg/kg/d, totally 21 days.Relaxain group：Rat skin lower injection be administered 2ug/kg/d, totally 21 days. Therapeutic alliance group：Rat antisterone gastric infusion, 30mg/kg/d, relaxain subcutaneous administrations 2ug/kg/d (antisterones With relaxain in mass ratio 15000：1), totally 21 days.

4) each group cardiac function is evaluated：After rat weight, 1% yellow Jackets intraperitoneal injection of anesthesia.Rat is fixed on Mouse plate, neck unhairing preserved skin separate right carotid, and conduit of the insertion full of 0.05% heparin-saline is another to left ventricle Terminate pressure transducer input multimedia bio signal recorder, record analysis LVSP, LVEDP ,+dp/dtmax and-dp/ dtmax。

5) left and right ventricles performance figure is measured：Rat extracting blood rear left ventricles enter the physiological saline of 4 DEG C of precoolings to heart After being lost color with kidney, take out heart and weigh.Left ventricular mass divided by body weight are calculated as left ventricular mass index (LVWI), right ventricle Quality divided by body weight are calculated as right ventricle performance figure (RVWI).

6) each group myocardiac histology is observed：Cardiac muscular tissue row HE is dyed, and observes its pathological change.Take left ventricle apex few Tissue (about 80mg) is measured, formaldehyde is fixed, conventional dehydration, FFPE.Remainder loads sterile cryopreservation tube and is immediately placed in liquid Quick-frozen preservation in nitrogen, -80 DEG C of refrigerators are transferred to for other detection projects.

FFPE process：

(1) 4 DEG C, 4% paraformaldehyde is fixed, 24h；

(2) clear water rinses；

(3) 50% ethanol 30min；

(4) 75% ethanol 30min；

(5) 85% ethanol 30min；

(6) 95% ethanol 40min；

(7) 95% ethanol 40min；

(8) the 40min of 100% ethanol I；

(9) the 40min of 100% ethanol II；

(10) dimethylbenzene I soaks 30min；Dimethylbenzene II soaks 30min；

(11) paraffin I, waxdip 2h；Paraffin II, waxdip 2h；

(12) sample embeds；

HE staining procedures：

(1) paraffin section makes, about 4 μm of thickness；

Conventional dewaxing aquation (100% dimethylbenzene liquid I, II dewaxes each 15min, 100% ethanol I, II, 95% ethanol I, II, 90%, 80% ethanol aquation each 5min, ultrapure washing 2min*2 times)；

(3) haematoxylin dyeing 3min；

(4) ultrapure washing 5 minutes；

(5) 1% hydrochloride alcohol breaks up about 5 seconds；

(6) microscopy after ultrapure water, nuclear targeting situation is observed；

(7) ultrapure washing 5 minutes；

(8) eosin stains 5min；

(9) ultrapure water；

(10) it is dehydrated, transparent, mounting；

7) each group cardiac muscular tissue area of collagen detects：

Cardiac muscular tissue paraffin section row Masson is dyed, and cardiac muscle cell takes on a red color under Masson dyeing, and collagen is in blueness, Nucleus is in black.Micro- Microscopic observation Myocardial Interstitial Fibrosis simultaneously randomly selects 5 unduplicated visuals field under 200 times of visuals field And take pictures.Using Image-Pro Plus softwares, myocardial collagen area is measured, is averaged, numerical value is bigger, and fibrosis is more obvious.

Masson staining procedures：

(1) paraffin section makes, about 4 μm of thickness；

(2) conventional dewaxing aquation；

(3) 60 DEG C of packet mode liquid fix 2h；

(4) it is ultrapure to wash 5 minutes * 2 times；

(5) bush uniformly dyeing 5 minutes；

(6) it is ultrapure to wash 5 minutes * 2 times；

(7) Ponceaux acid fuchsin contaminates 3 minutes；

(8) ultrapure washing 3 minutes；

(9) contaminated 10 minutes in 1% phosphomolybdic acid；

(10) it is not washed with water and directly enters aniline blue liquid 10 minutes, Microscopic observation fibr tissue color；

(11) ultrapure washing 3 minutes；

(12) fast dewatering, dimethylbenzene is transparent, sealing；

8) collagen I and collagen III assays in each group cardiac muscular tissue：100mg cardiac muscular tissues shred simultaneously Add PBS (PH7.4) 1ml, fully homogenate.Centrifuge 20 minutes or so (3000 revs/min).Collect supernatant.Said by ELISA kit Bright book is measured：

(1) prepare：Kit is taken out from refrigerator, room temperature rewarming balances 30 minutes；

(2) standard items and sample to be measured are added：Setting standard sample wells, blank control wells and sample to be tested hole, standard sample wells Middle addition standard items 50 μ L；The μ L of sample to be tested 10 are added in sample to be tested hole, it is (i.e. that sample is dilute to add the μ L of Sample dilution 40 Release 5 times)；Blank control wells are not added with；

(3) incubate：Insulating box incubates 30min；

(4) board-washing：Cleaning solution washs 4-6 times repeatedly, to filter paper on print it is dry；

(5) enzyme-added mark working solution：The μ L of enzyme mark working solution 50 are added per hole, blank control wells are not added with；

(6) incubate：Insulating box incubates 30min；

(7) board-washing：Ibid；

(8) develop the color：The μ L of developer A liquid 50 are added per hole, add the μ L of developer B liquid 50, gently concussion mixes 30s；

(9) terminate：Add the μ L of terminate liquid 50, terminating reaction per hole；

(10) determine：Returned to zero with blank well, after reaction terminating in 30 minutes, with the light absorption value in each hole of 450nm wavelength measurements；

(11) calculate：According to standard concentration and corresponding OD values, standard curve linear regression equation is calculated, according to sample OD values, utilize the concentration of regression equation calculation sample；Ultimate density is that practical measurement concentration is multiplied by extension rate；

9) each group kidney performance figure is evaluated:Double kidneys are taken to weigh, the ratio between average value (mg) and weight (g) are as kidney Performance figure (mg/g).

10) each group renal histology is observed：HE decoration methods observation renal histology changes.Kidney is taken to organize on a small quantity (about 80mg), formaldehyde is fixed, conventional dehydration, FFPE.Remainder is fitted into sterile cryopreservation tube and is immediately placed in quick-frozen guarantor in liquid nitrogen Deposit, be transferred to -80 DEG C of refrigerators for other detection projects.

HE staining procedures：

(1) paraffin section makes, about 4 μm of thickness；

Conventional dewaxing aquation (100% dimethylbenzene liquid I, II dewaxes each 15min, 100% ethanol I, II, 95% ethanol I, II, 90%, 80% ethanol aquation each 5min, ultrapure washing 2min*2 times)；

(3) haematoxylin dyeing 3min；

(4) ultrapure washing 5 minutes；

(5) 1% hydrochloride alcohol breaks up about 5 seconds；

(6) microscopy after ultrapure water, nuclear targeting situation is observed；

(7) ultrapure washing 5 minutes；

(8) eosin stains 5min；

(9) ultrapure water；

(10) it is dehydrated, transparent, mounting；

11) each group renal tissue area of collagen detects：Kidney paraffin section row Masson is dyed, measurement renal collagen face Product, averages, numerical value is bigger, and fibrosis is more obvious.

12) collagen I and collagen III assays in each group renal tissue.100mg renal tissues shred simultaneously Add PBS (PH7.4) 1ml, fully homogenate.Centrifuge 20 minutes or so (3000 revs/min).Collect supernatant.Said by ELISA kit Bright book is measured.

13) evaluation of each group kidney endothelial cell interstitial：

(1) fluorescence immunoassay observation each group renal tissue CD31, α-SMA fluorescence signals.This research immuno-fluorescence assay α-sma (fibroid labelled protein) and CD31 (endothelial lining labelled protein) expression are to observe EndMT phenomenons in endothelial cell.

Immunofluorescence step：

1. paraffin section makes, about 4 μm of thickness；

2. 60 DEG C of baking 2h of paraffin section；

3. conventional dewaxing aquation；

Citrate antigen retrieval buffers 4. (pH=6.0) high pressure antigen retrieval 3min, natural cooling, 0.1M PBS are washed 2min*3 times；

5. 3%H2O2 is incubated 10min, 0.1M PBS are washed 2min*3 times；

6. 10% hyclone is incubated at room temperature 30min, dry；

7. mixing primary antibody (α-sma antibody, Bo Shide, diluted concentration 1 is added dropwise:100；CD31 antibody, Abcam, diluted concentration 1：30), stay overnight for 4 DEG C, 0.1M PBS are washed 5min*3 times；

8. mixing fluorescence secondary antibody (1 is added dropwise:300), 37 DEG C of lucifuges are incubated 2h, and 0.1M PBS are washed 5min*6 times, and anti-fluorescence is quenched Mountant of going out mounting.

(2) WB surveys α-sma, CD31, TGF-β expression.

Main solution is prepared：

1. 6% separation gel 5ml

Ultra-pure water 2.0ml

30%Acr-Bis (29:1)1.0ml

1M Tris,pH8.8 1.9ml

10%SDS 0.05ml

10% ammonium persulfate 0.05ml

TEMED 0.004ml

Used immediately after mixing

2. 10% glue 5ml

Ultra-pure water 1.3ml

30%Acr-Bis (29:1)1.7ml

1M Tris,pH8.8 1.9ml

10%SDS 0.05ml

10% ammonium persulfate 0.05ml

TEMED 0.002ml

Used immediately after mixing

3. 5% concentration glue 2ml

Ultra-pure water 1.4ml

30%Acr-Bis (29:1)0.33ml

1M Tris,pH6.8 0.25ml

10%SDS 0.02ml

10% ammonium persulfate 0.02ml

TEMED 0.002ml

Used immediately after mixing

4. electrophoretic buffer：

Tris 3.03g

Glycine 14.4g

SDS lg

Ultra-pure water is dissolved to 1000ml.

5. transfering buffering liquid：

Tris 3g

SDS lg

Glycine 14.4g

Methanol 200mL

Ultra-pure water is settled to lOOOmL, at most reuses 4 times.

6. 5XTBS buffer solutions：

Tris 12.1g

NaCl 40g

Ultra-pure water dissolves, and dense HCl adjusts pH value to be settled to lOOOmL to 7 or so.

7. TBST buffer solutions

20%Tween20 1ml

TBS 100ml

It can be used after mixing, best matching while using.

8. 5% skim milk

Skimmed milk power 5g

TBST 100ml

4 DEG C of preservations after dissolving.It is in use, disposable after recovering room temperature, filtering.

WB experimental procedures

(1) total protein extraction in organizing：

1. taking 100mg tissue blocks to be placed in the spherical position of 1~2ml homogenizers, will organize to shred as far as possible with scissors.

2. plus 1000 μ l lysates split and 10ul PMSF are in homogenizer, are placed in and are fully homogenized on ice.

3. cracking 1h on ice, it is transferred in 1.5ml centrifuge tubes, 12000rpm centrifuges 5min at 4 DEG C, takes supernatant to be sub-packed in - 80 DEG C of preservations after 0.5ml centrifuge tubes.

(2) BCA methods measure total protein contains

(3) SDS-PAGE electrophoresis

Prepare 6% or 10%SDS-PAGE separation gels and be used for different Protein Detections, the concentration glue of configuration 4%, slowly irrigate system Glue, solidification is stood at room temperature.The gel glass plate prepared is fixed on gel-electrophoretic apparatus, the difference of two grooves up and down of electrophoresis apparatus Add electrophoretic buffer.It is careful to take out sample-adding comb.It is applied sample amount to calculate the liquor capacity containing 50 μ g albumen.Add 5*SDS loadings Buffer solution makes albumen fully be denatured to final concentration of 1*, boiling water bath 10min.It is separately added into each sample and protein molecular weight Marker, with 1* sample buffer trim loading volumes.It is initially after 70V constant pressures electrophoresis is layered to maker albumen and changes 120V perseverances Electrophoretic velocity is accelerated in piezoelectricity swimming.Electrophoresis to bromjophenol blue runs out of gel.

(4) transferring film：Take out offset plate, excision concentration glue.Clip with glue size identical pvdf membrane, by negative electrode-filter paper-glue- Pvdf membrane-filter paper-anode order is positioned in Protein transfer groove clamping plate, and with 300mA constant currents, electricity turns 90min.

(5) close：5% skim milk closes 1h.

(6) immune response：After closing terminates, film is washed 10min*3 times with TBST, film is moved into cleaning antibody incubation box, added Enter primary antibody dilution (CD31 antibody, Santa, diluted concentration 1:1000；Diluted concentration 1:1000；α-SMA antibody, Wuhanboshide, diluted concentration 1:1000；The antibody of TGF-β 1, Bioworld Technology, diluted concentration 1:1000； GAPDH antibody, Bioworld Technology, diluted concentration 1:5000) lid, adhesive plaster sealing, are covered, 4 DEG C of shaking tables were incubated Night；TBST washes film 10min*3 times；Pvdf membrane is moved into cleaning antibody incubation box again, adds the horseradish peroxidase of dilution Secondary antibody (horseradish peroxidase-labeled goat anti-rabbit igg, Bioworld Technology, the diluted concentration 1 of enzyme mark:5000— 1：10000), it is incubated at room temperature 2h；TBST washes film 15min*3 times；Pvdf membrane is placed in ECL luminescent solutions, vibrated at room temperature 5min。

(7) develop：Labworks image acquisition and analysis software detects and records pvdf membrane imaging

(8) graphical analysis：

Gray analysis is carried out to the purpose band of scanned picture with Image-Pro Plus softwares.

14) statistical procedures

Data processing, the data obtained mean scholar's standard deviation are carried out using SPSS16.0 statistical analysis softwares Table Show, single argument measurement data sample average compares with one-way analysis of variance (One-wayANOVA), P<0.05 is that difference has system Meter learns meaning, P<0.01 is difference highly significant.

Above-described embodiment is used for illustrating the present invention, rather than limits the invention, the present invention spirit and In scope of the claims, to any modifications and changes of the invention made, protection scope of the present invention is both fallen within.

Claims (3)

1. a kind of pharmaceutical composition for protecting chronic heart failure renal function, it is characterised in that quality is pressed by antisterone and relaxain Than 15000：1 mixing composition.

2. the pharmaceutical composition of protection chronic heart failure renal function according to claim 1, it is characterised in that the drug regimen Thing protects heart function.

3. the pharmaceutical composition of protection chronic heart failure renal function according to claim 1, it is characterised in that the drug regimen Thing converts (Endothelial-to-mesenchymal transition, EndMT) mechanism guarantor by suppressing endothelium-mesenchyma Protect renal function.