Nutr Res Pract.  2025 Apr;19(2):170-185. 10.4162/nrp.2025.19.2.170.

Tagetes erecta Linn flower extract inhibits particulate matter 2.5-promoted epithelial-mesenchymal transition by attenuating reactive oxygen species generation in human retinal pigment epithelial ARPE-19 cells

• Park BS
• Bang E
• Lee H
• Kim GY
• Choi YH

• KMID: 2566807
• DOI: http://doi.org/10.4162/nrp.2025.19.2.170

Abstract

BACKGROUND/OBJECTIVES
Particulate matter 2.5 (PM2.5) exposure can promote epithelialmesenchymal transition (EMT) in human retinal pigment epithelial (RPE) cells. The flowers of Tagetes erecta Linn, commonly known as marigold, are rich in diverse flavonoids and carotenoids and play a significant role in preventing cellular damage induced by oxidative stress, but the role of their extracts in RPE cells has not been reported. This study aimed to evaluate the influence of an ethanol extract of T. erecta Linn flower (TE) on PM2.5-induced EMT processes in RPE ARPE-19 cells.
MATERIALS/METHODS
To investigate the protective effect of TE against ARPE-19 cell damage following PM2.5 treatment, cells were exposed to TE for 1 h before exposure to PM2.5 for 24 h. We investigated whether the efficacy of TE on suppressing PM2.5-induced EMT was related to antioxidant activity and the effect on the expression changes of factors involved in EMT regulation. Additionally, we further explored the role of intracellular signaling pathways associated with EMT inhibition.
RESULTS
TE significantly blocked PM2.5-induced cytotoxicity while effectively preventing mitochondrial dysfunction, increased reactive oxygen species (ROS) generation, and mitochondrial membrane potential disruption. TE inhibited PM2.5-induced EMT and inflammatory response by suppressing the ROS-mediated transforming growth factor-β/ suppressor of mothers against decapentaplegic/mitogen-activated protein kinases signaling pathway.
CONCLUSION
Our results suggest that marigold extract is a highly effective in protection against PM2.5-induced eye damage.

Keyword

Particulate matter; retinal pigment epitheliums; reactive oxygen species; epithelial-mesenchymal transition; transforming growth factors

Figure

• 

  Fig. 1 Effect of TE on the decrease in ARPE-19 cell viability following PM2.5 treatment. Cells were subjected to varying concentrations of PM2.5 and TE for 24 h (A, B) or treated with 5 μg/mL TE for 1 h before exposure to 25 μg/mL PM2.5 for 24 h (C, D). (A-C) Cell viability was assessed using the CCK-8 assay (D) Representative images depicting morphological cellular changes are displayed.PM2.5, particulate matter 2.5; TE, ethanol extract of Tagetes erecta Linn flower; CCK-8, Cell Counting Kit-8.*P < 0.05, **P < 0.01 and ***P < 0.001 vs. control; #P < 0.05 vs. PM2.5.

• 

  Fig. 2 Effect of TE on cell migration induced by PM2.5 in ARPE-19 cells. Cells were cultured in medium with or without 5 μg/mL TE for 1 h before exposure to 25 μg/mL PM2.5 for 24 h. (A) The scratch wound healing assay was employed, capturing images at 0 and 24 h post-scratch application. The solid lines delineate the wound edges for visual clarity. (B) The graph depicts the relative migration rate, standardized against the control group.TE, ethanol extract of Tagetes erecta Linn flower; PM2.5, particulate matter 2.5.***P < 0.001 vs. control; ##P < 0.01 vs. PM2.5.

• 

  Fig. 3 Effect of TE on PM2.5-triggered ROS generation in ARPE-19 cells. Cells were treated with 5 μg/mL TE for 1 h before exposure to 25 μg/mL PM2.5 for 1 h. (A) Intracellular ROS production was assessed through DCF-DA staining. (C) To evaluate whether mtROS was produced, cells were labeled with MitoSOX Red, a fluorescent probe for mitochondrial superoxide detection (scale bar, 50 μm). (B, D) Relative values of green and red fluorescence intensities representing intracellular ROS and mtROS levels are presented.TE, ethanol extract of Tagetes erecta Linn flower; PM2.5, particulate matter 2.5; ROS, reactive oxygen species; DCF-DA, 5,6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate; mtROS, mitochondrial ROS; DAPI: 4’6-diamidino-2-phenylindole.***P < 0.001 vs. control; ###P < 0.001 vs. PM2.5.

• 

  Fig. 4 Effect of TE on PM2.5-induced mitochondrial dysfunction in ARPE-19 cells. Cells were treated with 5 μg/mL TE for 1 h before exposure to 25 μg/mL PM2.5 for 24 h. (A) MitoSOX Red dye was used to evaluate mitochondrial activity. (C) JC-1 was used to determine the level of ΔΨm. After staining with the indicated dyes, nuclei were counterstained using DAPI (blue fluorescence), and fluorescence images were observed using a fluorescence microscope (scale bar, 50 μm). (B, D) Relative values of red fluorescence intensity of MitoSOX Red and fluorescence of JC-1 aggregates and monomers are presented.TE, ethanol extract of Tagetes erecta Linn flower; PM2.5, particulate matter 2.5; JC-1, 1,1′,3,3′-tetraethyl-5,5′,6,6′-tetrachloroimidacarbocyanine iodide; ΔΨm, mitochondrial membrane potential; DAPI, 4’6-diamidino-2-phenylindole.***P < 0.001 vs. control; ###P < 0.001 vs. PM2.5.

• 

  Fig. 5 Effect of TE on PM2.5-triggered changes in EMT regulator and inflammatory cytokine levels in ARPE-19 cells. Cells were cultured in medium with or without 5 μg/mL TE for 1 h before exposure to 25 μg/mL PM2.5 for 24 h. (A, E) After treatment, Western blot analysis was performed using the proteins extracted from cells and antibodies corresponding to the proteins to be analyzed. (B, F) Expression of each protein was quantified and normalized to actin, a reference control. (C, D) The expression of epithelial marker molecules E-cadherin (red fluorescence) and ZO-1 (green fluorescence) was monitored by immunofluorescence staining using the corresponding antibodies. The nuclear location was confirmed by DAPI staining.TE, ethanol extract of Tagetes erecta Linn flower; PM2.5, particulate matter 2.5; EMT, mesenchymal transition; α-SMA, α-smooth muscle actin; MMP, matrix metalloproteinase; ZO-1, Zonula occludens-1; DAPI, 4’6-diamidino-2-phenylindole; IFN, interferon; IL, interleukin.***P < 0.001 vs. control; #P < 0.05 and ###P < 0.001 vs. PM2.5.

• 

  Fig. 6 Effect of TE on TGF-β/Smad/MAPKs signaling activated by PM2.5 in ARPE-19 cells. Cells were treated with 5 μg/mL TE for 1 h before treatment with 25 μg/mL PM2.5 for 24 h. (A) TGF-β1 levels in cell supernatants cultured under different conditions were quantified using an ELISA kit. (B, D) After treatment, Western blot analysis was performed using the proteins extracted from cells and antibodies corresponding to the proteins to be analyzed. (C, E) Expression of each protein was quantified and normalized to actin, a reference control.TGF-β, transforming growth factor-β; TE, ethanol extract of Tagetes erecta Linn flower; PM2.5, particulate matter 2.5; Smad, smooth muscle actin; MAPK, mitogen-activated protein kinase; ELISA, enzyme-linked immunosorbent assay; JNK, c-Jun N-terminal kinase; ERK, extracellular signal regulated kinase.***P < 0.001 vs. control; ##P < 0.01 and ###P < 0.001 vs. PM2.5.

• 

  Fig. 7 Schematic diagram indicating that TE prevents PM2.5-triggered mitochondrial superoxide production and mitochondrial dysfunction leading to TGF-β/Smad/MAPKs pathway-mediated EMT in human RPE ARPE-19 cells.PM2.5, particulate matter 2.5; TGF-β, transforming growth factor-β; TE, ethanol extract of Tagetes erecta Linn flower; Smad, smooth muscle actin; MAPK, mitogen-activated protein kinase; EMT, epithelial-mesenchymal transition; RPE, retinal pigment epithelial; TGF-βR, transforming growth factor-β receptor; ROS, reactive oxygen species; Smad, suppressor of mothers against decapentaplegic; ERK, extracellular signal regulated kinase; JNK, c-Jun N-terminal kinase; IFN, interferon; IL, interleukin; ZO-1, Zonula occludens-1; MMP, matrix metalloproteinase; α-SMA, α-smooth muscle actin.

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