Hydrodynamics-based transfection of rat interleukin-10 gene attenuates porcine serum-induced liver fibrosis in rats by inhibiting the activation of hepatic stellate cells

Abstract

Liver fibrosis is the common pathological outcome for the majority of chronic liver diseases. Interleukin-10 (IL-10) is a cytokine that downregulates proinflammatory responses and has a modulatory effect on liver fibrogenesis. However, little is known regarding the effect of rat interleukin‑10 (rIL‑10) gene by hydrodynamics-based transfection (HBT) on liver fibrosis in rats. The aim of this study was to investigate the effect of the rIL-10 gene by HBT on the progression of liver fibrosis induced by porcine serum (PS) in rats and explore its possible mechanism. Plasmid‑expressing rIL-10 was transferred into rats by HBT and immunohistochemistry and RT-PCR were used to detect the major organ expressing rIL-10. Liver fibrosis was induced in rats by intraperitoneal administration of PS for 8 weeks. Plasmid pcDNA3-rIL-10 solution was administered weekly by HBT starting at the 5th week. Liver function and hepatic histology were examined. The possible molecular mechanisms of rIL-10 gene therapy were assessed in liver tissue and hepatic stellate cells (HSCs) co-cultured with BRL cells (a hepatocyte line) in vitro. The results showed rIL-10 expression occurred mainly in the liver following rIL-10 gene transfer by HBT. Maintaining a stable expression of rIL-10 in serum was assessed by repeated administration. The rIL-10 gene treatment attenuated liver inflammation and fibrosis in PS-induced fibrotic rats, reduced the deposition of collagen and the expression of α-smooth muscle actin (α-SMA) in fibrotic rats. The in vitro experiment showed that the expression of a-SMA and procollagen type I in HSCs co-cultured with the BRL‑transfected rIL-10 gene were significantly decreased. These findings indicate that rIL-10 gene therapy by HBT attenuates PS-induced liver fibrosis in rats and that its mechanism is associated with rIL-10 inhibiting the activation of HSCs and promoting the degeneration of collagen.

Figure 1

Distribution of rat interleukin-10 (rIL-10) mRNA after rIL-10 gene by hydrodynamics-based transfection (HBT). Lanes 1) heart; 2) lung; 3) kidney; 4) spleen; 5) day 14 liver; 6) day 7 liver; 7) day 1 liver; M) marker.

Figure 2

Protein expression of rat interleukin-10 (rIL-10) in the liver after rIL-10 gene by hydrodynamics-based transfection (HBT). Expression on (A) day 1 and (B) day 7.

Figure 3

Levels of rat interleukin-10 (rIL-10) in the serum. (A) Levels of rIL-10 after rIL-10 gene repeating injection. (B) Levels of rIL-10 in fibrotic rats treated with rIL-10 gene.

Figure 4

The therapeutic effects of rat interleukin-10 (rIL-10) gene treatment on liver fibrosis in rats. (A–D) Photomicrographs of liver tissue by hematoxylin and eosin (H&E) (magnification, x200) to show hepatic fibrosis in (A) normal control rats (CTRL) or (B) porcine serum (PS)-induced fibrotic rats treated with saline or (C) with pcDNA3 or (D) with pcDNA3-rIL-10.

Figure 5

Hepatic function in different groups in fibrotic rats after rat interleukin-10 (rIL-10) gene treatment. Data shown are expressed as the means ± SD from 6–7 rats per group. ^**P<0.01.

Figure 6

Effects of rat interleukin-10 (rIL-10) gene treatment on the deposition of collagen in fibrotic rats. Photomicrographs of liver tissue by Sirius red staining (magnification, x200) to show collagen deposition in (A) normal control rats (CTRL) and (B) in porcine serum (PS)-induced fibrotic rats treated with saline or (C) with pcDNA3 or with (D) pcDNA3-rIL-10.

Figure 7

Relative expression of collagen types I and III in liver tissue. Data shown are expressed as the means ± SD from 6–7 rats per group. ^**P<0.01.

Figure 8

Effects of rat interleukin-10 (rIL-10) gene treatment on HSC activation. α-SMA-positive expression in liver by immunohistochemistry (magnification, x200) in (A) normal control rats (CTRL) and in (B) porcine serum (PS)-induced fibrotic rats treated with saline or (C) with pcDNA3 or (D) with pcDNA3-rIL-10.

Figure 9

Relative expression of α-SMA in liver tissue. Data shown are expressed as the means ± SD from 6–7 rats per group. ^**P<0.01.

Figure 10

Expression of procollagen type I and α-SMA in co-culture hepatic stellate cells (HSCs). (A) Expression of procollagen type I; (B) expression of α-SMA. HSCs/BRL: HSCs co-cultured with BRL-transfected saline; HSCs/BRL-rat interleukin-10⁻ (rIL-10⁻): HSCs co-cultured with BRL-transfected pcDNA3; HSCs/BRL-rIL-10⁺: HSCs co-cultured with BRL-transfected pcDNA3-rIL-10. Data are expressed as the means ± SD. ^**P<0.01.