The Research Of Mechanism Of B-type Natriuretic Peptide Inhibition Proliferation Of Cardiac Fibroblasts And The Relationship Between BNP And TGF-β1,Natriuretic Peptide Receptors Signal Transduction

Objective:To investigate the effect of B-type natriuretic peptide in cardiac fibroblasts proliferation and the possible signal transduction pathways.Methods: Primary cardiac fibroblasts were isolated by trypsin digestion and purified, and were identified by morphology and immunocytochemistry. After a 24 h synchronization by serum-free DMEM treated with CT-1, cardiac fibroblasts were tread with BNP of different concentration(5×10-4mol/L, 5×10-5mol/L, 5×10-6mol/L, 5×10-7mol/L, 5×10-8mol/L, 5×10-9mol/L respectively)and Brdu proliferation assay was conducted. Cardiac fibroblasts were treated with BNP, SB-431542, CANF4-23, BNP+ SB-431542, and BNP+CANF4-23 , then cGMP level was measured by radioimmunoassay; Cardiac fibroblasts of synchronization treatment by serum-free DMEM treated with CT-1 for 24h, and tread with BNP or SB-431542 were divided into 4 groups: the normal control group, BNP group, SB-431542 group and BNP+SB-431542 group,then the cell culture Medium were collected for TGF-β1 determination. Cells were collected for RNA extraction. The expression of Smad4 mRNA was detected by real time quantitative PCR.Results:1. Primary cardiac fibroblasts were high purified and were of good live rate.2. The inhibition effect of different concentration BNP on the cardiac fibroblasts proliferation is different. The OD value of BNP group were higher than the control group. Effect of BNP in 5×10-6mol/L,5×10-7mol/L were remarkably variated.3. The effect of different groups on the cGMP of cardiac fibroblasts is different. cGMP in BNP group and BNP+SB-431542 group were 6.16±1.30pmol/ml and 6.01±1.17pmol/ml respectively, both of which were remarkably higher than the control group (4.57±1.25pmol/ml, P<0.05). Compared to the control group, no significant difference were found in the other groupsP>0.05,n =6).4. The Concentration of TGF-β1 in BNP group, SB-431542 group and BNP+SB-431542 group were significanty inhibited than that of the normal control group(P<0.05,n =6), whereas the concentration of TGF-β1 was no significant difference among different dosing groups(P>0.05,n =6).The expression of Smad4 mRNA in BNP group, SB-431542 group and BNP+SB-431542 group were significanty inhibited(P<0.05,n =6), and the expression of Smad4 mRNA in BNP +SB-431542 group were significantly lower than that of BNP or SB-431542 respective(P<0.01,n =6). There is no significant difference between BNP group and SB-431542 group(P<0.05,n =6).Conclusion:1. There is a dose-dependent relationship in the effect of BNP inhibition in cardiac fibroblasts proliferation induced with CT-1 . However, there was not simple proportional relationship. NPRs may be involed with the possible signal transduction pathways .2. cGMP in BNP group was remarkably higher than the control group. This effect may be related to.NPR-A .3.BNP suppressd the expression of Smad4 mRNA. In addition to smad2/3 of TGF-βSignal transduction pathway, regulatory mechanism of B-type natriuretic peptide inhibition proliferation of CFs aslo depend on no-smad2/3 of TGF-β1 Signal transduction pathway.