| Cardiac hypertrophy, which is mainly characterized by cardiacmyocyte hypertrophy and interstitial change, is a response of the activation and inactivation of gene and cell phenotype alteration intracellular by stimulating a series of extracellular signal transduction process. At early stage, it can maintain the cardiac output, keep room wall pressure balance and eliminate the influence of the initial stimulus, however, it undergo apoptosis, cardiac dilation and heart failure eventually. At the cellular and molecular level, there are mainly 3 ways of the molecular mechanisms in cardiac hypertrophy: extracellular and intracellular stimulus, gene transcription in the nucleus.Adipose tissue, secreting a variety of cytokines and bioactive substances, is the biggest endocrine and paracrine organ in the body. Not only can it control energy balance in vivo, but also play an important role in regulate insulin sensitivity, diabetes, coagulation, fibrinolysis, inflammation, atherosclerosis and so on. Tumor necrosis factor-α(TNF-α), interleukin(IL)-6, plasminogen activator inhibitor(PAI-1), leptin, resistin, chemerin, retinol binding protein(retinol binding prorein, RBP) are known as Pro-inflammatory cytokines, whereas resistin,adiponectin and Clq/tumor necrosis factor related protein(CTRP9) are of anti-inflammatory properties.Secreted frizzled-related protein 5, a member of secreted frizzled-related protein family, is a novel anti-inflammatory adipokine associated with obesity and insulin resistance(IR). It is mainly secreted by white adipose tissue, highly expressed in the retinal pigment epitheliumis and pancreas, and also weakly expressed in the liver and muscle tissue. Studies have confirmed that the secreted frizzled-related protein 1-4 expressed in myocardial tissue and increased in cardiac hypertrophy. Structurally, secreted frizzled related protein can compete with frizzled(Fz) receptor which is a specific receptor of wnt signaling pathway through cysteine rich district(CRD), and thereby inhibit wnt signaling pathway. Wnt proteins are a group of cysteine-rich glycosylated proteins involved in cell proliferation, differentiation, apoptosis, and cellular processes such as positioning control. Wnt signaling pathway is involved in the regulation of cell proliferation, differentiation, apoptosis and a variety of pathological immune and other physiological processes.Research have confirmed that Wnt pathways are associated with myocardial hypertrophy, inhibition of wnt pathway play a significant role in the reversing cardiac hypertrophy. Studies have shown that Wnt signaling pathway plays an important role in heart development and angiogenesis. It can activate Wnt/β-catenin signaling pathway, promote cardiac progenitor cells proliferation and migration at early phrase of cardiogenesis and inhibiting its activity to differentiation at late stage. As a anti-inflammatory factor, secreted frizzled-related protein 5may have a protective role in atherosclerosis.Angiotensin II stimulation enables cell proliferation, tissue remodeling in cardiovascular system and plays an important role in cardiac hypertrophy. The biological effects of Ang II is mediated by Ang II type 1 receptor(AT1R) and type Ang II 2 receptor(AT2R). Both of them belong to the G protein-coupled receptors, but the tissue distribution and intracellular signal transduction pathways are not the same. Ang II binding with AT1 receptor cause cardiac hypertrophy, accumulation of extracellular matrix proteins and promote secretion of aldosterone, however, Ang II coupled with AT2 receptor play opposite effect with AT1 receptor, like makeing the blood vessels dilated, suppressing vascular smooth muscle cells’(VSMC) proliferation and participating in tissue repairing. It has been confirmed that AT2 receptor express up-regulated in ventricular hypertrophy, myocardial infarction and heart failure and mediate major role of Ang II. ATl receptors are mainly distributed in the lung, vascular smooth muscle, liver, kidney and adrenal gland, AT2 receptors are distributed in the embryonic tissue, brain and reproductive organs. The purpose of this study is to demonstrate the specific receptor exactly which mediate the expression of secreted frizzled-related protein 5 in cardiac hypertrophy.Rho/ROCK(Rho-associated coiled-coil protein kinase) signaling pathway, having the effect of the "molecular switch", is prevalent in a variety of tissue cells. Generally, it mediate cytoplasmic signal and translate signal into the nucleus, thereby regulating the expression of genes and proteins. Numerous studies have shown that Rho/ROCK signaling pathway is widely involved in various pathophysiological processes of the cardiovascular system, such as myocardial contraction, migration, proliferation, apoptosis, cardiac hypertrophy, myocardial infarction and ventricular remodeling. Rho-kinase may take part in the regulation cardiac hypertrophy and vascular smooth muscle hypertrophy processes, which are induced by Ang II. In addition, Rho/ROCK pathway involved in Ang II types 1 receptor(AT1R) signal transduction.Mitogen activated protein kinase(MAPK) signaling pathway, the hub of the signal transmission from cell membrane to the nucleus, is one of the major pathways of intracellular signal transduction. It belongs to serine/threonine protein kinase, There are four subtypes: extracellular regulated protein kinase 1,2(extracellular signal-regulated kinase 1/2, ERK1/2), P38 MAPK, c-Jun N-terminal kinase(c-Jun N-terminal Kinase, JNK) and ERK5 currently. They interleave and play significant role in a series of different physiological processes like regulating cell growth, differentiation and apoptosis. It is considered that MAPK activation is the final passage from contractile to hypertrophic form in cardiomyocyte hypertrophy, and is closely associated with the occurrence of cardiac remodeling and heart failure. JNK signaling pathway is an important member in MAPKs family which are involved in the regulation of cell proliferation, differentiation and apoptosis. Studies have demonstrated that myocardial fibrosis may be mediated by activated Rho/ROCK pathway through JNK pathway.In summary, we speculate s FRP5 can be expressed in cardiomyocytes, and presents up-regulated in cardiac hypertrophy process. In this study, we observe the expression of secreted frizzled-related protein 5 in cultured neonatal SD rat cardiomyocytes through Western Blot and Real-time PCR and explore the effect of Ang II-AT1R?Rho?ROCK?JNK signal pathway in the secreted frizzled-related protein 5 expression. It follows by three parts: Part one The expression of secreted frizzled-related protein 5 incardiomyocytesObjectives: To explore whether or not secreted frizzled-related protein 5 can express in cardiomyocytes.Methods: Ventricular cardiomyocytes were dissociated from the 2 to 3-day old neonatal SD rats. Forty-eight hours after seeding, the cardiomyocytes were cultured with the serum-free culture medium for another twenty-four hours. Afterthat, secreted Frizzled-related protein 5 expression in cardiomyocytes were determined by RT-PCR and Western-Blot.Results: The m RNA and protein expression of secreted frizzled-related protein 5 were detected in cardiomyocytes.Conclusions: Cardiomyocytes can express secreted frizzled-related protein 5. Part two Secreted frizzled-related protein 5 expression in angiotensinII-induced cardiomyocyte hypertrophyObjectives: To investigate the expression of Secreted frizzled-related protein 5 in angiotensin II(Ang II)-induced cardiomyocyte hypertrophy in vitro by means of the cultured neonatal rat cardiomyocytes.Methods: After primary culture of 2-3 day-old Sprague-Dawley(SD) rat cardiomyocytes, cardiomyocytes were pretreated with Ang II at different concentrations and different time intervention. The expression of Secreted frizzled-related protein 5 was examined by means of RT-PCR and WesternBlot. The expression of brain natriuretic peptide(BNP) was examined through western-blot analysis.Results: Ang II treatment induced the increased expression of Secreted frizzled-related protein 5 and BNP in a dose-and time-dependent manner in cardiomyocytes.Conclusions: Exposure to Ang II significantly upregulated s FRP5 and BNP protein and m RNA levels increased in a dose-and time-dependent manner. Ang II increases s FRP5 and BNP expression at an optimal concentration of 10-6 mol/L at 48 hours. Part three Role of Angiotensin II receptors in secreted frizzled-relatedprotein 5 expressionObjectives: To investigate the Angiotensin II receptors in secreted frizzled-related protein 5 expression in cardiomyocyte hypertrophy in vitro by means of the cultured neonatal rat cardiomyocytes.Methods: After primary culture of 2-3 day-old Sprague-Dawley(SD) rat cardiomyocytes, cardiomyocytes were pretreated with Ang II. Ang II type-1 receptor(AT1R) antagonist telmisartan and Ang II type-2 receptor(AT2R) antagonist PD123319 were used to block effects of Ang II. The expression of Secreted frizzled-related protein 5 was examined by means of RT-PCR and Western-Blot.Results: Pretreat with AT1 R antagonist telmisartan can completely blocked Ang II-induced Secreted frizzled-related protein 5 expression increasement(P<0.05).Conclusions: Secreted frizzled-related protein 5 expression was increased mainly through the AT1 R in the process of Ang II-induced cardiomyocyte hypertrophy. Part four Molecular mechanisms of Secreted frizzled-related protein 5 expression in angiotensin II induced cardiomyocyte hypertrophyObjectives: To observe the role and their relationships between Rho/ROCK and MAPK signaling pathway in Secreted frizzled-related protein 5 expression.Methods: After extracting and culturing primary 2-3 day-old SpragueDawley(SD) rat cardiomyocytes, cardiomyocytes were pretreated with Ang II. Y27632 and telmisartan were used to block effects of Rho/ROCK signal pathway and AT1 R. After that,SB203580, PD98059 and SP600125 were used to inhibit p38 mitogen-activated protein kinase(p38MAPK), extracellular signal-regulated protein kinases 1 and 2(ERK1/2) and c-Jun NH2-terminal kinase(JNK) pathway, respectively. The expression of Secreted frizzledrelated protein 5 was examined by means of RT-PCR and western-blot. The expression of brain natriuretic peptide(BNP), total c-Jun NH2-terminal kinase(JNK), p-JNK, total(myosin phosphatase target subunit 1(MYPT1)and p-MYPT1.Results: The levels of secreted frizzled-related protein 5 m RNA and protein expression were significantly lowered compared to Ang II group after treated with Y27632,(P <0.05). The total MYPT1 and total JNK expression had no obvious change. However, the p-MYPT1, p-JNK, p-MYPT1/total MYPT1, p-JNK/total JNK levels were significantly increased,(P<0.05). After intervented with AT1 R blocker telmisartan for 48 hours, the p-MYPT1/total MYPT1 levels were significantly downregulated,(P<0.05), the p-JNK/JNK levels were obviously decreased as well(P<0.05).Conclusions: Rho/ROCK pathway is necessary secreted frizzled- related protein expression in Ang II-induced cardiomyocyte hypertrophy, and it is the downstream of AT1 R pathway. Ang II increases s FRP5 expression through AT1R/Rho/ROCK/JNK signaling in cardiomyocyte. |
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