Andrographolide sodium bisulfate attenuates UV‑induced photo‑damage by activating the keap1/Nrf2 pathway and downregulating the NF‑κB pathway in HaCaT keratinocytes

Abstract

Oxidative and inflammatory damage has been suggested to play important roles in the pathogenesis of skin photoaging. Andrographolide sodium bisulfate (ASB) is a soluble derivative of andrographolide and has known antioxidant and anti‑inflammatory properties. In the present study, cellular experiments were designed to investigate the molecular mechanisms underlying the effect of ASB in relieving ultraviolet (UV)‑induced photo‑damage. Following ASB pretreatment and UV irradiation, the apoptosis and necrosis of HaCaT cells were investigated by Hoechst 33342/propidium iodide staining. Reactive oxygen species (ROS) production was investigated using a DCFH‑DA fluorescence probe. Furthermore, the protein expression levels of p65, NF‑κB inhibitor‑α, nuclear factor E2‑related factor 2 (Nrf2) and kelch‑like ECH‑associated protein 1 (keap1) were measured via western blotting and immunofluorescence analyses. Furthermore, NF‑κB‑mediated cytokines were assessed by ELISA, and Nrf2‑mediated genes were detected by reverse transcription‑quantitative PCR. Pretreatment with ASB markedly increased cell viability, decreased cell apoptosis and decreased UV‑induced excess ROS levels. In addition, ASB activated the production of Nrf2 and increased the mRNA expression levels of glutamate‑cysteine ligase catalytic subunit and NAD(P)H quinone oxidoreductase 1, while ASB downregulated the protein expression of p65 and decreased the production of interleukin (IL)‑1β, IL‑6 and tumor necrosis factor‑α. These results suggested that ASB attenuates UV‑induced photo‑damage by activating the keap1/Nrf2 pathway and downregulating the NF‑κB pathway in HaCaT keratinocytes.

Figure 1

Protective effect of ASB on UV-induced HaCaT cells. (A) The cells were treated with 0, 0.01, 0.1, 1, 10, 100, 500, 1,000 and 2,000 µM ASB for 24 h. The cell viability was determined by MTT assay (n=5 for each group). (B) The cells were preincubated with ASB (10, 30 and 100 µM) and irradiated with UV (90 mJ/cm²). Cell viability was measured by MTT assay (n=5 for each group). (C) The cells were preincubated with ASB (10, 30 and 100 µM) and irradiated with UV (90 mJ/cm²). Cell apoptosis and death were measured by staining with Hoechst 33342 and PI. The blue arrowheads indicate apoptotic cells, and the red arrowheads indicate dead cells. The images were examined by bright field and fluorescence microscopy. The results are expressed as the mean ± SD. ##P<0.01 vs. NC group; ^**P<0.01 vs. UV-alone group. UV, ultraviolet; ASB, andrographolide sodium bisulfite; NC, normal control; PI, propidium iodide.

Figure 2

ASB decreases oxidative stress and proinflammatory cytokines in UV-irradiated HaCaT cells. The cells were preincubated with ASB (10, 30 and 100 µM) and irradiated with UV (90 mJ/cm²). (A) The intracellular ROS in HaCaT cells was assessed by DCFH-DA. The appearance of green fluorescence represents the intensity of the generated ROS. (B) The fluorescence intensity was quantified with Image J software. The mRNA expression levels of (C) GCLC and (D) NQO1 in HaCaT cells were measured by reverse transcription-quantitative PCR. The production of (E) IL-1β, (F) IL-6 and (G) TNF-α was measured by ELISA (n=4 for each group). The results are expressed as the mean ± SD. ^#P<0.05, ^##P<0.01 vs. NC group; ^*P<0.05, ^**P<0.01 vs. UV-alone group. UV, ultraviolet; ASB, andrographolide sodium bisulfite; ROS, reactive oxygen species; GCLC, glutamate-cysteine ligase catalytic subunit; NQO1, NAD(P)H quinone oxidoreductase 1; IL-1β, interleukin-1β; IL-6, interleukin-6; TNF-α, tumor necrosis factor-α; NC, normal control.

Figure 3

Changes in the Nrf2 and NF-κB signaling pathways in HaCaT cells after UV irradiation. (A) Nuclear and cytoplasmic proteins were extracted from the cultured cells at 0, 0.5, 1, 2, 3, 4, 5 and 6 h after UV irradiation (90 mJ/cm²). The protein expression levels of NF-κB-mediated p65, and IκBα and Nrf2-mediated Nrf2 and keap1, were measured by western blotting. (B) Relative changes in protein intensities were quantified by densitometric analysis and are presented as bar diagrams (n=3 for each group). The results are expressed as the mean ± SD. ^aP<0.05 vs. the 0 h group; ^bP<0.05 vs. the 0.5 h group; ^cP<0.05 vs. the 1 h group; ^dP<0.05 vs. the 2 h group; ^eP<0.05 vs. the 3 h group; ^fP<0.05 vs. the 4 h group; ^gP<0.05 vs. the 5 h group. UV, ultraviolet; IκBα, NF-κB inhibitor-α; keap1, kelch-like ECH-associated protein 1; Nrf2, nuclear factor E2-related factor 2.

Figure 4

ASB activates the Nrf2 signaling pathway in UV-induced HaCaT cells. The cells were preincubated with ASB (10, 30 and 100 µM) and irradiated with UV (90 mJ/cm²). (A) Nuclear and cytoplasmic proteins were extracted; Nrf2 and keap1 proteins were measured by western blotting. Relative changes in protein intensity were quantified for (B) keap1, (C) cytosolic Nrf2 and (D) nuclear Nrf2 by densitometric analysis, and are presented as bar diagrams (n=3 for each group). ^#P<0.05 vs. NC group; ^*P<0.05, ^**P<0.01 vs. UV-alone group. ASB, andrographolide sodium bisulfite; UV, ultraviolet; keap1, kelch-like ECH-associated protein 1; Nrf2, nuclear factor E2-related factor 2; NC, normal control.

Figure 5

ASB increases the nuclear expression of Nrf2 in UV-induced HaCaT cells. The cells were preincubated with ASB (10, 30 and 100 µM) and irradiated with UV (90 mJ/cm²). The fluorescence localization of Nrf2 was measured with immunofluorescence. An anti-Nrf2 antibody was used to detect Nrf2 localization (green) using a fluorescence microscope. DAPI staining indicated the locations of the nuclei (blue). ASB, andrographolide sodium bisulfite; UV, ultraviolet; Nrf2, nuclear factor E2-related factor 2; NC, normal control.

Figure 6

ASB inhibits the NF-κB signaling pathway in UV-induced HaCaT cells. The cells were preincubated with ASB (10, 30 and 100 µM) and irradiated by UV (90 mJ/cm²). (A) Nuclear and cytoplasmic proteins were extracted; p65 and IκBα proteins were measured by western blotting. Relative changes in protein intensity were quantified for (B) IκBα, (C) cytosolic p65 and (D) nuclear p65 by densitometric analysis, and are presented as bar diagrams (n=3 for each group). ^#P<0.05, ^##P<0.01 vs. NC group; ^*P<0.05, ^**P<0.01 vs. UV-alone group. ASB, andrographolide sodium bisulfite; UV, ultraviolet; IκBα, NF-κB inhibitor-α; NC, normal control.

Figure 7

ASB decreases the nuclear expression of p65 in UV-induced HaCaT cells. The cells were preincubated with ASB (10, 30 and 100 µM) and irradiated by UV (90 mJ/cm²). The fluorescence localization of p65 was measured with immunofluorescence. An anti-p65 antibody was used to detect p65 localization (green) using a fluorescence microscope. DAPI staining indicated the locations of nuclei (blue). ASB, andrographolide sodium bisulfite; UV, ultraviolet; NC, normal control.

Figure 8

Schematic diagram of the mechanism of action of ASB in UV-induced photo-damage in HaCaT cells. UV, ultraviolet; ASB, androgra-pholide sodium bisulfite; ROS, reactive oxygen species; keap1, kelch-like ECH-associated protein 1; Nrf2, nuclear factor E2-related factor 2; IL, interleukin; TNF-α, tumor necrosis factor-α; GCLC, glutamate-cysteine ligase catalytic subunit; NQO1, NAD(P)H quinone oxidoreductase 1; IκBα, NF-κB inhibitor-α; ARE, antioxidant response element.