ABSTRACT
Background Dry eye is increasing gradually recently,but its etiology and manifestation are very diverse.Studies showed that menopause of adult females was one of the risk factors of dry eye.In addition,some inflammatory factors also participate in the pathogenesis and development.But the study on the relationship of sex hormone with inflammation and ocular surface damage is still below understanding.Objective This study was to investigate the expressing changes of interleukin (IL) and tumor necrosis factor-α (TNF-α) in conjunctiva and the manifestation of ocular surface in ovariectomized rat model.Methods Twenty clear female SD rats were randomized into the ovariectomized group and the sham operative group according to randomized number table.Ovariectomy was performed in the ovariectomized group,and abdominal myotomy without ovariectomy was performed in the sham operative group.Serum estrogen and androgen levels were detected by radiation immunoassay 3 months after operation.Schirmer Ⅰ test (S Ⅰ t) and corneal fluorescence staining were carried out in the rats before operation and 1 month,2 and 3 months after operation.The morphology of conjunctival epithelial cells was examined by hematoxylin & eosin staining at the 3rd month after operation.The expressions of IL-17A,IL-1 β,IL-6 and TNF-α in conjunctiva were semi-quantitative analyzed by immunohistochemistry and Western blot.The use and care of the animals complied with State Science and Technology Commission Regulations for the Administration of Affair Concerning Experimental Animals.Results Serum estrogen levels were (23.53 ± 1.65) pg/L and (47.89 ± 1.05) pg/L 3 months after surgery in the ovariectomized group and the sham operative group,respectively; the serum androgen levels were (1.84±0.09) ng/L and (2.47±0.12)ng/L in the ovariectomized group and the sham operative group,respectively,showing a significant decline of serum estrogen and androgen levels in the ovariectomized group compared with the sham operative group (t=-35.37,-12.13,both at P＜0.01).No significant differences were seen in S Ⅰ t between the two groups among various time points (Fgroup =0.38,P =0.55 ; Ftime =0.13,P =0.72 ; Finteraction =0.39,P =0.76).No obvious fluorescence staining was seen in the cornea of both the ovariectomized group and the sham operative group.The histopathological examination showed that the layers of rat conjunctival epithelial cells increased with the disordered arrangement in the ovariectomized group.Immunochemistry showed that the expressions of IL-17A,IL-1β,IL-6 and TNF-α (A values) were significantly higher in the ovariectomized group than those in the sham operative group (IL-17A:t=8.22,P＜0.01 ;IL-1β:t=16.43,P＜0.01 ;IL-6:t=13.44,P＜0.01 ;TNF-α:t=16.26,P＜0.01).Western blot assay showed the similar results (IL-17A:t=19.41,P＜0.01 ;IL-1β:t=12.63,P＜0.01 ;IL-6:t=17.17,P＜0.01 ;TNF-α:t=15.19,P＜0.01).Conclusions Serum estrogen and androgen levels drop obviously,and there is an up-regulation of IL and TNF-α expression in conjunctiva tissue in the ovariectromized SD rats.However,no obvious dry eye-related sign occurs.
ABSTRACT
Background Retinal Muller cells participate in the pathological process of diabetic retinopathy (DR) through expressing vascular endothelial growth factor (VEGF).It is reported that FK506 inhibits the expression of VEGF in solid tumors and experimental corneal neovascularization,but whether FK506 exerts its role on retinal Müller cells or not is still unclear.Objective This study aimed to investigate how FK506 affects the expression vascular endothelial growth factor (VEGF) in rat retinal Müller cells under the condition of high glucose.Methods Immortalized rat retinal Müller cell line was regularly cultivated and logarithmic phase of cells were incubated in 96-well plate with the cell density of 1 × 104/ml.Different concentrations of FK506 (800.00,400.00,200.00,100.00,75.00,50.00,25.00,12.50 and 6.25 pg/ml) (100 μl/well) were added into the culture medium to determine the half maximal inhibitory concentration (IC50) of FK506 by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).The cell lines were cultured with DMEM medium (containing D-glucose of 5.5 mmol/L) or high glucose DMEM (containing D-glucose of 50 mmol/L),and 75 pg/ml FK506 were added into DMEM,respectively,and the cells were divided into the normal control group,FK506 group,high glucose culture group and high glucose + FK506 group.ELISA was employed to assay the content of VEGF protein in the cell supernatant.The expressions of VEGF mRNA and protein in the cells were detected by reverse transcription PCR (RT-PCR) and Western blot,respectively.Results The cells grew well in the normal control group,FK506 group,high glucose culture group and high glucose+FK506 group in 12,24 and 48 hours after culture with the polygon-like shape.The IC50 of FK506 was 75 pg/ml.The contents of FK506 in the cell supernatant were (966.46± 13.59) pg/ml,(1 059.42±67.43) pg/ml,(16 243.11 ±3 926.38) pg/ml and (9 467.25± 1 525.56) pg/ml in the normal control group,FK506 group,high glucose culture group and high glucose+FK506 group,respectively,showing a significant difference among the four groups (F =20.51,P =0.00).The VEGF levels in cell supernatant were significantly higher in the high glucose group than those of the normal group and the high FK506 group (P =0.00,P =0.02),but no significant difference was found in the VEGF level in cell supernatant between the control group and FK506 group (P =0.08).The expressions of VEGF mRNA and protein in the cells were significantly different among the four groups (F=126.06,P=0.00;F=5.44,P=0.01),and the relative expressing values of VEGF mRNA and protein in the cells of the high glucose group were significantly higher than those of the control group and the high+ FK506 group (all at P＜0.01).The relative expressing values of VEGF mRNA and protein were 0.64±0.09 and 0.68±0.18 in the FK506 group,which were lower than those of the normal control group (0.84±0.07 and 0.75± 0.03).However,no significant differences were seen between the two groups (P =0.05,0.07).Conclusions The expression of VEGF in rat retinal Müller cells up-regulates under the high glucose condition.FK506 plays inhibitory effects on VEGF expression to certain extent in vitro.