The Effects And Mechanism Of Activin A On Oval Cell Proliferation

Part 1The Expression of ActivinA Signaling Pathway Components in 2-AAF/PH ModelObjective To detect the variation of activinA pathway in 2-AAF/PH model and the relationship between activinA pathway and oval cell proliferation.Methods Rat AAF/PH models were made and rats were sacrificed at indicated time points. The expression of activinA, activinA inhibitor follistatin, activin receptor type I and activinA receptor typeⅡmRNA in liver tissue were detected by real time PCR. Pan-CK positive oval cells and the expression of activin A and follistatin were detected by immunohistochemistry.Result In 2-AAF/PHx model, some high nuclei/cytoplasm ratio pan-CK positive cell (hepatic progenitor cells HPCs) firstly appeared close to pre-exist bile duct in or around portal tract, then became more numerous with time and reached its peak at the 9th day after PH. After 12th day, the tubular structures dramatically decreased, the pan-CK positive HPCs further decreased or disappeared from 18th day to 21st day. ActivinβA mRNA production decreased below the initial level from 6 hour to 9 day following PHx, increased from 12 to 15 day when the number of pan-CK positive HPCs began decreased(P＜0.01). After PHx, follistatin mRNA increased as soon as 6 hour (P＜0.05), and reached the peak at 24 hours after PHx (P＜0.05), declined below the initial level from 2day to 9day (P＜0.01), then restored to initiated level at 12 day. AVR1 mRNA increased at 9th day after Partial hepatectomy, declined to control level at 18th day after hepatectomy. AVR2 mRNA decreased immediately after hepatectomy. It gradually increased after 1st day and reached its peak at 15th day after hepatectomy.Conclusion In the early stage of oval cell proliferation after PH, the activity of activinA pathway was low because of high level of follistatin and low level of activinA and activin receptors. The activity of activinA pathway was enhanced in the terminal stage of oval cell proliferation. Part 2ActivinA Inhibits Hepatic Oval Cell Proliferationvia Smad Dependent PathwayObjectives To explore the effect and the mechamism of activin A on oval cell proliferation in vitro.Methods HPCs cell line LE6 cells were treated with indicated dose of activinA or follistatin, proliferation and apoptosis of LE6 cells were detected by CCK-8 assay, Brdu incorporation assay and Annexin V/PI assay; The activation of smads pathway, ERK, P38, JNK pathways and the change of phosphorylated Rb, cyclinDl, cyclinE, p21WAF1/Cip1and p15INK4B was detected by western blot. We monitored the effect of Activin A on smad4-knockdown LE6 cells.Results ActivinA inhibited growth of LE6 cells without impact their apoptosis. Phosphorylated Rb, cyclinD1, cyclinE was down-regulated by activinA, and p21WAF1/Cip1 and p15INK4B were up-regulated in LE6 cells treated with activin A. The antiproliferative effect of activin A was mediated by smads dependent pathways but not by MAPK pathways. Blocking smads dependent pathway by follistatin or knockdown smad4 in LE6 cells could interrupt antiproliferative effect of activinA.Conclusion ActivinA is a negative regulate cytokine of HPCs proliferation, and smads dependent pathway is mainly responsible for this anti-proliferation effect Part 3 Inportal Administration of Follistatin Promote Hepatic Oval Cells Mediated Liver RegenerationObjectives To study the effect of intro-portal administration of follistatin on hepatic oval cell mediated liver regeneration.Methods 2-acetylaminofluorene/partial hepatectomy(2-AAF/PH) oval cell mediated liver regeneration model was established.1μg follistatin (follistatin group) or normal saline (control NS group) was infused into portal vein immediately after 70% partial hepatectomy. Animals were sacrificed at indicated time points. Liver regeneration rate was measured by liver weight/body weight ratio. Changes of Pan-CK positive oval cell number, nuclear bromodeoxyuridine labeling were detected by immunohistochemistry.Results Compared to NS group More Pan-CK positive hepatic progenitor cells present in follistatin treated rats at 9,12,15 days after PH. But no significant different was detected in rats at 4days and 21 days after PH. Then we detected the proliferation cells in both group by Brdu label assay. More Brdu positive cells were detected in follistatin treated group than in NS group at 4,9,12 days after PH. And there was no significant different between those two groups at 15 and 21 days after PH.Conclusions Follistatin, an activinA antagonist, could enhanced and prolonged hepatic progenitor cell amplification in vivo. These result confirmed the anti-proliferation effect of activinA on hepatic progenitor cells in vivo.