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.

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.

Comparative analysis of cytomegalovirus retinitis and microvascular retinopathy in patients with acquired immunodeficiency syndrome

AIM：To compare the clinical manifestation of cytomegalovirus（CMV）retinitis and microvascular retinopathy（MVR）in patients with acquired immunodeficiency syndrome（AIDS）in China.METHODS：A total of 93 consecutive patients with AIDS,including 41 cases of CMV retinitis and 52 cases of MVR were retrospectively reviewed.Highly active antiretroviral therapy（HAART）status was recorded.HIV and CMV immunoassay were also tested.CD4＋T-lymphocyte count and blood CMV-DNA test were performed in all patients.Aqueous humor CMV-DNA test was completed in 39patients.Ophthalmological examinations including best corrected visual acuity（BCVA,by International Standard Vision Chart）,intraocular pressure（IOP）,slit-lamp biomicroscopy,indirect ophthalmoscopy were performed.RESULTS：In MVR group,the anterior segment examination was normal in all patients with a mean BCVA of 0.93±0.13.Blood CMV-DNA was 0（0,269 000）and 42 patients（80.77%）did not receive HAART.In CMV retinitis group,13 patients（31.71%）had anterior segment abnormality.The mean BCVA was 0.64±0.35 and blood CMV-DNA was 3470（0,1 450 000）.Nineteen patients（46.34%）had not received HAART.MVR group and CMV retinitis group the positive rates of aqueous CMV-DNA were 0 and 50%,respectively.Two patients with MVR progressed to CMV retinitis during the follow-up period.CONCLUSION：In comparison of CMV,patients with MVR have relatively mild visual function impairment.Careful ophthalmological examination and close follow-up are mandatory,especially for patients who have systemic complications,positive CMV-DNA test and without received HAART.

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.

青光眼中视网膜神经血管单元损伤的研究进展

青光眼是全球范围内引起视力丧失的主要原因之一。越来越多的研究表明,青光眼是一种复杂的视网膜神经血管疾病,神经元、胶质细胞和微血管细胞构成的视网膜神经血管单元(RNVU)的稳态失衡不仅诱导微血管结构和胶质细胞的变化,还会影响视网膜的神经组织,最终引起视力下降,目前尚无有效的治疗措施逆转这种视力下降。了解RNVU的细胞组成、分子结构以及各部分的生理功能,深入研究青光眼中正常细胞环境和细胞间联系的破坏机制,对于探究青光眼的发生发展,寻找有效的防治措施具有重要的意义。本文就青光眼疾病中的RNVU结构改变和功能障碍的研究进展进行综述,探寻青光眼潜在的发病机制,以期为青光眼的干预治疗提供新的思路。

妊娠期高血压和子痫前期患者视网膜微血管的变化

目的:利用光学相干断层扫描血管成像(OCTA)评估妊娠期高血压和子痫前期患者视网膜表层黄斑区和视乳头区微血管的变化。方法:回顾性研究。收集2020-09/2021-01就诊于山西医科大学第一医院产科的妊娠期高血压和子痫前期患者25例,其中妊娠期高血压10例、轻度子痫前期9例、重度子痫前期6例;另收集同期就诊于我院的健康孕妇25例、健康未孕女性25例。应用OCTA测量视网膜黄斑区和视乳头区血管密度(VD)、灌注密度(PD)。分析比较各组受检者右眼视网膜微血管各参数的变化。结果:妊娠期高血压和子痫前期患者黄斑区中心VD、内环VD、中心PD、内环PD均低于健康孕妇和健康未孕女性(均P<0.05)。妊娠期高血压和子痫前期患者视乳头内环PD低于健康孕妇和健康未孕女性(均P<0.05)。妊娠期高血压患者、轻度子痫前期患者、重度子痫前期患者黄斑区和视乳头VD、PD比较均无差异(均P>0.05)。健康孕妇和健康未孕女性黄斑区和视乳头VD、PD比较均无差异(均P>0.05)。结论:OCTA可较好地显示妊娠期高血压和子痫前期患者视网膜表层微血管的变化,并在未出现明显视网膜病变前已经观察到妊娠期高血压和子痫前期患者黄斑区和视乳头VD、PD下降,OCTA可能对早期识别妊娠期高血压和子痫前期患者微血管变化提供一定帮助。

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。

OCTA检测青少年不同程度近视患者视网膜神经纤维层厚度和视乳头周围脉络膜毛细血管的微观结构改变

目的利用光学相干断层扫描血管造影术(OCTA)测定青少年不同程度近视眼视网膜神经纤维厚度(RNFL)及视乳头周围脉络膜毛细血管的微结构改变。方法回顾性将2022年1月至2023年1月的惠州市中心人民医院收治的90例5~15岁青少年(共计180只眼睛)作为研究对象,按等效球镜度数(SE)分为正视组(n=30),轻度近视组(n=44),中度近视组(n=56),高度近视组(n=50)。用OCTA测定各组的RNFL、视盘旁萎缩弧β区(PPA-β)及视盘旁萎缩弧α区(PPA-α)面积、PPA-β及PPA-α区微血管丢失(MVD)面积、PPA-β及PPA-α区血流密度值,同时收集各组的SE和眼轴长度(AL)进行比较,用Spearman相关性分析PPA-β区MVD面积与SE、AL、RNFL及PPA-β区面积的相关性。结果4组的SD、AL值比较,差异均有统计学意义(P<0.05),且随着近视程度越重SD、AL值越高。4组的上方、下方、鼻侧及颞侧RNFLT、平均RNFLT比较,差异均有统计学意义(P<0.05);轻度近视组与正视组的上方、下方、鼻侧及颞侧RNFLT、平均RNFLT差异均无统计学意义(P>0.05);高度近视组的上方、下方、颞侧和平均RNFLT均显著低于中度近视组和轻度近视组,差异均有统计学意义(P<0.05)。150只近视眼中有141只(94.00%)的PPA-β区存在MvD。高度近视组PPA-β区的MvD面积、PPA-β面积分别为(0.239±0.106)、(1.104±0.270)mm^(2),均显著高于中度近视组[(0.095±0.041)、(0.612±0.264)mm^(2)]和轻度近视组[(0.071±0.030)、(0.507±0.241)mm^(2)],而PPA-β区血流密度为198.24±95.89,低于中度近视组(349.07±126.32)和轻度近视组(402.63±132.09),差异均有统计学意义(P<0.05)。高度近视组PPA-α区存在MvD占比为56.00%,显著高于中度近视组(26.79%)和轻度近视组(15.91%),差异有统计学意义(P<0.05),3组PPA-α区的MvD面积、PPA-α面积及PPA-α区血流密度差异均无统计学意义(P>0.05)。Spearman相关性分析显示,PPA-β区MvD面积与与SE、RNFLT呈负相关(r=-0.492、-0.574,P<0.05),与AL、PPA-β区面积呈正相关(r=0.480、0.798,P<0.05)。结论PPA-β区MvD是青少年高度近视患者视乳头周围脉络膜毛细血管的微血管改变的重要指标,且与SE、AL、RNFLT和PPA-β区面积相关。

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。

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.

常见鞍区肿瘤患者视网膜微血管密度与视野损害的相关性研究

目的:观察常见鞍区肿瘤患者视网膜微血管密度的变化,及与视野损伤的相关性,探索其在评估鞍区肿瘤患者视神经损伤中的应用价值。方法:横断面研究,选取2018-10/2022-05在首都医科大学附属北京天坛医院神经外科和眼科就诊的常见鞍区肿瘤患者157例292眼(垂体腺瘤82例152眼、颅咽管瘤75例140眼),收集同期就诊于首都医科大学附属北京天坛医院眼科患者的家属、本院学生及工作人员90例180眼作为对照组。所有受检者均进行OCTA检查。比较两组受检者视网膜微血管密度变化,及其与视野参数的相关性。结果:鞍区肿瘤组患者视乳头放射状毛细血管网(RPC)和黄斑区浅层毛细血管丛(SRCP)密度均较对照组降低[50.81%(46.49%,53.49%)vs 52.78%(50.73%,54.51%)和50.57%(48.13%,52.73%)vs 51.63%(49.78%,53.02%),均P<0.05]。颅咽管瘤组患者RPC密度较垂体腺瘤组患者更低[49.71%(44.33%,53.14%)vs 51.37%(47.42%,53.95%),P<0.05]。鞍区肿瘤组患者视野MD值为-4.33(-12.22,-1.85)dB,PSD值为3.37(1.91,8.82)dB,VFI为92%(65%,97%)。鞍区肿瘤患者各象限RPC密度与MD、VFI正相关,与PSD负相关,各象限SRCP密度与MD正相关,除Para-T外其余各象限与VFI正相关,与PSD负相关(均P<0.05)。结论:鞍区肿瘤患者的视网膜微血管密度降低,血管密度越低,视野损害越严重。在临床工作中,将OCTA检查与视野检查结合起来,更有助于发现患者的视神经损害。

miR-519d-3p靶向HIF-1α抑制高糖诱导的人视网膜微血管内皮细胞功能障碍及血管生成

目的:明确miR-519d-3p对高糖诱导的人视网膜微血管内皮细胞(HRMEC)功能障碍与血管生成的影响,并阐明其对低氧诱导因子-1α(HIF-1α)的调控机制。方法:通过5、30mmol/L葡萄糖分别诱导HRMEC建立正常(NG)和高糖(HG)细胞模型。将HRMEC分为对照组(HG细胞模型转染阴性对照模拟物)、甘露醇组(对照组加入25mmol/L甘露醇)、miR-519d-3p过表达组(HG细胞模型转染miR-519d-3p模拟物)、miR-519d-3p联合HIF-1α过表达组(HG细胞模型共转染miR-519d-3p模拟物和HIF-1α过表达载体)。实时荧光定量PCR法检测各组miR-519d-3p的表达情况。Western blotting法检测各组HIF-1α蛋白的表达情况。荧光素酶报告基因实验检测miR-519d-3p和HIF-1α的结合位点情况。CCK-8法检测各组细胞增殖情况。Hoechst 33342染色法检测各组细胞凋亡情况。ELISA法检测各组细胞外液炎症因子肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β、IL-6蛋白的表达情况。小管形成实验检测各组新生毛细血管管腔样结构形成情况。结果:与NG相比,HG细胞模型中miR-519d-3p表达显著减少,而HIF-1α蛋白表达显著增加(均P<0.01)。与对照组比较,miR-519d-3p过表达组中HIF-1α蛋白表达显著降低(P<0.01)。miR-519d-3p中“CGUGAAA”序列可以与HIF-1α3'-非编码区(3'-UTR)中“GCACUUU”序列特异性结合。与对照组比较,miR-519d-3p过表达组细胞24、48、72h吸光度值均显著增加,细胞凋亡率显著减少,细胞外液TNF-α、IL-1β、IL-6浓度均显著减少,新生毛细血管管腔样结构数量显著减少(均P<0.01)。与miR-519d-3p过表达组比较,miR-519d-3p联合HIF-1α过表达组细胞24、48、72h吸光度值均显著减少,细胞凋亡率显著增加,细胞外液TNF-α、IL-1β、IL-6浓度均显著增加,新生毛细血管管腔样结构数量显著增加(均P<0.01)。对照组和甘露醇组中上述各指标比较无差异(均P>0.05)。结论:高糖诱导HRMEC模型中miR-519d-3p表达下调,而HIF-1α蛋白表达上调。HIF-1α是miR-519d-3p的靶基因,miR-519d-3p靶向HIF-1α增加细胞增殖并降低细胞凋亡和炎症反应,从而减轻高糖诱导的HRMEC功能障碍并抑制血管生成。

miR-1-3p调控ANXA2表达对高糖诱导的视网膜微血管内皮细胞新生血管生成的影响

目的 探讨微小RNA-1-3p(miR-1-3p)/膜联蛋白A2(ANXA2)分子轴在高糖诱导的人视网膜微血管内皮细胞(HRMECs)新生血管生成过程中的作用机制。方法 体外培养HRMECs并采用高糖(HG)处理细胞建立细胞损伤模型。HRMECs分组处理:Con组(含体积分数10%胎牛血清的DMEM培养)、HG组(25 mmol·L^(-1)D-葡萄糖培养)、HG+miR-NC组(转染miR-NC)、HG+miR-1-3p组(转染miR-1-3p mimics)、HG+sh-NC组(转染sh-NC)、HG+sh-ANXA2组(转染sh-ANXA2)、HG+miR-1-3p+pcDNA组(转染miR-1-3p mimics+pcDNA)、HG+miR-1-3p+pcDNA-ANXA2组(转染miR-1-3p mimics+pcDNA-ANXA2)。转染48 h后收集细胞,采用25 mmol·L^(-1)的D-葡萄糖培养基培养HRMECs 24 h。采用MTT、Transwell小室实验分别检测细胞活力、迁移细胞数。管腔形成实验检测管腔形成数。双荧光素酶报告实验检测miR-1-3p与ANXA2的靶向关系。Western blot检测VEGF、MMP-2蛋白水平。结果 与Con组比较,HG组miR-1-3p表达水平降低,ANXA2 mRNA及蛋白水平均升高,差异均有统计学意义(均为P<0.05)。与Con组比较,HG组细胞活力升高,迁移细胞数、管腔形成数增多,VEGF、MMP-2蛋白水平升高,差异均有统计学意义(均为P<0.05)。与HG+miR-NC组比较,HG+miR-1-3p组细胞活力降低,迁移细胞数、管腔形成数减少,VEGF、MMP-2蛋白水平降低,差异均有统计学意义(均为P<0.05)。与HG+sh-NC组比较,HG+sh-ANXA2组细胞活力降低,迁移细胞数、管腔形成数减少,VEGF、MMP-2蛋白水平降低,差异均有统计学意义(均为P<0.05)。与HG+miR-1-3p+pcDNA组比较,HG+miR-1-3p+pcDNA-ANXA2组细胞活力升高,迁移细胞数、管腔形成数增多,VEGF、MMP-2蛋白水平升高,差异均有统计学意义(均为P<0.05)。结论 miR-1-3p过表达可通过靶向调控ANXA2表达抑制HRMECs的增殖、迁移及新生血管生成。

基于PI3K/AKT信号通路探讨益气养阴活血利水方抑制早期糖尿病视网膜病变大鼠微血管周细胞凋亡的作用机制

目的观察益气养阴活血利水方对早期糖尿病视网膜病变(diabetic retinopathy,DR)大鼠视网膜微血管周细胞中磷酸肌醇3-激酶蛋白(phosphatidylinositol 3-kiases,PI3K)/丝氨酸/苏氨酸蛋白激酶(phospho-alpha serine/threonine-protein kinase,AKT)信号通路相关因子表达的影响,探讨益气养阴活血利水方抑制早期DR视网膜微血管周细胞凋亡的作用机制。方法138只雄性SPF级SD大鼠随机分成空白组(等体积蒸馏水),模型组,羟苯磺酸钙组[150 mg/(kg·d)],益气养阴活血利水方低剂量组[6.2 g/(kg·d)]、中剂量组[12.4 g/(kg·d)]、高剂量组[24.8 g/(kg·d)],每组23只。除空白组外,各组均采用链佐脲菌素腹腔注射并维持10周高血糖状态,诱导早期DR大鼠模型。造模成功后给药4周,取大鼠眼球进行检测。采用HE染色观察视网膜病理变化,采用TUNEL染色检测大鼠视网膜微血管周细胞凋亡,采用免疫组织化学法及RT-PCR法检测PI3K、AKT、Bcl-2相关X蛋白(Bcl2-associated X protein,Bax)、胱天蛋白酶-8(cysteine aspartic acid specific protease-8,Caspase-8)、胱天蛋白酶-3(cysteine aspartic acid specific protease-3,Caspase-3)、细胞凋亡死亡激动剂BID蛋白(apoptotic death agonist BID protein,Bid)在视网膜微血管周细胞中的蛋白和mRNA表达水平。结果与空白组比较,模型组大鼠血糖显著升高(P<0.01),体质量显著降低(P<0.01),视网膜层次结构不清,各层细胞排列疏松,可见大量凋亡细胞,视网膜中PI3K、AKT mRNA表达水平下降(P<0.01),视网膜细胞凋亡数及Bax、Caspase-8、Caspase-3、Bid mRNA表达水平明显升高(P<0.05)。与模型组比较,益气养阴活血利水方中剂量、高剂量组大鼠血糖均降低(P<0.01);羟苯磺酸钙组、益气养阴活血利水方各剂量组大鼠体质量均升高(P<0.01);益气养阴活血利水方各剂量组大鼠视网膜组织层次结构较清楚,细胞排列相对紧密,视网膜微血管周细胞凋亡数下降(P<0.05);益气养阴活血利水方高剂量组大鼠视网膜微血管周细胞中PI3K、AKT蛋白和mRNA表达水平均明显升高(P<0.01),Bax、Caspase-8、Caspase-3、Bid蛋白和mRNA表达水平均明显下降(P<0.05)。结论益气养阴活血利水方可能通过调控PI3K/AKT信号通路,上调PI3K、AKT,下调Bax、Caspase-8、Caspase-3、Bid的表达,抑制早期DR大鼠视网膜微血管周细胞的凋亡,从而发挥其对早期DR视网膜微血管周细胞凋亡的保护作用。

Piezo拮抗剂GsMTx4通过激活Yap1/STAT3信号通路促进人视网膜微血管内皮细胞的血管生成

目的探讨Piezo拮抗剂GsMTx4对人视网膜微血管内皮细胞(hRMECs)血管生成能力的影响与机制。方法体外培养hRMECs,随机分为对照组、GsMTx4组、GsMTx4+维替泊芬(VP)组、GsMTx4+Stattic组。GsMTx4组用2.5μmol·L^(-1)的GsMTx4处理细胞24 h,GsMTx4+VP组用GsMTx4联合Yap1的拮抗剂VP(4μmol·L^(-1))处理hRMECs 24 h,GsMTx4+Stattic组则用GsMTx4联合STAT3的拮抗剂Stattic(1.5μmol·L^(-1))处理hRMECs 24 h。CCK-8法检测各组hRMECs的增殖能力。小管形成实验观察各组hRMECs的血管生成能力,记录小管分支数。qRT-PCR和Western blot检测对照组和GsMTx4组hRMECs中Piezo、Yap1、STAT3 mRNA和蛋白的表达水平。使用免疫共沉淀技术检测各组hRMECs中Yap1和STAT3的结合作用。结果与对照组相比,GsMTx4组hRMECs的增殖率和小管分支数均明显增加(均为P<0.05),Piezo蛋白表达下调,而Yap1、STAT3蛋白表达均上调,且Yap1和STAT3的结合作用明显增加(均为P<0.05)。与GsMTx4组相比,GsMTx4+VP组和GsMTx4+Stattic组hRMECs中Yap1和STAT3的蛋白表达均下调,且Yap1和STAT3的结合作用减弱(均为P<0.05)。与GsMTx4组相比,GsMTx4+VP组和GsMTx4+Stattic组hRMECs增殖率和小管分支数均降低(均为P<0.05)。结论Piezo拮抗剂GsMTx4通过激活Yap1/STAT3信号通路促进hRMECs的血管生成。

低浓度阿托品滴眼液对近视儿童青少年视网膜与脉络膜厚度及微循环的影响

目的 研究0.1 g·L^(-1)低浓度阿托品滴眼液对近视儿童青少年脉络膜、视网膜厚度(RT)及血管系统的影响,分析低浓度阿托品影响近视的机制。方法 选取经散瞳检影验光等效球镜(SE)≤-0.50 D的近视儿童青少年123例,随机分为用药组(白天配戴框架眼镜,每晚0.1 g·L^(-1)低浓度阿托品滴眼1滴)70例以及对照组(仅白天配戴框架眼镜)61例。两组患儿基线时进行SE、眼轴长度(AL)、眼压(IOP)、黄斑中心凹下脉络膜厚度(SFCT)、浅层血管复合体(SVC)、深层血管复合体(DVC)及脉络膜毛细血管(CC)血流密度、RT等检查,并在3个月和6个月时进行两次随访和检查数据比较。结果 两组患儿基线年龄、性别比、IOP、SE、AL、SFCT、黄斑部微血管系统(SVC、DVC及CC)血流密度及平均RT比较,差异均无统计学意义(均为P>0.05)。两组患儿随访3个月和6个月时的SE、AL及SFCT比较,差异均有统计学意义(均为P<0.05)。与基线数据相比,对照组患儿随访6个月时的AL增长,用药组患儿随访3个月和6个月时的SFCT均增厚,差异均有统计学意义(均为P<0.05)。用药组患儿3个月、6个月随访时SVC、DVC及CC血流密度均高于对照组及基线数据,差异均有统计学意义(均为P<0.05)。用药组患儿与对照组比较,3个月、6个月随访时内环鼻侧RT较厚,6个月随访时外环上侧及外环鼻侧RT较厚,3个月、6个月随访时外环颞侧RT较厚,差异均有统计学意义(均为P<0.05)。结论 近视儿童青少年局部使用0.1 g·L^(-1)低浓度阿托品滴眼液可以改善视网膜脉络膜微循环,使脉络膜明显增厚,视网膜轻度增厚,从而延缓近视的进展。

树鼩视网膜微血管内皮细胞永生化细胞株的建立及鉴定

目的建立树鼩视网膜来源的微血管内皮细胞的体外分离培养技术以及永生化细胞株,为体外利用树鼩视网膜微血管内皮细胞开展相关研究提供新的实验材料。方法利用Ⅱ型胶原酶、分散酶和DNaseⅠ酶消化法分离培养出原代视网膜微血管内皮细胞,利用差速消化法纯化内皮细胞,然后利用携带SV40T基因的慢病毒转染细胞,再挑取单克隆后进行传代培养。对传至50代以上的细胞进行形态学观察、免疫荧光鉴定以及核型鉴定。结果采用混合酶消化法能够分离获得微血管内皮细胞,纯化后的细胞呈不规则多角形和梭形。经慢病毒转染后的细胞传代培养后细胞形态一致,到第50代时细胞形态结构仍较好。细胞免疫荧光结果显示标志性蛋白VWF、CD34、Claudin1、ZO-1和永生化SV40T表达阳性。生长曲线结果显示:细胞生长旺盛,第2~4天时处于对数生长期,第4天进入平台期。细胞核型结果显示:染色体数与树鼩染色体相同,即所获取的细胞为永生化的树鼩视网膜微血管内皮细胞。结论成功建立的树鼩视网膜微血管内皮细胞永生化细胞株具有较好的形态结构与功能,为视网膜病变及眼科相关疾病的研究提供了新的实验材料。

全域扫频OCTA与荧光素眼底血管造影在糖尿病视网膜病变应用中的对比分析

目的比较全域扫频光学相干断层扫描血管造影(OCTA)与荧光素眼底血管造影(FFA)对糖尿病视网膜病变(DR)的诊断价值。方法选取2022年2月至2022年4月就诊于安徽医科大学第二附属医院眼科门诊的糖尿病视网膜病变患者35例(70眼),每位患者均使用超广角彩色眼底照相(UWF CFP)检查。采用全域扫频OCTA和FFA分别检查受试者视网膜,并分析两者对视网膜微血管瘤(MA)、视网膜新生血管(RNV)、视网膜无灌注区(NPAs)、视网膜内微血管异常(IRMA)、糖尿病性黄斑水肿(DME)的检出率差异。结果全域扫频OCTA中MA、RNV、NPAs、IRMA、DME的检出率与FFA相比,差异无统计学意义(P>0.05),全域扫频OCTA与FFA对DR的筛查、诊断、分类一致性良好(Kappa值>0.75)。结论全域扫频OCTA在DR应用中具有广阔的前景,可作为DR无创性筛查、诊断、随访的重要依据。