Efficacy Comparison Study of Human Epidermal Growth Factor (EGF) between Heberprot-P® and Easyef® in Adult Zebrafish and Embryo under Presence or Absence Combination of Diabetic Condition and Hyperlipidemia to Mimic Elderly Patients.

Abstract

Recombinant human epidermal growth factor (EGF) has been used to treat adult diabetic foot ulcers and pediatric burns by facilitating wound healing and epithelization, especially for elderly patients. Several formulation types of EGF from different expression hosts are clinically available, such as intralesional injection and topical application. On the other hand, no study has compared the in vivo efficacy of EGF products directly in terms of tissue regeneration and wound healing activity. The present study compared two commercial products, Heberprot-P75® and Easyef®, in terms of their tissue regeneration activity in adult zebrafish and the developmental speed of zebrafish embryos. Fluorescence spectroscopy showed that the two EGF products had different Trp fluorescence emission spectra: Easyef® showed a wavelength of maximum fluorescence (WMF) of 337 nm with weak fluorescence intensity (FI), while Heberprot-P75® showed WMF of 349 nm with a 4.1 times stronger FI than that of Easyef®. The WMF of Heberprot-P75® was quenched by adding singlet oxygen in ozonated oil, while the WMF of Easyef® was not quenched. Treatment with Heberprot-P75® induced greater embryo development speed with a higher survival rate after exposure to EGF in water and microinjection into embryos. Under normal diet (ND) consumption, Heberprot-P75® showed a 1.4 times higher tail fin regeneration activity than Easyef® during seven days from the intraperitoneal injection (10 μL, 50 μg/mL) after amputating the tail fin. Under ND consumption and diabetic condition caused by streptozotocin (STZ) treatment, Heberprot-P75® showed 2.1 times higher tail fin regeneration activity than Easyef® from the same injection and amputation protocol. Under a high-cholesterol diet (HCD) alone, Heberprot-P75® showed 1.2 times higher tail fin regeneration activity than the Easyef® group and PBS group from the same injection and amputation. Under diabetic conditions (STZ-injected) and HCD consumption, the Heberprot-P75® group showed 1.7 and 1.5 times higher tail fin regeneration activity than the Easyef® group and PBS group, respectively, with a distinct and clean regeneration pattern. In contrast, the Easyef® group and PBS group showed ambiguous regeneration patterns with a severe fissure of the tail fin, which is a typical symptom of a diabetic foot. In conclusion, Heberprot-P75® and Easyef® have different Trp fluorescence properties in terms of the WMF and fluorescence quenching. Treatment of Heberprot-P75® induced a greater developmental speed of zebrafish embryos in both water exposure and microinjection. Heberprot-P75® induced significantly higher wound healing and tissue regeneration activity than Easyef® and PBS in the presence or absence of diabetic conditions and cholesterol supplementation.