TRAF3 of blunt snout bream participates in host innate immune response to pathogenic bacteria via NF-κB signaling pathway.

Tumor necrosis factor receptor-associated factor 3 (TRAF3) is a multifunctional adaptor protein primarily involved in both bacterial defense and antiviral immunity in living organisms. However, the knowledge on TRAF3 in blunt snout bream (Megalobrama amblycephala), a freshwater fish with economic values, remained unclear. In the present study, we identified and characterized successfully Traf3 gene from M. amblycephala (maTraf3). The maTraf3 cDNA contained a 1722 bp open reading frame that encoded a protein of 573 amino acid residues. The deduced amino acid sequence comprised of a RING finger domain, two zinc finger motifs, a coiled-coil region and a MATH domain. Analysis of the transcriptional patterns of maTraf3 revealed that it was ubiquitously distributed in various tissues tested from M. amblycephala, with the abundance of expression in spleen and muscle. Following a challenge with Aeromonas hydrophila and lipopolysaccharide stimulation, the expression of maTraf3 was strongly enhanced at different time points in vitro and in vivo. MaTRAF3 was identified as a cytosolic protein and suggested to form aggregates or be associated with vesicles scattering in the cytoplasm. NF-κB transcription was activated by maTraf3 in reporter assay. The overexpression of maTraf3 produced high levels of pro-inflammatory cytokines such as IL-1ß, IL-6, IL-8 and TNF-α, implying its immune-regulatory role in M. amblycephala. Taken together, our results obtained in this study demonstrated the crucial role of maTraf3 in mediating host innate immune response to pathogen invasion via NF-κB signaling pathway, which might indicate a novel therapeutic approach to combat bacterial infection in fish.

Expression of mucins MUC5AC and MUC19 on the ocular surface in dry eye syndrome model of ovariectomized female rabbits.

BACKGROUND: Dry eye syndrome (DES) is a common symptom of tear film instability and ocular surface damage due to an abnormal quality and quantity of tears, including the sensation of foreign objects and blurred vision. Among all factors for tear film stability, MUC5AC and MUC19 are very important; the levels of both mucins are associated with the pathogenesis of DES. OBJECTIVES: The aim of this study was to explore the expression of MUC5AC and MUC19 on the ocular surface in a DES model of ovariectomized female rabbits. MATERIAL AND METHODS: Healthy female New Zealand white rabbits (n = 18; age: 1 year, weight: 2.5 ±0.6 kg) were randomly assigned to a test group and a control group. The DES model was constructed in ovariectomized female rabbits. Indicators of ocular surface injury, such as Schirmer's test, corneal fluorescence staining, a conjunctival imprinting cytology test, and the expression of MUC5AC and MUC19 in conjunctival tissues were evaluated by immunohistochemistry in week 1, week 2 and week 4. RESULTS: Both the length of soaked test paper and the total scores of corneal fluorescence staining at all time-points were significantly lower in the test group than in the control group, and they decreased over time (p < 0.05). The grades of imprinted cells at all time-points were significantly higher in the test group than in the control group, and they increased over time (p < 0.05). The percentage of goblet cells was significantly lower in the test group than in the control group, and it decreased over time (p < 0.05). The percentages of cells with a positive expression of MUC5AC and MUC19 at all time-points were significantly lower in the test group than in the control group, and they decreased over time (p < 0.05). CONCLUSIONS: The pathogenesis of DES is associated with an increased grade of imprinted cells, decreased goblet cells, and a decreased expression of MUC5AC and MUC19.

TUG1 promotes lens epithelial cell apoptosis by regulating miR-421/caspase-3 axis in age-related cataract.

BACKGROUND: Age-related cataract is among the most common chronic disorders of ageing and the apoptosis of lens epithelial cells contributes to non-congenital cataract development. We amid to explore the role of TUG1 and miR-421 in the age-related cataract. METHODS: The expression level of TUG1, miR-421 and caspase-3 were detected by RT-qPCR. The apoptotic-related protein, caspase-3, Bax and blc-2 were analyzed by western blot. We performed ultraviolet (UV) irradiation to induce SAR01/04 cell apoptosis which was analyzed by flow cytometry. RIP pull-down and luciferase reporter assay were used to verified the combination and regulating among TUG1, miR-421 and caspase-3. RESULTS: Here, we observed that the expression level of TUG1 and caspase-3 in the anterior lens capsules of age-related cataract were significantly higher and miR-421 was significantly lower than that in the normal anterior lens capsules. The apoptosis-related protein, caspase-3, Bax and blc-2 were abnormal expression in the anterior lens capsules of age-related cataract tissue. Our data showed that the expression level of TUG1 and caspase-3 and cell apoptosis rate in SAR01/04 cells treated with UV irradiation was remarkably higher than that in the control. TUG1 negatively regulated miR-421 expression and promoted UV irradiation-induced SAR01/04 cell apoptosis. However, miR-421 inhibitor and pcDNA-caspase-3 could reverse the action of the SRA01/04 cell apoptosis by si-TUG1, which suggested TUG1 promoted UV irradiation-induced apoptosis through downregulating miR-421 expression. Furthermore, this study confirmed TUG1 could been in combination with miR-421, and TUG1 and caspase-3 were both a directly target of miR-421. CONCLUSION: TUG1 modulated lens epithelial cell apoptosis through miR-421/caspase-3 axis. These findings will offer a novel insight into the pathogenesis of cataract.

Biocompatibility and drug release behavior of chitosan/poly (vinyl alcohol) corneal shield in vivo.

UNLABELLED: Backgound: Chitosan/polyvinyl alcohol corneal cap has good biocompatibility and drug slow release characteristics, which provided new treatment method for anterior segment disease. Our study was to evaluate biocompatibility of poly (vinyl alcohol)/chitosan corneal shield's intraocular and investigate its feasibility to treat ocular surface disorders. METHODS: Thirty-six white rabbits were randomly divided into four groups. Slit lamp observation were conducted at 1, 3, 7 and 10 days after operation. Corneal and conjunctiva tissue harvested from the experimental groups was observed by HE staining 10 days after operation. The aqueous humor was aspirated from the anterior chamber at each designated time point (1, 3, 7 and 10 days). The cornea and conjunctive were collected at 10 days. The concentration of each tissue was analyzed by ultra-performance liquid chromatography and microscope observation. RESULTS: In all groups, mild hyperemia was observed 1 day after operation, and there was no obvious inflammatory reaction occurring on the seventh and tenth day. No corneal edema and inflammatory reaction of anterior chamber occurred till the tenth day. For histopathology, there was no obviously mild chronic and inflammatory reaction occurred, and no significant difference between the corneal shield with-in groups and with-out groups. The drug concentrations in corneal and conjunctival in group (A, B) were significantly lower than eye drops in the control group (C, D), and blank corneal cover in group C was significantly sham operation in group D. CONCLUSION: The results indicated that the proposed membrane combined with ophthalmic solution has substantial potential as ocular delivery system.