| Myocardial hypertrophy is an adaptive response of the heart to virtually all forms of cardiac diseases, including those arising from hypertension, mechanical load, myocardial infarction, cardiac arrhythmias and endocrine disorders. While the hypertrophic response is initially a compensatory mechanism that augments cardiac output, hypertrophy can lead to dilated cardiomyopathy, heart failure, and sudden death. Cardiac hypertrophy was an absolute and dangerous factor of cardiovascular diseases.Two principal factors influence the genesis of myocardial hypertrophy: one belongs to mechanical causes, for example: pressure load and capacity load; the other includes nervous and body fluid effects such as renin-angiotensin-aldosterone system (RAAS), sympathetic nervous system, adrenal medulla hormone, thyrine, endothelin, myocardial hypertrophy peptide, aldosterone, atriopeptins, and nitrogen monoxide et al. Previous researches demonstrated that RAAS played an important role in the maintenance of normal cardiovascular function, moreover, this system also contributed to the occurrence of myocardial hypertrophy. AngiotensinⅡ(AngⅡ) manifests positive inotropic action and intensive vasoconstriction. It stimulates cellular protein synthesis, and involves in the early transduction of signaling pathway which eventually induces myocardial hypertrophy. The enlarged volume of cardiomyocytes and proliferation of cardio interstitial cells comprise the two elemental processes of myocardial hypertrophy. One of the main tasks in current cardiovascular researches is to develop effective therapeutical agents to treat myocardial hypertrophy. Numerous evidences indicate that Centipede can be used to improve myocardial ischemia, and artherosclerosis. In vitro studies confirm that Centipede Acidic Protein (CAP) had extensive cardiovascular activities including enhancing the contractilities of cardiac muscle and increasing the frequency of heart beat, all these effects were effective in dose-dependent manner.The aim of the present study was to investigate the cardioprotective effects of CAP on myocardial hypertrophy and the potential underlying mechanisms involved. The methods such as immunhistochemistry, flow cytometry (FCM), confocal laser scanning microscope (CLSM), and molecular biology techniques (Western-blot, RT-PCR) were applied, the experiments were designed to explore: (1) Cardioprotective effects of CAP against pressure-overload induced myocardial hypertrophy and possible mechanisms; (2) Effects of CAP on apoptosis of rat cardiomyocytes induced by AngⅡin vitro; (3) Influences of CAP on the proliferation of cultured neonatal rat cardiac fibroblast stimulated by AngⅡ; (4) Effects of CAP on cardiac function of rats with acute heart failure.Part 1 Cardioprotective effects of Centipede Acidic Protein against pressure-overload induced myocardial hypertrophy and mechanisms involvedObjective: To investigate the effects of CAP on pressure-overload induced myocardial hypertrophy (POH) and potential underlying mechanisms involved.Methods: Rats were divided into 4 groups: sham group (saline, 1ml·100g-1, i.p.), POH group (saline, 1ml·100g-1, i.p.), captopril group (captopril, 30mg·kg-1, i.g.), CAP group (CAP, 2.0g·kg-1, i.p.). POH was induced by partial abdominal aorta constriction (AC) in rats. The hemodynamics, the LVMI and the contents of AngⅡ, ET, ALD, ANP, NO, NOS, SOD were measured after 8 weeks of AC. The middle cardiac tissue of left ventricle was cut up, and fixed in 10% formaldehyde, sliced up, dyed by HE and Masson methods, changes of cardiac pathological histology were observed by microscope, MD and CVF were measured. Results:1. HR was 384.0±21.5, and SBP, DBP, MBP were 28.12±1.75, 18.24±1.18, 24.78±1.27 respectively in POH group, which was significantly higher than that in sham group (P<0.01). HR was 353.8±22.8, SBP and MBP were 25.84±1.54 and 22.16±1.95 respectively in CAP 2.0g·kg-1 group, which was significantly lower than that in POH group (P<0.05 or P<0.01).2. Values of LVSP and LVEDP were 24.52±1.89 and 2.86±0.84 respectively in POH group, which was significantly higher than that in sham group (P<0.01). Values of +dp/dtmax and -dp/dtmax were 1032.6±116.2 and 874.2±51.3 respectively, which was significantly lower than that in sham group (P<0.01). LVEDP was 1.98±0.57 in CAP group, which was significantly lower than that in POH group (P<0.05). Values of +dp/dtmax and -dp/dtmax were 1213.0±119.1 and 978.6±65.0 respectively, which was significantly higher than that in POH group (P<0.05).3. Values of LVMI, MD and CVF were 3.11±0.14, 44.80±4.03 and 21.86±0.62 respectively in POH group, which was significantly higher than that in sham group (P<0.01). Values of LVMI, MD and CVF were 2.51±0.22, 34.20±2.17 and 20.48±0.60 respectively in CAP group, which was significantly lower than that in POH group (P<0.05 or P<0.01).4. Levels of AngⅡ, ALD, ET and ANP were 122.93±17.42, 156.34±30.42, 150.44±13.24 and 563.38±77.20 respectively in POH group, which was significantly higher than that in sham group (P<0.05 or P<0.01). Levels of AngⅡ, ALD, ET and ANP were 102.95±20.56, 127.90±26.66, 130.82±8.41 and 491.97±56.74 respectively in CAP group, which was significantly lower than that in POH group (P<0.05).5. Contents of NOS, NO and SOD were 16.13±2.27, 134.71±38.48 and 40.55±5.95 respectively in POH group, which was significantly lower than that in POH group (P<0.01). Contents of NOS, NO, SOD were 19.32±1.66, 175.22±45.83 and 47.66±5.51, respectively in CAP group, which was significantly higher than that in POH group (P<0.05).Conclusions: These results indicate that CAP can inhibit the progress of POH mediated by combined mechanisms of regulation of RAAS, NO system.Part 2 Effects of Centipede Acidic Protein on apoptosis of rat cardiomyocytes induced by AngⅡin vitroObjective: To study the protective effects of CAP on AngⅡ-induced apoptosis of rat cardiomyocyte in vitro and the possible mechanisms.Methods: Cultured cardiomyocytes from neonatal rats were divided into 4 groups: Control group (DMEM without serum), AngⅡgroup (10-7mol·L-1), CAP high-dose group (AngⅡ10-7mol·L-1+CAP 4.8mg·L-1), CAP low-dose group (AngⅡ10-7mol·L-1+CAP 0.48mg·L-1). Cell viability was measured by MTT. Apoptosis was observed by using confocal laser scanning microscope (CLSM). The activity of caspase-3 was detected by caspase-3 Activity Assay Kit. The expression of Bcl-2, Bax were observed. The c-fos expression was examined by semi-quantitative RT-PCR analysis. The protein expression of calcineurin (CaN) was determined with Western blot.Results:1. AngⅡsignificantly decreased cell viability. The MTT values were 0.2648±0.0420, (P<0.01 vs control) in AngⅡgroup. The myocyte viabilities of CAP high- and low-dose groups were enhanced significantly. The MTT values were 0.4306±0.1610 and 0.3118±0.0853 respectively in CAP high- and low-dose groups (P<0.05, vs AngⅡgroup).2. The activity of caspase-3 was 2.8±0.7 in AngⅡgroup, which was significantly higher than that in control group (P<0.05). The activities of caspase-3 were 1.2±0.4 and 1.8±0.5 respectively in CAP high- and low-dose groups, significantly lower than that in AngⅡgroup (P<0.05).3. The expression of Bax was 13.64±4.32 in AngⅡgroup, which was significantly higher than that in control group (P<0.01). The expressions of Bax were 5.85±2.04 and 10.89±3.95 respectively, in CAP high- and low-dose groups, significantly lower than that in AngⅡgroup (P<0.05 or P<0.01). The expression of Bcl-2 was 12.62±2.39 in AngⅡgroup, which was significantly lower than that in control group (P<0.01). The expressions of Bcl-2 were 21.87±3.24 and 16.33±2.22 respectively in CAP high- and low-dose groups, significantly higher than that in AngⅡgroup (P<0.05 or P<0.01).4. The c-fos mRNA expression was 0.82±0.05 in AngⅡgroup, which were significantly higher than that in control group (P<0.05). The c-fos mRNA expressions were 0.51±0.11 and 0.66±0.07 respectively in CAP high- and low-dose groups, significantly lower than that in AngⅡgroup (P<0.05).5. The protein expression of CaN was 19.38±4.72, in AngⅡgroup, which was significantly higher than that in control group (P<0.01). The protein expressions of CaN were 11.28±2.68 and 14.82±3.67 respectively in CAP high- and low-dose groups, significantly lower than that in AngⅡgroup (P<0.05 or P<0.01).Conclusions: CAP can efficiently inhibit apoptosis of neonatal cardiomyocytes induced by AngⅡ. Its mechanisms may be involved in inhibition of the activity of caspase-3, increase in the expression of Bcl-2, down-regulation of the expressions of Bax, c-fos and CaN.Part 3 Influence and Centipede Acidic Protein on the proliferation of cultured neonatal rat cardiac fibroblast stimulated by AngⅡObjective: To study the influence of CAP on the proliferation and collagen synthesis of cultured neonatal rat cardiac fibroblasts (CFb) stimulated by AngⅡ, and to explore the mechanisms involved.Methods: Isolated and cultured CFb of neonatal Sprague-Dawley (SD) rats were divided into 4 groups: Control group (DMEM without serum), AngⅡgroup (10-7mol·L-1), CAP high-dose group (AngⅡ10-7mol·L-1+CAP 4.8mg·L-1), CAP low-dose group (AngⅡ10-7mol·L-1+CAP 0.48mg·L-1). The effects of CAP on proliferation of CFb were observed by MTT colorimetric assay, synthesis of collagen was observed by the hydroxyproline concentration. The NO contents were measured by nitric acid reductase method. The c-myc expression was examined by semi-quantitative RT-PCR analysis. Cell cycle distribution was determined with flow cytometer (FCM).Results:1. In this assay, the growth and proliferation of the fibroblasts were measured, AngⅡsignificantly increased proliferation of the fibroblasts. The MTT value was 0.6702±0.1104in AngⅡgroup (P<0.01 vs control). CAP at 4.8mg·L-1 and 0.48mg·L-1 inhibited markedly CFb proliferation induced by AngⅡ. The MTT values were 0.4306±0.1610 and 0.3118±0.0853 respectively in CAP groups (P<0.05, vs AngⅡgroup).2. AngⅡincreased production of collagen. The value was 0.85±0.06 (P<0.01 vs control). CAP at 4.8mg·L-1, 0.48mg·L-1 could inhibit the production of collagen induced by AngⅡ. The productions of collagen were 0.72±0.13 and 0.55±0.08 respectively in CAP groups (P<0.05, vs AngⅡgroup).3. The percentage of cells in G0/G1, S, G2/M phases was (65.0±6.7)%, (19.3±2.5)% and (15.7±4.5)% respectively in AngⅡgroup. The percentage of cells in G0/G1 phase was significantly lower than that in control group. The percentage of cells in S and G1/M phases was significantly higher than that in control group (P<0.05 or P<0.01). The percentage of cells in G0/G1 phase was (76.8±4.4)% and (75.2±6.0)% in CAP (4.8mg·L-1, 0.48mg·L-1) groups, all significantly higher than that in AngⅡgroup (P<0.01); The percentage of cells in S phase was (14.6±1.4)% and (14.4±1.4)% respectively, in CAP (4.8mg·L-1, 0.48mg·L-1) groups, which was significantly lower than that in AngⅡgroup, (P<0.05); The percentage of cells in G2/M phase was (8.6±4.4)% and (10.4±4.5)% respectively in CAP high- and low-dose groups, which was significantly lower than that in AngⅡgroup (P<0.05 or P<0.01); The proliferation index (PI) was (35.0±6.7)% in AngⅡgroup, which was significantly higher than that in control group (P<0.01). The proliferation index (PI) was (23.2±4.4)% and (24.9±6.0)% respectively in CAP high- and low-dose groups, which was significantly lower than that in AngⅡgroup (P<0.01).4. The contents of NO were 15.35±3.06,in AngⅡgroup, which was significantly lower than that in control group (P<0.01). The contents of NO were 24.44±3.23 and 20.92±2.36 respectively, in CAP high- and low-dose groups, significantly higher than that in AngⅡgroup (P<0.05).5. The c-myc mRNA expression was 0.79±0.06 in AngⅡgroup, which was significantly higher than that in control group (P<0.05). The c-myc mRNA expressions were 0.48±0.13 and 0.63±0.08 respectively in CAP high- and low-dose groups, significantly lower than that in AngⅡgroup (P<0.05).Conclusions:The results suggest that CAP significantly can inhibit cardiac fibrosis by suppression of CFb proliferation and collagen synthesis stimulated induced by AngⅡ, and its mechanisms may contribute to increase in NO contents and down-regulation of c-myc mRNA expression.Part 4 Effects of Centipede Acidic Protein on cardiac function in rats with acute heart failureObjective: To study the effects of CAP on cardiac function in rats with acute heart failure induced by doxorubicin hydrochloride (DH).Methods: Rats were randomly divided into 4 groups: Control group, DH group, CAP high-dose group and CAP low-dose group. CAP high- and low-dose group were given intraperitoneally CAP 2.0g·kg-1 and 1.0g·kg-1 for 3 days. The other 2 groups were given saline 1ml·100g-1 in the same way. On the fourth day, DH (10mg·kg-1) was administered intraperitoneally once to induce acute heart failure. On the fifth day, the parameters of hemodynamics and the contents of NOS, NO, MDA, SOD were measured, and the changes of cardiac pathological histology were observed by microscope.Results:1. The values of LVSP, LVEDP were 92.4±15.7 and 36.8±12.9 respectively in DH group, which was significantly lower than that in control group (P<0.05). The values of +dp/dtmax, -dp/dtmax were 4355.2±860.6 and 3097.6±162.1, which was significantly lower than that in control group (P<0.05). The values of LVSP, LVEDP were 122.2±8.9 and 23.3±9.8 in CAP 2.0g·kg-1 group, which was significantly higher than that in DH group (P<0.05). The values of +dp/dtmax, -dp/dtmax were 8930.8±1928 and 4463.6±968.5, which was significantly higher than that in DH group (P<0.05).2. The contents of NOS, NO, MDA were 23.90±0.51, 190.05±18.30 and 8.53±1.01 respectively in DH group, which was significantly higher than that in control group (P<0.05). The levels of SOD was 62.05±4.97, which was significantly lower than that in control group (P<0.05). The contents of NOS, NO, MDA were 22.16±0.44, 53.90±19.59 and 5.76±0.73 respectively in CAP 2.0g·kg-1 group, which was significantly lower than that in DH group (P<0.05). The levels of SOD was 89.38±4.07, which was significantly higher than that in DH group (P<0.05).3. The myocardial cells in the DH group appeared obviously edema, degeneration and the nucleuses were thin and long compared with control group. The myocardial cells in the CAP 2.0g·kg-1 group appeared normal on the whole, and the nucleuses were normal, compared with DH group.Conclusions: These results indicate that CAP can obviously improve the cardiac function and protect the myocardium from the damage of DH through regulation of oxyradical in body and protection of cardiac myofibrilla.CONCLUSIONS1. CAP can inhibit the progress of myocardial hypertrophy in rats in vivo, and its mechanisms may partly depend on changes in RAAS, NO system.2. CAP can efficiently inhibit apoptosis of neonatal cardiomyocytes induced by AngⅡ. Its mechanisms may be involved in inhibition of the activity of caspase-3, increase in the expression of Bcl-2, down-regulation of the expressions of Bax, c-fos and CaN.3. CAP can significantly inhibit CFb proliferation and collagen synthesis stimulated by AngⅡ, and its mechanisms may contribute to increase in the NO contents and down-regulation of c-myc mRNA expression.4. CAP can obviously improve the cardiac function and protect the myocardium from the damage of DH, which may partly depend on regulation of oxyradical in body and protection of cardiac myofibrilla.
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