Adiponectin up-regulates the expression of T-cadherin in cardiomyocytes injured by hypoxia/reoxygenation / 生理学报

The aim of the present study was to investigate the effects of adiponectin (APN) on the expression of T-cadherin in cultured Sprague-Dawley (SD) rat cardiomyocytes injured by hypoxia/reoxygenation (H/R). Primary myocardial cells from neonatal rats were obtained by enzymatic digestion. The cells were divided into control group, H/R group and H/R+APN (3, 10, 20 and 30 μg/mL) groups. The H/R group was incubated in anoxic environment (anoxic solution saturated with high concentration N2) for 3 h, and then in the reoxygenation environment (the reoxygenation solution saturated with pure oxygen) for 1 h. The H/R+APN group was pretreated with different concentrations of APN for 24 h prior to the initiation of H/R. The content of lactate dehydrogenase (LDH) was measured by chemistry chromatometry. Cellular apoptosis was analyzed by flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). The expression of T-cadherin was detected by RT-PCR and Western blotting. The results showed that, compared with control group, the apoptotic rate and release of LDH were significantly increased in the H/R group, whereas the expressions of T-cad mRNA and protein were decreased. Pretreating with APN significantly and dose-dependently decreased apoptotic rate and LDH release, and up-regulated T-cad mRNA and protein level in rat neonatal cardiomyocytes under H/R conditions. These results suggest that APN may protect cardiomyocytes against H/R-induced injury by up-regulating H/R-decreased T-cad expression.

Glucagon like peptide-1 inhibits high glucose-induced injury of oxidative stress in cardiomyocytes of neonatal rats / 生理学报

The present study was to investigate the effect of glucagon like peptide-1 (GLP-1) on high glucose-induced oxidative stress of cardiomyocytes and the possible role of the PI3K-Akt signal path in this process in the neonatal SD rats. With enzymatic digestion and immunofluorescence identification, cardiomyocytes after 72-96 h of primary culture were used in experiment. The cells were divided into 5 groups: normal control group, high glucose group, high glucose + GLP-1 group, high glucose + GLP-1 + LY294002 group and high osmolarity control group. The content of MDA was detected by TBA colouration method. The content of SOD was detected by xanthine oxidase method. The change of NADPH P47phox subunit mRNA quantity was detected by PCR gel electrophoresis. The level of ROS was detected by flow cytometry, and was also observed by fluorescence microscope. The DNA ladder was examined by agarose gel electrophoresis, and the cell apoptosis was determined by Annexin-V-FITC/PI flow cytometry, and the phosphorylation of Akt was determined by Western blotting. Compared with those in the normal control group, in the high glucose group, the cells grew poorly, and the beating rate was significantly lower (P < 0.05); The apoptotic rate was significantly increased (P < 0.05); The MDA content was increased (P < 0.05); It showed the typical DNA ladder, which is the characteristic of apoptosis; The SOD activity was decreased (P < 0.05); The level of intracellular ROS increased (P < 0.05); And the expression of NADPH P47phox subunit mRNA was increased; However the phosphorylation level of Akt was decreased. Pretreatment with GLP-1 improved the above-mentioned parameters and decreased the expression of NADPH P47phox subunit mRNA (P < 0.05). However, compared with the high glucose + GLP-1 group, LY294002, an inhibitor of PI3K-Akt signal path, attenuated the protective effect of GLP-1 in the high glucose + GLP-1 + LY294002 group. It is suggested that GLP-1 plays a protective role in the high glucose-induced injury and apoptosis of cardiomyocytes, and the PI3K-Akt signal path is involved in this process.

Protective effects of adiponectin against hypoxia/reoxygenation injury in neonatal rat cardiomyocytes / 生理学报

The aim of the present study is to investigate the effects of adiponectin (APN) on hypoxia/reoxygenation (H/R) injury in cultured cardiomyocytes. Primary cardiomyocytes were obtained from neonatal rats by enzymatic digestion method and identified by immunofluorescent technique. Primary cells cultured for 72 h were used in experiment and divided into 5 groups randomly: Control group, H/R group, H/R+APN group, H/R+APN+adenine 9-beta-D-arabinfuranoside (AraA, AMPK inhibitor) group, and H/R+AraA group. The cardiocyte morphology and beating rate were observed under inverted microscope. The DNA ladder was examined by agarose gel electrophoresis, and the cell apoptosis was determined by flow cytometry. Moreover, the malondialchehyche (MDA) content in myocardial cells and the superoxide dismutase (SOD) activity in the supernatant were measured using kits, the fluorescence intensity of intracellular Ca2+ was observed by laser scanning confocal microscope, and the phosphorylation of AMPK was determined by Western blotting. Compared with control group, H/R group showed increased apoptotic rate, oxidative stress level, intracellular Ca2+ concentration and phosphorylation level of AMPK (P<0.05), while significant ameliorations in the above indices were seen in H/R+APN group. On the contrast, AraA attenuated the protective effect of APN and decreased the phosphorylation of AMPK. These results suggest that adiponectin can protect cardiomyocytes from H/R-induced oxidative stress and apoptosis through AMPK pathway.

Effects of RNA interference targeting angiotensin 1 receptor and angiotensin-converting enzyme on blood pressure and myocardial remodeling in spontaneous hypertensive rats / 中华心血管病杂志

<p><b>OBJECTIVE</b>To investigate the effects of RNA interference (RNAi) targeting angiotensin II Type 1 receptor (ATlR) and angiotensin-converting enzyme (ACE) on blood pressure and myocardial remodeling in spontaneous hypertensive rats (SHRs).</p><p><b>METHODS</b>Saline (control), adenovirus (Ad5) and recombinant adenoviral vectors (Ad5-ACE-shRNA, Ad5-AT1R-shRNA and Ad5-ACE-AT1R-shRNA expressing ACE, AT1R, ACE and AT1R gene-specific shRNA, respectively) were randomly administered by caudal intravasation to SHRs (n = 12 each group) at day 1 and 17. Normotensive Wistar-Kyoto rats (WKY) served as normal controls. Systolic blood pressure (SBP) of the caudal artery was measured daily. Expression of ACE and AT1R at mRNA levels in ventricle and aorta were evaluated by fluorescence quantitative PCR. Angiotension II serum concentration was measured by ELISA at day 3 (n = 6 each group). The ratio of left ventricular to body weight (LVW/BW) and myocardial collagen content were measured, myocardial ultrastructure observed under transmission electron microscope at the study end.</p><p><b>RESULTS</b>The caudal artery pressure of saline and Ad5 group was equally increased by about 26 mm Hg(1 mm Hg = 0.133 kPa) compared to baseline (both P < 0.05). Ad5-ACE-shRNA, Ad5-AT1R-shRNA and Ad5-ACE-AT1R-shRNA injection significantly reduced SBP (-24 mm Hg, -22 mm Hg and -26 mm Hg respectively, all P < 0.05 vs. baseline) and the antihypertensive effect could last at least 15 days post each injection. SBP was not affected by saline and Ad5 injections. ACE and AT1 mRNA expressions at ventricle and aorta were significantly decreased in Ad5-ACE-shRNA, Ad5-ACE-AT1R-shRNA and Ad5-AT1R-shRNA, Ad5-ACE-AT1R-shRNA treated SHRs compared to those in saline and Ad5 groups (all P < 0.05) and was comparable to that in WKY group (P > 0.05). The LVW/BW ratio [(2.22 +/- 0.18) microg/mg, (2.23 +/- 0.19) microg/mg, (2.17 +/- 0.16) microg/mg] and myocardial collagen content [(1.291 +/- 0.019) microg/mg, (1.298 +/- 0.019) microg/mg, (1.276 +/- 0.019) microg/mg] in Ad5-ACE-shRNA, Ad5-AT1R-shRNA and Ad5-ACE-AT1R-shRNA treated SHRs were also significantly lower than those in saline treated [(3.23 +/- 0.13) microg/mg and(1.683 +/- 0.013) microg/mg, both P < 0.05] and Ad5 treated SHRs [(3.25 +/- 0.12) microg/mg and(1.693 +/- 0.013) microg/mg, both P < 0.05], but still higher than those of WKY group [(2.06 +/- 0.12) microg/mg and (1.258 +/- 0.019) microg/mg, both P < 0.05]. Myocardial ultrastructure was also significantly improved in all SHRs underwent RNAi treatments compared to saline and Ad5 treated SHRs.</p><p><b>CONCLUSION</b>RNAi targeting ACE and AT1R gene significantly inhibited myocardial and aortic ACE and AT1R mRNA expressions and resulted in prolonged antihypertensive effects and myocardial ultrastructure improvements in SHRsl. The RNAi technology may be a potential new strategy of gene therapy for hypertension.</p>

Effects of RNA interference targeting angiotensin-converting enzyme on the blood pressure and myocardial remodeling in spontaneously hypertensive rats / 中华心血管病杂志

<p><b>OBJECTIVE</b>To investigate the effects of RNA interference (RNAi) targeting angiotensin-converting enzyme (ACE) on the blood pressure and myocardial remodeling in spontaneously hypertensive rats (SHRs).</p><p><b>METHODS</b>Saline (control), adenovirus (Ad5) and recombinant adenoviral vectors (Ad5-ACE-shRNA expressing ACE gene-specific shRN) were randomly administered by caudal intravasation to SHRs (n = 12 each group) at day 1 and day 16. Normotensive Wistar-Kyoto rats (WKY) served as normal controls. Systolic blood pressure (SBP) of the caudal artery was measured daily. Expressions of ACE at mRNA and protein levels in myocardium and aorta were evaluated by RT-PCR and Western blot respectively, ACE serum concentration was measured by ELISA at day 3 (n = 6 each group). The ratio of left ventricular to body weight (LVW/BW), myocardial collagen content were measured and myocardial ultrastructure observed under transmission electron microscope at the study end.</p><p><b>RESULTS</b>Ad5-ACE-shRNA injection significantly reduced SBP (-22 mm Hg, 1 mm Hg = 0.133 kPa) and the antihypertensive effect could last at least 14 days post each injection. SBP was not affected by saline and Ad5 injections. ACE expressions at mRNA and protein levels at myocardium and aorta as well as serum ACE were significantly decreased in Ad5-ACE-shRNA treated SHRs compared to that in saline and Ad5 groups (all P < 0.05) and was comparable to that in WKY group (P > 0.05). The LVW/BW ratio (2.24 +/- 0.19) and myocardial collagen content [(1.283 +/- 0.019) microg/mg] in Ad5-ACE-shRNA treated SHRs were also significantly lower than those in saline treated [3.21 +/- 0.13 and (1.686 +/- 0.013) microg/mg, both P < 0.05] and Ad5 treated SHRs [3.13 +/- 0.12, (1.682 +/- 0.009) microg/mg, both P < 0.05] but still higher than those of WKY group [2.06 +/- 0.11, (1.257 +/- 0.019) microg/mg, both P < 0.05]. Myocardial ultrastructure was also significantly improved in Ad5-ACE-shRNA treated SHRs compared to saline and Ad5 treated SHRs.</p><p><b>CONCLUSION</b>RNAi targeting ACE gene significantly inhibited the expressions of ACE at mRNA and protein levels and resulted in prolonged antihypertensive effects and myocardial ultrastructure improvements in this SHR model.</p>

A Study in Construction of Short Hairpin Small Interfering RNA Expression Vector Target Lectin Like Oxidized Low Density Lipoprotein Receptor-1 Gene and Its Effect on Foam Cells / 中国生物工程杂志

Aim:To construct the short hairpin small interfering RNA(shRNA) eukaryotic expression vector specific to mouse lectin like oxidized low density lipoprotein receptor 1(LOX-1) gene and to observe its silencing effect on LOX-1 in RAW264.7 cells.Methods:(1)The pLOX-1-shRNA expression vector was constructed by gene recombination,Then transfected into the cultured RAW264.7 cells.At 48 h after Transfection,the expression of LOX-1 mRNA in RAW264.7 cells were determined by semi-quantitative RT-PCR,the expression of LOX-1 proteins examined by Western blot.(2) Oil Red O Dyeing experiment was used to show the cellular lipid droplets in lipid-loaded cells.The method of cholesterol oxidase analysis was performed to determine the content of cellular cholesterol.The ability of uptake Dil-ox-LDL in RAW264.7 cells was assayed by fluorescence microscopy.Results: pLOX-1-shRNA expression vector was successfully constructed.Transfection of pLOX-1-shRNA expression vector into RAW264.7 cells down regulaled the expression level of LOX-1 gene,as compared with the control group,transfection of the RAW264.7 cells with LOX 1-shRNA led to a remarkable reduction of the number macrophages transformed into foam cell,and could suppress the uptake of ox-LDL.Conclusion:The pLOX-1-shRNA expression vector can inhibit the expression of LOX 1 in RAW264.7 cells and the transformation of the macrophages into foam,which may he beneficial in searching new gene therapy of atherosclerosis.

Effects of Adenovirus-mediated Angiotensin-Converting Enzyme-shRNA on Spontaneously Hypertensive Rats / 中华高血压杂志

Background Hypertension is a multigenetic inheritable disease.Gene therapy with long-term effects and less side effects by regulating gene expression has been shown to be a potential and exciting prospect. Objective To investigate the effects of RNA interference(RNAi)targeting angiotensin-converting enzyme(ACE)on the blood pressure and ACE expression in kidney of spontaneously hypertensive rats(SHR).Methods SHR were randomly to receive placebo(n=12)or control adenovirus Ad5-EGFP)or a single injection of recombinant adenovi- ral vectors,Ad5-EGFP-ACE-shRNA(n=12,iv).Normotensive Wistar-Kyoto rats(WKY)were served as normal control group.SBP was measured before and after the intervention.Aorta,lung,myocardium and kidney were studied using fluorescence microscope to identify the sites of Ad5-EGFP-ACE-shRNA.Expressions of ACE mRNA and protein in kidney were evaluated by RT-PCR and Western blot.Results SBP of the treat group was effectively reduced by 19.0?3.2 mmHg at the 3rd day,and 22.1?3.3 mmHg at the 13th day of the experiment.The anti- hypertensive effect significant remained at least for 14 days.On the contrary,increase in BP was shown in placebo and the adenovirus control group.Compared with placebo or adenovirus control rats,ACE mRNA expression level in kidney of the treated rats was lower by 61.1% and 62.3% respectively,with ACE protein expression level lower- ing by 56.2% and 53.30% as well(ail P0.05). Conclusion RNA interference targeting ACE gene inhibits the expressions of ACE mRNA and protein.A single dose injection resulted in a prolonged decrease in BP.The evidence of strong antihypertensive effect by genetic therapy justifies efforts for further investigation.