| Diabetes is a progressive chronic metabolic disease characterized by hyperglycemia.At present,there are about 463 million adults with diabetes in the world,which will increase to 600 million by 2045.Diabetes has become a major chronic disease affecting people’s physical and mental health.Diabetes can cause complications such as vascular and neuropathy,and manifest in the eyes as diabetic retinopathy,cataract and keratopathy,but the effect and mechanism of diabetes on corneal endothelial cells are still unclear.Corneal endothelial cells are a single layer of hexagonal cells on the inner surface of the cornea,which rely on tight junctions and Na+-K+ion pumps to maintain corneal transparency and nutrition.Human corneal endothelial density decreases with age,and the loss rate is about 0.5%per year.Corneal trauma,intraocular surgery,glaucoma,Fuchs corneal endothelial dystrophy,etc.can all cause endothelial cell loss.Due to the extremely limited regenerative capacity,cell loss can only rely on adjacent cells to expand and migrate.Once too much is lost,the corneal endothelial barrier function is destroyed,resulting in increased corneal water content and corneal edema,unable to maintain the normal thickness and transparency of the cornea.The endoplasmic reticulum(ER)is a dynamic,membrane-bound organelle.The endoplasmic reticulum is connected to the nuclear membrane and extends throughout the cytoplasm in the form of a network of connecting sacs and branching tubules.It is an important organelle involved in protein synthesis,processing and modification,and is of great significance for maintaining the homeostasis of the eukaryotic intracellular environment.Endoplasmic reticulum synthesis of proteins is regulated by a series of mechanisms responsible for coordinating the folding,modification and unfolding of secreted integral membrane proteins.Under the stimulation of various physical and chemical factors,the protein folding ability of ER cannot maintain the normal needs of cells,resulting in the misfolding of unfolded proteins,causing the unfolded protein response(UPR),and causing endoplasmic reticulum stress(ERS).ERS is one of the main sources of mitochondrial dysfunction,and previous studies have shown that ERS and mitochondrial dysfunction coexist in various pathological states.Mitophagy can selectively remove damaged mitochondria,which is of great significance for maintaining the stability of the intracellular environment and the normal physiological functions of cells.Among them,mitochondrial autophagy mediated by Pink1/Parkin signaling pathway is the most classical regulatory mechanism.Under normal circumstances,mitochondrial Pink1 is hydrolyzed by proteolytic enzymes and has a low expression level;when certain factors lead to damage to mitochondrial function,the mitochondrial membrane potential decreases or disappears,the activity of proteolytic enzymes is inhibited,and Pink1 cannot be degraded by mitochondria in time.And a large number of them accumulate in the mitochondrial outer membrane and induce mitophagy.Abnormal mitophagy is closely related to the occurrence of various diseases,such as neurodegenerative diseases,cardiovascular diseases,fibrotic diseases and metabolic diseases.Therefore,it is of great significance to explore the mechanism of mitophagy in diabetic corneal endothelial disease.In summary,we mainly investigated the expression of ERS and mitophagy-related markers by studying the corneal endothelial cells of diabetic mice with different durations and human corneal endothelial cells(HCECs)cultured under high glucose environment in vitro.Further,by establishing a corneal endothelial injury model,the role and mechanism of ERS and mitophagy in diabetic corneal endothelial lesions were explored.The research is divided into three parts:(1)establishment of diabetic corneal endothelial disease model;(2)study on the mechanism of ERS regulating diabetic corneal endothelial dysfunction;(3)research on the mechanism of mitophagy regulating diabetic corneal endothelial dysfunction.Part Ⅰ Establishment of a model of diabetic corneal endothelial dysfunctionObjective A diabetic mouse model and endothelial injury model were established to detect changes in corneal thickness,endothelial cell morphology,density and gene expression,and to study the effect of diabetes on corneal endothelial cells.Methods 1.Establishment of Type Ⅰ Diabetic Mice:6-week-old C57BL/6J male mice were injected intraperitoneally with streptozotocin(STZ)for 5 consecutive days to establish a type I diabetes model.Body weight and blood glucose were measured every week after the last inj ection.Mice with an average blood glucose value greater than 300 mg/dL(16.7 mmol/L)twice were included in the diabetes group.The corneal endothelium of normal mice and diabetic mice was collected for morphological and related functional tests.2.Establishment of cell model in vitro:The passaged human corneal endothelial cells(HCECs)were seeded into the cell culture plate at 4 × 105 cells/mL.After 24 hours,the adherence of cells was observed,and the medium was changed to medium containing 30 mM mannose or glucose.The culture was continued,and cells were collected for endothelial function assays.3.Establishment of corneal endothelial injury model:The mice were anesthetized by intraperitoneal injection of 50 mg/kg pentobarbital,compound tropicamide eye drops and prilocaine hydrochloride eye drops for local anesthesia,and a 34 g microsyringe needle was used to enter the anterior chamber through the corneal tunnel.It was connected to a 500 mL saline injection container and injected for 30 minutes to construct a corneal endothelial injury model caused by acute intraocular hypertension.The changes of corneal transparency,corneal thickness and corneal endothelial density were observed after operation.Results This study firstly observed the corneal thickness of diabetic mice with different course of disease,and found that the corneal thickness of the 6-month diabetic mice increased significantly,and the decrease of the density of corneal endothelial cells was also observed.A compensatory increase in endothelial barrier function-related genes was found at an early stage,while chronic hyperglycemia led to a decrease in endothelial function gene expression.In vitro cell experiments also confirmed that long-term high glucose stimulation led to the decline of HCECs functional genes and the delay of injury healing.Acute intraocular hypertension can destroy the normal structure and function of corneal endothelial cells,causing corneal endothelial damage.Compared with normal mice,diabetic mice developed corneal opacity,edema,and loss of endothelial cells after endothelial injury.Conclusion 1.Long-term hyperglycemia leads to increased corneal thickness,decreased endothelial cell density and functional gene expression in mice,while short-term hyperglycemia causes a compensatory increase in endothelial barrier function genes;2.In vitro and in vivo high glucose environment can cause delayed endothelial cell injury healing;3.Induction of intraocular pressure in mice by anterior chamber perfusion can be used as a research model for diabetic corneal endothelial disease.in high glucose group and high glucose+4-PBA group were further detected.Part Ⅱ: Role of endoplasmic reticulum stress during diabetic corneal endothelial dysfunction Objective The expression of endoplasmic reticulum stress(ERS)-related genes was detected,and its role and mechanism in diabetic corneal endothelial disease were further verified.Methods1.Detection of ERS expression in diabetic corneal endothelium:The corneal endothelium of 4 months of type 1 diabetic mice was collected to detect the changes of ERS-related indexes.At the same time,the corneas of normal eye bank and diabetic patients were collected,and the expression of ERS-related indexes in human corneal endothelium was detected by immunohistochemistry.2.The role of ERS in diabetic corneal endothelial dysfunction:After injection of the ERS activator thapsigargin(TG)into the anterior chamber of normal mice,the expression of mouse corneal transparency,corneal thickness and endothelial function genes was observed.An endothelial injury model was established in a diabetic mouse model.The experimental group was given an ERS inhibitor,4-phenylbutyric acid(4-PBA).Changes in corneal transparency,corneal thickness and endothelial function genes were detected by other methods.HCECs were cultured in vitro and divided into normal culture group,TG group,high glucose treatment group and high glucose + 4-PBA treatment group,and the changes of endothelial cell function genes were detected.3.The mechanism of ERS in diabetic corneal endothelial dysfunction:HCECs were cultured in vitro and divided into normal culture group and TG treatment group,and the changes of mitochondrial bioenergetics under high glucose environment were detected.The changes of mitochondrial bioenergetics and ATP ratein high glucose group and high glucose+4-PBA group were further detected.Results In this experiment,we first found that the expression of ERS in the endothelium of diabetic mice with a course of 4 months was significantly increased,without an increase in corneal thickness and a decrease in endothelial function.The corneal endothelium of normal and diabetic patients was also observed.The expression of ERS was increased.Further normal mice were given anterior chamber injection of ERSinducing agent TG,which developed corneal opacity,increased corneal thickness and decreased endothelial function expression.In an endothelial injury model,subconjunctival injection and the addition of the ERS inhibitor 4-PBA to the perfusate both attenuated corneal edema and reduced endothelial cell loss in diabetic mice.In vitro experiments confirmed that both activation of ERS and high-glucose environment can lead to mitochondrial dysfunction,and administration of ERS inhibitor 4-PBA in high-glucose environment can protect mitochondrial function and change ATP production from anaerobic oxidation pathway to oxidative phosphorylation pathway..It was further found that mitochondria-targeted antioxidants can also protect the function of the corneal endothelium in diabetic mice.Conclusion1.The occurrence of ERS is earlier than the morphological changes of the diabetic endothelium,and activation of ERS leads to corneal endothelial lesions;2.The application of ERS inhibitors and mitochondria-targeted antioxidants can protect the corneal endothelium of diabetic mice,and the subconjunctival injection and the administration of ERS inhibitors to the perfusate have similar effects;3.Activation of ERS and high glucose can induce mitochondrial dysfunction,and inhibition of ERS protects the function of the diabetic corneal endothelium through the mitochondrial pathway.Part Ⅲ:Role of mitophagy during diabetic corneal endothelial dysfunctionObjective Detect the expression of mitophagy,and further verify its role and mechanism in diabetic corneal endothelial dysfunction.Methods 1.Changes in mitochondrial structure and function:HCECs were cultured for 72 h under 30 mM high glucose and mannose.The cells were collected to observe the structure of mitochondria by transmission electron microscope,and the expression of reactive oxygen species(ROS)in cells and mitochondria was observed by H2DCF-DA and MitoSOX staining.The changes of mitochondrial membrane potential were further detected by TMRM staining and flow cytometry.2.Expression changes of mitophagy levels:HCECs were cultured for 72 h under 30 mM high glucose and mannose.Total cell protein was extracted and the changes of mitophagy level were detected by WB.Corneal endothelial tissue was extracted from 6-month-old diabetic mice and agematched normal control mice to detect changes in mitophagy levels.3.The role of mitophagy in diabetic corneal endothelial disease:HCECs were cultured in a high-glucose environment with or without mitophagy activator(Carbonyl cyanide m-chlorophenylhydrazone,CCCP)for 72 hours,and the levels of mitophagy and the expression changes of endothelial function genes were detected.Endothelial injury models were established in 6-month-old diabetic mice and age-matched normal mice.The experimental group was given CCCP to observe changes in corneal transparency,corneal thickness and endothelial function genes.Results In this study,the increase of ROS in endothelial cells and mitochondria was first observed in vitro under high glucose environment,and the results of transmission electron microscopy showed that the structure of mitochondrial bilayer membrane was damaged.The results of TMRM staining showed that the mitochondrial membrane potential decreased.It was then detected that mitochondrial dynamics were unbalanced and the level of mitophagy decreased in the corneal endothelium of high-glucosetreated HCECs and 6-month diabetic mice.In contrast,mitophagy levels were increased in the corneal endothelium of 4-month-old diabetic mice.Administration of the mitophagy activator CCCP in high-glucose-treated HCECs in vitro up-regulated mitophagy levels,reduced ROS production and restored mitochondrial dynamics in high-glucose environments.Finally,a corneal endothelial injury model was established,and administration of the mitophagy activator CCCP could significantly alleviate corneal edema and reduce endothelial cell loss in diabetic mice.Conclusion 1.The increase of mitochondrial dysfunction may be the main mechanisms of corneal endothelial lesions caused by hyperglycemia;2.Decreased levels of mitophagy in diabetic corneal endotheliopathy,resulting in the accumulation of damaged mitochondria;3.Up-regulation of mitophagy levels can reduce corneal thickness and endothelial cell loss in diabetic mice. |
