2.2. Effect of BDL on Function and Expression of Mrp1 at Rat BRB

Retina distributions of fluorescein and 2,4-dinitrophenyl-S-glutathione (DNP-SG), substrates of Mrp1, were calculated to assess the function of retina Mrp1. It is generally accepted that the retinal concentrations of substrates are affected by plasma concentrations [21], and the retina-to-plasma concentration ratios (K_r/p) of substrates serve as the index of retinal penetration to eliminate the effects of plasma substrate levels on retinal substrate distribution. The results showed that BDL significantly increased fluorescein concentration in the retina (Figure 1a) without affecting plasma fluorescein levels (Figure 1b). Remarkably increased K_r/p of fluorescein was found in BDL rats (Figure 1c). Similarly, BDL did not affect DNP-SG concentration in the retina (Figure 1d) but strikingly decreased DNP-SG concentration in plasma (Figure 1e), leading to remarkably higher K_r/p of DNP-SG (Figure 1f) compared with Sham rats.

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Figure 1

Effect of BDL on the function of Mrp1 in rat retina. (a) Retina fluorescein levels, (b) plasma fluorescein levels, and (c) retina-to-plasma ratio (K_r/p) of fluorescein at 30 min following intravenous (i.v.) fluorescein (2 mg/kg) to rats. (d) Retina DNP-SG levels, (e) plasma DNP-SG levels, and (f) K_r/p of DNP-SG at 15 min following i.v. 1-chloro-2,4-dinitrobenzene (5 mg/kg) to rats. (g) Plasma UCB levels and (h) retina UCB levels in Sham and BDL rats. Lower limit of quantitation (LLOQ) = 0.67 pmol/mg protein. Data are expressed as means ± S.D. (n = 6). ** p < 0.01 vs. Sham rats.

UCB is an endogenous substrate of MRP1/Mrp1. Thus, levels of UCB in plasma and retina were also measured. As expected, BDL significantly increased the concentration of UCB in both plasma (Figure 1g) and retina (Figure 1h). The retina UCB levels were significantly increased from less than the lower limit of quantitation (LLOQ = 0.67 pmol/mg protein) in Sham rats to 2.72 ± 1.49 pmol/mg protein in BDL rats.

The mRNA levels of Abcc1~6, Abcb1a/1b, and Abcg2 were detected in eyecups of normal rats. The results showed that Abcc1 had the highest expression, followed by Abcc5 (Figure 2a). To investigate whether the increased retina distributions of fluorescein and DNP-SG in BDL rats come from impairment of Mrp1 expression, Mrp1 protein expression in eyecups of BDL and Sham rats was further measured. Western blot showed BDL significantly decreased Mrp1 protein expression in eyecups by approximately 60% (Figure 2b).

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Figure 2

Effect of BDL on expression of Mrp1 in rat retina. (a) The mRNA levels of Abcc1~6, Abcb1a/1b, and Abcg2 in eyecups of normal rats were measured by qRT-PCR and normalized to Actb (2^−ΔCt). (b) Expression of Mrp1 protein in eyecups of Sham and BDL rats (n = 6). (c) The location of Mrp1 (green) and Glut1 (red) in the eyecup of rats (Scale bar = 100 μm). Nuclei are DAPI-stained (blue). Regions marked 1 and 2 indicate microvessel and RPE, respectively, which are detailed in the upper right and lower right (Scale bar = 20 μm). Ap, apical membrane; bl, basolateral membrane. (d) Immunostaining analysis of Mrp1 and Glut1 in eyecups (Scale bar = 100 μm) and (e) its integrated fluorescence intensity of Mrp1 normalized by RPE length (n = 3). (f) Plasma FITC-dextran levels, (g) retina FITC-dextran levels, and (h) K_r/p of FITC-dextran in Sham and BDL rats at 30 min following i.v. 50 mg/kg FITC-dextran (n = 5). (i) Expressions of ZO-1 and claudin-5 (n = 6). (j) Immunoblots for pp38, p38, pAkt, Akt, pERK, ERK, pp65, p65, pJNK, and JNK and ratios of phosphorylated proteins to total protein levels (n = 6). (k) Expressions of pMK2 and total MK2 levels (n = 6). (l) Expression of Brn-3a protein (n = 6). Data are expressed as the mean ± S.D. * p < 0.05, ** p < 0.01 vs. Sham rats.

The location of Mrp1 in rat retina was detected using double immunofluorescence staining (Figure 2c). Glut1 was used to label retinal blood vessels and RPE [15,27]. As we can see, Glut1 (red) was mainly localized at retinal microvessels and RPE. In RPE, Glut1 protein was evidently detected on both apical and basolateral membranes. Mrp1 protein (green) was mainly colocated with Glut1 at the basolateral side of rat RPE. The apical side of rat RPE and retinal microvessels only showed very weak staining or no staining of Mrp1 protein. Very weak expression of Mrp1 also existed in retinal ganglion cells. This was in line with alterations in total protein levels of retina Mrp1 that BDL also remarkably decreased expression of Mrp1 at the basolateral membrane of rat RPE (Figure 2d,e).

To exclude the possibility that the increased retina distributions of the substrates result from the BRB breakdown, the integrity of BRB was measured using leakage of fluorescein isothiocyanate-conjugated dextran (FITC-dextran). It was found that although BDL rats showed a slight decrease in plasma FITC-dextran concentration (Figure 2f), retina concentrations of FITC-dextran and K_r/p of FITC-dextran in the retina of BDL were comparable to those of Sham rats (Figure 2g,h). Consistently, BDL barely affected expressions of tight junction proteins ZO-1 and claudin-5 in eyecups of rats (Figure 2i), indicating that BRB integrity was intact.

Several signaling pathways, including NF-κB (p65), PI3K/Akt, and mitogen-activated protein kinases (MAPK), have been demonstrated to be involved in the regulation of ABC transporter expressions [22,28,29,30]. Expressions of total and phosphorylated p38, Akt, ERK, p65, and JNK in rat eyecups were measured. It was found that BDL significantly increased phosphorylation of p38 and ERK but attenuated phosphorylation of JNK. However, levels of pp65 and pAkt were little altered by BDL (Figure 2j). MAPK-activated protein kinase 2 (MK2) is directly associated downstream to pp38 [31]. Here, phosphorylation of MK2 was also monitored to reflect activation of p38 MAPK. As expected, significantly increased levels of pMK2 (Figure 2k) were detected in eyecups of BDL rats. It was also noticed that BDL rats showed obviously lower protein expression of Brn-3a, a retinal ganglion cell (RGC) marker, inferring that BDL may lead to loss of RGC (Figure 2l).

2.3. Effects of Abnormally Altered Components in BDL Rat Serum on Function and Expression of MRP1 in ARPE-19 Cells

ARPE-19 cells serve as an in vitro outer BRB model to investigate the underlying mechanism by which BDL impairs the function and expression of retina MRP1. qRT-PCR analysis showed that, in line with findings in rat eyecups, high mRNA expression of ABCC1 was detected in ARPE-19 cells (Figure 3a). The functionality of MRP1 was characterized by the intracellular accumulation of fluorescein and calcein with or without MK571 (50 μM), a widely used MRP inhibitor. Treatment with MK571 resulted in a 1.6-fold increase in intracellular fluorescein accumulation and a 2.1-fold increase in calcein accumulation in ARPE-19 cells, respectively (Figure 3b,c), confirming the functionality of MRP1. Coincubation with MK571 also obviously elevated intracellular UCB accumulation (Figure 3d), signifying that MRP1 contributes to UCB efflux in ARPE-19 cells. Next, the effect of serum from BDL rats on protein levels of MRP1 was evaluated. The results showed that incubation with medium containing 10% serum from BDL rats significantly decreased MRP1 protein expression (Figure 3e), implying that the abnormal components in BDL rat serum impaired MRP1 protein expression.

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Figure 3

Effect of UCB on the function and expression of MRP1 in ARPE-19 cells. (a) The mRNA levels of efflux transporters in ARPE-19 cells were normalized to the ACTB (2^−ΔCt) (n = 3). Effects of MRP1 inhibitor MK571 on cellular uptakes of (b) fluorescein (n = 6), (c) calcein (n = 6), and (d) UCB (n = 4) in ARPE-19 cells. (e) Effect of serum from BDL rats on MRP1 protein expression (n = 6). Effects of (f) UCB, (g) NH₄Cl, (h) ADMA, and (i) bile salt cocktail on MRP1 protein expressions (n = 6). (j) TEER values across ARPE-19 monolayer after seeding (n = 4). (k) Apical (A) -to-basolateral (B) transport of FITC-dextran across ARPE-19 monolayer and control insert membranes (n = 4). (l) A-to-B and B-to-A transport of fluorescein with or without MK571 (n = 6). (m) Cross-sectional view of the reconstructed z-stack images in x-z plane. Ap, apical; bl, basolateral. (n) A-to-B transport of fluorescein after pre-treatment with UCB for 72 h (n = 4). (o) Protein levels of MRP1 were accessed by immunofluorescence after incubation with UCB (Scale bar = 50 μm). (p) The mean MRP1 fluorescence intensity was normalized to the mean DAPI fluorescence intensity (n = 3). Data are expressed as the mean ± S.D. * p < 0.05, ** p < 0.01 vs. Sham serum, control (Ctrl) cells or P_{app, A-to-B} of Ctrl.

BDL rats often exhibit significant elevations in serum levels of UCB, ammonia, ADMA, and bile acids, which were also repeated in the study (Table 1). To investigate which altered components in BDL rat serum are factors impairing expression of MRP1, ARPE-19 cells were separately incubated with medium containing UCB, NH₄Cl, ADMA, and bile acid cocktail for 72 h. The results showed that among the tested factors, only UCB concentration-dependently downregulated MRP1 protein expression in ARPE-19 cells (Figure 3f–i).

ARPE-19 cells were seeded in the transwell insert to form polarized RPE cell monolayers, which are structurally and functionally similar to the in vivo outer BRB [32]. Transepithelial electrical resistance (TEER) measurements and FITC-dextran permeability assay were conducted to investigate the barrier function of ARPE-19 cell monolayers. It was found that TEER reached a plateau on Day 6 and stayed stable from Day 6 to 10 (Figure 3j). Thus at 6 days after seeding, the monolayers with TEER values exceeding 90 Ω×cm² were used for the following experiments. The permeability of FITC-dextran, the marker for the predominantly paracellular pathway transport, was measured. The P_{app, A-to-B} of FITC-dextran across the control insert membranes was 1.00 × 10⁻⁵ cm/s. However, in the presence of the ARPE-19 monolayers, the P_{app, A-to-B} of FITC-dextran was remarkably decreased to 1.18 × 10⁻⁶ cm/s (Figure 3k). These results revealed that ARPE-19 monolayers display the barrier function to substance transport.

It is generally accepted that MRP1 is mainly located at the basolateral membrane of RPE. Bidirectional transepithelial transport experiments with fluorescein were performed to confirm the MRP1 functionality of ARPE-19 cell monolayers. The results showed that the transport of fluorescein across ARPE-19 monolayers was asymmetric. P_{app, A-to-B} values (7.00 ± 1.94 × 10⁻⁶ cm/s) were significantly higher than P_{app, B-to-A} values (4.01 ± 0.82 × 10⁻⁶ cm/s), whose ratio (P_{app, A-to-B} /P_{app, B-to-A}) was 1.75 (Figure 3l). Furthermore, MK571 inhibited the transport of fluorescein in the A-to-B direction, not the B-to-A direction (Figure 3l), indicating that MRP1 is involved in the A-to-B transport of fluorescein in ARPE-19 cells. The location of MRP1 protein on the ARPE-19 monolayer was detected using a confocal laser scanning microscope. A cross-section of z-stack images was reconstructed. The cross-sectional view of the x-z plane confirmed that MRP1 was mainly localized on the basolateral membrane of ARPE-19 cells (Figure 3m). Moreover, 72 h incubation with UCB also concentration-dependently decreased the transport of fluorescein in the A-to-B direction (Figure 3n). Consistently, immunofluorescence staining demonstrated that UCB significantly downregulated MRP1 protein levels (Figure 3o,p).

2.4. Involvement of P38 MAPK Pathway in UCB-Induced Downregulation of MRP1 Expression in APRE-19 Cells

In vivo data showed that BDL significantly increased phosphorylation of ERK, p38, and MK2 but decreased phosphorylation of JNK, inferring that these signal pathways could be involved in UCB-induced impairment of MRP1 expression. In APRE-19 cells, incubations with UCB (2, 10, 50 µM) (Figure 4a) or 10% BDL serum (Figure 4b) remarkably increased the phosphorylation of p38, MK2, Akt, and ERK1/2. P38 inhibitors SB203580 or SB202190 but neither ERK1/2 inhibitor U0126 nor Akt inhibitor LY294002 reversed the downregulation of MRP1 protein expression by UCB (Figure 4c,d). UCB itself increased p38 phosphorylation (Figure 4e). Neither SB203580 nor SB202190 reversed the phosphorylation of p38 by UCB. In contrast, UCB and SB202190 showed synthetic effects on p38 phosphorylation (Figure 4e). UCB markedly increased MK2 phosphorylation, which was almost abolished by SB203580 or SB202190 (Figure 4f). This finding is in agreement with the previous studies that SB203580 and SB202190 inhibit p38 MAPK catalytic activity, not phosphorylation of p38 [33,34]. To further verify whether p38 MAPK is involved in the impairment of MRP1 expression, the effect of anisomycin, a well-known p38 activator, on MRP1 protein expression was documented. This was consistent with findings in treatment with UCB that neither SB202190 nor SB203580 reversed anisomycin-induced phosphorylation of p38 but almost abrogated anisomycin-induced upregulation of pMK2 levels and impairment of MRP1 expression (Figure 4g).

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Figure 4

Roles of signal pathways in impairment of MRP1 protein expression by UCB in APRE-19 cells. Effects of (a) UCB and (b) BDL rat serum on expressions of pp38/p38, pMK2/MK2, pAkt/Akt, pERK/ERK1/2, pp65/p65, and pJNK/JNK levels. Effects of (c) p38 inhibitors SB203580 (SB203) and SB202190 (SB202), (d) Akt inhibitor LY294002 (LY294), and ERK1/2 inhibitor U0126 on UCB-induced downregulation of MRP1 protein. Effects of SB203 and SB202 on increases in phosphorylation of (e) p38 and (f) MK2 levels by UCB. (g) Effects of SB203 and SB202 on anisomycin (ANI)-induced impairment of MRP1 protein expression and increases in phosphorylation of p38, MK2. (h) Validation of p38 siRNA. (i) Effects of p38 siRNA on UCB- and ANI-induced decreases in MRP1 protein expressions. (j) Effects of UCB on H₂O₂ -induced ROS formation. (k) Effects of NAC on UCB-induced ROS formation. (l) Effect of H₂O₂ on MRP1 protein levels. Data are expressed as the mean ± S.D. (n = 6). * p < 0.05, ** p < 0.01 vs. control (Ctrl) cells, Sham serum, si-negative control (si-NC) + Ctrl. ^# p < 0.05, ^## p< 0.01 vs. UCB (50 μM), ANI or si-NC + UCB. ^$$ p < 0.01 vs. si-NC + ANI or H₂O₂.

The role of p38 MAPK in UCB-induced MRP1 downregulation was further confirmed using p38 knockdown with siRNA. Transfection of p38 siRNA markedly attenuated p38 protein expression, which was 45% of cells transfected with negative control siRNA, demonstrating successful silencing of p38 (Figure 4h). Both UCB and anisomycin no longer decreased MRP1 protein levels in cells transfected with p38 siRNA (Figure 4i).

Roles of reactive oxygen species (ROS) in UCB-induced MRP1 downregulation were also documented. APRE-19 cells were incubated with medium containing UCB, and H₂O₂ (0.1 mM) served as positive control. ROS levels were determined using fluorescent probe DCFH-DA according to the manufacturer’s instruction (Beyotime, Shanghai, China) with excitation/emission wavelengths of 480/535 nm on a SpectraMax Gemini XPS microplate fluorometer. The results showed that UCB at the tested concentrations (2~50 μM) did not prevent H₂O₂-induced ROS formation and did not show any antioxidant effects (Figure 4j). On the contrary, 50 μM UCB enhanced formation of ROS, which was attenuated by N-acetyl-L-cysteine (NAC, 8 mM) (Figure 4k). UCB-induced downregulation of MRP1 protein was not attenuated by NAC (Figure 4l). Furthermore, H₂O₂ did not affect expressions of MRP1 protein, indicating that downregulation of MRP1 protein by UCB was independent of ROS.

4.2. Animals

Male Sprague–Dawley rats, weighing 180 g to 200 g, were from Sino-British Sippr/BK Laboratory Animal Ltd. (Shanghai, China). The rats were housed in a room with controlled temperature (24 ± 2 °C) and humidity (50 ± 5%) under a 12 h light/dark cycle. Animals were fed with a commercial chow diet and water ad libitum. The animal studies were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health) and were approved by the Animal Ethics Committee of China Pharmaceutical University (Approval Number: 202004010).

4.3. Development of BDL Rats

Development of BDL rats was performed as described previously [18]. Briefly, rats were anesthetized using an intraperitoneal dose of pentobarbital (40 mg/kg), and then the common bile duct was exposed and ligated with 4-0 silk. Sham rats underwent a similar procedure without ligation. Following closing abdominal incision, the rats were kept on warm pads (37 °C) to recover. Two weeks following surgery, BDL and Sham rats were selected for the following experiments.

4.4. Distributions of Fluorescein and DNP-SG in Rat Retina

Distributions of fluorescein and DNP-SG, two typical substrates of Mrp1 [17,59,60], were utilized to assess the function of Mrp1 at the BRB of rats. Briefly, experimental rats were sacrificed under light ether anesthesia at 30 min following i.v. administration of 2 mg/kg fluorescein or at 15 min following i.v. administration of 5 mg/kg 1-chloro-2,4-dinitrobenzene (a precursor of DNP-SG). Blood samples and tissue (liver, spleen, and eye) were collected. Plasma and serum samples were obtained to analyze substrates, ADMA, SDMA, and serum biochemical parameters. Serum biochemical parameters such as ALT, AST, ALP, total bilirubin, ammonia, and total bile acids were measured using commercially available kits (Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. Eyecups (RPE/choroid/sclera and neural retina) were prepared by removing the cornea, lens, and vitreous from enucleated eyes [61]. For substrate analysis, the neural retina was further dissected from the eyecup.

Another batch of rats was used to assess BRB permeability using FITC-dextran (3–5 kDa) as a probe, according to the previous report [62]. In brief, the experimental rats were sacrificed under light ether anesthesia at 30 min following i.v. FITC-dextran (50 mg/kg). Fluorescence was determined (excitation 485 nm, emission 525 nm) with a SpectraMax Gemini XPS microplate fluorometer (Sunnyvale, CA, USA), and the amount of FITC-dextran in retina and plasma was calculated based on the corresponding standard curve.

4.5. Drug Assays

Plasma ADMA and SDMA were measured by HPLC with ortho-phthalaldehyde derivatization [63]. Concentrations of fluorescein in plasma and retina were measured by HPLC-MS following the previously established method [28]. Concentrations of UCB in plasma and retina were measured using the HPLC-UV method [64]. Retina UCB values were normalized to protein content and expressed as picomole per milligram protein.

The HPLC-MS/MS method was developed to measure concentrations of DNP-SG in plasma and retina. For the retina, the neural retina was homogenized on ice in 300 μL 50% acetonitrile, and 100 μL of homogenates were mixed with 50 μL acetonitrile containing 25 ng/mL internal standard (4-hydroxydiclodenac). For plasma, 50 μL of plasma was mixed with 100 μL acetonitrile containing 500 ng/mL internal standard. The above mixture was centrifuged at 15,000× g for 10 min at 4 °C. Two μL of the supernatant was injected into the HPLC-MS/MS system. Analysis was performed by negative ion LC-MS/MS electrospray (ABSciex QTrap 4500) on a Shim-pack VP-ODS column (5 µm, 150 L × 2.0 mm, Shimadzu, Japan). For the gradient elution, buffer A was 0.001% formic acid in the water, and buffer B was acetonitrile. The flow rate was 0.3 mL/min. A gradient program started at 20% B and changed linearly to 70% B at 1.5 min, then linearly to 90% B at 4 min, where it was run isocratically until 4.2 min. From 4.2 min to 4.3 min, the gradient was linearly switched back to starting conditions, keeping isocratically until 6.2 min. Calibration ranges were from 62.5 to 8000 ng/mL for plasma and from 0.98 to 125 ng/mL for retina homogenate (equivalent to 0.195~25 ng/mg protein), respectively.

4.6. Immunofluorescence Staining

Paraffin-embedded sections (3 μm) of rat eyecups were deparaffinized in dimethyl benzene and rehydrated in decreasing ethanol. The sections were blocked with 5% goat serum and incubated with mixed primary antibodies of mouse anti-Mrp1 (1:100, Abcam, Cambridge, UK) and rabbit anti-Glut1 (1:400, Servicebio) overnight at 4 °C. Sections, following washing with phosphate-buffered saline (PBS) three times, were incubated for 1h at room temperature with Alexa Fluor 488-conjugated goat anti-mouse IgG (1:1000, Invitrogen, Waltham, MA, USA) and Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:1000, Invitrogen). The cell nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI, Servicebio). Images were captured on LSM700 confocal laser scanning microscope (Zeiss). Integrated densities for Mrp1 in the RPE were quantified using ImageJ software (NIH, Bethesda, MD, USA) and normalized by RPE length.

ARPE-19 cells grown on the filter were fixed with 4% formaldehyde for 15 min at room temperature. Then, cells with the filter were cut out and stained with anti-MRP1 primary antibody (1:100, CST) at 4 °C overnight, followed by secondary antibody and DAPI. Finally, cells were mounted on the slide and viewed under confocal microscopy. The fluorescence intensity of MRP1/DAPI was quantified by ImageJ.

4.7. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

Total RNA was isolated from eyecups of normal rats and ARPE-19 cells (Cellcook, Guangzhou, China) using RNAiso Plus Reagent and reverse-transcribed into cDNA using HiScript^® III RT SuperMix (Vazyme, Nanjing, China). Expressions of mRNA, including Abcb1a/1b/ABCB1, Abcg2/ABCG2, and Abcc1~6/ABCC1-6, were determined by qRT-PCR. Actb/ACTB was used as a housekeeping gene to normalize the detected gene. qRT-PCR was conducted with SYBR Master Mix (Yeasen) on an Applied Biosystems QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific). Analyses used the 2^−ΔCt method [65], where ΔCt = Ct (target gene) − Ct (Actb). Expression levels (2^−ΔCt) of these genes were plotted on a logarithmic scale. The sequences of rat and human primers are presented in Table 3.

4.8. Cell Culture and Drug Treatment

ARPE-19 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM)/F12 medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Invitrogen), 100 IU/mL penicillin and 100 μg/mL streptomycin under 5% CO₂ in an incubator (37 °C). The cells were seeded in 12-well plates at a density of 1 × 10⁵ cells/well. At 70% confluence, ARPE-19 cells were exposed to culture medium containing 10% serum from rats or the tested factors, including NH₄Cl (0.2, 1, and 5 mM), UCB (2, 10, and 50 μM), ADMA (2, 10, and 50 μM), and bile acid cocktail (50 and 100 μM), respectively. Levels of NH₄Cl and UCB were referenced from the literature [18,22]. Levels of ADMA and bile acid cocktail were designed according to the measured levels in BDL rats. Bile acid cocktail consisted of sodium cholate, sodium chenodeoxycholate, sodium deoxycholate, and sodium hyodeoxycholate (30:18:8:5), which was obtained from a previous report [66]. CCK 8 test showed that these agents at tested concentrations used in the study had no damage to the viability of cultured cells. Following 72 h incubation, the cells were used to assess the expression and function of MRP1 protein.

4.9. P38 Knockdown with siRNA

ARPE-19 cells were transfected with p38 siRNA (90 pmol per 12-well) using lipofectamine 3000 (Invitrogen) according to instructions. The sequence of p38 siRNA was 5′- GCAUAAUGGCCGAGCUGUUTT -3′, which was referenced from the literature [67]. Both siRNA targeting p38 and scramble siRNA control were synthesized by GenePharma (Shanghai, China). After being transfected with siRNA for 24 h, cells were exposed to UCB (50 μM) or anisomycin (50 nM) for another 72 h. Then, Western blot was performed to detect the p38 and MRP1 expressions.

4.10. Uptake of MRP1 Substrates by ARPE-19 Cells

MRP1 functionality of ARPE-19 cells was assessed using accumulation of fluorescein and calcein. Briefly, cells were washed with Hank’s balanced salt solution (HBSS) 2 times and incubated with HBSS containing fluorescein (50 μM) or calcein-AM (0.5 μM) in the presence or absence of MRP1 inhibitor MK571 (50 μM). Following 30 min incubation, uptake reaction was stopped via aspirating solution, followed by washing thrice with ice-cold HBSS. Fluorescence intensity of intracellular calcein (excitation 494nm; emission 514 nm) was measured by SpectraMax Gemini XPS microplate fluorometer (Sunnyvale, CA, USA) and normalized by cellular protein content. Intracellular fluorescein content was determined by HPLC-MS [28] and also normalized by cellular protein content. Protein contents were determined by the Coomassie protein assay reagent (Beyotime).

Whether MRP1 mediated the transport of UCB in ARPE-19 cells was also investigated. Cells were incubated with HBSS containing UCB (4 and 10 μM) for 10 min with or without MK571 (50 μM) [68], and cellular amount of UCB was measured using the HPLC-UV method [64].

4.11. Transport of Fluorescein across ARPE-19 Monolayer

ARPE-19 cells were seeded at 1 × 10⁵ cells/well on transwell inserts (PET membrane, 0.4 μm pore, SPL 37024, Korea). Cell monolayer integrity and paracellular permeability were assessed by TEER and permeability of FITC-dextran, respectively [69]. TEER was measured using a Millicell ERS-2 Voltohmmeter (Merck, Kenilworth, NJ, USA). The measured resistance value was multiplied by the surface area of the membrane (0.33 cm²) to obtain TEER (Ω × cm²). Monolayer with TEER over 90 Ω × cm² was used for further experiments. For FITC-dextran permeability assays, 0.35 mL of FITC-dextran (1 mg/mL) was added to the apical compartment, and 1.3 mL of HBSS solution was added to the basolateral compartment. The samples in the lower chambers were taken after 30 min incubation and the amount of FITC-dextran was determined by the SpectraMax Gemini XPS microplate fluorometer (excitation 480 nm, emission 520 nm). Apparent permeability coefficients (P_app) were calculated as P_app = (A_receptor/1800)/(Area × C_0,donor) [70], where A_receptor/1800, Area, and C_0,donor are flux rate (μg/s), the surface area of membrane (0.33 cm²), and the initial concentration of substrate in donor chamber (μg/mL), respectively.

Bidirectional transport of fluorescein across the ARPE-19 monolayer was carried out. Fluorescein (50 μM) was added to the apical compartment or basolateral compartment in the absence or presence of MK571 (50 μM). Concentrations of fluorescein in the receiver were measured following 30 min incubation. Both P_app, _A-to-B and P_app, _B-to-A values were estimated.

4.12. Western Blot

Protein expressions in eyecups or cells were measured using Western blot. Total protein was extracted using the Blue Loading Buffer Pack (CST) containing protease inhibitor cocktail (Yeasen) and phenylmethyl sulfonyl fluoride. Proteins were separated on 8~12% SDS-PAGE and transferred to a nitrocellulose membrane (Pall Life Science, New York, NY, USA). Following blocking for 2 h at room temperature with 5% non-fat dry milk, the blots were incubated overnight at 4 °C with primary antibodies: anti-Mrp1 (1:500, Abcam), anti-MRP1 (1:1000, CST), anti-ERK1/2 (1:1000, CST), anti-phospho(p)ERK1/2 (1:1000, CST), anti-p38 MAPK (1:1000, CST), anti-pp38 MAPK (1: 1000, CST), anti- NF-κB p65 (1:1000, CST), anti-pp65 (1:1000, CST), anti-Akt (1: 1000, CST), anti-pAkt (1:1000, CST), anti-JNK1/2/3 (1:500, Huaan Biotechnology), anti-p-JNK1/2/3 (1: 500, Huaan Biotechnology), anti-MK2 (1:500, CST), anti-pMK2 (1: 500, CST), anti-ZO-1 (1:1000, Wanleibio), anti-claudin-5 (1:1000, Wanleibio), anti-Brn-3a (1:200, Santa), and β-actin (1:8000, Proteintech). After washing with TBST, blots were incubated with HRP-conjugated anti-rabbit IgG antibody (1:3000, CST) or HRP-conjugated anti-mouse IgG antibody (1:3000, CST) for 1.5 h at room temperature. Finally, protein amount was detected by the ECL reagent (Vazyme Biotech, Nanjing, China). Densitometric analysis was performed using Image J. Protein levels were normalized to either total protein or β-actin levels.

4.13. Development of HB Rats

HB rats were developed according to the previous report [18]. Briefly, 12 rats were randomly divided into bilirubin-treated and control groups. Bilirubin-treated rats received i.p. bilirubin (85.5 μmol/kg/d) injections consecutively for 14 days. Control rats only received vehicle. At 24 h following last dose, expression and function of Mrp1 in the retina of rats were assessed as described above.