Effects Of IL-10 On TGF-β1 Stimulated Hepatic Stellate Cell Activation And Its Intracellular Signal Transduction Via Smads

Aim: To investigate hepatic stellate cells (HSC) responses at different differentiation stages on transforming growth factor-β1 , and to elucidate the mechanisms of interleukin-10(IL-10) against hepatic fibrosis , relating to interference with TGF-β1 stimulated HSC activation and intracellular signal transduction via Smads.Methods: The liver of adult male Sprague-Dawley rat was perfused through portal vein with pronase E and type IV collagenase. Cells suspension was purified by 11% Nycodenz density gradient centrifugation to isolate HSCs. The viability of HSCs was determined by trypan blue exclusion staining. The purity of HSCs was identified by the typical appearance and the expression of desmin using immunocytochemistry. HSCs were primarily cultured in uncoated plastics for 2,3,4,5,6,7days respectively. Then cells were incubated with 5μg/L exotic transforming growth factorβ1 for 24 hours. Morphological features of cells were observed under inverted microscope.α-smooth muscle actin(α-SMA) expression was assayed by western blot. Then cells at one definite differentiation stage that responded most sensitively to transforming growth factorβ1 were selected as the cell model for the following study. 0,10,20,40μg/L exotic IL-10 was incubated with 5μg/L TGF-β1 stimulated HSCs for 48 hours respectively,α-SMA expression were checked by western blot. Then one definite concentration of IL-10 to inhibited TGFβ1 stimulated HSC activation was selected for the following study. The cells in following study were divided into four groups:①control group(group C)②IL-10 management group(group I)③TGFβ1 management group(group T)④Multiple management group(group M).The managements of all groups are lasting for 48 hours. Then the expressions ofα-SMA, Procollagen Type I, TGFβR-Ⅰ,TGFβR-Ⅱ, Smad3, Smad4 and Smad7 were measured by western blot.Results: The reliable and available method for isolation of HSC was successfully established for further research. The viability of HSC was over 90% with purity above 90%. As culture duration prolonged, HSC phenotypes underwent activation gradually, accompanied by the increase ofα-SMA expression. While TGF-β1 stimulated HSCs expressingα-SMA when cells had been cultured for 2,3,4,5days,but had no the same influence on HSCs cultured for 6,7days.The cells cultured for 3 days were the most sensitive with the level ofα-SMA increasing by 78.05% of the control, and HSCs cultured for 3 days were used as cell model for the following study. 10μg/L IL-10 had no influence onα-SMA expression of TGF-β1 stimulated HSC by the control., while 20,40μg/L IL-10 remarkably inhibited the-SMA expression by the control. 20μg/L IL-10 was used for the following study. The expressions ofα-SMA, Procollagen Type I and Smad3 increased in group T compared to group C (p<0.05). The level ofα-SMA, Procollagen Type I and Smad3 in group M was lower than group T (p<0.05). The levels of TGFβR-Ⅰ,TGFβR-Ⅱ, Smad4 and Smad7 among the four groups had no significant difference(P>0.05).Conclusions: The reliable and available method for isolation of HSC was successfully established for further research. The activation of partially-activated HSC could be promoted by TGFβ1, while fully-activated HSCs lost this response.IL-10 obviously inhibited TGF-β1 stimulated HSC activation and matrix protein expression ,and decreases stimulated HSC Smad 3 protein expression , but not influenced the expressions of TGFβR-Ⅰ,TGFβR-Ⅱ,Smad4 and Smad7.The inhibition of TGF-β1 signaling in HSC and its biological responses is the important mechanism of IL-10 against hepatic fibrosis.