[Development and assessment of a dry eye questionnaire applicable to the Chinese population].

OBJECTIVE: To develop and assess a new dry eye questionnaire applicable to the Chinese population. METHODS: Based on literature review and clinical practice, a dry eye questionnaire was developed and optimized to apply to Chinese dry eye patients in the language expression and culture background. Participants (78 patients with dry eye and 82 controls) completed the dry eye questionnaire and the ocular surface disease index (OSDI) questionnaire, and ophthalmic examinations were performed, including slit lamp examination, tear breakup time, fluorescein staining, Schirmer I test and meibomian gland assessment. The original questionnaire was optimized with factor analysis according to the answers from respondents and clinical evaluations. The Cronbach α and intraclass correlation coefficient (ICC) were used to evaluate the internal consistency reliability and test-retest reliability. Factor analysis was used to assess the construct validity, concurrent validity was obtained by Spearman correlation analysis, and discriminant validity was obtained by ANOVA and Wilcoxon rank sum test. Receiver operator characteristics curves were generated to identify the sensitivity and specificity of each questionnaire for diagnosis of dry eye. RESULTS: The questionnaire was optimized to 12 items by factor analysis. The response rate from respondents to the dry eye questionnaire and the OSDI was 100% and 91.25%, respectively. The Cronbachαof the dry eye questionnaire and the OSDI was 0.794 and 0.925, respectively, whilst the ICC of both questionnaires was 0.99, indicating good to excellent reliability. The factor analysis suggested that these two questionnaires had good construct validity. The Spearman correlation analysis indicated that the dry eye questionnaire score correlated positively with the OSDI score (r = 0.812, P < 0.01) and had a greater correlation relationship with the clinical evaluations compared with the OSDI score (r for each was 0.613 and 0.605, P < 0.01). The discriminant validity analysis suggested that there was significant difference in the dry eye questionnaire score between the dry eye group and non-dry eye group (P < 0.01). When the dry eye questionnaire score of 7 was used as the diagnostic threshold, the sensitivity and specificity were 83.33% and 70.73%, respectively, and the area under roc curve was 0.814, which was higher than 0.772 of the OSDI (P < 0.01). CONCLUSION: The dry eye questionnaire we developed is applicable to the Chinese population with Chinese culture characteristics, high reliability, validity, specificity, and sensitivity, and holds a better diagnostic value than the OSDI for Chinese patients with dry eye.

SERPINA3K plays antioxidant roles in cultured pterygial epithelial cells through regulating ROS system.

We recently demonstrated that SERPINA3K, a serine proteinase inhibitor, has antioxidant activity in the cornea. Here we investigated the antioxidant effects of SERPINA3K on the pterygial, which is partially caused by oxidative stress in pathogenesis. The head part of primary pterygial tissue was dissected and then cultured in keratinocyte serum-free defined medium (KSFM). The cultured pterygial epithelial cells (PECs) were treated with SERPINA3K. The cell proliferation and migration of PECs were measured and analyzed. Western blot and quantitative real-time polymerase chain reaction (PCR) assay were performed. It showed that SERPINA3K significantly suppressed the cell proliferation of PECs in a concentration-dependent manner, compared with cultured human conjunctival epithelial cells. SERPINA3K also inhibited the cell migration of PECs. Towards its underlying mechanism, SERPINA3K had antioxidant activities on the PECs by significantly inhibiting NADPH oxidase 4 (NOX4), which is an important enzyme of ROS generation, and by elevating the levels of key antioxidant factors of ROS: such as NAD(P)H dehydrogenase (quinone 1) (NQO1), NF-E2-related factor-2 (NRF2) and superoxide dismutases (SOD2). Meanwhile, SERPINA3K down-regulated the key effectors of Wnt signaling pathway: ß-catenin, nonphospho-ß-catenin, and low-density lipoprotein receptor-related protein 6 (LRP6). We provided novel evidence that SERPINA3K had inhibitory effects on pterygium and SERPINA3K played antioxidant role via regulating the ROS system and antioxidants.

Amniotic membrane extract ameliorates benzalkonium chloride-induced dry eye in a murine model.

Human amniotic membrane (AM) is avascular but contains various beneficial bioactive factors, its extract (AE) is also effective in treating many ocular surface disorders. In this study, we for the first time evaluated the therapeutic effects of AE on dry eye induced by benzalkonium chloride in a BALB/c mouse model. Topical application of AE (1.5 and 3 µg/eye/day) resulted in significantly longer tear break-up time on Day 3 and 6, lower fluorescein staining scores on Day 3, and lower inflammatory index on Day 6. AE reduced corneal epithelial K10 expression, inflammatory infiltration, and levels of TNF-α, IL-1ß and IL-6 in BAC treated mice than that in the control mice. Moreover, decreased TUNEL positive cells in cornea and increased goblet cells in conjunctiva were also observed in AE treated corneas. Finally, AE induced more Ki-67 positive cells in corneal epithelium of dry eye mouse. Taken together, our data provide further support for BAC induced dry eye model as a valuable for dry eye study and suggest a great potential for AE as a therapeutic agent in the clinical treatment of dry eye.

Increased importin 13 activity is associated with the pathogenesis of pterygium.

PURPOSE: We previously reported that importin 13 (IPO13), a member of the importin-ß family of nuclear import proteins, regulates nuclear import of the glucocorticoid receptor in airway epithelial cells, IPO13 serves as a potential marker for corneal epithelial progenitor cells, and IPO13 is associated with corneal cell proliferation. Here we investigated the role of IPO13 in the pathogenesis of pterygium and the underlying mechanism including interaction with other cell proliferation-related factors: keratin 17 (K17), a lesional protein and a member of the type I keratins, and c-Jun, a protein of the activator protein-1 complex. METHODS: Tissue samples were collected from primary pterygia, recurrent pterygia, and normal conjunctiva to perform the following experiments: immunohistochemical measurement of IPO13 and K17. Pterygium epithelial cells (PECs) were cultured in keratinocyte serum-free defined medium to examine the expression of IPO13 and K17. Lentivirus-mediated silencing and overexpression IPO13 testing was conducted, and K17 alternation was evaluated with western blot and immunostaining. In addition, the translocation of c-Jun (a K17 regulator) was further examined after IPO13 was silenced. RESULTS: IPO13 activity was significantly increased in the basal layer of the epithelium of the pterygium. In cultured PECs, overexpression or knockdown of the IPO13 gene increased or decreased PEC proliferation, respectively. IPO13 was colocalized with K17 in the epithelium of the pterygium, and overexpression or knockdown of the IPO13 gene induced upregulation or downregulation of K17 expression in PECs, respectively. In addition, silencing of the IPO13 gene blocked nuclear translocation of c-Jun. CONCLUSIONS: We provided novel evidence that IPO13 may contribute to the pathogenesis of pterygium via modulation of K17 and c-Jun.

[Abnormal of tear lipid layer and recent advances in clinical study of dry eye].

Dry eye is a common disease in the ophthalmological clinic, which is related to the dysfunction of tear film. The tear film is composed of lipid layer, aqueous layer and mucin layer (or lipid layer, aqueous/mucin layer). The lipid of the outmost layer derived from Meibomian gland and distributed on the tear film after blinking can decrease the evaporation and stabilize the tear film. The thickness, quality, and distribution of lipid layer are impaired in many dry eye patients, hence restoring the physiological function of lipid layer may be crucial for the treatment of this kind of dry eye. The lipid artificial tears manifest great effects on increasing lipid layer thickness, stabilizing tear film, improving Meibomian gland dysfunction, and promoting tear film distribution.

SERPINA3K protects against oxidative stress via modulating ROS generation/degradation and KEAP1-NRF2 pathway in the corneal epithelium.

PURPOSE: We recently reported that SERPINA3K (SA3K), a member of the serine proteinase inhibitor (SERPIN) family, has antiangiogenic and anti-inflammatory activities. Here we investigated the antioxidant effects of SA3K in the corneal epithelium and the mechanism underlying its action. METHODS: We established the oxidative stress models induced by hydrogen peroxide (H2O2) in cultured human corneal epithelial (HCE) cells and in rat corneal epithelium in vivo. Cell viability, flow cytometry, and TUNEL analysis were conducted to detect viable cells and cell death; reactive oxygen species (ROS) and 3-Nitrotyrosine fluorescent assay was applied to measure ROS levels. Activity assay, immunostaining, Western blot, and quantitative RT-PCR were performed to analyze the factors of the ROS generation/degradation system and pathway. RESULTS: SA3K protected the HCE cells from H2O2-induced oxidative stress in a dose- and time-dependent manner. SA3K also significantly reduced the production of ROS. Regarding the mechanism underlying these effects, SA3K downregulated ROS generation by inhibiting NOX4 and upregulated ROS degradation by increasing the activity of superoxide dismutases and catalase. Furthermore, H2O2 induced activation of the Kelch-like ECH-associated protein 1 (KEAP1)/NF-E2-related factor-2 (NRF2) pathway, while SA3K inhibited H2O2-induced activation of KEAP1 and NRF2 and their downstream factors, including NAD(P)H quinone oxidoreductase and glutathione S-transferase. In the H2O2-induced rat corneal epithelium, SA3K alleviated the oxidative stress and downregulated NOX4 and NRF2. CONCLUSIONS: Collectively, SA3K protects against oxidative stress by targeting the ROS generation/degradation system and modulating the KEAP1-NRF2 signaling pathway.

Therapeutic effects of epidermal growth factor on benzalkonium chloride-induced dry eye in a mouse model.

PURPOSE: To investigate the therapeutic effects and possible mechanisms of epidermal growth factor (EGF) on the mouse dry eye model induced by benzalkonium chloride (BAC). METHODS: The eye drop containing EGF was topically administered (3 ng per day) on a BAC-induced dry eye model. The following clinical indications of dry eye were evaluated on Days 2, 4, and 6: tear break-up time (BUT), corneal fluorescein staining, inflammatory index, and tear volume. Global specimens were collected on Day 6 and then the following examinations were performed: histologic investigation, TUNEL assay to measure the dead cells, periodic acid-schiff (PAS) assay to detect goblet cells, and immunostaining of antibodies of Ki-67, EGF receptor (EGFR), and MUC1 in the corneas. The levels of EGFR and p-ERK of the corneas were also measured by Western blot analysis. RESULTS: EGF resulted in longer BUTs on Days 2 and 6, lower fluorescein staining scores on Days 4 and 6, while no significant changes in inflammatory index or tear volume. EGF induced higher EGFR expression in corneal tissues by immunofluorescent staining and Western blot analysis. EGF also upregulated p-ERK, increased Ki-67 positive cells, and decreased TUNEL positive cells. In addition, EGF significantly increased the goblet cells number and MUC1 expression in the epithelium. CONCLUSIONS: Topical application of EGF presented clinical improvements on dry eye by stabilizing the tear film and maintaining the integrity of epithelium. The results indicate that EGF has potential as a therapeutic agent in clinical treatment of dry eye.