Signaling Pathways And Effection Of Cardiotrophin-1 On The Proliferation Of Cardiac Fibroblasts Under High Hydrostatic Pressure

OBJECTIVE: We study the role of CT-1 in the proliferation of cardiac fibroblasts under high hydrostatic pressure , assess the effections of the three signaling pathways; singal transducer and activator of transcription3(JAK-STAT3) and effection of extracellular signal regulated kinases1/2(ERK1/2). Phosphatidylinositol 3-kinase(PI3-K) pathway in the cardiac fibroblasts proliferation induced by CT-1.METHODS: Cardiac fibroblasts were isolated from the ventricles of 1-3 days Sprague-Dawley neonatal rats and primarily cultured, then passaged 3-4 generations for experiment. Cells were divided into eight groups according to the different intervention factors:(1)group C : Control ;(2)group HHP : cultured under 160mmHg high hydrostatic pressure; (3)group SODN: interfered with 10μmol/L CT-1 sense oligodeoxynucleotides;(4)group ASODN: incubated with 10μmol/L CT-1 anti-sense oligodeoxynucleotides (;5)Group AG490: interfered with 25μmol/L AG490 ; (6)Group PD98059:interfered with 20μmol/L PD98059 ;(7) Group LY294002 : interfered with 10μmol/L LY 294002; (8)group DMSO:interfered with 100mg/ml×10-3 DMSO .Cell proliferation was quantified by MTT, Semi- quantitative RT-PCR and Western-blotting were employed to assess the expression of STAT3,ERK1/2 and PI3-Krespectively.RESULTS: (1)Compared with C, group HHP stimulated the proliferation of cardiac fibroblasts(0.153±0.011 vs 0.115±0.012,P<0.05), Compared with group HHP, group ASODN inhibited the proliferation of cardiac fibroblasts(0.132±0.013 vs0.153±0.011,P <0.05).(2) Compared with group HHP, Group ASOND could extensively inhibit the expressions of STAT3,ERK1/2 and PI3-K at mRNA level,(0.452±0.015 vs 0.855±0.012,P<0.05; 0.355±0.042vs0.553±0.061,P<0.05; 0.489±0.044 vs 0.647±0.051,P<0.05; 0.486±0.060 vs 0.635±0.062, P<0.05)respectively, and the expression of STAT3, ERK1 and PI3-K at protein level(2.09±0.25 vs 2.47±0.28, P<0.05; 1.13±0.19 vs1.61±0.22,P<0.05;1.25±0.23vs1.71±0.25, P<0.05) resp- ectively. (3)AG490, a JAK-STAT3 inhibitor , reversed the increase of the proliferat- ion of cardiac fibroblasts stimulated by high hydrostatic pressure(0.118±0.018 vs 0.155±0.010, P <0.05)and the expression of ERK1 protein(1.85±0.18vs 1.45±0.23,P <0.05). (4)PD98059,a MAPK-ERK1/2inhibitor , increased the proliferation of cardiac fibroblasts stimulated by high hydrostatic pressure ( 0.185±0.011 vs 0.155±0.010,P <0.05)and the expression of STAT3 protei(n1.83±0.23vs 1.58±0.22,P <0.05). (5)LY294002, an PI3-K inhibitor, had no effection on the proliferation of cardiac fibroblasts stimulated byhigh hydrostatic pressure(0.157±0.015 vs0.155±0.010,P >0.05). While there are no differences of the expression of the above factors group SOND(0.151±0.010 vs 0.154±0.011, P >0.05) and group DMSO (0.110±0.015 vs 0.113±0.012 ,0.141±0.017 vs 0.155±0.010,P>0.05) compared with the control group.CONCLUSION:(1)CT-1 can activate the three signaling pathways as STAT3,ERK1/2 and PI3-K in the proliferation of cardiac fibroblasts.(2)The proliferation of cardiac fibroblasts is essentially mediated by STAT3 ,and independent of PI3-K ,which is negatively regulated by ERK1/2 under high hydrostatic pressure.(3)The proliferation of cardiac fibroblasts is negatively regulated by ERK1/2 via inhibiting STAT3 that declare a crosstalk between STAT3 and ERK1/2 ,which may assist CT-1 to develop adequate cardiac fibroblasts proliferation.