D 35578

A549 cells were seeded in sterile 6-well plates (1.2 × 10⁶ cells/well) and grown overnight in the incubator. Cells were treated with HML (400 μg/mL). When the cells have grown to 90–100% confluence in the monolayer, draw a straight line on the cell surface with a sterile pipette tip. Wounds were photographed under an inverted research microscope (Leica D-35578, Wetzlar, Germany) at 0, 24, 48, and 72 h after injury. Image J was used to analyze the scratch area.

Golgi staining was performed to investigate the spine density in each group (n = 6) using Golgi-Cox OptimStain Kit (Hitobiotec, USA). Briefly, as described in the manufacturer’s instructions, the brains of mice were removed, cut into 1 cubic centimeter of tissue pieces, and placed in Golgi-Cox solution (containing Solutions 1 and 2) at room temperature for 14 days in the dark. Subsequently, brain samples were immersed in Solution 3 for 48 h at 4 ℃. After that, brain tissues were sliced into 150-µm-thick coronal slices by a vibratome (Leica CM1950, Germany). Then, the sections were placed onto gelatin-coated microscope slides and stained with solutions 4 and 5 after natural drying. Afterward, the brain slices were dehydrated with increasing concentrations of ethanol (50% ethanol for 5 min, 70% ethanol for 5 s, 95% ethanol for 5 s, and twice 100% ethanol for 5 s), then incubated in xylene for 5 min twice and coverslipped with permount. The images of spine density were captured using microscope and Leica microsystems (Leica D35578, Germany). Secondary dendritic segments of 10 mm length from 4 to 5 neurons in the CA1 area from each animal were analyzed for the number of dendritic spines.

Transwell assays were performed by using transwell plates with 8 μm pore (Corning; 3422). For the migration assay, 1 × 10⁴ Cells were resuspended in serum-free RPMI-1640 medium and plated onto the 24-well upper chamber with the lower chamber filled with complete medium. For the invasion assay, the 24 transwell plate with matrigel (BD biosciences) polymerized at the 24-well upper chamber for 2 h at 37 °C as the intervening invasive barrier. After 24 h incubation at 37 °C, the cells on the upper chamber were removed and the number of cells that migrated or invaded to the lower side were fixed in 4% paraformaldehyde and stained with 1% crystal violet and counted at × 200 magnification in 10 different fields of microscope (Leica; D-35578). The results were determined from three repeated experiments.