Authors: Zhang Huiqin, Li Ying, Feng Lina, Xu Yonggen
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Background
Studies showed that inflammation is associated with the pathogenesis and development of dry eyes, and tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) are key inflammatory factors.Thymosin β4 (Tβ4) plays a promoting effect on the migration of epithelial cells and anti-inflammatory action.However, the influences of Tβ4 on the repair of ocular surface in dry eyes are unelucidated.
Objective
This study was to investigate the regulation of Tβ4 to the expressions of TNF-α and IFN-γ and its effect on the recovery of ocular surface in rat dry eye models.
Methods
The dry eye models were induced by topically administered of benzalkonium chloride (BAC) for consecutive 7 days in the left eyes of 50 SPF male SD rats, and 36 successful models were used in the experiment.Tβ4 solution (9 μg/ml), recombinant human epithelial growth factor (rhEGF) and sterile PBS at 5 μl was topically administered three times for consecutive 7 days in the Tβ4 group, rhEGF group and PBS group, and no drug was used in the model control group.The normal right eyes of rats served as the normal control group.The break-up time of tear film (BUT), corneal fluorescein staining score and Schirmer Ⅰ test (SⅠt) were examined and evaluated in the rats on the seventh day after administration of drugs.Then the rats were sacrificed by excessive anesthesia and the sections of the ocular surface were prepared.The morphology of the specimens was examined by hematoxylin and eosin staining, and the number of conjunctival gobelt cells was counted by periodic acid-schiff staining.The ultrastructure of the corneal and conjunctival cells was examined under the transmission electron microscope.The expressions of TNF-α mRNA and IFN-γ mRNA and their proteins in conjunctiva tissue were quantified by quantitative real-time PCR and Western blot, respectively.The use and care of the animals followed by Regulation for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.
Results
The BUT was (10.42±0.66), (7.46±0.49), (8.71±0.50), (9.59±0.35) and (8.63±0.68) seconds in the normal control group, model control group, rhEGF group, Tβ4 group and BUT group, showing a significant difference among the groups (F=5.65, P=0.00), and the BUT was evidently shortened in the model control group compared with the normal control group, while the BUT was significantly extended in the rhEGF group and Tβ4 group in comparison with the model control group (all at P<0.05). No significant differences were found in the corneal fluorescence score and SⅠt among the groups (F=0.42, P=0.79; F=136.77, P=0.00). The corneal and conjunctival epithelium defect and corneal stromal edema were seen in the model control group, and the proliferation of the epithelial cells were found in the rhEGF group and Tβ4 group, with the irregulated arrangement of the cells.A considerable difference was seen in the number of conjunctival goblet cells among the groups (F=3.16, P=0.04), and the number of conjunctival goblet cells in the rhEGF group and model control group was significantly less than that in the normal control group (all at P<0.05), and no statistically significant difference was seen between Tβ4 group and normal control group (P>0.05). The swelling, mergence, crispation, rupture and decrease of the microvilli and micro fold were found in the model control group, and the repair of the cell microvilli was seen in the Tβ4 group.The expressions of the TNF-α mRNA, IFN-γ mRNA and their proteins in the conjunctiva were significantly different among the groups (F=43.08, 371.69, 34.27, 43.52, all at P=0.00), the expressions of the inflammatory factors were significantly higher in the model control group compared with the normal control group, and these expressions were evidently lower in the Tβ4 group in comparison with the model control group and rhEGF group (all at P<0.05).
Conclusions
The topical administration of Tβ4 solution can promote the repair of ocular surface by down-regulating the expression of TNF-α and IFN-γ in conjunctiva and stablize the tear film in rat dry eyes.