The Protective Effect of Oral Application of Corni Fructus on the Disorders of the Cornea, Conjunctiva, Lacrimal Gland and Retina by Topical Particulate Matter 2.5

Particulate matter 2.5 (PM2.5) may aggravate dry eye disease (DED). Corni Fructus (CF), which is fruit of Cornus officinalis Sieb. et Zucc., has been reported to have various beneficial pharmacological effects, whereas the effect of CF on the eye is still unknown. Therefore, in this study, we investigated the effect of oral administration of water extract of CF (CFW) on the eye, hematology, and biochemistry in a DED model induced by topical exposure to PM2.5. Furthermore, the efficacy of CFW compared with cyclosporine (CsA), an anti-inflammatory agent, and lutein, the posterior eye-protective agent. Sprague-Dawley rats were topically administered 5 mg/mL PM2.5 in both eyes four times daily for 14 days. During the same period, CFW (200 mg/kg and 400 mg/kg) and lutein (4.1 mg/kg) were orally administered once a day. All eyes of rats in the 0.05% cyclosporine A (CsA)-treated group were topically exposed to 20 μL of CsA, twice daily for 14 days. Oral administration of CFW attenuated the PM2.5-induced reduction of tear secretion and corneal epithelial damage. In addition, CFW protected against goblet cell loss in conjunctiva and overexpression of inflammatory factors in the lacrimal gland following topical exposure to PM2.5. Furthermore, CFW markedly prevented PM2.5-induced ganglion cell loss and recovered the thickness of inner plexiform layer. Meanwhile, CFW treatment decreased the levels of total cholesterol and low-density lipoprotein cholesterol in serum induced by PM2.5. Importantly, the efficacy of CFW was superior or similar to that of CsA and lutein. Taken together, oral administration of CFW may have protective effects against PM2.5-induced DED symptoms via stabilization of the tear film and suppression of inflammation. Furthermore, CFW may in part contribute to improving retinal function and lipid metabolism disorder.


Introduction
Air pollution is a serious health issue and consists of thousands of solid particles, gases, and liquid droplets [1]. Among the various biologically toxic substances of air pollutants, particulate matter 2.5 is considered the most important harmful substrate of health threat [2]. PM 2.5 refers to particulate matter with diameter less than 2.5 µm [2]. Several epidemiological and biochemical studies have shown that chronic or acute exposure to PM 2.5 may be attributed to dry eye disease (DED), also known as keratoconjunctivitis sicca [3][4][5][6][7]. DED is a multifactorial disease of the ocular surface that involves tear film instability,

Tear Production
On days 0, 7, and 14 post-treatment, tear volume was measured using phenol red tear threads (Jingming Ltd., Tianjin, China) [35]. Briefly, threads were inserted into the lateral canthus of the lower eyelid for 1 min. The length of the red portion of the threads was measured, and the tear volume was expressed in millimeters (mm).

Hematoxylin and Eosin (H&E) Staining
Fixed eyeballs and lacrimal glands were embedded in paraffin and cut into 5 µm sections using a microtome (Leica RM2245, Leica Biosystems, Heidelberg, Germany). The sections were deparaffinized, hydrated, and stained with hematoxylin and eosin (YD Diagnostics Co., Yongin, Korea) The stained slides were observed using the EVOS FL Auto 2 imaging system (Thermo Fisher Scientific, Waltham, MA, USA).

Periodic Acid-Schiff (PAS) Staining
Paraffin-embedded sections of the entire globe were cut into 5 µm thickness, and stained using a PAS kit (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) according to the manufacturer's protocol. Images of violet PAS-positive goblet cells were acquired using a microscope (Carl Zeiss, Oberkochen, Germany) at the Core Facility Center for Tissue Regeneration (Dong-eui University, Busan, Korea).

Statistical Analysis
Data are presented as the mean ± standard deviation. One-way analysis of variance (ANOVA) and post hoc analyses were performed for comparisons between groups using GraphPad Prism 5.03 (GraphPad Software Inc., La Jolla, CA, USA). Statistical significance was set at p < 0.05.

Effect of CFW on the Physiological Changes in PM 2.5 -Exposed Sprague-Dawley (SD) Rats
Body weight was measured in all groups at 0 and 14 days after topical exposure to PM 2.5 , with or without treatment. As shown in Table 1, no significant differences were observed in body weight gain and organ weight between the normal group and PM 2.5 -exposed groups. Table 1. Effect of oral administration of CFW on the changes of the body weight and organ weight in PM 2.5 -exposed SD rats. The hematological analysis results showed no differences among the groups (Table 2). In addition, no biochemical abnormalities were observed among the groups. However, the levels of total cholesterol (TC) were markedly increased to 74.47 mg/dL following topical exposure to PM 2.5 , and the levels significantly decreased to control levels after topical administration of 400 mg/kg CFW, which was similar to the levels observed after CsA topical administration. Moreover, low-density lipoprotein cholesterol (LDL-C) levels were also significantly upregulated in the control group, and the levels were downregulated following 200 mg/kg and 400 mg/kg CFW administration. However, oral administration of lutein and topical administration of CsA did not improved serum LDL-C levels. Meanwhile, the levels of high-density lipoprotein cholesterol, triglyceride (TG), and free fatty acid were not different among the groups.

Effect of CFW on Tear Secretion after Topical Exposure to PM 2.5 in SD Rats
We investigated the effect of the oral administration of CFW on the changes in tear production in PM 2.5 -applied SD rats. On days 0, 7, and 14, the tear volume was measured using phenol red tear threads. At day 0, no significant difference was observed in tear production among all groups ( Figure 1). During the entire study period, the control group showed no significant difference in tear production. At day 7, tear volume was markedly suppressed in the PM 2.5 -treated DED group (4.90 ± 1.14 mm) compared with that in the control group (6.70 ± 1.05 mm). Although the CFW 200 group (5.08 ± 0.66 mm) and the CFW 400 group (5.83 ± 0.75 mm) showed no significant difference in tear volume compared with the DED group, the tear production gradually increased in the CFW 400 group. At day 14, PM 2.5 treatment greatly suppressed tear secretion to 3.83 ± 0.61 mm, but oral supplements of 200 mg/kg CFW and 400 mg/kg CFW significantly increased tear production to 5.10 ± 0.86 mm and 5.65 ± 0.71 mm, respectively. Meanwhile, tear production in the lutein group and CsA group was also markedly enhanced compared to that in the DED group at day 14 after treatment. The data are expressed as mean ± standard deviation (n = 5). ** p < 0.01 and *** p < 0.001 compared to the control group. # p < 0.05 and ## p < 0.01 compared to PM 2.5 -induced DED group. RBC, red blood cell; WBC, white blood cell; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, MCH concentration; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; BUN, blood urea nitrogen; TC, total cholesterol; TG, triglyceride; HDL-C, high-density lipoprotein cholesterol; LDL, low-density lipoprotein cholesterol; FFA, free fatty acid.
FOR PEER REVIEW 6 of 15 Figure 1. Effect of oral supplements of CFW on tear secretion after topical exposure to PM2.5 in SD rats. On days 0, 7, and 14, tear volume was determined using phenol red tear threads and the length of color-changed thread was measured. The data are expressed as the means ± standard deviation (n = 5). * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the control group. # p < 0.05, ## p < 0.01, and ### p < 0.001 compared to the PM2.5-induced DED group.

Effect of CFW on Detachment of Corneal Epithelium in PM2.5-Induced DED Rat Model
To investigate whether topical application of PM2.5 changes the corneal epithelium Figure 1. Effect of oral supplements of CFW on tear secretion after topical exposure to PM 2.5 in SD rats. On days 0, 7, and 14, tear volume was determined using phenol red tear threads and the length of color-changed thread was measured. The data are expressed as the means ± standard deviation (n = 5). * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the control group. # p < 0.05, ## p < 0.01, and ### p < 0.001 compared to the PM 2.5 -induced DED group.

Effect of CFW on Detachment of Corneal Epithelium in PM 2.5 -Induced DED Rat Model
To investigate whether topical application of PM 2.5 changes the corneal epithelium and the effect of CFW on PM 2.5 -mediated epithelium alteration, we performed H&E staining. Figure 2A shows that the detachment and swelling of corneal epithelium were more frequently observed in the DED group; however, CFW 200, CFW 400, lutein, and CsA treatments were markedly protect against PM2.5-induced the alteration. Figure 2B shows that the quantitative values of the detached epithelium are indicated as number per 100 µm 2 . Topical exposure to PM 2.5 markedly enhanced the detachment of corneal epithelium to 4.13 ± 0.63/100 µm 2 . However, the population of detachment epithelium was markedly decreased to 2.63 ± 0.48/100 µm 2 in the CFW 200 group. Additionally, the detachment of corneal epithelium in the CFW 400 group was greatly suppressed to 1.38 ± 0.47/100 µm 2 compared with that in the DED group. Meanwhile, the efficacy of administration of lutein and CsA on the corneal epithelium detachment was similar, and the levels were suppressed. This result indicated that oral administration of CFW had a protective effect on the detachment of corneal epithelium by topical exposure to PM 2.5 , and its efficacy was superior to that of lutein and CsA.

Effect of CFW on Detachment of Corneal Epithelium in PM2.5-Induced DED Rat Model
To investigate whether topical application of PM2.5 changes the corneal epitheliu and the effect of CFW on PM2.5-mediated epithelium alteration, we performed H&E stai ing. Figure 2A shows that the detachment and swelling of corneal epithelium were mo frequently observed in the DED group; however, CFW 200, CFW 400, lutein, and Cs treatments were markedly protect against PM2.5-induced the alteration. Figure 2B show that the quantitative values of the detached epithelium are indicated as number per 1 μm 2 . Topical exposure to PM2.5 markedly enhanced the detachment of corneal epitheliu to 4.13 ± 0.63/100 μm 2 . However, the population of detachment epithelium was marked decreased to 2.63 ± 0.48/100 μm 2 in the CFW 200 group. Additionally, the detachment corneal epithelium in the CFW 400 group was greatly suppressed to 1.38 ± 0.47/100 μm compared with that in the DED group. Meanwhile, the efficacy of administration of lute and CsA on the corneal epithelium detachment was similar, and the levels were su pressed. This result indicated that oral administration of CFW had a protective effect o the detachment of corneal epithelium by topical exposure to PM2.5, and its efficacy w superior to that of lutein and CsA.  The data are expressed as the means ± standard deviation (n = 10). * p < 0.05 and *** p < 0.001 compared to the control group. # p < 0.05 and ### p < 0.001 compared to the PM 2.5 -induced DED group.

Effect of CFW on Conjunctival Goblet Cell Population in PM 2.5 -Induced DED Rat Model
To evaluate the population of goblet cells that secrete gel-forming mucins in the conjunctiva, we performed PAS staining. In control rats, a large number of violet PAS-positive goblet cells were observed in the conjunctival tissue ( Figure 3A). However, topical exposure to PM 2.5 greatly suppressed the frequency of PAS-stained goblet cells, but this was markedly enhanced following CFW 400 administration. Figure 3B shows the quantitative values of the conjunctival goblet cell population, and these are expressed as number per 100 µm 2 . The number of conjunctival goblet cells greatly reduced from 4.00 ± 0.82/100 µm 2 to 0.26 ± 0.55/100 µm 2 following exposure to PM 2.5 . Nevertheless, PM 2.5 -mediated conjunctival goblet cell loss was substantially recovered by oral administration of CFW in a dose-dependent manner. Furthermore, the density of conjunctival goblet cells in the CFW 400 group was higher than that in the CsA group. Meanwhile, oral administration of lutein did not improve conjunctival goblet cells loss. This result suggested that conjunctival goblet cell loss, a DED-mediated event, was markedly induced following exposure to PM 2.5 , and this alteration was significantly prevented by oral administration of CFW.
dose-dependent manner. Furthermore, the density of conjunctival goblet cells in the C 400 group was higher than that in the CsA group. Meanwhile, oral administration o tein did not improve conjunctival goblet cells loss. This result suggested that conjunc goblet cell loss, a DED-mediated event, was markedly induced following exposur PM2.5, and this alteration was significantly prevented by oral administration of CFW.

Effect of CFW on Inflammation of Lacrimal Gland in PM2.5-Induced DED Rats
We assessed the effect of CFW on the pathological changes in the lacrimal glan rats with DED following exposure to PM2.5. Figure 4A shows normal secretory gland tology, including tight acini and ducts in control rats. However, exposure to PM2.5 le inflammatory cell infiltration, sizable interstitial edema with abnormal acini, and the mation of neo-vessels around lobules. In contrast, oral administration of CFW preven PM2.5-mediated histopathological changes in the lacrimal gland, in a dose-depen manner. Meanwhile, lutein administration also inhibited infiltration of inflammatory and edema with abnormal acini, but neo-vessels around lobules were still slightly pres To further investigate whether CD4 + T cell immune responses are involved in PM2.5diated histopathological alteration of the lacrimal gland, we performed immunoh chemical staining for specific antibodies, such as CD4, IL-17, and TNF-α. The top pa of Figure 4B show that the predominant immune cell subset in the lacrimal gland PM2.5-exposed rats consisted of CD4 + T cells. However, the overexpression of CD4 + T by PM2.5 was significantly down-regulated to control levels by oral administration of C 400 and cyclosporine ( Figure 4B,C). In addition, the expression of IL-17 and TNF-α in lesions of the lacrimal gland was significantly increased to 2.10-and 2.36-fold that of

Effect of CFW on Inflammation of Lacrimal Gland in PM 2.5 -Induced DED Rats
We assessed the effect of CFW on the pathological changes in the lacrimal gland in rats with DED following exposure to PM 2.5 . Figure 4A shows normal secretory gland histology, including tight acini and ducts in control rats. However, exposure to PM 2.5 led to inflammatory cell infiltration, sizable interstitial edema with abnormal acini, and the formation of neo-vessels around lobules. In contrast, oral administration of CFW prevented PM 2.5 -mediated histopathological changes in the lacrimal gland, in a dose-dependent manner. Meanwhile, lutein administration also inhibited infiltration of inflammatory cells and edema with abnormal acini, but neo-vessels around lobules were still slightly present. To further investigate whether CD4 + T cell immune responses are involved in PM 2.5 -mediated histopathological alteration of the lacrimal gland, we performed immunohistochemical staining for specific antibodies, such as CD4, IL-17, and TNF-α. The top panels of Figure 4B show that the predominant immune cell subset in the lacrimal glands of PM 2.5 -exposed rats consisted of CD4 + T cells. However, the overexpression of CD4 + T cells by PM 2.5 was significantly down-regulated to control levels by oral administration of CFW 400 and cyclosporine ( Figure 4B,C). In addition, the expression of IL-17 and TNF-α in the lesions of the lacrimal gland was significantly increased to 2.10-and 2.36-fold that of control by PM 2.5 topical exposure. However, the upregulated expression was substantially attenuated following CFW 400 and cyclosporine treatment, and was similar to control levels following treatment. However, treatment was less effective on the expression of CD4, IL-17, and TNF-α in lutein group. These results suggest that PM 2.5 exposure leads to pathological changes in the lacrimal gland, including inflammation, neovascularization, and abnormal acini, due to CD4 + T cell immune responses. Nevertheless, oral administration of CFW markedly prevented these alterations of the lacrimal gland by PM 2.5 , and the efficacy of spermidine was similar to that of cyclosporine treatment. following treatment. However, treatment was less effective on the expression of CD4, IL-17, and TNF-α in lutein group. These results suggest that PM2.5 exposure leads to pathological changes in the lacrimal gland, including inflammation, neovascularization, and abnormal acini, due to CD4 + T cell immune responses. Nevertheless, oral administration of CFW markedly prevented these alterations of the lacrimal gland by PM2.5, and the efficacy of spermidine was similar to that of cyclosporine treatment. The stained area of the photograph was quantitative analyzed using ImageJ ® and calculated in terms of the fold of the control. ** p < 0.01 and *** p < 0.001 compared to the control group. The data are expressed as the means ± standard deviation (n = 3). # p < 0.05, ## p < 0.01 and ### p < 0.001 compared to the PM2.5-induced DED group. The stained area of the photograph was quantitative analyzed using ImageJ ® and calculated in terms of the fold of the control. ** p < 0.01 and *** p < 0.001 compared to the control group. The data are expressed as the means ± standard deviation (n = 3). # p < 0.05, ## p < 0.01 and ### p < 0.001 compared to the PM 2.5 -induced DED group.

Effect of CFW on Histological Changes of the Retina after Topical Exposure to PM 2.5 in SD Rats
Next, we investigated the harmful effects on the retina following topical exposure to PM 2.5 , and the efficacy of CFW on PM 2.5 -mediated retinal alteration. As a result of H&E staining in the retinal section, the thickness of the nerve fiber layer (NFL), ganglion cell layer (GCL), and inner plexiform layer (IPL) markedly decreased by exposure to PM 2.5 , but this was markedly prevented by CFW 400, lutein, and CsA administration ( Figure 5A,B). Meanwhile, the thickness of the inner nuclear layer (INL) and outer nuclear layer (ONL) did not differ among the groups. In addition, the population of ganglion cells in GCL was markedly decreased by exposure to topical PM 2.5 ( Figure 5A). However, the population of ganglion cells was greatly increased after CFW treatment. Figure 5C shows that the number of ganglion cells was significantly decreased in the retina of the DED group (7.42 ± 1.08/100 µm 2 ) compared with that in the retina of the control group (16.50 ± 1.98/100 µm 2 ). In contrast, oral administration of 200 mg/kg and 400 mg/kg CFW significantly enhanced the ganglion cell population to 11.71 ± 1.80/100 µm 2 and 15.55 ± 1.93/100 µm 2 , respectively. The efficacy of CFW 400 on the improvement of ganglion cells was superior to that of cyclosporine, and similar to that of the lutein treatment.
staining in the retinal section, the thickness of the nerve fiber layer (NFL), ganglion layer (GCL), and inner plexiform layer (IPL) markedly decreased by exposure to PM but this was markedly prevented by CFW 400, lutein, and CsA administration ( Fig  5A,B). Meanwhile, the thickness of the inner nuclear layer (INL) and outer nuclear la (ONL) did not differ among the groups. In addition, the population of ganglion cells GCL was markedly decreased by exposure to topical PM2.5 ( Figure 5A). However, the p ulation of ganglion cells was greatly increased after CFW treatment. Figure 5C shows t the number of ganglion cells was significantly decreased in the retina of the DED gro (7.42 ± 1.08/100 μm 2 ) compared with that in the retina of the control group (16.50 ± 1.98/ μm 2 ). In contrast, oral administration of 200 mg/kg and 400 mg/kg CFW significantly hanced the ganglion cell population to 11.71 ± 1.80/100 μm 2 and 15.55 ± 1.93/100 μm 2 , spectively. The efficacy of CFW 400 on the improvement of ganglion cells was superio that of cyclosporine, and similar to that of the lutein treatment.  (B,C) The data are expressed as the means ± standard deviation (n = 10). * p < 0.05 and *** p < 0.001 compared to the normal group. ## p < 0.01 and ### p < 0.001 compared to the control group.

Discussion
In this study, we investigated the effect of oral administration of CFW on the changes of the eyes, as well as on the hematology and biochemistry in a DED model induced by topical exposure to PM 2.5 . Interestingly, topical exposure to PM 2.5 led to partial abnormality of the serum lipid profile, including TC and LDL-C. However, our findings showed that CFW contributes to the normalization of TC and LDL-C levels. Increasing epidemiological studies have recently shown that PM 2.5 may have a hazardous influence on the metabolic system, including an increased risk of dyslipidemia [36,37]. One cohort study demonstrated that exposure to PM 2.5 is associated with worsening LDL-C levels [36]. In this cohort study, researchers obtained atmospheric monitoring data for the daily mean concentration of hourly measured PM 2.5 from the Ministry of Environment of Korea, and multiple regression analyses were conducted to assess the associations between exposure to PM 2.5 and changes in lipid profiles at two-year intervals. Another cohort study investigated individual daily exposure to fine PM that was estimated by as spatiotemporal model, and analyzed the correlation between daily exposure levels of fine PM and blood lipid [37]. They suggested that high levels of fine PM exposure were closely correlated with increasing TC and LDL-C levels. Even though these few studies reported that exposure to ambient PM 2.5 may have a negative effect on lipid profiles, such as TC and LDL-C, no studies have investigated the influence of topical exposure to PM 2.5 on serum lipid profiles. In this context, our findings were very meaningful in that CFW treatment contributed to decrease serum TC and LDL-C levels that were markedly enhanced by topical exposure to PM 2.5. Actually, several studies demonstrated that CF and its bioactive components improved dyslipidemia [38][39][40][41]. Park et al. [38] suggested that CF decreased serum TC levels with a suppress in esterified cholesterol in hypercholesterolemic rat. In addition, Zhang et al. [39] demonstrated that polysaccharide isolated from CF attenuated the cholesterol accumulation in oxidized LDL-stimulated macrophages and suppressed LDL-C, TC, and TG levels in high-fat-diet-fed mice. Furthermore, it has been reported that 7-O-galloyl-D-sedoheptulose, isolated from CF, improved the levels of lipid profile in type 2 diabetic mice [40]. Another study demonstrated that oral administration of iridoid glycoside from CF regulated lipid metabolism in db/db mice [41].
The ocular surface is composed of the corneal and conjunctival epithelium, corneoscleral limbus, nerves, and tear film [42]. The ocular surface serves as a barrier to chemicals, microbes, and other airborne matter and provides anatomic, physiologic, and immunologic protective functions [42,43]. Accumulated epidemiological and biochemical studies demonstrate that exposure to PM 2.5 may contribute to DED, which is a multifactorial ocular surface disease that involves tear film instability, hyperosmolarity, inflammation, and damage to the ocular surface [6,8,9,11,12]. The tear film is composed of three distinct layers, a mucin layer, an aqueous layer, and a lipid layer, and performs many physiological functions [44]. Compromise of the tear film triggers tear film destabilization, promotes exposure of the corneal epithelium to air, and potentially contributes to dry eye symptoms [45]. A mucin layer composed of mucins produced by conjunctival epithelial cells provides an easily wettable ocular surface and assists in tear re-spreading after blinking [46,47]. The conjunctival epithelium houses mucin-producing goblet cells [48]. Recently, several animal studies have demonstrated that topical administration of PM 2.5 presented dry eye phenotypes, accompanied by a decreased tear production, damaged corneal epithelium, reduced conjunctival goblet cells, and an abnormal corneal structure [5,12,13,35]. In this context, the clinical characteristic of DED can be diagnosed the tear break up time (TBUT), the tear secretion test and goblet cell counting [5]. In this study, we also reproduced a DED murine model induced by topical instillation of PM 2.5 , which was accompanied by decreased tear secretion, induced detachment of the corneal epithelium, and loss of conjunctival goblet cells. However, oral administration of CFW significantly protected tear reduction, corneal epithelial detachment, and conjunctival goblet cell loss following PM 2.5 exposure. Recently, a newly discovered DED subtype is the short TBUT that is related with ocular neuropathic pain and eye strain [49], The TBUT analysis was developed to judge the abnormalities of the tear film [50]. Actually, several previous studies reported that PM 2.5 markedly decreased TBUT in DED murine models [5,12,13,35]. Although our current result suggested that CFW treatment involved in increment of tear production, the further studies are need to verify the efficacy of CFW on TBUT in PM 2.5 -exposed DED model. Nevertheless, our present finding showed that recovery of conjunctival goblet cells by oral administration of CFW may contribute to the stabilization of the mucin layer and lead to tear film stability. Therefore, this finding supported that oral application of CFW increased tear production and induced tear film stability through reinforcement of the mucin layer and this was ultimately caused by ocular surface stability.
The major pathogenesis of DED is an inflammation, including infiltrated immune cells in the conjunctiva and lacrimal glands, increased density of dendritic cells in the cornea, and increased secretion levels of tear cytokines [51,52]. Inflammation of the ocular surface in DED is sustained by the ongoing activation and infiltration of pathogenic immune cells, primarily of CD4 + T cells [53]. The main proliferating subset of dry eye T cell effectors in the presence of T regulatory cells is IL-17-secreting CD4 + T cells [54]. IL-17 induces the secretion of pro-inflammatory cytokines such as IL-1, IL-6, and TNF-α, and these cytokines are upregulated in DED [55]. In this regard, one study reported that the topical application of PM 2.5 elevated dendritic cell maturation and the expression of TNF-α, IL-1β, and IL-6 in a murine model [56]. Another study also reported that the expression of TNF-α and IL-6 was increased in the PM 2.5 -induced DED rat [57]. Although the role of CD4 + T cell-mediated immune responses in DED is well-established, there are no studies on the relationship between CD4 + T cell-mediated immune responses in PM 2.5 -induced DED. Just one study verified that PM 2.5 -stimulated CD4 + T cells potently increased the mRNA and protein levels of pro-inflammatory cytokines and induced the death of human bronchial epithelial cells [58]. Herein, we found that topical exposure to PM 2.5 was caused by the pathological changes in the lacrimal gland, including inflammation, neovascularization, and abnormal acini. Additionally, PM 2.5 increased the expression of CD4 + T cells, IL-17, and TNF-α in the lacrimal gland. However, oral administration of 400 mg/kg CFW markedly protected these PM 2.5 -mediated inflammatory responses of the lacrimal gland, and the efficacy of CFW was similar to that of cyclosporine treatment and superior to that of lutein. These results indicate that oral supplements of CFW may contribute to the suppression of CD4 + T cell immune responses induced by PM 2.5 . Furthermore, we considered that the protective effect of CFW on lacrimal gland inflammation in PM 2.5 -exposed rats was due to its anti-inflammatory potential [22,27].
The blood-retina barrier (BRB) is available for diffusion and permeabilization of PM 2.5 , and its function in BRB could potentially play a role in PM 2.5 -mediated retinal pathogenesis [59,60]. Several epidemiological studies have suggested that exposure to PM 2.5 correlates with retinal damage, including retinal atherosclerosis, retinal edema, and retinal vessel narrowing [59][60][61]. One study demonstrated that topical exposure to PM 2.5 markedly decreased the NFL/GCL and increased the expression of glial fibrillary acidic protein [4]. Furthermore, a correlation between PM 2.5 and changes in retinal structure features in subjects exposed to higher levels of PM 2.5 was reported [59]. Previously, we suggested that PM 2.5 mediates retinal dysfunction through ROS-mediated epithelialmesenchymal transition and necrotic and autophagic cell death in retinal pigment epithelial cells [62,63]. Our present findings showed that topical exposure to PM 2.5 led to the loss of retinal ganglion cells and the decrease of the NFL/GCL + IPL thickness; however, oral administration of CFW contributed to the normalization of retinal ganglion cell density and retinal constitution.
In the present study, we used two positive controls: CsA and lutein. CsA is a T cell immunomodulatory agent that is used to suppress ocular surface inflammation and improve tear film dynamics [32,33]. To date, the effect of CsA on PM 2.5 -exposed eyes is yet to be reported. Our results showed that CsA attenuated the DED symptoms, including decreasing tear secretion, epithelium detachment, loss of conjunctival goblet cells, and inflammation of lacrimal gland following topical exposure to PM 2.5 . In addition, CsA im-proved serum TC levels and restored retinal ganglion cell population and NFL/GCL + IPL thickness, which is the first finding for the effect of CsA on the posterior segment of the eye following exposure to PM 2.5 . However, this efficacy of CsA is less or similar than that of CFW. It is also noteworthy that CsA is an immunosuppressant drug widely used oral medications in organ transplant recipients and patients with autoimmune disorders. Some studies reported that long-term treatment with oral administration of CsA was associated with hyperlipidemia and an increased risk of atherosclerosis, but the mechanisms by which cyclosporin A causes hyperlipidemia are unclear [64,65]. However, these previous reports about the side effects of oral CsA administration are contrary with our present finding. The differences between previous study and the present study are the administered period and route. Therefore, we consider that further studies are needed to verify whether improvement of TC levels by CsA eye drops is a temporary situation, and to evaluate the effect of long-term topical application of CsA.
Meanwhile, lutein is one of the xanthophyll carotenoids and has posterior eyeprotective properties through anti-oxidative and anti-inflammatory effects [31]. Numerous clinical studies suggested that lutein supplements reduce neovascular age-related macular degeneration (AMD) risk, improve visual function, and increase macular pigment optical density [66][67][68]. However, the effect of lutein on PM 2.5 -exposed eyes, especially ocular surface, is yet to be reported. In the present study, we found that lutein had slightly improved efficacy or no efficacy on the anterior segment of eye in PM 2.5 -exposed rat, but very superior efficacy on the posterior segment of eye. Oral administration of lutein caused a minor increase in tear secretion and corneal epithelium stabilization, and inhibited inflammation of the lacrimal gland, while it had no effect on the conjunctival goblet cell loss. On the other hand, the lutein supplement markedly increased the retinal ganglion cell population and NFL/GCL + IPL thickness following exposure to PM 2.5 . Interestingly, the efficacy of CFW on the retina was similar to that of lutein. Overall, in the present study, we found that oral administration of CFW protected against PM 2.5 -induced DED symptoms, retinal disorder, and dyslipidemia. More importantly, the efficacy of CFW was superior and/or similar to that of cyclosporine A, an anti-inflammatory agent, and lutein, the posterior eye-protective agent.
Numerous reports demonstrated that women are disproportionately affected by DED, are diagnosed at a younger age, and experience more severe symptoms compared with men [69,70]. Similar to the human studies, several animal studies suggested female sex is a risk factor for DED [5,71,72]. McClellan et al. [71] reported that female C57BL/6 mice developed greater corneal barrier disruption than age-matched male mice did, although other features of DED such as low goblet cell density and LG infiltration were similar in both sexes. Recently, Song et al. [72] used six-week-old female SD rats to assess the effect of natural products on a PM 2.5 -induced DED model. Yang et al. [5] also used 6-8-week-old female C57BL/6 mice to evaluated the harmful effect of PM 2.5 on the eye. Based on this knowledge, we demonstrated that CFW prevented PM 2.5 -indued DED in female SD rats. Although we assessed the effect of CFW on PM 2.5 -exposed female SD rats, we consider that further studies are needed to evaluate the influence of topical exposure to PM 2.5 , and to confirm the efficacy of CFW in males.

Conclusions
Taken together, our present findings indicated that oral administration of CFW contribute to normalize the levels of serum TC and LDL-C in the topical PM 2.5 -exposed rat. Furthermore, oral administration of CFW protected tear film destabilization, inflammation of the lacrimal gland, and histological changes in the retinal NFL/GCL and IPL in the PM 2.5 -induced DED. Therefore, the present findings may provide an experimental basis for the potential application of CFW in preventing air pollution-related dry eye symptoms, retinal disorders, and lipid metabolism disorder.