Effects Of IL-10 On TGFβ₁ Stimulated Hepatic Stellate Cell Activation And Its Intracellular Signal Transduction Via ERK

AIM:To investigate HSC response on TGFβ1,and to elucidate the effects of IL-10 on TGFβ1 stimulated hepatic stellate cell activation and its intracellular signal transduction via ERK.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.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) 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. The cells in following study were divided into four groups:①control group(group C): The cultured media were changed by 2%(v/v)fetal calf serum (FCS)/DMEM;②IL-10 management group(group I):The cultured media were changed by 2%(v/v) FCS/DMEM including IL-10(20μg/L)③TGFβ1 management group(group T): The cultured media were changed by 2%(v/v) FCS/DMEM including TGFβ1(5μg/L).④Multiple management group(group M): The cultured media were changed by 2%(v/v) FCS/DMEM including TGFβ1(5μg/L) and IL-10(20μg/L).The managements of all groups were lasting for 48 hours.Then the expressions ofα-SMA,TβR-Ⅱ,TβR-Ⅰ,ERK1/2 and phosphorylated ERK1/2 were assayed 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%. 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. The expressions ofα-SMA and phosphorylated ERK increased in group T compared to group C (p<0.05). The level ofα-SMA in group M was lower than group T (p<0.05). The levels of TβR-Ⅱ,TβR-Ⅰ,ERK1/2 among the four groups had no significant difference, so did the expression of phosphorylated ERK1/2 between group T and group M.Conclusions: The reliable and available method for isolation of HSC was successfully established for further research. The activation and the expression of phosphorylated ERK of partially-activated HSC could be promoted by TGFβ1, while fully-activated HSCs lost this response. IL-10 inhibited TGFβ1 stimulated HSC activation not through influencing the expressions of TβR-Ⅱ,TβR-Ⅰ,ERK and phosphorylated ERK.