Role of endoplasmic reticulum stress in the loss of retinal ganglion cells in diabetic retinopathy

Endoplasmic reticulum stress is closely involved in the early stage of diabetic retinopathy. In the present study, a streptozotocin-induced diabetic animal model was given an intraperitoneal injection of tauroursodeoxycholic acid. Results from immunofluorescent co-localization experiments showed that both caspase-12 protein and c-Jun N-terminal kinase 1 phosphorylation levels significantly in- creased, which was associated with retinal ganglion cell death in diabetic retinas. The C/ERB ho- mologous protein pathway directly contributed to glial reactivity, and was subsequently responsible for neuronal loss and vascular abnormalities in diabetic retinopathy. Our experimental findings in- dicate that endoplasmic reticulum stress plays an important role in diabetes-induced retinal neu- ronal loss and vascular abnormalities, and that inhibiting the activation of the endoplasmic reticulum stress pathway provides effective protection against diabetic retinopathy.

Alterations in the polysialylated neural cell adhesion molecule and retinal ganglion cell density in mice with diabetic retinopathy

AIM：To investigate the impact of polysialylated neural cell adhesion molecule（PSA-NCAM）on the survival of retinal ganglion cells（RGCs）in the experimentally induced diabetes in mice.METHODS：Diabetes was induced in 2.5 months old Swiss Webster mice by intraperitoneal injection of streptozotocin（STZ,90 mg/kg）once daily for two consecutive days.Examination of the proteins of interest in the retinas from diabetic mice at 2mo after diabetes induction was performed using immunohistochemistry and Western blot analysis.RGCs were counted in the wholemounted retinas,and Brn3a marker was used.RESULTS：Examination of retinas from diabetic mice at 2mo after diabetes induction revealed a considerable reduction in RGC density.Our experiments also demonstrated a redistribution of PSA-NCAM in the retina of diabetic animals.PSA-NCAM immunoreactivity was diminished in the inner part of the retina where RGCs were located.In contrast,an enhanced PSA-NCAM immunoreactivity was detected in the outer layers of the retina.PSA-NCAM signal was co-localized with glial fibrillary acidic protein immunoreactivity in the Müller cell branches.Previous studies have shown that matrix metalloproteinase-9（MMP-9）is responsible for the reduction in PSA-NCAM levels in neuronal cells.The reduced levels of PSA-NCAM in inner layers（nerve fiber layer,ganglion cell layer）were accompanied by the increased expression of MMP-9.In contrast,in the outer retinal layers,the expression of MMP-9 was much less pronounced.CONCLUSION：MMP-9 induces PSA-NCAM shedding in the inner part of the retina and the decreased level of PSA-NCAM in the inner part of the retina might be,at least in part,responsible for the loss of RGCs in diabetic mice.

Inflammation in diabetic retinopathy: possible roles in pathogenesis and potential implications for therapy

Diabetic retinopathy, characterized as a microangiopathy and neurodegenerative disease, is the leading cause of visual impairment in diabetic patients. Many clinical features observed in diabetic retinopathy, such as capillary occlusion, acellular capillaries and retinal non-perfusion, aggregate retinal ischemia and represent relatively late events in diabetic retinopathy. In fact, retinal microvascular injury is an early event in diabetic retinopathy involving multiple biochemical alterations, and is manifested by changes to the retinal neurovascular unit and its cellular components. Currently, intravitreal anti-vascular endothelial growth factor therapy is the firstline treatment for diabetic macular edema, and benefits the patient by decreasing the edema and improving visual acuity. However, a significant proportion of patients respond poorly to anti-vascular endothelial growth factor treatments, indicating that factors other than vascular endothelial growth factor are involved in the pathogenesis of diabetic macular edema. Accumulating evidence confirms that low-grade inflammation plays a critical role in the pathogenesis and development of diabetic retinopathy as multiple inflammatory factors, such as interleukin-1β, monocyte chemotactic protein-1 and tumor necrosis factor-α, are increased in the vitreous and retina of diabetic retinopathy patients. These inflammatory factors, together with growth factors such as vascular endothelial growth factor, contribute to blood-retinal barrier breakdown, vascular damage and neuroinflammation, as well as pathological angiogenesis in diabetic retinopathy, complicated by diabetic macular edema and proliferative diabetic retinopathy. In addition, retinal cell types including microglia, Müller glia, astrocytes, retinal pigment epithelial cells, and others are activated, to secrete inflammatory mediators, aggravating cell apoptosis and subsequent vascular leakage. New therapies, targeting these inflammatory molecules or related signaling pathways, have the potential to inhibit retinal inflammation and prevent diabetic retinopathy progression. Here, we review the relevant literature to date, summarize the inflammatory mechanisms underlying the pathogenesis of diabetic retinopathy, and propose inflammation-based treatments for diabetic retinopathy and diabetic macular edema.

Effects of nuclear factor κB expression on retinal neovascularization and apoptosis in a diabetic retinopathy rat model

AIM: To investigate the expression and role of nuclear factor κB(NF-κB) in diabetic retinopathy(DR) and its relationship with neovascularization and retinal cell apoptosis. METHODS: A total of 80 male Wistar rats were randomly assigned to control(4, 8, 12 and 16 wk, n =10 in each group) and diabetes mellitus(DM) groups(4, 8, 12 and 16wk, n =10 in each group). A diabetic rat model was established by intraperitoneal injection of streptozotocin(60 mg/kg). After 4, 8, 12 and 16 wk, rats were sacrificed.Retinal layers and retinal neovascularization growth were stained with hematoxylin-eosin and examined under light microscopy. Cell apoptosis in the retina was detected by Td T-mediated d UTP nick end labeling, and NF-κB distribution and expression in the retina was determined using immunohistochemistry. RESULTS: DM model success rate up to 100%.Diabetes model at each time point after the experimental groupcompared with the control group, the blood glucose was significantly increased, decreased body weight, each time point showed significant differences compared with the control group(P 【0.01). After 12 wk other pathological changes in the retina of diabetic rats were observed; after 16 wk, neovascularization were observed. After 1mo, retinal cell apoptosis was observed.Compared with the control group, NF-κB expression in the DM group significantly increased with disease duration.CONCLUSION: With the prolonging of DM progression,the expression NF-κB increases. NF-κB may be related to retinal cell apoptosis and neovascularization.

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.

Effect of Sonic hedgehog gene-modified bone marrow mesenchymal stem cells on graft-induced retinal gliosis and retinal ganglion cells survival in diabetic mice

AIM:To investigate the effects of Sonic hedgehog(Shh)gene-modified bone marrow mesenchymal stem cells(MSCs)on graft-induced retinal gliosis and retinal ganglion cells(RGCs)survival in diabetic mice.METHODS:Bone marrow-derived MSCs were genetically modified with the Shh gene to generate a stably transfected cell line of Shh-modified MSCs(MSC-Shh).Intravitreal injections of MSC-Shh and green fluorescent protein-modified MSCs(MSC-Gfp;control)were administered in diabetic mice.After 4wk,the effects of MSC-Shh on retinal gliosis were evaluated using fundus photography,and markers of gliosis were examined by immunofluorescence and Western blotting.The neurotrophic factors expression and RGCs survival in the host retina were evaluated using Western blotting and immunofluorescence.The mechanisms underlying the effects of MSC-Shh was investigated.RESULTS:A significant reduction of proliferative vitreoretinopathy(PVR)was observed after intravitreal injection of MSC-Shh compared to MSC-Gfp.Significant downregulation of glial fibrillary acidic protein(GFAP)was demonstrated in the host retina after MSC-Shh administration compared to MSC-Gfp.The extracellular signal-regulated kinase 1/2(ERK1/2),protein kinase B(AKT)and phosphatidylin-ositol-3-kinase(PI3K)pathways were significantly downregulated after MSC-Shh administration compared to MSC-Gfp.Brain-derived neurotrophic factor(BDNF)and ciliary neurotrophic factor(CNTF)levels were significantly increased in the host retina,and RGCs loss was significantly prevented after MSC-Shh administration.CONCLUSION:MSC-Shh administration reduces graft-induced reactive gliosis following intravitreal injection in diabetic mice.The ERK1/2,AKT and PI3K pathways are involved in this process.MSC-Shh also increases the levels of neurotrophic factors in the host retina and promoted RGCs survival in diabetic mice.

Association between retinal neuronal degeneration and visual function impairment in type 2 diabetic patients without diabetic retinopathy

The changes in retinal thickness and visual function in type 2 diabetic patients without clinical evidence of diabetic retinopathy were evaluated. A total of 141 diabetic subjects without retinopathy and 158 healthy subjects were enrolled in this study. Superior macular ganglion cell complex thicknesses were significantly decreased in diabetic cases, and no significant peripapillary retinal nerve fiber layer thickness changes were observed. The contrast sensitivities at all space frequencies were significantly different between diabetic patients and controls. The mean P50 amplitude from pattern electroretinogram results was reduced significantly in the diabetic group. In the diabetic group, average superior ganglion cell complex thicknesses positively correlated with both contrast sensitivities at high spatial frequencies and P50 amplitudes. The results indicated that ganglion cell complex thickness and visual function changes could be observed in diabetic subjects before the onset of any significant diabetic retinopathy. Macular ganglion cell complex reduction occurred much earlier than peripapillary retinal nerve fiber layer thinning in diabetic patients without retinopathy.

Diabetic retinopathy:Role of inflammation and potential therapies for anti-inflammation

Diabetic retinopathy is a leading cause of blindness among working-age adults.Despite many years of research,treatment options for diabetic retinopathy remain limited and with adverse effects.Discovery of new molecular entities with adequate clinical activity for diabetic retinopathy remains one of the key research priorities in ophthalmology.This review is focused on the therapeutic effects of cannabidiol(CBD),a nonpsychoactive native cannabinoid,as an emerging and novel therapeutic modality in ophthalmology based on systematic studies in animal models of inflammatory retinal diseases including diabetic retinopathy-a retinal disease associated with vascular-neuroinflammation.Special emphasis is placed on novel mechanisms which may shed light on the pharmacological activity asso c iated with CBD preclinically.These include a selfdefence system against inflammation and neurodegeneration mediated by inhibition of equilibrat ive nucleoside transporter and activation of adenosine receptor by treatment with CBD.

Gac fruit extracts ameliorate proliferation and modulate angiogenic markers of human retinal pigment epithelial cells under high glucose conditions

Objective: To investigate the impact of the extracts of Gac fruit parts(peel, pulp, seed, and aril) on the cell viability and angiogenesis markers of human retinal pigment epithelial(ARPE-19) cells under high glucose conditions. Methods: The effect of the extracts of Gac fruit peel, pulp, seed and aril on the ARPE-19 cells was determined using MTT viability assay, Trypan blue dye and morphological changes were observed using light microscopy. Enzyme-linked immunosorbent-based assay was performed to evaluate the effect of Gac fruit parts on the reactive oxygen species(ROS), vascular endothelial growth factor(VEGF) and pigmented epithelium-derived factor(PEDF) secretions. Results: High glucose(HG) at 30 mmol/L increased ARPE-19 cell viability and ROS and VEGF secretions. While, the exposure of ARPE-19 cells in high glucose condition to Gac fruit extracts led to inhibition of cell viability, induced morphological changes, decreased ROS and VEGF secretions, and increased PEDF level. Gac pulp, seed, and aril at 1 000 μg/mL showed significant inhibition activities [(7.5 ± 5.1)%,(2.7 ± 0.5)%,(3.2 ± 1.1)%, respectively] against HG-induced ARPE-19 cell viability. The findings also demonstrated that Gac aril at 250 μg/mL significantly decreased ROS and VEGF levels [(40.6 ± 3.3) pg/mL,(107.4 ± 48.3) pg/mL, respectively] compared to ROS [(71.7 ± 2.9) pg/mL ] and VEGF [(606.9 ± 81.1) pg/mL] in HG untreated cells. Moreover, 250 μg/mL of Gac peel dramatically increased PEDF level [(18.2 ± 0.3) ng/mL] compared to that in HG untreated cells [(0.48 ± 0.39) ng/mL]. Conclusions: This study indicates that the extracts of Gac peel, pulp, seed and aril reduced cell viability, minimized ROS generations and showed angiogenic activities. Therefore, our findings open new insights into the potentiality of Gac fruit against HG-related diabetic retinopathy disease.

Autophagy dysregulation mediates the damage of high glucose to retinal pigment epithelium cells

AIM:To observe the role and mechanism of autophagy in retinal pigment epithelial cell(RPE)damaged by high glucose,so as to offer a new idea for the treatment of diabetic retinopathy(DR).METHODS:ARPE-19,a human RPE cell line cultured in vitro was divided into the normal control(NC),autophagy inhibitor 3-methyladenine(3-MA),high-glucose(HG),and HG+3-MA groups.Cell viability was detected by CCK-8 assay and the apoptosis rate was measured by flow cytometry.The protein expressions of apoptosis markers,including Bax,Bcl-2,and Caspase-3,as well as autophagy marker including microtubule-related protein 1 light chain 3(LC3),p62,and mechanistic target of rapamycin(m TOR)were detected by Western blotting.Autophagic flux was detected by transfection with Ad-m Cherry-GFP-LC3 B.RESULTS:Under high glucose conditions,the viability of ARPE-19 was decreased,and the apoptosis rate increased,the protein expressions of Bax,Caspase-3,and LC3-II/LC3-I were all increased and the expressions of Bcl-2,p62 and p-m TOR decreased,and autophagic flux was increased compared with that of the controls.Treatment with 3-MA reversed all these changes caused by high glucose.CONCLUSION:The current study demonstrates the mechanisms of cell damage of ARPE-19 through high glucose/m TOR/autophagy/apoptosis pathway,and new strategies for DR may be developed based on autophagy regulation to manage cell death of RPE cells.

炎症在糖尿病视网膜病变中的作用:发病机制及治疗策略

糖尿病视网膜病变(DR)被认为是一种慢性中低度炎症性(微炎症)疾病。炎症贯穿DR发病全过程,表现为全身及眼局部炎症生物标志物增加。在DR患者眼中,促炎介质增加,如白细胞介素(IL)-1β、IL-6和肿瘤坏死因子-α(TNF-α)等增加;炎症细胞活化并增多,如视网膜中活化的小胶质细胞、Müller细胞和单核巨噬细胞浸润等。此外,免疫细胞也参与了DR的发病,如循环T淋巴细胞参与白细胞瘀滞。这些研究结果表明DR具有慢性炎症的病因,多种炎症相关因素共同参与、互相影响,导致血-视网膜屏障破坏和神经元损伤,加重DR的进展。因此,个体化抗炎治疗在DR治疗中具有重要意义。

小胶质细胞在糖尿病视网膜病变视网膜神经血管单元中的作用

糖尿病视网膜病变(DR)是工作年龄人群主要致盲眼病,血-视网膜屏障破坏是关键环节。近年研究显示,DR不再是单纯微血管病变,而是视网膜胶质细胞与神经退行性变、微血管病变的共同发展结果。DR病程早期视网膜神经血管单元(RNVU)中神经元的损伤可能早于血管内皮的变化,胶质细胞的激活加重血管屏障功能障碍。视网膜小胶质细胞是常驻视网膜的局部免疫细胞,参与长期高糖诱导的慢性炎症反应、高糖诱导其分泌多种炎症因子,破坏血-视网膜屏障结构、增加神经元凋亡、改变Müller细胞胶质化等,影响视网膜局部稳态平衡。RNVU作为一个单元结构研究,近年来受到越来越多的关注,本文将针对小胶质细胞在RNVU中的作用机制、研究进展进行综述。

Diabetes and high-glucose could upregulate the expression of receptor for activated C kinase 1 in retina

BACKGROUND Diabetic retinopathy(DR)is a major ocular complication of diabetes mellitus,leading to visual impairment.Retinal pigment epithelium(RPE)injury is a key component of the outer blood retinal barrier,and its damage is an important indicator of DR.Receptor for activated C kinase 1(RACK1)activates protein kinase C-ε(PKC-ε)to promote the generation of reactive oxygen species(ROS)in RPE cells,leading to apoptosis.Therefore,we hypothesize that the activation of RACK1 under hypoxic/high-glucose conditions may promote RPE cell apoptosis by modulating PKC-ε/ROS,thereby disrupting the barrier effect of the outer blood retinal barrier and contributing to the progression of DR.AIM To investigate the role and associated underlying mechanisms of RACK1 in the development of early DR.METHODS In this study,Sprague-Dawley rats and adult RPE cell line-19(ARPE-19)cells were used as in vivo and in vitro models,respectively,to explore the role of RACK1 in mediating PKC-εin early DR.Furthermore,the impact of RACK1 on apoptosis and barrier function of RPE cells was also investigated in the former model.RESULTS Streptozotocin-induced diabetic rats showed increased apoptosis and upregulated expression of RACK1 and PKC-εproteins in RPE cells following a prolonged modeling.Similarly,ARPE-19 cells exposed to high glucose and hypoxia displayed elevated mRNA and protein levels of RACK1 and PKC-ε,accompanied by an increases in ROS production,apoptosis rate,and monolayer permeability.However,silencing RACK1 significantly downregulated the expression of PKC-εand ROS,reduced cell apoptosis and permeability,and protected barrier function.CONCLUSION RACK1 plays a significant role in the development of early DR and might serve as a potential therapeutic target for DR by regulating RPE apoptosis and barrier function.

Potential therapeutic applications of mesenchymal stem cells for the treatment of eye diseases

Stem cell-based treatments have been extensively explored in the last few decades to develop therapeutic strategies aimed at providing effective alternatives for those human pathologies in which surgical or pharmacological therapies produce limited effects.Among stem cells of different sources,mesenchymal stem cells(MSCs)offer several advantages,such as the absence of ethical concerns,easy harvesting,low immunogenicity and reduced tumorigenesis risks.Other than a multipotent differentiation ability,MSCs can release extracellular vesicles conveying proteins,mRNA and microRNA.Thanks to these properties,new therapeutic approaches have been designed for the treatment of various pathologies,including ocular diseases.In this review,the use of different MSCs and different administration strategies are described for the treatment of diabetic retinopathy,glaucoma,and retinitis pigmentosa.In a large number of investigations,positive results have been obtained by in vitro experiments and by MSC administration in animal models.Most authors agree that beneficial effects are likely related to MSC paracrine activity.Based on these considerations,many clinical trials have already been carried out.Overall,although some adverse effects have been described,promising outcomes are reported.It can be assumed that in the near future,safer and more effective protocols will be developed for more numerous clinical applications to improve the quality of life of patients affected by eye diseases.

转甲状腺素蛋白介导STAT4/miR-223-3p/FBXW7通路对高糖诱导的人视网膜内皮细胞新生血管生成的影响

目的分析转甲状腺素蛋白(TTR)介导信号转导和转录激活因子4(STAT4)/miR-223-3p/F-box WD重复蛋白7(FBXW7)通路对高糖(HG)诱导的人视网膜内皮细胞(hRECs)新生血管生成的影响。方法将hRECs分为对照组、HG组、HG+TTR组、HG+NC mimic组、HG+miR-223-3p mimic组和HG+TTR+miR-223-3p mimic组。生物信息学分析STAT4与miR-223-3p的靶向关系,并采用双荧光素酶报告实验进行验证。ELISA检测细胞中TTR水平,RT-PCR检测miR-223-3p水平,CCK-8法检测细胞增殖,血管生成实验分析血管生成率,Western blot检测血管内皮生长因子(VEGF)、TTR、STAT4和FBXW7蛋白表达。结果与对照组相比,HG组hRECs中TTR水平升高(P<0.05)。培养24 h时,与HG组相比,HG+TTR组细胞活力明显下降(P<0.05)。与HG组血管生成率(38.12±4.91)%相比,HG+TTR组hRECs血管生成率[(19.46±2.12)%]明显较小(P<0.05)。与HG+NC mimic组相比,HG+miR-223-3p mimic组hRECs中miR-223-3p表达上调,细胞活力增加(均为P<0.05);与HG+miR-223-3p mimic组相比,HG+TTR+miR-223-3p mimic组hRECs中miR-223-3p表达下调,细胞活力下降(均为P<0.05)。与HG+NC mimic组血管生成率(40.11±4.10)%相比,HG+miR-223-3p mimic组hRECs血管生成率[(61.52±6.25)%]增多(P<0.05);与HG+miR-223-3p mimic组相比,HG+TTR+miR-223-3p mimic组hRECs血管生成率[(42.24±4.33)%]减少(P<0.05),且与HG+NC mimic组比较差异无统计学意义(P>0.05)。生物信息学分析结果显示,STAT4与miR-223-3p启动子区域相互作用。与HG组相比,HG+TTR组hRECs中miR-223-3p、STAT4蛋白表达均降低,FBXW7和VEGF蛋白表达均升高(均为P<0.05);与HG+TTR组相比,HG+miR-223-3p mimic组hRECs中miR-223-3p、STAT4蛋白表达均升高,FBXW7和VEGF蛋白表达均降低(均为P<0.05);与HG+miR-223-3p mimic组相比,HG+TTR+miR-223-3p mimic组hRECs中miR-223-3p、STAT4蛋白表达均降低,FBXW7和VEGF蛋白表达均升高(均为P<0.05);与HG+TTR组相比,HG+TTR+miR-223-3p mimic组hRECs中miR-223-3p、VEGF、STAT4和FBXW7蛋白表达差异均无统计学意义(均为P>0.05)。结论TTR可能通过调节STAT4/miR-223-3p/FBXW7通路抑制HG诱导的hRECs的血管生成。

当归多糖通过Akt/GSK-3β通路增强Nrf2信号传导对高糖诱导视网膜神经节细胞凋亡的抑制作用

目的:探讨当归多糖对高糖诱导的视网膜神经节细胞凋亡的抑制作用及其可能机制。方法:采用不同浓度当归多糖干预视网膜神经节细胞RGC-5,MTT法检测细胞存活情况并筛选合适浓度。培养RGC-5细胞并添加50 mmol/L葡萄糖建立高糖损伤模型后,分为高糖组、当归多糖组和当归多糖+Nrf2信号通路抑制剂(ML385)组,另设对照组。当归多糖组使用100μmol/L的当归多糖处理,当归多糖+ML385组使用100μmol/L的当归多糖联合20μmol/L的ML385处理,高糖组使用50 mmol/L葡萄糖处理,对照组加入等量完全培养基。CCK-8法检测细胞存活情况;流式细胞术检测细胞凋亡率;ELISA法检测各组细胞中丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-Px)、超氧化物岐化酶(SOD)含量;实时定量PCR法检测细胞中蛋白激酶B(Akt)mRNA、糖原合成酶激酶-3β(GSK-3β)mRNA和核因子E2相关因子2(Nrf2)mRNA表达情况;Western blotting检测凋亡蛋白B细胞淋巴瘤因子2(Bcl-2)、Bcl-2相关蛋白(Bax)、Caspase-3和Caspase-9蛋白表达情况。结果:当归多糖组细胞活力、GSH-Px含量、SOD含量、Nrf2 mRNA相对表达量、Bcl-2蛋白相对表达量均高于高糖组,细胞凋亡率、MDA含量、Akt mRNA相对表达量、GSK-3βmRNA相对表达量、Bax蛋白相对表达量、Caspase-3蛋白相对表达量、Caspase-9蛋白相对表达量均低于高糖组(P<0.05)。当归多糖+ML385组细胞活力、GSH-Px含量、SOD含量、Nrf2 mRNA相对表达量、Bcl-2蛋白相对表达量均低于当归多糖组,细胞凋亡率、MDA含量、Akt mRNA相对表达量、GSK-3βmRNA相对表达量、Bax蛋白相对表达量、Caspase-3蛋白相对表达量、Caspase-9蛋白相对表达量均高于当归多糖组(P<0.05)。结论:当归多糖能抑制高糖诱导的RGC-5细胞凋亡,降低细胞内氧化应激水平,可能机制为通过阻滞Akt/GSK-3β通路,提高细胞内抗氧化Nrf2表达水平。

糖尿病患者黄斑神经节细胞复合体、视盘周围视网膜神经纤维层厚度水平与视网膜病变的相关性分析

目的分析糖尿病(diabetes mellitus,DM)患者黄斑神经节细胞复合体(ganglion cell complex,GCC)、视盘周围视网膜神经纤维层(retinal nerve fiber layer,RNFL)的厚度水平与视网膜病变(diabetic retinopathy,DR)的关系。方法选择2020年2月至2022年3月首都医科大学宣武医院收治的DM患者110例145眼作为研究对象,按照是否发生DR分为DR组(51例,69眼)和非DR组(59例,76眼),依据DR不同严重程度分期将DR组分为非增殖性DR组(NPDR组,38例,47眼)和增殖性DR组(PDR组,13例,22眼)。两组患者均进行光学相干断层扫描血管成像技术(optical coherence tomography angiography,OCTA)检查,比较不同分组DM患者黄斑部GCC和视盘周围RNFL厚度变化及与DR分期的关系。结果DR组GCC总体厚度(total GCC thickness,tGCC)、GCC上方厚度(superior GCC thickness,sGCC)和GCC下方厚度(inferior GCC thickness,iGCC)均低于非DR组(P<0.05);PDR组tGCC、sGCC和iGCC均低于轻中度NPDR组(mNPDR)和重度NPDR组(sNPDR),且sNPDR组tGCC、sGCC和iGCC均低于mNPDR(P<0.05);NPDR组视盘周围RNFL上方和下方以及平均厚度均低于非DR组和PDR组(P<0.05),PDR组视盘周围RNFL各部位厚度均高于非DR组和NPDR组(P<0.05);sNPDR组视盘周围上方、下方、颞侧、鼻侧及平均厚度均低于mNPDR组(P<0.05),PDR轻中度组和重度组视盘周围各部位RNFL厚度均高于mNPDR组和sNPDR组,且PDR重度组各部位RNFL厚度均高于轻中度组(P<0.05);Spearman相关性分析结果显示,DR患者黄斑tGCC、sGCC和iGCC厚度与DR严重程度呈负相关(r分别为-0.485、-0.496、-0.501,P<0.05),DR患者视盘周围各部位RNFL厚度与NPDR分期呈负相关(r分别为-0.519、-0.527、-0.498、-0.507、-0.511,P<0.05),与PDR严重程度呈正相关(r分别为0.531、0.514、0.501、0.512、0.499,P<0.05)。结论DR患者早期已经出现视网膜神经的损伤,黄斑GCC厚度随着DR进展明显下降;NPDR患者视盘周围RNFL厚度呈现下降趋势,随着DR进展至PDR,视盘周围RNFL厚度有所增加,黄斑GCC和视盘周围RNFL厚度的动态变化能够为糖尿病患者视网膜神经结构的损伤和DR的进展提供依据。

白花蛇舌草对高糖诱导的人视网膜血管内皮细胞增殖、凋亡、活性氧水平的影响研究

目的探讨白花蛇舌草注射液(HDI)对高糖诱导的人视网膜血管内皮细胞(HRCECs)增殖、凋亡、氧化应激的影响及机制。方法2019年10月至2020年6月,体外培养HRCECs。将HRCECs分为对照组(5.5 mmol/L葡萄糖处理细胞)、高渗对照组(19.5 mmol/L甘露醇及5.5 mmol/L葡萄糖处理细胞)、高糖对照组(25 mmol/L葡萄糖处理细胞)、白花蛇舌草组(HDI组)(6.25 mL/L、12.5 mL/L、25 mL/L、50 mL/L HDI和25 mmol/L的葡萄糖处理细胞)。处理细胞48 h,通过CCK8法检测细胞活力;流式细胞术检测细胞周期及凋亡率;激光扫描共聚焦显微镜检测活性氧水平。蛋白质印迹法检测增殖细胞核抗原(PCNA)、细胞周期蛋白A1(CyclinA1)和活化胱天蛋白酶-3(cleaved caspase-3)蛋白表达。结果与对照组比较,高糖组细胞活性明显升高[(1.116±0.105)比(0.684±0.072)](P<0.05)。与高糖组比较,12.5 mL/L、25 mL/L和50 mL/L的白花蛇舌草组细胞活性明显降低[(0.847±0.076)、(0.639±0.058)、(0.421±0.042)比(1.116±0.105)](P<0.05)。选择50 mL/L的白花蛇舌草作为研究对象。与对照组比较,高糖组G_(0)/G期细胞百分比[(60.02±5.01)%比(54.72±4.31)%]、细胞凋亡率[(17.11±1.01)%比(3.32±0.47)%]、活性氧水平[(96.18±5.22)比(42.14±3.56)]及PCNA、CyclinA1和cleaved caspase-3表达均明显升高,G_(2)/M期和S期细胞百分比明显降低(P<0.05)。与高糖组比较,HDI组G_(0)/G期细胞百分比[(32.31±3.42)%比(60.02±5.01)%]、细胞凋亡率[(9.72±0.73)%比(17.11±1.01)%]、活性氧水平[(67.75±4.83)比(96.18±5.22)]及PCNA、CyclinA1和cleaved caspase-3表达均明显降低,G_(2)/M期和S期细胞百分比明显升高(P<0.05)。结论白花蛇舌草可降低高糖诱导的HRCECs增殖、凋亡及活性氧水平,阻滞细胞周期,机制可能与调节PCNA、CyclinA1和cleaved caspase-3表达有关。

补阳还五汤对高糖培养的人视网膜微血管内皮细胞自噬和血管形成的干预作用

目的探究补阳还五汤(BYHW)对高糖培养的人视网膜微血管内皮细胞(HRCECs)自噬影响和血管形成的干预作用。方法将HRCECs随机分为对照组(CG)、模型组(MG)、BYHW组、自噬抑制剂3-甲基腺嘌呤(3-MA)组。除CG组外,其余各组细胞于25 g/L葡萄糖环境中培养建立高糖损伤模型,BYHW组在MG组的基础上加入5 mg/mL的BYHW水提液,3-MA组加入40μM的3-MA,各组细胞均孵育24 h。采用血管形成实验检测HRCECs血管生成情况,划痕实验检测细胞迁移能力,实时荧光定量PCR(RT-qPCR)和Western Blot法分别检测细胞中微管相关蛋白1A/1B-轻链3(LC3)、苄氯素1(Becline-1)的mRNA和蛋白表达水平。结果(1)血管形成:与CG组比较,MG组血管形成数升高(t=7.747,P=0.000);与MG组比较,BYHW、3-MA组血管形成数降低(t_(BYHW)=-6.303,P=0.000;t_(3-MA)=-4.596,P=0.002),差异均有统计学意义。而BYHW、3-MA组血管形成数比较,差异无统计学意义(P>0.05)。(2)迁移能力:与CG组比较,MG组HRCECs迁移率升高(t=14.035,P=0.000);与MG组比较,BYHW、3-MA组HRCECs迁移率降低(t_(BYHW)=-9.247,t_(3-MA)=-5.358,均P=0.000);与BYHW组比较,3-MA组HRCECs迁移率升高(t=-3.890,P=0.003),差异均有统计学意义。(3)自噬:与CG组比较,MG组LC3 mRNA、蛋白表达水平升高(t_(mRNA)=8.249,t_(蛋白)=14.890,均P=0.000),Beclin-1蛋白表达水平升高(t=7.114,P=0.000);与MG组比较,BYHW、3-MA组的LC3 mRNA和蛋白表达水平均降低(BYHW:t_(mRNA)=-9.298,t_(蛋白)=-11.727,均P=0.000。3-MA:t_(mRNA)=-3.718,P=0.006;t_(蛋白)=-7.511,P=0.000),BYHW组的Beclin-1蛋白表达水平下降(t=-4.115,P=0.003);与BYHW组比较,3-MA组HRCECs的Beclin-1蛋白表达水平升高(t=-2.448,P=0.038),差异均有统计学意义。结论BYHW延缓非增殖期糖尿病视网膜病的发展可能与其调控HRCECs自噬减少血管形成和迁移有关。

二甲双胍对高糖诱导的RF/6A细胞增殖、迁移和血管形成的影响及其可能机制

目的研究二甲双胍(MET)对高糖诱导的恒河猴脉络膜⁃视网膜内皮细胞RF/6A增殖、迁移和血管形成的影响,并初步探讨其作用机制。方法RF/6A细胞随机分为5组:NC组、HG组、HG+15 mmol·L^(-1) MET组、HG+30 mmol·L^(-1) MET组、HG+45 mmol·L^(-1) MET组,CCK⁃8法检测细胞增殖情况。将RF/6A细胞随机分为4组:NC组和NC+15 mmol·L^(-1) MET组、NC+30 mmol·L^(-1) MET组、NC+45 mmol·L^(-1) MET组,CCK⁃8法检测MET的细胞毒性。将RF/6A细胞随机分为3组:NC组、HG组和HG+15 mmol·L^(-1) MET组,细胞划痕法和Transwell法检测细胞迁移,Matrigengel法检测细胞血管形成,RT⁃PCR、Western blot检测Hippo信号通路的核心成分YAP、TEAD1的mRNA及蛋白表达。结果CCK⁃8法检测结果显示,与NC组比较,HG组RF/6A细胞的增殖活性升高(P<0.001);与HG组比较,HG+15 mmol·L^(-1) MET组、HG+30 mmol·L^(-1) MET组和HG+45 mmol·L^(-1) MET组RF/6A细胞的增殖活性均降低(均为P<0.001)。与NC组比较,NC+15 mmol·L^(-1) MET组RF/6A细胞增殖活性无变化(P=0.2273),因此选择15 mmol·L^(-1) MET进行后续实验。细胞划痕法和Transwell法检测结果显示,与NC组比较,HG组RF/6A细胞的迁移率升高、迁移个数增加(均为P<0.01);与HG组比较,HG+15 mmol·L^(-1) MET组RF/6A细胞的迁移率降低、迁移个数减少(均为P<0.01)。Matrigengel法检测结果显示,与NC组比较,HG组RF/6A细胞的血管相对总长度增加(P<0.05);与HG组比较,HG+15 mmol·L^(-1) MET组RF/6A细胞的血管相对总长度减少(P<0.01)。RT⁃PCR检测结果显示,与NC组比较,HG组RF/6A细胞的YAP和TEAD1 mRNA相对表达量均增加(均为P<0.05);与HG组比较,HG+15 mmol·L^(-1) MET组RF/6A细胞的YAP和TEAD1 mRNA相对表达量均减少(均为P<0.05)。Western blot检测结果显示,与NC组比较,HG组RF/6A细胞的YAP和TEAD1蛋白相对表达量均增加(均为P<0.05);与HG组比较,HG+15 mmol·L^(-1) MET组RF/6A细胞的YAP和TEAD1蛋白相对表达量均减少(均为P<0.05)。结论MET可抑制高糖诱导的RF/6A细胞增殖、迁移和血管形成,下调YAP、TEAD1的mRNA及蛋白表达,推测其作用机制与Hippo信号通路有关。