The Use of Antifibrotic Recombinant nAG Protein in a Rat Liver Fibrosis Model.

OBJECTIVES: The "nAG" protein is the key protein mediating the regeneration of amputated limbs in salamanders. The senior author (MMA) developed the original hypothesis that since "nAG" is a "regenerative" protein, it must be also an "antifibrotic' protein. The antifibrotic properties were later confirmed in a rabbit skin hypertrophic scar model as well as in a rat spinal cord injury model. The aim of this study is to evaluate the potential therapeutic properties of the nAG protein in a rat liver fibrosis model. METHODOLOGY: Liver fibrosis was induced using intraperitoneal injections of carbon tetrachloride (CCL4). A total of 45 rats were divided equally into 3 groups: Group I (the control group) received normal saline injections for 8 weeks, Group II received CCL4 for 8 weeks, and Group III received CCL4 and nAG for 8 weeks. At the end of the experiment, the serum levels of 6 proteins (hyaluronic acid, PDGF-AB, TIMP-1, laminin, procollagen III N-terminal peptide, and collagen IV-alpha 1 chain) were measured. Liver biopsies were also taken and the stages of live fibrosis were assessed histologically. RESULTS: The CCL4 treatment resulted in a significant increase in the serum levels of all 6 measured proteins. The nAG treatment significantly reduced these high levels. The degree of liver fibrosis was also significantly reduced in the CCL4/nAG group compared to the CCL4 group. CONCLUSIONS: nAG treatment was able to significantly reduce the serum levels of several protein markers of liver fibrosis and also significantly reduced the histological degree of liver fibrosis.

Salamander-derived, human-optimized nAG protein suppresses collagen synthesis and increases collagen degradation in primary human fibroblasts.

Unlike humans, salamanders regrow their amputated limbs. Regeneration depends on the presence of regenerating axons which upregulate the expression of newt anterior gradient (nAG) protein. We had the hypothesis that nAG might have an inhibitory effect on collagen production since excessive collagen production results in scarring, which is a major enemy to regeneration. nAG gene was designed, synthesized, and cloned. The cloned vector was then transfected into primary human fibroblasts. The results showed that the expression of nAG protein in primary human fibroblast cells suppresses the expression of collagen I and III, with or without TGF- ß 1 stimulation. This suppression is due to a dual effect of nAG both by decreasing collagen synthesis and by increasing collagen degradation. Furthermore, nAG had an inhibitory effect on proliferation of transfected fibroblasts. It was concluded that nAG suppresses collagen through multiple effects.