ABSTRACT
Background Studies showed that macophenolic acid (MPA)down-regulates and inhibits the expression and secretion of tissue growth factor and inflammatory factor,and further impacts the proliferation and inflammation process.Pterygium is an inflammatory and proliferative lesion.Whether MPA has an inhibitory effect on pterygium is unclear.Objective This study was to investigate the antifibrotic effects of macophenolic acid on pterygium fibroblasts(PFBs) in vitro and discuss its mechanism.Methods Pterygium tissue was obtained from pterygium patient during the surgery.PFBs were cultured using explants and identified with vimentin immunohistochemisty.0,0.125,0.250,0.500,1.000 μmol/L MPA were added into the culture medium,respectively,and the cells were cultured in the medium without MPA as the control group.MTT colorimetry was used to find the optimization effective concentration of MPA and evaluate their inhibitory effect on PFBs,and BrdU fluorescence staining was used to assess the growth statue of PFBs.Expressions of nuclear factor-κB(NF-κB),p65 and inhibitor of NF-κB-α(IκB-α) in the cells were detected by Western blot.Results The cells was spindle in shape 3 days after cultured and showed the vortex and radial arrangement with the positive response to vimentin.With the increase of MPA,the proliferative value of PFBs (A560)showed gradually decline,with a significant difference among the five groups (F =42.874,P＜0.01).In addition,the proliferative value of PFBs (A560) significantly lowed as the prolong of MPA active time(F=26.038,P＜0.01).BrdU fluorescence staining showed a significant decrease of DNA synthesis of PFBs with the elevation of MPA dose among the five groups(F=175.279,P＜0.05),and the A560of PFBs DNA synthesis in different concentrations of MPA groups was lower than that of the control group (all at P＜0.05).No apoptotic and necrotic cell was found after MPA action by DAPI staining.The expression level of p65 in the PFBs was 0.886±0.072 and 1.542±0.124 in the MPA group and the control group,indicating a declined value in the MPA group(P＜0.05).However,the expression value of IκB-α in the cytoplasm PFBs was significantly higher in the MPA group compared with the control group(2.141 ±0.305 vs.1.559±0.267) (P＜0.05).Conclusions MPA has an inhibitory effect on the growth of PFBs,which probably is related to the arresting of NF-κB pathway.
ABSTRACT
Background Proliferation of the human Tenon capsule fibroblasts(HTFs) is a main cause of failure of filtering surgery.To search the drug of inhibiting the growth of the HTFs is essential for the improvement of successful rate of filtering surgery.Objective The aim of this study was to investigate the inhibitory effect of apigenin on HTFs and its mechanism.Methods Human Tenon capsular tissue was obtained during the strabismus correction surgery.HTFs was primarily cultured using explant method and identified using vimentin by immunochemistry.The 3-5 generation of cells were incubated to 96-well plate.Apigenin of 0,20,40,80,160 μmol/L was added into the medium,respectively,for 24,48,72 hours,and the proliferation of HTFs was detected by sulfonyl chloride (SRB) at the wavelength of 560 nm (A560).Bromodeoxyuridine (BrdU) of 10 μg/L was added to culture the cells for 48 hours to calculate the labeling rate of BrdU.The morphology of the cells was observed using Hoechst 33258 staining,and apoptosis and cells cycle were evaluated by flow cytometry.Results Cultured cells grew well with the positive response for vimentin,showing the green fluorescence in cytoplasm.SRB assay showed that the A560 value was gradually declined with the increase of the dosage of apigenin and prolong of time (Fgroup =480.306,P =0.000 ; Ftime =555.144,P =0.000).The labeling rate after 0,40,80 μmol/L apigenin acted for 48 hours was (87.860 ±0.632)％,(61.520±4.306)％ and (23.480±4.472)％,showing a significant difference among the three groups (F =299.347,P =0.000).The labeling rate of HTFs for BrdU was significantly decreased in the 40 and 80 μmol/L apigenin groups compared with the 0 μmol/L apigenin group (P＜0.05).Hoechse 33258 staining found that the number of the HTFs was gradually decreased and the cell number of karyopyknosis and nuclear deformation was increased with the increase of apigenin dosage.Percentage of cells in G0/G1 phase were raised and that in S and G2/M phase were declined in the higher dosage apigenin group,with a significant difference among the different groups (FG0/G1 =58.621,P=0.000;Fs =32.357,P=0.001 ;FG2/M =83.998,P=0.000).In the 72nd hour after acted by 0,40,80,160 μmol/L apigenin,the apoptosis rate of HTFs was (4.77±0.21) ％,(13.24±1.35)％,(18.33±1.86) ％,(31.58 ± 2.77) ％,respectively,with a statistically significant difference among the four groups (F =204.791,P＜0.05).Conclusions Apigenin restrains the growth of HTFs by evoking G0/G1 cell cycle arrest and inducing apoptosis in a dosage-and time-dependent manner.