Overexpression of TRPV1 activates autophagy in human lens epithelial cells under hyperosmotic stress through Ca^(2+)-dependent AMPK/mTOR pathway

●AIM:To explore whether autophagy functions as a cellular adaptation mechanism in lens epithelial cells(LECs)under hyperosmotic stress.●METHODS:LECs were treated with hyperosmotic stress at the concentration of 270,300,400,500,or 600 mOsm for 6,12,18,24h in vitro.Polymerase chain reaction(PCR)was employed for the mRNA expression of autophagyrelated genes,while Western blotting detected the targeted protein expression.The transfection of stub-RFP-sens-GFPLC3 autophagy-related double fluorescence lentivirus was conducted to detect the level of autophagy flux.Scanning electron microscopy was used to detect the existence of autolysosome.Short interfering RNA of autophagy-related gene(ATG)7,transient receptor potential vanilloid(TRPV)1 overexpression plasmid,related agonists and inhibitors were employed to their influence on autophagy related pathway.Flow cytometry was employed to test the apoptosis and intracellular Ca^(2+)level.Mitochondrial membrane potential was measured by JC-1 staining.The cell counting kit-8 assay was used to calculate the cellular viability.The wound healing assay was used to evaluate the wound closure rate.GraphPad 6.0 software was utilized to evaluate the data.●RESULTS:The hyperosmotic stress activated autophagy in a pressure-and time-dependent manner in LECs.Beclin 1 protein expression and conversion of LC3B II to LC3B I increased,whereas sequestosome-1(SQSTM1)protein expression decreased.Transient Ca^(2+)influx was stimulated caused by hyperosmotic stress,levels of mammalian target of rapamycin(mTOR)phosphorylation decreased,and the level of AMP-activated protein kinase(AMPK)phosphorylation increased in the early stage.Based on this evidence,autophagy activation through the Ca^(2+)-dependent AMPK/mTOR pathway might represent an adaptation process in LECs under hyperosmotic stress.Hyperosmotic stress decreased cellular viability and accelerated apoptosis in LECs and cellular migration decreased.Inhibition of autophagy by ATG7 knockdown had similar results.TRPV1 overexpression increased autophagy and might be crucial in the occurrence of autophagy promoted by hyperosmotic stress.●CONCLUSION:A combination of hyperosmotic stress and autophagy inhibition may be a promising approach to decrease the number of LECs in the capsular bag and pave the way for improving prevention of posterior capsular opacification and capsular fibrosis.

Alu antisense RNA ameliorates methylglyoxal-induced human lens epithelial cell apoptosis by enhancing antioxidant defense

AIM:To determine whether an antisense RNA corresponding to the human Alu transposable element(Aluas RNA)can protect human lens epithelial cells(HLECs)from methylglyoxal-induced apoptosis.METHODS:Cell counting kit-8(CCK-8)and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assays were used to assess HLEC viability.HLEC viability/death was detected using a Calcein-AM/PI double staining kit;the annexin V-FITC method was used to detect HLEC apoptosis.The cytosolic reactive oxygen species(ROS)levels in HLECs were determined using a reactive species assay kit.The levels of malondialdehyde(MDA)and the antioxidant activities of total-superoxide dismutase(T-SOD)and glutathione peroxidase(GSH-Px)were assessed in HLECs using their respective kits.RT-q PCR and Western blotting were used to measure m RNA and protein expression levels of the genes.RESULTS:Aluas RNA rescued methylglyoxal-induced apoptosis in HLECs and ameliorated both the methylglyoxalinduced decrease in Bcl-2 m RNA and the methylglyoxalinduced increase in Bax m RNA.In addition,Aluas RNA inhibited the methylglyoxal-induced increase in Alu sense RNA expression.Aluas RNA inhibited the production of ROS induced by methylglyoxal,restored T-SOD and GSHPx activity,and moderated the increase in MDA content after treatment with methylglyoxal.Aluas RNA significantly restored the methylglyoxal-induced down-regulation of Nrf2 gene and antioxidant defense genes,including glutathione peroxidase,heme oxygenase 1,γ-glutamylcysteine synthetase and quinone oxidoreductase 1.Aluas RNA ameliorated methylglyoxal-induced increases of the m RNA and protein expression of Keap1 that is the negative regulator of Nrf2.CONCLUSION:Aluas RNA reduces apoptosis induced by methylglyoxal by enhancing antioxidant defense.

Role of reactive oxygen species in epithelial-mesenchymal transition and apoptosis of human lens epithelial cells

AIM:To investigate the role of reactive oxygen species(ROS)in epithelial–mesenchymal transition(EMT)and apoptosis of human lens epithelial cells(HLECs).METHODS:Flow cytometry was used to assess ROS production after transforming growth factorβ2(TGF-β2)induction.Apoptosis of HLECs after H_(2)O_(2) and TGF-β2 interference with or without ROS scavenger N-acetylcysteine(NAC)were assessed by flow cytometry.The corresponding protein expression levels of the EMT markerα-smooth muscle actin(α-SMA),the extracellular matrix(ECM),marker fibronectin(Fn),and apoptosis-associated proteins were detected by using Western blotting in the presence of an ROS scavenger(NAC).Wound-healing and Transwell assays were used to assess the migration capability of HLECs.RESULTS:TGF-β2 stimulates ROS production within 8h in HLECs.Additionally,TGF-β2 induced HLECs cell apoptosis,EMT/ECM synthesis protein markers expression,and pro-apoptotic proteins production;nonetheless,NAC treatment prevented these responses.Similarly,TGF-β2 promoted HLECs cell migration,whereas NAC inhibited cell migration.We further determined that although ROS initiated apoptosis,it only induced the accumulation of the EMT markerα-SMA protein,but not COL-1 or Fn.CONCLUSION:ROS contribute to TGF-β2-induced EMT/ECM synthesis and cell apoptosis of HLECs;however,ROS alone are not sufficient for EMT/ECM synthesis.

Effects of Sodium Salicylate on the Expression of HSP27 Protein during Oxidative Stress in Tissue-cultured Human Lens Epithelial Cells

The effects of sodium salicylate on the expression of heat shock protein 27 （HSP27） during oxidative stress in tissue-cultured human lens epithelial cells were investigated. Cultured human lens epithelial cells （HLB-3） were divided into 3 groups： control group （group A）, oxidation injury group （group B） and sodium salicylate group （group C）. Apoptosis of human lens epithelial cells cultured in vitro was induced in the presence of 150 μmol/L H2O2. Cells viability and the expression of HSP27 were analyzed. Viability of the cells was measured by methyl thiazole tetrazolium （MTT） chromatometry. The expression of HSP27 in HLB-3 cells was detected by using immunohistochemistry and image analysis system, Sodium salicylate could induce the expression of HSP27, and the cells viability in group C was significantly higher than in group B （0.2667±0.01414 vs 0.2150±0.01080, P=0.012〈0.05）. The average gray value of HSP27 in group B was less than that in group C （P=0.000〈0.05）. The increased expression of HSP27 by sodium salicylate might play an important role in the protection of hydrogen peroxide-induced injury of human lens epithelial cells, suggesting that sodium salicylate could suppress, at least in part, the apoptosis of human lens epithelial cells.

Growth Inhibition, Induction of Apoptosis by Green Tea Constituent (-)-Epigallocatechin-3-gallate in Cultured Rabbit Lens Epithelial Cells

Purpose: To evaluate effect of green tea extract (-)-Epigallocatechin-3-gallate (EGCG) in cultured rabbit lens epithelial cells in order to pave a new way to postcapsular opacity (PCO) prevention.Methods: Cell survival rate was measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) coloimetric assay. Cell apoptosis was detected by electron microscopy, Hochest 33258 stain and flow cytometer. DNA fragment was detected using agarose gel electrophoresis.Result: Proliferation of the cultured rabbit lens epithelia cells was inhibited by EGCG in a dose and time dependent manner. Morphologic study showed that the cells became shrunk, round shaped with their nuclei condensed and broken. Apoptotic bodies were also seen under electron microscope and in Hochest 33258 stain assay 24 hours after EGCG was added to the medium. DNA ladders were shown in agarose gel eletrophoresis. In flow cytometry assay, apoptosis peak was also evident.Conclusion: Green Tea Constituent(-)

The Biological Study of the Cultured Human Lens Epithelial Cells in Vitro

The human lens epithelial cells (HLE) cultured in vitro was established in normal and cataractous lenses. The biological feature, histological characteristics and the ultrastructure of the cultured HLE cells were investigated. The results reveal that the proliferative capacity of the culutured HLE cells is reversely proportional to the donour age; the cultured HLE cells has the limited proliferative capacity in vitro. The relieve of the contact inhibition is the effective trigger of the HLE cell prolife...

Alpha lipoic acid protects lens from H_2O_2-induced cataract by inhibiting apoptosis of lens epithelial cells and inducing activation of anti-oxidative enzymes

Objective:To determine whether alpha lipoic acid（LA）can effectively protect lenses from hydrogen peroxide（H2O2）-induced cataract.Methods:Lens from adult Sprague-Dawley rats were cultured in 24-well plates and treated without or with 0.2 mM of H2O2,0.2 mM of H2O2 plus 0.5 mM.1.0 mM.or 2.0 mM of LA for 24 h.Cataract was assessed using cross line grey scale measurement.Superoxide dismutase（SOD）.glutathione（GSH-Px）.lactate dehydrogenase（LDH）. and maloudialdehyde（MDA）activity or level in lens homogenates was measured.Apoptosis of lens epithelial cells in each group were detected by Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling（TUNEL） Assay.Results:A total of 0.2 mM of H2O2 induced obvious cataract formation and apoptosis in lens’ epithelial cells,but 0.5-2.0 mM of LA could block the effect of 0.2 mM H2O2 in inducing cataract and apoptosis.Furthermore.0.2 mM ol H2O2 significantly decreased SOD.GSH-Px,and LDH activity and significant increased MDA level in the lens,but 0.5-2.0 mM of LA blocked the effect of 0.2 mM H2O2.One mM of LA was found to be the most effective. Conclusions:LA can protect lens from H2O2-induced cataract.LA exerts protective effects through inhibition of lens’ epithelial cell apoptosis and activation of anti-oxidative enzymes.

MicroRNA-34a promoting apoptosis of human lens epithelial cells through down-regulation of B-cell lymphoma-2 and silent information regulator

AIM： To investigate the role of micro RNA-34a（mi R-34a） in the induction of apoptosis of human lens epithelial（HLE-B3） cells. METHODS： The apoptosis of HLE-B3 cells was detected by Annexin V-PE apoptosis detection kit after the treatment with 200 μmol/L H2O2 for 24h and lentiviral mi R-34 a vector transfection. The expression of mi R-34 a in the cells was quantified by quantitative real time polymerase chain reaction（q RT-PCR） in response to H2O2 exposure and the vector transfection. The effects of overexpression of mi R-34 a on the expression of B-cell lymphoma-2（Bcl-2） and silent information regulator 1（SIRT1） was determined by q RT-PCR and Western blot. RESULTS： The expression of mi R-34 a was up-regulated by the treatment of H2O2 in HLE-B3 cells. The increased expression of mi R-34 a is accompanied with the cell apoptosis. Consistence with the H2O2 exposure,ectopic overexpression of mi R-34 a in HLE-B3 cells promoted cells apoptosis. Importantly the anti-apoptosis factors Bcl-2 and SIRT1 were reduced significantly by up-regulation of mi R-34 a in HLE-B3 cells.CONCLUSION： Mi R-34 a promotes the apoptosis of HLE-B3 cells by down-regulating Bcl-2 and SIRT1,suggesting that mi R-34 a may involve in the pathogenesis of cataract formation and targeting mi R-34 a may be a potentially therapeutic approach for treatment of cataract.

Effects of Rapamycin on Expression of Bcl-2 and Bax in Human Lens Epithelial Cells and Cell Cycle in Rats

The effects of rapamycin on the expression of Bcl-2 and Bax protein in in vitro cultured human lens epithelial cells（LECs） and cell cycle were investigated in order to provide the theoretical basis for the development of new inhibitory drugs for clinical prevention and treatment of after-cataract.The cultured LECs of second and third passages were collected and treated with rapamycin.The LECs were transferred into 96-well culture plates and divided into 6 groups,and each group was set to have 8 duplicate wells.In the negative control group,the LECs were given culture medium only,and in the blank control group,only culture medium was given.In the four rapamycin-treated groups,different concentrations（20,40,60 and 80 ng/mL） of rapamycin were given.After treatment for 24,48 and 72 h,the absorbance（A） values in each well were determined by MTT assay.The cell cycles of all groups were detected by using flow cytometry.Real-time fluorescent quantitative polymerase chain reaction（RFQ-PCR） and Western blot were used to detect the mRNA and protein expression of Bcl-2 and Bax respectively.MTT assay showed that rapamycin could inhibit proliferation of LECs in a time-and dose-dependent manner.Flow cytometry revealed that rapamycin could block the conversion of LECs from G1 phase to S phase,resulting in the increase of cells in G1 phase and the decrease of the cells in S phase.RFQ-PCR indicated that rapamycin could down-regulate the expression of Bcl-2 mRNA,but up-regulate the expression of Bax mRNA,suggesting it could induce apoptosis of LECs.Western blot demonstrated that rapamycin could suppress the expression of Bcl-2 protein,but promote the expression of Bax protein.It is concluded that rapamycin could inhibit proliferation of LECs probably not only by blocking the progression of cell cycle,but also by promoting the induction of apoptosis.

Mechanisms of inhibition of elemene on human lens epithelial cell proliferation in vitro

AIM: To study the effects of elemene (Ele) on proliferation and cell cycle of human lens epithelial cells 63 (HLE-B3) and the mechanisms of its signal transduction. METHODS: Recombinant human basic fibroblast growth factor (rhbFGF) was used to induce proliferation of HLE-B3 cells, which were incubated with 80mg/L Ele for 24 hours. The inhibitory effects of Ele on the proliferation of HLE-B3 cells were evaluated by MU method. The effect of Ele on HLE-B3 cell cycle was analyzed by flow cytometry (FCM). The expressions of protein kinase A (PKA) and protein kinase G (PKG) of HLE-B3 were also analyzed by FCM. RESULTS: Ele altered the cell cycle of HLE-B3 and effectively inhibited HLE-B3 cell proliferation induced by rhbFGF. Ele up-regulated PKA and down-regulated the expression of PKG in HLE-B3 cell. CONCLUSION: Ele inhibits HLE-B3 proliferation, making it an attractive potential agent in regimens to treat after-cataracts.

High glucose: activating autophagy and affecting the biological behavior of human lens epithelial cells

AIM: To clarify the effect of autophagy on human lens epithelial cells (HLECs) under high glucose conditions.METHODS: HLECs were cultured with different concentrations of glucose and 3-methyladenine (3-MA);the expression of autophagy-related protein LC3B was detected by Western blotting and immunofluorescence histochemistry. The migration of HLECs was quantified by scratch wound assay and the expression of transforming growth factor-β(TGF-β) was measured by real-time polymerase chain reaction.RESULTS: Compared with 5 mmol/L normal glucose treatment, 40 mmol/L glucose treatment can significantly increase the gen eration of autophagosome in HLECs, which could be inhibited by 0.375 mmol/L 3-MA treatment. The migration of HLECs and the expression of TGF-β in HLECs induced by high glucose were significantly suppressed by 0.375 mmol/L 3-MA treatment.CONCLUSION: Autophagy promotes HLECs cell migration and increases the expression of TGF-β after exposed to high glucose, which may relate to the development of diabetic cataract.

In vitro inhibition of proliferation,migration and epithelial-mesenchymal transition of human lens epithelial cells by fasudil

AIM： To study the potential role of fasudil as a treatment for posterior capsular opacification（PCO） of the human crystalline lens.METHODS： Human lens epithelial cells（HLECs; line SRA01/04） was exposed to transforming growth factor-β2（TGF-β2） to induce the process of epithelial-mesenchymal transition（EMT）. Fasudil was applied to the cell samples. Its effect on overall HLECs proliferation and migration was studied, as was its influence on EMT induction by TGF-β2 using cell migration assay, MTT colorimetric assay and Western blot assay.RESULTS： Fasudil inhibited the proliferation of SRA01/04. Its effect was time-and concentration-dependent. The migration of SRA01/04 cells was significantly reduced 24-72 h after fasudil treatment, and the half maximal inhibitory concentration（IC50） was 22.37 μmol/mL at 72 h. Reversal of the elongated, fibroblast-like shape changes induced by TGF-β2 in SRA01/04 cells was observed. Fasudil up-regulated the expression of Connexin43 protein and down-regulated the expression of α-SMA protein compared with the cells treated with TGF-β2. Furthermore, when exposed to fasudil, the phosphorylation of Rhoassociated protein kinase（Rock） and myosin light chain（MLC） could not be activated in the cell preparations.CONCLUSION： Fasudil suppresses the proliferation and migration of SRA01/04 cells, and inhibits the process of EMT induced by TGF-β2. These results suggest that fasudil may serve as a therapeutic agent for PCO.

SUMOylation and deacetylation affect NF-κB p65 activity induced by high glucose in human lens epithelial cells

AIM: To explore the effects of IκBα SUMOylation and NF-κB p65 deacetylation on NF-κB p65 activity induced by high glucose in cultured human lens epithelial cells(HLECs).METHODS: HLECs(SRA01/04) were cultured with 5.5, 25, and 50 mmol/L glucose media for 24 h, and with 50 mmol/L glucose media for 0, 12, and 24 h respectively. SUMO1 and SIRT1 expressions were detected by reverse transcriptionpolymerase chain reaction(RT-PCR) and Western blot(WB). IκBα and NF-κB p65 expressions were detected by WB. With NAC, DTT, MG132 or Resveratrol(RSV) treatment, SUMO1 and SIRT1 expressions were detected by WB. Protein expression localizations were examined by immunofluorescence and co-immunofluorescence. The effects of SUMO1 or SIRT1 overexpression, as well as MG132 and RSV, on the nuclear expression and activity of IκBα and NF-κB p65 were analyzed by immunoblot and dual luciferase reporter gene assay.RESULTS: SUMO1 and SIRT1 expressions were influenced by high glucose in mRNA and protein levels, which could be blocked by NAC or DTT. SUMO1 was down-regulated by using MG132, and SIRT1 was up-regulated under RSV treatment. IκBα nuclear expression was attenuated and NF-κB p65 was opposite under high glucose, while IκBα and NF-κB p65 location was transferred to the nucleus. SUMO1 or SIRT1 overexpression and MG132 or RSV treatment affected the nuclear expression and activity of IκBα and NF-κB p65 under high glucose condition.CONCLUSION: IκBα SUMOylation and NF-κB p65 deacetylation affect NF-κB p65 activity in cultured HLECs under high glucose, and presumably play a significant role in controlling diabetic cataract.

Parthenolide protects human lens epithelial cells from oxidative stress-induced apoptosis via inhibition of activation of caspase-3 and caspase-9

透镜的 apoptosis 上皮的房间作为奔流形成的普通基础被建议了，与是的氧化应力主要原因。这研究被执行对人的透镜的导致 oxidativestress 的 apoptosis 调查草药的成分 parthenolide 的保护的效果上皮(HLE ) 房间和可能的分子的机制包含了。HLE 房间(SRA01-04 ) 在 parthenolide (10， 20 和 50 microM ) 的不同剂量的缺席或存在与 50 microM H (2 ) O (2 ) 被孵化。到学习 apoptosis，房间被词法检查和 Annexin V-propidium 碘化物估计两倍染色的流动 cytometry；调查内在的分子的机制， caspase-3 和 caspase-9 的表示旁边是 assayed 西方的污点和量的 RT-PCR，和 caspase-3 和 caspase-9 的活动我们由 Chemicon caspase 的 remeasured 比色的活动试金工具包。为 18 h 与 H (2 ) O (2 ) 刺激了，， HLE 房间的高部分面对不同集中的 parthenolide 经历了 apoptosis， HLE 房间 apoptosis 堵住的剂量依赖者被观察。H (2 ) O (2 ) 在 HLE 房间导致的表示 ofcaspase-3 和 caspase-9 被 parthenolideboth 显著地在蛋白质和信使 rna 层次减少，并且 caspase-3 和 caspase-9 的激活被 parthenolide 也以一种剂量依赖者方式压制。在结论， parthenolide 通过 caspase-3 andcaspase-9 的激活的抑制阻止 HLEcells 氧化导致压力的 apoptosis，建议对奔流形成的潜在的保护的效果。

Effects of transforming growth factor β2 and connective tissue growth factor on induction of epithelial mesenchymal transition and extracellular matrix synthesis in human lens epithelial cells

AIM:To Investigate the effects of transforming growth factorβ2(TGF-β2)and connective tissue growth factor(CTGF)on transdifferentiation of human lens epithelial cells(HLECs)cultured in vitro and synthesis of extracellular matrix(ECM).METHODS:HLECs were treated with TGF-β2(0,0.5,1.0,5,10μg/L)and CTGF(0,15,30,60,100μg/L)for different times(0,24,48,72h)in vitro and the expression ofα-smooth muscle actin(α-SMA),the main component of the extracellular matrix typeⅠcollagen(Col-1)and fibronectin(Fn)were measured by using real-time polymerase chain reaction(PCR)and western-blot.RESULTS:TGF-β2 and CTGF significantly increased expression ofα-SMA mRNA and protein(P【0.05,P【0.001),Fn mRNA and protein(P【0.001),Col-1 mRNA and protein(P【0.001).TGF-β2 could induce HLECs expression of CTGF mRNA and protein in dosedependent manner(P【0.05,P【0.001).TGF-β2 and CTGF could induce HLECs to expressα-SMA,Fn and Col-1 in time-dependent manner.Each time of TGF-β2and CTGF induced HELCs expression ofα-SMA,Fn,Col-1 mRNA and protein was significant increase compared with control(P【0.05,P【0.001).CONCLUSION:TGF-β2 and CTGF could induce HLECs epithelial mesenchymal transition and ECM synthesis.

Human lens epithelial cell apoptosis and epithelial to mesenchymal transition in femtosecond laser-assisted cataract surgery

AIM： To evaluate human lens epithelium cell apoptosis and epithelial to mesenchymal transition （EMT） induced by femtosecond laser in femtosecond laser assisted cataract surgery （FLACS）. METHODS： Sixty cataract patients with N2 to N3 stage according to the LOCS III were enrolled in this study and divided into three groups randomly： FLACSl group （cataract surgery by FLACS with LenSx）, FLACS2 group （cataract surgery by FLACS with LensAR） and manual group （cataract surgery by phacoemulsification）. Patients in two FLACS groups performed anterior capsulotomy by LenSx or LensAR laser system. Patients in the manual group were performed continuous curvilinear capsulorrhexis （CCC） manually. The anterior capsules were fixed right after moved out of eye. Hematoxylin-eosine staining, immunofluorescence staining and real-time PCR were performed in order to observe human lens epithelium cells changes after cataract surgery. RESULTS： The capsule cutting edge was shown irregularity and roughness in two FLACS groups and smooth edge in manual capsulotomy by pathologic staining. Irregularities of the cell configuration with partly swollen and destroyed nuclei were observed in two FLACS groups. Femtosecond laser could induce a significantly higher cell apoptosis in human lens epithelium cell than manually performed CCC （P〈0.05）. Lens epithelium cells apoptosis were correlated with femtosecond laser duration according to Pearson correlation analysis. Decreased N-cadherin expression, alpha-SMA and FSP-1 level in two FLACS groups showed the inhibition of cell EMT. CONCLUSION： Femtosecond laser may affect the apoptosis and EMT of lens epithelium cells which are under the peeled central lens capsule.

Effects of lentiviral RNA interference-mediated downregulation of integrin-linked kinase on biological behaviors of human lens epithelial cells

AIM：To investigate the effects of lentivirus（LV）mediated integrin-linked kinase（ILK）RNA interference（RNAi）on biological behaviors of human lens epithelial cells（LECs）.·METHODS：Human cataract LECs and immortalized human LEC line,human lens epithelial（HLE）B-3 cells were transfected by lentiviral vector expressing ILKspecific short hairpin RNA（sh RNA）and then stimulated by transforming growth factor-β（TGF-β）,the silencing of ILK gene and protein was identified by reverse transcription-polymerase chain reaction（RT-PCR）and Western blot methods;biological behaviors including cell cycle and apoptosis,cell morphology,α-smooth muscle actin（SMA）stress fiber formation and cell migration were examined.·RESULTS：Remarkable decreases of ILK protein expression were detected in LECs carrying lentiviral ILK-sh RNA vector;flow cytometry revealed arresting of cell cycle progression through the G1/S transition and higher apoptosis rate in ILK-RNAi-LV transfected cells.Lessα-SMA stress fiber formation and migration was observed in ILK-RNAi-LV transfected LECs.·CONCLUSION：The present study demonstrated that ILK was an important regulator for LECs proliferation and migration.LV mediated ILK RNAi is an effective way todecrease ILK-regulated cell growth by arresting cell cycle progression and increasing cell apoptosis,as well as,to prevent cell migration by inhibiting TGF-βinducedα-SMA stress fiber formation.Thus,LV mediated ILK RNAi might be useful to prevent posterior capsular opacification.

Mitochondrial proteomic analysis of ecdysterone protection against oxidative damage in human lens epithelial cells

AIM: To investigate the protective effects of the natural medicinal monomer ecdysterone(ECR) with estrogenic activity against oxidative damage in human lens epithelial cells B3(HLE-B3) caused by hydrogen peroxide 21(H2 O2) and to pursue the possible mitochondrial proteomic regularity of the protective effects. · METHODS: HLE-B3 cells were treated with H2O2(300μmol/L),β-estuarial(E2; 10-8mol/L) and H2 O2,ECR(10-6mol/L) and H2 O2,or left untreated. Altered expression of all mitochondrial proteins was analyzed by protein array and surface-enhanced laser desorption ionization time of flight mass spectrometry(SELDI-TOF-MS). The mass/charge(M/Z) ratios of each peak were tested by the Kruskal-Wallis rank sum test,and the protein peak value of the M/Z ratio for each treatment by pair comparison was analyzed with the Nemenyi test. ·RESULTS: H2O2 up-regulated expression of two protein spots(with M/Z of 6 532 and 6 809). When E2 mitigated the oxidative damage,the expression of one protein spot(M/Z 6 532) was down-regulated. In contrast,ECR downregulated both of protein spots(M/Z 6 532 and 6 809). · CONCLUSION: ECR could effectively inhibite H2O2 induced oxidative damage in HLE-B3 cells. The protein spot at M/Z of 6 532 might be the target spot of ECR against oxidative damage induced by H2 O2.

Expression of Transforming Growth Factor-β in Cultured Normal Human Lens Epithelia Cells

Summary: In order to investigate whether cultured normal human lens epithelial cells (LEC) express transforming growth factor β (TGF-β), reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemical methods were used for detection of TGF-β mRNA and protein in cultured normal human LEC. The results showed that a single RT-PCR amplified product about 310bp was obtained, and the sequence was homologous to the known sequence. TGF-β immunostain was positive in the plasma of LEC. It was suggested that normal human LEC could produce TGF-β, and LEC could be affected by TGF-β through autocrine action.

Sustained-release genistein from nanostructured lipid carrier suppresses human lens epithelial cell growth

AIM： To design and investigate the efficacy of a modified nanostructured lipid carrier loaded with genistein（Gen-NLC） to inhibit human lens epithelial cells（HLECs） proliferation.·METHODS： Gen-NLC was made by melt emulsification method. The morphology, particle size（PS）, zeta potentials（ZP）, encapsulation efficiency（EE） and in vitro release were characterized. The inhibition effect of nanostructured lipid carrier（NLC）, genistein（Gen） and Gen-NLC on HLECs proliferation was evaluated by cell counting kit-8（CCK-8） assay, gene and protein expression of the proliferation marker Ki67 were evaluated with real-time quantitative polymerase chain reaction（RT-q PCR） and immunofluorescence analyses.·RESULTS： The mean PS of Gen-NLC was 80.12±1.55 nm with a mean polydispersity index of 0.11±0.02. The mean ZP was-7.14 ±0.38 m V and the EE of Gen in the nanoparticles was 92.3% ±0.73%. Transmission electron microscopy showed that Gen-NLC displayed spherical-shaped particles covered by an outer-layer structure. In vitro release experiments demonstrated a prolonged drug release for 72 h. The CCK-8 assay results showed the NLC had no inhibitory effect on HLECs and Gen-NLC displayed a much more prominent inhibitory effect on cellular growth compared to Gen of the same concentration. The m RNA and protein expression of Ki67 in LECs decreased significantly in Gen-NLC group.·CONCLUSION： Sustained drug release by Gen-NLCs may impede HLEC growth.