Protection of the liver against CCl4-induced injury by intramuscular electrotransfer of a kallistatin-encoding plasmid

Abstract

Aim: To investigate the effect of transgenic expression of kallistatin (Kal) on carbon tetrachloride (CCl(4))-induced liver injury by intramuscular (im) electrotransfer of a Kal-encoding plasmid formulated with poly-L-glutamate (PLG).

Methods: The pKal plasmid encoding Kal gene was formulated with PLG and electrotransferred into mice skeletal muscle before the administration of CCl4. The expression level of Kal was measured. The serum biomarker levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), malonyldialdehyde (MDA), and tumor necrosis factor (TNF)-α were monitored. The extent of CCl4-induced liver injury was analyzed histopathologically.

Results: The transgene of Kal was sufficiently expressed after an im injection of plasmid formulated with PLG followed by electroporation. In the Kal gene-transferred mice, protection against CCl4-induced liver injury was reflected by significantly decreased serum ALT, AST, MDA and TNF-α levels compared to those in control mice (P<0.01 to 0.05 in a dose-dependent manner). Histological observations also revealed that hepatocyte necrosis, hemorrhage, vacuolar change and hydropic degeneration were apparent in mice after CCl4 administration. In contrast, the damage was markedly attenuated in the Kal gene-transferred mice. The expression of hepatic fibrogenesis marker transforming growth factor-β1 was also reduced in the pKal transferred mice.

Conclusion: Intramuscular electrotransfer of plasmid pKal which was formulated with PLG significantly alleviated the CCl4-induced oxidative stress and inflammatory response, and reduced the liver damage in a mouse model.

Keywords: Drug formulation; Electroporation; Gene delivery systems; Kallistatin; Liver injury.

Figure 1

Time-course study of the effect of the poly-L-glutamate formulation on transgene expression of pLac in the tibialis anterior muscle after electroporation transfer. The plasmids were transferred with (square) and without (triangle) poly-L-glutamate (PLG) formulation. The values shown are mean ± SD, n = 8.

Figure 2

Dose-dependent kallistatin concentrations in the serum responded to intramuscular electrotransfer of a plasmid pKal in poly-L-glutamate formulation. The values shown are mean ± SD, n = 8. Kal: Kallistatin.

Figure 3

Protective effects of kallistatin expression against carbon tetrachloride-induced liver injury in mice. HE staining was performed on paraffin embedded sections of the liver tissues. Representative sections are shown for each group. A: Control; B: Carbon tetrachloride (CCl₄); C: pLac + CCl₄; D: pKal (100 μg) + CCl₄; E: pKal (50 μg) + CCl₄; F: pKal (25 μg) + CCl₄; G: Necroinflammatory scores. ^bP < 0.01 vs control group; ^cP < 0.05, ^dP < 0.01 vs CCl₄ group.

Figure 4

Immunohistological analysis of transforming growth factor-β1 (brown) in the mice livers treated with carbon tetrachloride. A: Control; B: Carbon tetrachloride (CCl₄); C: pLac + CCl₄; D: pKal (100 μg) + CCl₄; E: pKal (50 μg) + CCl₄; F: pKal (25 μg) + CCl₄.

Figure 5

Inflammatory response and muscle damage arising from plasmid injection and electroporation. A: Saline; B: pLac.