Effect of HLP on TNF-α-induced ROS production in VSMC. (a) A7r5 cells were treated with TNF-α (10 ng/mL) in the absence or presence of 0.2 mg/mL of HLP for 24 and 48 h. The treated cells were then labeled with fluorescent probe, dichlorofluorescin diacetate (DCFH-DA), and reactive oxygen species (ROS) production was measured using Muse™ Cell Analyzer. M1: DCF-negative cells. M2: DCF-positive cells. The results are presented as mean ± SD (n = 3) from three independent experiments. ## p < 0.01 compared with the control. ** p < 0.01 compared with the TNF-α group. +, added; −, non-added. (b) Schematic representation of TNF-α-antagonist potential of HLP on VSMCs. TNF-α induces intracellular ROS production, leading cell migration and proliferation through Akt/AP-1/MMP-9 signaling and cyclin E/cdk2-mediated Rb phosphorylation in A7r5 cells. HLP functions against TNF-α via downregulation of Akt/MMP-9 and upregulation of p53 signals that subsequently inhibit VSMC migration and proliferation. Red arrows represent the changes in response to TNF-α stimulation; blue arrows represent changes in TNF-α-exposed VSMCs receiving HLP intervention.