Bone marrow-derived mesenchymal stem cells protect against experimental liver fibrosis in rats

Abstract

Aim: Recent reports have shown the capacity of mesenchymal stem cells (MSCs) to differentiate into hepatocytes in vitro and in vivo. MSCs administration could repair injured liver, lung, or heart through reducing inflammation, collagen deposition, and remodeling. These results provide a clue to treatment of liver fibrosis. The aim of this study was to investigate the effect of infusion of bone marrow (BM)-derived MSCs on the experimental liver fibrosis in rats.

Methods: MSCs isolated from BM in male Fischer 344 rats were infused to female Wistar rats induced with carbon tetrachloride (CCl4) or dimethylnitrosamine (DMN). There were two random groups on the 42nd d of CCl4:CCl4/MSCs, to infuse a dose of MSCs alone; CCl4/saline, to infuse the same volume of saline as control. There were another three random groups after exposure to DMN: DMN10/MSCs, to infuse the same dose of MSCs on d 10; DMN10/saline, to infuse the same volume of saline on d 10; DMN20/MSCs, to infuse the same dose of MSCs on d 20. The morphological and behavioral changes of rats were monitored everyday. After 4-6 wk of MSCs administration, all rats were killed and fibrosis index were assessed by histopathology and radioimmunoassay. Smooth muscle alpha-actin (alpha-SMA) of liver were tested by immunohistochemistry and quantified by IBAS 2.5 software. Male rats sex determination region on the Y chromosome (sry) gene were explored by PCR.

Results: Compared to controls, infusion of MSCs reduced the mortality rates of incidence in CCl4-induced model (10% vs 20%) and in DMN-induced model (20-40% vs 90%). The amount of collagen deposition and alpha-SMA staining was about 40-50% lower in liver of rats with MSCs than that of rats without MSCs. The similar results were observed in fibrosis index. And the effect of the inhibition of fibrogenesis was greater in DMN10/MSCs than in DMN20/MSCs. The sry gene was positive in the liver of rats with MSCs treatment by PCR.

Conclusion: MSCs treatment can protect against experimental liver fibrosis in CCl4-induced or DMN-induced rats and the mechanisms of the anti-fibrosis by MSCs will be studied further.

Figure 1

MSCs are induced to differentiate into osteoblasts and stained red color by Chinalizarin (A). Under adipogenic conditions, MSCs accumulate lipid vacuoles, which are positively stained by Oil Red O assay (B). Under neurogenic conditions, MSCs differentiate into neuronal-like cells and stained positively for NSE by immunocytochemical analysis (C). (Original magnification, ×200.)

Figure 2

Average weekly weight of rats induced with CCl₄ (A) or DMN (B). A: Paraffin/saline, n = 10; CCl₄/saline, n = 10-8; CCl₄/MSCs, n = 10-9; B: saline, n = 10; DMN/saline, n = 40-4; DMN10/MSCs, n = 10-8; DMN20/MSCs, n = 10-6.

Figure 3

Analysis of liver histopathology and immunohistochemistry in rats induced with CCl₄. A: Paraffin/saline; B: CCl₄×6 wk; C: CCl₄/MSCs; D: CCl₄/saline; 1: HE staining, original magnification ×100; 2: MT staining, original magnification ×40; 3: immunohistochemistry for α-SMA, DAB staining, original magnification ×400; E: MT and α-SMA staining was quantified using IBAS 2.5 software. Data represent the fold-increase in positive staining vs paraffin/saline. Values are presented as mean±SD. ^bP<0.01 vs CCl₄/saline. Not significantly different between values of CCl₄/saline and CCl₄×6 wk.

Figure 4

Survival of rats in control group and DMN-induced group with or without MSCs administration. Analysis of survival was conducted by a log-rank test based on the Kaplan–Meier method.

Figure 5

Analysis of liver histopathology and immunohistochemistry in rats induced with DMN. A: DMN10; B: DMN20; C: DMN10/MSCs; D: DMN20/MSCs; E: DMN10/saline; 1: HE staining, original magnification ×100; 2: MT staining, original magnification ×100; 3: immunohistochemistry for α-SMA, DAB staining, original magnification ×400; F: MT and α-SMA staining was quantified using IBAS 2.5 software. Data represent the fold-increase in positive staining vs saline. Values are presented as mean±SD. ^aP<0.05 vs DMN20/MSCs, ^bP<0.01 vs DMN10/saline respectively.

Figure 6

PCR signals of sry gene and β-actin gene in liver of rats. sry, 104 bp; β-actin, 206 bp; M: 100-bp DNA maker; lane 1: CCl₄/MSCs; lane 2: CCl₄×6 wk; lane 3: DMN10/saline; lane 4: DMN10/MSCs; lane 5: DMN20/MSCs; lane 6: a male Fischer rat 344 as positive control; lane 7: a female Wistar rat as negative control.

Figure 7

Signals of SRY by FISH with Rat 12/Y whole chromosome probes. Red color represent chromosome Y, green color represent chromosome 12, blue color represent nuclear. Original magnification ×40.

Figure 8

Morphology of hepatic stellate cells co-culture with or without mesenchymal stem cells by transwell in vitro for 72 h. A: Hepatic stellate cells co-culture with mesenchymal stem cells; B: hepatic stellate cells co-culture with hepatoceyte cells line; C: hepatic stellate cells only culture. Original magnification ×10.

Figure 9

Analysis of hepatic stellate cells by FACS. A: Hepatic stellate cells co-culture with mesenchymal stem cells; B: hepatic stellate cells only culture.