Preliminary Study On Factors Related To Meibomian Gland Dysfunction Induced By Hyperglycemia And Dry Environment In Mice

ObjectiveThe pathogenesis of diabetic-related dry eye is complicated,including of both aqueous deficient and evaporative dry eye.Meibomian gland dysfunction(MGD)is the main cause of evaporative dry eye.Meibomian glands are adjust by nerves and hormones which are closely related to diabetes.In this study,we observe the expressions of related factors that may cause MGD in diabetic-related dry eye animal models,which are simulated by hyperglycemia and the desiccating stress environment,in order to provide theoretical treatment.MethodsA total of 100 SPF 8-week-old C57BL/6 male mice(body weight 18-20 g)were randomly divided into normal control group 20(Nor group),diabetes group 30(DM group),dry eye group 20(Dry group)and diabetic related dry eye group 30(DD group).1.Diabetes models were intraperitoneal injected by 2% streptozotocin(STZ)method,monitor blood glucose weekly,and corneal fluorescein staining test every 4weeks.Sixteen weeks later,all mice were sacrificed.MG sections were subjected to H&E staining,oil red O staining and immunostaining.Real time-PCR and Westernblot were performed to detect the relative gene expression in MGs;2.20 animal models of dry group were prepared using the environmental intelligent control drying system(ICES).Corneal fluorescein staining and tear secretion tests were performed every 4 weeks.The animals were sacrificed after 16 weeks of feeding.MGs tissues were subjected to H&E staining,immunohistochemical staining,oil red O staining and TUNEL staining;real time-PCR and Western-blot were performed to detect the relative gene expression in MGs;3.30 diabetes mice were placed in ICES,so as to build diabetic-related dry eye animal models(DD group).Weekly blood glucose monitoring,corneal fluorescence staining and Schirmer’s test were performed every 4 weeks.Sixteen weeks later,allmice were sacrificed.MG sections were subjected HE staining,oil red O staining and immunohistochemical among DD group,DM group,Dry group and Nor group;TUNEL staining was compared between DD group and Nor group;real-time PCR and Western-Blot were used to detect the expression of relevant factors in meibomian gland tissue,and statistical analysis was performed.Results1.The corneal fluorescein staining scores of the DM group increased compared with the Nor group,and the difference was statistically significant(P<0.05).In the DM group,HE staining showed that the duct wall of MG became thinner,the lumen enlarged,and the acinar swelled.Immunohistochemical staining showed the expression of PPARγ increased in the cytoplasm and nucleus of meibomian glands.Oil red O staining showed oil red staining in a large number of acinar tissues.Real-time PCR detected DM group compared with Nor group,TNF-α mRNA increased,PEDF mRNA decreased,PPARγ mRNA increased,the differences were statistically significant(P<0.01).Western-Blot detected the relative expression of PEDF and PPARγ protein and mRNA,but the difference was no statistically significance.2.The corneal fluorescein staining score in Dry group increased compared with the Nor group,and the difference was statistically significant(P<0.05).The secretion of tear decreased with time in Dry gourp,and the difference of at 16 W decreased significantly(P<0.05).HE staining showed that the duct wall of MG became thinner,the lumen expanded.Immunohistochemical staining increased the expression of PPARγ in the cytoplasm and nucleus of meibomian glands.Oil red O staining showed lipid accumulated in the acinus and the open end of MGs.TUNEL suggests apoptosis of ductal epithelial cells.The difference of Ki67 mRNA detected by real-time PCR was statistically significant(P<0.05).3.The corneal fluorescein staining of the DD group,DM group,and Dry group compared with the Nor group were damaged,and the degree of damage increased with the extension of the course of the disease.There was no difference between the DM group and Dry group.The combination of the two factors in the early stage willaggravate the injury,and there is no significant differences between the three groups after 16 weeks.In the DD group,HE staining showed that the duct wall of MGs became thinner,lumen atrophy,and acinar swelled.The expression of PPARγ in the cytoplasm and nucleus of the meibomian gland tissue was significantly increased by immunohistochemical staining;Oil red O staining showed lipid accumulated in the acinus and the open end of MGs.TUNEL suggests apoptosis of ductal epithelial cells.Real-time PCR detects the difference in the relative expression of PPARγ mRNA,Ki67 mRNA,ADFP mRNA significances(P<0.05);Western-Blot detection of PEDF and PPARγ protein and mRNA relative expression levels are consistent,but the difference is no statistical significance.Conclusions1.The combined stimulation of hyperglycemia and dry stress builds the clinical diabetic-related dry eye model,resulting in serious corneal epithelial damage,tear film secretion decreased,and aggravated with the extension of the disease course.2.Hyperglycemia and dry environment can cause MGD,but the pathogenesis is different.The MGD stimulated by hyperglycemia mainly because of inflammation.The main reason for the abnormal function of MGs in dry environment is the abnormal function of the catheter.Because of inflammation,abnormal duct function,increased secretion of meibomate,all of which aggravate MGD of diabetic-related dry eye.3.PEDF and PPARγ,as upstream factors,regulates related factors during the pathogenesis of meibomian glands in diabetes-related dry eyes.These factors are related to inflammation,lipid metabolism regulation,apoptosis,and so on.So as to treat of MGD in diabetic-related dry eyes.