CN105641715A

CN105641715A - Application of Pin1 siRNA in preparation of targeted medicine for treating alcoholic cardiomyopathy

Info

Publication number: CN105641715A
Application number: CN201610081941.1A
Authority: CN
Inventors: 王越红; 杨巍; 李子卓; 孙晶; 田娟娟
Original assignee: Harbin Medical University
Current assignee: Harbin Engineering University; Harbin Medical University
Priority date: 2016-02-04
Filing date: 2016-02-04
Publication date: 2016-06-08

Abstract

The invention discloses application of Pin1 siRNA in preparation of a targeted medicine for treating the alcoholic cardiomyopathy, and belongs to the field of prevention and treatment of the alcoholic cardiomyopathy. It is proved through experiments that Pin1 takes participate in the cardiac muscle cell apoptosis process caused by alcohol and high-dosage alcohol stimulates the expression and activity of Pin1. By means of Pin1 gene knock-out mediated by Pin1 siRNA, cardiac muscle cell apoptosis mediated by alcohol can be remarkably restrained; due to the over-expression of Pin1, the number of apoptosis muscle cells is further increased. It is further proved that Pin1 promotes mitochondria oxidative stress mediated by alcohol and restrains the expression of endothelial nitric oxide synthase. Through Pin1 gene knock-out mediated by Pin1 siRNA, mitochondria oxidative stress mediated by alcohol, the production decrease of NO and the level decrease of eNOS can be remarkably restrained, and therefore the occurrence and development process of the alcoholic cardiomyopathy is further delayed. An effective technological means is put forward to prevent and treat the alcoholic cardiomyopathy.

Description

Pin1 siRNA application in the targeted drug of preparation treatment alcoholic cardiomyopathy

Technical field

The present invention relates to the novel medical use of Pin1siRNA, particularly relate to it and prevent and treat the purposes in the targeted drug of alcoholic cardiomyopathy in preparation, belong to prevention and the treatment field of alcoholic cardiomyopathy.

Background technology

Up to the present, heart failure remains an important public health problem. In the U.S., no matter male or women, long-term heavy drinking is considered as Ischemic dilated cardiomyopathy, i.e. the main cause of " alcoholic cardiomyopathy " (ACM). Generally, asymptomatic ACM, if every day, drinking amount was more than 90 grams, the time more than 5 years, is then likely to develop into Symptomatic ACM, and the sign of heart failure occurs.

In the asymptomatic ACM stage, being usually expressed as left ventricle dilatation, left ventricular mass increases, and left ventricular wall thickness reduces or normal. On pathology, between former research display ACM and apoptosis of cardiac muscle, there is very strong dependency. In the cardiac muscle of chronic alcoholism patient, the expression of apoptosis of cardiac muscle and BAX and BCL-2 detected. Research of Animal Model for Study it is also shown that chronic alcohol int akes can induced oxidation stress and apoptosis of cardiac muscle. At primary myocardial cell culture model, ethanol is found the apoptosis of induced activity oxygen mediation, and in the mode of dose dependent in the scope of 0-100mM. But, the molecular mechanism of ethanol inducing cardiomyocytes apoptosis need further research.

Peptidyl prolyl cis-trans isomerization enzyme (peptidyl-prolylcis/transisomerase, Pin1), it it is a member in the parvulin family of PPIase enzyme, it is able to the motif of the specific phosphorylation serine/threonine-Pro of isomerization peptidyl-prolyl key, this may change its activity, stability, phosphorylation state, and protein protein interaction. Pin1 is considered as at first in yeast and people's cell, necessary to cell division. Research later shows, Pin1 participates in other cell processes many, such as genetic transcription, and cell proliferation, in differentiation and apoptosis, and play important regulating and controlling effect.Further, since phosphorylated protein is an important signal transduction mechanism, Pin1 take part in Ras signal path and the activation of Wnt signal path.

Regulation and control in apoptotic, have research to confirm, Pin1 inhibits apoptosis in hepatoma carcinoma cell and colorectal cancer SW620 cell. In the present invention, we further study Pin1 effect (shown in Fig. 6) in the apoptosis of cardiac muscle that high dose ethanol brings out regulates, and finding, in Primary mouse myocardial cell, ethanol induction Pin1 expresses and activates and exist in dose-dependent mode. We further demonstrate that Pin1siRNA makes the apoptosis that myocardial cell is induced from high dose ethanol, stress play a role with the expression of endothelial nitric oxide synthase (NOS) by regulating mitochondrial oxidation.

There is no the targeted drug for alcoholic cardiomyopathy at present. The preventing and treating for alcoholic cardiomyopathy that proposes of the present invention provides new technological means.

Summary of the invention

The technical problem to be solved determines that Pin1siRNA effect in alcoholic cardiomyopathy pathogenesis, and it can be used as a kind of novel targeted drug for preventing and treating alcoholic cardiomyopathy, is applied among prevention and treatment alcoholic cardiomyopathy.

In order to achieve the above object, present invention employs techniques below means:

In order to prove relation between the apoptosis of cardiac muscle that Pin1 and ethanol cause, inventor by being exposed to the Pin1 expression of ethanol (ethanol be respectively as follows: 0,50, the 100 or 200mM) post analysis of variable concentrations by myocardial cell. Further, the present invention have studied the myocardial cell of process LAN Pin1 and Pin1 strikes the low myocardial cell suppression situation to the apoptosis of cardiac muscle that ethanol mediates, and to the myocardial cell of process LAN Pin1 and Pin1 strikes the oxidative stress of low myocardial cell Mitochondria and myocardial cell NO produces and eNOS expresses and studied. Above result of study display Pin1 take part in the apoptosis of cardiac muscle process that ethanol causes. The ethanol of high dose stimulates expression and the activity of Pin1. The Pin1 Knockdown of Pin1siRNA mediation can substantially suppress the apoptosis of the myocardial cell that ethanol mediates, and the process LAN of Pin1 then increases the number of the myocardial cell of apoptosis further. We also further prove Pin1 promote ethanol mediation mitochondrial oxidation stress and the loss of mitochondrial membrane potential, and suppress the expression of eNOS. And the Pin1 Knockdown of Pin1siRNA mediation can substantially suppress mitochondrial oxidation that ethanol mediates stress and the loss of mitochondrial membrane potential, the level reduced with eNOS that produces of NO reduces, thus having delayed the generation development process of alcoholic cardiomyopathy further.

Therefore, on the basis of the studies above, the present invention proposes Pin1siRNA application in the targeted drug of preparation treatment alcoholic cardiomyopathy.

Further, the invention allows for Pin1 application in the targeted drug treating alcoholic cardiomyopathy as Sites Screening. Described targeted drug can suppress the expression of Pin1.

The preventing and treating that the proposition of the present invention is alcoholic cardiomyopathy provides a kind of effective technological means.

Accompanying drawing explanation

Fig. 1 is shown in the myocardial cell of ethanol postincubation, and the expression of Pin1 and activity raise;

After variable concentrations ethanol (0,50,100, or 200mM) processes myocardial cell 24 hours: (a) qRT-PCR method records the expression of Pin1mRNA; The expression of (b) Westernblot method detection Pin1;The determination of activity of (c) myocardial cell Pin1; * P < 0.05 and * * P < 0.01 compares with non-ethanol postincubation group;

Fig. 2 shows that Pin1 knocks out the i.e. Pin1siRNA apoptosis suppressing the myocardial cell of ethanol mediation;

By the protein expression of Westernblot method detection Pin1 after (a) Pin1siRNA transfection; (b-d) myocardial cell after Pin1siRNA transfection, non-ethanol or ethanol (200mM) measure after processing 24 hours: the activity (b) of cell, caspase-9 and caspase-3 activity (c), TUNEL dyes (d); * P < 0.05, * * P < 0.01 and * * * P < 0.001.

Fig. 3 shows that the process LAN of Pin1 promotes the apoptosis of the myocardial cell of ethanol mediation;

By the protein expression of Westernblot method detection Pin1 after (a) Pin1 plasmid transfection; (b-d) myocardial cell of Pin1 process LAN, non-ethanol or ethanol (50mM) measure after processing 24 hours: the activity (b) of cell, caspase-9 and caspase-3 activity (c), TUNEL dyes (d). * P < 0.05, * * P < 0.01 and * * * P < 0.001;

Fig. 4 shows that the myocardial cell mitochondrial oxidation of Pin1 enhancing ethanol mediation stress;

In myocardial cell after Pin1Pin1siRNA transfection, respectively after the ethanol postincubation 24 hours of non-ethanol and 200mM, Westernblot method detection cytochrome C derivant (mCyt.C) (a), mitochondrial membrane potential (b) and mitochondria activity oxygen (mROS) level (c). The myocardial cell of Pin1 process LAN, non-ethanol or ethanol (50mM) measure after processing 24 hours: mCyt.C (d), mitochondrial membrane potential (e) and mitochondria activity oxygen (mROS) level (f). * P < 0.05, * * P < 0.01; The myocardial cell of Pin1 process LAN, ethanol (50mM), NAC (1 ��M) andMito-TEMPO (50 ��Ms) measure after processing: cytoactive (g), caspase-3 activity (h); * P < 0.05, * * P < 0.01 compares with non-ethanol group. The ethanol group cell of #P < 0.05 and Pin1 process LAN compares; I () non-ethanol or ethanol (200mM) process 24 hours after, Westernblot method measures p-p66Shc level in the mitochondrion of matched group and Pin1siRNA transfection group; (j) non-ethanol or ethanol (50mM) process 24 hours after Westernblot method measure Pin1 process LAN myocardial cell mitochondrion in p-p66Shc level;

Fig. 5 shows that ethanol suppresses Pin1 to reduce myocardial cell NO and produces the level with eNOS; After variable concentrations ethanol (0,50,100 or 200mM) processes myocardial cell 24 hours: the level that (a) NO produces level, (b) Westernblot method detection eNOS expresses; In myocardial cell after Pin1siRNA transfection, respectively after the ethanol postincubation 24 hours of non-ethanol and 200mM, the level that (c) NO produces level, (d) eNOS expresses; The level that after the myocardial cell of Pin1 process LAN, non-ethanol or ethanol (50mM) process 24 hours, (e) NO produces level, (f) eNOS expresses; * P < 0.05 and * * P < 0.01.

Fig. 6 is the diagram of the Pin1 apoptosis of cardiac muscle process mediated at ethanol.

Detailed description of the invention

Below in conjunction with specific embodiment further describe the present invention, advantages of the present invention and feature will be with describe and apparent. The scope of the present invention, only for purpose of illustration, is never constituted any restriction by these embodiments.

The embodiment one: Pin1 expression in the myocardial cell of ethanol postincubation and activation

First ethanol (ethanol be respectively as follows: 0,50,100 or 200mM) the post analysis Pin1 that myocardial cell is exposed to variable concentrations is made to express.Such as Fig. 1 a, b, can be seen that ethanol can promote that the mRNA of Pin1 and protein level raise from this result, and in dose dependent. Additionally, ethanol increases the activity of Pin1 in dose-dependent mode (Fig. 1 c).

Embodiment two: Pin1siRNA suppresses the apoptosis of cardiac muscle of ethanol induction

Method:

1, cell is cultivated and cell transfecting

Isolate the myocardial cell of newborn mice. Heart tissue is shredded and digests, sedimentation, remove fibroblast and endotheliocyte, in the culture dish that collagen is coated with (every square centimeter about 1.5 �� 10 by myocardial cells culture⁵Individual cell). Pin1siRNA (purchased from Invitrogen (Carlsbad)), Pin1 plasmid (purchased from Addgene (Cambridge)) and ScramblesiRNA use the transfection of LipofectamineRNAiMAX (Invitrogen) transfection reagent. Liposome LipofectamineLTX (Invitrogen) uses according to description. Myocardial cell (5 �� 10⁴Cells/well) it is inoculated into 24 orifice plates, and grow and overnight converge to about 80%. Then cell 30 picomole siRNA or 500 nanograms of plasmid co-culture 48 hours, adopt westernblot method to identify transfection efficiency.

2, quantitative reverse transcription polymerase chain reaction (quantitative RT-PCR)

Using TRIzol reagent to extract total serum IgE from myocardial cell, using method is according to description. SuperScriptIII the first chain synthesis system is used to synthesize cDNA from 1 microgram from the total serum IgE of each sample. The expression of Pin1mRNA is measured by qRT-PCR, and uses 2^{-��Ct}Method calculates, and is standardized as the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Primer is as follows:

5'-CCGGAATTCATGGCGGACGAGGAGAAG-3'(forward) and

5'-TGCTCTAGATCATTCTGTGCGCAGGAT-3'(is reverse), it is used for expanding Pin1;

5'-TGGACTCCACGACGTACTCAG-3'(forward) and 5'-GGGAAGCTTGTCATCAATGGAA-3'(reverse) be used for expanding GAPDH.

3��WesternBlot

Results myocardial cell, RIPA buffer cracking 30 minutes (120 mMs of NaCl, 40 mMs of Tris [pH is 8.0], and the NP40 of 0.1%) adds protease/inhibitors of phosphatases on ice. Lysate is centrifuged 18000 grams 15 minutes at 4 DEG C. Collect supernatant, and protein concentration is measured by BCA method (Pierce). The aliquot of described lysate carries out electrophoresis at 10% SDS-polyacrylamide acrylamide gel (SDS-PAGE). Resolve protein transduction move on to nitrocellulose filter, hatch primary antibodie subsequently, followed by horseradish peroxidase put together two resist. Use enhanced chemiluminescence western blot detection kit (Pierce) development, subsequently film is exposed on x-ray film, detect protein band.

4, PIN1 determination of activity

Myocardial cell is by the supersound process lysis buffer cracking (N-2-hydroxyethyl piperazine-N'-2-ethyl sulfonic acid of 50mM, 100mM sodium chloride, 0.25%3-[(3-gallbladder amido propyl) dimethylamino]-1-propane (CHAPS), 5mM sodium fluoride, the �� glycerophophate of 1mM, and the ethylene glycol tetraacetic of 1mM) at 4 DEG C. Then, we are prepared for N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid buffer (50mMN-2-hydroxyethyl piperazine-N'-2-ethyl sulfonic acid (pH value 7.8) containing 93 microlitres, the NaCl of 100mM, the DTT of 2mM, the bovine serum albumin of 0.04 mg/ml, 5 Fl of Cell Lysis liquid (10⁵The Pin1 of cell or the restructuring of 0.25 nanomole) and the mixture of trypsin solution (20 mg/ml) of 2 microlitres.Secondly reactant is started by adding (NeoMPS) of 50 microlitres (720 ��Ms of peptides TRP-PHE-tyrosine-serine (PO3H2's))-ProArg-pNA, is the absorption detecting 4 minutes of p-Nitraniline. at 390nm.

5, cell viability measures

3-(4,5-lutidines-2-base)-2,5-diphenyl bromination (MTT) method is used to measure myocardial viability rate. By cell in 96 well culture plates with every hole 3 �� 10³Individual cell seeding is in 100 �� L culture medium. Aseptic MTT dyestuff (20 microlitre) (Sigma) is added in each hole. After hatching 4 hours at 37 DEG C, the MTT mixture of culture medium is removed, and the mixture in 200 �� l dimethyl sulfoxides is added in each hole. Recording numerical value at 490nm wavelength place and represent the absorbance of living cells, cell survival rate calculates relative ratios as a control group.

6, Caspase determination of activity

Cell (1 �� 10⁴Individual/hole) it is inoculated in 96 orifice plate 24 hours, and the determination of activity that use carries out according to the explanation measuring test kit of manufacturer caspase-3 and caspase-9. Using spectrophotometer to carry out caspase-3 and caspase-9 active level at 405nm place, data normalization is in matched group.

7, TUNEL dyeing

Scheme according to manufacturer uses DeadEnd^TMFluorometricTUNEL system (Promega) carries out TUNEL dyeing.

Result:

Fig. 2 (a) passes through the protein expression of Westernblot method detection Pin1 for Pin1siRNA after transfecting; Myocardial cell after Fig. 2 (b-d) respectively Pin1siRNA transfection, non-ethanol or ethanol (200mM) measure the activity (b) of cell after processing 24 hours, the result of caspase-9 and caspase-3 activity (c) and TUNEL dyeing (d).

Fig. 3 (a) for detecting the protein expression of Pin1 by Westernblot method after Pin1 plasmid transfection; The myocardial cell of Fig. 3 (b-d) respectively Pin1 process LAN, non-ethanol or ethanol (50mM) measure the activity (b) of cell after processing 24 hours, the result of caspase-9 and caspase-3 activity (c) and TUNEL dyeing (d).

Can be seen that from the above compared with matched group, Pin1 strikes low cytoactive and decreases more than 50%. But, the myocardial cell activity that the Pin1 of ethanol postincubation lowers have dropped 20% (Fig. 2 b). Caspase-9 and Caspase-3 determination of activity display consistent with TUNEL dyeing, Pin1 lowers the apoptosis (Fig. 2 c and Fig. 2 d) that ethanol can be suppressed to cause. Pin1 process LAN then further enhances apoptosis and cell viability loss (Fig. 3 a-d) of ethanol induction. The above is it is shown that Pin1 plays an important role in the apoptosis of cardiac muscle promoting ethanol induction.

The knocking out of embodiment three: Pin1 (Pin1siRNA) reduces the myocardial cell mitochondrial oxidation of ethanol mediation stress

The apoptosis of ethanol induction depends on mitochondrial oxidation signal, and in diabetics, the downward of Pin1 is possible to prevent the mitochondrial oxidation stress. Therefore, whether we study Pin1 and lower and can suppress the myocardial cell mitochondrial oxidation that ethanol is induced stress. In the myocardial cell that Pin1 lowers, in or without the ethanol of 200mM, mCyt.C, mitochondrial membrane potential and mitochondria activity oxygen (mROS) detection are carried out respectively.

Method:

1, the measurement of mitochondrial membrane potential

Use TMRE mitochondrial membrane potential to measure test kit mitochondrial membrane potential (�� m) is assessed.

2, measure mitochondrial cytochrome c and be discharged into endochylema

The cytochrome C western blot that is released through from mitochondrion to cytosol measures.

3, the measurement of the generation of NO and mitochondria activity oxygen

By the nitric oxide production level of the oxidised form in measuring samples (nitrite and nitrate), Analysis of NO test kit is used to be estimated. Use EliteTM mitochondria activity oxygen activity to measure test kit (eEnzyme) mitochondria activity oxygen content has been analyzed. Harvesting, and react 60 minutes with 100 �� LEliteROSDeepRedstainsolution at 96 orifice plates at 37 DEG C. Fluorescence intensity is measured at EX/EM=650/675 nanometer.

4, mitochondrial protein separates

370g centrifuge cell 10 minutes, and washing three times in lavation buffer solution (1 mM of Tris hydrochloric acid, pH is 7.4,0.13M sodium chloride, the potassium chloride of 5mM, the MgCl2 of 7.5mM). Then, by cell in homogenisation buffer (Tris-HCl of 10mM, the 10mM potassium chloride of pH value 6.76 erythrocyte volume settling flux, 0.15 milli magnesium chloride, the DTT of PMSF, the 1mM of 1mM), homogenize on ice 10 minutes. Homogenate is transferred in the conical centrifuge tube containing 1 unit 2M sucrose solution volume. 1,200g is centrifuged 5 minutes twice, removes uncracked cell, nucleus and big foreign material. 7000g collects mitochondrion in centrifugal 10 minutes. Mitochondrial pellet is resuspended in 3 unit cell volumes of mitochondrion buffer suspension liquid (10 mMs of Tris hydrochloric acid, pH value 6.7,0.15 milli magnesium chloride, the sucrose of 0.25mM, the DTT of PMSF, the 1mM of 1mM) immunoblotting assay further.

Result:

Lower the Pin1 mCyt.C release significantly having reversed under ethanol induction, the mitochondrial membrane potential (Fig. 4 a-c) of minimizing. Next we study the myocardial cell of Pin1 process LAN. As expected, ethanol causes mCyt.C to discharge, and mitochondrial membrane potential reduces and ROS produces, and this is to be further enhanced (Fig. 4 d-f) by the Pin1 of process LAN. Additionally, the cardiomyocyte viability whether removing of the ROC of our test can reverse ethanol postincubation declines and apoptosis. As shown in Fig. 4 g-h, two ROS scavengers (NACandMito-TEMPO), in Pin1 process LAN myocardial cell, partly suppress cell death and the apoptosis of ethanol induction. For the downstream signal of the apoptosis of cardiac muscle caused at ethanol of Pin1 in studying further, we lower at the Pin1 of unused Ethanol Treatment or analyze p-p66Shc level in mitochondrion in process LAN myocardial cell. Being expected as us, Pin1 lowers and decreases p66Shc level in mitochondrion, and Pin1 process LAN adds p66Shc level in mitochondrion (Fig. 4 i-j).

Embodiment four: Pin1 regulates the myocardial cell NO of ethanol mediation and produces and eNOS expression

Owing to nitric oxide (NO) plays an important role in apoptosis of cardiac muscle, we study whether ethanol mediates endothelium eNOS expression. In Fig. 5 a-c, ethanol suppresses NO to produce and eNOS level in dose-dependent mode. We also test generation and the eNOS expression of the NO that ethanol causes in the Pin1 myocardial cell lowered. Comparing compared with control cells, ethanol significantly reduces NO and produces, and Pin1 downward part reverses it and produces to reduce (Fig. 5 c). Under the existence of ethanol, Pin lowers and can continue to suppress the expression of eNOS to reduce (Fig. 5 d). In order to study the function of Pin1 in the NO of ethanol mediation produces and eNOS expresses further, our process LAN Pin1 myocardial cell, the level of NO and eNOS is processed and assesses with ethanol (50 mMs). In Fig. 5 (e) and 5 (f), Pin1 process LAN suppresses NO to produce further and eNOS expresses, and this shows in myocardial cell, and the NO of Pin1 adjustment ethanol mediation produces and eNOS expresses.

Above said content is only the preferred embodiments of the present invention, is merely illustrative for the present invention, and not in any limiting sense. It will be understood by those skilled in the art that and the details of technical solution of the present invention and form can be modified or replace lower without departing from the spirit and scope of the present invention, but fall within protection scope of the present invention.

Claims

1.Pin1siRNA application in the targeted drug of preparation treatment alcoholic cardiomyopathy.

2.Pin1 application in the targeted drug treating alcoholic cardiomyopathy as Sites Screening.

3. apply as claimed in claim 2, it is characterised in that described targeted drug suppresses the expression of Pin1.