The Effect Of Estrogen And Androgen On Lacrimal Secretion And The Expression In Apoptosis Gene Bax, Bcl-2, TGF-β₁, MMP-2 And Its Inhibitor TIMP-2 In Lachrymal Gland Of Ovariectomized Rats

Ocular surface(cornea,conjunctiva,accessory lacrimal gland and meibomian gland),lachrymal gland and their nerve conjunction setting up a function unit for their intimate anatomy and function relationship, is called"ocular surface-lachrymal gland feedback system".They play an important role in regulating lacrimal secretion and tear-film formation together and maintaining the health of ocular surface. The damage of every element will induce to destroy the integrality and function of tear film and lead to dry eye. It is multiplicity about the pathogenesy of dry eye.Now it is sugested that dry eye is related to inflammation, apoptosis and the abnormality of neuromodulation and the imbalance of sex hormone. Dry eye is most often in old women. It is sugested that sex hormone is one of the pathogenesies of dry eye. The change of internal hormone is the important factor in post-menopause women.It remains to be ascertained, however, whether dry eye relates to estrogen excess or deficiency, androgen deficiency and/or estrogen/androgen imbalance.Apoptosis,a special cell death, is a phenomenon that in definite physiology and pathology condition cells end the life following their own program. Apoptosis is a normal physiology phenomenon,through which the body clear infect and damage and retain the body normal physiological functions.However, in certain conditions, apoptosis is a pathology phenomenon.Apoptosis coordinated regulation through many signals. The most important regulation factor is bcl-2 and its related gene. Apoptosis plays most important role in immune diseases.Apoptosis in lacrimal gland and ocular surface epithelium increase abnormally,but in local lymphocytic cell is inhibitted in dry eye . The expression of matrix metalloproteinase(MMPs) is regulated by many factors such as cytokine, growth factor, sex hormone, neurohormone,the change of cell appearance,the interaction between cells and cell transformation.Elevated levels of both MMP-2 and MMP-9, which have been found in the tears and saliva of patients with systemic dry eye disease and non-systemic dry eye disease, as well as in the lacrimal gland and cornea of a rabbit model with lacrimal gland inflammation-induced dry eye, suggest that both MMP-2 and MMP-9 may be implicated in the pathogenesis of this disease. Transforming growth factor-β1 is a kind of bioactive cytokine. It participates in regulating cell growth and differentiation. It is major rejection capability molecule in immune system. TGF-β1 stimulates the synthesis of extracellular matrix such as collagen protein, fibrous joint protein, laminin and proteoglycan and inhibits the production of MMPs. It promotes the expression of connexin receptor and the combination of ECM with these receptors. However, it is not clear that whether sex hormone regulates the expression of MMP-2 and it's inhibitor TIMP-2 in lachrymal gland and that the relationship among MMPs,sex hormone and TGF-β1.The research indicated that 17β-estradiol up-regulation proinflammatory cytokine and MMPs in human corneal epithelial cells.There is a close relationship between MMPs and dry eye.Investigators have paid more attention to dry eye and the role of sex hormone recently.Therefore,this investigation as follows: sexual mature rats had been done ovariectomy(OVX), modelling artificial menopause state, administrated systemic and local eyedrop application with estrogen and androgen. The estrogen and androgen levels in serum and lacrimal secretion, tear film break-up time and corneal fluorescence staining were measured.Lachrymal gland was obtained for pathohistological observation. The expressions of Bax, bcl-2, TGF-β1, MMP-2 and it's inhibitor TIMP-2 in lachrymal gland and relationship were studied. The effect of estrogen and androgen on lacrimal secretion and lachrymal gland function in ovariectomized rats and mechanism were investigated.It provided experiment evidence and rationale for the treatment of dry eye with estrogen and androgen to clinician. This experiment devided into 3 parts.Part 1 Effect of estrogen and androgen on lacrimal secretion and the histological and ultramicrostructure change in lacrimal gland and ocular surface in ovariectomized ratsObjective:To investigate the effect of estrogen and androgen on lacrimal secretion and the histological and ultramicrostructure change of lacrimal gland and ocular surface in ovariectomized rat. Methods: sixty-four female Wistar rats were devided into contol group, sham operation group and experiment group randomly. In sham operation group, rats were only cut little fat in abdominal cavity.In experiment group, rats had been done ovariectomy(OVX). Lacrimal secretion (SⅠt), tear film break-up time (BUT test) and corneal fluorescence staining were measured in all groups before the operation and 1,2,3,4, 5 months after the operation.Five months later, rats of OVX experiment group were treated with systemic application and eyedrop application with corn oil, estrogen and androgen and devided into experiment group 1, experiment group 2, experiment group 3, experiment group 4, experiment group 5, experiment group 6.There was no any intervention in experiment group 1.The rats in experiment group 2 were given hypodermical injection of oil (100ul )every 3 days. The rats in experiment group 3 were given hypodermical injection of estradiol benzoate (200ug/kg)every 3 days. The rats in experiment group 4 were given eyedrop of estradiol benzoate 4 times every day. The rats in experiment group 5 were given intramuscular injection of testosterone (3.75mg/kg)every 3 days. The rats in experiment group 6 were given eyedrop of testosterone 4 times every day.Six weeks later, SIt and BUT test and corneal fluorescence staining were measured.Then all the rats were sacrificed. The lachrymal gland, cornea and conjunctiva were obtained for pathohistological ultramicrostructure observation. The serum estrogen and androgen were measured before and 5 months after the operation,as well as before death. Results: 1 The result of BUT in the experiment group 1 month after OVX was shortter than that before OVX (P<0.01). The result of BUT in the experiment group 3 with estradiol enzoate injected hypodermically for 6 weeks was shortter than that before injection (P<0.01).While the BUT of the experiment group 3 with testosterone intramuscular injected for 6 weeks was longer than that before injection (P<0.01). There was no difference in other groups (P>0.05).2 The result of SⅠt 3 months after OVX decreased to 50% of the result before OVX (P<0.01). The result of SⅠt in experiment group 3 was shorter than that before injection(P<0.05). The result of SⅠt in experiment group 5 was longer than that before injection(P<0.05). There was no difference in other groups (P>0.05). 3 The score of corneal fluorescence staining 4 months after OVX increased compared with the score before OVX(P<0.05). The score of corneal fluorescence staining in experiment group 3 increased compared with the score before injection(P<0.05). The score of corneal fluorescence staining in experiment group 5 decreased compared with the score before injection(P<0.05). There was no difference in other groups(P>0.05).4 The histological change and ultrastructure of lacrimal gland and ocular surface:There had been obvious improvement in experiment group 5 than experiment group 1. There had been more serious in experiment group 3 than experiment group 1. 5 The estrogen and androgen levels in serum decreased after OVX,compared with those before OVX. After estrogen or androgen injected, both the serum estrogen level in experiment group 3 and the serum androgen level in experiment group 5 increased.Conclusion:1 The sexual mature rat had been done ovariectomy(OVX), modelling artificial menopause state , the level of estrogen and androgen in serum decreased, lacrimal secretion decreased, the stability of tear-film decreased.In systemic androgen treatment group, lacrimal secretion increased, the stability of tear-film increased. In systemic estrogen treatment group, lacrimal secretion decreased, the stability of tear-film decreased. It indicated that the decrease of lacrimal secretion and stability of tear-film is related to the decrease of the level of androgen in serum. 2 Systemic androgen treatment can improve atrophy in the lacrimal gland caused by OVX It relieve the damage of ocular surface and increase lacrimal secretion and the stability of tear-film. 3 Systemic estrogen treatment can aggravate the atrophy in the lacrimal gland and the damage of ocular surface and decrease lacrimal secretion and the stability of tear-film. We remind clinician that it must be cautious when they applicate hormone replacement therapy.Part 2 Effect of estrogen and androgen on the expression of apoptosis in lachrymal gland in ovariectomized ratsObjective: To investigate the effect of estrogen and androgen on the expression in apoptosis gene in lachrymal gland in ovariectomized rats. Methods: The rats handled as same as part one. Six weeks after treatment, all rats were sacrificed, and the lachrymal glands were obtained. The expression of Bax and bcl-2 were detected by immunohistochemical staining in different groups. Results: 1 The optical density(OD) of Bax in lacrimal epithelium cell :The OD in experiment group 1 rats was higher than A group (P<0.01), The OD in experiment group 3 rats was higher than experiment group 1 (P<0.01).The OD in experiment group 5 rats was lower than experiment group 1 (P<0.05).The OD of bcl-2 in lacrimal epithelium cell: The OD in experiment group 1 rats was lower than A group (P<0.01), The OD in experiment group 3 rats was lower than experiment group 1(P<0.01).The OD in experiment group 5 rats was higher than experiment group 1(P<0.01).2 Apoptosis index of lacrimal epithelium:The percent of positive cell in experiment group 1 was higher than A group (P<0.01),The percent of positive cell in experiment group 3 higher than experiment group 1(P<0.01). The percent of positive cell in experiment group 5 was lower than experiment group 1 (P<0.01). Conclusions: There was expression on apoptosis of Bax in normal lacrimal epithelium. After OVX, the expression of Bax increased and the expression of bcl-2 decreased in lacrimal epithelium.The apoptosis in lachrymal gland epithelium may be one of mechanisms of action. Apoptosis in lachrymal gland epithelium became more serious in systemic estrogen group and relieved in systemic androgen group. It suggested that androgen treatment made lacrimal secretion and the stability of tear-film increase through relieving apoptosis. However, the role of estrogen is opposite.Part 3: The experiment study of estrogen and androgen on expression of TGF-β1,MMP-2,it's inhibitor TIMP-2 and relationship to TGF-β1 in lachrymal gland in ovariectomized rats.Objectives: To investigate the effect of estrogen and androgen on the expression of MMP-2mRNA,TIMP-2mRNA and MMP-2 protein in lachrymal gland in ovariectomized rats.To make correlation analysis of TGF-β1,MMP-2 and TIMP-2.To explore the function of MMP-2 and TIMP-2 in dry eye.Methods: The rats handled as same as part one. Six weeks after treatment, all rats were sacrificed, and the lachrymal gland was obtained.The expression of MMP-2mRNA and TIMP-2mRNA was detected by RT-PCR.The expression of MMP-2 protein was detected by Western-blot. The level of TGF-β1was detected by ELISA.Results:1 The expression of MMP-2mRNA in lachrymal gland:Six weeks after treatment, there was significant differences on the expression of MMP-2mRNA in lachrymal gland in different groups(P<0.01).Experiment group 1 was hihger than A group(P<0.05).Experiment group 3 was hihger than experiment group 1(P<0.05).Experiment group 5 was lower than experiment group 1(P<0.01).2 The expression of TIMP-2 mRNA in lachrymal gland: Six weeks after treatment, there was significant differences of the expression of TIMP-2 mRNA in lachrymal gland in different groups(P<0.01). Experiment group 1 was higher than A group (P<0.05).Eexperiment group 3 was hihger than experiment group 1(P<0.05).Experiment group 5 was lower than experiment group 1(P<0.01).3 The expression of MMP-2 protein in lachrymal gland: Six weeks after treatment, there were significant difference of the expression of MMP-2 protein in lachrymal gland in different groups(P<0.01). Experiment group 1 was higher than A group(P<0.05).Experiment group 3 was higher than experiment group 1(P<0.05).Experiment group 5 was lower than experiment group 1(P<0.01). 4 The expression of TGF-β1 in lachrymal gland: There was significant difference in different groups after treatment for 6 weeks about mass concentration of TGF-β1 in supernatant of lachrymal gland homogenate(P<0.01). There was significant difference between experiment group 5 and experiment group 1 after treatment for 6 weeks about mass concentration of TGF-β1 in supernatant of lachrymal gland homogenate(P<0.01).5 The correlation analysis among TGF-β1,MMP-2mRNA and TIMP-2mRNA:There was no correlation between TGF-β1 and MMP-2mRNA(r=0.089,P=0.587).There was no correlation between TGF-β1 and TIMP-2 mRNA(r=0.106,P=0.515).There was correlation between MMP-2 mRNA and TIMP-2 mRNA(r=0.445,P = 0.004). Conclusion:1 After OVX the level of MMP-2 mRNA and TIMP-2 mRNA in lachrymal gland is higher than normal group.Estrogen up-regulate the expression of MMP-2mRNA and TIMP-2mRNA in lachrymal gland. Androgen down-regulate the expression of MMP-2mRNAand TIMP-2mRNA in lachrymal gland. There was correlation between MMP-2 mRNA and TIMP-2 mRNA.2 After OVX the level of MMP-2 protein in lachrymal gland is higher than normal group.Estrogen up-regulate the expression of MMP-2 protein in lachrymal gland. Androgen down-regulate the expression of MMP-2 protein in lachrymal gland. Androgen can promote lacrimal secretion and the expression of TGF-β1 in lacrimal gland in OVX rats.It suggested that estrogen induced the decrease of lacrimal secretion and the stability of tear-film through up-regulation of MMP-2. The role of androgen is opposite.In one hand,it increase the level of TGF-β1 and inhibit immune reaction in lachrymal gland. In another hand, it inhibit the expression of MMP-2.