The effect and possible mechanism of the Rho/Rock signaling pathway during TGF-β1 -induced ISMCs pheno-typic modulation in rats

CAO Fei-peng; HUANG Shu-wen; MAI Shu-rong

Abstract：Objective To investigate the effects and possible mechanism of the Rho/Rock signaling pathway on TGF-β1 -induced of rat ISMCs phenotypic modulation.Methods The rat ISMCs were randomly divided into two groups:blank control group and TGF-β1 stimulation group, in which TGF-β1 of different concentrations (1, 5, 10, 20 and 50 ng/mL) were given for 24 h.Transgelin ( SM22α) and osteopontin ( OPN) mRNA expression was assessed by flu-orescence quantitative real-time polymerase chain reaction ( FQ-RT-PCR) .The derived optimal concentration was ap-plied to stimulate cells, which were collected at different times (6, 12, 24 and 48 h).After 24 h of serum-free DMEM high glucose medium culture, ISMCs were randomly divided into 3 groups: blank control group, TGF-β1 (10ng/mL) stimulation group, TGF-β1 (10 ng/mL)+Fasudil (10, 30 and 50 μmol/L) groups.The expression levels of SM22α, OPN, RhoA, Rock-1 mRNA were assessed with FQ-RT-PCR;and SM22αand OPN expression levels were assessed using Western Blot.The ultrastructure of the cells was observed by transmission electron microscopy.Results TGF-β1 in 10 ng/mL stimulation of ISMCs reached the summit at 24 h.SM22 mRNA and protein expression levels were signifi-cantly increased in TGF-β1 +Fasudil group (P<0.01).Significant reduction in OPN mRNA was observed in TGF-β1+Fasudil group in dose-dependent manners, and reached lowest effects with Fasudil of 30μmol/L (P<0.05).So was the protein expression of OPN according to Western blot.According to FQ-RT-PCR, the mRNA expression of RhoA and Rock-1 and Rock-1 was significantly increased in TGF-β1 group ( P<0.01 ) , and it was significantly lower in TGF-β1 +Fasudil (30 μmol/L) group than TGF-β1 group (P<0.05).Under electron microscope, normal Golgi ISMC hypertrophy, mitochondria and rough endoplasmic reticulum were observed in control group ( ×20 000) , while in-creased intracellular mitochondria was observed in TGF-β1 group, which was reversed by Fasudil.Conclusion TGF-β1 of 10ng/mL stimulation in ISMCs for 24 h can be successfully induced phenotype modulation.Rho/Rock signaling pathway inhibitors Fasudil increases of TGF-β1 -induced SM22αexpression, loweres OPN expression, thus inhibits ISMCs phenotypic modulation.