Abstract: Objective To explore the protective effect of oxymatrine(OMT)on oxidative damage of keratinocytes in hypoxic-ischemic environment. Methods HaCaT cells cultured in vitro were divided into normal control(NC)group, hypoxia-ischemia(HI)group, low-dose OMT group(0.05 g/L)and high-dose OMT group(0.1 g/L). The proliferation and vitality of keratinocytes were detected with CCK-8 assay; the apoptosis was detected with Annexin V; the mitochondrial membrane potential was detected with mitochondrial membrane potential probe; the reactive oxygen species(ROS)level was detected with DCFH-DA fluorescent probe; the activities of superoxide dismutase(SOD), glutathione peroxidase(GSH-Px)and total antioxidant capacity(T-AOC)in the supernatant of HaCAT cells were determined with colorimetry; the expression levels of Caspase 3, Cleaved-Caspase 3, B lymphoma 2(Bcl-2)and TGF-β1/Smad3 signaling pathway were detected with Western blotting. Results Compared with the NC group, the HI group had reduced Ki67 positive rate ［(13.52±2.89)%, P<0.001］ and lower mitochondrial membrane potential(0.54±0.03, P<0.001), increased apoptotic cells(13.83±0.81, P<0.001)and ROS level(164.31±16.93, P<0.001), and reduced expression levels of GSH-Px(0.96±0.05, P<0.001), SOD(0.67±0.06, P<0.001)and T-AOC(1.90±0.02, P<0.001). Low-dose OMT treatment resulted in increased Ki67 positive rate ［(57.98±9.81)%, P<0.001］, higher mitochondrial membrane potential(0.81±0.04, P<0.001), reduced apoptotic cells(8.10±0.53, P<0.001), reduced ROS level(175.94±15.75, P<0.001), but enhanced expression levels of GSH-Px(1.04±0.05, P<0.001), SOD(0.86±0.04, P<0.001)and T-AOC(2.08±0.03, P<0.001). Western blotting showed that HI treatment increased the expression levels of TGF-β1(1.15±0.14, P=0.010)and p-SMAD3(0.13±0.03, P=0.112), and increased the relative expression levels of Caspase-3(0.37±0.045, P=0.001)and Cleaved-Caspase 3(0.54±0.03, P=0.108). Compared with HI group, low-dose OMT group had reduced relative expressions of TGF-β1(0.69±0.13, P=0.005), p-SMAD3(0.07±0.01, P<0.001), Caspase-3(0.21±0.041, P=0.006)and Cleaved-Caspase 3(0.29±0.054, P=0.016), but increased relative expression of Bcl-2(0.35±0.013, P=0.015). Between the low-dose and high-dose OMT groups, there were only significant differences in Ki67 positive rate(P<0.001), SOD activity(P<0.001)and Bcl-2 expression(P=0.045), and no differences in the other parameters(P>0.05). Conclusion Treatment with 0.05-0.1 g/L can reduce mitochondrial dysfunction, oxidative damage and apoptosis induced by hypoxia-ischemia by inhibiting TGFβ1/ Smad3 pathway, and thus protect the survival of keratinocytes.

Key words: Oxymatrine, Keratinocytes, Apoptosis, TGFβ1/SMAD3, Oxidative stress