1.Inhibition effects of sulforaphane-induced autophagy flux to cell proliferation on ex vivo human capsular bags
Hanruo, LIU ; Bowei, YUAN ; Ying, AN ; Xiuhua, WAN
Chinese Journal of Experimental Ophthalmology 2017;35(3):226-232
Background Sulforaphane (SFN) is an effective chemopreventive agent and can regulate the biological molecular mechanisms to inhibit the overgrowth of cells.Autophagy is a biological process of maintaining cellular internal environment.Understanding the affection of SFN to biological behavior of human lens epithelial cells (LECs) and the association of SFN with autophagy is helpful for the prevention and target treatment of posterior capsule opacification (PCO).Objective This study was to investigate the eradication effeccts of SFN on residual lens cell population in vitro posterior capsule opacification (PCO) model and evaluate the mechanism of SFN-induced cell death.Methods In vitro human capsular bag models were generated from fresh donor eyes by phacoemulsification and were cultured in EMEM containing 2% fetal bovine serum (FBS).Different concentrations of SFN (0,1,10 and 100 μ mol) were added in the medium for 30 days respectively according to grouping,and the growth of LECs was observed by optical microscope and immunofluorescence technique.FHL124,a human LEC line,was cultured with EMEM containing 5% FBS and divided into 0,1,10,30 and 100 μmol SFN groups.Lactate dehydrogenase (LDH) release rate in the medium was detected to evaluate cell damage/death.The migration of the cells on capsular bags was assessed by scratch test.The ultrastructure and number of autophagosomes were examined under the transmission electron microscope.The expression of LC3 in the cells were detected using Western blot in the presence or absence of autophagy inhibitors.Results The cell coverage rates on the capsular bags were significantly lower in the 10 and 100 μ mol/L SFN groups than those in the 0 and 1 μmol/L SFN groups,with a statistically significant difference among the groups (F =48.57,P < 0.01).Immunofluorescence showed that the density of F-actin-and Vimentin-positive cells was evidently decreased in the 10 and 100 μmol/L SFN groups compared with 0 and 1 μ mol/L SFN groups.The releasing levels of LDH (absorbancy) were 0.19± 0.03,0.39±0.06,0.56±0.07,0.68±0.08 and 0.89±0.09 in the 0,1,10,30 and 100 μ mol/L SFN groups,respectively,and the releasing level of LDH was gradually increased in the 10 and 100 μ mol/L SFN groups in comparison with the 1 μmol/LSFN group (all at P<0.01).With the increase of SFN concentration,the reduction rate of scratched area decreased with the increase of SFN concentration,and the decrease of scratch area was significantly lower than that of adjacent low mass concentration group and the differences were statistically significant (P<0.05).The relative expressions of LC3-Ⅱ protein were 0.423±0.003,14.543±0.024,0.668±0.024 and 0.576±0.056 in the blank control group,SFN group,SFN + 3-MA group and 3-MA group,respectively,and the relative expressions of LC3-Ⅱ protein were significantly lower in the SFN+3-MA group and 3-MA group than those in the SFN group (all at P<0.01).The number of autophagosomes was 4.07±0.32,4.13±0.34,9.21 ±0.53 and 21.02± 1.34 in the blank control group,and 1,10,100 μmol/L SFN groups,and the number of autophagosomes in the 10 and 100 μ mol/L SFN groups was significantly higher than that in the blank control group and 1 μmol/L SFN group (all at P<0.01).Conclusions SFN mediates LECs death by promoting autophagy in ex vivo capsular bags,and SFN may be a novel agent of potential chemopreventive and target treatment for PCO.
2.Change and significance of optic nerve sheath pulsatile dynamics in normal-tension glaucoma
Ruiqi PANG ; Hanruo LIU ; Teng MA ; Wenyuan SHI ; Kai CAO ; Diya YANG ; Qiang ZHU ; Ningli WANG
Chinese Journal of Experimental Ophthalmology 2020;38(5):427-432
Objective:To analyze the value and difference of the optic nerve sheath pulse dynamic deformation index (DI) in normal-tension glaucoma (NTG) and high-pressure primary open angle glaucoma (POAG).Methods:A cross-sectional study was conducted to collect clinical data at the Eye Center of Beijing Tongren Hospital from June 2016 to March 2017, 32 patients with NTG and 35 patients with high-pressure POAG were sampled.For all subjects, their basic information, body mass index (BMI), mean arterial blood pressure (MAP), 24 hours intraocular pressure, and ophthalmologic examinations required for diagnosis were recorded.All subjects underwent transorbital ultrasonography and for each 15 seconds of consecutive ultrasound images were taken.The dynamic post-processing technique was used to calculate the DI.The difference in DI between the two groups and the correlation of DI with other variables were analyzed.The study protocol was approved by the Ethics Committee of Beijing Tongren Hospital.Written informed consent was obtained from all subjects prior to their entering the study cohort and receiving the transorbital ultrasound examination.Results:The median level of DI in the NTG group was 0.51 (0.48, 0.54), which was higher than that in the high-pressure POAG group (0.23[0.20, 0.25]), exhibiting a significant difference ( Z=-7.01, P<0.01). The mean BMI in the NTG group was lower than that in the high-pressure POAG group([21.29±4.64]kg/m 2vs. [23.53±3.40]kg/m 2), the mean MAP in the NTG group was lower than that in the high-pressure POAG group([91.44±14.30]mmHg vs. [104.05±13.96] mmHg), the differences between the two groups were statistically significant ( t=-2.30, P<0.05; t=-3.65, P<0.01). There was no statistical association between the two groups of DI and age, MAP, BMI, mean intraocular pressure and maximum intraocular pressure (all at P>0.05). Conclusions:The DI of the NTG patient is higher than that of the POAG patient, which indicates that the optic nerve sheath subarachnoid pressure and optic nerve sheath stiffness in NTG patients are lower than those in POAG patients.Therefore, the DI is a potential indicator of non-invasive intracranial pressure and translaminar cribrosa pressure difference detection in ophthalmology.