| Part I cAMP Regulate Myocardial Fibrosis after Myocardial Infraction by Inhibiting CTGF Expression Level in MiceObjective To observe the regulatory effects of cyclic adenosine monophosphate (cAMP) on connective tissue growth factor (CTGF) and myocardial fibrosis.Methods72SPF C57BL mices were random divided into three groups:sham operation group (MS group), myocardial infarction group (MI group) and drug treatment group (MT group). MT group were injected meglumine adenosine cyclophosphate (MAC) by intraperitoneal for4weeks after7days postoperation.5weeks after operation,10mices in each group were examined by transthoracic echocardiography and then harvested heart tissues for HE, masson and immunohistochemical (IHC) staining. The left mices were harvested heart tissues and random divided into three groups, separated the infracted zone and non-infracted zone of left ventricle, then detected the expression level of CTGF mRNA, TGF-β1mRNA, CTGF protein and hydroxyproline separately.Results Heart ultrasound showed the heart function indexes (EF%, FS%and SV) in MT group were higher than MI group [EF (19.83±3.95)%vs.(13.65±2.14)%; FS (9.07±1.9)%vs.(6.22±1.03)%; SV (26.26±3.57μL) vs.(18.82±2.71μ L)](P<0.05), the heart morphological indexes (wall thickness and diameter) of left ventricular between MT group and MI group has no statistically significant differences (P>0.05). Masson staining examination shows the degree of collagenous fiber infiltration from infracted zone to non-infracted zone in MT group was lower than MI group. Compared with MI group, the expression level of CTGF protein, CTGF mRNA and hydroxyproline in MT group (decreased after intervening by MAC (P<0.05). But the TGF-β mRNA expression level has no statistically significant differences both in infracted zone and non-infracted zone between MT group and MI group (P>0.05)Conclusion cAMP can reduce the collagenous fiber expression level by inhibiting CTGF expression level, decrease the infiltration degree of collagenous fiber from infracted zone to non-infracted zone and improve the heart function after myocardial infarction in mice. Part Ⅱ Activating the cAMP Signaling Pathway to Regulate CTGF Expression Level in Mice Cardiac FibroblastsObjective To observe the inhibiting effect of cyclic adenosine monophosphate (cAMP) on connective tissue growth factor (CTGF) in mice cardiac fibroblasts (MCFs) and discuss the signal transduction pathway.Methods Isolated and primarily cultured MCFs from C57BL newborn mice’s heart within1week old and used the third generation cells for experiment. MCFs were stimulated by transforming growth factor β1(TGF-β1) to simulate the high CTGF expression after myocardial infarction in mice. Then MCFs were cultured with Forskolin for increasing the intracellular content of cAMP, then dectected the expression level of CTGF mRNA, CTGF protein, PKA, p44/42MAPK and phospho-p44/42MAPK. PD98509and Rp-cAMPS were used to inhibit the phospho-p44/42MAPK and PKA expression level respectively and detected the expression change of the PKA, p44/42MAPK, phospho-p44/42MAPK and CTGF.Results TGF-β1can stimulated the MCFs to express high level of CTGF. After intervened by Forskolin, the content of cAMP in MCFs increased and the cell viability decreased while the expression level of PKA and CTGF increased, the phosphorylation level of p44/42MAPK declined. When phospho-p44/42MAPK was inhibited by PD98509, the PKA expression level increased while the expression level of CTGF decreased, but after PKA was inhibited by Rp-cAMPS, the phosphorylation level of p44/42MAPK and CTGF expression level upregulated.Conclusion Forskolin can inhibit the CTGF expression level by increasing the intracellular content of cAMP. The signal transduction pathway is the high concentrations of cAMP upregulate the PKA expression level which reduce the phosphorylation level of p44/42MAPK and then inhibit the CTGF expression level. |
PDF Full Text Request