Rebamipide promotes lacrimal duct epithelial cell survival via protecting barrier function

Nasolacrimal duct obstruction (NLDO) is thought to be due to inflammation and fibrosis of lacrimal duct epithelial cells (LDECs). Here we investigated the effect of rebamipide, a drug that is used for the protection of the mucosa and the treatment of gastritis and gastroduodenal ulcers, on LDECs, both in vitro and in vivo. In this study, LDECs were cultured from rabbit lacrimal duct tissues, and the barrier function of LEDCs was examined in vitro via transepithelial electrical resistance (TER) measurement, with or without interleukin (IL)-6 and/or rebamipide. For the in vivo examination, benzalkonium chloride (BAC) was injected into the rabbit lacrimal ducts, followed by the application of rebamipide or a placebo vehicle alone. The results of the in vitro examination revealed a significant decrease in TER in the group treated with IL-6 alone compared with the placebo-vehicle group (p < 0.05) and the group treated with IL-6 and rebamipide (p < 0.01). The results of the in vivo examination revealed that the infiltration of neutrophils under the basement membrane and the disruption of tight junction proteins with BAC injection and rebamipide attenuates the disturbance of tissue construction. These results suggest that rebamipide protects LDECs via an anti-inflammatory effect and preserves the barrier function of those cells.

It has been theorized that inflammation and age-related fibrosis of the lacrimal-duct epithelium are involved in the pathology of idiopathic nasolacrimal duct obstruction (NLDO) 1 . The common symptoms of NLDO include epiphora, discharges and dacryocystitis, an infection or inflammation of the lacrimal sac. Lacrimal stent intubation or dacryocystorhinostomy improves the symptoms, but these treatments are accompanied by surgical stress.
It has recently been reported that various tear cytokines, such as interleukin (IL)-2, IL-6, and IL-10, are involved in the pathology of primary acquired nasolacrimal duct obstruction (PANDO) 2 , and that IL-6 is also involved in congenital NLDO (CNLDO) 3 . Thus, the suppression of the level of inflammatory cytokines in tears is considered to be a key factor in the development of new methods for the treatment of NLDO.
Rebamipide, an oral drug that is used for the protection of the mucosa and the treatment of gastritis and gastroduodenal ulcers, is currently marketed in Japan as an ophthalmic suspension (Mucosta ® ; Otsuka Pharmaceutical Co., Ltd, Tokyo, Japan) for the treatment of dry eye. We previously reported that rebamipide has an anti-inflammatory effect and enhances the barrier function of cultured human corneal epithelial cells (CECs) 4 . In a previous study by Mimura et al., it was reported that the postoperative administration of rebamipide increases the rate of surgical success in patients who undergo lacrimal stent intubation for the treatment of PANDO 5 . However, the exact mechanism for the anatomical success in that study was not revealed. In this present study, we investigated the barrier function and protective effect of rebamipide on rabbit lacrimal duct epithelial cells (LDECs), both in vitro and in vivo.
Second, the LDECs were subcultured on 24-well dishes until cell confluence was achieved. The cells were stimulated with 1% benzalkonium chloride (BAC) with 2-mM rebamipide or the vehicle for 72-hours and fixed in 4% paraformaldehyde, and investigated with immunohistochemistry. As a control, the cells were incubated with medium (n = 2 each).
In vivo examination. We next investigated whether BAC affected the nasolacrimal duct epithelium in vivo, and whether rebamipide might prevent the damage of epithelium with BAC treatment. For the in vivo examination, the rabbits were first placed under general anesthesia via the injection of a cocktail containing ketamine and xylazine. Next, an LDEC damage model was created by injecting 250 µl of 1% BAC into the rabbit's lacrimal duct once daily for 3 consecutive days. After verification that the LEDC damage model was successfully created, 250 μl of 2% rebamipide ophthalmic solution (Mucosta ® ophthalmic suspension UD2%) or the vehicle of rebamipide was injected (n = 3 each) simultaneously with the injection of 1% BAC. As a control, phosphate buffered saline (PBS) was injected into the lacrimal duct of 3 rabbits.
Next, lacrimal duct tissue was obtained and embedded in optimal cutting temperature compound (Tissue-Tek ® O.C.T.; Sakura ® Finetek Japan Co., Ltd., Tokyo, Japan). The tissue was then cut into 7-um sections, fixed in 4% paraformaldehyde, and investigated with histological examination and immunohistochemistry (n = 3 each). The thickness of the LDECs was then measured via the results of the histological examinations by another investigator, and the measurements were compared between the 3 groups.

Analysis via scanning electron microscopy (SeM).
For SEM analysis, the obtained tissue samples that were first fixed in 0.1 mol phosphate buffered 2% glutaraldehyde, then subsequently post-fixed in 2% osmium tetroxide for 2 hours in an ice bath. The specimens were then dehydrated in a graded ethanol and dried by frozen t-butyl alcohol in a vacuum. The freeze-dried specimens then underwent SEM examination (JEM-7500F; JEOL Ltd., Tokyo, Japan) after being coated with an osmium plasma ion coater device.
Statistical analysis. Statistical analysis of the data was performed and basic descriptive statistics were calculated on all of the gathered data, with the values reported as mean ± SD. Results were compared among three groups by Bonferroni correction. A p value of <0.05 was considered statistically significant.

Effect of rebamipide on damaged LDECs in vivo.
We next investigated the effect of rebamipide on 1% BAC-damaged LDECs in vivo (Fig. 2). We found that the LDECs were disrupted and the neutrophils were accumulated under the connective tissue in the group injected with 1% BAC and the vehicle of rebamipide (Fig. 2b). However, those inflammation-associated tissue disorders were not observed in the group injected with 1% BAC and rebamipide (Fig. 2c). The thickness of the LDECs in the control group, the 1% BAC and the vehicle of rebamipide injected group, and the rebamipide injected group was 76.5 ± 8.1 μm, 8.41 ± 0.53 μm, and 63.3 ± 4.4 μm, respectively (p < 0.05) (Fig. 2d). Immunohistological examination findings confirmed the expression of tight junction proteins ZO-1, CLDN1, and CLDN7 between the epithelial cells in the control group and in the 1% BAC and rebamipide injected group, yet disruption in the 1% BAC and vehicle of rebamipide injected group (Fig. 3).
Analysis of the SEM images revealed healthy microvilli on the surface of the LDECs in the group injected with 1% BAC and rebamipide, similar to the findings observed in the control group. On the other hand, healthy    www.nature.com/scientificreports www.nature.com/scientificreports/ microvilli were not observed accompanied with a disruption of the LDECs in the group injected with 1% BAC and the vehicle of rebamipide (Fig. 4).

Discussion
In this study, we successfully cultivated LDECs in vitro and established a rabbit lacrimal-duct-damage model with BAC in vivo. Our findings revealed that rebamipide attenuates the IL-6-induced decrease of barrier function between rabbit LDECs in vitro, and that it has protective effects on rabbit LDECs in a BAC-induced lacrimal-duct-damage model that disrupts the tight junctions between the cells in vivo. These results suggest that rebamipide suppresses the inflammatory-induced barrier function damage and helps to maintain the LDEC structure, thus protecting against tissue disruption.
Although numerous clinical research and anatomy-related studies focusing on lacrimal ducts have previously been performed, there have been few published basic-research reports on the related cell biology. In this present study, we were able to successfully cultivate LDECs until confluence, and subculture the LDECs using the method previously described by Xie et al. 6 .
Inflammation is thought to be a pathology of NLDO, and it has been reported that several inflammatory cytokines, such as IL-2, IL-6, IL-10, vascular endothelial growth factor, and fibroblast growth factor 2, are increased in the tear fluid of patients with PANDO 2 , and that IL-6, specifically, is increased in the tear fluid of patients with CNLDO 3 . As the findings in several previous reports have suggested that IL-6 attenuates the barrier function between cells 7,8 , our results revealed that IL-6 induced barrier dysfunction of cultured LDECs. In this present study, our findings suggest that the anti-inflammatory effect of rebamipide can maintain intercellular barrier function by suppressing IL-6 inflammation. Based on these results, anti-inflammatory drugs might possibly be one key element in the treatment of lacrimal-duct disease.
Several studies on the anatomy of lacrimal ducts in various animals have been reported, and the findings in those studies have shown that rabbit lacrimal ducts are suitable for creating models to clarify disease pathology, as the anatomy of those ducts is similar to human lacrimal ducts 6,9 . Via the injection of BAC, we were able to successfully create a rabbit lacrimal duct damage model. Histological examination revealed obvious disruption of LDECs with the injection of 1% or more BAC. Thus, 1% was selected as the appropriate percentage of BAC to create the lacrimal duct damage model (Supplemental Fig. 2). BAC is frequently used as an ophthalmic preservative, and it is also known to cause ocular tissue damage 10,11 . In fact, the long-term use of anti-glaucoma eye drops, specifically, is known to cause inflammation and fibrosis-related changes in conjunctiva 12 , and is also known to be a risk factor for the development of NLDO 13 . Those changes may be related to the preservatives in the commercial preparations, and/or the medications themselves 14 . Hence, in this study, we chose to use a rabbit lacrimal duct damage model treated with BAC, and the infiltration of neutrophils was observed in addition to epithelial disorder in this model, thus indicating that BAC injection resulted in acute inflammation in the lacrimal duct. Moreover, our findings revealed that rebamipide also antagonizes the BAC-induced shedding of LDECs with anti-inflammatory actions.
It has been reported that rebamipide was first developed as a gastroprotective drug due to its ability to increase gastric mucus production 15,16 and suppress gastric mucosal inflammation 17,18 . In Japan, it has also been used as an ophthalmic suspension for the treatment of dry eye. Previous studies have reported that rebamipide has an anti-inflammation effect on CECs 4 , corneal fibroblasts 19 , and conjunctival epithelial cells (CjECs) 20 . It has also been reported that rebamipide helps to maintain the microvilli of CECs and CjECs against N-acetylcysteine 21 and the barrier function between CECs 4 .
Moreover, it has been reported that human LDECs are faced with microvilli 22 , the same as in rabbit LDECs. In this present study, we found that rebamipide also helps to maintain the microvilli of LDECs. In regard to lacrimal duct disease, it has been reported that rebamipide clinically improved postoperative results after tube intubation for PANDO 5 , however, the specific underlying mechanisms for that improvement are still unknown. In this present study, our findings showed that rebamipide improves the disruption of barrier function of LDECs, thus suggesting that its use post-surgery might improve surgical outcomes.
In summary, the findings in this study show that rebamipide has a protective effect against inflammatory cytokines that helps to recover the barrier function of LDECs in vitro, and that pre-administered rebamipide suppressed BAC-induced cell damage in vivo. Although rebamipide already has a long and well-recognized history of being applied for the treatment of corneal and gastro-intestinal diseases, maybe it can possibility now be repositioned as an effective drug for the protection of LDECs as well.