Down-regulation Of Smad3 Gene By SiRNA And Its Effect On HSC And Hepatic-fibrosis Model Induced By Carbon Tetrachloride In Rats

Objective(1)Activation of hepatic stellate cells is a key step for the process of liver fibrosis, and TGF-β/Smads signaling is most important to the activation of hepatic stellate. Among the Smads family, Smad3 is the most important proteins in the TGF-β/Smads signaling, and it performs a significant role during the whole process of TGF-βsignal transfer into intra-cellular and activiating hepatic stellate cells. In this study, interference vectors of rat Smad3 gene that could silence Smad3 effectively were constructed first.(2)Effective Smad3 siRNA Expression vectors with lipofectamine 2000 encapsulated plasmids were transfected into rat HSC-T6 cells. In order to observe the effect of activaction of HSC in vitro, and judge the effect on anti-fibrosis after knocking down the expression of Smad3 gene and blocking TGF-β/Smad3 signaling.( 3 ) Effective Smad3 siRNA Expression vectors with lipofectamine 2000-encapsulated plasmids were transfected into carbon tetrachloride hepatic fibrosis model. The influence on these indices of hepatic fibrosis was studied when the Smad3 gene was knocked down in rat model of hepatic fibrosis induced by carbon tetrachloride. Thereby, a new therapeutic approach to hepatic fibrosis was explored.(4)In order to probe the effect on TGFβ1-Smad3 signaling by ppGalNAc-T2 gene in hepatic setellate cells (HSC), we observed the influence on the expression of genes related to TGFβ1-Smad3 activation pathway after ppGalNAc-T2 gene was down-regulated by the ppGalNAc-T2 siRNA expression vector in rat HSC.Methods(1)Smad3 gene sequence was accessed by http://www.ncbi.nlm.nih.gov/genebank, Three possible small interfering RNAs of Smad3 gene were designed with ambion corporation’s siRNA design tools, then recombined with the vector pRNAT-U6.1/Neo. Three constructed vectors were confirmed by PCR and sequencing. Using RT-PCR and Western-Blot methods to screen a optimal Smad3 RNAi-vector in HSC after transfected with the three different Smad3 RNAi-vector(pRNAT-siSmad3-1、pRNAT-siSmad3-2、pRNAT-siSmad3-3)labled GFP respectively.(2)Effective Smad3 siRNA Expression vector(pRNAT-siSmad3-2)with lipofectamine 2000-encapsulated plasmids were transfected into Rat HSC-T6 cells(1.0μg plasmid/106 cells) in the logarithmic growth phase, At 48 h post-transfection, total RNA was extracted from the HSCs with trizol according to manufacturer instructions. The effects of siRNAs on the mRNA levels of Smad2, Smad3, Smad4 and collagens I-α1, III-α1 and IV-α1 (Colα1, Col3α1, Col4α1, respectively) were tested by RT-PCR.(3)Eighty adult male Sprague-Dawley rats were randomly divided into three groups. Twice a week for 8 weeks, the untreated hepatic fibrosis model (N=30) and treatment the treated group (N=20) were injected subcutaneously with 40% (v/v) carbon tetrachloride (CCl4)-olive oil (3 ml/kg), and the normal control group (N=30) was injected with olive oil (3 ml/kg). In the 4th week, the treated rats were injected subcutaneously with liposome-encapsulated plasmids (150μg/kg) into the right liver lobe under general anesthesia once every 2 weeks, and the model group untreated rats were injected with the same volume of buffer. At the end of the 6th and 8th weeks, liver tissue and sera were collected. The Pathological changes were assessed via by a semi-quantitative scoring system (SSS), and a radioimmunoassay was used to establish a serum liver fibrosis index (type III procollagen, type IV collagen, laminin and hyaluronic acid). The mRNA expression levels of the above cited genes were reduced in the HSCs transfected with the siRNA expression plasmids.(4)After the transfection of plasmid of ppGalNAc-T2 siRNA in HSC, the mRNA expression of genes related to TGFβ1-Smad3 activation pathway was detected by RT-PCR and the Smad3 protein was detected by Western blot and Immuno-fluorescence.Results(1)Three different siRNA expression vectors (pRNAT-SiSmad3-1, pRNAT -SiSmad3-2, pRNAT-SiSmad3-3) were constructed successfully. After being screened, the vector of pRNAT-SiSmad3-2 was the optimal Smad3 RNAi-vector. (2)The mRNA levels of Smad2, Smad3, Smad4 and Colα1, Col3α1, Col4α1, were reduced in the HSCs transfected with the siRNA expression plasmids.(3)Pathological changes were assessed by a semi-quantitative scoring system (SSS) was significantly reduced (P < 0.05), and a radioimmunoassay was used to establish a serum liver fibrosis index (type III procollagen, type IV collagen, laminin and hyaluronic acid) were greatly improved (P < 0.01).(4)The knock-down of ppGalNAc-T2 had no significant effect on the mRNA expression of Smad 2, Smad 3, Smad 4, Col3α1, Tbrg4, TGFβ, TGFβ2 and TGFβr3 and the protein expression of Smad3 (P﹥0.05). However, the mRNA expression of Tgfβr1 and TGFβr2 was significantly down-regulated(P<0.05).Conclusions(1)Smad3 siRNA expression plasmids, which knocked down the expression of Smad3 gene and blocked TGF-β/Smad3 signaling, have an anti-fibrotic effect when transfected into rat HSCs and rat Hepatic fibrosis model. It supported the feasibility of the application of siRNA in hepatic fibrosis.(2) In HSC,some genes expressions related to TGFβ1-Smad3 activation pathway in HSC are not varied after the ppGalNAc-T2 is down-regulated. The function of ppGalNAc-T2 in HSC should be studied further.