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.

SIRT1 inhibits apoptosis of human lens epithelial cells through suppressing endoplasmic reticulum stress in vitro and in vivo

AIM:To explore the effect of silent information regulator factor 2-related enzyme 1(SIRT1)on modulating apoptosis of human lens epithelial cells(HLECs)and alleviating lens opacification of rats through suppressing endoplasmic reticulum(ER)stress.METHODS:HLECs(SRA01/04)were treated with varying concentrations of tunicamycin(TM)for 24h,and the expression of SIRT1 and C/EBP homologous protein(CHOP)was assessed using real-time quantitative polymerase chain reaction(RT-PCR),Western blotting,and immunofluorescence.Cell morphology and proliferation was evaluated using an inverted microscope and cell counting kit-8(CCK-8)assay,respectively.In the SRA01/04 cell apoptosis model,which underwent siRNA transfection for SIRT1 knockdown and SRT1720 treatment for its activation,the expression levels of SIRT1,CHOP,glucose regulated protein 78(GRP78),and activating transcription factor 4(ATF4)were examined.The potential reversal of SIRT1 knockdown effects by 4-phenyl butyric acid(4-PBA;an ER stress inhibitor)was investigated.In vivo,age-related cataract(ARC)rat models were induced by sodium selenite injection,and the protective role of SIRT1,activated by SRT1720 intraperitoneal injections,was evaluated through morphology observation,hematoxylin and eosin(H&E)staining,Western blotting,and RT-PCR.RESULTS:SIRT1 expression was downregulated in TMinduced SRA01/04 cells.Besides,in SRA01/04 cells,both cell apoptosis and CHOP expression increased with the rising doses of TM.ER stress was stimulated by TM,as evidenced by the increased GRP78 and ATF4 in the SRA01/04 cell apoptosis model.Inhibition of SIRT1 by siRNA knockdown increased ER stress activation,whereas SRT1720 treatment had opposite results.4-PBA partly reverse the adverse effect of SIRT1 knockdown on apoptosis.In vivo,SRT1720 attenuated the lens opacification and weakened the ER stress activation in ARC rat models.CONCLUSION:SIRT1 plays a protective role against TM-induced apoptosis in HLECs and slows the progression of cataract in rats by inhibiting ER stress.These findings suggest a novel strategy for cataract treatment focused on targeting ER stress,highlighting the therapeutic potential of SIRT1 modulation in ARC development.

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.

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.

A Study of Rabbit Lens Epithelial Cells Survival and Growth on the Rabbit Capsular Bag in Vitro

Objective: To study the proliferation, migration and metaplasm of residual rabbit lens epithelial cells (LECs) after extracapsular cataract extraction(ECCE)based on the rabbit capsular bag model in vitro. Methods: Sham cataract surgery, including anterior capsulorhexis, nucleus hydroexpression and aspiration of lens fibers, was performed on 20 rabbit lens. The capsular bags were isolated and pinned to sterile non-toxic silicone rings on petri dishes. The capsular bags were incubated with Eagle's minimum essential medium (DMEM) supplemented with 10% fetal calf serum (FCS) and monitored for 3 weeks by phase-contrast microscopy, after which light microscopy was performed on them.Results: After a latent period of 2-3 d, outgrowth was observed across the posterior capsule. Growth proceeded rapidly so that the posterior capsule was totally covered by a confluent monolayer of cell at 6-8 day. Capsular wrinkles became increasingly apparent as time progressed, causing a marked rise in light scatter. An increase in capsular tension also came.Conclusion: This model exhibits many of the in vito characteristics of the lens capsule after extracapsular surgery and may prove useful in further elucidating the cellular mechanisms of posterior capsule opacification and developing strategies for inhibiting cell growth with this system.

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.

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

The apoptosis of lens epithehal cells has been proposed as the common basis of cataract formation, with oxidative stress as the major cause. This study was performed to investigate the protective effect of the herbal constituent parthenolide against oxidative stress-induced apoptosis of human lens epithelial （HLE） cells and the possible molecular mechanisms involved. HLE cells （SRA01-04） were incubated with 50 μM H2O2 in the absence or presence of different doses of parthenolide （10, 20 and 50 μM）. To study apoptosis, the cells were assessed by morphologic examination and Annexin V-propidium iodide double staining flow cytometry; to investigate the underlying molecular mechanisms, the expression of caspase-3 and caspase-9 were assayed by Western blot and quantitative RT-PCR, and the activities of caspase-3 and caspase-9 were measured by a Chemicon caspase colorimetric activity assay kit. Stimulated with H202 for 18 h, a high fraction of riLE cells underwent apoptosis, while in the presence ofparthenolide of different concentrations, dose-dependent blocking of HLE cell apoptosis was observed. The expression of caspase-3 and caspase-9 induced by H202 in HLE cells was significantly reduced by parthenolide both at the protein and mRNA levels, and the activation ofcaspase-3 and caspase-9 was also suppressed by parthenolide in a dose-dependent manner. In conclusion, parthenolide prevents HLE cells from oxidative stress-induced apoptosis through inhibition of the activation ofcaspase-3 and caspase-9, suggesting a potential protective effect against cataract formation.

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.

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.

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.

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.

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.

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.

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.

Comparison of FGFR1 expression on lens epithelial cells between adults and fetuses

AIM: To study the differences of fibroblast growth factor receptor 1 (FGFR1) gene on human lens epithelial cells (HLECs) of adults and fetuses. METHODS: Indirect in situ RT-PCR was adopted for detection of FGFR1 gene. The cDNA of the nnRNA in the paraffin sections of fetus and adult HLEC was synthesized by reverse transcription reaction. After PCR amplification, in situ hybridization test was performed with synthesized oligonucleotide probe and relative quantification was carried out using image analysis. RESULTS: HLECs of adults and fetuses expressed FGFR1 gene, the expression level was higher in fetuses than in adults. The difference between them had significance (P<0.05). CONCLUSION: FGFR1 Exist in HLEC and the expression is age-related, which could be one of causes of the high occurrence of post operational after-cataract in children.

Effects of Mitogen-activated Protein Kinase Signal Pathway on Heat Shock Protein 27 Expression in Human Lens Epithelial Cells Exposed to Sodium Salicylate in vitro

The roles of mitogen-activated protein kinase （MAPK） signal pathway in sodium salieylate-induced expression of heat shock protein 27 （HSP27） in human lens epithelial cells （HLECs-B3） in vitro were investigated. HLECs-B3 were incubated in the fresh media containing sodium salicylate at different concentrations for different durations, and allowed to be recovered in fresh medium without sodium salicylate for different durations with or without pretreatment with p38MAPK inhibitor （SB203580）, ERK1/2 inhibitor （PD98059） and JNK/SAPK inhibitor （SP600125）. The expression of P38MAPK, ERK1/2, JNK/SAPK, phosphorylated P38MAPK, phosphorylated ERK1/2, phosphorylated JNK/SAPK and HSP27 was detected by Western blot. The expression of HSP27 mRNA and protein was detected by RT-PCR and immunohistochemistry respectively. It was found there was only weak expression of HSP27 in normal HLECs. The expression of HSP27 was not detectable in HLECs-B3 that were exposed to sodium salicylate （55 retool/L） for 1-5 h. It was indicated that recovery from sodium salicylate （〉35 mmol/L） significantly increased the synthesis of HSP27. The expression of HSP27 was up-regulated in HLECs-B3 under sodium salicylate recovery for 3 h, reached the peak level for 6 h, and returned to the level of control cells by 24 h. Activation of P38MAPK from sodium salicylate stimulation occurred at 30th rain, and increased significantly at 1st h, then declined and renamed to baseline level at 3rd h under sodium salicylate recovery. Activation of ERK1/2 occurred at 1st h and reached the peak level at 6th h under sodium salicylate recovery. However, JNK/SAPK was inactivated by sodium salicylate. The expression of HSP27 could be down-regulated with the pretreatment of SB203580 and PD98059 jointly. It is concluded that sodium salicylate can induce the expression of HSP27 in HLECs-B3. The effects are mediated, at least in part, through the activation of P38MAPK and ERK1/2 signaling pathway.

Surgical stress and cytoskeletal changes in lens epithelial cells following manual and femtosecond laser-assisted capsulotomy

AIM:To study the effect of mechanical stress on the cytoskeleton in lens epithelial cells following conventional phacoemulsification surgery(CPS)and femtosecond laserassisted cataract surgery(FLACS).METHODS:The cytoskeleton of the epithelial cells of the anterior lens capsules(ALC)removed by CPS and FLACS was examined by immunohistochemistry.Expression of the intermediate filament,glial fibrillary acidic protein(GFAP),and glutamine synthetase(GS)immunoreactivity were detected.In order to map the actin network of cells,fluorescently labeled phalloidin was used.The samples were examined using confocal laser scanning microscopy.RESULTS:GFAP expression was visible in a larger number of the epithelial cells after CPS compared to FLACS.In CPS sample’s epithelial cells,GFAP immunoreactivity indicated robust morphological change.Regarding the actin filaments,the presence of tubular elements connecting epithelial cells,regular actin pattern and marked cortical network after CPS were found.Following FLACS,the actin cytoskeleton of the epithelial cells remained densely structured,and the tubular elements were undetectable,however,the above-mentioned regular actin pattern and the marked cortical network were visible.CONCLUSION:The conventional removal of the ALC induces more robust changes of the cytoskeleton of the lens epithelial cells.

Effects of Lutein on the Growth and Migration of Bovine Lens Epithelial Cells In Vitro

The effects of lutein on the growth and migration of bovine lens epithelial cells （BLECs） in vitro were observed in an attempt to find a drug that can prevent after-cataract. BLECs were cultured in vitro and different concentrations of lutein were added to the BLECs cultures of the second and third generations. The effects of lutein on the proliferation of BLECs in vitro were examined by the MTT method, and the migration of BLECs was evaluated by a scratch wound assay. The results showed that： （1） Lutein at concentrations of 1 to 16 μmol/L could inhibit the proliferation of BLECs in a dose-and time-dependent manner （P〈0.01）; （2） The migration of BLECs was evaluated by wound healing rate. As compared with the control group, the wound healing rate in the experimental groups was decreased from 0.672±0.164 to -0.234±0.144 and -0.597±0.063 （P〈0.01） at 1 and 2 μmol/L lutein, respectively. It was concluded that lutein at concentration of 〉1 μmol/L inhibited the proliferation and migration of BLECs in vitro, Lutein may become an effective drug to prevent after-cataract.