Infigratinib

Plumbagin Inhibits Proliferation, Migration, and Invasion of Retinal Pigment Epithelial Cells Induced by FGF-2

Yang Zhang a, Ri Wang c, He Zhang b, Liya Liu b, Jianbin An b, Jun Hao d, Jingxue Ma b,*
a Department of Ophthalmology, The second Hospital of Hebei Medical University, Shijiazhuang, 050000, China b Department of Ophthalmology, The second Hospital of Hebei Medical University, Shijiazhuang, 05000, China c College of Integrated Chinese and Western Medicine, Hebei Medical University, Shijiazhuang, 05000, China
d Department of Pathology, Hebei Medical University, Shijiazhuang, 05000, China

A B S T R A C T

Proliferative vitreoretinopathy (PVR) is a serious ophthalmic disease and characterized by the formation of proliferative membranes by retinal pigment epithelial (RPE) cells. In PVR, the contraction and traction of the fibrocellular membranes cause retinal detachment, which can cause reduction surgery for retinal detachment to fail. Fibroblast growth factor-2 (FGF-2) causes RPE cells to form extracellular matriX (ECM), promotes chemo- taxis, mitosis, and positively promotes the disease process of PVR. Plumbagin (PLB) is a plant small molecule naphthoquinone compound. It has the functions in anti-tumor, anti-inflammatory, inhibit proliferation. We tried to investigate the possible effects of PLB on the biological behavior of ARPE-19 cells induced by FGF-2 and its underlying mechanisms. Our study confirmed that proliferation, migration, and invasion of ARPE-19 cells induced by FGF-2 (10 ng/ml) were significantly inhibited by PLB. PLB also significantly inhibits the expression of MMP-2/-9, collagen I Alpha 1 (Col1A1), collagen IV Alpha 1 (Col4A1), collagen VI Alpha 1 (Col6A1), and the phosphorylation of FGF receptor (FGFR)-1, FGFR-2, ERK, p38, JNK of FGF-2-induced ARPE-19 cells. In summary, PLB inhibits FGF-2-stimulated proliferation, migration, and invasion of ARPE-19 cells, which may take place through inhibiting the expression of MMP-2/-9, Col1A1, Col4A1, Col6A1, and the mitogen-activated protein kinase (MAPK) pathway. PLB may have a preventive effect on proliferation, migration, and invasion of FGF-2- induced ARPE-19 cells.

Keywords: Plumbagin RPE
Migration Invasion Proliferation Proliferative vitreoretinopathy

1. Introduction

PVR is a disease characterized by the formation of fibrocellular membranes on the anterior and posterior surfaces of the retina and in the vitreous body (The Retina Society Terminology Committee, 1983). Some epiretinal membranes may contract and undergo traction which can cause retinal detachment. PVR is commonly observed upon rheg- matogenous retinal detachment, severe ocular trauma, and after retinal repair surgery (Pennock et al., 2014). Epiretinal membranes can be secondary (Iannetti et al., 2011; Oberstein et al., 2011) or idiopathic (Snead et al., 2008; Tosi et al., 2020). Epiretinal membranes are also commonly found in other ocular diseases, such as hypertensive reti- nopathy (Iannetti et al., 2011; Oberstein et al., 2011). The pathogenesis of PVR is complex and may involve many kinds of cells. RPE cells may be an important cell type that affects the progression of the disease (Chiba, 2014). Under physiological conditions, the retinal pigment epithelium resembles a typical paving stone. It is rich in pigment. The cells form a stable single-cell layer through intercellular junctions, help photore- ceptor cells complete their function, and recycle visual pigments. When retinal detachment occurs, RPE cells are exposed to various kinds of cytokines, possibly because of damage to the blood-retinal barrier (Pastor et al., 2002). Under these conditions, RPE cells may undergo pathological changes, such as proliferation, migration, and EMT, and become involved in the formation of PVR membranes (Pastor et al., 2016). In this process, growth factors have been proved to have positive effects on the formation of fibrocellular membranes (Ciprian, 2015; Pennock et al., 2014).
FGF-2 is one of the key factors to promote RPE cell proliferation and migration. It causes chemotaxis and can promote the mitosis of RPE cells and regulate the formation of ECM (Hoffmann et al., 2005; Pastor et al., 2016). Chen et al. (Chen et al., 2012) investigated the effects of epidermal growth factor (EGF), FGF-2, and transforming growth factor-β1 (TGF-β1) on the proliferation of ARPE-19 cells (He et al., 2017). They found that EGF and FGF-2 may promote cell proliferation and epithelial-mesenchymal transition (EMT) through the Wnt/β-catenin signaling pathway. In contrast, TGF-β1 inhibits cell proliferation.
Hoffmann et al. (Hoffmann et al., 2005) found that FGF-2 and PDGF-BB promoted proliferation of human RPE cells, which may have been partially caused by the up-regulation of integrin receptors αVβ3 and αVβ5, which triggers the cell signaling pathway. Integrin and growth factors act synergistically on the cell signaling pathway to promote the proliferation of RPE cells (Clark and Brugge, 1995). In this study, we used FGF-2 to induce RPE cells in order to establish a pathological environment mimicking the development of PVR.
Currently, mainstream clinical treatment of PVR is still vitreous retinal surgery, and surgical treatment is still not ideal. Hence, the development of new safe and effective drugs for treatment of PVR is a looming. PLB is likely to be an effective treatment for this disease. It is a small -molecule naphthoquinone compound from plants and is also a vitamin K3 analogue (Rajalakshmi et al., 2018; Sharma et al., 1991). It can be extracted from the roots of Plumbagenaeace, Ebenceae, and Dro- seraceae (Liu et al., 2017). PLB has many pharmacological activities, such as anti-tumor, anti-inflammatory, anti-fungal, anti-parasite, and nerve protective effects (Liu et al., 2017; Tripathi et al., 2019). The mechanism underlying its anti-tumoral properties may be related to inhibition of cell proliferation, migration, and promotion of autophagy (Wang et al., 2015; Yan et al., 2013), and it has no obvious toXic effects or side effects. Our team has shown that PLB inhibits epithelial mesen- chymal transformation in rabbit RPE cells (Chen et al., 2018a). It in- hibits ARPE-19 cell proliferation, possibly by blocking the cell cycle in G2/M phase and down-regulating PI3K and p38MAPK (Chen et al., 2018b). However, because the experiment did not use growth factors to induce RPE cells, conditions mimicking more closely what occurs in real PVR could not be assessed at that time.
For these reasons, we explored whether PLB can inhibit the patho- logical processes of RPE cells induced by FGF-2 and investigated the effects and associated signaling pathways to develop valuable ideas for treating patients with PVR.

2. Materials and methods

2.1. Cell culture and treatment

ARPE-19 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) and 1% penicillin and streptomycin. Cells were cultivated in a humidified incubator containing 5% CO2 at 37 ◦C. When the cells reached 90%–95% confluence, they were starved in serum-free medium for 24 h before further experiments. In the subsequent experi- mental treatment, cells were maintained in DMEM/F12 medium supplemented with 1% FBS, for exclusion the effect of growth factors of 10% FBS on the inducer FGF-2. We dissolved PLB (PLB, Sigma-Aldrich, St. Louis, MO, USA) and FGF-2 (PeproTech, Cranbury, NJ, USA) in accordance with the instructions for the reagents. The final concentration of DMSO in PLB dilutions was <0.1%. NVP-BGJ398 and GM6001 were purchased from Molnova CO. (Ann Arbor, MI, USA). A BeyoClickTM EdU Cell Proliferation Kit with Alexa Fluor 488 was used to assess cell proliferation and purchased from Beyotime Co. (Shanghai, China). Transwell and Matrigel were used to determine cell invasion and purchased from Corning Co. (NY, USA). CWbio RT Kit and SYBR Green I Real Time PCR Kit were used to assess mRNA levels of ARPE-19 cells. The kit and Trizol reagent were pur- chased from Tiangen Co. (Beijing, China) and Invitrogen Co. (Carlsbad, USA), respectively. A BCA protein assay kit was used to detect protein concentration and purchased from Cwbiotech Co. (Beijing, China). The primary antibodies are shown in Table 1. 2.2. CCK-8 assay Cell viability and proliferation were assessed by CCK-8 assay. ARPE- 19 cells (100 μl/well; 4000 cells/well) were seeded in 96-well plates and incubated for 24 h. For each group, three wells were set up. The blank wells without cells were also set up. FGF-2 (10 ng/ml), PLB (1.25, 2.5, 5, or 7.5 μM), FGFR inhibitor NVP-BGJ398 (3, 5 or 10 μM), and MMP in- hibitor GM6001 (5, 10 or 20 μM) were added or not added to the wells and incubated for 24 h. Then, each well was treated with 10 μl CCK-8 reagent and put into an incubator for 4 h. The 96-well plates were measured by a microplate reader at 450 nm absorbance. Cell viability [(experimental well - blank well) / (control well - blank well)] 100%. Cell proliferation was detected according to the CCK-8 instructions. The experiment was performed in triplicate. 2.3. EdU assay The EdU assay is the most accurate assay used to assess cell prolif- eration. The ARPE-19 cells (1 105 cells/well) were seeded in 6-well plates overnight. The cells were cultured with or without FGF-2 (10 ng/ml) and PLB (2.5 or 5 μM) for 24 h. EdU assay was performed using a BeyoClickTM EdU Cell Proliferation Kit with Alexa Fluor 488 according to the instructions. A laser scanning confocal microscope (IX83, Olympus, Japan) was used to take photos and count the prolif- erating cells. EdU+ cells = (green fluorescent cells/blue fluorescent cells) × 100%. The experiment was performed in triplicate. 2.4. Wound healing assay Wound healing assays are used to analyze tumor invasion and cell migration. ARPE-19 cells (1 × 105 cells/ml) were seeded in 6-well plates and cultured the cell confluence exceeded 90%. A 200 μl plastic straw tip perpendicular to the 6-well plate was used to draw a straight line. The cell monolayer was washed three times with phosphate-buffered saline. FGF-2 (10 ng/ml), PLB (1.25 or 2.5 μM), FGFR inhibitor NVP- BGJ398 (5 μM), and MMP inhibitor GM6001 (10 μM) were added to the 6-well plates for 24 h. We took photos and counted the cells that migrated to the damaged area using a phase contrast microscope (Leica, Germany). The experiment was performed in triplicate. 2.5. Transwell assay Transwell assays are usually used to assess cell invasion. ARPE-19 cells were cultured with or without FGF-2 (10 ng/ml) and PLB (1.25 or 2.5 μM) for 24 h. In the upper chambers of each Transwell (8 μm pore size) which contains Matrigel (0.5 mg/ml) added to ARPE-19 cells (100 μl/well, 1 × 105 cells/ml) containing serum-free medium. We added 600 μl medium containing 10% FBS to each well in the lower chambers of the Transwell. Then, the Transwell was placed in a cell incubator for 24 h. Cells which did not pass through the Transwell membrane were wiped away with a cotton swab. Then, the upper chamber was fiXed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet for 20 min. The numbers of invasive cells were photographed and counted using a phase contrast microscope (Leica, Germany). The experiment was performed in triplicate. 2.6. Real-time PCR Changes in mRNA transcription were detected by RT-PCR. ARPE-19 cells were treated with or without FGF-2 (10 ng/ml) and PLB (1.25, 2.5, 5 μM) for 24 h. The primer sequences for the genes of interest are shown in Table 1. Total RNA was extracted from cells with Trizol reagent, and 5 μl RNA was extracted. The integrity of RNA was determined by 1% agar-gel electrophoresis. A CWbio RT Kit was used for reverse System. The relative expression of the protein was analyzed using Vision Works LS software. The experiment was performed in triplicate. (Tables 1–3) 2.8. Statistical analysis The above experiments were performed at least three times, and SPSS 26.0 software was used for statistical analysis. The data were expressed as mean standard deviation (Mean SD). Differences among groups were evaluated using one-way analysis of variance (ANOVA) followed by a post-hoc test with the least significant difference (LSD). In each experiment, P＜0.05 was considered statistically significant. 3. Results 3.1. PLB inhibits proliferation of ARPE-19 cells induced by FGF-2 First, we investigated whether PLB can inhibit ARPE-19 cell viability. The results indicated that 1.25 and 2.5 μM PLB did not indicate a sig-transcription, and the experimental operation was performed in accor- dance with the product instructions. A SYBR Green I Real Time PCR Kit was used for fluorescent quantitative PCR reaction. The fluorescent quantitative PCR instrument (Bio-Rad, USA) was used for relative quantitative analysis of the data by the 2-ΔΔCt method. The experiment was performed in triplicate. 2.7. Western blotting Western blotting was a crucial method for detection of changes protein expression. The steps of cell treatment in the experiments are generally as follows. FGF-2 (10 ng/ml) and PLB (1.25, 2.5, 5 μM) were added or not added to ARPE-19 cells for 24 h. However, in the pre- induction drug assay, cells were pretreated with PLB (5 μM) for 30 min or 4 h before adding FGF-2 (10 ng/ml). The ARPE-19 cells were lysed with radio-immunoprecipitation (RIPA) protein extraction reagent and protease inhibitor (Roche). The supernatant was obtained by ul- trasonic treatment for 3 min and then centrifugation (13,000 rpm, 4 ◦C) for 20 min. The BCA protein quantitative kit was used to measure the protein concentration according to the instructions. The total protein was isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and then transferred to PVDF membrane. The PVDF membrane was completely immersed in 5% BSA-TBST and incubated for 1 h. Primary antibody was diluted with 5% BSA-TBST (dilution ratio: 1:1000) and incubated in a horizontal container at 4 ◦C overnight. The membrane was washed three times in TBST, 10 min each time. The secondary antibody was diluted with 5% BSA-TBST and incubated for 40 min. Bands were visualized using a UVP Bio Spectrum Imaging nificant effect on cell viability, while 5 and 7.5 μM PLB significantly reduced the cell viability (Fig. 1A). Therefore, 1.25 and 2.5 μM PLB were used in subsequent cell migration and invasion experiments to eliminate the effects of inhibited cell viability. The next we wanted to study whether PLB inhibits FGF-2-induced proliferation, so we carried out an EdU assay. Our results indicated that FGF-2 promoted the proliferation of ARPE-19 cells. 5 μM PLB inhibited the proliferation of cells significantly relative to the untreated control group or FGF-2 control group (Fig. 1B and C). As is known to all, proliferating cell nuclear antigen (PCNA) is a marker of cell proliferation (Jurikova et al., 2016). The results showed that 5 μM PLB blocked the proliferation of RPE cells, as indicated by comparison to an untreated control group (Fig. 1D and E). It is clearly observable that with increasing concentration of PLB, the protein expression level of PCNA decreased gradually in a dose-dependent manner compared with the FGF-2 control group. Therefore, we proved PLB could block proliferation of ARPE-19 cells which induced by FGF-2. 3.2. PLB inhibited migration and invasion of ARPE-19 cells induced by FGF-2 With regard to migration and invasion of ARPE-19 cells, we specu- lated that PLB might play an inhibitive effect. Hence, a wound healing assay and Transwell assay were conducted on cells. Figs. 2A and B indicated that relative to the untreated control group and FGF-2 control group, PLB significantly inhibited cell migration in the wound healing assay. As shown in Fig. 2C and D, relative to the untreated control group and FGF-2 control group, PLB significantly inhibited cell invasion in the Transwell assay. 3.3. PLB, NVP-BGJ398, and GM6001 inhibit proliferation and migration in ARPE-19 cells induced by FGF-2 To make this study robust, we included two positive control drugs, namely FGFR inhibitor NVP-BGJ398 and MMP inhibitor GM6001, in the CCK-8 assay and the wound healing assay. The results showed that FGF- 2-induced ARPE-19 cell proliferation was inhibited by FGFR inhibitor (NVP-BGJ398) and MMP inhibitor (GM6001) in a concentration- dependent manner (Fig. 3A-B). The CCK-8 assay showed that PLB (5 μM), BVP-BGJ398 (5 μM), and GM6001 (10 μM) inhibited FGF-2- induced ARPE-19 cell proliferation (Fig. 3C). The results of the wound healing assay showed that PLB (2.5 μM), BVP-BGJ398 (5 μM), and GM6001 (10 μM) inhibited FGF-2-induced migration of ARPE-19 cells (Fig. 3D-E). 3.4. PLB inhibits mRNA and protein expression of MMP-2 and MMP-9 in ARPE-19 cells induced by FGF-2 MatriX metalloproteinase-related proteins (MMP-related proteins) are a category of enzymes that are closely related to migration and in- vasion. MMPs are elevated in PVR and could promote RPE cell migra- tion. We hypothesized that the inhibitory effect of PLB on migration and invasion might be associated with MMPs. For this reason, we assessed the level of expression of MMP-2 and MMP-9 of ARPE-19 cells. The re- sults confirmed our belief that PLB significantly inhibited mRNA expression and reduced protein levels of MMP-2 and MMP-9 (Fig. 4C and E) in a dose-dependent manner, as indicated by comparison to the untreated control group and FGF-2 control group (Fig. 4A-B). 3.5. PLB inhibited mRNA and protein expression of Col1A1, Col4A1, and Col6A1 in FGF-2-induced ARPE-19 cells The ECM is an important component of the epiretinal membrane and has many functions. It can affect cell proliferation, morphology, migration, contraction, and other processes (Gamulescu et al., 2006; Kimoto et al., 2004; Pastor et al., 2002). Studies have shown that the ECM of the epiretinal membrane contains different amounts of collagens I, II, III, IV, and VI, among which collagens I, IV, and VI may be the key factors in the development of the epiretinal membrane (Jerdan et al., 1989; Morino et al., 1990; Regoli et al., 2020; Tosi et al., 2020). We hypothesized that the inhibition of cell proliferation, migration, and invasion might be related to collagens I, IV, and VI. Col1A1 (Heffer et al., 2019; Yin et al., 2019), Col4A1 (Jin et al., 2017; Wang et al., 2020), and Col6A1 (Owusu-Ansah et al., 2019; Uhlen et al., 2017) are all related to cell growth, metastasis, and invasion. Therefore, expression levels of such extracellular matriX proteins were assessed in FGF-2-induced ARPE-19 cells using RT-PCR and western blotting. As shown in Fig. 5, FGF-2 can promote mRNA and protein expression of Col1A1, Col4A1, and Col6A1, which is significantly different from untreated control group. The experimental groups were compared to untreated control group and FGF-2 control group, and expression of Col1A1, Col4A1, and Col6A1 mRNA and protein resulted inhibited by PLB in a dose-dependent manner. 3.6. PLB inhibits FGFR and MAPK pathways in ARPE-19 cells induced by FGF-2 To further determine whether the ability of PLB to block prolifera- tion, migration, and invasion of ARPE-19 cells induced by FGF-2 was related to the signaling pathway induced by FGF-2, we assessed the phosphorylation of FGFR-1/-2, ERK, p38, and JNK. These three mole- cules are particularly important to cellular proliferation and migration. Our results showed that FGFR-1 and FGFR-2 phosphorylation levels were significantly increased in cells after FGF-2 treatment, while PLB significantly decreased FGFR-1 and FGFR-2 phosphorylation in a dose- dependent manner (Fig. 6C and D). FGF-2 stimulation also signifi- cantly increased the phosphorylation levels of ERK, P38, and JNK. However, treatment with gradually increasing concentrations of PLB was associated with significant decreases of ERK, P38, and JNK phos- phorylation levels (Fig. 6E-G). One mechanism by which PLB might exert its inhibitory effect on proliferation, migration, and invasion of FGF-2-induced ARPE-19 cells might involve FGFR1, FGFR2, and MAPK pathways. The results in Fig. 6 showed that PLB only affected phosphorylation after FGF-2 induction. In order to observe this drug whether had a preventive effect on proliferation and migration of FGF-2-induced ARPE-19 cells, we added PLB and conducted a western blot before FGF-2 induction. Before adding FGF-2 (10 ng/ml), ARPE-19 cells were pretreated with 5 μM of PLB for 30 min or 4 h. The results showed that compared with FGF-2 control group, PLB pretreatment for 30 min or 4 h significantly inhibited phosphorylation of FGFR1/R2, ERK, p38, and JNK (Fig. 7). Therefore, PLB inhibits FGFR and MAPK pathways in FGF- 2-induced ARPE-19 cells. 4. Discussion The mechanisms underlying PVR development are not well under- stood. It is thought to be closely related to growth factors, cytokines, signaling mediators, and certain receptors. FGF-2 is an effective mitogen that can stimulate RPE cells and it is involved in the formation of epi- retinal membrane. It is considered an important factor closely related to the severity of PVR (Chiquet and Rouberol, 2014; Dempsey et al., 2000). FGF-2 and other growth factors, such as TGF-β and PDGF, are overexpressed in the vitreoretinal subretinal fluid and epiretinal membranes in PVR patients (Kita et al., 2008; Ricker et al., 2011; Tosi et al., 2020). In vitro, FGF-2 stimulated EMT in RPE cells (Romo et al., 2011). TGF-β is a multi-functional cytokine family that can regulate apoptosis transition and other important biological activities (Massague, 1998). Both TGF-β1 and TGF-β2 can promote RPE EMT and ECM deposition, and they can induce cell contraction in vitro to simulate the contraction of PVR membrane (Carrington et al., 2000; Dvashi et al., 2015). There are four genes in the PDGF family, namely PDGF-A, -B, -C, and -D. Bioactive PDGF has four homodimers (PDGF-AA, -BB, -CC, -DD) and one hetero- dimer (PDGF- AB). In vitro, PDGF has been shown to stimulate the proliferation and chemotaxis of RPE cells (Yang et al., 2020). Although the vitreous fluid of PVR patients and PVR animal models contained higher levels of PDGF than those of other growth factors, complete neutralization of PDGF did not inhibit the development of PVR (Lei et al., 2009). Hence, the pathogenesis of other growth factors that play an important role in the progression of PVR should also be investigated. For this reason, in this study, FGF-2 was applied to ARPE-19 cells to determine whether PLB has an inhibitory effect on FGF-2. PLB is a small molecule naphthoquinone compound with many biological activities. It can inhibit bacterial and tumorous growth and has anti-inflammatory and other effects (Aziz et al., 2008; Pan- ichayupakaranant and Ahmad, 2016). PLB has potential therapeutic effects on many diseases such as breast tumors, prostate tumors, and squamous cell carcinoma. The molecular mechanisms underlying its effects include autophagy, apoptosis, cell cycle arrest, migration, and invasion (Tripathi et al., 2019).In this work, we observed that PLB markedly inhibited proliferation, migration, and invasion of ARPE-19 cells induced by FGF-2 (Fig. 1B and Fig. 2A and C). To make this study rigorous, we selected FGFR inhibitor NVP-BGJ398 and MMP in- hibitor GM6001 as two positive control drug groups added to the CCK-8 assay and the wound healing assay. The results showed that PLB, NVP-BGJ398, and GM6001 could inhibit FGF-2-induced proliferation and migration of ARPE-19 cells (Fig. 3). In order to investigate the specific mechanism of action of PLB, we used RT-PCR and western blotting to determine whether the expression of MMP-2 and MMP-9 of cells could be inhibited by it. MMPs play a vital part in the degradation of ECM and their overexpression is an essential step in causing cell migration and invasion (Huang et al., 2005; Symeonidis et al., 2007). Large amounts of evidence have established MMP-2 and MMP-9 increase after retinal detachment, which promotes cell movement by degrading most ECM components (Ciprian, 2015). Therefore, we take MMP-2 and MMP-9 as the indicators to reflect the ability of migration and invasion. EXperiments showed that, after treatment of PLB, the mRNA and protein expression of MMP-2 and MMP-9 significantly decrease (Fig. 4). RT-PCR and western blot analyses were also used to determine whether FGF-2 and PLB could affect mRNA or protein expression of Col1A1, Col4A1, and Col6A1. Collagen is the most abundant component in the PVR membranes. RPE cells are probably the main cells involved in collagen synthesis (Hollborn et al., 2004; Kimoto et al., 2004). RPE cells may produce ECM components in vitreous cavity and eventually epi- retinal membranes (Martini et al., 1992; Williams and Burke, 1990). The contraction of the PVR membranes may damage the retinal structure, causing retinal re-detachment and loss of vision (Tosi et al., 2014). In the ECM of PVR membrane, collagen I is the most abundant. It is the main collagen involved in fibrosis (Kimoto et al., 2004; Pastor et al., 2002). Collagen IV is present in normal retinal tissues, but its expression is significantly enhanced in the retina of eyes affected by PVR (Hollborn et al., 2004). Both collagens IV and VI are abundant in the idiopathic epiretinal membranes (Kritzenberger et al., 2011; Regoli et al., 2020). Moreover, collagens I, IV, and VI may be key factors in the development of epiretinal membranes (Regoli et al., 2020). Col1A1, Col4A1, and Col6A1 promote cell proliferation, migration, and invasion. Therefore, we evaluated whether FGF-2 and PLB had any effect on Col1A1, Col4A1, and Col6A1 of cells. EXperiments showed that FGF-2 promoted the expression of Col1A1, Col4A1, and Col6A1 mRNA and protein by ARPE-19 cells. PLB precisely inhibited their expression (Fig. 5). In order to further establish the molecular mechanism underlying PLB, we performed a western blot assay to determine whether the FGFR and MAPK pathways were inhibited by PLB. A range of studies reported that FGF-2 interacted with FGFRs to activate downstream signaling molecules and promote cell transformation, EMT, angiogenesis, prolif- eration, and migration (Chlebova et al., 2009; Yu et al., 2007). Within the FGFR family, FGFR-1 and FGFR-2 have been identified as the most momentous molecular targets for a variety of cells (Ishiwata, 2018; Mariz et al., 2018). For this reason, we selected the two to verify their role in the mechanism of this study. The MAPK pathway is also of vital importance for proliferation and migration in a variety of cells. It is mainly composed of ERK, JNK, and P38 MAPK. It is a crucial cell pro- liferation signaling pathway (Liu et al., 2018). We here hypothesized that FGFR phosphorylation and MAPK pathway activation may play a vital part in proliferation and migration of RPE cells. Western blot re- sults showed that FGF-2 binds to FGFR-1 and FGFR-2 receptors in ARPE-19 cells, which significantly increases FGFR-1, FGFR-2, ERK, P38, and JNK phosphorylation levels and activates the MAPK pathway (Fig. 6). Nevertheless, PLB significantly decreased the phosphorylation effect of FGFR-1/-2, ERK, p38, and JNK in FGF-2-induced RPE cells (Fig. 6). In Fig. 7, we found that PLB cell pretreatment for 30 min or 4 h significantly inhibited phosphorylation of FGFR1/R2, ERK, p38, and JNK, which indicated PLB inhibited FGFR and MAPK pathways in ARPE-19 cells induced by FGF-2. Therefore, PLB has a preventive effect on proliferation, migration, and invasion of FGF-2-induced ARPE-19 cells. In conclusion, for the first time, our study suggests that PLB inhibits FGF-2-induced proliferation, migration, and invasion of ARPE-19 cells. 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