AIM: To develop a novel gene-delivery therapeutic based on CRISPR/Cas9 genome editing technology capable of specifically targeting and knocking out the VEGFA gene, thereby achieving sustained suppression of VEGFA expression in retinal pigment epithelial(RPE)cells and providing a new strategy for gene therapy in retinal neovascular diseases.METHODS:Single guide RNAs targeting the human VEGFA gene for knockout were designed, and corresponding recombinant plasmids were constructed. A novel polymer(PTEE)was used to encapsulate the plasmids to prepare a PTEE-loaded anti-VEGFA plasmid(PLAP)gene delivery system. PTEE materials at concentrations of 0.1, 0.2, 0.4, 0.8, and 1.6 μg/μL were co-incubated with ARPE-19 cells, and the biocompatibility of PTEE was evaluated using the cell counting kit-8(CCK-8)assay. Recombinant plasmids expressing green fluorescent protein(GFP)were constructed. Lipofectamine 3000 and jetOPTIMUS®DNA transfection reagents were used as control groups, and PTEE nanomaterials were used as the experimental group to encapsulate the plasmids. When the cell confluence reached 80%, the formulations were transfected into ARPE-19 and 293T cells. GFP expression was observed under light microscopy, and the transfection efficiencies of each group were compared. ARPE-19 cells were induced under hypoxia, and PLAP was transfected into the cells. The expression level of VEGFA was detected by enzyme-linked immunosorbent assay(ELISA)to evaluate the efficacy of this novel gene delivery system.RESULTS: After co-incubation of ARPE-19 cells with different concentrations of PTEE for 24 h and 48 h, no significant effect on cell viability was observed in any group. The transfection efficiency of PLAP in ARPE-19 cells was higher than that in the Lipo3000 and jetOPTIMUS groups, with statistically significant differences(P<0.01). Hypoxia for 6 h significantly induced the upregulation of VEGFA mRNA expression in ARPE-19 cells, and under hypoxic conditions, the PTEE group exhibited a significant inhibitory effect on VEGFA expression(P<0.01).CONCLUSION:PLAP exhibits favorable biocompatibility and prominent VEGFA inhibitory effects in vitro, making it a potential candidate drug for gene therapy of retinal neovascular diseases.

With the rapid advancement of artificial intelligence (AI) technology, particularly breakthroughs in large models, AI applications in healthcare are becoming increasingly widespread.Ophthalmology, as a critical branch of medicine, has emerged as a clinical specialty with comprehensive AI research and applications, especially in the AI-driven analysis of ophthalmic imaging.Currently, AI in ophthalmology is undergoing profound transformation driven by large model technology.AI large models, with their robust data comprehension and multimodal interaction capabilities, offer new possibilities for the diagnosis, treatment, and health education of ophthalmic diseases, powerfully propelling the evolution of current AI paradigms in ophthalmology.This article explores the novel opportunities brought by the extensive application of AI large models in ophthalmic research and practice, and provides recommendations for ophthalmologists and insights for industry development.

With the rapid advancement of artificial intelligence (AI) technology, particularly breakthroughs in large models, AI applications in healthcare are becoming increasingly widespread.Ophthalmology, as a critical branch of medicine, has emerged as a clinical specialty with comprehensive AI research and applications, especially in the AI-driven analysis of ophthalmic imaging.Currently, AI in ophthalmology is undergoing profound transformation driven by large model technology.AI large models, with their robust data comprehension and multimodal interaction capabilities, offer new possibilities for the diagnosis, treatment, and health education of ophthalmic diseases, powerfully propelling the evolution of current AI paradigms in ophthalmology.This article explores the novel opportunities brought by the extensive application of AI large models in ophthalmic research and practice, and provides recommendations for ophthalmologists and insights for industry development.

Diabetic retinopathy(DR)is one of the most common and severe microvascular complications in diabetic patients and has become one of the leading causes of blindness worldwide. With the continuous rise in the prevalence of diabetes, in-depth exploration of the pathogenesis of DR and effective intervention measures is of great clinical significance. The mechanistic target of rapamycin(mTOR), as a protein kinase, is widely involved in cellular processes such as growth, metabolism, and autophagy. Research indicates that the mTOR signaling pathway plays a crucial regulatory role in the pathological progression of DR, and its abnormal activity can disrupt retinal cell autophagy function, thereby accelerating cellular damage and disease progression. Autophagy, as an important regulatory mechanism for cellular homeostasis, maintains cellular functional balance by clearing damaged organelles and protein aggregates. This article provides a systematic review of the structural and functional aspects of the mTOR signaling pathway, the molecular regulatory mechanisms of autophagy, and their roles in retinal pathological changes. By summarizing current research findings, the article aims to clarify the key regulatory role of the mTOR-autophagy axis in DR, providing theoretical support for elucidating the molecular pathogenesis of DR and offering potential targets and research directions for developing novel targeted therapeutic strategies, thereby holding significant scientific and clinical value.

Diabetic retinopathy(DR)is one of the common causes of visual impairment and blindness in adults, which is caused by various pathogenesis. Although the mechanism of DR has not been elucidated yet, the destruction of blood-retinal barrier is a key process. As a highly endothelial-specific factor in promoting the growth of vascular endothelial cell, vascular endothelial growth factor(VEGF)plays a crucial role in the formation of pathological retinal neovascularization and the destruction of blood-retinal barrier. Therefore, a comprehensive understanding of the etiology and pathogenesis of blood-retinal barrier damage promoted by VEGF is critical for exploring the pathogenesis of DR. In this study, the underlying relationship between VEGF and the mechanism of blood-retinal barrier damage, including retinal vascular endothelial cell permeability, vascular inflammatory response, apoptosis, oxidative stress, mitochondrial damage and endoplasmic reticulum stress, with a view to providing a reference for the study in VEGF in the pathogenesis of blood-retinal barrier damage in DR.

Objective To investigate the relationship between Notch1 and autophagy under high glucose conditions and to explore the effects of Notch1 inhibitor DAPT and autophagy inhibitor 3-MA on human retinal pigment epithelial cells cultured in high glucose conditions.Methods Via preliminary experiment,25 mmol·L-1 glucose was used as the high glucose culture medium of adult retinal pigment epithelial(ARPE)-19 cells,and 5 mmol·L-1 3-MA was adopted as the au-tophagy inhibitor.ARPE-19 cells cultured in vitro were randomly divided into four groups:control group(treated with 5 mmol·L-1 glucose for 48 h),high glucose group(treated with 25 mmol·L-1 glucose for 48 h),high glucose+DAPT group(treated with 40 μmol·L-1 DAPT for 2 h and then 25 mmol·L-1 glucose for 48 h),and high glucose+3-MA group(treated with 5 mmol·L-1 3-MA for 2 h and then 25 mmol·L-1 glucose for 48 h).A transmission electron microscope was used to observe the ultrastructure of cells in each group.Cell proliferation and migration were observed using Cell Counting Kit-8 and scratch assays.Western blot was used to detect the protein expression levels of Notch1 and autophagy-related proteins LC3 and Beclin1.Reverse transcription-polymerase chain reaction was used to measure the relative messenger ri-bonucleic acid(mRNA)expression levels of Notch1,LC3 and Beclin1 of cells in each group.Results Transmission elec-tron microscope showed that cells in the control group had normal structures,with round or oval nuclei and a few autopha-gosomes.In the high glucose group,cells exhibited slightly obvious injury,with uneven cytoplasm and numerous autolyso-somes.Compared to the control group,ARPE-19 cells in the high glucose group had increased proliferation and migration abilities,and higher mRNA and protein expression levels of Notch1,LC3 and Beclin1(all P＜0.05).Compared to the high glucose group,ARPE-19 cells in the high glucose+DAPT group showed decreased proliferation and migration abilities,and lower mRNA and protein expression levels of Notch1,LC3 and Beclin1(all P＜0.05).The high glucose+3-MA group showed reduced proliferation and migration abilities,as well as decreased mRNA and protein expression levels of LC3 and Beclin1(all P＜0.05)compared to the high glucose group.Conclusion High glucose can activate Notch1 and the auto-phagy process,promoting the proliferation of ARPE-19 cells.In the high glucose+DAPT group and high glucose+3-MA group,the autophagy process is inhibited to a certain extent,thereby restraining cell proliferation.

Objective To investigate if Lycium barbarum polysaccharide(LBP)could inhibit the high glucose-in-duced human retinal microvascular endothelial cell(HRMEC)injury by regulating the NOD-like receptor family pyrin do-main containing protein 3(NLRP3)/Caspase-1 pyroptosis pathway.Methods HRMECs cultured in vitro were randomly divided into the control group(5.5 mmol·L-1 glucose),the high glucose group(55.5 mmol·L-1 glucose),the low LBP group(55.5 mmol·L-1 glucose+100 mg·L-1 LBP),the medium LBP group(55.5 mmol·L-1 glucose+500 mg·L-1 LBP),the high LBP group(55.5 mmol·L-1 glucose+1 000 mg·L-1 LBP),the si-NC group(55.5 mmol·L-1glucose after transfection with 20 pmol·L-1 si-NC)and the si-NLRP3 group(55.5 mmol·L-1 glucose after transfection with 20μmol·L-1si-NLRP3).The Cell Counting Kit-8 was used to detect the proliferation of HRMECs in each group and flow cy-tometry was adopted to measure the pyroptosis of HRMECs in each group.The reverse transcription-polymerase chain reac-tion was used to detect the relative messenger ribonucleic acid(mRNA)expression levels of NLRP3,Caspase-1,nuclear factor(NF)-κB,Gasdermin-D(GSDMD)and vascular endothelial growth factor(VEGF)in the HRMECs of each group,Western blot was adopted to detect the relative protein expression levels of HRMEC pyroptosis-related NLRP3,Caspase-1,NF-κB,GSDMD and VEGF in each group,and enzyme-linked immunosorbent assay was used to detect the interleukin(IL)-1β and IL-18 expression levels in downstream pyroptosis in the HRMEC supernatant of each group.Results Com-pared with the control group,the proliferation rate of HRMECs decreased,the pyroptosis rate increased,the relative mR-NA and protein expression levels of NLRP3,Caspase-1,NF-κB,GSDMD and VEGF increased,and the expressions of IL-1βand IL-18 increased in the high glucose group(all P＜0.05).Compared with the high glucose group,the proliferation rate of HRMECs increased,the pyroptosis rate decreased,the relative mRNA and protein expression levels of NLRP3,Caspase-1,NF-κB,GSDMD and VEGF decreased,and the expressions of IL-1β and IL-18 decreased in the si-NLRP3 group(all P＜0.05).There were no significant differences in cell proliferation rate,pyroptosis rate,mRNA and protein expression levels of NLRP3,Caspase-1,NF-κB,GSDMD and VEGF,as well as levels of IL-1β and IL-18,in the si-NC group compared with the high glucose group(all P＞0.05).Compared with the high glucose group,the medium LBP group and high LBP group had increased proliferation rates,lower pyroptosis rates,and declined mRNA and protein expression levels of NLRP3,Caspase-1,NF-κB,GSDMD and VEGF as well as expressions of IL-1β and IL-18(all P＜0.05).Compared with the high glucose group,there was no significant difference in the proliferation rate of HRMECs and various protein expression levels in the low LBP group(all P＞0.05),and other indicators were consistent with those in the medium LBP group and high LBP group.Conclusion LBP has a protective effect on HRMEC injury induced by high glucose,can promote cell prolif-eration and inhibit pyroptosis,and its mechanism is related to inhibiting the activation of NLRP3/Caspase-1 signaling path-way and reducing the expression of related inflammatory factors.

Age-related macular degeneration (AMD) is one of the main causes of irreversible vision loss in the elderly worldwide.Its main pathological features are the degeneration of retinal pigment epithelium (RPE) and the irreversible damage or loss of photoreceptor cells.Extracellular vesicles (EVs) are a class of heterogeneous nanovesicles with lipid bilayer membranes, including exosomes, microvesicles and apoptotic bodies, which exert biological effects by transmitting molecules such as RNA and protein.In this review, RPE-derived extracellular vesicles (RPE-EVs) are involved in the regulation of oxidative stress, inflammation, and neovascularization in AMD.RPE-EVs derived Apaf1, HDAC6, miR-494-3p, miR-138-5p, miR-21, miR-543 and miR-302a-3p can be used as candidate molecular targets for the diagnosis and treatment of AMD, but their mechanisms of action have not been elucidated.Due to the unique advantages of high biocompatibility, low immunogenicity, low toxicity, targeting, stability, and specificity of RPE-EVs, it is necessary to further study the role of RPE-EVs in the pathogenesis of AMD, and focus on the role of RPE-EVs in the diagnosis and treatment of AMD, so as to realize the clinical transformation of RPE-EVs, and open up new ways for the diagnosis and treatment of AMD.

Objective:To analyze the clinical manifestations and explore the etiology in a family with congenital aniridia and to analyze the influence of candidate variants on the protein structure.Methods:A pedigree investigation was performed.A Han Chinese family with congenital aniridia of two generations consisting of three members from Henan Province, including one patient diagnosed with congenital aniridia, was identified and studied following their admission to Henan Eye Hospital in June 2023.A thorough medical history was obtained for the patient and their family members.Comprehensive ophthalmologic examinations were conducted, including visual acuity, intraocular pressure, anterior segment photography, color fundus photography, ultrasound biomicroscopy, and optical coherence tomography, etc.Peripheral blood samples were obtained from the family members and whole exome sequencing (WES) was performed on the patient and validated by Sanger sequencing for other members.The pathogenicity and protein structure of newly identified variant sites were analyzed.This study adhered to the Declaration of Helsinki.The study protocol was approved by the Ethics Committee of Henan Eye Hospital (No.HNEECKY-2023[06]).Written informed consent was obtained from each subject.Results:The proband is a 23-year-old male presenting with poor binocular vision, aniridia, corneal degeneration, mild lens opacity, shallow anterior chamber, elevated intraocular pressure, peripheral retinal degeneration, and macular dysplasia.The clinical phenotype of the proband's parents did not show any significant abnormality.WES identified a heterozygous frameshift and nonsense varint c. 734_735del (p.Arg245Asnfs*20) in exon 10 of the PAX6 gene, which consisited of two bases deletion at positions 734 to 735, resulting in the mutation of its arginine at position 245 to asparagine and the early appearance of a termination codon at the next 19 amino acids.The variant had not been identified in the HGMD, Clinvar, 1 000 Genomes, and gnomAD databases.Neither of the proband's parents carried the variant, consistent with the pattern of family co-segregation.Substructural analysis using the SMART tool indicated that the variant is situated within the HOX domain.Amino acid conservation analysis demonstrated that the arginine residue at position 245 in the PAX6 gene is highly conserved across multiple species, including human, house mouse, domestic dog, African clawed frog, and macaque.The variant was classified as pathogenic (PVS1+ PS2+ PM2+ PP3) based on the ACMG standards and guidelines for the interpretation of sequence variants.Protein structure analysis revealed the absence of both the homologous domain and the proline-serine-threonine-rich domain in the PAX6 protein. Conclusions:A novel pathogenic variant, c.734_735del (p.Arg245Asnfs*20), in the PAX6 gene has been identified in a family affected by congenital aniridia.This variant results in the deletion of both the PAX6 protein homology domain and the proline-serine-threonine-rich domain.

With the accelerated aging society in China, the incidence of biliary surgical diseases in the elderly has increased significantly. The clinical characteristics of these patients indicate that improving treatment outcomes and realizing healthy aging are worthy of attention. How to effectively improve the treatment effect of geriatric biliary surgical diseases has attracted widespread attention. This paper reviews and comments on the hotspots and difficulties of biliary surgery in older patients from six aspects: (1) higher morbidity associated with an aging society, (2) prevention and control of pre-operative risks, (3) extending the indications of laparoscopic surgery, (4) urgent standardization of minimally invasive surgery, (5) precise technological progress in hepatobiliary surgery, and (6) guarantee of peri-operative safety. It is of great significance to fully understand the focus of controversy, actively make use of its favorable factors, and effectively avoid its unfavorable factors, for further improving the therapeutic effects of geriatric biliary surgical diseases, and thus benefits the vast older patients with biliary surgical diseases. Accordingly, a historical record with the highest age of 93 years for laparoscopic transcystic common bile duct exploration has been created by us recently.
Humans ; Aged ; Aged, 80 and over ; Biliary Tract Surgical Procedures ; Gallstones ; Laparoscopy ; Treatment Outcome ; Aging ; Retrospective Studies