Protecting HaCaT cells from ionizing radiation using persimmon tannin- Aloe gel composite

Abstract

Context: Persimmon tannin (extract of Diospyros kaki L.f [Ebenaceae]) and Aloe gel (extract of Aloe vera (L.) Burm.f. [Asphodelaceae]) are known as anti-radiation agents. However, radiation resistance of the persimmon tannin-Aloe gel composite remains inconclusive.Objective: To investigate the capacity of the persimmon tannin-Aloe gel composite to protect against ionising radiation at the cellular level.Materials and methods: HaCaT (human epidermal keratinocytes) cells were pre-treated with PT-A-1 (the mass ratio of persimmon tannin and Aloe gel was 2:1) or the single component (persimmon tannin or Aloe gel) at various concentrations (0, 50, 100, 200, 400, 800 μg/mL. Control group: medium with no HaCaT cells), and then radiated with X-rays (radiation dose: 4, 8, 12, 16, and 20 Gy). Cell viability, cell apoptosis, and radiation-induced intracellular reactive oxygen species (ROS) generation were analysed by CCK-8, Hoechst 33258 staining/flow cytometry, and 2',7'-dichlorfluorescein diacetate (DCFH-DA) assay, respectively, for 12 or 24 h incubation after radiation.Results: The optimal radiation dose and post-radiation incubation period were determined to be 8 Gy and 12 h. CCK-8 activity detection showed that the cell activity was 77.85% (p < 0.05, IC₅₀ = 55.67 μg/mL). The apoptotic rate was the lowest (4.32%) at 200 μg/mL of PT-A-1 towards HaCaT cells. ROS production was the most effectively suppressed by 200 μg/mL PT-A-1 towards HaCaT cells.Discussion and conclusions: The persimmon tannin-Aloe gel composite has good radioprotective effect, and which will facilitate its clinic application as a potential natural anti-radiation agent in future.

Keywords: Human epidermal keratinocytes cells; Radiation resistance; X-rays.

Figure 1.

The HaCaT cells morphology with different ingredients at a concentration of 200 μg/mL after 8 Gy radiation pre-treatment for 12 h incubation (400×).

Figure 2.

Cellular viability (%) of HaCaT cells with PT-A-1 for different concentrations and different radiation doses for 12 h incubation after X-ray irradiation (a); Cellular viability (%) of HaCaT cells with PT-A-1 for different concentrations and different radiation doses for 24 h incubation after X-ray irradiation (b). All above values are presented as the median from analysis of three independent experiments and the error bars indicate standard deviation (n = 3, p < 0.05). Symbol* represents p < 0.05 vs. blank group (Concentration of 0 g/mL).

Figure 3.

Hoechst 33258 fluorescent dyeing of HaCat cells pre-treated with different concentrations of PT-A, PT, and Aloe gel materials for 12 h incubation after 8 Gy X-ray irradiation (200×).

Figure 4.

Flow cytometry detecting the apoptosis rate of HaCaT cells pre-treated with different concentrations of PT-A, PT, and Aloe gel materials for 12 h incubation after 8 Gy X-ray irradiation. All above values are presented as the median from analysis of three independent experiments and the error bars indicate standard deviation (n = 3), Symbol* represents p < 0.05 vs. Control group.

Figure 5.

The inhibition effects of PT-A-1 pre-treatment prior to radiation with 8 Gy X-ray irradiation on the production of intracellular reactive oxygen species in HaCat cells. All above values are presented as the median from analysis of three independent experiments and the error bars indicate standard deviation (n = 3), Symbol* represents p < 0.05 vs. Control group.