The Protective Effect of Aesculin Eye Drops on Dry Eye Syndrome Mice

Background: The objective of this study was to test the effectiveness of the novel aesculin (AES) eye drops on treating dry eye syndrome (DES), using murine model. Methods: Dry eye was induced in murine eye with the use of an intelligent controlled environmental system (ICES). High-ow air which had been desiccated within the ICES, induced the DES. Treatment included Aesculin eye drops for 14 days. After expiration, corneal uorescein staining was examined and stained with an endothelial maker created for tracking of lymph: Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1). Subsequently, real-time PCR and ELISA were completed to quantify secretion of TNF-α (cid:0) IFN-γ (cid:0) IL-1β and IL-8 in serum, ocular surface and lacrimal gland. Results: Aesculin eye drops signicantly repaired ICES-induced corneal injury in murine models Statistically signicant increase in lymph angiogenesis was observed at day 14 of observation. Consequently, DES in treatment group was reduced using aesculin eye drops. aesculin eye drops can signicantly inhibit lymphangiogenesis and reduce inammatory cytokines levels in mice. The percentage of Th1 and Th17 cells were decreased after treating with aesculin eye drops, while the percentage of Treg was increased. Injection of VEGF-C into mice can reduce the ecacy of aesculin eye drops in ICES-induced DES. Conclusion: as treatment group of murine models indicated signicant reduction of inammation in the cornea associated with DES, treatment of DES with aesculin eye drops, should be considered a viable treatment method for DES.


Background
Keratoconjunctivitis sicca, commonly known as dry eye syndrome (DES), refers to a progressive multifactorial disorder ocular surface, which results in patient discomfort and disruption in visual acuity (McDonald et al., 2016). Dry eye syndrome is a medical condition in which those affected experience chronic lack of moisture within the eye. Although, cases of DES in singular eyes are reported, most patients are infected in both eyes (P ugfelder and de Paiva, 2017). When patients contract DES, they are likely to present with an array of symptoms.
When individuals are affected by DES, patients' eyes are often easily fatigued, and patients present with redness, lacrimal stringy discharge, and blurred vision (Barabino et al., 2016). Blurred vision is frequently accompanied by pressure behind the eye and sensitivity to light (Tsubota et al., 2017). Additional symptoms of DES include a burning sensation and feelings of sandy irritation, or dust in the eye (McDonald et al., 2016). When DES infection is novel, symptoms are generally mild. Mild symptomology is often easily resolved, with little or no long-term effects (Yamanishi et al., 2019). However, if DES advances untreated, patients are likely to experience increased damage of the eye, including scarring on the cornea, resulting in permanent impairment to vision and in rare cases, loss of vision .
Dry eye syndrome occurs for three main reasons, inadequate tear production, rapid tear evaporation or inadequate blinking re exes (Tsubota et al., 2017). Underperformance, or overperformance, of blinking re ex can create a variety of issues for persons with DES (Tsubota et al., 2017). Underperformance of the blink re ex is often caused by overuse of the eye . Typically, individuals that have occupations that require prolonged focus of the eyes are prone to DES (Lambert, 2018). Conversely, when eyes are irritated tears are created at an elevated rate, creating an overabundance of tears (Lambert, 2018). Although irritated eyes create an excess of tears, increased tear production counterintuitively facilities drier eyes. Dry eye syndrome is accelerated with persistent tearing, as re exive tears of this nature does not have the lubricating quality of tears created when irritated (Tsubota et al., 2017).
Environmental factors also impact the prevalence of DES (Lambert, 2018). When individuals reside in environments that boast elevated wind, dust or smoke, DES is more apt to occur (Tsubota et al., 2017).
Moreover, environments that are higher in altitude, lower in humidity or the climate is arti cially regulated are also more prone to exacerbate symptoms associated with inadequate tear production (Lambert, 2018).
Inadequate tear production occurs primarily from lacrimal hyposecretion, while rapid evaporation of tears does not allow lacrimal secretion to coat the eye properly. In both cases, the aqueous tear layer (ATL) is impacted. The ATL is a discrete layer that, along with two other layers, comprise the tear lm. The tear lm is amalgamation of three discrete layers the mucin layer, lipid layer. and aqueous layer (Sridhar, 2018). The most proximal layer of the tear lm is the mucin layer, which abuts the cornea (Werkmeister, 2017). The primary function of the mucin layer is to nourish the cornea, and to aid in cornea functioning.
The mucin layer of the tear lm also allows the tears to slide evenly over the ocular surface and allows for even distribution of tears (Werkmeister, 2017). The mucin layer of the tear lm also allows the tears to slide evenly over the ocular surface and allows for even distribution of tears (Sridhar, 2018). The middle layer of the tear lm is the aqueous layer (Rolando and Zierhut, 2001). The aqueous tear layer lubricates the eye and allows the clearing of particles and prevent infection (Sridhar, 2018). Finally, the most distal layer of the tear lm is lipid layer (Cwiklik, 2016). he lipid layer allows for the sealing of the tear lm, which reduces evaporation and ensure the eye remains hydrated (Sridhar, 2018).
The positioning of the lm layers is uniform, however the composition of the three layers of the tear lms are unique amongst individuals (Bai et al., 2019). Thickness of mucin, aqueous, and lipid layers are dependent on environment, diet and biological factors, creating uctuations in ocular tear lm e cacy.
Thickness of mucin, aqueous, and lipid layers are dependent on environment, diet and biological factors, creating uctuations in ocular tear lm e cacy (King-Smith et al., 2000). The thickness of the human precorneal tear lm: evidence from re ection (Rolando and Zierhut, 2001). Other risk factors for dry eye syndrome. In addition to tear lm thickness, and proper blink response, other factors likely contribute to the presentation of DES (Lambert, 2018). Other risk factors for dry eye syndrome. In addition to tear lm thickness, and proper blink response, other factors likely contribute to the presentation of DES . Menopausal women are most at risk for contracting DES, provided environmental .
In addition to environmental factors, DES is also associated with ocular surface in ammation caused by immune response (Stern and P ugfelder, 2004;Stern et al., 2013). Until relatively recently, the eye was believed to lack all lymphatic vessels, except for the conjunctiva (Schroedl et al., 2014). Advancements in ophthalmic instruments allowed for the classi cation of a lymphatic network using endothelial markers especially Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), as well as lymphangiogenic factors such as vascular endothelial growth factor c (VEGF-C). Even more recent studies have established that the lacrimal gland, orbital meninges, extraocular muscles and the corneal limbus all are connect to the lymphatic system (Nakao et al., 2012). Although little is known about lymphatic uid of the ocular region, research has established that lymphatic pathway might contribute to overall ocular uid homeostasis.
The identi cation of the lymphatic pathway in uid homeostasis generated data that indicated that lymphangiogenesis in uences a variety of pathological conditions within the eye. Lymphangiogenesis has been associated with corneal transplant complication and subsequent corneal rejection, ocular tumor growth and various ocular infection (Cursiefen et al., 2003). The role of lymphangiogenesis in DES remains unknown. As such, studies on corneal lymphangiogenesis using a murine model could create better understanding of the role of the lymphangiogenesis in ocular in ammation and DES.
A better understanding of lymphatic and lymphangiogenesis in the eye will open new therapeutic opportunities to prevent vision loss in ocular diseases. Previous research suggests that T-cells are present within both murine and human models of DES, especially within proximal lymph nodes. The acquisition of speci c chemokine markers near the ocular regions allow for the congregation of T-cells into the in amed and irritated ocular surfaces, commonly found in in mice with DES in laboratory studies.
Autoimmunity was also demonstrated within the murine model within the context of DES when lymphatic vessels were drained (Chauhan et al., 2009). Aesculin ( Figure. 1A), a natural product from the traditional and widely-used Chinese medicine named Qinpi, Fraxinus rhynchophylla. Previous studies found that aesculin have effectiveness in anti-in ammation and analgesia. Aesculin eye drops have already been put to clinical use for curing conjunctivitis from last century. Within this study, we attempt to determine the growth of lymphatic vessels in the cornea with the presentation and progression of DES. The expression levels of regulatory T cells (Tregs) and T helper cells Th1 and Th17 were also investigated after aesculin eye drops treatment. We pleasantly found that aesculin eye drops might have signi cant effectiveness on treating DES.

Animals for Murine Modeling
This animal experiment was approved by the Animal Care and Ethics Committee of Shanghai University of Traditional Chinese Medicine. All care for murine models adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research to reinforce ethical behavior and care. Six female mice (C57BL) aged 4-6 weeks were used within the study.

The Intelligently Controlled Environmental System
The intelligent controlled environmental system (ICES), included one box. The box utilized for this study was a dry and sealed box (500L capacity) with an adjustable temperature humidi er, as well as an intelligent humidi er. Dual humidi ers were used to create a two-step process to reduce moisture within study environment.
Industrial temperature humidi er (ZD-890c, Ouvi Company, Hangzhou, China) was used rst to lower humidity 40% − 50%. Once humidity was reduced within the sealed box, an intelligent dehumidifying device will be used. The intelligent dehumidifying device (MS, 13X; Hengye Company, Shanghai, China) was used to lower humidity within study environment further. The MS humidi er was selected as it promotes a new environmentally friendly material, referred to molecule lter. Molecular lter is toxic free and allows for the active control of moisture within the ICE.
The last component of the ICE is a noise free ventilator (LFA-40, 539; Jiaxin Company, Wenzhou, China).
Ventilator was placed 20 cm from subjects (speed 0-5m/s). Noise free ventilator was chosen to allow for ambient noise to re ect the same level of the noise within the ICE. With the noise free ventilator and two humidi ers, researchers were able to control temperature, humidity and din within the ICE.
Finally, an anemometer used to measure air ow, (AR816; Haoxin Company, Shanghai, China) was used to stabilize air ow between 0 to 30 m/s. The anemometer had was selected as it boasted measures within 5% accuracy. Thus, the humidity and temperature in the ICES were monitored and subsequently displayed by a control system found within the intelligent dehumidifying device.
All mice were placed into one of three groups which were randomly assigned: control, environmental control and treatment group. The control group was housed in RH 60-80%, 21-23 degrees Celsius. Mice within the experimental groups are sealed within container in noise, humidity and temperature controls.

Dry Eye Model Testing
Aesculin (purity > 98.5%) was purchased from Meilune (Dalian, China). Distilled water was added to the powder to create the eye drop used in the experiment. The eyes of mice were treated twice a day with 80 µg of aesculin (8 µL of 10 g/L solution) or vehicle (saline). Daily treatment with aesculin started 2 days before ICES and continued up until the time animals were euthanized and cornea tissue was collected for testing. All mice were humanely slain with a lethal overdose of ketamine and xylazine. Area of the superior conjunctivas were marked by researchers with black ink after the animals expired. Each eye was xed in 10% formalin for dehydration.
Taking out the right eyeball after death, remove cornea at 2 mm and placed in 10% in formaldehyde. Tear production in murine model was measured in 6 eyes using the phenol red thread test. Testing lasted 60 seconds per eye. Phenol red threads were held by researchers using one pair of forceps throughout the entirety of testing. Forceps were applied to the lateral cantus of the conjunctival fornix 6 right eyes for 60 seconds under slit lamp biomicroscopy. The length of the wet cotton thread was measured using a millimeter scale and recorded.

Corneal Fluorescein Staining
Corneal Fluorescein (CF) Staining was used to measure new lymph growth on the ocular surface. Corneal Fluorescein staining was evaluated in 6 right eyes by injecting 0.5 L of ve percent uorescein solution into the inferior conjunctival sac using a sterilized micropipette. The cornea of each eye was examined utilizing slit-lamp microscopy under cobalt blue light 10 minutes subsequent after uorescein instillation. The corneal surface was then split into ve distinct regions and then graded on a system from 0 to 3. New lymph growth electronic microscope observation: sterile condition, put sample in 2.5%

Immuno uorescence
Frozen sections were brie y blocked by goat serum for 60 minutes. When blocking the specimen, prepare the primary antibody in the antibody dilution buffer according to the dilution ratio recommended in the data sheet(LYVE-1: ab10278, 1:100; VEGFC: ab83905, 1:100; VEGFR-3:ab10284, 1:100, Abcam Inc.).Aspirate the blocking buffer and add the diluted primary antibody. Incubate at 4 ° C overnight. Rinse three times with 1X PBS for 5 minutes each. After diluting the uorescently labeled secondary antibody with antibody dilution buffer, incubate the specimen in the dark at room temperature for 1-2 hours. Rinse three times with 1X PBS for 5 minutes each. Use Prolong® Gold Antifade Reagent with DAPI (CST, 8961) and cover the section with a coverslip. Sections were imaged with a confocal microscope.

RNA isolation and quantitative real-time PCR (qRT-PCR)
The RNA from the tissue was isolated using TRIzol reagent (Invitrogen) according to manufacturer's instructions and reverse-transcribed using an miScript Reverse Transcription Kit (QIAGEN). The qRT-PCR was performed using a SYBR Premium Ex Taq II kit (TaKaRa) on an ABI PRISM 7500 Sequence Detection System (Applied Biosystems). All reactions were performed in triplicate and the mean value was used to calculate the relative level of expression after normalization against β-actin.

Corneal epithelial OGD staining
Corneal epithelial staining with Oregon Green dextran (OGD; 70,000 MW; Invitrogen Inc., Grand Island, NY) was assessed in the 5 different groups. Brie y, 0.5 µl of 50 µg/ml OGD was instilled in the ocular surface 1 min before euthanasia. Corneas were rinsed with 2 ml PBS and photographed with a stereoscopic zoom microscope (V20; Zeiss with kryptonargon and He-Ne laser; Carl Zeiss Meditec, Ltd., Thornwood, NY) under uorescence excitation at 470 nm. Images were obtained 2 h after instillation of the last treatment drop and were processed. The uorescein solution contained 1 mg uorescein sodium in 0.5 ml PBS. The severity of corneal OGD staining shown in digital images was graded by two masked observers, using the Baylor grading scheme for corneal uorescent staining. The number of uorescein staining dots were graded in the 1-mm central cornea zone of each eye, on a standardized ve-point scale (0 dot, grade 0; 1-5 dots, grade 1; 6-15 dots, grade 2; 16-30 dots, grade 3; 30 dots, grade 4). One point was added to the score if there was one area of con uent staining, and two points were added for two or more areas of con uence.

Statistical analysis
Data are expressed as the mean ± standard error of the mean (SEM). Signi cant differences were determined by One-Way ANOVAs with LSD post hoc tests; and P < 0.05 is considered to be statistically signi cant.

Results
Aesculin eye drops signi cantly repaired ICES-induced corneal injury in murine models ICES were used for induce DES. Corneal uorescein staining was applied for assessing the corneal integrity. It is commonly accepted that uorescein staining represents compromised corneal epithelium (Dundas et al., 2001). The mean corneal staining results of mice in the NC, ICES1W, ICES2W, Aesculin eye drops and vehicle groups were quanti ed and compared. Results showed that ICES timedependently led to higher corneal staining score ( Figure. 1B). Aesculin eye drops topical treatment signi cantly reduced corneal staining score compared to ICES groups and control groups ( Figure. 1A). To further con rm the corneal staining results, corneal epithelial staining with OGD was then carried out to detect the injury levels in 5 groups. Similar results were obtained after OGD staining, the speci c ICES environment directly increased corneal damage while Aesculin eye drops treatment noticeably inhibit the level of corneal injury (Figure. 1C). These results suggested that Aesculin eye drops have remarkable recovery effects on curing corneal injury in ICES-induced DES murine models.

Aesculin eye drops inhibited lymphangiogenesis caused by ICES in mice
It has been revealed that corneal lymphangiogenesis played an important part in DES (Goyal et al., 2010;Ji et al., 2018). Lymphatic growth within the vascularized vessels is illustrated by facilitation and promotion of blood vessels. Corneal were stained with lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), vascular endothelial growth factor c (VEGF-C) and its receptor VEGFR-3 for indicating lymphangiogenesis of murine model. It is assumed lymphatic vessels are facilitated from the limbal vascular arcade proximal to the cornea, especially during early time point measurements (day 0, day 1, and day 7) in all three groups, which was shown to increase throughout the trial. Advanced growth was observed more typically towards the median of the murine cornea, especially with DES progression.
Immuno uorescence results of the frozen sections from all 5 groups showed that the expression of LYVE-1 signi cantly increased and continued to progress exponentially until day 14 ( Figures. 2A and B). With Aesculin eye drops treatment, LYVE-1 was reduced remarkably compared to the ICES group ( Figures. 2A  and B). Development of lymphatic vessels is regulated by factors common to lymphangiogenesis. Signi cant overexpression of VEGF-C and VEGFR-3 was observed in ICES group by day 14 (Figures. 2C -F). Topical Aesculin eye drops treatment reduced the mean uorescence intensity ( Figures. 2C -F). These results combinedly indicated that ICES-induced DES promoted the growth of lymphangiogenesis while Aesculin eye drops can inhibit the promoting effects.

Aesculin eye drops topical treatment reduced the expression of in ammatory factors
To further detect the ocular and systemic in ammation, samples from serum, ocular surface and lacrimal gland were collected and investigated. The detect objectives were TNF-α, IFN-γ, IL-1β and IL-8, which had been regarded as cell signaling cytokines involved in systemic in ammation response (Chung and Benveniste, 1990;Doherty et al., 1992). ELISA results revealed that all 4 factors increased signi cantly in ICES groups, and Aesculin eye drops treatment reversed the expression to levels similar with NC groups while vehicle treatment had no exceptional in uence (Fig. 3A). In addition, RT-PCR results showed that the gene expression of TNF-α, IFN-γ, IL-1β and IL-8 saw a rapid rise in ICES groups by day 14, both in ocular surface and in lacrimal gland ( Figures. 3B and C). However, treatment with Aesculin eye drops signi cantly inhibited the overexpression of all in ammatory factors, especially IL-1β ( Figures. 3B and C). It has been reported in many researches that the core mechanism of DES is in ammation (Hessen et al., 2014;Stern and P ugfelder, 2004;Wei et al., 2014). These results suggested that Aesculin eye drops topical treatment signi cantly inhibited ICES-induced in ammation, which implied that Aesculin eye drops might have great effectiveness in controlling DES-induced ocular in ammation.

Cellular compositional changes of draining lymph nodes by dry eye induction in ICES mice
The immune response to foreign antigens requires a perfect coordination between sensor and effector cells. CD4 + T cells, also known as Th cells, play a central role in immune protection (P ugfelder et al., 2013). IFN-γ production in T cells induce CD4 + Th precursor cells to differentiate into Th1 cells (Stern et al., 2005). Th1 cells secrete IFN-γ that activates macrophages that functioning eradicating intracellular microorganisms, such as mycobacteria (P ugfelder et al., 2013). Th17 cells secrete IL-17, which induces production of proin ammatory molecules (Bettelli et al., 2006).
Flow cytometry analysis of the Th1-type cytokines IFN-γ in CD4 + T cells revealed that the percentage of cells expressing IFN-γ increased rapidly in ICES groups, from 5.38 to 15.1 (Fig. 4A). A dramatic reduction in the proportion of IFN-γ-expressed Th cells starting from 15.1 to 7.99 in Aesculin group was detectable (Fig. 4A). Analysis of IL-17A-expressed CD4 + cells presented results similar with IFN-γ groups. The proportion of Th cells expressing IL-17A saw a rapid rise after ICES day 14 (from 0.21 to 2.6) while Aesculin eye drops treatment signi cantly suppressed the expression (Fig .4B). These results indicated that Th1 cells and Th17 cells increased in DES condition and Aesculin eye drops had great inhibitory effects on these cells.
It has been revealed that the resistance of Th17 to Treg suppression played an important role in the autoimmune mechanisms of DES (Chauhan et al., 2009). We analyzed FOXP3-expressing CD25 cells to determine the proportion of Treg cells. Flow cytometry showed that ICES effectively suppressed the level of Treg cells from 4.85 to 1.39 (Fig. 4C). In Aesculin group, the percentage of Treg cells increased from 1.39 to 3.45 (Fig. 4C). These results suggested that ICES-induced dry eye gave a rise to the proportion of Th1 cells and Th17 cells while inhibited the level of Treg cells. Additionally, Aesculin eye drops treatment signi cantly suppressed the effects of ICES on all 3 kinds of T cells.
Aesculin eye drops treatment suppressed in ammation, Th1 and Th17 cells via inhibiting lymphangiogenesis in ICES-induce dry eye mice To determine whether the therapeutic effects of Aesculin eye drops was related with its ability to suppress lymphangiogenesis, the mice were injected with VEGF-C through tail vein. The serum samples were collected and used for ELISA. Results showed that even though Aesculin eye drops treatment inhibited the expression of in ammatory factors TNF-α, IFN-γ, IL-1β and IL-8, injection with VEGF-C totally reversed the inhibitory effects and gave a rise to the factors (Figure. 5A).
As for the cellular compositional changes draining lymph nodes, ow cytometry analysis con rmed that the percentage of Th1 and Th17 cells rose rapidly in ICES group and fell dramatically after Aesculin eye drops treatment ( Figures. 5A and B). VEGF-C injection wiped out the inhibitory effects of Aesculin eye drops on Th1 and Th17 cells ( Figures. 5A and B). A similar result was obtained from Treg groups: Aesculin eye drops treatment gave a rise to the decreased percentage of Treg cells in mice (from 1.54 to 3.56), while VEGF-C reversed the promoting effect. These results indicated that the therapeutic effects of Aesculin eye drops on suppressing in ammation might come from its function in inhibiting lymphangiogenesis.

Discussion
Previous studies have shown that DES was associated with lymphangiogenesis, in ammation and changes in T cell composition. Lymphatic vessels which indicated the presence of DES were present in the cornea of both the environmentally controlled and experimental murine models. Statistically signi cant increase in lymphangiogenesis was observed (p < 0.001) at 14 days of observation. DES was observed in 4 in control group, 4 in environmental control group. Dry eye syndrome was also observed in 4 subjects in treatment group with Aesculin eye drops. Consequently, DES in treatment group was reduced (p < 0.001) through the use of Aesculin eye drops. Study was completed on murine model in an ICES which was controlled for temperature, humidity, air ow and noise. These control variables were selected as DES is commonly associated with higher temperature, reduced humidity and increased air ow (Barabino et al., 2016). Additionally, ambient noise was kept at a minimum not to stress the mice.
The use of the ICES allows for maintenance of environments, and the experimental treatment. Moreover, use of ICES allowed for the establishment of DES in both environmental control and experimental groups.
Mice within the ICES environment in both the experimental and environmental control group experienced reduced tear loss and development of DES within approximately 14 days when compared to control group. Dry eye syndrome increased in symptomology throughout the course of experiment.
Mechanisms involved in DES in the present study is not exhaustive of DES in other models. However, DES symptomology including in ammation and cellular compositional changes were reduced within experimental group. Treatment with Aesculin eye drops was associated with reduced defects in ocular surface. We rstly detected the potentially role of lymphangiogenesis signaling pathway in cellular compositional changes of T cells. Moreover, Aesculin eye drops treatment allowed for mitigation of hyperproliferation in conjunctival epithelium.

Conclusion
Aesculin eye drops should be considered for use within future studies on other models, including in vivo trials and is a promising novel treatment for DES. Availability of data and materials Please contact corresponding authors for data requests.

Competing interests
The authors declare that they have no competing interests.  Aesculin eye drops signi cantly repaired ICES-induced corneal injury in murine models Corneal Fluorescein (CF) Staining was used to measure new lymph growth on the ocular surface. A. Structural formula of Aesculin. The molecular formula and weight of API is C15H18O10 and 358.297 g/mol. B. CF staining results of murine eyes from control, ICES1W, ICES2W, ICES2W+Aesculin and ICES2W+vehicle groups and the quanti ed results. C. CF staining with OGD of murine eyes from all 5 groups and the quanti ed results. Data are presented as the mean ± SD. P < 0.001 versus control group.

Figure 1
Aesculin eye drops signi cantly repaired ICES-induced corneal injury in murine models Corneal Fluorescein (CF) Staining was used to measure new lymph growth on the ocular surface. A. Structural formula of Aesculin. The molecular formula and weight of API is C15H18O10 and 358.297 g/mol. B. CF staining results of murine eyes from control, ICES1W, ICES2W, ICES2W+Aesculin and ICES2W+vehicle groups and the quanti ed results. C. CF staining with OGD of murine eyes from all 5 groups and the quanti ed results. Data are presented as the mean ± SD. P < 0.001 versus control group.

Figure 2
Aesculin eye drops inhibited lymphangiogenesis caused by ICES in mice Immuno uorescence assay were applied for the detection of LYVE-1, VEGF-C and VEGFR-3 in all 5 groups. A. Immuno uorescence results of LYVE-1 in control, ICES1W, ICES2W, ICES2W+Aesculin and ICES2W+vehicle groups. B. Quanti ed results of LYVE-1 mean uorescence intensity. C. Immuno uorescence results of VEGF-C in all 5 groups. D. Quanti ed results of VEGF-C mean uorescence intensity. E. Immuno uorescence results of VEGFR-3 in all 5 groups. F. Quanti ed results of VEGFR-3 mean uorescence intensity. Data are presented as the mean ± SD. P < 0.001.

Figure 2
Aesculin eye drops inhibited lymphangiogenesis caused by ICES in mice Immuno uorescence assay were applied for the detection of LYVE-1, VEGF-C and VEGFR-3 in all 5 groups. A. Immuno uorescence results of LYVE-1 in control, ICES1W, ICES2W, ICES2W+Aesculin and ICES2W+vehicle groups. B. Quanti ed results of LYVE-1 mean uorescence intensity. C. Immuno uorescence results of VEGF-C in all 5 groups. D. Quanti ed results of VEGF-C mean uorescence intensity. E. Immuno uorescence results of VEGFR-3 in all 5 groups. F. Quanti ed results of VEGFR-3 mean uorescence intensity. Data are presented as the mean ± SD. P < 0.001.  Aesculin eye drops topical treatment reduced the expression of in ammatory factors Samples from serum, ocular surface and lacrimal gland were collected and investigated for the level of TNF-α, IFN-γ, IL-1β and IL-8. A. ELISA results of TNF-α, IFN-γ, IL-1β and IL-8 in serum in groups with different treatment. B.
RT-PCR results of TNF-α, IFN-γ, IL-1β and IL-8 expression level on ocular surface in all 5 groups. C. RT-PCR results of TNF-α, IFN-γ, IL-1β and IL-8 expression level in lacrimal gland in all 5 groups. Data are presented as the mean ± SD. P < 0.001.

Figure 4
Cellular compositional changes of draining lymph nodes by dry eye induction in ICES mice Th cells play a central role in immune protection. We used ow cytometry for assessment of the proportion of Th1, Th17 and Treg cells. A B. Flow cytometry analysis and quanti ed results of the CD4+ cells expressing IFN-γ or IL-17A in negative control, ICES2W, ICES2W+Aesculin and vehicle groups, which indicated the percentage of Th1 and Th17 cells, respectively. C. Flow cytometry analysis and quanti ed results of the CD25 cells expressing FOXP3 in all 4 groups. Data are presented as the mean ± SD. P < 0.001.

Figure 4
Cellular compositional changes of draining lymph nodes by dry eye induction in ICES mice Th cells play a central role in immune protection. We used ow cytometry for assessment of the proportion of Th1, Th17 and Treg cells. A B. Flow cytometry analysis and quanti ed results of the CD4+ cells expressing IFN-γ or IL-17A in negative control, ICES2W, ICES2W+Aesculin and vehicle groups, which indicated the percentage of Th1 and Th17 cells, respectively. C. Flow cytometry analysis and quanti ed results of the CD25 cells expressing FOXP3 in all 4 groups. Data are presented as the mean ± SD. P < 0.001.

Figure 5
Aesculin eye drops treatment suppressed in ammation, Th1 and Th17 cells via inhibiting lymphangiogenesis in ICES-induce dry eye mice The mice were injected with VEGF-C to determine whether the therapeutic effects of Aesculin eye drops was related with its ability to suppress lymphangiogenesis. A. The expression of TNF-α, IFN-γ, IL-1β and IL-8 in serum of mice before and after VEGF-C injection. B C. Flow cytometry analysis and quanti ed results of the CD4+ cells expressing IFN-γ or IL-17A in groups with or without VEGF-C injection. D. Flow cytometry analysis and quanti ed results of the CD25 cells expressing FOXP3 in all 4 groups. Data are presented as the mean ± SD. P < 0.001.

Figure 5
Aesculin eye drops treatment suppressed in ammation, Th1 and Th17 cells via inhibiting lymphangiogenesis in ICES-induce dry eye mice The mice were injected with VEGF-C to determine whether the therapeutic effects of Aesculin eye drops was related with its ability to suppress