<p>Activity and Effectiveness of Recombinant hEGF Excreted by <em>Escherichia coli</em> BL21 on Wound Healing in Induced Diabetic Mice</p>

Abstract

Context: Human epidermal growth factor (hEGF) has biological activities and can be used in medicines and cosmetics. A high level of effectiveness of hEGF can be obtained when three disulfide bonds fold perfectly. Extracellular secretion from E. coli BL21 using the PelB signal peptide is a new way to obtain hEGF with a structure that folds appropriately. Object: This study aimed to determine the activity and effectiveness of recombinant hEGF excreted by E. coli BL21 on wound healing in induced diabetic mice. Methods: Cell proliferation and migration tests were performed on NIH3T3 cells, followed by wound healing tests in induced diabetic mice, along with histological and endotoxin test at various hEGF concentrations (25, 50, and 75 µg/mL). Results: Based on the results, hEGF at a level of 50 μg/mL showed optimal proliferation and migration activities. Wound healing in induced diabetic mice showed faster-wound closure within 12 days at hEGF 50 and 75 µg/mL with a percentage wound closure of 95% and 98.5%, respectively, which was significant versus control. In the histology test, the number of fibroblasts showed an increase and was significant at hEGF 75 µg/mL compared to the control group. The single test vial (STV) showed that hEGF solution was free of endotoxin. Conclusion: Recombinant hEGF produced by extracellular secretion using E. coli BL21 has optimal diabetic wound healing activity through increased fibroblast proliferation.

Refolding of bioactive human epidermal growth factor from E. coli BL21(DE3) inclusion bodies & evaluations on its in vitro & in vivo bioactivity

“…On the 8 th day, all treatments provided a higher wound closure percentage than the control group while in 10 th day all treatment have complete wound closure and significantly different from the control group. Mallory-azan staining to distinguish extracellular tissue components ( Sriwidodo et al., 2020 ) revealed that the mice treated with the hEGF-liposome-chitosan patch had thick epithelial formation, equally fibroblasts, and better collagenisation ( Figure 8 ), indicating that the refolded EGF induced wound closure.

Figure 7 Refolded EGF activity in inducing wound closure.

…”

“…The wound was visualised using a “USB microscope” and the wound area was measured by ImageJ software on days 0, 4, 8, 10, and 12 after treatment. The percentage of wound closure was calculated using the following equation ( Sriwidodo et al., 2020 ): …”

“…Human epidermal growth factor (hEGF) is a 6.2 kDa protein consisting of 53 amino acid residues and three intramolecular disulphide bonds ( Tang et al., 2016 ; Zheng et al., 2016 ). It promotes cell proliferation, differentiation, and migration ( Carpenter and Cohen, 1990 ; Schultz et al., 1991 ), exhibits mitogenic properties in accelerating the wound healing process and is effective in preventing skin ageing ( Sriwidodo et al., 2020 ). It is also used as a therapeutic protein in the pharmaceutical and cosmetic industries ( Aldag et al., 2016 ; Sriwidodo et al., 2019 ).…”

Mechanism of the Anti‐Influenza Functions of Guizhi Granules Based on Network Pharmacology, Molecular Docking, and in Vitro Experiments

“…Suspended cells were mixed with PCM and centrifuged, and the pellet was dissolved in fresh PCM and seeded into fresh flasks. When the adhesive surface of the culture vessel is completely covered with cultured cells and there is no room for cells to grow as a monolayer, it is considered to be confluent [29] . At 80 % confluence, the cells were harvested in a centrifuge tube with trypsin/EGTA (0.25/0.2 %, 3 min, 37 °C) and centrifuged at 1200 r/min, 5 min.…”

“…When the adhesive surface of the culture vessel is completely covered with cultured cells and there is no room for cells to grow as a monolayer, it is considered to be confluent. [29] At 80 % confluence, the cells were harvested in a centrifuge tube with trypsin/EGTA (0.25/0.2 %, 3 min, 37 °C) and centrifuged at 1200 r/min, 5 min. Cell suspensions were prepared using low-FBS/PCM (containing 0.2 % FBS and the same remaining components as PCM) instead of PCM in 6-well plates at 4 × 10 4 cells/mL suspension.…”