Allicin attenuates UVB-induced photodamage of keratinocytes by inhibiting NLRP3 inflammasomes and activating the PI3K/Akt pathway

Abstract

Allicin is a sulfide extracted from garlic bulbs responsible for various physiological and pathophysiological effects, including antioxidant, antibacterial, and anti-parasite activities. However, its efficacy and mechanism of protecting UVB-induced photodamage have not been studied. The research explores Allicin's protective roles and underlying mechanisms in UVB-induced photodamage of keratinocytes. UVB was employed to generate photodamage in the HaCaT cell line. DCFH-DA fluorescent and Biochemical analyses were carried out to evaluate reactive oxygen species (ROS) and oxidative stress on UVB-induced photodamage to HaCaT cells. RT-qPCR and western blot were performed to measure mRNA and protein expression. Allicin pretreatment (10 and 25 µM) improved cell proliferation and reduced apoptotic rates in UVB-induced HaCaT cells. Allicin (10 and 25 µM) inhibited tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1 beta (IL-1β) expressions (all, P < 0.001). Allicin reduced the intracellular ROS level and attenuated oxidative stress, with reduced malondialdehyde (MDA) level while increasing the levels of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSh-Px) (all, P < 0.001) in UVB-induced HaCaT cells. Allicin pretreatment inhibited autophagy and reduced the protein expression of Beclin-1 while increasing the p62 protein expression (all, P < 0.001). We also observed that Allicin pretreatment reduced the NLRP3-related protein, such as Caspase-1 (P < 0.001) and increased the protein expressions of the PI3K/Akt pathway molecules, such as PI3K and Akt (all, P < 0.001). Our research data demonstrated that Allicin might inhibit UVB-induced photodamage of keratinocytes via inhibiting NLRP3 inflammasomes and activating the PI3K/Akt pathway.

Keywords: Allicin; Autophagy; NLRP3 inflammasomes; Oxidative stress; ROS.

Fig. 1

Allicin attenuated keratinocyte proliferation and apoptosis induced by UVB. A Structural formula, molecular weight, chemical formula and CAS number of Allicin. B HaCaT cells were incubated with different concentrations of Allicin (0, 5, 10, 25, 50, and 100 μM) for 24 h, and the cell viability was assessed using an MTT assay. C HaCaT cells were pretreated with Allicin (10 and 25 µM) for 2 h, followed by UVB irradiation (50 mJ/cm²) and incubated for further 6, 12, 24, 48, and 72 h, and the effect of Allicin on the viability of UVB-exposed HaCaT cells was assessed using an MTT assay. D HaCaT cells were stained with TUNEL and DAPI, and cell apoptotic rate was assessed by calculating the percentage of TUNEL-positive cells to all DAPI-positive cells. E Representative images of microphotographs of HaCaT cells with TUNEL and DAPI staining. Data are presented as mean ± SD in triplicates and analyzed using one-way ANOVA, and LSD was used for the post-hoc test. Scale 50 µm. *P < 0.05, ***P < 0.001 vs. control group; ###P < 0.001 vs. UVB group

Fig. 2

Allicin suppresses UVB-induced inflammation of keratinocytes. A, B, C RT-qPCR was performed to determine the mRNA levels of pro-inflammatory cytokines in HaCaT cells, including TNF-α, IL-1β, and IL-6. D-F. D, E, F ELISA was used to determine the release of TNF-α, IL-1β, and IL-6 in the culture media of HaCaT cells. Data are presented as mean ± SD in triplicates and analyzed using one-way ANOVA, and LSD was used for the post-hoc test. ***P < 0.001 vs. control group; ###P < 0.001 vs. UVB group

Fig. 3

Allicin inhibits intracellular ROS generation and oxidative stress in UVB-induced keratinocytes. A HaCaT cells were stained with DCFH-DA, and the representative images were shown (200 ×). B Quantitative analysis of the relative DCFH-DA fluorescence intensity of HaCaT cells. The cell lysate of HaCaT cells was used to measure the oxidative stress markers for C MDA, D SOD, E CAT and F GSH-Px. Data are presented as mean ± SD in triplicates and analyzed using one-way ANOVA, and LSD was used for the post-hoc test. Scale 50 µm. ***P < 0.001 vs. control group; ###P < 0.001 vs. UVB group

Fig. 4

Allicin inhibits autophagy in UVB-induced HaCaT cells. A HaCaT cells were stained with LC3-II antibody. The nucleus was stained with DAPI (blue). B Quantifying relative LC3-II fluorescence intensity, as presented by cells with LC3-II dots to DAPI + cells. C Representative blots of autophagy-related proteins by western blot analysis. Allicin significantly increases the protein expression of D Beclin-1 and decreases the protein expression of (F) p62. Data are presented as mean ± SD in triplicates and analyzed using one-way ANOVA, and LSD was used for the post-hoc test. Scale 50 µm. *P < 0.05, **P < 0.01, ***P < 0.001 vs control group; ###P < 0.001 vs UVB group

Fig. 5

Allicin activates the PI3K/Akt pathway and inhibits NLRP3 inflammasome in UVB-induced keratinocytes. A Representative gel blots of phosphorylated PI3K and Akt, and NLRP3-related protein by Western blotting. Quantification analysis of (B) phosphorylated PI3K (normalized to total PI3K), C phosphorylated Akt (normalized to total Akt), D NLRP3 (normalized to GAPDH), and E Caspase-1 (normalized to GAPDH) protein bands. F HaCaT cells were pretreated with Allicin (25 μM) or PI3K/Akt pathway inhibitor LY294002 (10 μM), followed by irradiated with UVB for further 24 h. Cell viability was evaluated with MTT assay. Data are presented as mean ± SD in triplicates and analyzed using one-way ANOVA, and LSD was used for the post-hoc test. ***P < 0.001 vs. control group; ###P < 0.001 vs. UVB group; $$P < 0.001 vs. UVB + Allicin 25 group

Fig. 6

Schematic illustration of the protective effects of Allicin against UVB-induced photodamage of keratinocytes by inhibiting NLRP3 inflammasomes and activating the PI3K/Akt/autophagy pathway