| ObjectivesSirt3(sirtuin3) is one of the silent information regulator factor2(SIR2)family members,which located in the mitochondrial inner membrane and has deacetylase activity.This protein is reported to involve in the regulation of many cellular processes, such as cell metabolism,cell cycle, poptosis,cell differentiation and cell life.Sirt3whose increase expression has been linked to the longevity of humans.In recent years,more and more research has focused on the relationship with cardivocascular disease.In the present,the relationship sirt3with hypertension-induced left bentricular hypertrophy is not clear.In this study,we obserbved the expression of sirt3in the hearts of spontaneously hypertensive rats (SHR) and the role of it in left ventricular hypertrophy(LVH)caused by hypertension.Methods1.Experimental animals and design29-week-old SHRs(n=26) were randomly divided into two groups:30-week-old SHR group (fed1week) and38-week-old SHR group (fed9weeks);29-week-old Wistar-Kyoto(WKY) rats(n=20) were used as controls and these control rats were randomly divided into30-week-old WKY group (fed1week) and38-week-old WKY group (fed9weeks).The rats were killed in batches at30-week and38-week. The systolic blood pressure(SBP) of tail artery and left ventricular mass/body mass (LVM/BM) were measured. The cardiac apex(about1/2of ventricular) was reserved. After tin foil wrapped these tissue, liquid nitrogen frozed them.Then,they were stored in -80℃for qRT-PCR and Western.The other tissue was fixated in4%neutrality phosphate paraformaldehyde liquid for48hours and then was imbedded with paraffin for immunohistochemical staining.2. EchocardiographyApparatus:PHILIPS IE33, S12-5ultrasound detector(5MHz frequency). LVEDd, LVESd, LVPWD, IVSD, LVFS were measured to evaluate the cardiac structure and heart function.3. Masson stainingSlice conventionally dewaxed to water. Masson trichrome staining’s kit was staining them.Graded ethanol dehydration,xylene transparent,mounting with neutral resin.And automatic image analysis system was adopted,randomly selected five fields of vision in the light microscope and measured of myocardial collagen volume fraction(CVF).CVF=the area of collagen/total area(the area of collagen did not include the perivascular collagen area).4.Immunohistochemical stainingTaking tissue slice,sirt3of immunohistochemical staining was conducted. Each experimental animal selected five sections, randomly analysis three fields of vision. Image used the Image-Pro Express6.0software to analyze protein expression, results expressed through the intergrated optical density values.5.Western blot method showed the protein expression of sirt3in the tissueTaking tissue from stored at-80℃,at first, extraction of total protein,then determined protein concentration by BCA method. Separation gel and stacking gel collocated by conventional methods,electrophoresis、transmembrane、sealing off、 first-antibody overnight、second-antibody incubation、exposure、development、fuser, bands of gray value analysed with Image-Pro Express6.0software.6.Quantitative reverse transcription PCR (qRT-PCR)Total RNA was extracted by Trizol Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) was used to detect the relative mRNA expression. Sirt3specific primers were designed by Premer5.0, GAPDH for reference.7.Statistics Statistical analysis was performed using SPSS17.0software, measurement data were expressed as means±SD, mean of multiple samples were compared using single factor analysis of variance, pairwise comparisons used the SNK test. Linear correlation (Person-related) analysis studied the correlation between two variables. Values of p<0.05were considered statistically significant.Results1.Two rats died in the experiment, all in38-week-old SHR group, eventually,44rats completed the experiment, including eleven in30-week-old SHR group, thirteen in38-week-old SHR group and twenty WKY rats of controls.2.Compared to the control groups,30-and38-week-old SHRs showed SBP、 LVM/BM、LVESd、IVSD、LVPWD、sirt3mRNA and protein expression of SHRs were significantly increased (all P<0.05); while LVFS and LVEDd were significantly decreased (all P<0.05).Thus,the SHRs showed obvious left ventricular hypertrophy and left ventricular dysfunction which aggravated with time (all P<0.05)3. sirt3mRNA expression of the SHR myocardial correlation analysis with left ventricular hypertrophy.Conclusions1.The high expression of sirt3in myocardium may be closely related to the left ventricular hypertrophy.2.1n SHR rats,with the extension of weeks of age,myocardial hypertrophy aggravated,the expression of sirt3increased further. |
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