Effects of haem oxygenase-1 expression on oxidative injury and biological behaviours of rat dermal fibroblasts.

Abstract

This study investigated the effects of high haem oxygenase-1 (HO-1) expression on oxidative injury and the biological behaviours of rat dermal fibroblasts, under high glucose conditions. Rat dermal fibroblasts were cultured in normal glucose (1.0g/l), high glucose (4.5g/l) or haemin (5μm). A bilirubin kit, real-time polymerase chain reaction (RT-PCR) and Western blotting measured the protease activity, mRNA, and protein levels of HO-1, respectively. An enzyme-linked immunosorbent assay (ELISA) kit measured media levels of 8-hydroxydeoxyguanosine (8-OHdG), reactive oxygen species (ROS) and collagen (hydroxyproline) secretion. Cell proliferation was measured using flow cytometry. Cell apoptosis was measured using Hoechst 33258 staining and flow cytometry. The transwell method and scratch test evaluated cell migration. HO-1 expression exhibited a time-dependent change that was lowest in the high glucose (HG) group at 96 hours compared with the normal glucose (NG) group. In the HG group, the 8-OHdG, ROS and cell apoptosis were increased, and collagen secretion, cell proliferation and cell migration (horizontal and vertical) were decreased compared with the NG group at 96 hours. Haemin treatment sustained high HO-1 expression for at least 96 hours, and the cells exhibited decreased 8-OHdG and ROS, increased collagen synthesis, improved proliferation and migration ability, and decreased apoptosis in the NG and haemin (NH) group/HG and haemin (HH) group compared with the NG/HG groups. These cells recovered from oxidative injury and biological behaviours dysfunction. Haemin induces HO-1 expression in fibroblasts and it may influence the oxidative injury and biological behaviours of fibroblasts. These findings suggest that HO-1 may accelerate the healing of diabetic wounds via alleviation of oxidative injury and improvement of biological behaviours of fibroblasts.