OBJECTIVETo observe the effects of Wnt3a on proliferation and, activation of hepatic stellate cells (HSCs) and their the expression of the transforming growth factor beta (TGFb) and /Smad signaling factors of rat hepatic stellate cells line in vitro using a rat HSC line.

METHODSSynchronized HSC-T6 cells were stimulated with various concentrations of recombinant Wnt3a (50, 100, 200, 250 and 300 ng/mL). Unstimulated cells served as controls. Edu Effects on proliferation were determined by EdU (5-ethynyl-2'-deoxyuridine) incorporation assay and fluorescence microscopy.analysis was used to observe the proliferation of the hepatic stellate cells stimulated by different concentration of recombinant Wnt3a, and the Effects on the protein expression of TGFb/Smad signaling factors was assessed by western blot detection (gray-value analysis) of alpha-smooth muscle actin (a-SMA), a-SMA, TGFb1, Smad3, and and Smad7; glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was detected as the normalization control in the hepatic stellate cells was observed by Western blot analysis .The correlation was also observed. The significance of inter-group differences was assessed by one-way ANOVA, and correlations were determined using bivariate statistical modeling.

RESULTSIn general, HSC The proliferation of hepatic stellate cells increased after the addition of in response to Wnt3a stimulation for 24 h, reaching its peak at the maximum proliferation rate was observed with the 200 ng/mL Wnt3a concentration (63.00+/-2.30%), and it increased dramatically compared with those in which was significantly higher than the proliferation rates of the unstimulated control cells, and the cells stimulated with 50, 100 and 150 ng/mLl group (P less than 0.05), but the increase was not significantly different from that in the compared cells stimulated with 250 and 300 ng/mLl group,it had no obvious increase(P more than 0.05).; The Wnt3a stimulation also led to time-dependent increases in the protein expressions of a-SMA, TGFb1, and Smad3 increased with the addition of Wnt3a and the extension of time . For all three, The maximal amount of increased protein expression all reached to the was maximal produced by stimulation when hepatic stellate cells were treated by with 300 ng/mLl Wnt3a for 48 h hours,and the rations of(normalized gray- values:s of a-SMA, 1.0860+/-0.0101; TGFb1, 1.0346+/-0.0118; Smad3, to GAPDH were 1.0860+/-0.0101, 1.0346+/-0.0118, 1.0306+/-0.0122)respectively. However in contrast, the Wnt3a stimulation led to concentration- and time-dependent decreases in Smad7 expression varied inversely, with to them with the minimal ration of it to GAPDH the maximal decrease occurring with 300 ng/mL Wnt3a for 48 h (0.7736+/-0.0139) after being treated by 300 ng/ml Wnt3a for 48h. The comparison was remarkably discrepant, (P less than 0.05).There were positive correlations between a-SMA expression and was found to be positively correlated to TGFb1, Smad3 (r=0.968, P less than 0.05) and; Smad3 (r=0.997, P less than 0.01), but a-SMA and Smad7 had negatively correlated to Smad7 ion(r=0.960, P less than 0.05).

CONCLUSIONWnt3a can increase the stimulates proliferation as well as and activation of rat the hepatic stellate cells HSCs , and upregulate modifies the expression of TGFb/Smad signaling factors, of the hepatic stellate cells, and which may promote the hepatic fibrosis.