The effect of modified turtle shell decoction on the expression of TGFβ1, Smad3 and Smad7 in rat hepatic stellate cell

BAI Ganping; ZHANG Ronghua; YAN Guohe; WANG Guojian; WAN Ping

doi:10.13431/j.cnki.immunol.j.20170137

Abstract：Hepatic fibrosis (HF) will develop into cirrhosis when it continues for long periods of time.Activated hepatic stellate cells (HSC) are involved in the formation of HF and structural remodeling of the liver through the proliferation and secretion of extracellular matrix.Modified turtle shell decoction (MTSD) has a certain inhibitory effect on liver fibrosis in rats,but its mechanism is not clear.Then we plan to investigate the effect of MTSD on the expression of TGFβ1,Smad3 and Smad7 in hepatic stellate cell line T6 (HSC-T6) of rat.In this study,HSC-T6 was treated with MTSD containing crude drug 0.71 g/ml (experimental group),treated with turtle shell pills (TSP) at the same dose (positive control),or cultured routinely (normal control).The morphological changes of HSC-T6 in different group were observed and compared under the inverted microscope;the growth of HSC-T6 cells in each group was tested by cell count kit-8 (CCK-8),and the growth curve was draw.Real time-PCR,immunofluorescence and Western blot were employed to detect the expression changes of TGFβ1,Smad3 and Smad7 genes and their coding proteins in each group of HSC-T6,respectively.Inverted microscope observation showed that HSC-T6 treated with MTSD for 48 h became small,and showed partial cell death and fragmentation,as compared with the control cells;CCK-8 assay identified that MTSD could inhibit HSC-T6 cells growth,and the growth of the HSC-T6 cell line treated with MTSD were lower than that of controls and the differences were statistically significant after the cells were treated for 48 h (P＜0.01).Real time-PCR,immunofluorescence and Western blotting revealed that MTSD could significantly down-regulate the expression of TGFβ1 and Smad3 in mRNA and protein levels,but up-regulated the expression of Smad7 in HSC-T6 (P＜0.01).In conclusion,MTSD can significantly inhibit the growth of HSC-T6,down-regulate the expression of TGF beta 1 and Smad3,and up-regulated the expression of Smad7 at the same time,and the inhibition effects of MTSD on the growth of HSC-T6 may be closely related to interference of the TGF beta 1/Smad3 signaling pathway.