Journal of Shandong University (Health Sciences) ›› 2021, Vol. 59 ›› Issue (3): 26-34.doi: 10.6040/j.issn.1671-7554.0.2020.1557

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Oxymatrine ameliorates oxidative stress injury of HaCaT cells induced by hypoxia ischemia

LIU Shudan, ZHANG Feiyan, GUO Songlin, LIANG Xueyun, CHEN Dongmei   

  1. Human Stem Cell Institute, General Hospital of Ningxia Medical University;
    Ningxia Key Laboratory of Stem Cell and Regenerative Medicine, Yinchuan 750004, Ningxia, China
  • Published:2021-04-06

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

CLC Number: 

  • R-932
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