ObjectiveTo observe the effect of complement receptor 1 (CR1) on barrier of cultured human retinal epithelial cells (hRPE) under complement-activated oxidative stress.
MethodsThe third to fifth passage of hRPE cultured on Transwell insert were used to establish a stable hRPE monolayer barrier. The hRPE monolayer barrier was exposed to 500 μmol/L ten-butyl hydroperoxide and 10% normal human serum to establish the hRPE monolayer barrier model of complement-activated oxidative stress in vitro. hRPE monolayer barriers under complement-activated oxidative stress were divided into two groups including model group and CR1 treatment (1 μg/ml) group. Model group and CR1 treatment group were treated with 1 μl phosphate buffer solution (PBS) or CR1 for 4 hours. Normal hRPE monolayer barrier were used as control in transepithelial resistance (TER) measurement experiment. TER was measured to evaluate the barrier function of hRPE. The hRPE-secreted vascular endothelial growth factor (VEGF) and chemokine (C-C Motif) Ligand 2 (CCL2), together with complement bioactive fragments (C3a, C5a) and membrane-attack complex (MAC) in the supernatant were detected by enzyme-linked immune sorbent assay.
ResultsStable hRPE monolayer barrier was established 3 weeks after hRPE seeded on Transwell insert. Complement-activated oxidative stress resulted in a sharp decrease of TER to 54.51% compared with normal hRPE barrier. CR1 treatment could significantly improve TER of barrier under complement-activated oxidative stress to 63.48% compared with normal hRPE barrier(t=21.60, P < 0.05). Compared with model group, CR1 treatment could significantly decrease the concentration of VEGF and CCL2 by 11.48% and 23.47% secreted by hRPE under complement-activated oxidative stress (t=3.26, 2.43; P < 0.05). Compared with model group, CR1 treatment could also decreased the concentration of C3a, C5a and MAC by 24.00%, 27.87%, 22.44%.The difference were statistically significant (t=9.86, 2.63, 6.94; P < 0.05).
ConclusionsCR1 could protect the barrier function of hRPE cells against complement-activated oxidative stress. The underlying mechanism may involve inhibiting complement activation and down-regulating the expression of VEGF and CCL2.
ObjectiveTo evaluate the photoreceptor-protective effects of Cdk5 inhibitor Roscovitine on retinal degeneration in Royal College of Surgeons (RCS) rat.
MethodsThe RCS rats were divided into three groups according to postnatal days: the early (17 days), medium (25 days) and late intervention group (35 days). Cdk5 inhibitor Roscovitine were used in the right eyes by intravitreal injection as experimental eyes and Roscovitine solvent dimethylsulfoxide were used in the left as control at postnatal 17, 25, 35 days. Hematoxylin-eosin (HE) staining was used to observe the thickness of outer nuclear layer. The expression of Cdk5 P25 and cleave-caspase 3 in the retina was evaluated by immunohistochemistry. The protein expression of cleave-caspase 3 in the retina was determined by Western blot. The apoptosis of retinal cells was examined by terminal-deoxynucleotidyl transferase mediated nick end labeling.
ResultsHE staining showed that thickness of outer nuclear layer in the early and medium intervention groups were significantly thicker than that in the control group (P < 0.05), particularly in the early intervention group. And there was no significant change in the late intervention group (P > 0.05). The expression level of Cdk5, p25, cleave-caspase 3 in the outer nuclear layer in three intervention groups were lower than that in the control group (P < 0.05), especially in the early intervention group.
ConclusionCdk5 inhibitor Roscovitine can delay the retinitis pigmentosa process in RCS rats by early, medium interventional therapy and may have a certain degree of photoreceptor-protective effects.
Objective To evaluate the effectiveness of GnRH antagonist on vitro fertilization-embryo transfer (IVF-ET). Methods We searched CBMdisc (1979 to 2010), Wanfang (1994 to 2010), CNKI (1994 to 2010), VIP (1989 to 2010), PubMed (1997 to 2010), PML (1997 to 2010), FMJS (2000-2010), and 9 related journals to identify randomized controlled trials (RCTs) on the comparison between GnRH antagonist (GnRHA) and GnRH agonist (GnRHa). The quality of included trials was critically appraised. RevMan 4.2.7 software was used for statistical analysis. Results Six published RCTs involving 1 208 participants were included. Compared with the GnRHa group, stimulation duration in the GnRHA group was lower (WMD= –1.07, 95%CI –1.38 to –0.76), dose of gonadotrophins (Gns) in the GnRHA group was slightly lower (WMD= –0.49, 95%CI –1.63 to 0.66), endometrial thickness at the time of HCG administration was no significant difference in the two groups (WMD= –0.09, 95%CI –0.42 to 0.24), number of oocytes retrieved in the GnRHA group was lower (WMD= –1.80, 95%CI –2.48 to –1.12), OHSS rate in the GnRHA group was slightly lower (Peto OR= 0.77, 95%CI 0.35 to 1.72), pregnancy rate in the GnRHA group was slightly lower (Peto OR= 0.83, 95%CI 0.65 to 1.05), miscarraige rate as no significant difference in the two groups (Peto OR= 1.49, 95%CI 0.79 to 2.82). Conclusions Compared with GnRHa, GnRHA requires shorter stimulation duration and less Gn, less affected the pregnancy rate, and reduces the incidence of OHSS. The use of GnRHA in clinical practice is relatively flexible with good acceptability. GnRHA for the superovulation IVF-ET offers an alternative treatment. The above conclusion still needs more well-designed, multi-center, and large-scale RCTs to confirm and update.
Chronic central serous chorioretinopathy (CSC) usually demonstrates frequent recurrence, diffuse leakage and persistent subretinal fluid, which cannot be absorbed, thus lead to photoreceptor damage and poor visual acuity. As glucocorticoids have been implicated in the pathogenesis of chronic CSC, various anti-glucocorticoids oral drugs were used in the clinic to promote retinal fluid absorption and reduce the central retinal thickness of the macula and improve the vision outcomes. In addition, the 5α-reductase-specific inhibitor finasteride, the P450-3A4 inducer rifampicin, circadian rhythmic regulator melatonin, and systemic anti-inflammatory drug methotrexate have also been put into clinical trials for chronic CSC, and achieved certain effects. However, most of the clinical studies on these oral drugs were case reports, but not multi-center randomized clinical trials. The long-term effects of these oral drugs need to be observed and studied further.
ObjectiveTo investigate the effect of laparoscopic surgery combined with the following treatment of gonadotropin-releasing hormone agonist (GnRH-a) on the patients with endometriotic infertility and the value of endometriosis fertility index (EFI) system on forecasting the pregnancy outcome.
MethodsFrom January 2005 to July 2011, the clinical data of 15 patients with endometriotic infertility patients were analyzed retrospectively. All the patients underwent laparoscopic surgery, and the effect was evaluated according to the endometriosis fertility index (EFI). Then all the patients were divided randomly into two groups on the basis of the different assisted treatment after the laparoscopic surgery:the control group (without any other therapy) and the GnRh-a group (combined with GnRH-a). Eventually, the pregnant rates were calculated respectively in the different groups or according to the different EFI.
ResultsAt last, 103 cases finished the follow-up. The pregnancy rate in the control group after 6 months, 1 year and 3 years therapy were 12.5%, 31.2%, and 41.7%, respectively; while in the GnRh-a group were 0%, 16.3%, and 40.0%, respectively. There were no difference between the two groups after the 1 year and 3 years therapy (P>0.05). Besides, all of the 55 cases in the GnRh-a group had side-effects, but no severe adverse effect was encountered. All the side-effects were disappeared after stopping the treatment. The pregnancy rate of the patients with the EFI score of 8-10 was respectively 31.3% 1 year and 62.5% 3 years after the treatment. However, the pregnancy rate of the patients with the EFI score of 5-7 was respectively 15.2% 1 year and 26.0% 3 years after the treatment. There were only 9 patients with the EFI score of 0-4, and all of them were not pregnant. The EFI score had positive correlation with the pregnancy rate 1 year and 3 years after the treatment (rs=0.204, P=0.039; rs=0.437, P<0.001).
ConclusionThe treatment of GnRh-a after the laparoscopic surgery can not only increase the rate of the pregnancy, but also delay the pregnancy occasion and increase the occurrence of the side-effects. The EFI may be valuable for forecasting the rate of pregnancy in patients with endometriotic infertility. The patients with EFI score higher than 8 may expect the pregnancy, while the ones with below 7 probably have much lower rate of natural pregnancy rate.
ObjectiveTo preliminarily investigate the mechanism of MMP-9 blocking CD73 detachment from RPE cells surface and preventing and treating experimental autoimmune pigment membranitis (EAU).MethodsRPE cells isolated from wild-type C57BL/6 and CD73 gene knockout (CD73-/-) mice were cultured in vitro, and treated with lipopolysaccharide and TNF-α to induce CD73 detachment from RPE surface. According to whether MMP-9 inhibitor CTK8G1150 was added at the same time (the final concentration was 5.0 mol/L) or not, RPE cells cultured in the two types of mice were respectively set as MMP-9 inhibitor intervention group and non-intervention control group. The cells in each group were treated with the intervention of a solvent, 1 μmol/L adenosine monophosphate (AMP), 1 μmol/L AMP, and 3 μmol/L 5' -α,β-methylene adenosine diphosphate (APCP) (AMP+APCP). The stimulating effect of RPE cells in different groups on CD4+ T cell proliferation was detected by tritiated thymidine incorporation. Adoptive immune induced EAU in wild-type B6 mice and CD73-/- mice, respectively. The receptor mice were randomly divided into the MMP-9 inhibitor intervention group and the non-intervention control group, and CTK8G1150 or the solvent were injected into the subretinal cavity 4, 7 and 10 days after adoptive immunity. CD73 mRNA and protein expression in RPE cells of recipient mice were detected by real-time quantitative PCR (RT-PCR) and Western blot. One-way ANOVA was used to analyze all experimental data.ResultsWhen the stimulation mode was AMP, the proliferation of CD4+ T cells in the C57BL/6 MMP-9 inhibitor intervention group decreased significantly compared with the non-intervention group (F=13.28, P＜0.01). When the stimulation mode was solvent and AMP+APCP, there was no statistically significant difference in the proliferation capacity of CD4+ T cells between the two groups (F=7.78, 6.58; P＞0.05). There was no statistically significant difference in the proliferation capacity of CD4+ T cells between the CD73-/- MMP-9 inhibitor intervention group and the non-intervention group (F=5.24, 6.12, 7.04; P＞0.05). RT-PCR results showed that there was no statistically significant difference in the relative expression of CD73 mRNA in RPE cells between the MMP-9 inhibitor group and the non-intervention control group (F=6.54, P>0.05). Western blot results showed that the expression of CD73 protein in RPE cells in the MMP-9 inhibitor group of B6 receptor mice was significantly increased compared with the control group (F=15.24, P＜0.01).ConclusionMMP-9 inhibitor blocks CD73 detachment from RPE cells surface and has a protective effect on EAU.
ObjectiveTo screen and identify an ideal lead compound with potential inhibitory effects on N-methyl-D-aspartate receptor (NMDAR) from the ZINC15 drug database, promoting drug design and development to improve epilepsy treatment. Methods Potential NMDAR inhibitors were identified through a series of computer-aided structural and chemical virtual screening techniques (Discovery Studio). Structure-based virtual screening was used to predict and further filter candidate compounds based on physicochemical, pharmacological, and toxicological properties. The binding affinity and chemical bond distribution between selected compounds and NMDAR were then analyzed, and the stability of the ligand-NMDAR complex in a natural environment was evaluated. Results The study identified one novel natural compound from the ZINC15 database, with ZINC000096085903 showing low rodent carcinogenicity, no Ames mutagenicity, no developmental toxicity, and ideal physicochemical properties. This compound demonstrated high binding affinity and favorable interaction energy, with the ZINC000096085903-NMDAR complex exhibiting more favorable potential energy than the complex formed by NMDAR and the reference ligand ketamine. Furthermore, molecular dynamics simulation indicated that this complex remains stable in vivo and can inhibit NMDAR similarly to ketamine. Conclusion ZINC000096085903 is an ideal lead compound for NMDAR inhibition. With higher binding affinity and stability when bound to NMDAR, as well as slower metabolism, ZINC000096085903 showed significant potential for long-term epilepsy treatment.
ObjectiveTo study how CD73 is shed from the retinal pigment epithelium (RPE) surface.MethodsCD73 shedding was induced by treating RPE with lipopolysaccharides (LPS) and TNF-α. After Phospholipase C (PLC) or pan matrix metalloproteinase (MMP) inhibitors were added, surface amount of CD73 was evaluated by flow cytometry (FACS). Then selective inhibitors or their corresponding siRNAs of MMP-2 and MMP-9 were applied to the treatments of RPE; and their effects on induced CD73 shedding were evaluated by FACS. By site directed mutagenesis, mutations were introduced to Lys547-Phe548 coding sites of CD73 cDNA, which was cloned in a pcDNA mammalian expression vector. Both wt-CD73 and mutated-CD73 were over expressed in CD73-/- RPE and their induced shedding was compared.ResultsLPS and TNF-α induced CD73 shedding from RPE was completely blocked by the addition of pan MMP inhibitor but not PLC inhibitor. Selective MPP-9, but not MMP-2, inhibitor or its siRNA blocked CD73 shedding. In CD73-/- RPE induced CD73 shedding was happened to overexpressed wt-CD73 but not Lys547-Phe548 sites mutant CD73.ConclusionMMP-9 is responsible for shedding CD73 from RPE through hydrolyzing its Lys547 -Phe548 sites.
Objective To observe the effect of ginsenoside Rg3 on the proliferation, migration, and tube formation of human retinal capillary endothelial cell (HRCEC) cultured in normal and hypoxia condition. Methods HRCEC was cultured in normal condition and treated with 0.0 mmol/L (group A), 0.1 mmol/L (group B) and 0.5 mmol/L (group C) ginsenoside Rg3. HRCEC was also cultured in hypoxia condition and treated with 0.0 mmol/L (group D), 0.1 mmol/L (group E) and 0.5 mmol/L (group F) ginsenoside Rg3. The effects of ginsenoside Rg3 on HRCEC proliferation were measured by methylthiazoletrazolium assay in 24, 48 and 72 hours after culture. In 24 hours after culture, the effect of cell migration was evaluated by transwell chamber; the effect of tube formation was evaluated by Matrigel; the expression of vascular endothelial growth factor (VEGF) protein and mRNA were detected by Western blot and real-time quantitative reverse transcription-polymerase chain reaction. Results Ginsenoside Rg3 could inhibit proliferation of HRCEC, depending on the concentration (F=30.331 and 33.402 in normal and hypoxia condition, respectively; P＜0.05) and time (F=85.462 and 136.045 in normal and hypoxia condition, respectively; P＜0.05). The number of cell migration was 103.33plusmn;3.54, 92..25plusmn;3.68, 78.64plusmn;4.66 in group A, B and C, the difference among three groups was statistically significant (F=28.801, P＜0.05). The number of cell migration was 125.76plusmn;3.11, 90.27plusmn;3.55, 77.81plusmn;5.01 in group D, E and F, the difference among three groups was statistically significant (F=117.594, P＜0.05). The number of tube formed in Matrigel was 24.3plusmn;2.2, 15.7plusmn;1.7, 10.1plusmn;2.3 in group A, B and C, the difference among three groups was statistically significant (F=35.364, P＜0.05). The number of tube formed in Matrigel was 26.2plusmn;1.9, 15.1plusmn;2.6, 8.6plusmn;1.9 in group D, E and F, the difference among three groups was statistically significant (F=50.989, P＜0.05). The expression of VEGF mRNA was 1.00plusmn;0.06, 0.79plusmn;0.06, 0.68plusmn;0.02 in group A, B and C, the difference among three groups was statistically significant (F=31.303, P＜0.05). The expression of VEGF mRNA was 3.88plusmn;0.12, 2.83plusmn;0.09, 1.15plusmn;0.05 in group D, E and F, the difference among three groups was statistically significant (F=682.668, P＜0.05). The expression of VEGF protein was 0.62plusmn;0.03, 0.41plusmn;0.02, 0.32plusmn;0.02 in group A, B and C, the difference among three groups was statistically significant (F=125.471, P＜0.05). The expression of VEGF protein was 0.91plusmn;0.03, 0.82plusmn;0.03, 0.71plusmn;0.02 in group D, E and F, the difference among three groups was statistically significant (F=41.045, P＜0.05). Conclusion Ginsenoside Rg3 can inhibit the proliferation, migration, and tube formation of HRCEC through the inhibition of VEGF expression.
Objective To observe the inhibitory effect of kallikrein-binding protein (KBP) on choroidal neovascularization. Methods Forty Brown Norway rats were randomly divided into the KBP groups and the control group, 20 rats in each group, the right eye as the experimental eye. The rats were photocoagulated by 532 nm laser to induce CNV model. One week after laser photocoagulation, the rats were received FFA examination. At the second day after FFA examination, the rats of KBP group were received an intravitreal injection of KBP 5 mu;l (4 mg/ml KBP). The same volume of deionized water was injected into the rats in the control group. The rats of two groups received FFA examination at one, two and three weeks after injection. The expressions of vascular endothelial growth factor and pigment epithelium derived factor were observed using hematoxylin and eosin stain and immunohistochemistry stain. CNV leakage area and the cumulative absorbance of laser spot area were analyzed by Image-Pro plus 6.0 software. Results FFA examination showed that there were CNV and fluorescence leakage at one week after laser photocoagulation; one, two and three weeks after injection, the leakage decreased gradually in KBP group, but increased with time in control group. Compared with control group, the spot area and CNV in KBP group reduced gradually, but CNV was always there in control group. The differences of VEGF (F=1.29) and PEDF (F=6.29) expressions at one week after laser photocoagulation were not statistically significant (P＞0.05). The differences of VEGF and PEDF expressions at one, two and three weeks after injection were statistically significant（VEGF:F=14.16,66.89,24.34; PEDF：F=4.22,62.04,233.05;P＜0.001）.Conclusion Intravitreal injection with KBP can inhibit CNV.
