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Objective To study the effect and mechanisms of chloride channel blocker 5-Nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) on thansforming growth factor β1 (TGF-β1) induced human conjunctival fibroblasts (HConF) fibrosis.Methods Cell counting kit (CCK-8) was used to screen out the optimal TGF-β1 treatment time and the optimal NPPB concentration.The cells were divided into control group,TGF-β1 treatment group and TGF-β1+NPPB group.Cell proliferation and cell cycle were detected by CCK-8 and flow cytometer,respectively.Cell migration ability were observed by scratch and transwell migration assays.Western blot and Real time-PCR were used to detect the expression of collagen Ⅰ (COL-Ⅰ),fibronectin (FN) and α-smooth muscle actin (α-SMA).The phosphorylation level of PI3K and Akt were measured by Western blot.Results TGF-β1 promotes cell proliferation in a time-dependent manner.There was no statistically significant difference in A values between 48 hours and 72 hours after TGF-β1 treatment (P =0.064).Forty-eight hours was selected as the most appropriate time for TGF-β1 treatment.NPPB inhibited HConF cell proliferation in a concentration-dependent manner.Compared with the control group,the proliferation A values of cells in the 50 mol/L and 100 mol/L NPPB groups were significantly reduced (P =0.020,0.000),and 100 mol/L was selected as the optimal concentration of NPPB.The cell proliferation A value,migration area and migration cell number of TGF-β1 +NPPB group were significantly lower than those of TGF-β1 treatment group (all at P＜0.05).Compared with the control group and TGF-β1 +NPPB group,the proportion of G1 phase cells in the TGF-β1 treatment group was reduced,and the proportion of cells in the S phase and G2/M phase were increased,with statistically significant differences between them (all at P ＜ 0.05).The protein and mRNA expression of α-SMA,COL-Ⅰ and FN in the TGF-β1 treatment group were higher than those in the control group and TGF-β1+NPPB group,with statistically significant differences between them(all at P＜0.05);the ratios of p-PI3K/PI3K and p-Akt/Akt in the TGF-β1 treatment group were significantly higher than those in the control group and TGF-β1 +NPPB group,with statistically significant differences between them (all at P＜0.05).Conclusions NPPB may inhibit TGF-β1 induced HConF fibrosis process by inhibiting phosphorylation of PI3K and Akt.
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Objective To study the inhibitive effects of matrine on the formation of glaucoma filtration bleb scar.Methods A total of 40 New Zealand white rabbit were chosen and randomly divided into four groups(group A,B,C and D).Both eyes in four groups were treated with glaucoma filtration surgery.The cotton pieces under the sclera flap were made:1.0 g · L-1 matrine cotton piece in group A,0.4 g · L-1 matrine cotton piece in group B,0.2 mg · L-1 mitomycin cotton piece in group C and PBS cotton piece in group D.The intraocular pressure,filtering bleb,anterior chamber reaction and complications were observed at preoperative and postoperative 1 day,3 days,7 days,14 days,28 days in all groups.Two rabbits selected randomly from every group were killed at 1 day,3 days,7 days,14 days and 28 days after surgery.Tissue was harvested from the bleb area and made into pathological section,immunestaining and Masson trichromatic dyeing were used to observe the inflammatory cells,collagen,fibroblasts,the change of muscle fibroblasts and new collagen fibers under light microscope.The cornea was observed at 7 days under transmission electron microscope for drug toxic reaction,Results At postoperative 7 days,14 days,28 days,there were statistical difference in IOP between group A,B,C and group D group (P ＜ 0.05),and there were statistical difference between group A and group B (P ＜ 0.05).At postoperative 7 days,14 days,28 days,there were statistical difference in fibroblasts counting between group A,B,C and group D group (P ＜0.01),and there were statistical differences between group A and group B (P ＜ 0.05).Conclusion Matrine can inhibit fibroblasts hyperplasia,reduce the scar of filtration area and has no side effects on eye tissues.
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Abstract? AlM: To investigate the inhibition effect of bevacizumab on human Tenon capsule fibroblasts ( HTFs ) and discuss the countermeasures of bleb scarringscarring in glaucoma surgery countermeasures.? METHODS: Adopted cell recovery method and followed the aseptic principles, we performed the culture of HTFs which came from the Central Laboratory of Shaanxi People's Hospital cell library. Wound Healing assay:We scraped a cell-free zone on the cell surface when the cells reached confluence at 80%. The control group was added to serum-free DMEM medium. The HTFs of bevacizumab group were stimulated with 1mg/mL concentrations without DEME for 0, 24, 48, and 72h. The scratch width was observed and measured.? RESULTS: HTFs were long fusiform shape under microscope, the nucleus is in the center of the cell with larger nucleus, abundant cytoplasm, were arranged in a whorled growth out of shape, strong ability to proliferate, conform to general forms of fibroblast. The morphological and biological characteristics of cells after cryopreservation and resuscitation remain unchanged. Wound healing assay: 0h, equal to the initial width of the two groups, 24h when the migration distance of the two groups of cells are basically the same, 48h when the control group cell migration distance is greater than that in the bevacizumab processing group, 72h when the control group scratches basichealing, bevacizumab treated cells migrate closer than 48h no significant change, and a lot of cells died.?CONCLUSlON:Cell recovery method can successfully cultured HTFs, which was stability on morphology and biological properties, laying the cellular basis for experimental research. Fibroblast itself has a strong ability to migrate, outside - derived bevacizumab can inhibit HTFs migration evidently and it will cause excessive cell death when. Bevacizumab has certain extent inhibitory effect on HTFs migration, and it is likely to become one of the important drugs for creating bleb scarring after glaucoma surgery in the future.
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Objective@#To determine the effect of topically administered bevacizumab on bleb survival and histology after trabeculectomy in rabbit eyes.@*Methods@#This is an experimental interventional comparative animal study. Sixteen rabbit eyes underwent trabeculectomy, 8 of which were enhanced with intraoperative mitomycin-C. Eyes were randomized to receive either topical balanced salt solution (BSS) or topical bevacizumab at a concentration of 12.5 mg/mL. Intraocular pressure, bleb dimensions and vascularity grading were measured. IOP was recorded as a ratio of IOP of the experimental operated eye divided by the IOP of the contralateral control eye (IOPratio) as a function of time. Bleb morphology was recorded as a percentage of the maximum estimated bleb volume (% bleb) as a function of time. Bleb failure occurred if IOPratio ≥0.8, or if % bleb=0. The eyes were then submitted for histopathological analysis after bleb failure has occurred.@*Results@#In plain trabeculectomy, the mean bleb survival in terms of IOP were 6.3 and 9.2 days in the BSS and topical bevacizumab groups respectively (ρ=0.25). In mitomycin-C-enhanced trabeculectomy, the mean bleb survival was 16 and 18.2 days respectively (ρ=0.40). In plain trabeculectomy, mean bleb survival in terms of bleb morphology were 8 and 12.2 days for the BSS and bevacizumab groups respectively (ρ=0.08). In enhanced trabeculectomy, mean bleb survival were 19.5 and 20 days respectively (ρ=0.99). Mean vascularity grading were 2 and 1.9 for the BSS groups, and 1.6 and 1.4 for the bevacizumab groups.@*Conclusion@#Topical bevacizumab as adjunctive therapy after trabeculectomy, whether plain or enhanced with mitomycin-C, showed a trend towards prolonged bleb survival, even though the results of this study were not statistically significant.