Intrinsic apoptotic pathway and G2/M cell cycle arrest involved in tubeimoside I-induced EC109 cell death

Objective： Squamous esophageal carcinoma is highly prevalent in developing countries, especially in China. Tu Bei Mu （TBM）, a traditional folk medicine, has been used to treat esophageal squamous cell carcinoma （ESCC） for a long term. tubeimoside I （TBMS1） is the main component of TBM, exhibiting great anticancer potential. In this study, we investigated the mechanism of TBMS1 cytotoxic effect on EC109 cells. Methods： Comparative nuclear proteomic approach was applied in the current study and we identified several altered protein spots. Further biochemical studies were carried out to detect the mitochondrial membrane potential, cell cycle and corresponding proteins＇ expression and location. Results： Subcellular proteomic study in the nucleus from EC109 cells revealed that altered proteins were associated with mitochondrial function and cell proliferation. Further biochemical studies showed that TBMSl-induced molecular events were related to mitochondria-induced intrinsic apoptosis and P21-cyclin B 1/cdc2 complex-related G2/M cell cycle arrest. Conclusions： Considering the conventional application of TBM in esophageal cancer, TBMS1 therefore may have a great potential as a chemotherapeutic drug candidate for ESCC.

Intestinal M cells:The fallible sentinels?

The gastrointestinal tract represents the largest mucosal membrane surface in the human body. The immune system in the gut is the first line of host defense against mucosal microbial pathogens and it plays a crucial role in maintaining mucosal homeostasis. Membranous or microfold cells, commonly referred to as microfold cells, are specialized epithelial cells of the gut-associated lymphoid tissues (GALT) and they play a sentinel role for the intestinal immune system by delivering luminal antigens through the follicle-associated epithelium to the underlying immune cells. M cells sample and uptake antigens at their apical membrane, encase them in vesicles to transport them to the basolateral membrane of M cells, and from there deliver antigens to the nearby lymphocytes. On the flip side, some intestinal pathogens exploit M cells as their portal of entry to invade the host and cause infections. In this article, we briefly review our current knowledge on the morphology, development, and function of M cells, with an emphasis on their dual role in the pathogenesis of gut infection and in the development of host mucosal immunity.

Prion Protein Binds to Aldolase A Produced by Bovine Intestinal M Cells

Microfold (M) cells are a kind of intestinal epithelial cell in the follicle-associated epithelium (FAE) of Peyer’s patches. They can transport antigens and microorganisms to lymphoid tissues. Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative disorder in cattle. It is linked to variant Creutzfeldt-Jakob disease in humans. Although it is thought that M cells transport the BSE agent, the exact mechanism by which it crosses the intestinal barrier is not clear. We have bovine intestinal epithelial cell line (BIE cells), which can differentiate into the M cell type in vitro after stimulation, and which is able to transport the BSE agent. We show here that M cells are able to incorporate large numbers of PrP coated magnetic particles into intracellular vesicles, which we collected. The results of 2-DE show a specific protein associated with the PrP-coated particles, compared with non-coated particles. This protein was identified as aldolase A, a glycolytic pathway enzyme, using LC-MS/MS analysis. Aldolase A was synthesized and secreted by BIE cells, and increased during M cell differentiation. In the villi of the bovine intestine, aldolase A was detected on the surface of the epithelium and in the mucus droplet of goblet cells. In the FAE of bovine jejunal and ileal Peyer’s patches, aldolase A was localized on the surface and the apical part of the M cells. The binding of rbPrP to aldolase A was clearly detected and inhibited by pre-treatment of anti-aldolase A antibody. Aldolase A was co-stained with incorporated PrPSc in M-BIE cells. These results suggest that bovine M cells and goblet cells synthesize aldolase A, and that aldolase A may have the ability to bind PrP and associate with PrP in cellular vesicles. Therefore, aldolase A-positive M cells may play a key role in the invasion of BSE into the body.

Overexpressing NeuroD1 reprograms Müller cells into various types of retinal neurons

The onset of retinal degenerative disease is often associated with neuronal loss. Therefore, how to regenerate new neurons to restore vision is an important issue. NeuroD1 is a neural transcription factor with the ability to reprogram brain astrocytes into neurons in vivo. Here, we demonstrate that in adult mice, NeuroD1 can reprogram Müller cells, the principal glial cell type in the retina, to become retinal neurons. Most strikingly, ectopic expression of NeuroD1 using two different viral vectors converted Müller cells into different cell types. Specifically, AAV7 m8 GFAP681::GFP-ND1 converted Müller cells into inner retinal neurons, including amacrine cells and ganglion cells. In contrast, AAV9 GFAP104::ND1-GFP converted Müller cells into outer retinal neurons such as photoreceptors and horizontal cells, with higher conversion efficiency. Furthermore, we demonstrate that Müller cell conversion induced by AAV9 GFAP104::ND1-GFP displayed clear dose-and time-dependence. These results indicate that Müller cells in adult mice are highly plastic and can be reprogrammed into various subtypes of retinal neurons.

Homer1a reduces inflammatory response after retinal ischemia/reperfusion injury

Elevated intraocular pressure(IOP)is one of the causes of retinal ischemia/reperfusion injury,which results in NRP3 inflammasome activation and leads to visual damage.Homerla is repo rted to play a protective role in neuroinflammation in the cerebrum.However,the effects of Homerla on NLRP3inflammasomes in retinal ischemia/reperfusion injury caused by elevated IOP remain unknown.In our study,animal models we re constructed using C57BL/6J and Homer1^(flox/-)/Homerla^(+/-)/Nestin-Cre^(+/-)mice with elevated IOP-induced retinal ischemia/repe rfusion injury.For in vitro expe riments,the oxygen-glucose deprivation/repe rfusion injury model was constructed with M uller cells.We found that Homerla ove rexpression amelio rated the decreases in retinal thickness and Muller cell viability after ischemia/reperfusion injury.Furthermore,Homerla knockdown promoted NF-κB P65^(Ser536)activation via caspase-8,NF-κB P65 nuclear translocation,NLRP3 inflammasome formation,and the production and processing of interleukin-1βand inte rleukin-18.The opposite results we re observed with Homerla ove rexpression.Finally,the combined administration of Homerla protein and JSH-23 significantly inhibited the reduction in retinal thickness in Homer1^(flox/-)Homer1a^(+/-)/Nestin-Cre^(+/-)mice and apoptosis in M uller cells after ischemia/reperfusion injury.Taken together,these studies demonstrate that Homer1a exerts protective effects on retinal tissue and M uller cells via the caspase-8/NF-KB P65/NLRP3 pathway after I/R injury.

稳定表达PRRSV M蛋白的MARC-145^(ORF6)细胞系的构建及其对PRRSV增殖的影响

为了给深入研究猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndromevirus,PRRSV)ORF6基因编码的M蛋白的生物学功能提供重要试验材料,本研究首先利用慢病毒包装系统构建了过表达PRRSVORF6基因的重组慢病毒质粒,将该质粒连同辅助质粒共同转染至HEK293T细胞获得重组慢病毒;之后将重组慢病毒感染MARC-145细胞,利用嘌呤霉素结合有限稀释法进行筛选,连续筛选3轮后建立了稳定表达PRRSVM蛋白的MARC-145ORF6细胞系;并使用CCK-8试验评估过表达PRRSVM蛋白对MARC-145细胞生长的影响。利用RT-PCR、蛋白免疫印迹(Westernblot)和间接免疫荧光(IFA)评估MARC-145ORF6细胞系的传代稳定性并鉴定M蛋白的亚细胞定位,进一步利用RT-qPCR评估过表达M蛋白对MARC-145细胞的干扰素及相关调节基因的影响;此外,还测定了PRRSV在MARC-145ORF6细胞系、MARC-145Flag细胞系和MARC-145细胞中的病毒滴度并绘制多步生长曲线以比较其差异。CCK-8试验结果表明,过表达PRRSVM蛋白对MARC-145细胞活力无显著影响;RT-qPCR、Westernblot和IFA等试验结果表明,MARC-145ORF6细胞系能够表达PRRSV的M蛋白且在传代过程中稳定。此外,稳定表达PRRSVM蛋白显著下调了细胞系的Ⅰ型干扰素及其相关调节基因;多步生长曲线表明,MARC-145ORF6细胞系促进PRRSV增殖,提高其病毒滴度。综上,本研究构建了可以稳定表达PRRSVM蛋白的MARC-145ORF6细胞系,发现其Ⅰ型干扰素水平显著下调且促进PRRSV复制。本研究构建的MARC-145ORF6细胞系将为M蛋白功能的深入研究提供重要生物材料。

m^(6)A修饰在非小细胞肺癌中作用的研究进展

肺癌是全世界常见的恶性肿瘤,也是恶性肿瘤导致死亡的常见原因。根据肺癌的分化程度和形态特征,目前将肺癌分为两大类,即小细胞肺癌和非小细胞肺癌(non-small-cell lung cancer,NSCLC),其中常见的是NSCLC,约占肺癌的85%^([1])。尽管近几十年来诊断和治疗技术得到了改进,但NSCLC的不良预后导致患者的五年生存率始终低于20%^([2]),严重危害人类健康,是人类未解决的难题,因此,还需要对其发生发展的分子机制有更深入的认识。近年,RNA的N^(6)-甲基腺嘌呤(N^(6)-methyladenosine,m^(6)A)修饰与肿瘤的关系得到了广泛关注,大量研究揭示m^(6)A修饰参与包括NSCLC在内的各种肿瘤的发生发展过程,本文总结了近年m^(6)A修饰各种相关调节因子在NSCLC中发挥的细胞生物学功能及分子机制的研究进展。

姜黄素介导的骨髓间充质干细胞外泌体对ACC-M细胞增殖、迁移和侵袭的影响

目的:探讨姜黄素介导的骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)外泌体对ACC-M细胞增殖和转移的影响及其作用机制。方法:将7.5μmmol/L的姜黄素与人BMSCs共培养72 h后,分离外泌体并进行鉴定,将外泌体与ACC-M细胞进行共培养,并将ACC-M细胞分为control组(正常培养)、BMSC-Exo组(未经姜黄素处理的BMSCs外泌体与ACC-M细胞共培养)、Cur-BMSC-Exo组(经姜黄素处理的BMSCs外泌体与ACC-M细胞共培养)及Cur组(经7.5μmmol/L的姜黄素处理细胞);培养48 h后使用蛋白质印迹法(Western blotting)实验检测外泌体肿瘤易感基因101(tumor susceptibility gene 101,TSG-101)、CD63、CD9和重组人钙连蛋白(calnexin,CANX)及ACC-M细胞上皮钙黏素(E-cadherin)、神经钙黏素(N-cadherin)、波形蛋白(vimentin)和转化生长因子β1(transforming growth factor-β1,TGF-β1)、细胞外信号调节激酶(extracellular signal-regulated kinase,ERK)的蛋白表达情况;使用细胞计数试剂盒-8(cell counting kit-8,CCK8)实验检测ACC-M细胞存活率;使用transwell实验检测ACC-M细胞迁移和侵袭情况;使用实时定量聚合酶链反应(real-time quantitative polymerase chain reaction,RT-qPCR)检测ACC-M细胞TGF-β1和ERK mRNA相对表达量。结果:与control组和BMSC-Exo组比较,Cur-BMSC-Exo组和Cur组的细胞活力、细胞迁移和侵袭数量及N-cadherin、vimentin、TGF-β1和ERK表达水平均显著降低(P<0.05),而E-cadherin蛋白水平显著增加(P<0.05);与Cur-BMSC-Exo组比较,Cur组细胞活力、细胞迁移和侵袭数量及N-cadherin、vimentin、TGF-β1和ERK表达水平均显著增加(P<0.05),而E-cadherin蛋白水平显著降低(P<0.05)。结论:BMSCs分泌的外泌体可作为姜黄素的载体,抑制ACC-M细胞的增殖和转移并调控TGF-β1/ERK信号通路。

Absence of ephrin-A2/A3 increases retinal regenerative potential for Müller cells in Rhodopsin knockout mice

Müller cells(MC) are considered dormant retinal progenitor cells in mammals.Previous studies demonstrated ephrin-As act as negative regulators of neural progenitor cells in the retina and brain.It remains unclear whether the lack of ephrin-A2/A3 is sufficient to promote the neurogenic potential of MC.Here we investigated whether the MC is the primary retinal cell type expressing ephrin-A2/A3 and their role on the neurogenic potential of Müller cells.In this study, we showed that ephrin-A2/A3 and their receptor EphA4 were expressed in retina and especially enriched in MC.The level of ephrin As/EphA4 expression increased as the retina matured that is correlated with the reduced proliferative and progenitor cell potential of MC.Next, we investigated the proliferation in primary MC cultures isolated from wild-type and A2~(–/–) A3~(–/–) mice by 5-ethynyl-2′-deoxyuridine(EdU) incorporation.We detected a significant increase of EdU~+ cells in MC derived from A2~(–/–) A3~(–/–) mice.Next, we investigated the role of ephrin-A2/A3 in mice undergoing photoreceptor degeneration such as Rhodopsin knockout(Rho~(–/–)) mice.To further evaluate the role of ephrin-A2/A3 in MC proliferation in vivo, EdU was injected intraperitoneally to adult wild-type, A2~(–/–) A3~(–/–), Rho~(–/–) and Rho~(–/–) A2~(–/–) A3~(–/–) mice and the numbers of EdU~+ cells distributed among different layers of the retina.Ephrin As/EphA4 expression was upregulated in the retina of Rho~(–/–) mice compared to the wild-type mice.In addition, cultured MC derived from ephrin-A2~(–/–) A3~(–/–) mice also expressed higher levels of progenitor cell markers and exhibited higher proliferation potential than those from wild-type mice.Interestingly, we detected a significant increase of EdU~+ cells in the retinas of adult ephrin-A2~(–/–) A3~(–/–) mice mainly in the inner nuclear layer;and these EdU~+ cells were co-localized with MC marker, cellular retinaldehyde-binding protein, suggesting some proliferating cells are from MC.In Rhodopsin knockout mice(Rho~(–/–) A2~(–/–) A3~(–/–) mice), a significantly greater amount of EdU~+ cells were located in the ciliary body, retina and RPE than that of Rho~(–/–) mice.Comparing between 6 and 12 weeks old Rho~(–/–) A2~(–/–) A3~(–/–) mice, we recorded more EdU~+ cells in the outer nuclear layer in the 12-week-old mice undergoing severe retinal degeneration.Taken together, Ephrin-A2/A3 are negative regulators of the proliferative and neurogenic potentials of MC.Absence of ephrin-A2/A3 promotes the migration of proliferating cells into the outer nuclear layer and may lead to retinal cell regeneration.All experimental procedures were approved by the Animal Care and Use Committee at Schepens Eye Research Institute, USA(approval No.S-353-0715) on October 24, 2012.

The construction of in vitro nasal cavity-mimic M-cell model,design of M cell-targeting nanoparticles and evaluation of mucosal vaccination by nasal administration

In order to better evaluate the transport effect of nanoparticles through the nasal mucosa,an in vitro nasal cavity-mimic model was designed based on M cells.The differentiation of M cells was induced by co-culture of Calu-3 and Raji cells in invert model.The ZO-1 protein staining and the transport of fluorescein sodium and dexamethasone showed that the inverted co-culture model formed a dense monolayer and possessed the transport ability.The differentiation of M cells was observed by upregulated expression of Sialyl Lewis A antigen(SLAA)and integrinβ1,and down-regulated activity of alkaline phosphatase.After targeting M cells with iRGD peptide(cRGDKGPDC),the transport of nanoparticles increased.In vivo,the co-administration of iRGD could result in the increase of nanoparticles transported to the brain through the nasal cavity after intranasal administration.In the evaluation of immune effect in vivo,the nasal administration of OVA-PLGA/iRGD led to more release of IgG,IFN-γ,IL-2 and secretory IgA(sIgA)compared with OVA@PLGA group.Collectively,the study constructed in vitro M cell model,and proved the enhanced effect of targeting towards M cell with iRGD on improving nasal immunity.

弥漫大B细胞淋巴瘤合并淋巴浆细胞淋巴瘤/华氏巨球蛋白血症伴IgM及IgG共存的临床研究

目的 探讨弥漫大B细胞淋巴瘤合并淋巴浆细胞淋巴瘤/华氏巨球蛋白血症(LPL/WM)伴免疫球蛋白M(IgM)及免疫球蛋白G(IgG)共存的发病机制、诊断标准、治疗方案及预后。方法 分析1例弥漫大B细胞淋巴瘤合并LPL/WM伴IgM及IgG共存患者的临床资料,以“弥漫大B细胞淋巴瘤”“免疫球蛋白”“单克隆”“IgM”“IgG”“淋巴浆细胞淋巴瘤”“华氏巨球蛋白血症”“diffuse large B-cell lymphoma”“immune globulin”“monoclonal”“lymphoplasmacytic lymphoma”“Waldenstr9m macroglobulinemia”为检索词,对中国知网数据库、万方知识服务平台及PubMed数据库中2017年1月至2023年6月发表的文献进行检索。结果 该例患者明确诊断为弥漫大B细胞淋巴瘤合并LPL/WM伴IgM及IgG共存,确诊后经环磷酰胺+多柔比星+长春新碱+泼尼松(CHOP)方案、环磷酰胺+长春新碱+泼尼松(COP)方案治疗有效,但治疗期间病情进展,从发病到死亡存活10个月。依照上述检索条件并经过阅读文献题目与摘要筛选出7篇文献。阅读全文共筛出11例双克隆免疫球蛋白共存LPL/WM患者,其中IgM伴IgG双克隆6例,IgM伴免疫球蛋白A(IgA)双克隆2例,IgG伴IgA双克隆2例及IgMκ伴IgMλ双克隆1例。文献中提到的治疗方案主要包括硼替佐米+利妥昔单抗方案、COP方案、CHOP方案等,上述方案联合化疗均有效,但该病的发病机制尚不明确,尚无标准的治疗方案及确切的生存期。结论 弥漫大B细胞淋巴瘤合并LPL/WM伴IgM及IgG共存系首次报告,因该病发病率极低,发病机制尚不明确,因此目前尚无统一的治疗方案,预后尚不明确,临床需进一步提高对该病的认识,未来对于其发病机制及新型分子靶向药物的应用进行深入研究,将会大大改善患者的预后。

An Ultrastructural Study of Phagocytosis and Transport of Formalized Campylobacter jejuni By M Cells into Mouse Peyer's Patches

Suspension of formalized Campylobacter jejunt (2×10 CFU/ml)was injected into a bothend-ligated intestinal loop of ileum which contained some Peyer's patches from non-immunized adult mice after laparotomy under anesthesia.After 1-hour post inoculation, the specimen was taken out and prepared for TEM and SEM observation.The results showed that bacteria adhered specifically on the surface of M cells of Peyer's patches.The microvilli and microfolds of the M cells fused to capture the bacteria and to form a large endocytotic vesicle in the cytoplasm of the M cell,then transported inward,and, at last.released into the central cavity between lymphocytes.Occasionally, we found some of them were phagocytosed by lymphocytes.

Cucurbitacin B-induced G2/M cell cycle arrest of conjunctival melanoma cells mediated by GRP78-FOXM1-KIF20A pathway

Conjunctival melanoma(CM) is a rare and fatal malignant eye tumor. In this study, we deciphered a novel anti-CM mechanism of a natural tetracyclic compound named as cucurbitacin B(CuB). We found that CuB remarkably inhibited the proliferation of CM cells including CM-AS16,CRMM1, CRMM2 and CM2005.1, without toxicity to normal cells. CuB can also induce CM cells G2/M cell cycle arrest. RNA-seq screening identified KIF20A, a key downstream effector of FOXM1 pathway, was abolished by CuB treatment. Further target identification by activity-based protein profiling chemoproteomic approach revealed that GRP78 is a potential target of CuB. Several lines of evidence demonstrated that CuB interacted with GRP78 and bound with a Kdvalue of0.11 μmol/L. Furthermore, ATPase activity evaluation showed that CuB suppressed GRP78 both in human recombinant GRP78 protein and cellular lysates. Knockdown of the GRP78 gene significantly induced the downregulation of FOXM1 and related pathway proteins including KIF20A, underlying an interesting therapeutic perspective. Finally, CuB significantly inhibited tumor progression in NCG mice without causing obvious side effects in vivo. Taken together, our current work proved that GRP78-FOXM1-KIF20A as a promising pathway for CM therapy, and the traditional medicine CuB as a candidate drug to hinder this pathway.

Genistein-induced Anticancer Effects on Acute Leukemia Cells Involve the Regulation of Wnt Signaling Pathway Through H4K20mel Rather Than DNA Demethylation

Objective:To investigate the effects and mechanisms of genistein on the gene expression in the Wnt pathway in acute leukemia(AL)cells.Methods:The expression of Wnt pathway genes and cell cycle-related genes were analyzed in two AL cell lines.Pyrophosphate sequencing was performed to determine the methylation degree.Then,the enrichment of H4K20mel and H3K9ac was determined using ChIP-qPCR.Flow cytometry was used to analyze the cell cycle.Results:The IC_(50) of genistein in the two AL cell lines was lower than that for the bone marrow mesenchymal stem cell line.Genistein upregulated H4K20mel,KMT5A and Wnt suppressor genes,including Wnt5a,and downregulated the downstream target genes of Wnt,such as c-myc and β-catenin.The methylation degree and H3K9ac enrichment in the Wnt5a promoter region remained unchanged.However,the enrichment of H4K20mel in the Wnt5a promoter and coding regions increased.In addition,genistein upregulated Phospho-cdc2,Mytl,Cyclin A,Cyclin E2,p21 and Phospho-histone H3,but downregulated Phospho-weel.Cell cycle arrest was induced in the G2/M phase.Conclusion:Genistein inhibits the activation of the Wnt pathway by promoting the expression of Wnt5a through the activation of KMT5A and enrichment of H4K20mel in the Wnt5a gene promoter and coding regions,rather than demethylation.Genistein also blocks the cell cycle in the G2/M phase.Therefore,genistein is a potential anti-leukemia drug.

Protective effects of curcumin on retinal Müller cell in early diabetic rats

·AIM: To explore the effects and potential mechanisms of curcumin on retinal Müller cell in early diabetic rats. ·METHODS: Diabetic rats were induced by a single intraperitoneal injection of streptozotocin (STZ). Male Sprague-Dawley (SD) rats were randomly assigned into 4 groups: control group (nave SD rats administered with a single intraperitoneal injection of citric buffer), diabetic group (STZ -diabetic rats), dimethyl sulfoxide (DMSO) group (diabetic rats intraperitoneally administered with mixture of DMSO and normal saline, once a day) and curcumin group (diabetic rats intraperitoneally administered with curcumin, 80mg/kg, once a day). Three months after diabetes onset, malondialdehyde (MDA, indication of oxidative stress level) and reduced glutathione (GSH) in retina were detected with kits, glial fibrillary acidic protein (GFAP) in retina was revealed by immunohistochemistry and Western blot, and retinal glutamine synthetase (GS) were observed by Western blot. ·RESULTS: Compared with control group, retinal MDA was increased, and GSH was decreased in diabetic and DMSO groups ( 【0.05, respectively). While, retinal MDA and GSH in curcumin group showed no difference compared with control group ( 】0.05). Furthermore, upregulation of retinal GFAP and down-regulation of retinal GS were detected in diabetic and DMSO groups, and no alteration could be observed in curcumin group revealed with Western blot. Compared with control group, retinal Müller cells showed significant increase in GFAP immunochemistry staining in diabetic and DMSO groups. Moreover, GFAP -positive staining was decreased in curcumin group compared with diabetic group. · CONCLUSION: Curcumin inhibits diabetic retinal oxidative stress, protects Müller cell, and prevents the down -regulation of GS in diabetic retina. Therefore, curcumin has a therapeutic potential in the treatment of diabetic retinopathy (DR).

Modulation of TGFβ_2 and dopamine by PKC in retinal Müller cells of guinea pig myopic eye

AIM: To investigate the effect of protein kinase C (PKC) on transforming growth factor-β2 (TGFβ2) and dopamine in retinal Müller cells of guinea pig myopic eye. METHODS: Myopia was induced by translucent goggles in guinea pig, whose retinal Müller cells were cultured using the enzyme-digesting method. Retinal Müller cells were divided into 5 groups: normal control, myopia, myopia plus GF109203X, myopia plus PMA, myopia plus DMSO. PKC activities were detected by the non-radioactive methods. TGFβ2 and tyrosine hydroxylase (TH) proteins were analyzed by Western Blotting in retinal Müller cells. Dopamine was determined by the high-performance liquid chromatography- electrochemical detection in suspensions. RESULTS: After 14 days deprived, the occluded eyes became myopic with ocular axle elongating. Müller cells of guinea pigs were obtained using enzyme digestion. Compared with normal control group, the increase in PKC activity and the up-regulation in TGFβ2 expression were found in retinal Müller cells of myopic eyes, with the decrease of TH and dopamine content (P <0.05). After PKC activated by PMA, TGFβ2 and TH content were up-regulated with the increase of dopamine content (P <0.05). While the PKC activities was inhibited by GF109203X, proteins of TGFβ2 and TH were down-regulated in the myopic eyes, with the decrease of dopamine content (P <0.05). CONCLUSION: TGFβ2 and dopamine are modulated by PKC in Müller cells of the myopic eyes in guinea pig.

Potential role of Müller cells in the pathogenesis of macropsia associated with epiretinal membrane:a hypothesis revisited

Pathophysiological explanations for metamorphopsia associated with retinal pathologies generally focus on photoreceptor organization disruption. However, the retinal microarchitecture is complicated, and we hypothesize that other retinal cells may also be involved. Metamorphopsia has been widely studied in eyes with epiretinal membranes and we revisit the idea that Müller cell displacement causes retinal macropsia. A Pub Med query and related article search for the macula ultrastructure under normal and pathological conditions revealed an enormous amount of information, particularly ultrahigh definition optical coherence tomography and other retinal imaging modality studies. Findings of these imaging studies support our hypothesis that Müller cells, and not cone photoreceptors, are primarily responsible for macropsia in eyes with epiretinal membranes. More specifically, we conclude that displacement of Müller cell endfeet, and not photoreceptor cones, is a more likely the explanation for retinal macropsia associated with epiretinal membranes.

The ROCK pathway inhibitor Y-27632 mitigates hypoxia and oxidative stress-induced injury to retinal Müller cells

Rho kinase （ROCK） was the first downstream Rho effector found to mediate RhoA-induced actin cytoskeletal changes through effects on myosin light chain phosphorylation. There is abundant evidence that the ROCK pathway participates in the pathogenesis of retinal endothelial injury and proliferative epiretinal membrane traction. In this study, we investigated the effect of the ROCK pathway inhibitor Y-27632 on retinal Müller cells subjected to hypoxia or oxidative stress. Müller cells were subjected to hypoxia or oxidative stress by exposure to CoCl2 or H2O2. After a 24-hour treatment with Y-27632, the 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyl tetrazolium bromide assay was used to assess the survival of Müller cells. Hoechst 33258 was used to detect apoptosis, while 2′,7′-dichlorodihydrofluorescein diacetate was used to measure reactive oxygen species generation. A transwell chamber system was used to examine the migration ability of Müller cells. Western blot assay was used to detect the expression levels of α-smooth muscle actin, glutamine synthetase and vimentin. After treatment with Y-27632, Müller cells subjected to hypoxia or oxidative stress exhibited a morphology similar to control cells. Y-27632 reduced apoptosis, α-smooth muscle actin expression and reactive oxygen species generation under oxidative stress, and it reduced cell migration under hypoxia. Y-27632 also upregulated glutamine synthetase expression under hypoxia but did not impact vimentin expression. These findings suggest that Y-27632 protects Müller cells against cellular injury caused by oxidative stress and hypoxia by inhibiting the ROCK pathway.

pigment epithelium-derived factor protects the morphological structure of retinal Müller cells in diabetic rats

AIM: To investigate if pigment epithelium-derived factor(PEDF) has any protective effect on the retinal Müller cells of Sprague-Dawley rats suffering from diabetes mellitus.METHODS: Sixty Sprague-Dawley rats were randomly divided into a negative control group, a group receiving0.1 μg/μL PEDF, another group receiving 0.2 μg/μL PEDF,and a group receiving balanced salt solution(BSS). Rats in both the PEDF and BSS groups were treated intravitreally based on previously established diabetic models. After 4wk of treatment, morphological alterations of Müller cells and protein expression of glutamine synthase(GS) and glial fibrillary acidic protein(GFAP)were analyzed.RESULTS:PEDFateither0.1μg/μLor0.2μg/μLsignificantly improved the structures of both nuclei and organelles of Müller cells compared to the BSS-treated group.Expression of GS was significantly higher in the 0.2 μg/μL PEDF group than that in the BSS group(P =0.012), but expression of GFAP was significantly lower in the 0.2 μg/μL PEDF group than that in the BSS group(P =0.000);however, there were no significant differences in expression of these proteins between the 0.1 μg/μL PEDF group and the BSS group(P =0.608, P =0.152). CONCLUSION: PEDF protects the morphological ultrastructure of Müller cells, improves the expression of glutamate synthase and prevents cell gliosis.

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.