Background Studies confirmed that hydroxycamptothecin cause the apoptosis of human Tenon capsule fibroblasts (HTFs) by protein kinase R-like endoplasmic reticulum stress kinase (PERK) single pathway.Autophagy and apoptosis are programmed cell death following stress reaction,so they remain a close association.However,the effect of hydroxycamptothecin on the autophagy of HTFs and its mechanism are still unclear.Objective This study was to explore the promoting effect of PERK signal pathway on hydroxycamptothecin inducing the autophagy of HTFs.Methods This study procedure was approval by Ethic Committee of Nanjing Medical University.Human Tenon capsule tissue was obtained from fresh adult donors.HTFs were cultured and passaged by explant-culture method and identified by immunofluorescence for vimentin and keratin.pLVX-PERK lentiviral packed by 293T cells was transfected into HTFs to obtain stable PERK-knockout cell line by puromycin selection.Then the HTFs were treated with 0.10 g/L of hydroxycamptothecin for 5 minutes and consecutively cultivated for 24 hours,and the untreated cells were used as the control group.Western blot assay was used to detect the expressions of autophagy specific proteins in the cells,including autophagy related gene 5 (ATG-5),Beclin-1,light chain 3 (LC-3).Cyto-ID staining was used to identify the autophagosome in the cells.The experimental results were analyzed and compared between different treating groups.Results The gray scales for the expressions of Beclin 1,ATG-5,LC-3-Ⅰ and LC-3-Ⅱ proteins in HTFs were 0.365:±0.045,0.765 ±0.055,0.120±0.030 and 0.215 ±0.035 in the control group,and those in the hydroxycamptothecin treated group were 0.980±0.070,1.495±0.095,0.585±0.025 and 0.785±0.055,showing a significant decline in the hydroxycamptothecin treated group(P=0.018,0.022,0.007,0.013).The green fluorescence of the autophagosome was stronger in the hydroxycamptothecin treated group compared with the control group.Western blot revealed that the gray scale of PERK expression in the cells was 0.130±0.030 in the PERK-knockout group,with a significant reduce in comparison with 0.765 ±0.055 of the control group (P =0.010).However,no obvious distinctions were seen in the band intensities of the expressions of Beclin-1,ATG-5 and LC-3 proteins between the two groups.Western blot indicated that the grey scale of the PERK expression in the cells was 1.790± 0.060 in the 0.10 g/L hydroxycamptothecin group,which was significantly higher than 0.880 ± 0.070 of the control group (P =0.010).Expression levels (gray scales) of Beclin-1,ATG-5,LC-3-Ⅰand LC-3-Ⅱ in the PERK-knockout+ 0.10 g/L hydroxycamptothecin group were 0.475 ± 0.045,0.390 ± 0.040,0.055 ± 0.015 and 0.075 ± 0.025,which were significantly lowed in comparison with 0.955 ± 0.065,0.765 ± 0.055,0.155 ± 0.015 and 0.280 ± 0.030 of the control+ 0.10 g/L hydroxycamptothecin group (P =0.026,0.031,0.042,0.034).In addition,the fluorescence intensity of autophagosomes was weaker in the PERK-knockout+0.10 g/L hydroxycamptothecin group compared with the control+0.10 g/L hydroxycamptothecin group.Conclusions Hydroxycamptothecin induces the autophagy of HTFs by PERK signal pathway.

Background Scarring of surgical area,the most important factor,leads to the failure of glaucoma filtering surgeries.Therefore,more and more attentions are paid to the causes and process of scar formation.Objective This study was to compare the differences of proliferation and migrating abilities of fibroblasts between filtering bleb scar tissue and normal Tenon capsular tissue,and to investigate the inhibitory effects of miRNA-200a (miR-200a) on biological behavior of conjunctival fibroblasts.Methods Normal Tenon capsular tissue and filtering bleb scar tissue were collected during the strabismus surgery and glaucoma filtering surgery,respectively for the primarily culture of fibroblasts.The proliferation (absorbency,A) of the cells was assayed by cell counting kit-8 (CCK8) method;the relative migrating distance of the cells was measured by cell scratch test;and the relative expressions of transforming growth factor-β1 (TGF-β1)mRNA and miR-200a mRNA in the cells were detected by realtime fluorescence quantitative PCR.TGF-β1 mimic of 0,1,2 and 5 ng/ml was added in the medium of human normal Tenon capsular-derived fibroblasts (HTFs),and 0.00,0.25,0.50,1.00 μg/ml TGF-β1 inhibitor was added in the medium of human scarring-derived fibroblasts (HSFs) for 24 hours,respectively,and CCK8 was used to evaluate the proliferation of the cells.The relative migrating distance as well as the relative expressions of miR-200a mRNA were analyzed in the 2 ng/ml TGF-β1 mimic-or 1.00 μg/ml TGF-β1 inhibitor-treated cells.Results The primary conjunctival presented the spindle and star-like in shape with large body and oval nuclei.The cells showed the positive response for keratin and vimentin antibodies.The A values were 1.476±0.110 in the HSFs and 0.958±0.074 in the HTFs,with a significant difference between them (t =24.900,P=0.016).The relative expressions of TGF-β1 mRNA were significantly higher in the HSFs than those in the HTFs,and the relative expressions of miR-200a were evidently lower in the HSFs than those in the HTFs,showing significant differences between them (t =6.358,P =0.024;t=7.394,P =0.018).Compared with the 2 ng/ml TGF-β1 mimic-treated HTFs,the relative migrating distance increased,while the expression level of miR-200a mRNA was significantly reduced in the 2 ng/ml TGF-β1 mimictreated HSFs (all at P＜0.05);Compared with the 1.00 μg/ml TGF-β1 inhibitor-treated HTFs,the relative migrating distance decreased,but the expression level of miR-200a mRNA was significantly elevated in the 1.00 μg/ml TGF-β1 inhibitor-treated HSFs (all at P＜0.05).Conclusions The proliferation and migrating abilities are stronger in the HSFs than those in the HTFs,which probably is regulated by the expression of miR-200a in the cells.The miR-200a plays a negative feedback for the effect of TGF-β1 promoting proliferation and migration of fibroblasts.

Background Our previous study showed that the expression level of glial fibrillary acid protein (GFAP) increases in astrocytes and Müller cells of retina in chronic hypertensive eye,and this change was clarified to be associated with the damage process of the retinal ganglion cells (RGCs).Aquaporin 4 (AQP4) exists in neural glial cells,so we conjecture AQP4 plays a role in the regulating GFAP expression in glaucomatous eye.Objectives This study was to investigate whether AQP4 gene can regulate the expression of GFAP in retina and explore the effect of AQP4 on RGCs damage of glaucoma.Methods Chronic ocular hypertensive eye models were established by cauterizing the scleral venous in the left eyes of 30 male AQP4-/-mice and 30 male wild type (WT) mice with the same background,and the right eyes served as control eyes.The intraocular pressure (IOP) was measured with Icare rebound tonometer at 1 day,3,7,14 and 28 days respectively and the retinas were isolated from 6 of each types of mice at the corresponding time points.The expression of GFAP in the retina was detected by Western blot.The use and care of the experimental animals followed ARVO Statement.Results The IOP was significantly higher in the model eyes than that of the control eyes 1 day,3,7,14 and 28 days in both AQP-/-mice (t =15.29,16.02,13.77,14.34,12.40,all at P＜0.05) and WT mice (t =17.65,14.91,15.97,13.41,12.53,all at P ＜0.05).GFAP was expressed in the control eyes both of the AQP4-/-mice and the WT mice.The expressing level of GFAP (GFAP/β-actin) in retinas was 1.00±0.00,1.99±0.29,4.05±0.69,4.47±0.48,3.21±0.35 and 3.25±0.53 in the control eyes and 1-,3-,7-,14-,28-day model eyes of WT mice; and those in the AQP4-/-mice were 1.00±0.00,1.69±0.31,2.27 ±0.55,2.79 ± 0.39,1.93 ± 0.31 and 1.54 ± 0.40,with a significant difference in the expressing level of GFAP in various time points (F =9.54,P＜0.05).In addition,significant gradually elevation of GFAP expression were seen in the WT mice and gradually decline of GFAP expression was found in the AQP4-/-mice with the lapse of time (all at P＜0.05).No significant difference was seen in the expression of GFAP in the control eyes between the WT mice and AQP4-/-mice (P＞0.05).However,the expression level of GFAP in retina was significantly higher in the WT mice than that of A QP4-/-mice 3,7,14 and 28 days after operation (t =4.51,7.95,6.12,5.76,all at P＜0.01).tonelusions In chronic high IOP mice,AQP4 gene plays an important role in retinal damage by upregulating the expression of GFAP in retina and promoting the activation of RGCs.AQP4 probably is a new target of treatment of glaucoma.

Background Previous study showed that both hydroxycamptothecin (HCPT) and etoposide (VP-16) can induce the apoptosis of human Tenon capsule fibroblasts (HTFs).However,whether the combination of HCPT with VP-16 enhance the efficacy of drugs is unknown.Olbjeetive This study was to investigate the synergistic effect and its mechanism of HCPT combined with VP-16 on apoptosis of HTFs.Methods Human Tenon capsule tissue was obtained from the eye bank of Jiangsu Province People's Hospital.HTFs were cultured in vitro using explant method and identified by immunofluorescence with vimentin.The fourth generation of cells were incubated in 96-well plate,and different concentrations of HCPT (1,5,10,50,100 mg/L),VP-16 (0.6,2.5,5.0,25.0,50.0 mg/L) and HCPT+VP-16 (2:1,final concentrations 0.80,3.75,7.50,37.50,75.00 mg/L) were added for 24 hours.The inhibiting rate of drugs to HTFs growth was detected using CCK-8 kit.The HTFs were divided into blank control group,HCPT (50 ng/L) treated group,VP-16 (25 mg/L) treated group and HCPT+ VP-16 (37.5 mg/L) treated group,and the apoptosis rates of HTFs in various groups were assayed by flow cytometry.The expressions of caspase-3,cleaved caspase-3,bax,bcl-2,JNK,p-JNK,Akt,p-Akt in the cells were detected by Western blot assay.Results Cultured cells grew well with the polygon shape and positive response for vimentin.The inhibiting rate was elevated with the increase of drug dosage 24 hours after addition of drugs (HCPT:F=41.34,P=0.00 ; VP-16:F =62.60,P =0.00 ; HCPT+VP-16:F =46.77,P =0.00).The half maximal inhibitory concentrations (IC50) of HCPT,VP-16,HCPT+VP-16 were 80.99,27.93,19.81 mg/L,respectively,and the combined index (CI) of HCPT with VP-16 was 0.399,showing a stronger synergistic action.The apoptotic rates of HTFs were (4.87±0.78) ％,(11.20± 1.94)％,(12.67±1.51)％ and (19.77±2.01)％ in the blank control group,HCPT treated group,VP-16 treated group and HCPT+VP-16 treated group,respectively,with a significant difference among them (F=18.23,P ＜ 0.01),and the apoptotic rate was significantly raised in the HCPT + VP-16 treated group,HCPT treated group and VP-16 treated group compared with the blank control group (q'=15.67,16.32,26.88,all at P＜0.01).Compared with the blank control group,the grey scale values of cleaved caspase-3,bax,p-JNK in the cells of HCPT+VP-16 treated group,HCPT treated group and VP-16 treated group were significantly increased (all at P＜0.01),and those in the HCPT+VP-16 treated group significant ascent in comparison with the HCPT treated group and VP-16 treated group (all at P＜0.01).However,the changes of caspase-3,JNK and Akt expression were insignificant.The grey scale values of bcl-2 and p-Akt in the HTFs of the HCPT,VP-16 and HCPT+VP-16 treated groups were significantly lower than those of the blank control group,with a dominant reducing in the HCPT+VP-16 treated group (all at P＜0.01).Conclusions HCPT and VP-16 induce the apoptosis of HTFs in vitro at a dose-dependent manner.The combination of HCPT with VP-16 has a stronger synergistic efficacy.The up-regulation of p-JNK and bax as well as the down-regulation of p-Akt and bcl-2 in HTFs are involved in the coaction of HCPT and VP-16.

Background The filtration surgery is the main method of treating glaucoma,which usually fails due to postoperative scarring.The study about application of anti-scarring agents in filtration surgery is a hotspot.Objective This study was to investigate whether topical administration of hydroxycamptothecin (HCPT) could be used to prevent postoperative scarring in after experimental glaucoma filtration surgery,and explore its optimal dose.Metbods Trabeculectomy was performed on the right eyes of 40 New Zealand white rabbits to establish the trabeculectomy animal models.The rabbits were then randomized into normal saline solution group,0.3 g/L mitomycin C(MMC) group,0.3 g/L HCPT group and 1.0 g/L HCPT group based on the intraoperative topical drugs using randomized number table method.The different drugs above-mentioned were put beneath the conjunctival flap and scleral flap for 5 minutes during the surgery.The intraocular pressure (IOP) was measured before and day 1,4,7,14,21 and 28 after the surgery with Icare tonometer,and the filtering area and height were measured under the slit lamp microscope to assess the efficacy of various drugs.The adverse effects were evaluated by examining the responses of the ocular anterior segment and retinal change.The specimens at operative zone were obtained in 7,14 and 28 days after surgery with the size 5 mm×5 mm for the hematoxylin-eosin staining and Masson trichrome staining to estimate the anti-fibrosis effect of various drugs,and to evaluate the survival time of functional bleb.Kaplan-Meier analysis was used to compare the survival time of functional bleb of different groups.The use and care of the animals complied with the Regulation for the Administration of Affair Concerning Experimental Animals by State Science and Technology Committee.Results The IOP was significantly different in the rabbits from different groups among various time points (Fgroup =20.79,P =0.00 ; Ftime =85.34,P =0.00 ; Fiion =2.13,P =0.01).From 1 day through 28 days after operation,the IOPs in MMC group and 1.0 g/L HCPT group were significantly lower than those before operation (all at P＜0.05).The survival time of functional bleb of different groups was (11.3 ±2.8),(19.5 ±2.4),(13.3 ±2.2) and (20.2 ± 4.5) days,respectively,showing a significant difference (log rank =11.92,P ＜ 0.01),with a considerable prolong in the 1.0 g/L HCPT group.No significant change was found in the bleb area and height among the four groups within 7 days after operation,but postoperative 7,14,28 days,the area and height values of bleb were significantly smaller in the normal saline solution group and 0.3 g/L HCPT group compared with the MMC group and 1.0 g/L HCPT group (all at P ＜ 0.05).Histopathological examination showed loosen subconjunctival tissue,less inflammatory cells and weaker collagenous fibrillary staining in the MMC group and 1.0 g/L HCPT group in comparison with the normal saline solution group and 0.3 g/L HCPT group.Conclusions The topical administration of 1.0 g/L HCPT inhibit the inflammatory response and collagen fibrosis and therefore prolong the survival time of functional bleb after glaucomatous filtering surgery.

Background Long-term administration of glucocorticoid drugs induces ocular hypertension in susceptible individuals probably.It has been verified that 1 1β-hydroxysteroid dehydrogenase type 1 (11β-HSD1),glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) can affect the generating of aqueous humor,but how they play the role in glucocorticoid-induced ocular hypertension is unclear.Objective This study was to investigate the relationship of expressions of 11β-HSD1 and steroids receptors in ciliary body and steroid-induced ocular hypertension.Methods Thirteen 12-16 week-old New Zealand albino rabbits were randomized to control group (5 rabbits) and experimental group (8 rabbits).Steroid-induced glaucoma models were induced by administration of subconjunctival injection of 5 mg dexamethasone solution(1 ml) and 0.5％ dexamethasone eye drops on alternate days in the left eyes for consecutive two months in the experimental group,and the equal volume of sterile normal saline solution was used in the same way in the control group.The successful criteria of model eyes was defined as rising of intraocular pressure (IOP) to ≥ 18 mmHg for over one week.Then,the animals were sacrificed by excessive anesthesia and the ciliary tissues were isolated for the assay of expressions of 1 1β-HSD1 protein by immunochemistry,and the expressions of 11β-HSD1 mRNA,GR mRNA and MR mRNA in ciliary body were semi-quantitatively detected by reverse transcription-PCR (RT-PCR).The experimental results were compared between the two groups.Results The IOP was normal in the first two weeks after administration of drugs,and no significant difference was found in IOP between the first week and the second week in the experimental group (q =0.469,P ＞0.05).From 3 through 5 weeks after injection,the IOP was gradually elevated,with the highest value of (18.87±0.77) mmHg in the fifth week.Significant differences were seen between the two groups at mentioned-above time points (q =10.535,20.353,28.681,all at P ＜ 0.01).11β-HSD1 protein was positively expressed in nonpigmented epithelial cells of ciliary tissue of rabbits in both groups,however,the expression intensity was weaker in the experimental group compared with the control group.The relative expressional values of MR mRNA,GR mRNA and 11β-HSD1 mRNA in the ciliary tissue were 2.22±0.78,0.64±0.11 and 0.47±0.16 in the experimental group,and those in the control group were 0.94±0.27,1.88±0.74 and 2.68±1.28,with significant differences between the two groups (t =6.070,P =0.004 ; t =5.170,P =0.007 ; t =5.540,P =0.005).Conclusions Corticosteroidinduced glaucoma probably is associated with the up-regulation of MR level and down-regulations of GR and 11β-HSD1 in ciliary body.

Background The fibrosis of filtering area caused by proliferation of human Tenon fibroblasts (HTFs) is one of failure causes following glaucoma surgery.Researches revealed that hydroxycamptothecin can induce the apoptosis of HTFs,but its influence on autophagy of HTFs is unclear.Objective This study attempted to investigate whether hydroxycamptothecin can cause an alteration of autophagic activity in HTFs.Methods Human Tenon capsular tissue was obtained from 3 patients during strabismus correction surgery under the informed consent of patients and their parents for the primary culture and passaged of HTFs in DMEM containing 10％ fetal bovine serum.The generation 3 to 6 cells then were incubated with 0.0,0.5,1.0,4.0,10.0 mg/L hydroxycamptothecin for 24 hours,respectively.A cell counting kit-8 (CCK-8) was used to detect the cell viability in different treated groups.The autophagic activity of HTFs was evaluated by a Cyto-ID autophagy detection kit,and then the autophagic flux was evaluated by counting the Cyto-ID positive cells under a fluorescence microscope,and the green fluorescence intensity was determined by flow cytometry.Quantitative reverse transcriptase PCR (qRT-PCR) and Western blot analysis were employed to assay the relative expressions of autophagic-associated genes and their proteins in HTFs,including Beclin-1,autophagy related gene 5 (ATG-5) and light chain 3 (LC-3).Results The cell viability of HTFs in the 0.0,0.5,1.0,4.0 and 10.0 mg/L hydroxycamptothecin groups were (100.00 ± 6.44) ％,(91.70 ± 6.36) ％,(81.47 ± 6.00) ％,(68.43 ± 6.69) ％ and (59.97 ± 6.98) ％ respectively,showing a gradually declining trend with the increase of hydroxycamptothecin doses,with a significant difference among them (F=19.040,P＜0.001),and the viability of HTFs in the 1.0,4.0 and 10.0 mg/L hydroxycamptothecin groups were significantly decreased than the control group (P＜0.05,P＜0.01,P＜0.01).qRT-PCR analysis revealed that the relative expression levels of Beclin-1 mRNA,ATG-5 mRNA and LC-3 mRNA in 4.0 mg/L hydroxycamptothecin group were (3.225 ±0.346),(2.839 ±0.418) and (3.761±0.224) folds higher than those of the control group.The expressions of Beclin-1 and ATG-5 proteins were significantly increased in the 4.0 mg/L hydroxycamptothecin group in comparison with the control group,and the expression intensity ratio of LC-3-Ⅱ/Ⅰ was 0.965±0.159 in the hydroxycamptothecin group,which was significantly higher than 0.275 ±0.860 of the control group (P =0.003).Cyto-ID staining showed that the percentage of autophagic cells increased dramatically from (11.333±4.010) ％ to (55.000±9.013) ％ upon the exposure of HTFs to 4.0 mg/L hydroxycamptothecin (P=0.002).Flow cytometry analysis showed that the green fluorescence intensity in the 4.0 mg/L hydroxycamptothecin group was (3.037 ±0.513) fold relative to that in the control group,showing a significant difference between the two groups (P =0.003).Conclusions Hydroxycamptothecin can induce autophagy in HTFs in vitro.

Based on the standards for mental developmental re-tardation set up by WHO in 1985,by applying IQ andadaptation to social behavior as indexes for observa-tion with testing methods for intelligence commonlyaccepted,we have observed the effects in 128 suchcases treated by combined use of acupuncture,adhe-sive—pressing therapy at auricular points and drugplastering at points.Results showed that our therapyis conducive to the improvement of intelligence in suchcases.

Objective : To investigate the mental health status of the pupils from Grade Four and Five in Jiading District of Shanghai, and to provide evidence for mental health intervention. Methods : All the students from Grade Four and Five in Jiading District, Shanghai were selected as survey objects. The MHT was used to evaluate their mental health status, and the influencing factors of mental health problems were analyzed by multivariate logistic regression model. Results : A total of 3 788 questionnaires were issued, of which 3 100 ones were valid and the effective rate was 81.84%. The detection rate of mental health problems was 17.06%. The detection rates of impulse tendency, terror tendency, physical symptoms, sensitivity tendency, self-blame tendency, loneliness tendency, anxiety towards people and learning anxiety were 11.77%, 15.55%, 12.97%, 16.19%, 10.10%, 4.81%, 13.16% and 14.00%, respectively. The results of multivariate logistic regression analysis showed that female students ( OR=1.617,95%CI: 1.325-1.974 ) , poor self-rated appearance ( OR=3.683, 95%CI: 2.428-5.586 ) ,only one or two friends ( OR=1.806, 95%CI: 1.342-2.428 ) , poor academic performance ( OR=1.626, 95%CI: 1.167-2.267 ) , primary school education or below of mother ( OR=1.539, 95%CI: 1.107-2.139 ) general or poor relationship with mother ( OR=1.785, 95%CI: 1.303-2.444 ) or father ( OR=1.745, 95%CI: 1.336-2.280 ) were risk factors for mental health problems. Conclusion The fourth-to fifth-grade primary school students in Jiading District of Shanghai have high detection rate of psychological problems. Gender, the number of friends, academic performance, education level of mother and the relation with parents are associated.

Objective To investigate the effects of matrine on proliferation and apoptosis of human Tenon capsule fibroblasts (HTFs) in.vitro.Methods After treated with 0,0.3,0.6 and 0.9 g/L matrine in vitro,cell counting kit-8 (CCK-8) method was used to assay the proliferation of HTFs at 24,48 and 72 hours,Western blot and PCR were performed to evaluate the expression of apoptosis-associated factor caspase-3 on both protein and RNA levels.Results The activity of human Tenon capsule fibroblast at 48 hours and 72 hours after treated with 0.3,0.6,0.9 g/L matrine was significantly inhibited when compared with the 0 g/L matrine group,and the inhibitory effect was dose-dependent and time-dependent (F ion =1 019.51,P =0.00;Ftime =5 848.66,P =0.00;Fi ion =147.45,P=0.00).After treated with 0,0.3,0.6 and 0.9 g/L matrine,the early apoptosis rate of HTFs was (2.68±0.30)％,(5.08±0.47)％,(6.97±0.69)％ and (10.30±1.20)％,the grey value ofcaspase-3 protein was 1.00±0.13,1.90±0.19,2.50±0.30 and 2.67±0.30,the relative expression of caspase-3 mRNA was 0.98 ±0.12,2.01 ±0.34,6.15 ± 0.60 and 11.40 ± 1.12,respectively,with significant differences among them (F =55.74,66.01,154.50;all at P＜0.01),the early apoptosis rate of HTFs,the grey value of caspase-3 protein and the relative expression of caspase-3 mRNA were all increased significantly as the concentration of matrine increased,with significant differences between any two groups (all at P＜0.05).Conclusions Matrine can inhibit the proliferation of HTFs and induce the apoptosis of HTFs in a time-and dose-depended manner.