Panax notoginseng saponins ameliorate oral submucous fibrosis by inhibiting ANE-induced oxidative stress in HaCaT cells via activating Nrf2/GCLC signaling pathway

Objective To investigate the anti-fibrotic effect of Panax notoginseng saponins in arecoline (ANE) -induced oral submucous fibrosis, and to analyze the effect of Panax notoginseng saponins on nuclear factor E2-related factor 2/glutamate-cysteine ligase catalytic subunit (Nrf2/GCLC) signaling pathway.Methods CCK-8 assay was used to evaluate the effects of different concentrations of Panax notoginseng saponins and arecoline on the proliferation of human immortalized keratinocyte cell line (HaCaT). The results of CCK-8 were used to select 75μg/ml arecoline and 25,50,100μg/ml Panax notoginseng saponins as subsequent experimental concentrations. The cells were set as blank control group, model group, and low, medium, and high doses (25, 50, 100 μg/ml) of Panax notoginseng saponins groups. Western blot and RT-qPCR were used to detect the expression of collagen-Ⅰ (COL-1), -smooth muscle actin (α-SMA), E-cadherin, Nrf2, GCLC protein and mRNA in each group of cells. Immunofluorescence method was used to detect the entry of Nrf2 into the nucleus; biochemical kit was used to detect the content of malondialdehyde (MDA) and superoxide dismutase (SOD) activity in each group of cells; DCFH-DA fluorescent probe was used to detect the content of intracellular reactive oxygen species (ROS).Results Compared with the blank control group, the expression of COL-I protein and mRNA in the model group was up-regulated, and the expression of E-cadherin, Nrf2, GCLC, nuclear Nrf2-related proteins and mRNA were down-regulated. The content of MDA and ROS in the cells increased, and the activity of SOD was significantly reduced;Compared with the model group, the expression of COL-I protein and mRNA was down-regulated, and the expression of E-cadherin, Nrf2, GCLC, nuclear Nrf2-related protein and mRNA were up-regulated in the Panax notoginseng saponins 50 and 100 μg/mL groups. Compared with the model group,the content of MDA and ROS in cells decreased, and the activity of SOD was significantly enhanced (all P < 0.05 or P < 0.01).Conclusion Panax notoginseng saponins have anti-fibrosis effects in HaCaT cells, and their mechanism may be related to the activation of Nrf2/GCLC signaling pathway, thereby resisting oxidative stress and improving oral submucosal fibrosis.