Protective Effect of Garlic on Cellular Senescence in UVB-Exposed HaCaT Human Keratinocytes

Abstract

Ultraviolet (UV) irradiation generates reactive oxygen species (ROS) in the cells, which induces the cellular senescence and photoaging. The present study investigated the protective effects of garlic on photo-damage and cellular senescence in UVB-exposed human keratinocytes, HaCaT cells. An in vitro cell free system was used to examine the scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals and nitric oxide (NO). The effect of garlic extract on ROS formation, MMP-1 protein and mRNA expressions, cytokines such as interleukin (IL)-1β and IL-6, senescence associated-β-galactosidase (SA-β-gal) activity, and silent information regulator T1 (SIRT1) activity were determined in UVB-irradiated HaCaT cells. Garlic exhibited strong DPPH radical and NO scavenging activity in cell free system exhibiting IC50 values of 2.50 mg/mL and 4.38 mg/mL, respectively. Garlic pretreatment attenuated the production of UVB-induced intracellular ROS. MMP-1 level, which has been known to be induced by ROS, was dramatically elevated by UVB irradiation, and UVB-induced MMP-1 mRNA and protein expressions were significantly reduced by garlic treatment (50 µg/mL) comparable to those of UV-unexposed control cells. UV-induced pro-inflammatory cytokine productions (IL-6 and IL-1β) were significantly inhibited by pretreatment with garlic in a dose-dependent manner. SA-β-gal activity, a classical biomarker of cellular senescence, and SIRT1 activity, which has attracted attention as an anti-aging factor in recent years, were ameliorated by garlic treatment in UV-irradiated HaCaT cells. The present study provides the first evidence of garlic inhibiting UVB-induced photoaging as a result of augmentation of cellular senescence in HaCaT human keratinocytes.

Keywords: HaCaT cells; MMP-1; SA-β-gal; SIRT1; UVB irradiation; garlic; pro-inflammatory cytokine; senescence.

Figure 1

Antioxidant effects of garlic. (A) DPPH and NO radical scavenging activity of garlic extract in a cell-free system. The level of DPPH radical was measured spectrophotometrically at 515 nm. The NO scavenging capacity was assessed by Griess assay. The IC₅₀ values for the DPPH radical and NO scavenging activities were 2.50 mg/mL and 4.38 mg/mL, respectively; (B) intracellular ROS levels induced by UVB were determined by the DCF–DA method. HaCaT cells, treated with garlic prior to UV irradiation (100 mJ/cm²), were incubated with 20 μM DCF–DA for 30 min, and harvested after 24 h. ROS formation was analyzed with a fluorometer (excitation; 486 nm, emission; 530 nm). Each bar represents the mean ± SD (n = 6). The bars with a different letter are significantly different from each other at the level of p < 0.05.

Figure 2

Effect of garlic on MMP-1 expressions in human skin keratinocytes. Cells were pretreated with garlic for 24 h prior to UVB irradiation (100 mJ/cm²) and harvested 24 h later. Expression of MMP-1 mRNA was determined by quantitative real time RT-PCR. GAPDH was used as an internal control. MMP-1 level was determined using an ELISA kit. Data are expressed as the percentage of control (Non-UV) group. MMP-1 level of HaCaT cells before UV irradiation was 0.12 ± 0.02 pg/mL (100%). Each bar represents the mean ± SD (n = 3). The bars with a different letter are significantly different from each other at the level of p < 0.05.

Figure 3

Effect of garlic on IL-1β and IL-6 productions in human skin keratinocytes. Cells were pretreated with garlic for 24 h prior to UVB irradiation and harvested 24 h later. The levels of IL-1β and IL-6 in culture media were determined by ELISA kit. Each bar represents the mean ± SD (n = 3). The bars with a different letter are significantly different from each other at the level of p < 0.05.

Figure 4

Effect of garlic on SA-β-gal activity in human skin keratinocytes. Cells were pretreated with garlic for 24 h prior to UVB irradiation (100 mJ/cm²) and cellular senescence was assessed by SA-β-gal activity staining. Each bar represents the mean ± SD (n = 3). The bars with a different letter are significantly different from each other at the level of p < 0.05.

Figure 5

Effect of garlic on SIRT1 activity in human skin keratinocytes. Cells were pretreated with garlic for 24 h prior to UVB irradiation (100 mJ/cm²) and cellular senescence was assessed by SIRT1 activity. Data are expressed as the percentage of control (Non-UV) group (13.2 ± 1.8 unit/μg protein; 100%). Each bar represents the mean ± SD (n = 3). The bars with a different letter are significantly different from each other at the level of p < 0.05.

Figure 6

HPLC profile of garlic extract. Allicin, diallyl disulfide (DADS), and diallyl trisulfide (DATS) were evaluated.