Adenoviral 15-lipoxygenase-1 gene transfer inhibits hypoxia-induced proliferation of retinal microvascular endothelial cells in vitro

AIM: To investigate whether 15-Lipoxygenase-1 (15-LOX-1) plays an important role in the regulation of angiogenesis, inhibiting hypoxia-induced proliferation of retinal microvascular endothelial cells (RMVECs) and the underlying mechanism. METHODS: Primary RMVECs were isolated from the retinas of C57/BL63 mice and identified by an evaluation for FITC-marked CD31. The hypoxia models were established with the Bio-bag and evaluated with a blood-gas analyzer. Experiments were performed using RMVECs treated with and without transfer. Ad-15-LOX-1 or Ad-vector both under hypoxia and normoxia condition at 12, 24, 48, 72 hours. The efficacy of the gene transfer was assessed by immunofluorescence staining. Cells proliferation was evaluated by the CCK-8 method. RNA and protein expressions of 15-LOX-1, VEGF-A, VEGFR-2, eNOs and PPAR-r were analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blot. RESULTS: Routine evaluation for FITC-marked CD31 showed that cells were pure. The results of blood-gas analysis showed that when the cultures were exposed to hypoxia for more than 2 hours, the Po2 was 4.5 to 5.4 Kpa. We verified RMVECs could be infected with Ad-LS-LOX-for Ad-vectorvia Fluorescence microscopy. CCK-8 analysis revealed that the proliferative capacities of RMVECs in hypoxic group were significantly higher at each time point than they were in normoxic group (P <0.05). In a hypoxic condition, the proliferative capacities of RMVECs in 15-LOX-1 group were significantly inhibited (P<0.05). Real-time RT-PCR analysis revealed that the expressions of VEGF-A, VEGF-R2 and eNOs mRNA increased in hypoxia group compared with normoxia group (P<0.01). However, the expressions of 15-LOX-1, PPAR-r mRNA decreased in hypoxia group compared with normoxia group (P<0.01). It also showed that in a hypoxic condition, the expressions of VEGF-A, VEGF-R2 and eNOs mRNA decreased significantly in 15-LOX-1 group compared with hypoxia group (P<0.01). However, 15-LOX-1 and PPAR-r mRNA increased significantly in 15-LOX-1 group compared with hypoxia group (P<0.01). There was no significant difference of the mRNA expressions between vector group and hypoxia group (P>0.05). Western blot analysis revealed that the expressions of relative proteins were also ranked in that order. CONCLUSION: Our results suggested that 15-LOX-1 and PPAR-r might act as a negative regulator of retinal angiogenesis. And the effect of 15-LOX-1 overexpression is an anti-angiogenic factor in hypoxia-induced retinal neovascularization (RNV). Overexpression 15-LOX-1 on RMVECs of hypoxia-induced RNV blocked signaling cascades by inhibiting hypoxia-induced increases in VEGF family. PPAR-r effect on VEGFR(2) could be an additional mechanism whereby 15-LOX-1 inhibited the hypoxia-induced RNV.

Autophagy activation and the mechanism of retinal microvascular endothelial cells in hypoxia

AIM： To explore the state of autophagy and related mechanisms in the murine retinal microvascular endothelial cells（RMECs） under hypoxia stimulation.METHODS： The murine RMECs were primarily cultured and randomly divided into three groups： hypoxia group（cultured in 1% O_2 environment）, hypoxia＋autophagy inhibition group [pretreated with 5 mmol/L 3-methyladenine（3-MA） for 4 h followed by incubation in 1% O_2] and control group（cultured under normoxic condition）. The state of autophagy in RMECs was examined by assaying the turnover of light chain 3 B（LC3BB） and expression of Beclin-1, Atg3 and Atg5 proteins with Western blotting, by detecting formation of autophagosomes with transmission electron microscopy（TEM） and by counting the number of GFP＋ puncta in RMECs. The protein levels of AMPK, P-AMPK, Akt, P-Akt, m-TOR and P-m TOR were also assayed by Western blotting.RESULTS： Primary murine RMECs were successfully cultured. Under hypoxic conditions, the ratio of LC3BB-Ⅱ/Ⅰ and the expression of Beclin-1, Atg3 and Atg5 proteins were increased when compared with the control group. In addition, the numbers of autophagosome and the GFP＋ puncta were also increased under hypoxia. However, pretreatment with 3-MA obviously attenuated these changes in autophagy in RMECs under hypoxia. Protein expression of P-Akt and P-AMPK was increased but P-m TOR level was decreased in cells exposed to hypoxia. CONCLUSION： In murine RMECs autophagy is activated under hypoxia possibly through activation of the AMPK/mTOR signaling pathway.

Role of apigenin in high glucose-induced retinal microvascular endothelial cell dysfunction via regulating NOX4/p38 MAPK pathway in vitro

AIM:To investigate the retinoprotective role of Apigenin(Api)against high glucose(HG)-induced human retinal microvascular endothelial cells(HRMECs),and to explore its regulatory mechanism.METHODS:HRMECs were stimulated by HG for 48h to establish the in vitro cell model.Different concentrations of Api(2.5,5,and 10μmol/L)were applied for treatment.Cell counting kit-8(CCK-8),Transwell,and tube formation assays were performed to examine the effects of Api on the viability,migration,and angiogenesis in HG-induced HRMECs.Vascular permeability was evaluated by Evans blue dye.The inflammatory cytokines and oxidative stress-related factors were measured using their commercial kits.Protein expression of nicotinamide adenine dinucleotide phosphate(NADPH)oxidase 4(NOX4)and p38 mitogen-activated protein kinase(MAPK)was measured by Western blot.RESULTS:Api prevented HG-induced HRMECs viability,migration,angiogenesis,and vascular permeability in a concentration-dependent manner.Meanwhile,Api also concentration-dependently inhibited inflammation and oxidative stress in HRMECs exposed to HG.In addition,HG caused an elevated expression of NOX4,which was retarded by Api treatment.HG stimulation facilitated the activation of p38 MAPK signaling in HRMECs,and Api could weaken this activation partly via downregulating NOX4 expression.Furthermore,overexpression of NOX4 or activation of p38 MAPK signaling greatly weakened the protective role of Api against HG-stimulated HRMECs.CONCLUSION:Api might exert a beneficial role in HGstimulated HRMECs through regulating NOX4/p38 MAPK pathway.

Down-regulation of histone deacetylase 7 reduces biological activities of retinal microvascular endothelial cells under high glucose condition and related mechanism

AIM:To investigate the expression and effect of histone deacetylase 7(HDAC7)in human retinal microvascular endothelial cells(HRMECs)under high glucose condition and related mechanism,and the expression of HDAC7 in the retinal tissue in diabetic rats.METHODS:The expression of HDAC7 in HRMECs under high glucose and the retinal tissue from normal or diabetic rats were detected with immunohistochemistry and Western blot.LV-shHDAC7 HRMECs were used to study the effect of HDAC7 on cell activities.Cell count kit-8(CCK-8),5-ethynyl2’-deoxyuridine(EdU),flow cytometry,scratch test,Transwell test and tube formation assay were used to examine the ability of cell proliferation,migration,and angiogenesis.Finally,a preliminary exploration of its mechanism was performed by Western blot.RESULTS:The expression of HDAC7 was both upregulated in retinal tissues of diabetic rats and high glucosetreated HRMECs.Down-regulation of HDAC7 expression significantly reduced the ability of proliferation,migration,and tube formation,and reversed the high glucose-induced high expression of CDK1/Cyclin B1 and vascular endothelial growth factor in high glucose-treated HRMECs.CONCLUSION:High glucose can up-regulate the expression of HDAC7 in HRMECs.Down-regulation of HDAC7 can inhibit HRMECs activities.HDAC7 is proposed to be involved in pathogenesis of diabetic retinopathy and a therapeutic target.

Trimethylamine N-oxide aggravates vascular permeability and endothelial cell dysfunction under diabetic condition:in vitro and in vivo study

AIM:To provide the direct evidence for the crucial role of trimethylamine N-oxide(TMAO)in vascular permeability and endothelial cell dysfunction under diabetic condition.METHODS:The role of TMAO on the in vitro biological effect of human retinal microvascular endothelial cells(HRMEC)under high glucose conditions was tested by a cell counting kit,wound healing,a transwell and a tube formation assay.The inflammation-related gene expression affected by TMAO was tested by real-time polymerase chain reaction(RT-PCR).The expression of the cell junction was measured by Western blotting(WB)and immunofluorescence staining.In addition,two groups of rat models,diabetic and non-diabetic,were fed with normal or 0.1%TMAO for 16wk,and their plasma levels of TMAO,vascular endothelial growth factor(VEGF),interleukin(IL)-6 and tumor necrosis factor(TNF)-αwere tested.The vascular permeability of rat retinas was measured using FITC-Dextran,and the expression of zonula occludens(ZO)-1 and claudin-5 in rat retinas was detected by WB or immunofluorescence staining.RESULTS:TMAO administration significantly increased the cell proliferation,migration,and tube formation of primary HRMEC either in normal or high-glucose conditions.RT-PCR showed elevated inflammation-related gene expression of HRMEC under TMAO stimulation,while WB or immunofluorescence staining indicated decreased cell junction ZO-1 and occludin expression after high-glucose and TMAO treatment.Diabetic rats showed higher plasma levels of TMAO as well as retinal vascular leakage,which were even higher in TMAO-feeding diabetic rats.Furthermore,TMAO administration increased the rat plasma levels of VEGF,IL-6 and TNF-αwhile decreasing the retinal expression levels of ZO-1 and claudin-5.CONCLUSION:TMAO enhances the proliferation,migration,and tube formation of HRMEC,as well as destroys their vascular integrity and tight connection.It also regulates the expression of VEGF,IL-6,and TNF-α.

枸杞多糖通过调控NLRP3/Caspase-1通路在抑制高糖诱导的HRMEC损伤中的作用

目的观察枸杞多糖(LBP)是否可以通过调控核苷酸寡聚化结构域样受体家族3/半胱天冬酶-1(NLRP3/Caspase-1)细胞焦亡途径抑制高糖诱导的人视网膜微血管内皮细胞(HRMEC)损伤。方法将体外培养的HRMEC随机分组,即正常组(给予5.5 mmol·L^(-1)葡萄糖)、高糖组(给予55.5 mmol·L^(-1)葡萄糖)、LBP低浓度组(给予55.5 mmol·L^(-1)葡萄糖+100 mg·L^(-1) LBP)、LBP中浓度组(给予55.5mmol·L^(-1)葡萄糖+500 mg·L^(-1) LBP)、LBP高浓度组(给予55.5 mmol·L^(-1)葡萄糖+1000 mg·L^(-1) LBP)、si-NC组(转染20μmol·L^(-1) si-NC后给予55.5 mmol·L^(-1)葡萄糖)和si-NLRP3组(转染20μmol·L^(-1) si-NLRP3后给予55.5 mmol·L^(-1)葡萄糖);利用CCK-8法检测各组HRMEC细胞增殖情况,流式细胞术检测各组HRMEC细胞焦亡情况,RT-PCR法检测各组HRMEC中NLRP3、Caspase-1、核因子(NF)-κB、Gasdermin-D(GSDMD)、血管内皮生长因子(VEGF)的mRNA相对表达水平,Western blot法检测各组HRMEC焦亡相关NLRP3、Caspase-1、NF-κB、GSDMD、VEGF的蛋白相对表达水平,ELISA检测各组HRMEC细胞上清液中细胞焦亡下游白细胞介素(IL)-1β和IL-18的表达水平。结果与正常组相比,高糖组HRMEC细胞增殖率降低,细胞焦亡率升高,NLRP3、Caspase-1、NF-κB、GSDMD、VEGF的mRNA和蛋白相对表达水平均升高,IL-1β和IL-18表达水平均升高(均为P<0.05);与高糖组相比,si-NLRP3组中HRMEC细胞增殖率升高,细胞焦亡率降低,NLRP3、Caspase-1、NF-κB、GSDMD、VEGF的mRNA和蛋白相对表达水平均降低,IL-1β和IL-18表达水平均降低(均为P<0.05);与高糖组相比,si-NC组的细胞增殖率,细胞焦亡率,NLRP3、Caspase-1、NF-κB、GSDMD、VEGF蛋白和mRNA以及IL-1β、IL-18的表达水平差异均无统计学意义(均为P>0.05);与高糖组相比,LBP中、高浓度组中HRMEC细胞增殖率升高,细胞焦亡率降低,NLRP3、Caspase-1、NF-κB、GSDMD、VEGF的mRNA和蛋白相对表达水平均降低,IL-1β和IL-18表达水平均降低(均为P<0.05);与高糖组相比,除LBP低浓度组HRMEC细胞增殖率、各蛋白相对表达水平差异均无统计学意义外(均为P>0.05),其余指标表现和LBP中、高浓度组一致。结论LBP对高糖诱导的HRMEC损伤具有保护作用,能够促进细胞增殖,抑制细胞焦亡,其作用机制与抑制NLRP3/Caspase-1信号通路的激活,降低相关炎症因子的表达有关。

脂联素预处理对高糖诱导下HRMECs细胞损伤的保护

目的观察脂联素(adiponectin,APN)预处理对高糖诱导下人视网膜微血管内皮细胞(HRMECs)凋亡及增殖的影响。方法将生长状况良好的HRMECs随机分为对照组(DMEM培养基,无干预)、高糖组(DMEM培养基+25 mmol/L D-葡萄糖)、高糖+APN组(细胞先用10μg/ml APN预处理2 h,然后用高糖培养基培养)。各组分别培养24 h后,Western blot检测HRMECs凋亡过程中Bax、Bcl-2以及Caspase-3的蛋白表达水平,流式检测HRMECs凋亡率,CCK-8法检测HRMECs增殖。结果与对照组比较,高糖组HRMECs的Bax、Caspase-3蛋白水平以及细胞凋亡率均明显增高,但Bcl-2的表达量及细胞增殖率均明显降低(均P<0.001)。与高糖组比较,高糖+APN组HRMECs的Bax、Caspase-3蛋白水平以及细胞凋亡率有所回落,Bcl-2的蛋白水平及细胞增殖率有所回升(均P<0.001)。结论高糖诱导下HRMECs凋亡增加,增殖减少,APN预处理可以抑制凋亡,促进增殖,提示APN对高糖下视网膜微血管内皮细胞损伤有保护作用。

Genipin relieves diabetic retinopathy by down-regulation of advanced glycation end products via the mitochondrial metabolism related signaling pathway

BACKGROUND Glycation is an important step in aging and oxidative stress,which can lead to endothelial dysfunction and cause severe damage to the eyes or kidneys of diabetics.Inhibition of the formation of advanced glycation end products(AGEs)and their cell toxicity can be a useful therapeutic strategy in the prevention of diabetic retinopathy(DR).Gardenia jasminoides Ellis(GJE)fruit is a selective inhibitor of AGEs.Genipin is an active compound of GJE fruit,which can be employed to treat diabetes.AIM To confirm the effect of genipin,a vital component of GJE fruit,in preventing human retinal microvascular endothelial cells(hRMECs)from AGEs damage in DR,to investigate the effect of genipin in the down-regulation of AGEs expression,and to explore the role of the CHGA/UCP2/glucose transporter 1(GLUT1)signal pathway in this process.METHODS In vitro,cell viability was tested to determine the effects of different doses of glucose and genipin in hRMECs.Cell Counting Kit-8(CCK-8),colony formation assay,flow cytometry,immunofluorescence,wound healing assay,transwell assay,and tube-forming assay were used to detect the effect of genipin on hRMECs cultured in high glucose conditions.In vivo,streptozotocin(STZ)induced mice were used,and genipin was administered by intraocular injection(IOI).To explore the effect and mechanism of genipin in diabetic-induced retinal dysfunction,reactive oxygen species(ROS),mitochondrial membrane potential(MMP),and 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose(2-NBDG)assays were performed to explore energy metabolism and oxidative stress damage in high glucose-induced hRMECs and STZ mouse retinas.Immunofluorescence and Western blot were used to investigate the expression of inflammatory cytokines[vascular endothelial growth factor(VEGF),SCG3,tumor necrosis factor-alpha(TNF-α),interleukin(IL)-1β,IL-18,and nucleotide-binding domain,leucine-rich-containing family,pyrin domain-containing 3(NLRP3)].The protein expression of the receptor of AGEs(RAGE)and the mitochondria-related signal molecules CHGA,GLUT1,and UCP2 in high glucose-induced hRMECs and STZ mouse retinas were measured and compared with the genipin-treated group.RESULTS The results of CCK-8 and colony formation assay showed that genipin promoted cell viability in high glucose(30 mmol/L D-Glucose)-induced hRMECs,especially at a 0.4μmol/L dose for 7 d.Flow cytometry results showed that high glucose can increase apoptosis rate by 30%,and genipin alleviated cell apoptosis in AGEs-induced hRMECs.A high glucose environment promoted ATP,ROS,MMP,and 2-NBDG levels,while genipin inhibited these phenotypic abnormalities in AGEs-induced hRMECs.Furthermore,genipin remarkably reduced the levels of the pro-inflammatory cytokines TNF-α,IL-1β,IL-18,and NLRP3 and impeded the expression of VEGF and SCG3 in AGEs-damaged hRMECs.These results showed that genipin can reverse high glucose induced damage with regard to cell proliferation and apoptosis in vitro,while reducing energy metabolism,oxidative stress,and inflammatory injury caused by high glucose.In addition,ROS levels and glucose uptake levels were higher in the retina from the untreated eye than in the genipin-treated eye of STZ mice.The expression of inflammatory cytokines and pathway protein in the untreated eye compared with the genipin-treated eye was significantly increased,as measured by Western blot.These results showed that IOI of genipin reduced the expression of CHGA,UCP2,and GLUT1,maintained the retinal structure,and decreased ROS,glucose uptake,and inflammation levels in vivo.In addition,we found that SCG3 expression might have a higher sensitivity in DR than VEGF as a diagnostic marker at the protein level.CONCLUSION Our study suggested that genipin ameliorates AGEs-induced hRMECs proliferation,apoptosis,energy metabolism,oxidative stress,and inflammatory injury,partially via the CHGA/UCP2/GLUT1 pathway.Control of advanced glycation by IOI of genipin may represent a strategy to prevent severe retinopathy and vision loss.

胃饥饿素对高糖下视网膜微血管内皮细胞氧化应激及铁死亡的抑制作用

目的研究胃饥饿素对高糖下人视网膜微血管内皮细胞(HRMEC)氧化应激及铁死亡的影响。方法将体外培养的HRMEC分为对照组、高糖组、高糖+胃饥饿素组,分别采用常规培养基、含30 mmol/L D-葡萄糖培养基、含30 mmol/L D-葡萄糖+10 nmol/L胃饥饿素培养基培养24 h。采用细胞计数试剂盒8检测细胞增生情况;采用流式细胞术检测细胞活性氧簇(ROS)水平;采用试剂盒检测细胞中氧化应激指标还原型谷胱甘肽(GSH)浓度、丙二醛(MDA)浓度、超氧化物歧化酶(SOD)活性及Fe^(2+)浓度;采用透射电子显微镜观察线粒体结构;采用Western blot法检测HRMEC中铁死亡关键分子谷胱甘肽过氧化合物酶4(GPX4)、溶质载体家族7成员11(SLC7A11)蛋白表达。结果对照组、高糖组、高糖+胃饥饿素组细胞增生率分别为(100.62±3.40)%、(63.74±4.25)%和(88.19±4.65)%,ROS荧光强度分别为15512.20±1347.53、46457.00±1072.65和22220.87±1669.20,GSH浓度分别为(68.52±7.61)、(21.45±1.57)和(55.68±5.15)μmol/L,MDA浓度分别为(0.79±0.10)、(2.47±0.27)和(1.08±0.15)μmol/L,SOD活性分别为(111.67±10.32)、(37.75±5.92)和(97.45±9.12)U/ml,Fe^(2+)浓度分别为(3.02±0.30)、(9.45±0.71)和(4.63±0.32)mmol/mgprot。各组间细胞增生率、ROS荧光强度、GSH浓度、MDA浓度、SOD活性及Fe^(2+)浓度总体比较,差异均有统计学意义(F=61.82、414.59、61.28、67.24、61.64,146.14,均P<0.001);与对照组相比,高糖组细胞增生率、GSH浓度和SOD活性均明显降低,ROS荧光强度、MDA浓度和Fe^(2+)浓度明显升高,差异均有统计学意义(均P<0.05);与高糖组相比,高糖+胃饥饿素组细胞增生率、GSH浓度和SOD活性明显升高,ROS荧光强度、MDA浓度和Fe^(2+)浓度明显降低,差异均有统计学意义(均P<0.05)。与对照组相比,高糖组细胞内线粒体铁死亡现象明显;与高糖组相比,高糖+胃饥饿素组线粒体状态明显改善。各组间细胞中GPX4、SLC7A11蛋白相对表达量总体比较,差异均有统计学意义(F=63.94、182.84,均P<0.001);与对照组相比,高糖组和高糖+胃饥饿素组细胞中GPX4、SLC7A11蛋白相对表达量均明显降低,差异均有统计学意义(均P<0.05);与高糖组相比,高糖+胃饥饿素组2种蛋白表达水平均升高,差异均有统计学意义(均P<0.01)。结论胃饥饿素可促进高糖下的HRMEC增生,抑制高糖诱导的氧化应激和铁死亡。

基于网络药理学、分子对接及实验验证探讨滋肾健脾化瘀片对糖尿病视网膜病变的干预机制

目的运用网络药理学方法和分子对接技术探讨滋肾健脾化瘀片(山萸肉、三七、黄芪、葛根、鸡血藤、生地黄)治疗糖尿病视网膜病变(DR)的作用机制,并通过体外实验进行验证。方法(1)利用中药系统药理学数据库与分析平台(TCMSP)及BATMAN-TCM数据库筛选滋肾健脾化瘀片的有效成分及其对应的靶点蛋白。利用GeneCards、OMIM及TTD数据库检索DR疾病相关靶点。利用VENNY 2.1.0平台对药物活性成分靶点与DR疾病相关靶点取交集(共同靶点),即为滋肾健脾化瘀片治疗DR的潜在作用靶点。构建“中药-活性成分-共同靶点”网络,筛选出滋肾健脾化瘀片治疗DR的关键活性成分。将共同靶点导入STRING数据库,获取PPI网络关系,并筛选出核心靶点。运用Metascape平台对共同靶点进行GO功能及KEGG通路富集分析。将关键活性成分及核心靶点通过Autodock 4软件进行分子对接验证。(2)制备滋肾健脾化瘀片含药血清及空白血清。将人视网膜微血管内皮细胞(HRmECs)随机分成5组:对照组(低糖DMEM培养基+10%空白血清)、高糖组(高糖DMEM培养基+10%空白血清)及滋肾健脾化瘀片含药血清低、中、高剂量组(高糖DMEM培养基+10%低、中、高剂量含药血清),培养48 h后进行检测。采用CCK-8法检测HRmECs细胞增殖活性;RT-qPCR法检测HRmECs细胞中IL-1β、AKT1、VEGFA、TP53 mRNA表达水平。结果(1)共筛选出滋肾健脾化瘀片治疗DR的潜在作用靶点(共同靶点)74个;9个关键活性成分包括槲皮素、芒柄花黄素、毛地黄黄酮、β谷甾醇、山柰酚、毛蕊异黄酮、γ-氨基丁酸、豆甾醇、异鼠李亭;12个核心靶点:IL-1β、PPARG、NOS3、CXCL8、IL-6、AKT1、TNF、INS、EGF、VEGFA、TP53、PTGS2。GO功能及KEGG富集分析显示核心靶点主要参与了炎症反应、蛋白质磷酸化的正调控、细胞迁移等生物过程,以及NF-κB信号通路、AGE-RAGE信号通路、HIF-1通路、TNF通路、PI3K-AKT通路等。关键活性成分与核心靶点均具有较好的结合亲和力。(2)与对照组比较,高糖组HRmECs细胞活性显著降低(P<0.01);细胞中VEGFA、TP53、IL-1βmRNA表达水平显著升高(P<0.01),AKT1 mRNA表达水平显著降低(P<0.01)。与高糖组比较,滋肾健脾化瘀片含药血清低、中、高剂量组的HRmECs细胞活性均显著升高(P<0.05,P<0.01),并呈浓度依赖性;含药血清高剂量组细胞的VEGFA、TP53、IL-1βmRNA表达水平显著降低(P<0.05,P<0.01),而AKT1 mRNA表达水平显著升高(P<0.01)。结论滋肾健脾化瘀片可能通过槲皮素、山柰酚、毛地黄黄酮等多种活性成分,作用于IL-1β、IL-6、VEGFA等核心靶点,以及NF-κB信号通路、AGE-RAGE信号通路、PI3K-AKT通路等关键通路,从而发挥对DR的治疗作用。

lncRNA SNHG6对高糖诱导的人视网膜微血管内皮细胞损伤的影响

目的:探讨lncRNA SNHG6对高糖诱导的人视网膜微血管内皮细胞(hRMECs)损伤的影响及其可能的作用机制。方法:采用高糖诱导hRMECs建立细胞损伤模型。高糖(HG)组将hRMECs培养在浓度为25 mmol/L D-葡萄糖的DMEM培养基24 h;正常(NG)组将hRMECs培养在浓度为5.5 mmol/L D-葡萄糖的DMEM培养基;按照实验设计分别将si-NC、si-SNHG6、si-SNHG6和anti-miR-NC、si-SNHG6和anti-miR-186-5p转染至hRMECs,随后25 mmol/L D-葡萄糖孵育24 h,分别记为HG+si-NC组、HG+si-SNHG6组、HG+si-SNHG6+anti-miR-NC组、HG+si-SNHG6+anti-miR-186-5p组。qRT-PCR法检测lncRNA SNHG6、miR-186-5p的表达量;双荧光素酶报告实验检测lncRNA SNHG6与miR-186-5p的靶向关系;MTT法与流式细胞术分别检测细胞增殖及凋亡;ELISA法检测IL-1β、TNF-α、IL-8、IL-10的水平;采用试剂盒检测SOD的活性和MDA的水平;Western blot检测cleaved-caspase3、Bax、Bcl-2蛋白表达量。结果:HG组与NG组比较lncRNA SNHG6表达升高,miR-186-5p表达降低(均P<0.05);lncRNA SNHG6可靶向结合miR-186-5p。转染si-SNHG6后细胞增殖抑制率、凋亡率、cleaved-caspase3、Bax蛋白水平,IL-1β、TNF-α、IL-8含量以及MDA活性降低(P<0.05),而Bcl-2蛋白、IL-10含量以及SOD的活性升高(P<0.05)。共转染si-SNHG6和anti-miR-186-5p后,细胞增殖抑制率、凋亡率、cleaved-caspase3、Bax、IL-1β、TNF-α、IL-8以及MDA升高(P<0.05),而Bcl-2蛋白、IL-10和SOD降低(P<0.05)。结论:干扰lncRNA SNHG6表达可通过促进miR-186-5p表达而抑制高糖诱导的hRMECs凋亡、炎症反应及氧化应激。

玄参提取物对糖尿病视网膜病变微血管内皮细胞损伤的影响及其作用机制

目的:探讨玄参提取物抑制微小RNA-646(miR-646)改善微血管内皮细胞损伤缓解糖尿病视网膜水肿的影响及其作用机制。方法:根据简单随机化法将人视网膜微血管内皮细胞(HRMEC)细胞分为对照组、模型组(30 mmol/L葡萄糖)、观察组(30 mmol/L葡萄糖+100μg/mL玄参提取物组)、模型+Anti-NC(转染Anti-NC+30 mmol/L葡萄糖)、模型+Anti-miR-646(转染Anti-miR-646+30 mmol/L葡萄糖)组;将30只小鼠根据简单随机化法分为对照组、模型组(60 mg/kg的链脲佐菌素)、观察组(60 mg/kg的链脲佐菌素+100 mg/kg-的玄参提取物),每组10只。比较各组细胞增殖,迁移及miR-646、血管内皮生长因子A(VEGFA)、细胞间黏附分子-1(ICAM-1)、血小板-内皮细胞黏附分子(CD31)表达;比较各组小鼠视网膜组织ICAM-1、CD31表达及视网膜组织水肿情况。结果:细胞实验结果显示,与对照组比较,模型组细胞miR-646、ICAM-1表达显著升高,VEGFA、CD31表达显著下降(均P<0.05);与模型组比较,观察组细胞miR-646、ICAM-1表达显著降低,VEGFA、CD31表达显著升高(均P<0.05);与模型+Anti-NC组比较,模型+Anti-miR-646组细胞增殖率、迁移细胞数及VEGFA表达显著增加,miR-646表达及凋亡率显著降低(均P<0.05)。动物实验结果显示,与对照组比较,模型组小鼠视网膜组织中miR-646、ICAM-1表达显著升高,VEGFA、CD31表达显著下降(均P<0.05),同时小鼠视网膜组织水肿情况加重;与模型组比较,观察组小鼠网膜组织中miR-646、ICAM-1表达显著降低,VEGFA、CD31表达显著升高(均P<0.05),同时小鼠视网膜组织水肿明显得到缓解。结论:玄参提取物可能通过miR-646/VEGFA机制调节HRMEC细胞增殖、迁移和凋亡,改善缓解糖尿病视网膜水肿。

二十烷二酸通过结合PPARδ介导ANGPTL4表达增多对人视网膜血管内皮细胞增殖和迁移的影响

目的 研究二十烷二酸(C20DC)对人视网膜内皮细胞(HREC)增殖、迁移能力的影响,并探讨其作用机制。方法 寻找C20DC干预人视网膜色素上皮-19(ARPE-19)细胞与HREC的最佳工作浓度,分别为30 mg·L^(-1)与25 mg·L^(-1)。将HREC分为C20DC处理组(C20DC干预HREC)和对照组[二甲基亚砜(DMSO)干预HREC],通过细胞增殖和迁移实验观察C20DC对HREC迁移及增殖能力的影响;采用分子对接方法模拟C20DC与PPARδ的结合能力;将ARPE-19细胞设为C20DC+ARPE-19组(C20DC干预ARPE-19细胞)与DMSO+ARPE-19组(DMSO干预ARPE-19细胞),采用Western blot检测ARPE-19细胞中PPARδ和血管生成素样蛋白4(ANGPTL4)蛋白,以及HREC中ANGPTL4蛋白表达水平;采用ELISA定量分析ARPE-19细胞与HREC中ANGPTL4蛋白表达水平。结果 与对照组相比,C20DC处理组细胞增殖、迁移能力均显著提高(均为P<0.05),且可与PPARδ结合稳定(结合能为-7.2 kcal·mol^(-1));Western blot检测结果提示,C20DC+ARPE-19组细胞中ANGPTL4蛋白表达量高于DMSO+ARPE-19组,差异有统计学意义(P<0.05),两组PPARδ受体蛋白表达量比较,差异无统计学意义(P>0.05);C20DC处理组细胞中ANGPTL4蛋白表达量高于对照组,差异有统计学意义(P<0.05)。ELISA定量分析检测结果显示,C20DC+ARPE-19组细胞中ANGPTL4表达量高于DMSO+ARPE-19组(P<0.001);C20DC处理组细胞中ANGPTL4的表达量高于对照组,差异有统计学意义(P<0.05)。结论 C20DC可结合PPARδ促进ANGPTL4蛋白的表达,导致视网膜相关细胞(HREC与ARPE-19细胞)增殖与迁移能力增强,其作用机制可能与早产儿视网膜病变新生血管生成增多有关。

基于腺苷酸活化蛋白激酶/哺乳动物雷帕霉素靶蛋白通路探讨柚皮素对视网膜微血管内皮细胞的损伤机制研究

目的 基于腺苷酸活化蛋白激酶(AMPK)/哺乳动物雷帕霉素靶蛋白(mTOR)通路探讨柚皮素(NAR)对人视网膜微血管内皮细胞(HRMECs)的损伤机制。方法 将HRMECs随机分为对照组、高糖(HG)组、HG+NAR组(3 mg/L NAR)、HG+激活剂(AICAR)组(1 mmol/L AICAR)、HG+NAR+AICAR组(3 mg/L NAR+1 mmol/L AICAR);除对照组向培养基中加入5 mmol/L的D-葡萄糖处理外,其他各组均向培养基中加入30 mmol/L的D-葡萄糖处理。采用CCK-8及Transwell分别检测细胞增殖及迁移情况;采用酶联免疫吸附试验(ELISA)检测上清液中白细胞介素(IL)-1β、IL-6和肿瘤坏死因子-α(TNF-α)水平;采用实时荧光定量聚合酶链反应(qRT-PCR)检测自噬因子LC3 mRNA、p62 mRNA表达水平;采用蛋白质免疫印迹(Western blot)检测AMPK/mTOR通路及自噬相关蛋白表达水平。结果 与对照组相比,HG组细胞活力,迁移数,IL-1β、IL-6和TNF-α水平,p-AMPK/AMPK,LC3Ⅱ/LC3Ⅰ,LC3 mRNA表达增加,p-mTOR/mTOR、p62蛋白及p62 mRNA表达下降,差异有统计学意义(P<0.05);与HG组相比,HG+NAR组细胞活力,迁移数,IL-1β、IL-6和TNF-α水平,p-AMPK/AMPK,LC3Ⅱ/LC3Ⅰ,LC3 mRNA表达下降,p-mTOR/mTOR、p62蛋白及p62 mRNA表达增加,但HG+AICAR组细胞活力,迁移数,IL-1β、IL-6和TNF-α水平,p-AMPK/AMPK,LC3Ⅱ/LC3Ⅰ表达增加,p-mTOR/mTOR、p62蛋白及p62 mRNA表达下降,差异有统计学意义(P<0.05);与HG+NAR组相比,HG+NAR+AICAR组细胞活力,迁移数,IL-1β、IL-6和TNF-α水平,p-AMPK/AMPK,LC3Ⅱ/LC3Ⅰ,LC3 mRNA表达增加,p-mTOR/mTOR、p62蛋白及p62 mRNA表达下降,差异有统计学意义(P<0.05);与HG+AICAR组相比,HG+NAR+AICAR组细胞活力,迁移数,IL-1β、IL-6和TNF-α水平,p-AMPK/AMPK,LC3Ⅱ/LC3Ⅰ,LC3 mRNA表达下降,p-mTOR/mTOR、p62蛋白及p62 mRNA表达增加,差异有统计学意义(P<0.05)。结论 NAR可减轻HG诱导的HRMECs损伤,其机制可能与抑制AMPK/mTOR通路介导的自噬有关。

改良的人视网膜血管内皮细胞的培养与鉴定方法

背景优化人视网膜血管内皮细胞的培养和鉴定方法在视网膜血管性疾病的研究中有重要作用，以往研究者已成功培养了人视网膜血管内皮细胞，但进一步简化其方法并达到获取量大而纯的细胞是基础研究和临床研究的基础。目的探索一种更为简便快速、收获量大且纯度高的视网膜血管内皮细胞的改良培养方法，并对目标细胞的抗原表达特点进行分析，比较眼科新生血管性疾病研究中常用的两种血管内皮细胞的标志性蛋白表达情况。方法利用正常人角膜移植供体眼球分离视网膜组织，采用质量分数2％胰蛋白酶和质量分数0．133％胶原酶I用二步消化法获取人视网膜血管内皮细胞，在传统培养方法中采用含质量分数10％胎牛血清的人血管内皮细胞培养液的基础上，添加内皮细胞生长因子一B（13-ECGF）和肝素钠，对分离的细胞进行体外培养，培养皿用纤维黏连蛋白（FN）包被以促进培养细胞的贴壁。观察收获的目标细胞的形态特征，采用活体显微镜进行形态学观察、常规组织病理学观察目标细胞的生长状态，免疫组织化学法检测Ⅷ因子、CD31、CD34在细胞中的表达以鉴定目标细胞。结果应用胰蛋白酶、胶原酶二步消化法可成功获取人视网膜血管内皮细胞，原代培养的细胞72h贴壁，第9～10天细胞达到融合状态，呈铺路石样。常规组织学观察显示，细胞核呈鲜亮蓝色，细胞质呈淡红色。培养的细胞对Ⅷ因子、CD31、CD34相关抗原表达呈阳性反应。结果显示培养的血管内皮细胞均有CD31、CD34同时表达，但其阳性染色程度低于Ⅷ因子相关抗原阳性反应，人脐静脉血管内皮细胞（UVECs）与人血管内皮细胞相同。结论在胰蛋白酶、胶原蛋白酶消化法的基础上用含10％优质胎牛血清的人内皮细胞培养基中添加生长因子和肝素钠，并用FN包被培养皿进行体外培养，可达到快速、大量分离和纯化人视网膜血管内皮细胞的目的。

苦参碱对大鼠视网膜微血管内皮细胞的增生及VEGF表达的影响

目的探讨苦参碱(Ma)对体外培养的大鼠视网膜微血管内皮细胞的增生抑制及对血管内皮细胞生长因子(VEGF)表达的影响。方法原代培养大鼠视网膜微血管内皮细胞,噻唑蓝比色法(MTT)检测不同质量浓度的Ma对其增生的抑制作用。将培养的细胞分为对照组、Ma(40mg/L)组、高糖(25mmol/L)组、Ma+高糖组4组。免疫细胞化学和Westernblot法检测细胞VEGF的表达。结果一定质量浓度范围内Ma可抑制视网膜微血管内皮细胞的增生(P<0.05),48h内呈质量浓度依赖性。与对照组和高糖组相比,Ma组48h后,VEGF表达明显下降(P<0.05),与对照组相比,高糖组VEGF的表达明显增加(P<0.05)。结论Ma可抑制视网膜微血管内皮细胞的增生,抑制主要是通过下调VEGF表达而发生作用的,Ma对高糖引起的VEGF表达上调同样也有抑制作用。

Tumstatin肽对视网膜微血管内皮细胞迁移及P38MAPK蛋白表达的影响

目的:观察tumstatin肽对体外培养的视网膜微血管内皮细胞迁移及P38MAPK蛋白表达的影响,初步探讨tumstatin肽抗视网膜内皮细胞迁移的机制。方法:采用细胞划痕实验测定tumstatin肽(T8肽)对血管内皮生长因子(VEGF)诱导下RF/6A细胞(恒河猴视网膜微血管内皮细胞)迁移的影响;Western blotting检测T8肽对VEGF刺激后15,30,45,60min的RF/6A细胞P38MAPK蛋白水平的变化。结果:Tumstatin肽对RF/6A细胞迁移具有抑制作用,且可抑制VEGF对RF/6A细胞的促迁移作用,呈剂量依赖性。正常情况下,RF/6A细胞无P38MAPK蛋白的表达,但VEGF可诱导其表达P38MAPK蛋白,而tumstatin可抑制VEGF诱导的RF/6A细胞P38MAPK蛋白的表达(加入20mg/LT8肽30,45,60min时蛋白表达受到显著抑制,差异有显著性意义,P<0.01)。结论:Tumstatin抑制视网膜微血管内皮细胞的迁移,其作用可能与P38MAPK通路有关。

缺氧条件下视网膜微血管内皮细胞中缺氧诱导因子1和血管内皮生长因子mRNA表达的研究

目的探讨视网膜微血管细胞在缺氧过程中缺氧诱导因子1(HIF1)和血管内皮生长因子(VEGF)基因mRNA水平表达的变化。方法视网膜微血管细胞(RECs)分别在正常氧、缺氧3h不同条件下培养后,应用反转录聚合酶链反应(RT-PCR)方法半定量测定HIF1α和VEGF基因的mRNA水平的表达。结果缺氧组RECsHIF1基因的mRNA水平表达为对照组的4.6倍。同时缺氧组RECs细胞的VEGF基因的mRNA水平表达为对照组的3倍。结论缺氧能引起视网膜微血管细胞的HIF-1及VEGF基因mRNA水平高表达,提示缺氧可能通过HIF1基因的转录途径来诱发VEGF基因的高表达导致视网膜新生血管形成。

共培养系统中视网膜微血管周细胞经KDR/Flk-1途径对内皮细胞增生的影响(英文)

目的:采用细胞共培养模型研究缺氧诱导视网膜新生血管生成过程中周细胞对微血管内皮细胞增生的作用及其相关血管内皮细胞生长因子受体2(KDR/Flk-1)调节机制。方法:免疫磁珠法原代分离培养大鼠视网膜微血管内皮细胞和周细胞,分别采用抗VIII因子、CD31抗体以及抗血小板源性生长因子受体β、结蛋白抗体免疫细胞化学法鉴定内皮细胞和周细胞。通过Millicell小室建立周细胞和内皮细胞共培养模型,使用MTT法和流式细胞仪检测内皮细胞增殖数量及周期,观察缺氧和常氧下周细胞对血管内皮细胞增生的影响,并采用RT-PCR法检测内皮细胞KDR/Flk-1的mRNA水平变化,探讨相关调节机制。结果:经免疫细胞化学法鉴定,免疫磁珠法可获得高纯度的视网膜微血管内皮细胞和周细胞。MTT法显示,缺氧3～9d内皮细胞增生明显,6d增生达高峰(24.9%,P<0.01);共培养时内皮细胞的增生可被周细胞抑制。流式细胞仪检测发现,缺氧6dS期内皮细胞数量明显增加(43.9%,P<0.01);常氧(3.6%,P<0.05)或缺氧(15.1%,P<0.01)共培养时,周细胞均可抑制内皮细胞增生。缺氧下内皮细胞KDR/Flk-1的mRNA水平是常氧下的1.3倍;常氧(45.1%,P<0.05)和缺氧(27.7%,P<0.05)共培养下,周细胞均可下调内皮细胞KDR/Flk-1的mRNA表达。结论:缺氧和常氧共培养时周细胞均可抑制微血管内皮细胞增生,此抑制作用部分是通过下调内皮细胞KDR/Flk-1mRNA表达实现。

静态压力对大鼠视网膜微血管内皮细胞内皮素-1和NO表达的影响

目的观察静态压力变化对大鼠视网膜微血管内皮细胞(RMECs)形态、增生活性及分泌血管活性物质内皮素-1(ET-1)和一氧化氮合酶(NOS)的影响。方法体外培养并鉴定大鼠RMECs,分为A(1.33kPa)、B(2.67kPa)、C(5.33kPa)和D(10.67kPa)组和未加压对照组。用倒置相差显微镜观察细胞形态的变化,常规方法进行细胞计数,锥虫蓝染色检测活细胞比例。用Griess硝酸还原酶法检测NO代谢产物NO2-/NO3-的含量,放射免疫法检测培养细胞的ET-1蛋白表达;用RT-PCR法半定量研究RMECs中ET-1、eNOS、iNOSmRNA的表达。结果B、C、D组RMECs细胞核膨大,胞体拉长,可见少量悬浮细胞;静态压力促进RMECs增生(F=12.205,P<0.01),C组和D组细胞数量高于对照组、A组和B组(P<0.01);静态压力升高造成各组细胞损伤,拒染率降低(F=11.180,P<0.01),B、C、D组细胞拒染率低于对照组(P<0.05)。B、C、D组ET-1浓度较对照组增加(P<0.01)。C组和D组RMECs培养液中NO2-/NO3-浓度高于对照组(P<0.01)。B、C、D组RMECs中ET-1mRNA表达较对照组升高,差异有统计学意义(P<0.01);C组和D组RMECs中eNOSmRNA和iNOSmRNA表达较对照组升高,差异有统计学意义(P<0.01)。结论高静态压力可直接导致RMECs的结构损伤,高静态压力下RMECs合成ET-1、NO增多可能是高眼压眼底病变的机制之一。