5-ALA/SFC Ameliorates Endotoxin-Induced Ocular Inflammation in Rats by Inhibiting the NF-κB Signaling Pathway and Activating the HO-1/Nrf2 Signaling Pathway

Sodium ferrous citrate (SFC) is involved in the metabolism of 5-aminolevulinic acid (5-ALA) and enhances its anti-inflammatory effects. The effects of 5-ALA/SFC on inflammation in rats with endotoxin-induced uveitis (EIU) have yet to be elucidated. In this study, during lipopolysaccharide injection, 5-ALA/SFC (10 mg/kg 5-ALA plus 15.7 mg/kg SFC) or 5-ALA (10 or 100 mg/kg) was administered via gastric gavage, wherein we saw that 5-ALA/SFC ameliorated ocular inflammation in EIU rats by suppressing clinical scores; by infiltrating cell counts, aqueous humor protein, and inflammatory cytokine levels; and by improving histopathological scores to the same extent as 100 mg/kg 5-ALA. Immunohistochemistry showed that 5-ALA/SFC suppressed iNOS and COX-2 expression, NF-κB activation, IκB-α degradation, and p-IKKα/β expression, and activated HO-1 and Nrf2 expression. Therefore, this study has investigated how 5-ALA/SFC reduces inflammation and revealed the pathways involved in EIU rats. 5-ALA/SFC is shown to inhibit ocular inflammation in EIU rats by inhibiting NF-κB and activating the HO-1/Nrf2 pathways.


Introduction
Subcutaneous injection of lipopolysaccharide (LPS) leads to endotoxin-induced uveitis (EIU) in rats, an animal model of acute anterior uveitis [1]. In this model, inflammatory cells infiltrate the disrupted blood-aqueous barrier, and proteins leak into the aqueous humor (AqH). Acute inflammatory responses start 4 h after LPS injection and peak in 18-24 h [2,3]. EIU is a commonly used animal model for studying drug pharmacological and immunological effects on intraocular inflammation [4][5][6]. It has been suggested that the elevated expression of inflammatory cytokines, such as TNF-α, IL-1β, IL-6, NO produced by iNOS, and PGE2 produced by COX-2, are significant in the pathogenesis of EIU [7][8][9]. Furthermore, inflammatory cells infiltrating the iris ciliary body (ICB) are known to be involved in the production of these cytokines [10,11]. The expression of these cytokines has been shown to be regulated by NF-κB in intraocular tissues, including ICB [12][13][14][15].
NF-κB, a dimer of p50, and p65 maintain a quiescent state in the cytoplasm by binding to IκB-α to prevent nuclear translocation signals [16,17]. Upon stimulation with LPS, IκB-α is phosphorylated by p-IKK and degraded, and the p65 subunit is transferred to the nucleus, where it induces inflammatory gene expression [18]. IKK consists of three subunits (IKKα, IKKβ, and IKKγ) and IKKβ is mainly involved in IκB-α phosphorylation. Inhibiting NF-κB activation-p65 nuclear translocation-may be a therapeutic target for ocular inflammation, where NF-κB activation is considered essential to EIU pathogenesis because NF-κB inhibitors reduce inflammation in EIU [19][20][21][22]. 2 of 12 HO-1 is a hematolytic enzyme induced by several stimuli, including LPS, oxidative stress, and ischemia-reperfusion [23,24]. Ohta et al. reported that hemin increases HO-1 expression in ICB and suppresses ocular inflammation in the EIU rats [25]; thus, the heme degradation products from HO-1, biliverdin, and iron, producing carbon monoxide, also contribute to anti-inflammation [26,27]. The anti-inflammatory effects of HO-1, which prevent nuclear translocation of NF-κB and suppress the production of inflammatory mediators [28], are thought to be due to the suppression of IκB-α degradation. Thus, enhancing HO-1 expression is considered anti-inflammatory. HO-1 is mainly expressed via Nrf2 [29], which remains quiescent in the cytoplasm and translocates into the nucleus when stimulated by LPS or other agents to activate antioxidant-responsive gene sequences such as HO-1 [30].
5-Aminolevulinic acid (5-ALA) has been detected in foods and has attracted attention in recent years due to its diverse actions [31][32][33]. In animal cells, 5-ALA synthesis is mediated by the mitochondrial aminolevulinic acid synthase from glycine and succinate CoA. Porphyrinogens, precursors of protoporphyrin IX (PpIX), are formed by condensation polymerization of eight molecules of 5-ALA. Heme is formed in the mitochondria of PpIX by inserting iron ions [34]. Interestingly, a combination of sodium ferrous citrate (SFC) and 5-ALA (5-ALA/SFC) has been suggested to be effective in rats, mice, and humans [35][36][37][38][39]. Recently, although the effects of 5-ALA/SFC have not been elucidated in this model, we found that 5-ALA alone exhibits potent anti-inflammatory effects by suppressing antiinflammatory mediators in EIU rats [40].
Previous studies have demonstrated the HO-1 expression upregulation function of 5-ALA/SFC compared to 5-ALA [31,41]. Additionally, 5-ALA/SFC has been shown to suppress NF-κB activation and subsequent inflammatory mediator expression in a murine fatty liver ischemia-reperfusion model [42]. With this study aiming to investigate the anti-inflammatory effects of 5-ALA/SFC and elucidate the pathways involved in EIU rats, which are yet to be elucidated, we hypothesize that the 5-ALA/SFC mechanism may also be related to the NF-κB and HO-/Nrf2 pathways in EIU rats.

5-ALA/SFC Downregulates the Expression of iNOS and COX-2 in ICB
The iNOS and COX-2 expression in the ICB of the LPS group increased 24 h after the LPS injection compared to the control group. Inhibition of iNOS and COX-2 expression by 5-ALA (100 mg/kg 5-ALA) and 5-ALA/SFC (100 mg/kg) was observed in ICB. Minimal iNOS and COX-2 expression was observed in the control group ( Figure 4).

5-ALA/SFC Downregulates the Expression of iNOS and COX-2 in ICB
The iNOS and COX-2 expression in the ICB of the LPS group increased 24 h after the LPS injection compared to the control group. Inhibition of iNOS and COX-2 expression by 5-ALA (100 mg/kg 5-ALA) and 5-ALA/SFC (100 mg/kg) was observed in ICB. Minimal iNOS and COX-2 expression was observed in the control group ( Figure 4).

5-ALA/SFC Upregulates the Nrf2/HO-1 Pathway
Immunoreactivity of activated Nrf2 and HO-1 was strongly expressed, and nuclear translocation was observed in the LPS group ICB 3 h after LPS injection. 5-ALA/SFC tended to activate Nrf2 and HO-1 expression in ICB compared to the LPS group. Minimal expression of Nrf2 and HO-1 in ICB was observed in the control group. With 100 mg/kg doses, 5-ALA regulated the Nrf2/HO-1 pathway similarly to 5-ALA/SFC ( Figure 6).

5-ALA/SFC Upregulates the Nrf2/HO-1 Pathway
Immunoreactivity of activated Nrf2 and HO-1 was strongly expressed, and nuclear translocation was observed in the LPS group ICB 3 h after LPS injection. 5-ALA/SFC tended to activate Nrf2 and HO-1 expression in ICB compared to the LPS group. Minimal expression of Nrf2 and HO-1 in ICB was observed in the control group. With 100 mg/kg doses, 5-ALA regulated the Nrf2/HO-1 pathway similarly to 5-ALA/SFC ( Figure 6).

5-ALA/SFC Upregulates the Nrf2/HO-1 Pathway
Immunoreactivity of activated Nrf2 and HO-1 was strongly expressed, and nuclear translocation was observed in the LPS group ICB 3 h after LPS injection. 5-ALA/SFC tended to activate Nrf2 and HO-1 expression in ICB compared to the LPS group. Minimal expression of Nrf2 and HO-1 in ICB was observed in the control group. With 100 mg/kg doses, 5-ALA regulated the Nrf2/HO-1 pathway similarly to 5-ALA/SFC ( Figure 6).

Discussion
This study demonstrates that 5-ALA/SFC is as effective as 100 mg/kg 5-ALA in reducing inflammation in EIU. The 5-ALA/SFC suppressed the clinical scores, number of infiltrating cells, and protein leakage into the AqH. In addition, it inhibited inflammatory cytokines (IL-6 and TNF-α) and mediators (PGE2 and NO) released into the AqH, and, in a manner comparable to 100 mg/kg 5-ALA alone and 1 mg/kg of prednisolone, improved histopathologic scores. 5-ALA/SFC inhibited NF-κB p65 nuclear translocation, IKKα/β phosphorylation, and IκB-α degradation in the ICB of EIU rats. In addition, in results suggesting that 5-ALA/SFC inhibits EIU development by NF-κB signaling suppression and Nrf2/HO-1 signaling activation, 5-ALA/SFC promoted nuclear translocation of Nrf2 in the ICB of EIU rats, which increased HO-1 expression. A similar mechanism was observed with 100 mg/kg 5-ALA.
The features of EIU include blood-ocular barrier disruption and increased total protein levels in the anterior chamber [4]. Thus, total protein concentration, infiltrating cells, and inflammatory cytokines in AqH indicate the severity of inflammation. Inflammatory cytokines, released by a wide variety of inflammatory cells, such as TNF-α, IL-6, NO, and PGE2, participate in EIU pathogenesis [43]. Our previous study revealed that 100 mg/kg 5-ALA has anti-inflammatory effects on EIU rats by inhibiting the subsequent production of inflammatory mediators [40], while this study demonstrated that 5-ALA/SFC could significantly attenuate TNF-α, IL-6, NO, and PGE2 production in the AqH, thereby indicating that 5-ALA/SFC suppressed these cytokines to the same extent as 100 mg/kg 5-ALA. Furthermore, this suggests that 5-ALA/SFC is more synergistic than the additive effect of 5-ALA alone.
NO and PGE2 play a role in EIU pathogenesis [10,44]. In rats, LPS injection increases iNOS and COX-2 expression in the ICB and is involved in NO and PGE2 production [2,45]. Inhibition of NO and PGE2 synthesis may have a therapeutic effect on uveitis due to the additive effects of iNOS/NO and COX-2/PGE2 synthesis in EIU [46]. 5-ALA/SFC suppressed iNOS and COX-2 expression in the ICB of EIU rats, and the levels of NO and PGE2 in AqH might be attributed to iNOS and COX-2 suppression in the ICB.
NF-κB activity is increased in the ICB of EIU rats, leading to the development of EIU due to the excessive production of inflammatory mediators in the ocular tissues [47,48]. Several reports show that NF-κB inhibitors prevent uveitis in EIU rats [49][50][51]. The 5-ALA may reduce NF-κB activity in a rat model of ischemia-reperfusion injury [41]. In this study, our results indicate that, by suppressing NF-κB activation, 5-ALA/SFC reduced iNOS and COX-2 protein expression and, subsequently, NO, and PGE2 production, with us having showed, for the first time, that 5-ALA/SFC suppresses LPS-induced phosphorylation of the IKK, degradation of IκB-α, and the subunit of NF-κB, thereby preventing further NF-κB translocation in ICB.
Enhanced HO-1 expression early after LPS injection is considered an important therapeutic target for uveitis [25], with HO-1 exhibiting anti-inflammatory effects in the EIU rat model. In this study, 5-ALA/SFC enhanced HO-1 expression in ICB 3 h after LPS injection in the EIU rat model, further suggesting that part of the anti-inflammatory mechanism of 5-ALA/SFC in the EIU rat model is its ability to enhance the nuclear translocation of Nrf2 and consequent HO-1 expression. Thus, 5-ALA/SFC enhanced the nuclear translocation of Nrf2 3 h after LPS injection. In addition, HO-1 has been reported to inhibit the degradation of IκB-α [26]. In the present study, 5-ALA/SFC enhanced HO-1 expression and inhibited IκB-α degradation in ICB 3 h after LPS injection, suggesting that the subsequent inhibition of NF-κB nuclear translocation may be involved in early HO-1 expression.
In this study, EIU was suppressed by administering 5-ALA/SFC simultaneously with EIU induction. To clarify the therapeutic effect of 5-ALA/SFC on ocular inflammation, additional experiments are needed, such as administering 5-ALA/SFC after inducing EIU. Therefore, further experiments with 5-ALA/SFC in EIU 4 h after LPS injection are needed.
In conclusion, this study suggests that 5-ALA/SFC treatment on EIU attenuates ocular inflammation by inhibiting the expression and release of inflammatory mediators and inhibiting the activated NF-κB and Nrf2/HO-1 pathways in ICB. Therefore, it is seen that 5-ALA/SFC has been demonstrated to be effective in uveitis, and further research may yield a novel treatment for uveitis.

Animals
The study used 180 male Sprague Dawley rats (6-week-old, 180-220 g). The rats were purchased from SLC (Hamamatsu, Japan) and maintained in an air-conditioned room with a 12 h light/12 h dark cycle. The ARVO Statement on the Care and Use of Laboratory Animals guided the experiments in this study. This study was approved by the Animal Care and Use Committee of Kitasato University (No. 20-068), and sample size calculations were carried out using the open-source software R version 4.2.2 [52] before the study began, confirming that the study had sufficient statistical power to detect between-group differences.

Induction of EIU Rats
EIU was injected subcutaneously into each footpad with 200 µg LPS diluted in 0.2 mL saline sterilized (100 µg each subcutaneously) under anesthesia with isoflurane (Mylan Inc., Canonsburg, PA, USA). In the 10 m/kg 5-ALA, 100 mg/kg 5-ALA, 5-ALA/SFC groups (10 mg/kg 5-ALA plus 15.7 mg/kg SFC), and the 1 mg/kg Pred group, each reagent dissolved in 10 mL/kg saline were administered intragastrically to each rat at the time of LPS injection. With the dose ratio of 5-ALA/SFC determined with reference to a previous study [35], in the LPS group, each rat received 10 mL/kg saline in the same schedule. In the control group, each rat received the same volume of saline and was injected subcutaneously with 0.2 mL saline without LPS. All rats were randomly divided into groups comprising five animals each.

Clinical Scoring
The clinical manifestations were recorded in images using a digital camera ( ® PENTAX Q-S1, RICOH Imagining, Co., Ltd., Tokyo, Japan) and evaluated in the left eye by two blinded observers 24 h after the LPS injection and before euthanization. The clinical scores were evaluated from 0 to 4 according to a previous study [53].

Number of Infiltrating Cells and Proteins in AqH
This analysis was performed as described in our previous report [39]. In brief, rats were euthanized with an overdose of isoflurane 24 h after LPS injection. AqH was collected from both eyes using a 30-gauge needle under the surgical microscope (OME-1000; Olympus Optical Co., Ltd., Tokyo, Japan). AqH samples from the control group were not diluted, whereas those from other groups were diluted 10-fold with sterile saline. AqH samples were added in equal parts to the Türk stain solution and counted using a hematocytometer (Bürker-Türk hemocytometer; Erma Inc., Tokyo, Japan). After counting, AqH samples were centrifuged (2500 rpm, 5 min, 4 • C). The supernatants were diluted 5 fold and 100 fold with sterilized saline in the control and other groups, respectively. A bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA) was used to measure the total protein concentration in AqH.

Histopathologic Evaluation
Rats were euthanized 24 h after the LPS injection. Both eyes were enucleated under a surgical microscope, fixed in 4% paraformaldehyde in PBS for 24 h at 4 • C, and embedded in paraffin. Sagittal sections (3 µm) were cut near the optic nerve head and stained with hematoxylin and eosin (H&E). Light microscopy was used to observe ICB masked in the anterior chamber. The histopathologic evaluation of inflammation was graded 0-3, as described previously [35,40]. Both eyes of the five rats from each group were analyzed. 4.7. The Levels of TNF-α, IL-6, PGE2, and NO in AqH The TNF-α, IL-6, and PGE2 levels in the AqH 24 h after LPS injection were assessed using a commercially available ELIS kit (TNF-α: KE20001; Proteintech Group Inc., Rosemont, IL, USA; IL-6: #BMS625; Thermo Fisher Scientific, Waltham, MA, USA; PGE2: 500141; Cayman Chemical Co., Ann Arbor, MI, USA) according to the manufacturer's instructions (n = 5). The total nitrate/nitrite in AqH was measured using a NO 2 /NO 3 colorimetric assay kit (NK05; Dojindo Molecular Technologies Inc., Kumamoto, Japan) (n = 5).

Statistical Analysis
StatMate V (ATMS Co., Ltd., Tokyo, Japan) was used for statistical analyses. Parametric data were analyzed using variance (ANOVA), and an ad hoc comparison between the two treatment groups was performed using the Tukey test. Nonparametric data were analyzed using the Kruskal-Wallis test, and an ad hoc comparison between the two treatment groups was performed using the Newman-Keuls test. All data are expressed as the mean ± standard deviations. A p-value of less than 0.05 was considered statistically significant.