Cav-1——AKF-PD Anti-pulmonary Fibrosis Treatment New Target

Objective:To investigate the inhibiting effect of AKF-PD on normal human lung fibroblasts activation and ECM synthesis induced by TGF-p land the role of caveolin-1(cav-1) in its anti-fibrosis mechanism.Methods:1. Human Tenon’s capsule fibroblasts culture:Taken normal human lung tissues from lobectomy of lung and cultured fibroblasts with tissue explants technique in vitro.Fibroblasts were subclutured in5%CO2,37℃condition with DMEM contained10%fetal bovine serum, then purification at the third passage cells was cryopreserved standby.2. The cells were cultured to3-7generation using. Firstly, the cultured cells were divided into5groupsrcontrol group,TGF-(31(10ng/ml) group、TGF-β1(10ng/ml)+AKF-PD(200μg/ml、400μg/ml、800μg/ml) groups. After12、24、36、48、64、72hours in culture, the protein level of α-SMA、FN and cav-1were measured by Western blot.3. Secondly, the cultured cells were divided into7groups:control group,TGF-β1(10ng/ml) group、Co-siRNA transfection group、 Co-siRNA transfection+TGF-β1(10ng/ml) group、cav1-siRNA transfection group、cavl-siRNA transfection+TGF-β1(10ng/ml) group、cavl-siRNA transfection+TGF-β1(10ng/ml)+AKF-PD (400μg/ml) group. After48hours in culture, the protein level of α-SMA、FN and cav-1were measured by Western blot. The normal cav1-SIRNA sequences were designed and prepared by Shanghai gemma pharmaceutical technology co., LTD.Results:1.The cells cultured in vitro proliferated actively.2. TGF-β1markedly decreased the expression of cav-1(p<0.05), and increased the expression of α-SMA、FN (p<0.05). It showed a time dependent way, and the best acting time is the point of48hours.3. AKF-PD effectively inhibited the effects of TGF-β1, which increased the expression of cav-1(p<0.05), and decreased the expression of α-SMA、FN (p<0.05). It showed a concentration dependent way, and the best concentration of AKF-PD is400μg/ml.4. Transfection of Co-siRNA:Compared with control group, it was no significant difference in expression of Cav-1, α-SMA and FN (P>0.05).5. Transfection of cavl-siRNA:Compared with control group, the expression of cav-1was almost disappeared (P<0.05), and it was no significant difference in expression of α-SMA and FN (P>0.05).6. Co-siRNA Transfection+TGF-β1(10ng/ml) group:Compared with Co-siRNA transfection group, the expression of cav-1was decreased obviously (P<0.05), and the expression of α-SMA, FN were increased significantly (P<0.05). Compared with TGF-β1(10ng/ml) group, it was no significant difference in expression of cav-1, α-SMA and FN (P>0.05).7. cavl-siRNA Transfection+TGF-β1(10ng/ml) group:Compared with cav1-siRNA transfection group, the expression of cav-1was decreased slightly (P>0.05), and the expression of α-SMA, FN was increased significantly (P<0.05). Compared with TGF-β1(10ng/ml) group, the expression of cav-1、α-SMA and FN were decreased obviously (P<0.05).8. cavl-siRNA transfection+TGF-β1(10ng/ml)+AKF-PD (400μg/ml) group:Compared with cavl-siRNA Transfection+TGF-β1(10ng/ml) group, it was no significant difference in expression of cav-1, α-SMA and FN (P>0.05).Conclusions:AKF-PD can inhibit cell activation and extracellular matrix synthesis in NHLFSs stimulated by TGF-β1,and its potential mechanisms are that partly restore the expression of cavl in NHLFSs, down-regulate the TGF-β1induced a-SMA and FN overexpression.