Mesenchymal stem cell-based Smad7 gene therapy for experimental liver cirrhosis

Abstract

Background: Bone mesenchymal stem cells (MSCs) can promote liver regeneration and inhibit inflammation and hepatic fibrosis. MSCs also can serve as a vehicle for gene therapy. Smad7 is an essential negative regulatory gene in the TGF-β1/Smad signalling pathway. Activation of TGF-β1/Smad signalling accelerates liver inflammation and fibrosis; we therefore hypothesized that MSCs overexpressing the Smad7 gene might be a new cell therapy approach for treating liver fibrosis via the inhibition of TGF-β1/Smad signalling.

Methods: MSCs were isolated from 6-week-old Wistar rats and transduced with the Smad7 gene using a lentivirus vector. Liver cirrhosis was induced by subcutaneous injection of carbon tetrachloride (CCl₄) for 8 weeks. The rats with established liver cirrhosis were treated with Smad7-MSCs by direct injection of cells into the main lobes of the liver. The expression of Smad7, Smad2/3 and fibrosis biomarkers or extracellular matrix proteins and histopathological change were assessed by quantitative PCR, ELISA and Western blotting and staining.

Results: The mRNA and protein level of Smad7 in the recipient liver and serum were increased after treating with Smad-MSCs for 7 and 21 days (P < 0.001). The serum levels of collagen I and III and collagenase I and III were significantly (P < 0.001) reduced after the treatment with Smad7-MSCs. The mRNA levels of TGF-β1, TGFBR1, α-SMA, TIMP-1, laminin and hyaluronic acid were decreased (P < 0.001), while MMP-1 increased (P < 0.001). The liver fibrosis score and liver function were significantly alleviated after the cell therapy.

Conclusions: The findings suggest that the MSC therapy with Smad7-MSCs is effective in the treatment of liver fibrosis in the CCl₄-induced liver cirrhosis model. Inhibition of TGF-β1 signalling pathway by enhancement of Smad-7 expression could be a feasible cell therapy approach to mitigate liver cirrhosis.

Keywords: Fibrosis; Gene therapy; Liver cirrhosis; Mesenchymal stem cells; Smad7; TGF-β.

Fig. 1

CCl₄-induced liver fibrosis assessed by Masson’s trichrome staining (× 400). a Normal rat liver tissue as control. b Rat liver tissue obtained at 4 weeks after injection of CCl₄ (prior to cirrhosis). c Typical liver cirrhosis tissue section after injection of CCl₄ for 8 weeks. d Histological scores for liver fibrosis (n = 5 for each group). ***P < 0.001 comparing with the control group

Fig. 2

Distribution of Smad7-EGFP-MSCs in the liver and the expression of Smads. a Example of direct injection of Smad7-EGFP-MSCs into the liver lobes. The distribution of Smad7-EGFP-MSCs (green fluorescence) in the liver tissue was detected on the frozen sections using a laser confocal microscope at the time points of 24 h, 7 days and 21 days after cell injection. b Smad7 mRNA quantification in the groups of control (vehicle control without CCl₄), cirrhosis group (with CCl₄ injection), cirrhosis group treated with MSCs and cirrhosis group treated with Smad7-MSCs. c Serum protein level of Smad7 detected using ELISA (n = 4). d Smad2 mRNA quantification using real-time PCR. GAPDH was set as housekeeping gene control for relative quantification (n = 6). e Smad3 mRNA quantification (n = 6). **P < 0.01 or ***P < 0.001 comparing with the control group; ^###P < 0.001 comparing with the cirrhosis group

Fig. 3

Detection of fibrosis biomarkers collagen and collagenase. a Real-time PCR detection of collagen I and collagen III expression in the liver tissues treated with Smad7-MSCs, comparing with the control (vehicle), cirrhosis group (with CCl₄ injection) and the cirrhosis group treated with MSCs (n = 6 for each group). b Quantification of collagen III mRNA (n = 6 for each group). c ELISA detection of collagenase 1 and collagenase III (d) in rat serum (n = 4 for each group). *P < 0.05 or ***P < 0.001 comparing with the control group; ^##P < 0.01 or ^###P < 0.001 comparing with the cirrhosis group

Fig. 4

Downregulation of TGF-β1, TGFBR1 and α-SMA after Smad7-MSC treatment. Real-time PCR detection of TGF-β1 (a), TGFBR1 (b) and α-SMA (c) expression in the rat liver tissues treated with Smad7-MSCs, comparing with the control (vehicle), cirrhosis group (with CCl₄ injection) and the cirrhosis group treated with MSCs (n = 6 for each group). The GAPDH was set as the housekeeping gene for relative quantification (n = 6 for each group). **P < 0.01 or ***P < 0.001 comparing with the control group; ^###P < 0.001 comparing with the cirrhosis group

Fig. 5

Changes in extracellular matrix protein laminin, hyaluronic acid, TIMP-1 and MMP-1. ELISA detection of laminin (a) and hyaluronic acid (b) in the sera from the rats treated with Smad7-MSCs for 7 and 21 days in comparison with the control group (vehicle), cirrhosis group (with CCl₄ injection) and the cirrhosis group treated with MSCs (n = 4 for each group). c, d Quantitative PCR for TIMP1 and MMP-1. GAPDH was used as the housekeeping gene for relative quantification (n = 6 for each group). e TIMP-1 and MMP-1 detected by Western blotting in the liver lysate (n = 5 for each group). *P < 0.05, **P < 0.01 or ***P < 0.001 comparing with the control group; ^###P < 0.001 comparing with the cirrhosis group

Fig. 6

Histopathological evaluation of liver fibrosis after Smad7-MSC therapy. a The rat liver tissue sections were stained using Masson’s trichrome stain method and assessed by fibrosis semi-quantitative scoring method (grades 0–5). The isolated collagen fibres (indicated by arrow) and collagen around the wall of central vein (V) were stained blue. Examples for the control group (vehicle), cirrhosis group (with CCl₄ injection) and the cirrhosis groups treated with MSCs or Smad7-MSCs for 21 days. b Mean ± SD for the scoring on severity of fibrosis (n = 5 for each group)