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Background Our previous study showed that curcumin suppresses the proliferation of human lens epithelial cells (LECs) in vitro and has an influence on the signal transduction of cyclic adenosine monophosphate (cAMP) and cyclic guanosinc monophosphate (cGMP).Actually,the regulation for biological behavior of LECs in vivo is complex.Objective This study was to investigate the changes of signal transduction of protein kinase (PK) in inhibition of curcumin on human LECs proliferation.Methods HLE-B3 was cultured and then divided into the blank control group,recombinant human basic fibroblast growth factor (rhbFGF) group and rhbFGF+ curcumin group.rhbFGF of 10 ng/ml was added in the medium in the rhbFGF group,and 20 mg/L curcumin was added into rhbFGF-induced cell medium for 24 hours in the rhbFGF+ curcumin group.The expression rates of PKA,PKC,PKG and calmodulin (CaM) in the cells were assayed using flow cytometry.Results The expression rates of PKA protein were (46.847± 1.673) ％,(33.250 ± 2.242) ％ and (71.645 ±2.432) ％ in the blank control group,rhbFGF group and the rhbFGF+ curcumin group,respectively,and the expression rate of PKA protein was significantly reduced in the rhbFGF group compared with the blank control group (t =11.904,P＜0.01),but the expression rate of PKA protein in the rhbFGF+ curcumin group was significnatly higher than that in the rhbFGF group (t=28.430,P＜0.01).In the blank control group,rhbFGF group and the rhbFGF+ curcumin group,the expression rates of PKC protein in the cells were (35.575± 1.937) ％,(50.652±2.068) ％ and (27.662t4.481) ％,those of PKG protein were (63.838±0.486) ％,(86.562 ± 1.325) ％ and (40.733 ± 2.175) ％,while those of CaM protein were (67.408± 1.391)％,(83.887±3.499)％ and (53.785 ± 1.942)％,the expression rates of PKC,PKG and CaM were significantly lower in the rhbFGF group in comparison with the blank control group (all at P＜0.01),and those in the rhbFGF+ curcumin group were significantly declined in comparison with the rhbFGF group (all at P＜0.01).Conclusions Suppression of curcumin on HLE-B3 proliferation probably is associated with the up-regulation of PKA expression and down-regulation of PKC,PKG and CaM expression in the cells.
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AIM: To investigate the effect of lipopolysaccharide (LPS) on the mRNA expression of Toll-like receptor 4 (TLR4) and CD14 in lens epithelial cells (LECs).METHODS: The cultured LECs were incubated with defferent concentrations of LPS for defferent times.The mRNA expression of TLR4 and CD14 in LECs were detected by reverse transcription polymerase chain reaction (RT-PCR).RESULTS: The mRNA expression of TLR4 in the LECs treated with LPS at concentrations of 50 μg/L, 100 μg/L, 200 μg/L, 500 μg/L or 1 000 μg/L was higher than that in the LECs without LPS treatment (P<0.01), respectively.The mRNA expression of TLR4 in the LECs incubated with 100 μg/L LPS for 24 h, 48 h or 72 h was higher than that in LECs without LPS treatment at same time points (P<0.01).The mRNA expression of CD14 in the LECs incubated with LPS at concentration of 100 μg/L for 24 h was also higher than that in the LECs without LPS treatment (P<0.01).CONCLUSION: LPS enhances the mRNA expression of TLR4 and CD14 in LECs, which may be related to intraocular response, as well as to the formation and development of after-cataract.
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Objective To suppress the proliferation of lens epithelial cells (LECs) is a primary goal in prevention of after cataract. Recent study demonstrated an effective inhibition of elemene(Ele) on tumor cells. Present study was to investigate the effects of Ele on proliferation and cell cycle of human LECs B3 (HLE-B3). Methods Recombinant human basic fibroblast growth factor(rhbFGF) was utilized to induce proliferation of HLE-B3. Proliferative HLE-B3 was incubated with 80 mg/L Ele in CO_2 incubator for 24 hours. Then the inhibitory effects of Ele on proliferation of HLE-B3 was evaluated by methyl thiazolyl tetrazolium(MTT). The effect of Ele on HLE-B3 morphology was observed under the optical microscope. The effect of Ele on HLE-B3 cell cycle was analyzed by flow cytometer(FCM). Results MTT test showed that the optical density (OD) value of rhbFGF group was remarkably higher than that of control group(0. 599 0 ± 0. 053 1 versus 0. 409 1 ± 0. 042 2) (P ＜ 0. 01), and that of Ele group(0. 450 0 ±0. 061 4) was obviously declined in comparison to rhbFGF group(P ＜0. 01) . The inhibitory rate of Ele was 24.90 %. In proliferation group, the number of HLE-B3 was increased with the normal cell structure and abundant cytoplasm under the optical microscope. However, in Ele group, the number of HLE-B3 was evidently decreased with less cytoplasm, undistinguished cell structure, condensed and aggregated nucleuses. The result of flow cytometer showed that the percentage of HLE-B3 in G_1 phase in rhbFGF group was 42. 062% ± 1. 270% and that in control group was 46. 422% ±3. 765% with a significant difference(P ＜ 0. 05). HLE-B3 in G_1 phase in Ele group (60. 665% ±2.069%) was evidently increased in comparison with rhbFGF group (P ＜ 0. 01). HLE-B3 in S phase in rhbFGF group compared with control group was increased (51.647% ±1.123% versus 31.842% ± 2. 798%) (P ＜ 0. 01), but that in S phase in Ele group(30. 222% ±3.429%) was lower than rhbFGF group (P ＜ 0. 01). HLE-B3 in G_2 phase in rhbFGF group was decreased in comparison with control group (6. 288% ± 0. 966% versus 21. 735% ± 3. 806%, P ＜ 0. 01), and that in G_2 phase in Ele group (9. 112% ± 1. 659%) compared with proliferation group was increased (P ＜ 0. 01). Conclusion Ele could alter the cell cycle of HLE-B3 and effectively inhibit the HLE-B3 proliferation induced by rhbFGF. Ele may be a reliable and effective drug for prevention and treatment of after cataract.
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OBJECTIVE: To investigate the inhibition effects of compound leech eye drops (Co-SZ) on apoptosis of lens epithelial cells (LECs) induced by hydrogen peroxide (H2O2) and the expressions of apoptosis-related genes Bcl-2 and Bax in rats. METHODS: All fresh transparent LECs in SD rats were bathed in culture medium with H2O2 in vitro, meanwhile Co-SZ were added in the culture medium. All LECs were incubated for 24 hours. The apoptosis rate of LECs was determined by terminal deoxynucleotidyl transferase mediated biotin-dUTP nick end labeling method (TUNEL). The changes of LEC ultrastructure and the formation of apoptotic body were observed by transmission electron microscopy. The expressions of apoptosis-related genes Bcl-2 and Bax were detected by streptavdin-peroxidase-biotin method. RESULTS: The apoptosis rate of LECs in the Co-SZ-treated group was significantly lower than that in the H2O2-treated group. The changes of apoptotic LEC ultrastructure in the Co-SZ-treated group were less than those in the H2O2-treated group. The expression of Bcl-2 protein was up-regulated and the expression of Bax protein was down-regulated in the Co-SZ-treated group as compared with the H2O2-treated group. CONCLUSION: The LEC apoptosis induced by H2O2 can be inhibited by Co-SZ. The molecular mechanisms of Co-SZ in inhibiting LEC apoptosis may be related to regulating the expressions of apoptosis-related genes Bcl-2 and Bax.
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OBJECTIVE: To investigate the signal transduction mechanism of curcumin in inhibiting the proliferation of bovine lens epithelial cell (LEC) induced by recombinant human epidermal growth factor (rhEGF). METHODS: There were three groups in this experiment, which were normal control group, untreated group and curcumin-treated group. Proliferation of LEC was induced by rhEGF (50 microg/L). The concentration of intracellular Ca(2+) ([Ca(2+)](i)) in LEC was measured with Fura-2/AM by spectrofluorimetry. The contents of intracellular cAMP and cGMP were assayed by radioimmunoassay. RESULTS: The [Ca(2+)]i in LEC was obviously increased in the untrated group as compared with that in the normal control group (P<0.01), and the [Ca(2+)](i) in LEC in the curcumin-treated group was highest among three groups (P<0.01). The content of intracellular cAMP in LEC was decreased while the content of intracellular cGMP was obviously increased in the untreated group as compared with those in the normal control group (P<0.01). The content of intracellular cAMP in LEC was higher in the curcumin-treated group than that in the untreated group, while the content of intracellular cGMP was lower than that in the untreated group (P<0.01). CONCLUSION: The antiproliferation effects of curcumin on LEC may relate to the regulations of multiple processes of signal transduction.
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AIM: To investigate the relationship between cytokine level and nitric oxide (NO) content in aqueous humor after intraocular lens implantation. METHODS: All New Zealand rabbits were divided randomly into three groups: (1) control group, (2) extracapsular cataract extraction group (ECCE), (3) extracapsular cataract extraction and posterior chamber intraocular lens implantation group (ECCE+IOL). The inflammation of all experimental rabbit eyes, including cornea edema and anterior chamber exudation were observed via zoom-photo slitlamp microscope 1,3,7,14,30 d postoperation. Meanwhile, aqueous humor was drawn for white blood cell (WBC) counting and classifying, and for NO-2／NO-3 and cytokine assay, including interleukin-2(IL-2), tumour necrosis factor-α(TNF-α). Statistics were taken by SPSS softwear. RESULTS: (1) Total WBC in aqueous humor were higher and anterior chamber exudation were more severe in ECCE+IOL group than that in ECCE group and control group. (2) The level of IL-2 and TNF-α and the content of NO-2／NO-3 in aqueous humor of ECCE+IOL group were higher than that in ECCE group and control group 1-14 d postoperation respectively, and it increased to peak value at 3-7 d postoperation and decreased gradually after two weeks postoperation. (3) The change regularity of IL-2, TNF-α and NO-2／NO-3 in each group were basically similar, i.e. when the level of cytokine (IL-2 and TNF-α) was normal, the content of NO-2／NO-3 was normal too, when the level of cytokine (IL-2 and TNF-α) increased, the content of NO-2／NO-3 increased too. CONCLUSION: The intraocular inflammation after ECCE+IOL was more severe than that after simple ECCE. NO, IL-2 and TNF-α play an important role in intraocular inflammation after intraocular lens implantation. The changes of IL-2 and TNF-α level was closely related with NO content in aqueous humor.
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AIM: To investigate the effects of Co-SZ eye drop on galactose cataract in rats. METHODS: Based on folk remedy, SZ eye drop was made from leech, as a modified SZ eye drop, Co-SZ eye drop was enriched in Zinc and Vitamin C. In the present study, animal model of galactose cataract in SD rats was used. All animals were randomly divided into 3 groups : control group(using 0.9 % NaCl instead of SZ and Co SZ), SZ group and Co-SZ group. Lens opacities were examined dynamically in each groups via FS-3V slit-lamp microscope. Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione (GSH) and soluble protein (SP) in the lenses were measured in 15 days. RESULTS: Both the Co-SZ and SZ eye drops could significantly delay and alleviate galactose cataract in rats, with better effect of Co-SZ than SZ eye drop. The antioxidant index indicated that SOD, GSH-Px, GSH in Co-SZ and SZ group were significantly higher than that in control group. Furthermore, SOD, GSH-Px in Co-SZ group were higher than that in SZ group significantly. CONCLUSION: Co-SZ eye drops could significantly delay and alleviate galactose cataract in rats, the effect is much better than SZ eye drops. The different effect between SZ and Co-SZ eye drops could be raised from the different content of Zinc, which is involved in anti-oxidation.
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AIM: To investigate the effect of sodium ferulate on expression of apoptosis-related genes, bcl-2 and bax , in rat lens epithelial cells (LEC) injured by oxidation.METHODS: Eyes of SD rats were divided randomly into four groups: control group, hydrogen peroxide group (H 2O 2), pirenoxine sodium group (PS) and sodium ferulate group (SF). Eyes were excised and lenses were separated under operating microscope and sterilized condition. Lenses were incubated in CO 2 incubator for 24 h with 300 ?mol?L -1 H 2O 2 and with or rithout 5 mmol?L -1 SF. The expression of Bcl-2 and Bax protein of LEC were measured and compared by tearing the LEC anterior capsule via immunohistochemical analysis.RESULTS: (1) There were Bcl-2 and Bax expression in normal lenses of SD rates, Bcl-2 expression was stronger than Bax. (2) Bcl-2 expression decreased and Bax expression increased markedly ( P
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AIM: To investigate the effects of Schisandrin B (Sch B) on apoptosis of lens epithelial cells (LEC) treated with H 2O 2. METHODS: Eyes in SD rats were excised and lenses were separated under operating microscope in sterilized condition. Lenses were divided randomly into four groups with different treatment: control group, hydrogen peroxide group (H 2O 2), pirenoxine sodium group (PS) and schisandrin B group (Sch B). Lenses were incubated in CO 2 incubator for 24 h with 300 ?mol?L -1 H 2O 2 and with or without 0 5 mmol?L -1 Sch B. LEC aoptosis and apoptosis rate were measured by TUNEL method. Ultrastructure changes and apoptosis bodies of LEC were observed via transmitted electron microscope. RESULTS: (1) Apoptosis rate in H 2O 2 group (92.0?2.6) was significantly higher than that in control group (3.5?1.8). Apoptosis rate in Sch B group (13.8?3.27) was remarkably lower than that in H 2O 2 group and PS group. (2) Ultrastructure observation indicated that apoptosis cells occurred in most LEC in H 2O 2 group and the changes were severe presenting different stages. While a few apoptosis cells were observed in Sch B group, the changes were slight and most of them were in early and middle stages. CONCLUSION: These data indicated that Sch B significantly inhibited apoptosis of LEC during experimental oxidative injury, the effects were stronger than PS.
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Oncosis is another form of cell death, which is different from apoptosis. The review will discuss the recent advances of oncosis on pathological morphology, nuclear biochemical changes and molecular mechanisms. [
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AIM: To investigate the relationship between the level of interleukin-2 (IL-2), tumour necrosis factor-?(TNF-?) and nitric oxide (NO) in aqueous humor after intraocular lens implantation. METHODS: New Zealand rabbits were divided randomly into three groups: (1)control group; (2)extracapsular cataract extraction group (ECCE); (3)extracapsular cataract extraction and posterior chamber intraocular lens implantation group (ECCE+IOL). The inflammation in all experimental rabbit eyes was observed via zoom-photo slit-lamp microscope on 1, 3, 7, 14 d and 30 d postoperation. Meanwhile, aqueous humor was drawn for white blood cell(WBC) counting and classifying , and for determining IL-2, TNF-? and NO 2 -/NO 3 - contents.RESULTS: (1)The level of IL-2 and TNF-? and NO 2 - / NO 3 - in aqueous humor of ECCE+IOL group were higher than that in ECCE and control at 1 to 14 days postoperation, respectively, it increased to peak value at 3 to 7 days postoperation and decreased gradually two weeks postoperation; (2) The changes in IL-2, TNF-? and NO 2 -/NO 3 - in each group were basically similer; (3) The changes of IL-2 and TNF-? level were closely related with NO content in aqueous humor( r=0.69, P
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AIM: To investigate the inhibitory effect of Co-SZ eye drops on apoptosis of lens epithelial cells(LEC) induced by H_2O_2 and to study the cellular and molecular mechanisms.METHODS:(1) All lenses of Sprague-Dawley rats were incubated with H_2O_2 and Co-SZ eye drops.The apoptosis rates of LEC were determined by TUNEL method.The changes of LEC ultrastructure and the formation of apoptotic body were observed by electron microscopy.(2) Bovine LEC were incubated with H_2O_2 and Co-SZ eye drops.The inhibitory of LEC apoptosis was detected by MTT after incubation.The changes of fractional DNA content in LEC were detected by flow cytometry(FCM).[Ca~(2+)]i,cAMP and cGMP of LEC were determined by spectrofluoremeter and radioimmunoassay,respectively.RESULTS: The LEC apoptosis rates in Co-SZ eye drops group were decreased significantly compared with H_2O_2 group by TUNEL.The ultrastructure changes in LEC of Co-SZ eye drops group were lighter than that in H_2O_2 group.The LEC apoptosis rates of Co-SZ eye drops group were dose-dependently decreased significantly compared with H_2O_2 groups via MTT assay.LEC apoptosis induced by H_2O_2 was inhibited by Co-SZ eye drops,and showing dose-dependent.The DNA contents in LEC of Co-SZ group were increased.The [Ca~(2+)]i and cAMP in Co-SZ group were decreased obviously.The cGMP was increased.CONCLUSION: The LEC apoptosis induced by H_2O_2 was inhibited by Co-SZ eye drops.The mechanism of apoptosis inhibition by Co-SZ eye drops maybe contribute to the increase in DNA content.The signal transduction mechanisms are related to the decrease in [Ca~(2+)]i and cAMP and the increase in cGMP.
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Objective To investigate the proliferative inhibition of lens epithelial cell (LEC) by arsenic trioxide (ATO) and rhizoma curcumae (RC), in order to provide scientific basis for pursuing safe and effective natural drugs to prevent and cure after cataract. Design Experimental study. Participants Cultured Bovine LEC in vitro. Methods Changes of cellular form were observed with microscope. Different concentration of ATO (2.5, 5, 10?mol/L) and RC(5, 10, 20mg/ml) were added to the proliferative LEC separately. The effects of proliferative inhibition by ATO and RC on the LEC were measured with methyl thia-zolyl tetrazolium (MTT) assay method after 72 hours incubation. Main Outcome Measures Cellular form, Absorbance. Results Under the microscope, good growth and quantity increase were found in proliferative control group. Slowing proliferation,poor growth, and few disintegration were observed in ATO group and RC group. MTT assay: Different concentration of ATO (2.5, 5, 10?mol/L)and RC(5, 10, 20mg/ml) can significantly inhibit the proliferation of LEC and these effects were dose dependent(P
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Apoptosis,a form of cell death,has generated considerable interest in recent years.Much progress has been made on the apoptosis induced by natural drgus,including component or mixture of plant,animal,mineral or marine materials,This review discusses some of the natural drugs that induce apoptosis and the possible mechanisms.
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AIM: To investigate the effect of natural medicinal monomer elemene (Ele) on apoptosis of lens epithelial cells (LEC) and its the cellular and molecular mechanism in vitro. METHODS: The bovine LEC were cultured with Ele and used to observe the ultrastructure changes under transmission electron microscope and to detect DNA content and mitochondrial transmenbrane potential (??m) changes by flow cytometry. RESULTS: The typical morphological changes of LEC apoptosis in Ele group were observed under transmission electron microscope, such as chromatin condensation and aggregation at the nuclear periphery, and nuclear fragmentation as well. The DNA content of LEC in Ele group decreased and showed time-dependent. It was significant lower than that in control group (P
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AIM: To investigate the effect of nitric oxide (NO) in ocular inflammation after intraocular lens implantation. METHODS: All New Zealand rabbits were divided randomly into three groups: 1. control group, 2. L-arginine (L-Arg) group, 3. N-nitro-L-arginine (L-NNA) group. Extracapsular cataract extraction (ECCE) and posterior chamber intraocular lens implantation (IOL) were operated in all animals of each groups. The inflammatory response of all rabbit eyes, including cornea edema and anterior chamber exudation were investigated 1, 3, 7, 14, 30 days afteroperation. Meanwhile, aqueous humor was drawn for white blood cell (WBC) counting and classifying, as well as for NO- 2/NO- 3 measurement. RESULTS:NO- 2/NO- 3 contents, total WBC and anterior chamber exudation in aqueous humor of L-Arg group were higher than that in control group. While that of L-NNA group were lower than that in control group.CONCLUSION:NO plays a role in intraocular inflammatory response after intraocular lens implantation. L-NNA, a nitric oxide synthase exhibitor, decreased NO contents, therefore it may reduce intraocular inflammatory response after intraocular lens implantation.
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AIM: To investigate the protective effect of five natural anti-oxidants (schisandrin B, Sch B; silibinin, SIB; propyl gallate, PG; sodium ferulate, SF; total flavonoids of hippophase, TFH) on experimental oxidative injuries of lens. METHODS: All fresh transparent lenses of rabbit eyes except control group were bathed in Fenton reaction system in order to produce a model of oxidative damages of lens, meanwhile Sch B, SIB, PG, SF, TFH and pirenoxine sodium (PS) were added in the reaction system in different groups respectively. Lenses were incubated for 24 hours. Then total protein (TP), soluble protein (SP), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione (GSH), vitamin C (Vit C), total activities of anti-oxidation (TAO) and malondiaoldehyde (MDA) in homogenized lenses were measured to observe the effects of five anti-oxidants on above index. RESULTS: Five anti-oxidants increased the anti-oxidative index and decreased MDA in lens of oxidative damages in different levels, the effects are better than that of PS, especially in group SF and Sch B. CONCLUSION: The five natural anti-oxidants protected lens against experimental oxidative injuries very well. There are wide prospects to pursue effective anti-cataract drugs from natural anti-oxidants.