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

Beom Su Park; EunJin Bang; Hyesook Lee; Gi-Young Kim; Yung Hyun Choi

doi:10.4162/nrp.2025.19.2.170

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Park, Bang, Lee, Kim, and Choi: 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

Original Research

Nutrition Research and Practice 2025; 19(2): 170-185.

Published online: 4 December 2024

DOI: https://doi.org/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

Beom Su Park^1,^2,^*

, EunJin Bang^1,^2,^*

, Hyesook Lee³

, Gi-Young Kim⁴

, Yung Hyun Choi^1,²

¹Basic Research Laboratory for the Regulation of Microplastic-Mediated Diseases and Anti-Aging Research Center, Dong-eui University, Busan 47227, Korea.

²Department of Biochemistry, College of Korean Medicine, Dong-eui University, Busan 47227, Korea.

³Department of Convergence Medicine, Pusan National University School of Medicine, Yangsan 50612, Korea.

⁴Laboratory of Immunobiology, Department of Marine Life Sciences, Jeju National University, Jeju 63243, Korea.

Corresponding Author: Yung Hyun Choi. Department of Biochemistry, College of Korean Medicine, Dong-eui University, 52-57 Yangjeong-ro, Busanjin-gu, Busan 47227, Korea. Tel. +82-51-890-3319, Fax. +82-51-890-3333, choiyh@deu.ac.kr

Equal contributor:

^*Beom Su Park and EunJin Bang contributed equally to this work.

Received 4 September 2024 Revised 15 October 2024 Accepted 12 November 2024

The Korean Nutrition Society and the Korean Society of Community Nutrition

https://creativecommons.org/licenses/by-nc/4.0/

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

BACKGROUND/OBJECTIVES

Particulate matter 2.5 (PM2.5) exposure can promote epithelial-mesenchymal 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.

Keywords: Particulate matter, retinal pigment epitheliums, reactive oxygen species, epithelial-mesenchymal transition, transforming growth factors

INTRODUCTION

Air pollution is widely recognized as a significant global threat to human health. Particulate matter (PM) is a critical component of air pollution and is commonly defined by particle size. The smaller the particle size, the greater the health impact. Particulate matter 2.5 (PM2.5) is fine PM with an aerodynamic diameter < 2.5 μm and could potentially harm human health [1]. Generally, PM2.5 is highly absorbed by the respiratory system and reaches the bloodstream through the lung alveoli [2]. PM2.5 has the potential to cross various biological barriers and translocate into different organs, such as the brain, heart, and placenta [3 4]. Although the eyes are directly exposed to the external environment and air, studies on the effects of PM2.5 on the eyes remain limited. PM2.5 poses a potential risk for ocular conditions such as conjunctivitis and keratitis, along with associated symptoms involving eye irritation, inflammation, itching, tearing, and stinging [5 6]. The retinal pigment epithelium comprises a monolayer of pigmented cells that creates a barrier between the retina and blood and plays a critical role in maintaining visual function [7]. Although exposure to PM2.5 has been associated with ocular diseases, studies on their molecular mechanism on retinal pigment epithelial (RPE) cells is limited.

Epithelial-mesenchymal transition (EMT) in RPE cells is an important factor in development of proliferative retinal disorders, such as age-related macular degeneration [8 9]. EMT is a biological process that involves epithelial cells transitioning into mesenchymal-like characteristics, accompanied by elevated expression levels of myofibroblast marker molecules such as α-smooth muscle actin (α-SMA), fibronectin, and vimentin [10 11]. Generally, EMT is induced by impaired tight junctions, accumulation of misfolded proteins, and dysregulation of major signaling pathways, such as transforming growth factor (TGF)-β and β-catenin/Wnt signaling pathways in RPE cells [12 13]. In association with EMT, the extracellular matrix is remodeled by the expression of matrix metalloproteinase (MMP) molecules [14 15]. Particularly, TGF-β signaling is a canonical pathway that promotes EMT by activating various downstream effector proteins, including mitogen-activated protein kinases (MAPKs) and suppressor of mothers against decapentaplegic (Smad) proteins [16 17]. The conversion of epithelial to mesenchymal cells is a critical risk factor for developing vitreoretinal diseases, proliferative retinopathy, and proliferative diabetic diseases [12]. Based on previous studies, PM2.5 exposure has been known as an inducer of EMT through phosphatidylinositol 3′-kinase/Akt/mammalian target of rapamycin pathway in RPE cells in vitro [18]. Furthermore, PM2.5 has been demonstrated to promote EMT in human RPE cells via reactive oxygen species (ROS) generation [19].

Tagetes erecta Linn, commonly referred to as marigold, is a member of the Asteraceae family that has been used in traditional herbal medicine for many years [20 21]. The dried flowers of this plant are widely consumed as tea in many parts of East Asia, including Korea. They contain antioxidant bioactive substances such as flavonoids, carotenoids, lutein, zeaxanthin, and vitamin A, which are known to prevent ocular diseases and improve vision loss [22 23]. Recently, we found that an ethanol extract of T. erecta Linn flower (TE) protected against dry eye syndrome caused by desiccation stress via stabilizing the tear film and suppressing inflammation [24]. In the current study, we explored the inhibitory effects of marigold flower extract on EMT induced by PM2.5 in RPE cells to reveal additional beneficial effects on eye diseases caused by harmful external environmental factors. We revealed that TE exhibited protective effects against PM2.5-induced ROS generation, mitochondrial impairment, and EMT in RPE cells at optimal concentrations. Therefore, TE can be used to prevent PM2.5-induced ocular EMT advancement and eye diseases associated with EMT, including age-related macular degeneration.

MATERIALS AND METHODS

Reagents of PM2.5

PM2.5 (Diesel exhaust PM2.5, NIST SRM 1650b) used in this study was obtained from Sigma-Aldrich (St. Louis, MO, USA). The constituents of diesel PM2.5 are black carbon, redox-active metals (e.g., Fe, Cu, Ni, Zn, Cr, As, and Mn), and polyaromatic hydrocarbons, including naphthalene, benzo-a-pyrene, and benzo-a-anthracene. PM2.5 was vortexed before cellular treatment to prevent particle aggregation [19].

Cell proliferation rate measurement

ARPE-19 cells, a human RPE cell line, were obtained from the American Type Culture Collection (Manassas, MD, USA) and grown in Dulbecco’s Modified Eagle’s Medium/F12 (WelGENE Inc., Gyeongsan, Korea) with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C in a 5% CO₂ incubator. Cell viability was measured using the Cell Counting Kit-8 (CCK-8) assay (Sigma-Aldrich) according to a previously described procedure [25].

Wound healing assay

A pipette tip of 200 μL was used to make a wound line. The cells were then treated with PM2.5 and incubated at different concentrations for 24 h. TE was applied for 1 h before exposure to PM2.5. Cells were photographed at 0 and 24 h using a microscope (Zeiss Oberkochen, Baden-Württemberg, Germany) installed at TRCORE, Dong-eui University (Busan, Korea).

Immunofluorescence

Cells were stimulated with PM2.5 treatment for 24 h with or without pretreatment with TE for 1 h. Subsequently, using a previously described method [26], cells were fixed, washed and reacted with appropriate primary antibodies overnight at 4°C. After staining with fluorescence-labeled secondary antibodies for 1 h, the cells were mounted, and fluorescence images for each antibody were observed using an EVOS fluorescence microscope (Thermo Fisher Scientific, Waltham, MA, USA).

Western blot analysis

Cells were collected and disrupted using lysis buffer for 30 min at 4°C to extract proteins [27]. Following protein quantification, equal quantities were separated and transferred onto polyvinylidene difluoride membranes. The transferred membranes were blocked with 5% skim milk for 1 h and subsequently reacted with primary antibodies overnight at 4°C. After washing with PBST (phosphate-buffered saline with Tween 20), the membrane was probed with peroxidase-conjugated secondary antibodies for 1 h at room temperature. The membranes were developed using chemiluminescent reagents, and changes in protein expression in response to the corresponding antibodies were detected using the Fusion FX Image system (Vilber Lourmat, Torcy, France).

Measurement of ROS

Following a previously described experimental method [28], ROS measurement was conducted. To investigate the production of intracellular and mitochondrial ROS (mtROS), cells were dyed with 5,6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA, Invitrogen, Carlsbad, CA, USA) and MitoSOX™ Red (Thermo Fisher Scientific) for 20 min, respectively. After counterstaining the nuclei with 4’6-diamidino-2-phenylindole (DAPI), the fluorescence images of each dye were captured using an EVOS microscope system (Thermo Fisher Scientific).

Measurement of mitochondrial dysfunction

To evaluate mitochondria dysfunction, cells cultured under various conditions were stained with fluorescent MitoTracker™ Red (Cell Signaling Technology, Danvers, MA, USA), a cell-permeable reagent that labels mitochondria in living cells. Additionally, alterations in mitochondrial membrane potential (ΔΨm) were investigated using 1,1′,3,3′-tetraethyl-5,5′,6,6′-tetrachloroimidacarbocyanine iodide (JC-1; Thermo Fisher Scientific), a cationic fluorescent carbocyanine dye, which accumulates in polarized mitochondria. Cells stained with each dye were observed under an EVOS microscope according to the procedures recommended by each manufacturer [29].

TGF-β level analysis

Supernatants were collected to analyze TGF-β levels in PM2.5-treated cells with or without TE. TGF-β1 levels were determined using the TGF-β1 enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer’s instructions.

Statistical analysis

Data were analyzed to confirm statistical significance using GraphPad Prism software (version 8.4.2; San Diego, CA, USA). Each cell group was analyzed using a one-way analysis of variance and Tukey’s post hoc test. Statistical significance was set at P < 0.05

RESULTS

TE inhibited cell damage caused by PM2.5

To examine the effects of TE and PM2.5 on the proliferation of ARPE-19 cells, a CCK-8 assay was conducted on ARPE-19 cells cultured for 24 h in medium containing varying treatment concentrations of TE and PM2.5. In the case of PM2.5 treatment, the cell proliferation rate was slightly reduced to 87.89 ± 3.39% in cells exposed to 25 μg/mL of PM2.5 compared with the control group (Fig. 1A). In ARPE-19 cells exposed to TE, cell viability was significantly inhibited in the group treated with ≥ 7.5 μg/mL of TE (Fig. 1B). Therefore, the concentration for inducing cytotoxicity by PM2.5 treatment was set at 25 μg/mL, and the concentration for evaluating the protective effect of TE against PM2.5 was set at 5 μg/mL. As presented in Fig. 1C, at a concentration of 5 μg/mL TE, the reduction in the cell proliferation rate induced by exposure to 25 μg/mL PM2.5 was notably restored. Additionally, in cells exposed to PM2.5, the accumulation of intracellular PM2.5 greatly increased, and the cells revealed a flat shape with an expanded cytoplasm, deviating from the typical epithelial cell shape (Fig. 1D). Although TE pretreatment did not prevent the accumulation of intracellular PM2.5 in ARPE-19 cells, the deformation of cell morphology was slightly alleviated.

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.

TE decreased PM2.5-stimulated cell migration

We investigated the link between the countervailing effect of TE on PM2.5-induced cytotoxicity and the suppression of cell migration, as PM2.5 was reported to promote the migration of ARPE-19 cells [18 19]. Based on the results of the wound healing assay, the migration of ARPE-19 cells exposed to PM2.5 toward the scratch area was increased, but under TE pretreatment conditions, PM2.5-induced cell mobility was significantly reduced (Fig. 2). These results suggest that the mitigation of PM2.5-induced suppression of ARPE-19 cell proliferation by TE is linked to a reduction in the rate of cell migration.

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.

TE reduced PM2.5-induced ROS production

PM2.5-promoted increase in cell motility is closely linked to ROS generation in various cell types [30 31 32]. Therefore, we investigated whether ROS generation was linked to the inhibitory effect of TE on the PM2.5-induced increase in the migration rate of ARPE-19 cells. DCF-DA staining was performed to detect intracellular ROS production. As indicated in Fig. 3A and B, DCF-DA fluorescence was markedly increased by PM2.5 treatment but greatly reduced in TE-pretreated cells. To determine whether there was an increase in ROS production following treatment with PM2.5, we performed MitoSOX Red staining, a mitochondrial superoxide indicator, and observed that the MitoSOX Red fluorescence intensity was markedly higher in PM2.5-exposed cells than in the control group (Fig. 3C and D). However, in cells exposed to PM2.5 after pretreatment with TE, the fluorescence intensity of MitoSOX Red was weaker than that in cells exposed to PM2.5 alone, suggesting that mitochondria may be a source of ROS generated by 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.

TE protected PM2.5-induced mitochondrial disruption

Subsequently, MitoTracker red dye, which labels active mitochondria in live cells, was used to ascertain whether PM2.5-induced mtROS generation was caused by impaired mitochondrial function. As depicted in Fig. 4A and B, MitoTracker red fluorescence greatly decreased in PM2.5-treated ARPE-19 cells. Additionally, changes in ΔΨm were investigated using the JC-1 probe, which is widely used to monitor mitochondrial health. Compared with the control group, in cells treated with PM2.5, red fluorescence emission, indicating aggregates of JC-1, decreased, whereas green fluorescence, indicating the monomeric form, increased (Fig. 4C and D). This decrease in the red/green fluorescence intensity ratio caused by PM2.5 indicates that mitochondrial depolarization has occurred. However, TE reversed the PM2.5-induced changes in the fluorescence intensity of MitoTracker and JC-1, suggesting that PM2.5-induced mtROS generation and mitochondrial damage were blocked by TE.

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.

TE attenuated PM2.5-induced EMT and inflammation

We investigated whether TE could inhibit PM2.5-triggered EMT as the increased mobility of various cell types, including ARPE-19 cells, induced by PM2.5, is linked to EMT induction mediated by oxidative stress [19 31 33]. As indicated in Fig. 5A and B, the expression of mesenchymal markers, including vimentin, α-SMA, MMP-2, and MMP-3, increased under PM2.5 exposure conditions at both transcriptional and translational levels, but intracellular expression of epithelial markers including E-cadherin and Zonula occludens-1 (ZO-1) decreased (Fig. 5C and D). These findings demonstrated that PM2.5 promoted EMT in ARPE-19 cells, but TE reversed PM2.5-induced EMT by interfering with all mesenchymal characteristics. Additionally, as observed in earlier studies [34 35 36], PM2.5 treatment increased the expression of inflammatory factors such as interferon-γ, interleukin (IL)-1β, IL-6, and IL-18, such that EMT induction by PM2.5 was accompanied by an inflammatory response; however, TE pretreatment restored the expression of these inflammatory markers (Fig. 5E and F), indicating that TE has antioxidant and anti-inflammatory effects.

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.

TE eliminated TGF-β/Smad/MAPKs signaling activated by PM2.5

Subsequently, we investigated the impact of TE on TGF-β signaling, which is crucial in triggering EMT by PM2.5 [19 34 35]. We evaluated the levels of TGF-β generated in ARPE-19 cells exposed to PM2.5 with or without TE using the TGF-β1 ELISA kit. As presented in Fig. 6A, secretion of TGF-β1 was notably elevated in cells treated with PM2.5; however, this level was significantly suppressed by TE pretreatment. Consistent with the results of ELISA, protein expression level of TGF-β was higher in PM2.5-exposed cells compared with the control group. However, TE significantly reduced this expression. TE also counteracted PM2.5-promoted upregulation of Snail and Slug, critical regulators of TGF-β-induced EMT (Fig. 6B and C). Additionally, as a result of examining changes in the activity of Smad and MAPKs, which are important factors in regulating EMT as TGF-β downstream signaling systems [37 38], phosphorylation of Smad2/3 and three main components of MAPKs, including p38, c-Jun N-terminal kinase and extracellular signal-regulated kinase, was increased by PM2.5 (Fig. 6B-E). However, their phosphorylation by PM2.5 was completely neutralized by TE, revealing that inhibition of PM2.5-induced EMT by TE was achieved by inactivation of TGF-β/Smad/MAPK signaling.

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.

DISCUSSION

Oxidative stress is a critical factor in the development of degenerative diseases in various organs, including the eyes [39 40]. Therefore, an oxidative stress scavenging strategy is an effective way to prevent ocular dysfunction. This is supported by the results of previous studies that have suggested that antioxidants can effectively reduce the incidence of retinal degeneration and various other eye diseases [41 42]. Numerous species of marigolds, members of the Asteraceae family, occur worldwide [43 44]. Diverse species of marigold have been used as herbal medicines to treat a range of conditions such as skin diseases, conjunctivitis and blurred vision, wounds and burns, inflammation, pain, and swelling [20 21 45]. The flower extracts or constituents of T. erecta Linn., a type of marigold, have also been reported to be beneficial to eye health [23 24 46], which may be owing to their antioxidant and anti-inflammatory properties [47 48]. In this study, we investigated the effects of T. erecta Linn flower extract in blocking PM2.5-induced damage to RPE cells.

Recent studies have established that exposure to PM2.5 is correlated with the development of EMT, which can cause various eye diseases [8 35]. The discovery that EMT is induced by PM2.5 in ARPE-19 cells, an RPE cell line, was based on the finding that PM2.5 enhances the expression levels of MMP-2 and MMP-9 in ARPE-19 cells, thereby enhancing their morbidity and migratory ability [18 19]. We treated ARPE-19 cells previously exposed to TE in the non-cytotoxic range with PM2.5 to investigate whether the cytotoxic effect of PM2.5 is decreased by TE. According to our results, TE significantly reduced cell mobility while alleviating the cytotoxic effects induced by PM2.5, which was associated with the suppression of MMP expression. PM2.5 was identified as a potent inducer of ROS from accumulated results. These PM particles directly increase ROS levels and promote redox reactions through direct emission or by participating in redox processes as reactants [49 50]. Metal ions such as are iron, copper and nickels are major constituents of PM2.5 and these metals are known to participate in Fenton reactions, generating ROS, which can cause lipid peroxidation, protein oxidation and DNA damage leading to cell death [51 52 53]. In addition, nickel can impair mitochondrial function leading to further ROS production [54]. And polycyclic aromatic hydrocarbons in PM2.5 could further contribute to cellular toxicity by forming DNA adducts, DNA mutations and DMA strands breaks leading to carcinogenesis [55]. The production of ROS induced by PM2.5 was reported to be linked to further mitochondrial damage, indicating that impaired mitochondria may contribute as a source of ROS generation [4 56 57]. Similar to previous studies, PM2.5 increased the production of intracellular ROS and mtROS while causing mitochondrial damage in ARPE-19 cells; however, TE significantly blocked these changes. These results suggest that TE preserved mitochondrial function in PM2.5-exposed ARPE-19 cells, thereby attenuating the cytotoxicity and cell motility associated with ROS production induced by PM2.5. Based on previous studies, flavonoids such as quercetin and kaempferol in TE exhibited scavenging activity for free radicals and chelating iron and copper, preventing further ROS production and demonstrated no significant cell cytotoxicity in dermal fibroblasts [58 59]. However, whether ROS generation by PM2.5 originates from damaged mitochondria and the exact mechanism by which TE suppresses it may be an interesting topic that requires further elucidation.

During EMT, epithelial cells exhibit a loss of cell-cell adhesion, cell polarity, and increased cell mobility. This increases cell motility and promotes the development of invasive phenotypes [60 61]. EMT is necessary for normal functions, such as embryonic development and wound healing; however, other functions of EMT have been linked to various pathological factors, including tumor metastasis and fibrosis [62 63]. Prior research has shown that EMT in RPE cells is the primary contributor to diverse intraocular fibrotic diseases. Ocular diseases, including age-related macular degeneration, diabetic retinopathy, and proliferative vitreoretinopathy, are also associated with EMT, which has a significant impact on visual impairment [8 64]. Increased MMP activity owing to decreased expression of tissue inhibitors of metalloproteases contributes to cell migration and invasion and is involved in the initiation of EMT; hence, they are recognized as mesenchymal markers along with α-SMA and vimentin [65 66]. Additionally, as chronic inflammation can act as an initiating and enhancing factor for PM-induced EMT [33 35]; therefore, attenuation of the inflammatory response may be a strategy for inhibiting EMT, along with antioxidant activity. In ARPE-19 cells, PM2.5 induces EMT by increasing the inflammatory response and expression of mesenchymal markers and reducing the expression of epithelial markers such as E-cadherin and ZO-1 [18 19]. In this study, TE prevented the PM2.5-induced increase in the expression of mesenchymal markers and various inflammatory cytokines. In contrast, in the presence of TE, PM2.5 did not increase the expression of epithelial markers, implying that TE blocked the expression of factors involved in EMT induction in ARPE-19 cells exposed to PM2.5.

ROS-mediated TGF-β/Smad signaling was an important pathway regulating PM2.5-induced EMT-related gene expression [19 35 67]. When the TGF-β receptor on the cell membrane forms a complex with TGF-β and is activated, Smad2 and Smad3 are phosphorylated and translocated to the nucleus. Activated Smad2 and Smad3 function as transcriptional regulators by binding and cooperating with the nuclear transcription factors Slug and Snail to regulate the transcriptional activity of EMT-associated target genes [16 68]. During EMT, TGFβ signaling also crosstalks with the MAPK pathway, as TGF-β downstream signaling [37 38]; moreover, they are activated in a ROS-mediated mechanism during PM2.5-promoted EMT of ARPE-19 cells [19]. In this study, PM2.5-induced TGF-β secretion and expression were significantly inhibited by TE pretreatment, increased expression of Slug and Snail, and phosphorylation of Smad2/3 was attenuated in the presence of TE. Furthermore, TE counteracted the activation of MAPKs by PM2.5. These results support that TE interfered with PM2.5-induced EMT by blocking TGF-β/Smad/MAPK signaling. Moreover, in various cell lines, including RPE cells, pathological damage such as apoptosis, necrosis, and autophagy, as well as PM2.5-induced TGF-β-mediated EMT, were typically ROS-dependent phenomena [19 31 33]. Moreover, it was revealed that the generation of ROS following PM2.5 treatment may be responsible for the increase in mtROS owing to mitochondrial impairment [32 55 69 70]. These findings suggest that the scavenging activity of TE against the generation of intracellular ROS and mtROS may have contributed to the inhibition of EMT in PM2.5-treated ARPE-19 cells. Based on our results, we speculate that TE eliminated TGF-β-induced EMT by suppressing mitochondrial dysfunction and ROS production triggered by PM2.5 in ARPE-19 cells (Fig. 7). Therefore, we propose that TE can be used to prevent or treat EMT-related ocular diseases caused by microenvironmental hazards such as PM2.5. However, additional research should be conducted to determine the components of TE contributing to blocking PM2.5-induced EMT and confirm its efficacy in an in vivo model.

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.

In conclusion, we observed that TE effectively attenuated the PM2.5-induced TGF-β-mediated EMT process by blocking mitochondrial dysfunction and ROS production in ARPE-19 cells. The reduction in PM2.5-induced intracellular ROS and mitochondrial superoxide production by TE was associated with preserving mitochondrial function. Additionally, we confirmed that TE attenuated PM2.5-induced inflammatory response, changes in mesenchymal and epithelial marker expression, and TGF-β/Smad/MAPKs signaling activity in ARPE-19 cells. Our results provide important evidence that TE can potentially prevent or treat PM2.5-promoted retinal dysfunction.

Notes

Funding: This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) grant funded by the Korea government (2021R1A2C2009549).

Conflict of Interest: The authors declare no potential conflicts of interests.

Author Contributions:

Conceptualization: Park BS, Bang EJ, Lee H, Kim GY, Choi YH.
Data curation: Bang EJ, Lee H.
Formal analysis: Park BS, Lee H.
Investigation: Park BS, Bang EJ, Lee H.
Methodology: Park BS, Bang EJ, Lee H.
Project administration: Kim GY, Choi YH.
Supervision: Choi YH.
Writing - original draft: Park BS, Bang EJ Kim GY, Choi YH.
Writing - review & editing: Park BS Kim GY, Choi YH.

References

1. Yang Y, Ruan Z, Wang X, Yang Y, Mason TG, Lin H, Tian L. Short-term and long-term exposures to fine particulate matter constituents and health: a systematic review and meta-analysis. Environ Pollut. 2019; 247:874–882. PMID: 30731313.

2. Yang WK, Lyu YR, Kim SH, Chae SW, Kim KM, Jung IC, Park YC. Protective effect of GHX02 extract on particulate matter-induced lung injury. J Med Food. 2020; 23:611–632. PMID: 32316823.

3. Wang J, Li M, Geng Z, Khattak S, Ji X, Wu D, Dang Y. Role of oxidative stress in retinal disease and the early intervention strategies: a review. Oxid Med Cell Longev. 2022; 2022:7836828. PMID: 36275903.

4. Li Z, Tian F, Ban H, Xia S, Cheng L, Ren X, Lyu Y, Zheng J. Energy metabolism disorders and oxidative stress in the SH-SY5Y cells following PM_2.5 air pollution exposure. Toxicol Lett. 2022; 369:25–33. PMID: 36007723.

5. Somayajulu M, Ekanayaka S, McClellan SA, Bessert D, Pitchaikannu A, Zhang K, Hazlett LD. Airborne particulates affect corneal homeostasis and immunity. Invest Ophthalmol Vis Sci. 2020; 61:23.

6. Zhu S, Gong L, Li Y, Xu H, Gu Z, Zhao Y. Safety assessment of nanomaterials to eyes: an important but neglected issue. Adv Sci (Weinh). 2019; 6:1802289. PMID: 31453052.

7. Yumnamcha T, Devi TS, Singh LP. Auranofin mediates mitochondrial dysregulation and inflammatory cell death in human retinal pigment epithelial cells: Implications of retinal neurodegenerative diseases. Front Neurosci. 2019; 13:1065. PMID: 31649499.

8. Gelat B, Rathaur P, Malaviya P, Patel B, Trivedi K, Johar K, Gelat R. The intervention of epithelial-mesenchymal transition in homeostasis of human retinal pigment epithelial cells: a review. J Histotechnol. 2022; 45:148–160. PMID: 36377481.

9. Shu DY, Butcher E, Saint-Geniez M. EMT and EndMT: emerging roles in age-related macular degeneration. Int J Mol Sci. 2020; 21:4271. PMID: 32560057.

10. Li L, Li H, Zhang Z, Zheng J, Shi Y, Liu J, Cao Y, Yuan X, Chu Y. Recombinant truncated TGF-β receptor II attenuates carbon tetrachloride-induced epithelial-mesenchymal transition and liver fibrosis in rats. Mol Med Rep. 2018; 17:315–321. PMID: 29115426.

11. Scanlon CS, Van Tubergen EA, Inglehart RC, D’Silva NJ. Biomarkers of epithelial-mesenchymal transition in squamous cell carcinoma. J Dent Res. 2013; 92:114–121. PMID: 23128109.

12. Zhou M, Geathers JS, Grillo SL, Weber SR, Wang W, Zhao Y, Sundstrom JM. Role of epithelial-mesenchymal transition in retinal pigment epithelium dysfunction. Front Cell Dev Biol. 2020; 8:501. PMID: 32671066.

13. Tamiya S, Kaplan HJ. Role of epithelial-mesenchymal transition in proliferative vitreoretinopathy. Exp Eye Res. 2016; 142:26–31. PMID: 26675400.

14. Caban M, Owczarek K, Lewandowska U. The role of metalloproteinases and their tissue inhibitors on ocular diseases: focusing on potential mechanisms. Int J Mol Sci. 2022; 23:4256. PMID: 35457074.

15. Saucedo L, Pfister IB, Zandi S, Gerhardt C, Garweg JG. Ocular TGF-b, matrix metalloproteinases, and TIMP-1 increase with the development and progression of diabetic retinopathy in type 2 diabetes mellitus. Mediators Inflamm. 2021; 2021:9811361. PMID: 34257518.

16. Runa F, Ortiz-Soto G, de Barros NR, Kelber JA. Targeting SMAD-dependent signaling: considerations in epithelial and mesenchymal solid tumors. Pharmaceuticals (Basel). 2024; 17:326. PMID: 38543112.

17. Nady ME, Abd El-Raouf OM, El-Sayed EM. Linagliptin mitigates TGF-β1 mediated epithelial-mesenchymal transition in tacrolimus-induced renal interstitial fibrosis via Smad/ERK/P38 and HIF-1a/LOXL2 signaling pathways. Biol Pharm Bull. 2024; 47:1008–1020. PMID: 38797693.

18. Lin HW, Shen TJ, Chen PY, Chen TC, Yeh JH, Tsou SC, Lai CY, Chen CH, Chang YY. Particulate matter 2.5 exposure induces epithelial-mesenchymal transition via PI3K/AKT/mTOR pathway in human retinal pigment epithelial ARPE-19 cells. Biochem Biophys Res Commun. 2022; 617:11–17. PMID: 35689837.

19. Lee H, Hwang-Bo H, Ji SY, Kim MY, Kim SY, Park C, Hong SH, Kim GY, Song KS, Hyun JW, et al. Diesel particulate matter2.5 promotes epithelial-mesenchymal transition of human retinal pigment epithelial cells via generation of reactive oxygen species. Environ Pollut. 2020; 262:114301. PMID: 32155554.

20. Kusman IT, Pradini GW, Ma’ruf IF, Fauziah N, Berbudi A, Achadiyani A, Wiraswati HL. The potentials of Ageratum conyzoides and other plants from Asteraceae as an antiplasmodial and insecticidal for malaria vector: an article review. Infect Drug Resist. 2023; 16:7109–7138. PMID: 37954507.

21. Salehi B, Valussi M, Morais-Braga MF, Carneiro JN, Leal AL, Coutinho HD, Vitalini S, Kręgiel D, Antolak H, Sharifi-Rad M, et al. Tagetes spp. essential oils and other extracts: chemical characterization and biological activity. Molecules. 2018; 23:2847. PMID: 30388858.

22. Sanjaya SS, Park MH, Karunarathne WA, Lee KT, Choi YH, Kang CH, Lee MH, Jung MJ, Ryu HW, Kim GY. Inhibition of α-melanocyte-stimulating hormone-induced melanogenesis and molecular mechanisms by polyphenol-enriched fraction of Tagetes erecta L. flower. Phytomedicine. 2024; 126:155442. PMID: 38394730.

23. Wang X, Liu X, Wang X, Wang H, Zhang LH, Yu H, Yang W, Wu HH. Carotenoid-derived norsesquiterpenoids and sesquiterpenoids from Tagetes erecta L. Phytochemistry. 2023; 215:113860. PMID: 37714249.

24. Lee H, Hwangbo H, Hyun JW, Shim JH, Leem SH, Kim GY, Choi YH. Ameliorative effects of Tagetes erecta Linn. flower against desiccation stress-induced dry eye symptoms in the mice model. Integr Med Res. 2024; 13:101038. PMID: 38716164.

25. Gao P, Duan W, Shi H, Wang Q. Silencing circPalm2 inhibits sepsis-induced acute lung injury by sponging miR-376b-3p and targeting MAP3K1. Toxicol Res. 2023; 39:275–294. PMID: 37008689.

26. Karima G, Shin K, Jeong J, Choi D, Hwang KG, Hong JW. Stem cell oriented exosomes regulate cell proliferation in hepatoma carcinoma. Biotechnol Bioprocess Eng; BBE. 2023; 28:263–273.

27. Jeon SJ, Jung GH, Choi EY, Han EJ, Lee JH, Han SH, Woo JS, Jung SH, Jung JY. Kaempferol induces apoptosis through the MAPK pathway and regulates JNK-mediated autophagy in MC-3 cells. Toxicol Res. 2023; 40:45–55. PMID: 38223666.

28. Hwangbo H, Park C, Bang E, Kim HS, Bae SJ, Kim E, Jung Y, Leem SH, Seo YR, Hong SH, et al. Morroniside protects C2C12 myoblasts from oxidative damage caused by ROS-mediated mitochondrial damage and induction of endoplasmic reticulum Stress. Biomol Ther (Seoul). 2024; 32:349–360. PMID: 38602043.

29. Park C, Kim DH, Kim TH, Jeong SU, Yoon JH, Moon SK, Kwon CY, Park SH, Hong SH, Shim JH, et al. Improvement of oxidative stress-induced cytotoxicity of Angelica keiskei (Miq.) Koidz. leaves extract through activation of heme oxygenase-1 in C2C12 murine myoblasts. Biotechnol Bioprocess Eng; BBE. 2023; 28:51–62.

30. Nguyen UT, Hsieh HY, Chin TY, Wu G, Lin YP, Lee CY, Hsu YC, Fan YJ. Evaluation of Pm2.5 influence on human lung cancer cells using a microfluidic platform. Int J Med Sci. 2024; 21:1117–1128. PMID: 38774761.

31. Zhang J, Xu X, Liang Y, Wu X, Qian Z, Zhang L, Wang T. Particulate matter promotes the epithelial to mesenchymal transition in human lung epithelial cells via the ROS pathway. Am J Transl Res. 2023; 15:5159–5167. PMID: 37692935.

32. Shin TH, Kim SG, Ji M, Kwon DH, Hwang JS, George NP, Ergando DS, Park CB, Paik MJ, Lee G. Diesel-derived PM_2.5 induces impairment of cardiac movement followed by mitochondria dysfunction in cardiomyocytes. Front Endocrinol (Lausanne). 2022; 13:999475. PMID: 36246901.

33. Molavinia S, Dayer D, Khodayar MJ, Goudarzi G, Salehcheh M. Suspended particulate matter promotes epithelial-to-mesenchymal transition in alveolar epithelial cells via TGF-β1-mediated ROS/IL-8/SMAD3 axis. J Environ Sci (China). 2024; 141:139–150. PMID: 38408815.

34. Li J, Zeng G, Zhang Z, Wang Y, Shao M, Li C, Lu Z, Zhao Y, Zhang F, Ding W. Urban airborne PM_2.5 induces pulmonary fibrosis through triggering glycolysis and subsequent modification of histone lactylation in macrophages. Ecotoxicol Environ Saf. 2024; 273:116162. PMID: 38458067.

35. Liu D, Zhang C, Zhang J, Xu GT, Zhang J. Molecular pathogenesis of subretinal fibrosis in neovascular AMD focusing on epithelial-mesenchymal transformation of retinal pigment epithelium. Neurobiol Dis. 2023; 185:106250. PMID: 37536385.

36. He X, Zhang L, Hu L, Liu S, Xiong A, Wang J, Xiong Y, Li G. PM2.5 aggravated OVA-induced epithelial tight junction disruption through Fas associated via death domain-dependent apoptosis in asthmatic mice. J Asthma Allergy. 2021; 14:1411–1423. PMID: 34848976.

37. Lee JH, Massagué J. TGF-β in developmental and fibrogenic EMTs. Semin Cancer Biol. 2022; 86:136–145. PMID: 36183999.

38. Avila-Carrasco L, Majano P, Sánchez-Toméro JA, Selgas R, López-Cabrera M, Aguilera A, González Mateo G. Natural plants compounds as modulators of epithelial-to-mesenchymal transition. Front Pharmacol. 2019; 10:715. PMID: 31417401.

39. Markitantova Y, Simirskii V. Endogenous and exogenous regulation of redox homeostasis in retinal pigment epithelium cells: an updated antioxidant perspective. Int J Mol Sci. 2023; 24:10776. PMID: 37445953.

40. Hsueh YJ, Chen YN, Tsao YT, Cheng CM, Wu WC, Chen HC. The pathomechanism, antioxidant biomarkers, and treatment of oxidative stress-related eye diseases. Int J Mol Sci. 2022; 23:1255. PMID: 35163178.

41. Maurya M, Bora K, Blomfield AK, Pavlovich MC, Huang S, Liu CH, Chen J. Oxidative stress in retinal pigment epithelium degeneration: from pathogenesis to therapeutic targets in dry age-related macular degeneration. Neural Regen Res. 2023; 18:2173–2181. PMID: 37056126.

42. Buonfiglio F, Böhm EW, Pfeiffer N, Gericke A. Oxidative stress: a suitable therapeutic target for optic nerve diseases? Antioxidants. 2023; 12:1465. PMID: 37508003.

43. Cicevan R, Sestras AF, Plazas M, Boscaiu M, Vilanova S, Gramazio P, Vicente O, Prohens J, Sestras RE. Biological traits and genetic relationships amongst cultivars of three species of Tagetes (Asteraceae). Plants. 2022; 11:760. PMID: 35336643.

44. Garcia-Oliveira P, Barral M, Carpena M, Gullón P, Fraga-Corral M, Otero P, Prieto MA, Simal-Gandara J. Traditional plants from Asteraceae family as potential candidates for functional food industry. Food Funct. 2021; 12:2850–2873. PMID: 33683253.

45. Shahane K, Kshirsagar M, Tambe S, Jain D, Rout S, Ferreira MK, Mali S, Amin P, Srivastav PP, Cruz J, et al. An updated review on the multifaceted therapeutic potential of Calendula officinalis L. Pharmaceuticals (Basel). 2023; 16:611. PMID: 37111369.

46. Madhavan J, Chandrasekharan S, Priya MK, Godavarthi A. Modulatory effect of carotenoid supplement constituting lutein and zeaxanthin (10:1) on anti-oxidant enzymes and macular pigments level in rats. Pharmacogn Mag. 2018; 14:268–274. PMID: 29720843.

47. Rivas-García L, Crespo-Antolín L, Forbes-Hernández TY, Romero-Márquez JM, Navarro-Hortal MD, Arredondo M, Llopis J, Quiles JL, Sánchez-González C. Bioactive properties of Tagetes erecta edible flowers: polyphenol and antioxidant characterization and therapeutic activity against ovarian tumoral cells and Caenorhabditis elegans tauopathy. Int J Mol Sci. 2023; 25:280. PMID: 38203451.

48. Meurer MC, Mees M, Mariano LN, Boeing T, Somensi LB, Mariott M, da Silva RC, Dos Santos AC, Longo B, Santos França TC, et al. Hydroalcoholic extract of Tagetes erecta L. flowers, rich in the carotenoid lutein, attenuates inflammatory cytokine secretion and improves the oxidative stress in an animal model of ulcerative colitis. Nutr Res. 2019; 66:95–106. PMID: 30979660.

49. Yan R, Ma D, Liu Y, Wang R, Fan L, Yan Q, Chen C, Wang W, Ren Z, Ku T, et al. Developmental toxicity of fine particulate matter: multifaceted exploration from epidemiological and laboratory perspectives. Toxics. 2024; 12:274. PMID: 38668497.

50. Garcia A, Santa-Helena E, De Falco A, de Paula Ribeiro J, Gioda A, Gioda CR. Toxicological effects of fine particulate matter (PM2.5): health risks and associated systemic injuries-systematic review. Water Air Soil Pollut. 2023; 234:346. PMID: 37250231.

51. Zhou Z, Zhang YY, Xin R, Huang XH, Li YL, Dong X, Zhou D, Zhu B, Qin L. Metal ion-mediated pro-oxidative reactions of different lipid molecules: revealed by nontargeted lipidomic approaches. J Agric Food Chem. 2022; 70:10284–10295. PMID: 35944096.

52. Qiao Y, Ma L. Quantification of metal ion induced DNA damage with single cell array based assay. Analyst (Lond). 2013; 138:5713–5718. PMID: 23892322.

53. Bridgewater JD, Lim J, Vachet RW. Using metal-catalyzed oxidation reactions and mass spectrometry to identify amino acid residues within 10 A of the metal in Cu-binding proteins. J Am Soc Mass Spectrom. 2006; 17:1552–1559. PMID: 16872838.

54. Yin H, Li X, Wang C, Li X, Liu J. Nickel induces mitochondrial damage in renal cells in vitro and in vivo through its effects on mitochondrial biogenesis, fusion, and fission. Chem Biol Interact. 2024; 394:110975. PMID: 38552765.

55. Das DN, Ravi N. Influences of polycyclic aromatic hydrocarbon on the epigenome toxicity and its applicability in human health risk assessment. Environ Res. 2022; 213:113677. PMID: 35714684.

56. Kim DH, Lee H, Hwangbo H, Kim SY, Ji SY, Kim MY, Park SK, Park SH, Kim MY, Kim GY, et al. Particulate matter 2.5 promotes inflammation and cellular dysfunction via reactive oxygen species/p38 MAPK pathway in primary rat corneal epithelial cells. Cutan Ocul Toxicol. 2022; 41:273–284. PMID: 36097682.

57. Wang B, Chan YL, Li G, Ho KF, Anwer AG, Smith BJ, Guo H, Jalaludin B, Herbert C, Thomas PS, et al. Maternal particulate matter exposure impairs lung health and is associated with mitochondrial damage. Antioxidants. 2021; 10:1029. PMID: 34202305.

58. Huang X, Gao W, Yun X, Qing Z, Zeng J. Effect of natural antioxidants from marigolds (Tagetes erecta L.) on the oxidative stability of soybean oil. Molecules. 2022; 27:2865. PMID: 35566214.

59. Burlec AF, Pecio Ł, Kozachok S, Mircea C, Corciovă A, Vereștiuc L, Cioancă O, Oleszek W, Hăncianu M. Phytochemical profile, antioxidant activity, and cytotoxicity assessment of tagetes erecta L. flowers. Molecules. 2021; 26:1201. PMID: 33668106.

60. Fujisaki H, Futaki S. Epithelial-mesenchymal transition induced in cancer cells by adhesion to type I collagen. Int J Mol Sci. 2022; 24:198. PMID: 36613638.

61. Amack JD. Cellular dynamics of EMT: lessons from live in vivo imaging of embryonic development. Cell Commun Signal. 2021; 19:79. PMID: 34294089.

62. Blake B, Ozdemir T. Developing fibrous biomaterials to modulate epithelial-to-mesenchymal transition. Cells Tissues Organs. 2023; 212:416–438. PMID: 37071982.

63. Jayachandran J, Srinivasan H, Mani KP. Molecular mechanism involved in epithelial to mesenchymal transition. Arch Biochem Biophys. 2021; 710:108984. PMID: 34252392.

64. Shu DY, Chaudhary S, Cho KS, Lennikov A, Miller WP, Thorn DC, Yang M, McKay TB. Role of oxidative stress in ocular diseases: a balancing act. Metabolites. 2023; 13:187. PMID: 36837806.

65. Agraval H, Kandhari K, Yadav UC. MMPs as potential molecular targets in epithelial-to-mesenchymal transition driven COPD progression. Life Sci. 2024; 352:122874. PMID: 38942362.

66. Sarrand J, Soyfoo MS. Involvement of epithelial-mesenchymal transition (EMT) in autoimmune diseases. Int J Mol Sci. 2023; 24:14481. PMID: 37833928.

67. Xu Z, Ding W, Deng X. PM2.5, Fine particulate matter: a novel player in the epithelial-mesenchymal transition? Front Physiol. 2019; 10:1404. PMID: 31849690.

68. Hao Y, Baker D, Ten Dijke P. TGF-b-mediated epithelial-mesenchymal transition and cancer metastasis. Int J Mol Sci. 2019; 20:2767. PMID: 31195692.

69. Ding S, Jiang J, Zhang G, Yu M, Zheng Y. Ambient particulate matter exposure plus chronic ethanol ingestion exacerbates hepatic fibrosis by triggering the mitochondrial ROS-ferroptosis signaling pathway in mice. Ecotoxicol Environ Saf. 2023; 256:114897. PMID: 37043943.

70. Yu D, Cai W, Shen T, Wu Y, Ren C, Li T, Hu C, Zhu M, Yu J. PM_2.5 exposure increases dry eye disease risks through corneal epithelial inflammation and mitochondrial dysfunctions. Cell Biol Toxicol. 2023; 39:2615–2630. PMID: 36786954.

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