ABSTRACT
Background Oxidative stress is a main cause of age-related macular degeneration (AMD).Lutein has a preventive role for AMD, but its antioxidant mechanism remains unclear.Objective Present study was to investigate the effect of lutein on oxidative stress of Müller cells and its signaling pathway.Methods Human Müller cells (human Müller cell strain) were cultured, and the cells at logarithmimic growth phase were incubated in 96 well plate overnightly.Oxidative stress cell models were established by adding 160 μmol/L H2O2, a median lethal dose for Müller cells.The models were divided into the model control group and 12.5,25.0,50.0 mg/L lutein groups,and the different concentrations of lutein were used to culture the cells for 24 hours, respectively.The routine cultured cells served as the blank control group.Growth of the cells was assayed by MTT method (absorbancy);the reactive oxygen species (ROS) content in the cells was assayed by flow cytometry;the mRNA and protein levels of nuclear factor-E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in the cells were detected by quantitative real-time PCR and Western blot, respectively.Results The inhibitory effects on the cells were gradually enhanced with the increase of H2O2 concentrations,showing a significant difference among the groups (F =43.890,P＜0.01).A significant difference was found in apoptotic rate of the cells among the blank control group,model control group and 12.5,25.0,50.0 mg/L lutein groups (F =346.770, P =0.000) , and the apoptosis rate was significant elevated with the increase of lutein dose (all at P＜0.05).The ROS contents in the cells were 1.92±0.18,64.89±2.86,52.70±2.80,32.61 ±4.20 and 5.68 ± 1.35 in the blank control group, model control group and 12.5,25.0,50.0 mg/L group, respectively, with significant difference among the groups (F =324.900, P =0.000), and the ROS content was gradually reduced as the increase of lutein dose (all at P＜0.05).The relative mRNA and protein expressions of Nrf2 and HO-1 were remarkedly higher in the 12.5,25.0,50.0 mg/L lutein groups than those in the model control group (F =236.960,242.620,186.830,263.120, all at P =0.000) , and no significant difference was seen in the relative expression level of nuclear Nrf2 protein among the groups (F =1.790, P =0.210).Conclusions Lutein can induce the expression of antioxidant enzymes by inducing the expression of nuclear translocation of Nrf2 and consequently inhibit the oxidative stress status.
ABSTRACT
Background Oxidative stress is a pathophysiological process of retina,so it is very important to explore a protective way against retinal oxidative stress.Studies determined that extract of ginkgo biloba (EGb) has antioxidant,anti-apoptosis,anti-thrombosis and anti-inflammatory effects,however,the effect of EGb on human Müller cells in oxidative stress is still below understood.Objective This study was to investigate the protection of EGb against oxidative stress of human retinal Müller cells induced by As2O3 in vitro.Methods Human retinal Müllercell line was cultured in DMEM/F12 with 10％ fetal bovine serum (FBS),2 μmol/L glutamine and 1％ antibiotics.As2O3 solution at the final concentration 5 mg/L was added in the medium for 24 hours to establish oxidative models,and then the EGb with the final concentrations of 5,10 and 20 mg/L was used to cell models for 24 hours,respectively.Cell viability was detected by MTT assay,and reactive oxygen species (ROS) levels in the cells were detected with CM-H2DCFDA fluorescent probe.The relative expression levels of caspase-3 mRNA in cytoplasm and cell nuclei were assayed by reverse-transcription PCR (RT-PCR),and the expressions of Nrf2 protein were quantitatively detected by Western blot.Results Müller cells adhered well 24 hours after cultured.At 6-7 days after culture,Müller cell body was large with abundant cytoplasm and mosaic-like arrangement.However,floating cells were seen after As2 O3 treatment.Cell viability (absorbance) was significantly different among the normal culture group,As2 O3-treated group,As2 O3 + 5 mg/L EGb group,As2 O3 + 10 mg/L EGb group and As2 O3 + 20 mg/L EGb group,with the strongest viability in the normal culture group and the weakest viability in the As2 O3-treated groups (F=163.57,P =0.00).The fluorescence intensity of ROS was the weakest in the normal culture group and the strongest in the As2 O3-treated group and was gradually weakened with the increase of EGb doses,showing a remarkable difference among the groups (F =4 013.61,P =0.00).The relative expression level of caspase-3 mRNA in the cells was gradually reduced with the increase of EGb doses,with a statistically significant difference among the groups (F =2 199.72,P =0.00).In addition,no considerable difference was seen in the expression level of Nrf2 protein (grey scale) in cytoplasm among the groups (F=15.42,P=0.40);while in the nuclei,the expression levels of Nrf2 protein were 100.01 ±0.04,46.59±0.63,54.51 ±0.62,59.93 ±0.17 and 67.60±0.24 in the normal culture group,As2 O3-treated group and As2O3+5 mg/L EGb group,As2O3+10 mg/L EGb group,As2O3+20 mg/L EGb group respectively,with a significant difference among them (F=7 271.72,P=0.00).Conclusions EGb can protect human retinal Müller cells against As2O3-induced damage in a dose-dependant manner by antioxidant and antiapoptotic effects in vitro,and the activities occur primarily in cell nucleus.
ABSTRACT
Objective To detect the expressions of BMPR Ⅰa of BMPs/BMPR/Smads signaling transduction pathway in human brain glioma ,to research the role of its in tumorigenesis and progression of brain glioma and its correlation to clinical pathology . Methods The mRNA and protein expressions of BMPR Ⅰa was detected in 20 normal brain tissues and 40 samples with human glioma by RT-PCR and SABC immunohistochemical methods ,repectively ,and analyzed the correlation with the patient′s age ,gender and pathological grade .Results Compared with normal brain tissues ,BMPR Ⅰa mRNA and protein expressions in human glioma were reduced significantly ,especially in Ⅲ and Ⅳ stage tumor tissues .The difference was not related with the patient′s age and gender .Conclusion In progression of glioma ,BMPR Ⅰa may play a role in restraining ,and has nothing to do with age and sex .
ABSTRACT
The study aimed to search for a new, rapid and effective treatment of ocular alkali burns,and a new free radical scavenging agent. Experimental corneal alkali burns in rabbits were treated with highly purified ?_2-maeroglobulin drops extracted from rabbit plasma. On the 1st, 2nd, 3rd, 4th, and 7th day after the burn, examinations of the outer eye were done, On the 2nd, 7th, and 14th day, samples of aqueous humor were drawn to test for theirtotal SOD content. On the 7th and the 14th day the total aqueous protein was also determined. The results showed that on the 1st, 2nd, and 3rd day after the burn, mixed congestion in the experimental group animals was markedly milder than that in the the control group, and on the 2nd and 3rd day, opacity and swelling of the cornea were also less severe in the experimental group. On the 2nd, 7th, and 14th day the tot al aqueous SOD contents of the experimental group were all higher than those of the eontrol samples, but the total aqueous protein which was determined twice showed no statistical significance in the two groups of animals, It was concluded that ?_2-macroglohulin had a marked inhibitory effect on the acute inflammatory reaction of the rabbit corne of alkali burns, and that it also had the power of doing away with superoxide free radieals in the damaged tissue and their metabolites.