Effects of lentiviral vector­mediated shRNA silencing of TGFß1 on the expression of Col1a1 in rat hepatic stellate cells.

The present study aimed to construct a lentiviral RNA interference (RNAi) vector targeting the transforming growth factor ß1 (TGFß1) gene of rats, in order to examine its effect on silencing of the TGFß1 gene and on the expression of collagen type 1 α1 (Col1a1) in HSC­T6 rat hepatic stellate cells. Three RNAi sites of the TGFß1 gene were selected according to its CDs sequence. Three pairs of small interfering RNA (siRNA) of these RNAi sites were synthesized and then transfected into HSC­T6 cells, respectively, to confirm the optimal siRNA sequence via reverse transcription­polymerase chain reaction analysis. Subsequently, shRNA targeting the sequence of the optimal siRNA was designed, synthesized and annealed to form a double­stranded structure. The annealed oligonucleotide fragment was cloned into pGreenPuro plasmids to establish the pGreenPuro/TGFß1 shRNA lentiviral vector, which was then transfected into 293T cells, following identification by restriction enzyme digestion and sequencing for the production of lentiviral particles exhibiting high reactivity. These particles were used to infect HSC­T6 cells, following which the expression of GFP in the transfected cells was observed under an inverted microscope. The effects on TGFß1 gene silencing and the expression levels of Colla1 were detected at the mRNA and protein levels. The results provided confirmation of the optimal siRNA sequence. Enzyme digestion and sequencing verified successful construction of the pGreenPuro/TGFß1 shRNA lentiviral vector. This lentiviral vector effectively silenced the TGFß1 gene in the HSC­T6 cells, and inhibited the expression of Col1a1 at the mRNA and protein levels. Taken together, the lentiviral RNAi vector targeting the TGFß1 gene of rats was successfully constructed, which effectively silenced the TGFß1 gene of the HSC­T6 cells and inhibited the expression of Col1a1.

Anti-hepatic fibrosis effects of a novel turtle shell decoction by inhibiting hepatic stellate cell proliferation and blocking TGF-ß1/Smad signaling pathway in rats.

Hepatic fibrosis (HF), a wound-healing response to a variety of chronic stimuli, is characterized by the excessive synthesis of extracellular matrix (ECM) proteins by hepatic stellate cells (HSC) and eventually the development of hepatic cirrhosis. Turtle shell pill (TSP) is a common traditional Chinese medicine used for preventing and treating HF and early hepatic cirrhosis, but its side-effects and the shortage of ingredients limit its clinical application. In addition, its mechanism of action is not clear. In the present study, we first improved the original formula of TSP to produce a novel turtle shell decoction (NTSD) with less toxicity and easier accessible materials. In a carbon tetrachloride (CCl4)-induced HF rat model, we observed that NTSD and TSP had similar effects on the improvement of liver functions in rats, including a decrease in serum alanine amino transferase (ALT) and aspartate amino transferase (AST) serum concentrations and increased albumin content in addition to a marked attenuation of CCl4-induced liver damage and fibrosis. NTSD containing rat serum inhibited rat liver stellate cell line HSC-T6 cell proliferation and induced cell apoptosis in vitro. Moreover, the NTSD treatment significantly decreased the transforming growth factor beta 1 (TGF-ß1) and Smad3 gene expression and increased inhibitory Smad7 gene expression in liver tissues of HF rats, suggesting that NTSD inhibited the ECM expression of HSC by downregulating the TGF-ß1/Smad signaling pathway. The results of our rat model study revealed that NTSD showed good in vitro and in vivo anti-HF effects via proliferation inhibition and the induction of apoptosis of HSCs and blocked the TGF-ß1/Smad signaling pathway.