川芎嗪对N-甲基-N-亚硝基脲致光感受器细胞损伤的保护作用及其机制川芎嗪对N-甲基-N-亚硝基脲致光感受器细胞损伤的保护作用及其机制

目的探讨川芎嗪对N-甲基-N-亚硝基脲(N-methyl-N-nitrosourea，MNU)致大鼠视网膜损伤的作用。方法 大鼠生后47 d，腹腔注射不同剂量的川芎嗪注射液；50 d注射MNU 60 mg·kg^-1。测量视网膜总厚度；TUNEL和RT-PCR法分别检测细胞凋亡、c-jun和c-fos的表达。结果川芎嗪注射液可剂量依赖性地增加周边视网膜总厚度和降低凋亡指数，并抑制MNU诱导的即早基因表达。结论川芎嗪通过下调基因c-jun和c-fos的表达，抑制光感受器细胞凋亡，从而部分保护MNU引起的视网膜损伤。     

非促分裂haFGF对MNU所致大鼠视网膜损伤的保护作用

目的研究非促分裂haFGF(nm-haFGF)对N-甲基-N-亚硝脲(MNU)所致大鼠视网膜损伤的保护作用及其作用机制。方法通过ip MNU (60 mg·kg^-1)复制大鼠视网膜损伤模型，0和12 h后，玻璃体腔内注射不同剂量nm-haFGF。24 h和7 d后，测量周边视网膜总厚度和外视网膜厚度、光感受器细胞凋亡指数及视网膜Bcl-2和Bax蛋白表达水平。结果MNU作用24 h后，nm-haFGF组周边视网膜的凋亡指数显著降低(P<0.01)；7 d后，nm-haFGF组周边视网膜光感受器细胞数明显增多，且周边视网膜总厚度和外视网膜厚度增加(P<0.01)；不同剂量nm-haFGF可调节Bcl-2和Bax蛋白表达水平。结论nm-haFGF 能部分抑制MNU引起的SD大鼠视网膜损伤，其作用机制可能与上调Bcl-2和下调Bax蛋白表达水平有关。     

灵芝孢子油对N-甲基-N-亚硝酸脲诱导的大鼠视网膜损伤中Caspase-3表达的影响

目的观察灵芝孢子油(ganoderma spore oil,GSO)对N-甲基-N亚硝酸脲(N-methyl-N-nitrosourea,MNU)诱导的视网膜外核层细胞损伤的作用及对Caspase-3时空表达的影响。方法50d♀SD大鼠随机分为正常对照(NC)组、MNU造模(MNU)组、二十二碳六烯酸处理(DHA)组和灵芝孢子油处理(GSO)组。各组分别于MNU造模前0h及造模后1d、3d、7d、10d处死大鼠取材,采用RT-PCR、免疫荧光技术检测视网膜中Caspase-3表达和分布的变化,TUNEL法检测视网膜细胞凋亡情况。结果①RT-PCR检测:与NC组比较,MNU组1d、3d、7d视网膜Caspase-3表达增强(P<0.01);GSO组和DHA组1d、3d低于MNU组(P<0.01),1dGSO组表达较DHA组低(P<0.01)。②免疫荧光检测:在3d,GSO组和DHA组Caspase-3表达水平低于MNU组,GSO组较DHA组稍弱。③TUNEL法检测:NC组在视网膜各层均未发现凋亡细胞,MNU造模后可见外核层细胞凋亡。GSO组和DHA组在1d、3d外核层细胞凋亡百分率少于对应的MNU组(P<0.01),3d GSO组低于DHA组(P<0.01)。结论灵芝孢子油可能通过下调视网膜Caspase-3的表达,阻抑MNU诱导的视网膜外核层光感受器细胞凋亡的作用。     

黄斑明目颗粒对N-甲基-N-亚硝脲诱导大鼠视网膜光感受器细胞损伤的防护作用

目的 探讨黄斑明目颗粒对N-甲基-N-亚硝脲(MNU)诱导的大鼠视网膜光感受器细胞变性动物模型的防护作用,为防治视网膜变性提供一种有价值的现代化中药.方法 将鼠龄为43 d的雌性SD大鼠33只随机分为3组.黄斑明目颗粒组(n=10)以黄斑明目颗粒溶解后灌胃,正常对照组(n=10)和模型对照组(n=10)以等量蒸馏水灌胃,bid,连续7 d.给药7 d后,黄斑明目颗粒组和模型对照组大鼠予MNU 40 mg/kg,ip;正常对照组注射等量生理盐水.观察MNU处理后12、24、48、72 h各组大鼠视网膜电图a波及b波的变化,并于MNU作用后7 d处死大鼠,取眼球测定视网膜全层及外核层厚度.结果 正常对照组大鼠不同时间点视网膜电图(ERG)a波及b波的振幅差异无统计学意义(P＞0.05);模型对照组在MNU处理后ERG a波及b波的振幅均呈持续性、进行性下降,黄斑明目颗粒可明显抑制ERG a波及b波振幅的降低(P＜0.05).眼病理结果,黄斑明目颗粒组与模型对照组比较,MNU诱导的大鼠视网膜光感受器细胞损伤明显减轻(P＜0.05).结论 黄斑明目颗粒可减轻MNU对大鼠视网膜光感受器细胞的损伤程度,促进MNU诱导的大鼠视网膜光感受器细胞损伤的功能恢复.     

三七皂苷Rg1、Rb1对大鼠局灶性脑缺血后c-fos、c-jun表达的抑制作用分析

目的探讨三七皂苷Rg1、Rb1对大鼠局灶性脑缺血后c-fos、c-jun的表达与细胞凋亡的关系。方法将雄性Wistar大鼠60只随机分为单纯缺血30min再灌流0、0.5、1、3、6、12、24、48天8个时点组和正常对照组、假手术组,每组6只大鼠。用线栓法造成大鼠局灶性脑梗死模型。用Tunel法检测脑细胞凋亡,用免疫组化法测定转录因子c-fos、c-jun在肝内的表达。结果 Tunel法检测出细胞凋亡在缺血再灌后24～48h达高峰,c-fos在3h达高峰,c-jun持续增加,24h达高峰。三七皂苷Rg1、Rb1显著抑制c-fos、c-jun的表达。结论 c-fos、c-jun的表达与细胞凋亡有关,提示三七皂苷Rg1、Rb1可通过抑制c-fos、c-jun的表达降低脑细胞凋亡。     

外伤性PVR状态大鼠视网膜前膜c-fos和c-jun的表达

目的观察与探讨实验性外伤致大鼠增殖性玻璃体视网膜病变时,大鼠视网膜前膜中c-fos和c-jun的表达及其相关性。方法采用S-P法在不同时间点对外伤性PVR大鼠视网膜前膜做c-fos和c-jun免疫组织化学染色,染色结果行图象分析。结果实验中c-fos和c-jun表达阳性细胞率和平均光密度值先升高后稍降低,14d组和21d组阳性率最高,与7d组比较有显著性差异,c-fos和c-jun表达之间有显著相关性。结论 c-fos和c-jun参与PVR视网膜前膜细胞增殖的调控,两者的变化呈高度相关性,是促进PVR细胞增埴过程中一个重要的早期分子事件,为用基因手段防治PVR以及治疗时间的选择提供实验依据。     

黄芪对脑缺血再灌注损伤c-fos/c-jun表达和细胞凋亡的影响

目的:探讨黄芪注射液对大鼠局灶性脑缺血再灌注损伤后的保护作用。方法:采用线栓法阻断大鼠一侧大脑中动脉(MCA)血流2h,再灌注24h制成局灶性脑缺血再灌注损伤模型。将30只Wistar雄性大鼠随机分成假手术组、缺血再灌注组、黄芪组3组。造模前1h时黄芪组给与黄芪200mg/kg腹腔注射。假手术组、缺血再灌注组给予等剂量生理盐水。再灌注24h后断头取脑、切片,进行HE染色、c-fos和c-jun免疫组化染色和细胞凋亡检测。结果:缺血2h再灌注24h后,黄芪组和缺血再灌注组大鼠缺血侧皮层可检测到凋亡细胞,黄芪组凋亡细胞数明显少于缺血再灌注组,假手术组未见凋亡细胞;黄芪组和缺血再灌注组大鼠缺血侧皮层c-fos/c-jun阳性细胞数均高于假手术组,与缺血再灌注组相比,黄芪组大鼠缺血侧皮层c-fos/c-jun表达降低。结论:黄芪可抑制缺血再灌注损伤后缺血侧皮质的细胞凋亡。     

Bcl-2、Bax在百草枯诱导大鼠视网膜损伤中的表达

目的观察百草枯对SD大鼠视网膜损伤的作用及对Bcl-2、Bax表达的影响,并探讨Bcl-2、Bax在百草枯诱导的视网膜细胞凋亡中的作用及意义。方法将大鼠随机分成对照组和模型组,对照组玻璃体腔注射0.1 mol.L-1 PBS,模型组玻璃体腔注射一定浓度的百草枯,分别在12 h、1 d、2 d、5 d处死动物,HE染色观察视网膜的损伤程度,TUNEL法检测视网膜细胞的凋亡情况,同时采用Western blot检测Bcl-2、Bax蛋白的表达。结果①HE染色观察随着时间的增加,外核层逐渐变得稀疏并观察到核固缩,内核层可观察到核固缩,神经节细胞出现空泡样,5 d时内丛状层消失,外核层内核层紊乱。②TUNEL法检测凋亡结果:对照组没有发现凋亡阳性细胞,PQ可以诱导视网膜细胞凋亡,2 d凋亡指数达到高峰③Western blot结果:百草枯诱导的大鼠视网膜损伤可上调Bcl-2的表达,在1 d时达到高峰,与对照组相比差异有显著性(P<0.01),2 d开始下降;同时上调Bax的表达,在2 d时达到高峰,5 d时下降,各组之间差异均具有显著性(P<0.01);下调Bcl-2/Bax的比值,在2 d时达到最低,与对照组相比差异有显著性(P<0.01),到5 d时升高,与对照组比仍较低(P<0.01)。结论百草枯诱导了视网膜细胞的凋亡,可能是通过调节Bcl-2和Bax的表达来诱导视网膜细胞的凋亡,Bcl-2/Bax也与凋亡的发生密切相关。     

葛根素对遗传性视网膜色素变性rd小鼠的干预作用及其抗凋亡机制研究

目的观察葛根素对视网膜色素变性rd小鼠外核层细胞和视网膜光感受器外段的病理、超微结构、凋亡和Bcl-2表达的影响,探讨葛根素对rd小鼠的干预作用和干预机制。方法rd新生小鼠随机分为2组,实验组和对照组。实验组小鼠从出生时腹腔注射葛根素80mg.kg-1,每天2次,至生后35d,对照组同时注射等量生理盐水。分别在用药后当日和3、7、14、21、28、35d取眼球,固定后,常规病理和电镜观察。用TUNEL方法检测视网膜光感受器细胞的凋亡,用免疫组化方法测定Bcl-2在视网膜的表达。结果病理结果显示,经葛根素治疗后14、21、28、35d,rd小鼠光感受器细胞层数与未用药的对照组相比较明显增加,P<0.01。电镜结果显示,葛根素可减缓rd小鼠光感受器细胞及其外段盘膜部位线粒体的病变,减少盘膜和外界膜的破坏。rd小鼠经葛根素药物治疗后d7、14、21、28、35,光感受器细胞的凋亡率比未用药的对照组明显减少,P<0.01;d3、7、14、21、28、35,Bcl-2在视网膜外核层及光感受器细胞外段的表达明显增强,P<0.05或P<0.01。结论葛根素可延缓rd小鼠视网膜外核层细胞和视网膜光感受器外段损伤。     

SD大鼠视网膜光损伤模型的建立

目的 研究中等强度的光照对大鼠视网膜的影响.方法 24只8～10周的雌性SD大鼠随机分6组.一组做对照,另5组分别置于12h亮、12h暗的1 500Lux白色氙灯循环光照下3、7、14、21、28天,对视网膜行光镜组织测厚检查.结果 视网膜光感受器细胞最早受累,外节段变短,外核层变薄.结论 1 500Lux光照大鼠视网膜可出现光感受器细胞层的退行性变性.     

Role of oxidative stress in retinal photoreceptor cell death in N-methyl-N-nitrosourea-treated mice

This study aimed to investigate whether oxidative stress contributes to retinal cell death in a mouse model of photoreceptor degeneration induced by N-methyl-N-nitrosourea (MNU). We measured in vitro MNU-induced radical production in retinal cell cultures of murine 661W photoreceptor-derived cells; RGC-5, a mouse ganglion cell line; and primary retinal cells. The addition of MNU induced oxidative radical generation in 661W and primary retinal cells, but not in RGC-5 cells. Edaravone, a free radical scavenger, at 1 μM reduced MNU-induced radical production in 661W and primary retinal cells. To induce in vivo retinal photoreceptor degeneration in mice, we administered 60 mg/kg MNU by intraperitoneal injection. We intravenously administered 1 mg/kg edaravone immediately and at 6 h after the MNU injection. Retinal photoreceptor degeneration was evaluated by measuring the thickness of the outer nuclear layer (ONL) by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining and by oxidative stress markers. MNU caused photoreceptor cell loss at 7 days after administration. Edaravone inhibited ONL thinning and reduced TUNEL-positive cells and the oxidative stress markers. These findings indicate that MNU leads to selective photoreceptor degradation via oxidative stress in vitro and in vivo and may help to understand the pathogenic mechanism of retinitis pigmentosa.     

Tetramethylpyrazine protected photoreceptor cells of rats by modulating nuclear translocation of NF-κB

Aim: To evaluate the effect of tetramethylpyrazine (TMP) injection on retinal damage induced by N-methyl-N-nitrosourea (MNU) in rats and on nuclear factorkappa B (NF-κB) family members. Methods: Female Sprague-Dawley (SD) rats were randomly divided into groups: (i), control group; (ii), model group; and (iii), TMP-injection groups, in which the rats were subdivided into 40 mg/kg, 80 mg/kg and 160 mg/kg groups. Drugs were injected ip into 47-day-old SD rats once a day. At 50 days of age, all rats in the model group and drug groups also received a single ip injection of 60 mg/kg MNU. Rats in group 1 received ip injection of physiological saline. All rats were killed at different times after MNU or physiological saline treatment. The apoptotic index of photoreceptor ceils was calculated by TUNEL labeling; retinal damage was evaluated based on retinal thickness and the expression of NF-nB family members was detected by Western blot. Results: TMP injections, in a dose-dependent manner, suppressed photoreceptor cell apoptosis and decreased its loss in the peripheral retina. As compared with the MNU-treated group, TMP injection at a dose of 160 mg/kg also timedependently upregulated the NF-κB/p65 protein level in the nucleus and downregulated the IκBα protein level in the cytoplasm. However, no protective effect of TMP injection on MNU-induced central retinal damage was found. Conclusion: TMP injection partially protects against MNU-induced retinal damage by upregulating the nuclear translocation of p65 to inhibit photoreceptor cells apoptosis.     

Puerarin reduces increased c-fos, c-jun, and type Ⅳ collagen expression caused by high glucose in glomerular mesangial cells

AIM: Increased expression of c-fos, c-jun and type IV collagen (CoIV) in glomerular mesangial cells (GMC) are important characteristics of diabetic nephropathy. Both c-fos and c-jun regulate the gene expression of extracellular matrix components, and CoIV is the main component of the extracellular matrix. It has been reported that puerarin inhibits aggregation of the extracellular matrix in diabetic rats by an as yet unknown mechanism. The aim of this study is to investigate the effect of puerarin on c-fos, c-jun and CoIV expression in GMC cultured in medium containing 5.6 or 27.8 mmol/L glucose. METHODS: The expressions of c-fos and c-jun were measured at the protein level using flow cytometry. CoIV content was detected using radioimmunoassay. Protein kinase C (PKC) activity was measured using liquid scintillation counting. RESULTS: Puerarin (10(-5) mmol/L) significantly ameliorated the high-glucose effect on c-fos, c-jun and CoIV expression. This effect is accompanied by a reduced PKC activity in these cells. CONCLUSION: Our results suggest that reduced PKC activity and expression of c-fos and c-jun in GMC might participate in the mechanisms underlying the therapeutic effect of puerarin on diabetic nephropathy.     

四环素-哌嗪雌酚酮上调骺板c-fos, c-jun 的mRNA及其表达产物水平

目的：研究四环素-哌嗪雌酚酮(XW630)对软骨细胞c-fos和c-jun基因的mRNA及其表达产物水平的影响,初步探讨其对骨的作用机制。方法：用原位杂交法,测定体外培养小鼠胚胎长骨骺板c-fos和c-jun mRNA的水平,同时用免疫组织化学法,测定了相应的表达产物。结果：10^-7 mol.L^-1 XW630可显著上调骺板静止区、增殖区和肥大区c-fos和c-jun的mRNA及其表达产物水平,且其作用比雌酚酮强。结论：XW630可能通过上调软骨细胞c-fos, c-jun的表达,促进软骨细胞中含有AP-1位点的、与骨生长发育有关的基因表达,从而促进软骨内骨形成。     

Selective down-regulation of c-jun gene expression by pentoxifylline and c-jun antisense interrupts platelet-derived growth factor signaling: pentoxifylline inhibits phosphorylation of c-Jun on serine 73

Platelet-derived growth factor (PDGF) signals through several pathways, including mitogen-activated protein (MAP) kinase, Jun kinase, and C kinase, and stimulates proliferation of fibroblasts. Pentoxifylline inhibits PDGF-driven proliferation of fibroblasts. We have reported that pentoxifylline did not inhibit binding of PDGF to its specific cell-surface receptors or PDGF receptor phosphorylation. In this study, we investigated the effect of PDGF on the expression of c-fos and c-jun, because c-fos and c-jun form activator protein-1 complexes that stimulate genes involved in proliferation. We determined whether pentoxifylline would alter the expression of c-fos and c-jun. Our results indicate that PDGF induced the expression of both c-fos and c-jun. Pentoxifylline effectively reduced c-jun gene expression, which had been up-regulated by PDGF, but did not alter c-fos gene expression. The lack of effect on c-fos supports other studies from this laboratory, which indicate that pentoxifylline did not inhibit PDGF activation of MAP kinase. Treatment of fibroblasts with a phosphothioate c-jun antisense oligodeoxynucleotide reduced the levels of c-Jun protein and blocked PDGF-stimulated proliferation, suggesting a critical role for c-jun in PDGF-mediated proliferation. Combination of pentoxifylline and c-jun antisense suggested that they were likely inhibiting PDGF-stimulated proliferation at a single site in the PDGF signaling pathway. These results suggest that pentoxifylline inhibits PDGF-stimulated proliferation by selectively decreasing c-jun expression. To further define the mechanism of action of pentoxifylline, we assessed the effect of pentoxifylline on c-Jun and phosphorylated c-Jun immunoreactivity in cells treated with PDGF and cells that were transfected with wild-type c-jun plasmid using immunocytochemistry and Western blot analyses, and our results indicate that pentoxifylline inhibited phosphorylation of c-Jun on serine 73.