The role of b1 methylation, physiologic replication independent endogenous dna double strand breaks (phy-rind-edsb) and laminin 511 e8 proteins in rat second degree burn wound healing

Abstract

Background: DNA methylation via short interspersed nuclear element (SINE) small interfering (si)RNA prevents DNA damage and promotes cell proliferation. Furthermore, laminin α5 β1 γ1(LM511) is an extracellular structural protein that can support epithelial cell adhesion and migration. Box A of high-mobility group box 1 protein (Box A of HMGB1) is a common nuclear protein in eukaryotic cells that can reduce DNA damage response toward burn injury.

Objective: To investigate whether treatment of burn wounds using B1 siRNA, Box A of HMGB1 and LM511-E8 fragment improved wound closure in a rat second-degree burn wound model.

Methods: I performed a cross-sectional analytical study using tissue and blood samples from post-burn and healthy patients (n = 23 each) to measure Alu methylation levels and patterns. In in vivo experiments, second-degree burn wounds were introduced on the backs of rats. The rats were then divided into control and experiment groups: a B1 siRNA-treated, Box A of HMGB1 protein and LM511-E8, saline-treated control, and calcium phosphate-nanoparticle-treated control group (n = 15–20/group). The wounds were imaged on days 0, 7, 14, 21, and 28 post-injuries. The tissue sections were processed for methylation and histological and immunohistochemical examination and scored based on the overall expression of histone H2AX phosphorylated on serine 139 (γH2AX), 8-hydroxy-2′-deoxyguanosine (8-OHdG), and presence of cytokeratin 10 and 14.

Results: Alu methylation levels were lower in hypertrophic scar tissues than in normal skin (29.4 ± 2.5% vs. 35.6± 3.2%, P = 0.0002). Burn wound closure improved in the B1 siRNA-treated group compared to that in the control group, especially from days 14 to 28 post-injury (P < 0.001). The overall pathological score and degree of B1 methylation in the B1 siRNA-treated group improved at days 14–28 days post-injury. The Box A of HMGB1 protein group demonstrated improvement in burn wound closure, starting from day 7th until day 28th after injury (P < 0.001). Furthermore, γH2AX and 8-OHdG expression in the HMGB1 plasmid-treated group was lower than that in the control group (P < 0.05). The re-epithelialisation in the LM511-E8-treated group was quicker than that of the control group at 7–28 days post-injury, with the largest improvement observed on days 7 and 14 (P < 0.001). The pathological score of the LM511-E8-treated group was higher than that of the control group at 14–28 days post-injury.

Conclusion: These results imply that LM511-E8 fragment, B1 siRNA, and Box A of High-mobility group box 1 protein (Box A of HMGB1) are promising therapeutic options for managing second-degree burns.