ABSTRACT
Background Researches showed that thymic stromal lymphopoietin (TSLP) is an interleukin-17-like inflammatory factor and plays important roles in the pathogenesis and development of allergic diseases.However,the study whether TSLP plays roles in allergic conjunctivitis is rare.Objective This study was to investigate the expression change of TSLP and IL-4 in ocular surface tissue and cervical lymph node in the mice with allergic conjunctivitis induced by artemisia annua,a common plant in China,and to explore the role of TSLP and IL-4 in the pathogenesis and development of allergic conjunctivitis.Methods Twenty female BALB/c mice aged 6-8 weeks were randomized into normal control group and model group.Antigen solution was prepared using 400 μl extracting solution of artemisia annua pollen with 400 μl antigen solvent.The mouse models of allergic conjunctivitis were established by injection of 50 μl antigen solution in footpad followed by ocular topical administration of extracting solution once a day from day 10 to 12 after injection,and no any intervention was given in the normal control group.The mice were sacrificed and eyeballs were obtained 13 days after modeling,and corneal epithelium,conjunctiva and cervical lymph nodes were harvested for the detection of TSLP mRNA and IL-4 mRNA by real-time PCR.Immunochemistry was employed to assay the expression of TSLP and IL-4 proteins in corneal,conjunctival and subconjunctival tissues.Results Ocular inflammatory signs appeared 0.5 hours after ocular topical administration of extracting solution,including eyelid edema,conjunctival congestion,tears,scratching eyelids,etc.The symptoms lasted for 6-24 hours,with the model successful rate 80％.Real-time PCR indicated that the expression of IL-4 was absent in corneal epithelium in both model group and normal control group.The relative expression levels of TSLP mRNA in the corneal epithelium,conjunctiva and cervical lymph nodes in the model group were more (1.63±0.20)times,(2.71±0.48) times and (1.48 ±0.05) times than the normal control group,showing significant differences between the two groups (t =4.44,14.16,5.01,all at P＜0.05).Compared with the normal control group,the relative expression levels of IL-4 mRNA in the model group increased (2.94±0.39) times and (1.74±0.09) times,with significant differences between the two groups (t =9.92,14.54,both at P＜0.05).Immunochemistry assay showed that the expression of TSLP protein in the corneal and conjunctival epithelial cells were enhanced in the model group compared with the normal control group.In addition,an intensive expression of IL-4 protein was seen in subconjunctival tissue in the model group,while the expression of IL-4 was absent in the normal control group.Conclusions TSLP is mainly expressed in the cornea,conjunctiva and cervical lymph nodes of the mice with allergic conjunctivitis,suggesting that TSLP promotes the inflammatory process of cornea and conjunctiva;IL-4 is mainly expressed in conjunctiva,showing IL-4 participates in conjunctival inflammatory process.TSLP and IL-4 play synergistic roles in promoting the inflammatory process of ocular surface in the mice with allergic conjunctivitis,which may be new therapeutic targets.
ABSTRACT
Background The primary pathologic mechanism of posterior capsular opacification (PCO) is proliferation,migration and epithelial-mesenchymal transformation of residuary lens epithelial cells (LECs) following cataract surgery.Matrix metalloproteinases (MMPs) play a role during the migration of LECs.Researches showed that GM6001,a broad inhibitor of MMPs,can arrest the migration of LECs,but as specific inhibitors of MMPs,the efficacy and safety of MMP-2/9 inhibitor Ⅰ and Ⅱ on LECs migration remain unclear.Objective This study was to determine and compare the inhibitory efficacy among GM6001,MMP-2/9 inhibitor Ⅰ and Ⅱ on human LECs and search the clinical medication to prevent PCO.Methods Human LECs were cultured and passaged in vitro,and the cells of 3-4 generation were incubated in 6-well plates.Then the cells of 70％ confluent monolayer were cultured in DMEM without fetal bovine serum for 12 hours.GM6001,MMP-2/9 inhibitor Ⅰ and Ⅱ at different concentrations (0.25,0.50,1.00,2.00,4.00,8.00,16.00,32.00,64.00,128.00 μmol/L) were added into the culture medium for 24 hours separately,and regularly cultured cells served as the control group.A bare area was made by a 200 μl sterile spear on the cell layer,and the migrated distance and inhibitory rate were calculated.The second or third generation of cells were incubated in 96-well plates at a density of 5×105/ml (200 μl/well).GM6001 (128.00 μmol/L),MMP-2/9 inhibitor Ⅰ (64.00 μmol/L) and Ⅱ (32.00 μmol/L) were added into the culture medium for 24 hours,and the cell viability was assayed by using MTT assay.Results Cultured cells grew well with irregular arrangement and presented the polygon in shape.The migrated distance was gradually reduced as the increase of concentrations of GM6001,MMP-2/9 inhibitor Ⅰ and Ⅱ,showing significant differences among the various concentration groups (GM6001:F=248.647,P＜0.05;MMP-2/9 inhibitor Ⅰ:F=357.125,P＜0.05;MP2/9 inhibitor Ⅱ:F=396.374,P＜ 0.05).The cell migrated distance in the control group was set to 1,the relative migrated distances were 0.478 ± 0.091,0.294±0.088 and 0.191 ±0.081 in the GM6001 group,MMP-2/9 inhibitor Ⅰ group and MMP-2/9 inhibitor Ⅱ group at the concentrations of 32.00 μmol/L,respectively,showing a significant difference among the groups (F =116.031,P＜0.01),and cell migrated distance was obviously shorter in the MMP-2/9 inhibitor Ⅱ group than that in the GM6001 group or MMP-2/9 inhibitor Ⅰ group (all at P＜0.01).The A values were 0.607±0.016,0.567±0.015,0.583±0.010 and 0.595 ±0.0138 in the control group,GM6001 group (128.00 μmol/L),MMP-2/9 inhibitor Ⅰ group (64.00 μmol/L) and MMP-2/9 inhibitor Ⅱ group (32.00 μmol/L),respectively,without significant difference among the groups (F=1.403,P＞0.05).Conclusions GM6001,MMP-2/9 inhibitor Ⅰ and Ⅱ reduce the mobility of human LECs effectively but do not affect the viability of the cells in vitro.MMP-2/9 inhibitor Ⅱ appears to be most dominant in inhibiting migration of human LECs.