| Objective To estabish a kind of rabbit dry eye model with insufficient lacrimal secretion by removing the lacrimal gland,Harderian gland and the third eyelid.To investigate the effects of recombinant human lysozyme eye drops on the ocular surface of the dry eye model,and to explore the mechanism of the action.Methods 36 healthy New Zealand rabbits were used to establish dry eye model with the left eye as the operative eye and the right eye without any treatment.Schirmer I test,tear ferning test and corneal fluorescence staining were performed before operation,7 days and 14 days after operation respectively.14 days after operation,they were randomly divided into model group,basal solution group,low,middle and high concentration lysozyme eye drops group and sodium hyaluronate group.The right eye of the model control group was divided into 7 groups and 42 rabbit eyes.No treatment was given to the model control group.Give the basal solution group basal solution(5 mmol/L phosphate buffer solution(pH6.5)containing 0.75% NaCl)2 drops per eye 3 times a day.The low,middle and high concentration groups were given 0.75mg/ml,1.50mg/ml and 3.00mg/ml recombinant human lysozyme eye drops(lysozyme basic solution)three times a day for 2 drops.In the sodium hyaluronate group,sodium hyaluronate eye drops were given 3 times a day,2 drops each time.And the eyes of each group were continuously given 28 d.Before treatment,7,14,21 and 28 days after treatment,Schirmer I test,tear ferning test and corneal fluorescence staining were performed.After 28 days of treatment,the cornea was observed under light microscope.The expression of TGF-β1 and TNF-α were detected by immunohistochemistry.Determination of lysozyme in rabbit eye house by SDS-PAGE and immunoblotting method.Results 1.On the 14 th day after operation,Schirmer I test values in the model group < 5mm/5min,and they in the right eye self-control > 10 mm.Through the tear ferning test,the dentate crystallinity diagram could hardly be observed.Only a few,unshaped crystals were observed in the tear ferning test.Fluorescein sodium staining showed corneal spots and lamellar staining.The statistical results showed that the lacrimal secretion in the model group was significantly lower than that in the normal group(P < 0.01),but the lacrimal crystal grade and corneal fluorescein staining were significantly increased(P < 0.01),showing a significant change in the "quantity" and "quality" of tear fluid in the model group.And previous communications The characteristics of the animal model of dry eye were similar,which indicated that the model was successful.2.On the 7th day after treatment,the Schirmer I value in the high concentration lysozyme eye drops group was significantly higher than that in the model group(P < 0.05).On the 14 th day after administration,the Schirmer I values in the middle and high concentration lysozyme eye drops group and sodium hyaluronate group were significantly higher than those in the model control group and the basal solution group(P < 0.05).The lacrimal secretion in the lysozyme treatment group and sodium hyaluronate group was significantly higher than that in the model control group(P < 0.05)on the 28 th day after administration of lysozyme eye drops in the low,middle and high concentration groups(P < 0.05).With the prolongation of the treatment time,the crystalline morphology of lacrimal fluid in each treatment group also showed a smaller interval.On the 28 th day after administration,the crystalline grade of the four groups in low concentration,middle concentration,high concentration and sodium hyaluronate group were significantly lower than that in model group(P < 0.05),mainly grade Ⅰ and grade Ⅱ;In the model control group,the basal liquid group was better than before,but it was mainly grade Ⅲ and grade Ⅳ.On the 7th day,the score of fluorescein staining in high concentration group was lower than that in model control group(P < 0.05).On the 14 th day,the scores of fluorescein staining in low concentration group,middle concentration group,high concentration group and sodium hyaluronate group were significantly lower than those in model control group(P < 0.05),and 28 days after administration,the scores of fluorescein staining were significantly lower than those in model control group(P < 0.05).The fluorescein staining scores in the lysozyme eye drops group and sodium hyaluronate group were significantly lower than those in the model control group and the basal solution group(P < 0.05).However,there was no significant difference among the four groups in low concentration group,middle concentration group,high concentration group and sodium hyaluronate group.3.Compared with the normal group,the expression of TNF-α and TGF-β1 in the model group was significantly higher(P <0.01).Compared with the model group,the expression of TNF-α in the lower and middle and high concentration lysozyme eye drops and the sodium hyaluronate group was significantly decreased(P <0.05),and the expression of TGF-β1 was significantly increased(P <0.05).The expression of TGF-β1 in the high concentration group was significantly higher than that of the sodium hyaluronate group(P <0.05).The expression of TNF-α in high concentration group was significantly lower(P <0.05).4.The contents of aqueous lysozyme in the model group were significantly lower than those in the normal group(P < 0.01).Although the number of lysozyme eye drops in the treatment group was more positive than that in the model group,the difference is not statistically significant.Conclusion 1.Through the method of removing the lacrimal gland,Harderian gland and the third eye,it is possible to successfully establish a stable and persistent rabbit’s dry eye model of lacrimal insufficiency.2.The concentration of lysozyme in aqueous humor of dry eye animals with insufficient secretion was significantly decreased.Supplementation of recombinant human lysozyme eye drops could effectively relieve dry eye symptoms and promote the repair of ocular surface injury,and in the key evaluation index(tear secretion,tear secretion),the lysozyme concentration in aqueous humor of dry eye animals was significantly decreased.The recovery rate of lacrimal crystal morphology was obviously better than that of sodium hyaluronate eye drops which had been used clinically.3.The recombinant human lysozyme eye drops can not only relieve and improve dry eye symptoms,but also play an important role in inhibiting inflammatory reaction,suggesting that its mechanism may be related to its inhibitory effect on inflammatory reaction.4.Adding synthetic lysozyme to artificial tear can significantly improve the therapeutic effect of dry eye.This study provides a new way to develop new therapeutic drugs for dry eye. |
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