Sodium Ferrous Citrate and 5-Aminolevulinic Acid Exert a Therapeutic Effect on Endotoxin-Induced Uveitis in Rats

The metabolism of 5-aminolevulinic acid (ALA) is more efficient when combined with sodium ferrous citrate (SFC). Our previous study revealed that oral administration of ALA, which has anti-inflammatory properties, and SFC (ALA/SFC) immediately before lipopolysaccharide (LPS) inoculation suppressed endotoxin-induced uveitis (EIU) in rats. However, the therapeutic effect of ALA/SFC post-administration remains unexplored. Hence, this study aimed to evaluate the therapeutic efficacy of ALA/SFC on EIU in rats, which were administered with a gastric gavage of ALA/SFC (100/157 mg/kg) or prednisolone (Pred, 10 mg/kg) after 4 h of LPS inoculation. The treatment groups showed ameliorated clinical scores, inflammatory cells, protein levels in the aqueous humor (AqH), and histopathologic evaluation 24 h after LPS inoculation. Furthermore, the treatment groups had reduced tumor necrosis factor-α, nitric oxide, prostaglandin E2, and interleukin-6 levels in the AqH. ALA/SFC demonstrated an anti-inflammatory effect equivalent to that demonstrated by Pred. These findings indicate that ALA/SFC exerts a therapeutic effect on EIU in rats, indicating its clinical usefulness in uveitis treatment.

Corticosteroids are the current mainstays of uveitis treatment to control the inflammatory process [8,9].Corticosteroids are effective for the acute and chronic treatment of noninfectious eye inflammations but can cause side effects, especially after long-term treatment of chronic and recurrent eye inflammation [10][11][12][13].Additionally, patients with uveitis having an infectious or corneal ulcer do not benefit from the treatment.Therefore, safe and effective novel therapies are highly desirable.
Endotoxin-induced uveitis (EIU) is induced in rats by injecting lipopolysaccharide (LPS) [14].Elevated inflammatory cytokines are also observed in EIU, causing cellular infiltration and protein leakage into the AqH 4 h after the LPS injection [5,15,16].The number of infiltrating cells and protein levels in the AqH peaks 18-24 h after the LPS injection [5,15,16].
The therapeutic efficacy of drugs administered after the onset of inflammation, which is 4-6 h after LPS inoculation, has been investigated in EIU rats to explore their potential as therapeutic agents for uveitis [17,18].
This study aimed to investigate the therapeutic efficacy of ALA/SFC in EIU rats and evaluate the anti-inflammatory potential of ALA/SFC compared to prednisolone (Pred).

Therapeutic Effect of ALA/SFC on Clinical Scoring and the Number of Infiltrating Cells and Protein Levels in AqH
Clinical scoring in the control group demonstrated no inflammatory response and normal iris vessels.The LPS group demonstrated intense severe iridial hyperemia with anterior chamber flare, fibrinous exudate in the pupillary margin, and a clinical score of 4.0 (median and interquartile range [IQR]; 3.0-4.0,n = 5).The groups treated with ALA/SFC (100/157 mg/kg) and Pred (10 mg/kg) demonstrated mild iridial hyperemia compared with the LPS group, and the clinical scores were significantly reduced to 2.0 (1.5-2.5, p < 0.05, vs. LPS) and 2.0 (1.0-2.0,p < 0.05, vs. LPS), respectively (Figure 1A,B).
The protein levels in the AqH were 1.20 ± 0.28 mg/mL and 36.41 ± 2.71 mg/mL (n = 5) in the control and LPS groups.ALA/SFC and Pred treatment significantly suppressed the protein concentrations in the AqH (ALA/SFC: 24.12 ± 3.3 mg/mL, p < 0.001; Pred at 10 mg/kg: 18.31 ± 1.85 mg/mL, p < 0.001).ALA/SFC had almost the same effect on the protein levels in the AqH as that for the Pred (Figure 1D).

ALA/SFC Improves Histopathological Evaluation
A histopathological evaluation of the control group revealed normal tissue.The mean histopathological score was 3.0 (3.0-3.0,n = 5) in the LPS group 24 h after the LPS injection, indicating severe uveitis with heavy inflammatory cell infiltration in the iris stroma, ciliary body, and anterior chamber.An improved inflammation response to LPS and a decreased number of infiltration cells resulted in significantly reduced histopathological scores in the ALA/SFC and Pred groups (ALA/SFC: 1.5 [1.0-2.0],p < 0.05; Pred at 10 mg/kg: 1.0 [1.0-1.5],p < 0.05, vs. LPS).The effect of ALA/SFC on the histopathological scores was almost comparable to Pred (Figure 2).

ALA/SFC Improves Histopathological Evaluation
A histopathological evaluation of the control group revealed normal tissue.The mean histopathological score was 3.0 (3.0-3.0,n = 5) in the LPS group 24 h after the LPS injection, indicating severe uveitis with heavy inflammatory cell infiltration in the iris stroma, ciliary body, and anterior chamber.An improved inflammation response to LPS and a decreased number of infiltration cells resulted in significantly reduced histopathological scores in the ALA/SFC and Pred groups (ALA/SFC: 1.5 [1.0-2.0],p < 0.05; Pred at 10 mg/kg: 1.0 [1.0-1.5],p < 0.05, vs. LPS).The effect of ALA/SFC on the histopathological scores was almost comparable to Pred (Figure 2).

Therapeutic Effect of ALA/SFC on TNF-α, IL-6, NO, and PGE 2 Levels in AqH
Increased inflammatory mediators of AqH, such as TNF-α, IL-6, NO, and PGE 2 , are a significant feature in EIU rats.Table 1 shows increases in these inflammatory mediator levels in the LPS group.ALA/SFC and Pred significantly inhibited these inflammatory mediator levels in the AqH (Table 1).

Discussion
ALA/SFC administration 4 h after the LPS injection significantly reduced the clinical uveitis score, LPS-induced cell infiltrate levels, AqH protein, and inflammatory mediators, and it decreased the histopathological score 24 h after the LPS injection.Moreover, ALA/SFC demonstrated comparable anti-inflammatory effects to Pred at 10 mg.
Numerous investigators have reported that ALA has multiple molecular targets, including transcription factors, cytokines, and apoptosis factors [33,34].In our previous study, ALA/SFC used as a preventive treatment suppressed inflammatory mediators in EIU rats via inhibition of the NF-κB signaling pathway and activation of the Nrf2/HO-1 signaling pathway [32].Inflammatory cytokines, such as TNF-α, IL-6, NO, and PGE 2 , which are released by various inflammatory cells, participate in the pathogenesis of EIU [5][6][7]35].This study revealed that ALA/SFC significantly attenuated ocular inflammation in EIU rats 24 h after the LPS injection.Inflammatory cell infiltration, protein leakage, and TNF-α, IL-6, NO, and PGE 2 production in the AqH were markedly reduced.Thus, the therapeutic effects of ALA/SFC may also be mediated by inhibiting inflammatory mediators.
This study used high ALA/SFC doses (100/157 mg/kg).Our previous study with a low ALA/SFC dose (10/15.7 mg/kg) revealed a protective anti-inflammatory effect in EIU rats [32].However, the dosage used in our previous preliminary studies did not show a therapeutic effect on EIU (Supplementary Table S1).A previous study revealed that administering a high dose of ALA/SFC (100/157 mg/kg) protected against binge alcoholinduced liver injury in HIV transgenic rats [27], and we based our dose of ALA/SFC on this study.The LD 50 for ALA alone single orally administrated to rats is >2000 mg/kg [36].The exact toxic dose of ALA/SFC orally administrated to rats is not yet known.However, no side effects or toxicity were observed within 20 h of ALA/SFC oral administration (100/157 mg/kg) in this study.Further studies are essential to evaluate the safety profile and toxicity of ALA/SFC, including its long-term use.As one way to address the issue of toxicity, we intend to elucidate the anti-inflammatory effects of ALA-containing eye drops in order to reduce the potential for systemic side effects.
In summary, to our knowledge, this is the first study to demonstrate the efficacy of ALA/SFC in ocular inflammation after the onset of EIU.Based on results of this study, ALA/SFC might have potential efficacy in treating uveitis.

Animals
This study used 6-week-old male Sprague-Dawley rats, weighing 180-220 g.The rats were purchased from SLC (Hamamatsu, Shizuoka, Japan).They were kept in airconditioned rooms with a 12 h light/dark cycle.All animal experiments were conducted according to the ARVO statement on the Care and Use of Laboratory Animals.This study was conducted following the Guidelines for Animal Experiments and the Animal Management Manual of the School of Veterinary Medicine, Kitasato University (Approval No. 22-017).

Endotoxin-Induced Uveitis
EIU was induced by subcutaneous injection of LPS at 200 µg diluted in 0.2 mL saline (100 µg each subcutaneously) into each footpad, under anesthesia with isoflurane (Mylan Inc., Canonsburg, PA, USA).A total of 120 rats were randomly divided into four groups, comprising 30 rats in each group: (i) the control group: subcutaneously injected with 0.2 mL of saline and received 10 mL/kg of saline; (ii) the LPS group: subcutaneously injected with 200 µg of LPS and received 10 mL/kg of saline; (iii) the ALA/SFC group: subcutaneously injected with 200 µg of LPS and received 100 mg/kg of ALA and 157 mg/kg of SFC; (iv) the Pred group: subcutaneously injected with 200 µg of LPS and received 10 mg/kg of Pred.Each drug was dissolved in 10 mL/kg of saline 4 h after the LPS injection and administered orally by the gastric tube.The ALA/SFC dosage ratio (molar ratio; ALA: SFC = 1:0.5)was determined based on previous studies [27,32].Clinical scoring was assessed 24 h after the LPS injection, and AqH and eye samples were collected and analyzed after euthanasia with isoflurane in deep anesthesia.

Evaluation of Clinical Scoring
The rats were evaluated for clinical scoring before euthanasia 24 h after the LPS injection.The clinical scores were assessed from scores 0-4, following a previously published system [32].Two observers who were not informed about the drug treatment assessed clinical scores of the left eye.Images of clinical symptoms were recorded with a digital camera ( ® PENTAX Q-S1, RICOH Imaging, Co, Ltd., Tokyo, Japan) (n = 5 in each group).

Infiltrating Cell Count and Protein Level Measurement in AqH
This analysis was performed as previously described [32].The rats were euthanized via overdose with isoflurane 24 h after the LPS injection.AqH in both eyes was obtained by a 30-gauge needle under a microscope (MEGA-21; Inami and Co., Ltd., Tokyo, Japan).AqH from the control group was not diluted for cell counting, but AqH from other groups was diluted 10-fold with sterile saline.The AqH samples were diluted 2-fold with Türk-staining solution and counted using a hematocytometer.Next, the AqH samples were centrifuged (2500 rpm, 5 min, 4 • C).The total protein concentration in the supernatant in AqH was determined using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL, USA) and diluted 5-fold with sterile saline for the control group and 100-fold for the other groups (n = 5 in each group).

Evaluation of Histopathologic Scores
The rats were euthanized 24 h after the LPS injection, and both eyes were promptly enucleated, stored in 4% paraformaldehyde in phosphate-buffered saline for 24 h at 4 • C, dehydrated in a graded ethanol series, and embedded in paraffin.Sagittal sections of 3 µm were cut near the optic disc and stained with hematoxylin and eosin (H&E).The histopathologic scores were graded 0-3, as previously described [32].A histopathological evaluation was performed on both eyes of 5 rats in each group, and the evaluators were unaware of the drug treatment.Each rat obtained a mean score for both eyes (n = 5 in each group).

Statistical Analysis
Nonparametric data, clinical and histopathologic scores, are expressed as median (IQR).Parametric data are expressed as mean ± SD.Statistical analyses were performed using EZR version 1.55, which is an open-source software [37].One-way analysis of variance and Tukey's post-hoc multiple comparison tests were used to analyze significant differences among groups in cell, protein, and cytokine levels in AqH.The Kruskal-Wallis and Steel-Dwass post-hoc multiple comparison tests were conducted to analyze significant differences in clinical and histopathologic scores between the two treatment groups.The significance was marked with p-values of <0.05 for all analyses.