Hesperidin Protects Human HaCaT Keratinocytes from Particulate Matter 2.5-Induced Apoptosis via the Inhibition of Oxidative Stress and Autophagy

Abstract

Numerous epidemiological studies have reported that particulate matter 2.5 (PM_2.5) causes skin aging and skin inflammation and impairs skin homeostasis. Hesperidin, a bioflavonoid that is abundant in citrus species, reportedly has anti-inflammatory properties. In this study, we evaluated the cytoprotective effect of hesperidin against PM_2.5-mediated damage in a human skin cell line (HaCaT). Hesperidin reduced PM_2.5-induced intracellular reactive oxygen species (ROS) generation and oxidative cellular/organelle damage. PM_2.5 increased the proportion of acridine orange-positive cells, levels of autophagy-related proteins, beclin-1 and microtubule-associated protein light chain 3, and apoptosis-related proteins, B-cell lymphoma-2-associated X protein, cleaved caspase-3, and cleaved caspase-9. However, hesperidin ameliorated PM_2.5-induced autophagy and apoptosis. PM_2.5 promoted cellular apoptosis via mitogen-activated protein kinase (MAPK) activation by promoting the phosphorylation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38. The MAPK inhibitors U0126, SP600125, and SB203580 along with hesperidin exerted a protective effect against PM_2.5-induced cellular apoptosis. Furthermore, hesperidin restored PM_2.5-mediated reduction in cell viability via Akt activation; this was also confirmed using LY294002 (a phosphoinositide 3-kinase inhibitor). Overall, hesperidin shows therapeutic potential against PM_2.5-induced skin damage by mitigating excessive ROS accumulation, autophagy, and apoptosis.

Keywords: apoptosis; autophagy; hesperidin; human keratinocyte; mitogen-activated protein kinase; particulate matter 2.5.

Figure 1

Hesperidin alleviated intracellular ROS generation and cell viability reduction. Cells were pretreated with hesperidin (50 µM) or NAC (1 mM) for 1 h, and then exposed to PM_2.5 (50 µg/mL) for another 24 h. Intracellular ROS generation was observed using (A) flow cytometry and (B) confocal microscopy after H₂DCFDA staining. (C) Apoptotic body formation was detected via Hoechst 33342 staining. Arrow indicates apoptotic body. (D) MTT and (E) trypan blue assays were used to determine cell viability. Arrows indicate the dead cells. * p < 0.05 and ^# p < 0.05 compared with the control cells and PM_2.5-treated cells, respectively.

Figure 1

Hesperidin alleviated intracellular ROS generation and cell viability reduction. Cells were pretreated with hesperidin (50 µM) or NAC (1 mM) for 1 h, and then exposed to PM_2.5 (50 µg/mL) for another 24 h. Intracellular ROS generation was observed using (A) flow cytometry and (B) confocal microscopy after H₂DCFDA staining. (C) Apoptotic body formation was detected via Hoechst 33342 staining. Arrow indicates apoptotic body. (D) MTT and (E) trypan blue assays were used to determine cell viability. Arrows indicate the dead cells. * p < 0.05 and ^# p < 0.05 compared with the control cells and PM_2.5-treated cells, respectively.

Figure 2

Hesperidin mitigated PM_2.5-induced lipid peroxidation, protein carbonylation, and DNA damage. Cells were pretreated with hesperidin (50 µM) for 1 h, and then exposed to PM_2.5 (50 µg/mL) for another 24 h. (A) Lipid peroxidation was detected via the image of DPPP staining. (B) Protein carbonylation was detected using the protein carbonyl ELISA kit. (C) Comet assay and (D) avidin-TRITC staining were used to assess DNA damage. * p < 0.05 and ^# p < 0.05 compared with the control cells and PM_2.5-treated cells, respectively.

Figure 3

Hesperidin prevented PM_2.5-induced intracellular Ca²⁺ accumulation, mitochondria dysfunction, and autophagy. Cells were pretreated with hesperidin (50 µM) for 1 h, and then exposed to PM_2.5 (50 µg/mL) for another 24 h. (A) Intracellular Ca²⁺ level was detected using confocal microscopy after Fluo-4-AM staining. (B) The mitochondrial membrane potential was detected using confocal microscopy after JC-1 staining. (C) Autophagy was detected using fluorescence microscopy after acridine orange staining. (D) Cell lysates were subjected to western blotting of target proteins (beclin-1, LC3, and actin).

Figure 4

Hesperidin prevented cellular apoptosis and reduced cell viability via the inhibition of PM_2.5-induced autophagy. Cells were pretreated with hesperidin (50 µM), BAF (10 nM), or both for 1 h, and then exposed to PM_2.5 (50 µg/mL) for another 24 h. (A) Autophagy was detected using images captured via fluorescence microscopy after staining with acridine orange. (B) Apoptotic bodies were observed using Hoechst 33342 staining, and the arrows indicate apoptotic bodies. Cell viability was assessed via (C) MTT assay and (D) trypan blue staining, and the arrows indicate the dead cells. * p < 0.05, ** p < 0.05, and ^# p < 0.05 compared with BAF-untreated control cells, BAF-untreated PM_2.5-exposed cells, and BAF-untreated hesperidin + PM_2.5-exposed cells, respectively.

Figure 5

Hesperidin mitigated PM_2.5-induced cell apoptosis and MAPK activation. Cells were pretreated with hesperidin (50 µM) for 1 h, and then exposed to PM_2.5 (50 µg/mL) for another 24 h. (A) Cell lysates were subjected to western blotting for Bax, Bcl-2, cleaved caspase-9, cleaved caspase-3, (B) phospho-ERK, ERK, phospho-JNK, JNK, phospho-p38, p38, phospho-Akt, Akt. Actin was used as the loading control. Cells were pretreated with hesperidin (50 µM), U0126 (50 nM), SP600125 (5 µM), SB203580 (10 µM), and LY294002 (50 µM) for 1 h, and then exposed to PM_2.5 (50 µg/mL) for 24 h. (C) Hoechst 33342 staining was used to assess cellular apoptosis, and the arrows indicate apoptotic bodies. Cell viability was assessed using (D) trypan blue, arrows indicating the dead cells, and (E,F) MTT assays. * p < 0.05, ^# p < 0.05, and ** p < 0.05 compared with the control cells, PM_2.5-exposed cells, and hesperidin + PM_2.5-exposed cells, respectively.

Figure 5

Hesperidin mitigated PM_2.5-induced cell apoptosis and MAPK activation. Cells were pretreated with hesperidin (50 µM) for 1 h, and then exposed to PM_2.5 (50 µg/mL) for another 24 h. (A) Cell lysates were subjected to western blotting for Bax, Bcl-2, cleaved caspase-9, cleaved caspase-3, (B) phospho-ERK, ERK, phospho-JNK, JNK, phospho-p38, p38, phospho-Akt, Akt. Actin was used as the loading control. Cells were pretreated with hesperidin (50 µM), U0126 (50 nM), SP600125 (5 µM), SB203580 (10 µM), and LY294002 (50 µM) for 1 h, and then exposed to PM_2.5 (50 µg/mL) for 24 h. (C) Hoechst 33342 staining was used to assess cellular apoptosis, and the arrows indicate apoptotic bodies. Cell viability was assessed using (D) trypan blue, arrows indicating the dead cells, and (E,F) MTT assays. * p < 0.05, ^# p < 0.05, and ** p < 0.05 compared with the control cells, PM_2.5-exposed cells, and hesperidin + PM_2.5-exposed cells, respectively.

Figure 6

Schematic diagram summarizing the protective mechanism of hesperidin against PM_2.5-induced cell damage. Hesperidin protects keratinocytes by suppressing PM_2.5-induced intracellular ROS generation, intracellular macromolecules damage, autophagy activation, and cell apoptosis. Hesperidin alleviates cell apoptosis via the inhibition of PM_2.5-induced MAPK activation as well as excessive autophagy activation, which may cause the uncontrolled degradation of intracellular components that eventually results in apoptosis and cell death. Furthermore, hesperidin restored the PM_2.5-mediated decrease in cell viability via the activation of the PI3K/Akt pathway.