2.Protective effect of epidermal growth factor on proliferation and migration of the human Müller cell from oxidative damage
Chun-li, CHEN ; Zhong-lou, ZHOU ; Dong-sheng, YAN ; Jing-wei, ZHENG ; Zong-ming, SONG
Chinese Journal of Experimental Ophthalmology 2012;30(8):715-720
Background Oxidative damage plays an important role in pathogenesis of age-related macular degeneration( AMD ),and its mechanism is the destroy of blood-retinal barrier.Müller cells is a primary component to stabilize the inner barrier of the blood-retina.Researches showed that epidermal growth factor(EGF) can promote the proliferation and migration of animal Müller cells,but less study was found in the effect of EGF on human Müller cells. Objective The present study was to investigate the effects of EGF on the proliferation and migration of human Müller cells and its molecular mechanism. Methods Human Müller cell line MIO-M1 cells were cultured and incubated,and cultured cells were identified using glial fibrillory acidic protein (GFAP),factor Ⅷ,α-smooth muscle actin( α-SMA ),keratin and S-100.Different concentrations of EGF( 0,1,10,30,100 mg/L)was added in freeserum DMEM,and the positive rate of the cells was calculated using 5-bromo-2-deoxyuridine(BrdU) method.The cells were divided into EGF group,H2 O2 group,EGF + H2 O2 group,glucose oxidase ( GO ) group,GO + EGF group,EGF + LY294002+H2O2 group according to the different intervention,and the effects of LY294002 on the proliferation of Müller cells (A590 )were detected by colorimetric assay for cellular growth and survival( MTT assay).The scratch test of Müller cells was used to assess the influence of EGF(0,1,10,30,100 mg/L)on H2 O2-induced damage of human Müller cell.Western blot was used to detect the cell proliferation under the protection of EGF on co-cultured cells using LY294002 and H2O2 and the activation of Akt signal pathways. Results The proliferative rates of the cells were 28.0%,32.9%,39.0% in 10,30,100 mg/L EGF groups respectively and obviously higher than those in 0,1 mg/L EGF groups (24.5 %,26.2 % ).Under the H2O2 culture,GO culture,respectively,the A570 value of the Müller cell in high concentrations of EGF groups was significantly increased in comparison with lower concentrations EGF groups with the statistical significance among the groups( F=23.582,P=0.000).Compared with EGF+H2O2 group,the A570value of the Müller cells was lowed in EGF+LY294002+H2O2 group.The maximum migration rate of Müller cells was found in 10 mg/L EGF group.Western blot revealed that the presence of H2O2 reinforced the expression of Akt in Müller cells,however,pretreatment with 100 mg/L EGF antagonized the harmful effect of H2O2 on Müller cells.Meanwhile,pretreatment with EGF and LY294002 reduced the expression of Akt in Müller cells. Conclusions EGF can induce the proliferation and migration of human Müller cells with the strongest effect in 10 mg/L.100 mg/L exogenous EGF has a stronger protection to the Müiller cells against H2O2-induced cell damage by activating the PI3KAkt cell survival pathway.
3.Effect of 935-MHz phone-simulating electromagnetic radiation on endometrial glandular cells during mouse embryo implantation.
Wenhui, LIU ; Xinmin, ZHENG ; Zaiqing, QU ; Ming, ZHANG ; Chun, ZHOU ; Ling, MA ; Yuanzhen, ZHANG
Journal of Huazhong University of Science and Technology (Medical Sciences) 2012;32(5):755-9
This study examined the impact of 935MHz phone-simulating electromagnetic radiation on embryo implantation of pregnant mice. Each 7-week-old Kunming (KM) female white mouse was set up with a KM male mouse in a single cage for mating overnight after induction of ovulation. In the first three days of pregnancy, the pregnant mice was exposed to electromagnetic radiation at low-intensity (150 μW/cm(2), ranging from 130 to 200 μW/cm(2), for 2- or 4-h exposure every day), mid-intensity (570 μW/cm(2), ranging from 400 to 700 μW/cm(2), for 2- or 4-h exposure every day) or high-intensity (1400 μW/cm(2), ranging from 1200 to 1500 μW/cm(2), for 2- or 4-h exposure every day), respectively. On the day 4 after gestation (known as the window of murine embryo implantation), the endometrium was collected and the suspension of endometrial glandular cells was made. Laser scanning microscopy was employed to detect the mitochondrial membrane potential and intracellular calcium ion concentration. In high-intensity, 2- and 4-h groups, mitochondrial membrane potential of endometrial glandular cells was significantly lower than that in the normal control group (P<0.05). The calcium ion concentration was increased in low-intensity 2-h group but decreased in high-intensity 4-h group as compared with the normal control group (P<0.05). However, no significant difference was found in mitochondrial membrane potential of endometrial glandular cells between low- or mid-intensity groups and the normal control group, indicating stronger intensity of the electromagnetic radiation and longer length of the radiation are required to inflict a remarkable functional and structural damage to mitochondrial membrane. Our data demonstrated that electromagnetic radiation with a 935-MHz phone for 4 h conspicuously decreased mitochondrial membrane potential and lowered the calcium ion concentration of endometrial glandular cells. It is suggested that high-intensity electromagnetic radiation is very likely to induce the death of embryonic cells and decrease the chance of their implantation, thereby posing a high risk to pregnancy.
4.Influence of Pranlukast on Expression of Eotaxin-3 in Bronchial Epithelia Cells
wei-lin, OU ; huan, WEI ; zhi-ying, FENG ; chun-jiang, ZHU ; ming-ci, ZHENG
Journal of Applied Clinical Pediatrics 2006;0(16):-
Objective To investigate whether leukotriene D4 (LTD4) regulates eotaxin-3 (Eot-3) expression in bronchial epithelial cells, and study effect of pranlukst on the regulation.Methods BEAS-2B cells and normal human bronchial epithelia cells were pre- treated with LTD4 for 1 hour,stimulated with interleukin-4, the cells were incubated for 24 hours. Eot-3 protein in supernatant were measured by enzyme linked immunosorbent assay(ELISA). The cells were pretreated with pranlukast in different concentration, then the above procedure was repeated. Results The untreated bronchial epithelial cell expressed Eot-3 protein on a very low level. After stimulating with IL-4 and incubating for 24 hours, Eot-3 production increased significantly. Pretreating the cells with LTD4 enhanced the inducing effect of IL-4. Pranlukast inverted the upregulation of LTD4. Conclusions Upregulating the expression of Eot-3 induced by IL-4 on bronchial epithelial cells may explain partially the mechanism of leukotrienes involving airway allergic inflammation of asthma. The invertion impact on upregulation of LTD4 by pranlukast may be one of mechanisms that leukotrienes receptor antagonist cure asthma.
6.Study on quality standard for Humulus scandens.
Fan XIE ; Ying-Chun WU ; Yi-Ming LI ; Hong XU ; Rui WANG ; Zheng-Tao WANG
China Journal of Chinese Materia Medica 2014;39(20):3986-3990
To control the quality of Humulus scandens, the quality standard was established in this study. According to the method recorded in the Appendix of Chinese Pharmacopoeia (2010 Edition) , the water and ash inspections were carried out. The component luteoloside and cosmosiin in Humulus scandens were identified and assayed by TLC and HPLC. The results showed a strong characteristics microscopic of Humulus scandens, and trichoromethane-methanol-formic acid (10: 3: 0. 3) as the mobile phase of TLC, the spots at 365 nm with a UV lamp was clear. The 16 batches of samples were analyzed by HPLC with a gradient elution of acetonitrile and phosphate solution (0.2%) at a flow rate of 1.0 mL · min(-1) and detected at 350 nm. The content of luteoloside was 0.015%- 0.651% (average 0.148%); the content of cosmosiin was 0.003%-0.118% (average 0.036%). The linear calibration curve of luteoloside and cosmosiin was acquired in the ranges of 0.011-0.364 g · L(-1) (r = 1.000 0) and 0.003-0.096 g · L(-1) (r = 1.000 0), respectively. The average recovery was 100.5% and 98.5%, respectively. The methods are convenient and reliable, which can be ap- plied for quality assessment of Humulus scandens.
China
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Chromatography, High Pressure Liquid
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Drugs, Chinese Herbal
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analysis
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standards
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Humulus
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anatomy & histology
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chemistry
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Quality Control
7.Changes of Aspartate and Glutamate in Hippocampus Formation and Cerebrospinal Fluid in Kainite Acid Induced Epilepsy Rat
chun-feng, WU ; song-ming, HUANG ; guo, ZHENG ; xiao-peng, LU
Journal of Applied Clinical Pediatrics 2006;0(24):-
Objective To explore the changes of contents of glutamate (Glu) and aspartate (Asp) in hippocampus formation and cerebrospinal fluid (CSF) in kainite acid(KA) induced epilepsy rats.Methods SD rats(n=40) were divided into 2 groups randomly:the KA group [intracerebroventricular injection(icv) of KA, 2 ?g/kg] and control group(icv of NS). KA group were divided into 4 groups at 6 h,1,3 and 7 d,each group 8 rats.High pressure liquid chromatgraphy(HPLC) was used to assess the concentrations of Glu and Asp in hippocampus formation and CSF.Results In the hippocampus, the contents of Glu and Asp increased continuously 1 d after seizure , but not different from those of control group.Three days later, only Glu became significantly different from control group. However, the contents of Glu and Asp in the CSF were significantly different from the control 6 h after seizure.Conclusion The contents of excite amino acid (especially Glu)in CSF increase immediately after KA injection, which are earlier than those in hippocampus formation.
8.Effect of Postoperative Continuous Epidural Analgesia with Sufentanil in Different Concentrations and Combined with Bupivacaine on Pain after Thoracotomy
Chun WANG ; Yue SU ; Wan-ming GENG ; Hui ZHENG ; Wei LIU ; Guangkuo GAO ; Zhengfang HU
Chinese Journal of Rehabilitation Theory and Practice 2006;12(4):352-354
ObjectiveTo observe the effect and safety of continuous epidural analgesia with sufentanil in different concentrations combined with 0.125% bupivacaine on pain after thoracotomy.Methods30 patients with ASA grade Ⅱ~Ⅲ and underwent thoracotomy were randomly divided into 3 groups treated with 0.125% bupivacaine combined with sufentanil 0.25 μg/ml (group A), 0.50 μg/ml (group B) and 0.75 μg/ml (group C) respectively. Before operation starting, epidural puncture was performed at T7~T8 and a catheter was put in. After operation, continuous epidural analgesia was performed by connecting the catheter and a analgesic pump. Analgesia effect was evaluated by visual analogous score (VAS) at sixth, twelfth, twenty-fourth and forty-eighth hours after operation. Dosage of assistant drug and side effects such as calmness, nausea, vomiting, skin pruritus and respiratory inhibition were also recorded.ResultsVAS scores and dosage of assistant drug of group B and group C were not different, but they were all lower than that of group A (P<0.05). Scores of skin pruritus of group A and group B were lower than that of group C (P<0.05), but there was no significant difference between group A and group B. No respiratory inhibition occurred in patients of all three groups.ConclusionContinuous epidural analgesia of 0.50 μg/ml sufentanil combined with 0.125% bupivacaine is safe and effective for patients after thoracotomy.
9.Inosine attenuates necrosis, but not apoptosis, of zinc-injured PC12 cells
Shi MING ; Zheng CHUN-XIA ; You SI-WEI
Neuroscience Bulletin 2005;21(2):158-164
Objective To explore the death types of PC12 cells injured by a high concentration of zinc, and effects of inosine on the types of zinc-induced cell death. Methods MTT assay was used to assess the viability of PC12 cells treated with different concentrations of zinc chloride (50, 100,200,400 μmoL/L) or inosine (0.1,0.5, 1.0, 2.0 mmol/L) for 12 h. Hoechst 33342 / PI double staining, Annexin-V binding assay and DNA agarose gel electrophoresis were employed to investigate the death forms of PC12 cells with treatment of 200 μmol/L zinc chloride or 2.0 mmol/L inosine for 12 h. Results Zinc at 100 μ mol/L and more reduced cell viability significantly. After treatment with 200 μmoL/L zinc,56.5, 24.4 and 19.1% of total PC12 cells were necrotic, survival and apoptotic. Inosine, from the concentration of 0.5 mmol/L, markedly increased cell viability of zinc-induced PC12 cells. However, additional exposure to 2.0 mmol/L inosine, necrotic, survival and apoptotic cells were 27.9, 33.8 and 38.4% of the total PC12 cells that were injured by zinc.Conclusion The viability of PC12 cells decreases when the concentration of zinc increases, and inosine protects zinc-induced PC12 cells at a dose-dependent manner. A high concentration of zinc causes both necrosis and apoptosis, and inosine attenuates necrosis, but not apoptosis, of zinc-injured PC12 cells.