Insufficient TRPM5 Mediates Lipotoxicity-induced Pancreaticβ-cell Dysfunction

Objective:While the reduction of transient receptor potential channel subfamily M member 5(TRPM5)has been reported in islet cells from type 2 diabetic(T2D)mouse models,its role in lipotoxicity-induced pancreaticβ-cell dysfunction remains unclear.This study aims to study its role.Methods:Pancreas slices were prepared from mice subjected to a high-fat-diet(HFD)at different time points,and TRPM5 expression in the pancreaticβcells was examined using immunofluorescence staining.Glucose-stimulated insulin secretion(GSIS)defects caused by lipotoxicity were mimicked by saturated fatty acid palmitate(Palm).Primary mouse islets and mouse insulinoma MIN6 cells were treated with Palm,and the TRPM5 expression was detected using qRT-PCR and Western blotting.Palm-induced GSIS defects were measured following siRNA-based Trpm5 knockdown.The detrimental effects of Palm on primary mouse islets were also assessed after overexpressing Trpm5 via an adenovirus-derived Trpm5(Ad-Trpm5).Results:HFD feeding decreased the mRNA levels and protein expression of TRPM5 in mouse pancreatic islets.Palm reduced TRPM5 protein expression in a time-and dose-dependent manner in MIN6 cells.Palm also inhibited TRPM5 expression in primary mouse islets.Knockdown of Trpm5 inhibited insulin secretion upon high glucose stimulation but had little effect on insulin biosynthesis.Overexpression of Trpm5 reversed Palm-induced GSIS defects and the production of functional maturation molecules unique toβcells.Conclusion:Our findings suggest that lipotoxicity inhibits TRPM5 expression in pancreaticβcells both in vivo and in vitro and,in turn,drivesβ-cell dysfunction.

Upregulation of α-ENaC induces pancreatic β-cell dysfunction,ER stress,and SIRT2 degradation

Islet beta cells(β-cells)produce insulin in response to high blood glucose levels,which is essential for preserving glucose homeostasis.Voltage-gated ion channels inβ-cells,including Na+,K+,and Ca2+channels,aid in the release of insulin.The epithelial sodium channel alpha subunit(α-ENaC),a voltage-independent sodium ion channel,is also expressed in human pancreatic endocrine cells.However,there is no reported study on the function of ENaC in theβ-cells.In the current study,we found thatα-ENaC was expressed in human pancreatic glandule and pancreatic isletβ-cells.In the pancreas of db/db mice and high-fat diet-induced mice,and in mouse isletβ-cells(MIN6 cells)treated with palmitate,α-ENaC expression was increased.Whenα-ENaC was overexpressed in MIN6 cells,insulin content and glucose-induced insulin secretion were significantly reduced.On the other hand,palmitate injured isletβ-cells and suppressed insulin synthesis and secretion,but increasedα-ENaC expression in MIN6 cells.However,α-ENaC knockout(Scnn1a−/−)in MIN6 cells attenuatedβ-cell disorder induced by palmitate.Furthermore,α-ENaC regulated the ubiquitylation and degradation of sirtuin 2 inβ-cells.α-ENaC also modulatedβ-cell function in correlation with the inositol-requiring enzyme 1 alpha/X-box binding protein 1(IRE1α/XBP1)and protein kinase RNA-like endoplasmic reticulum kinase/C/EBP homologous protein(PERK/CHOP)endoplasmic reticulum stress pathways.These results suggest thatα-ENaC may play a novel role in insulin synthesis and secretion in theβ-cells,and the upregulation ofα-ENaC promotes isletβ-cell dysfunction.In conclusion,α-ENaC may be a key regulator involved in isletβ-cell damage and a potential therapeutic target for type 2 diabetes mellitus.

Pancreaticβ-cell dysfunction in type 2 diabetes:Implications of inflammation and oxidative stress

Insulin resistance and pancreaticβ-cell dysfunction are major pathological mechanisms implicated in the development and progression of type 2 diabetes(T2D).Beyond the detrimental effects of insulin resistance,inflammation and oxidative stress have emerged as critical features of T2D that defineβ-cell dysfunction.Predominant markers of inflammation such as C-reactive protein,tumor necrosis factor alpha,and interleukin-1βare consistently associated withβ-cell failure in preclinical models and in people with T2D.Similarly,important markers of oxidative stress,such as increased reactive oxygen species and depleted intracellular antioxidants,are consistent with pancreaticβ-cell damage in conditions of T2D.Such effects illustrate a pathological relationship between an abnormal inflammatory response and generation of oxidative stress during the progression of T2D.The current review explores preclinical and clinical research on the pathological implications of inflammation and oxidative stress during the development ofβ-cell dysfunction in T2D.Moreover,important molecular mechanisms and relevant biomarkers involved in this process are discussed to divulge a pathological link between inflammation and oxidative stress duringβ-cell failure in T2D.Underpinning the clinical relevance of the review,a systematic analysis of evidence from randomized controlled trials is covered,on the potential therapeutic effects of some commonly used antidiabetic agents in modulating inflammatory makers to improveβ-cell function.

β-cell dysfunction: Its critical role in prevention and management of type 2 diabetes

Type 2 diabetes(T2DM) is characterized by insulin resistance and β-cell dysfunction. Although, in contrast to type 1 diabetes, insulin resistance is assumed to be a major pathophysiological feature of T2 DM, T2 DM never develops unless β-cells fail to compensate insulin resistance. Recent studies have revealed that a deficit of β-cell functional mass is an essential component of the pathophysiology of T2 DM, implying that β-cell deficit is a common feature of both type 1 and type 2 diabetes. β-cell dysfunction is present at the diagnosis of T2 DM and progressively worsens with disease duration. β-cell dysfunction is associated with worseningof glycemic control and treatment failure; thus, it is important to preserve or recover β-cell functional mass in the management of T2 DM. Since β-cell regenerative capacity appears somewhat limited in humans, reducing β-cell workload appears to be the most effective way to preserve β-cell functional mass to date, underpinning the importance of lifestyle modification and weight loss for the treatment and prevention of T2 DM. This review summarizes the current knowledge on β-cell functional mass in T2 DM and discusses the treatment strategy for T2 DM.

The relationship between insulin resistance/β-cell dysfunction and diabetic retinopathy in Chinese patients with type 2 diabetes mellitus： the Desheng Diabetic Eye Study

AIM： To investigate the relationship between insulin resistance （IR）/β-cell dysfunction and diabetic retinopathy （DR） in Chinese patients with type 2 diabetes mellitus （T2DM）, and to explore further whether there were differences in the relationship among diabetic patients with higher and lower body mass index （BMI）. METHODS： Cross-sectional study. A total of 1466 subjects with T2DM were recruited in a local Desheng Community of urban Beijing from November 2009 to June 2012 for the cohort of Beijing Desheng Diabetic Eye Study. Standardized evaluation was carried out for each participant, including questionnaire, ocular and anthropometric examinations, and laboratory tests. Seven fields 30° color fundus photographs were used for DR grading according to the Early Treatment Diabetic Retinopathy Study protocols. Homeostatis Model Assessment （HOMA） method was employed for IR and β-cell function assessment. RESULTS： After excluding those participants who were treated with insulin （n=352） or had missing data of fasting insulin （n=96）, and further excluding those with poor quality of retinal photographs （n=10）, a total of 1008 subjects were included for the final analysis, 406 （40.3%） were men and 602 （59.7%） were women, age ranging fiom 34 to 86 （64.87±8.28）y. Any DR （levels 14 and above） was present in 278 （27.6%） subjects. After adjusting for possible covariates, the presence of any DR did not correlate with HOMA IR [odds ratio （OR） 1.51, 95% confidence interval （Cl） 0.87-2.61, P=0.14] or HOMA β-cell （OR 0.71, 95%CI 0.40-1.26, P=0.25）. After stratification by BMI, the presence of any DR was associated positively with HOMA IR （OR 2.46, 95%CI： 1.18-5.12, P=0.016）, and negatively with HOMA β-cell （OR 0.40, 95%CI： 0.19-0.87, P=0.021） in the group of patients with higher BMI （225 kg/m2）. In the group of patients with lower BMI （〈25 kg/m2）, the presence of any DR was not associated with HOMA IR （OR 1.00, 95%C1： 0.43-2.33, P=I.00） or HOMA β-cell （OR 1.41, 95%CI： 0.60-3.32, P=0.43）. CONCLUSION： The data suggest that higher IR and lower 13-cell function are associated with the presence of DR in the subgroup of diabetic patients with higher BMI. However, this association is not statistically significant in diabetic patients with lower BMI.

Chronic alcohol consumption potentiates the development of diabetes through pancreatic β-cell dysfunction

Chronic ethanol consumption is well established as a major risk factor for type-2 diabetes(T2D), which is evidenced by impaired glucose metabolism and insulin resistance. However, the relationships between alcoholconsumption and the development of T2 D remain controversial. In particular, the direct effects of ethanol consumption on proliferation of pancreatic β-cell and the exact mechanisms associated with ethanolmediated β-cell dysfunction and apoptosis remain elusive. Although alcoholism and alcohol consumption are prevalent and represent crucial public health problems worldwide, many people believe that low-tomoderate ethanol consumption may protect against T2 D and cardiovascular diseases. However, the J- or U-shaped curves obtained from cross-sectional and large prospective studies have not fully explained the relationship between alcohol consumption and T2 D. This review provides evidence for the harmful effects of chronic ethanol consumption on the progressive development of T2 D, particularly with respect to pancreatic β-cell mass and function in association with insulin synthesis and secretion. This review also discusses a conceptual framework for how ethanolproduced peroxynitrite contributes to pancreatic β-cell dysfunction and metabolic syndrome.

Elaidic acid leads to mitochondrial dysfunction via mitochondria-associated membranes triggers disruption of mitochondrial calcium fluxes

Elaidic acid(EA)stimulation can lead to endoplasmic reticulum stress(ERS),accompanied by a large release of Ca^(2+),and ultimately the activation of NLRP3 inflammasome in Kupffer cells(KCs).Mitochondrial instability or dysfunction may be the key stimulating factors to activate NLRP3 inflammasome,and sustained Ca^(2+)transfer can result in mitochondrial dysfunction.We focused on KCs to explore the damage to mitochondria by EA.After EA stimulation,cells produced an oxidative stress(OS)response with a significant increase in ROS release.Immunoprecipitation experiments and the addition of inhibitors revealed that the increase in the level of intracellular Ca^(2+)led to Ca^(2+)accumulation in the mitochondrial matrix via mitochondria-associated membranes(MAMs).This was accompanied by a significant release of m ROS,loss of MMP and ATP,and a significant increase in mitochondrial permeability transition pore opening,ultimately leading to mitochondrial instability.These findings confirmed the mechanism that EA induced mitochondrial Ca^(2+)imbalance in KCs via MAM,ultimately leading to mitochondrial dysfunction.Meanwhile,EA induced OS and the decrease of MMP and ATP in rat liver,and significant lesions were found in liver mitochondria.Swelling of the inner mitochondrial cristae and mitochondrial vacuolization occurred,with a marked increase in lipid droplets.

Mitochondrial dysfunction and quality control lie at the heart of subarachnoid hemorrhage

The dramatic increase in intracranial pressure after subarachnoid hemorrhage leads to a decrease in cerebral perfusion pressure and a reduction in cerebral blood flow.Mitochondria are directly affected by direct factors such as ischemia,hypoxia,excitotoxicity,and toxicity of free hemoglobin and its degradation products,which trigger mitochondrial dysfunction.Dysfunctional mitochondria release large amounts of reactive oxygen species,inflammatory mediators,and apoptotic proteins that activate apoptotic pathways,further damaging cells.In response to this array of damage,cells have adopted multiple mitochondrial quality control mechanisms through evolution,including mitochondrial protein quality control,mitochondrial dynamics,mitophagy,mitochondrial biogenesis,and intercellular mitochondrial transfer,to maintain mitochondrial homeostasis under pathological conditions.Specific interventions targeting mitochondrial quality control mechanisms have emerged as promising therapeutic strategies for subarachnoid hemorrhage.This review provides an overview of recent research advances in mitochondrial pathophysiological processes after subarachnoid hemorrhage,particularly mitochondrial quality control mechanisms.It also presents potential therapeutic strategies to target mitochondrial quality control in subarachnoid hemorrhage.

Alpha-synuclein in mitochondrial dysfunction:opportunities or obstacles

Recent work suggests a link betweenα-synuclein(α-syn)and mitochondrial dysfunction;however,the mechanisms of howα-syn influences mitochondrial function are still unclear.Most notably,whetherα-syn plays a direct role during mitochondrial function and/or whether diseasedα-syn-mediated mitochondrial dysfunction is a potential modifiable risk factor in Parkinson’s disease(PD)is unknown.To date,mutations in more than eight genes cause familial PD(fPD)and have functions in diverse pathways including synaptic homeostasis,mitochondria maintenance,autophagy/lysosome,and ubiquitin-proteasome pathways.

Fibrinogen’s potential role in connecting cerebrovascular abnormalities with glymphatic dysfunction in Alzheimer’s disease

Alzheimer’s disease(AD)stands out as the primary manifestation of age-related dementia,portraying a chronic neurodegenerative disorder distinguished by the accumulation of fibrillar amyloid-β(Aβ)plaques and neurofibrillary tangles of hyperphosphorylated tau.However,from a clinical standpoint,AD presents itself as a complex condition with a spectrum of dysfunctions rather than a singular pathological mechanism.An often-overlooked aspect of the disease is the presence of extensive cerebrovascular abnormalities,given that the majority of AD patients experience altered cerebral blood flow,damaged vasculature,increased microinfarcts and microhemorrhages.Animal models of AD further support this observation,showing cerebrovascular dysfunction such as impaired cerebral blood flow and altered cerebrovascular reactivity(Tataryn et al.,2021;Gareau et al.,2023).

Anesthesia,Anesthetics,and Postoperative Cognitive Dysfunction in Elderly Patients

Postoperative cognitive dysfunction(POCD)remains a major issue that worsens the prognosis of elderly surgery patients.This article reviews the current research on the effect of different anesthesia methods and commonly utilized anesthetics on the incidence of POCD in elderly patients,aiming to provide an understanding of the underlying mechanisms contributing to this condition and facilitate the development of more reasonable anesthesia protocols,ultimately reducing the incidence of POCD in elderly surgery patients.

Efficacy of tadalafil on improvement of men with erectile dysfunction caused by COVID-19:A randomized placebo-controlled trial

Objective: According to the high prevalence of COVID-19 and the subsequent risk of men's sexual health, we decided to investigate the efficacy of tadalafil on improvement of men with erectile dysfunction caused by COVID-19.Methods: In this study, 70 outpatients who were recovered from COVID-19 without acute respiratory distress syndrome with negative polymerase chain reaction test and a complaint of erectile dysfunction were divided into two groups: 35 patients who received tadalafil 5 mg daily and 35 who received placebo. For each patient, basic assessment of sexual function was performed using the 5-item version of the International Index of Erectile Function (IIEF-5) questionnaire. Then, treatment was started from 2 months after complete recovery of COVID-19 with negative polymerase chain reaction test for 3 months. At the end of the treatments, the patients were re-evaluated for sexual function using the complete version of IIEF questionnaire. Finally, the results before and after treatment in the intervention group were compared with those of the control group.Results: Treatment with both tadalafil and placebo improved the patients' sexual function criteria compared to the baseline. However, this improvement was significantly higher in the intervention group with tadalafil than the control group with placebo (p<0.05).Conclusion: Daily administration of tadalafil 5 mg seems to be effective and safe for improvement of erectile dysfunction caused by COVID-19.

Olfactory dysfunction and its related molecular mechanisms in Parkinson’s disease

Changes in olfactory function are considered to be early biomarkers of Parkinson’s disease.Olfactory dysfunction is one of the earliest non-motor features of Parkinson’s disease,appearing in about 90%of patients with early-stage Parkinson’s disease,and can often predate the diagnosis by years.Therefore,olfactory dysfunction should be considered a reliable marker of the disease.However,the mechanisms responsible for olfactory dysfunction are currently unknown.In this article,we clearly explain the pathology and medical definition of olfactory function as a biomarker for early-stage Parkinson’s disease.On the basis of the findings of clinical olfactory function tests and animal model experiments as well as neurotransmitter expression levels,we further characterize the relationship between olfactory dysfunction and neurodegenerative diseases as well as the molecular mechanisms underlying olfactory dysfunction in the pathology of early-stage Parkinson’s disease.The findings highlighted in this review suggest that olfactory dysfunction is an important biomarker for preclinical-stage Parkinson’s disease.Therefore,therapeutic drugs targeting non-motor symptoms such as olfactory dysfunction in the early stage of Parkinson’s disease may prevent or delay dopaminergic neurodegeneration and reduce motor symptoms,highlighting the potential of identifying effective targets for treating Parkinson’s disease by inhibiting the deterioration of olfactory dysfunction.

The ILux^(®) compared to the mechanical meibomian gland expression for the treatment of moderate and severe meibomian gland dysfunction

AIM:To compare the safety and effectiveness of eyelid treatment with the ILux®-MGD Treatment System in one session versus five sessions of mechanical meibomian gland expression(MMGE)in patients with moderate to severe meibomian gland dysfunction(MGD).METHODS:A prospective,randomized,open-label,and controlled clinical trial that compared one session of the ILux®MGD Treatment System versus five sessions of MMGE in both eyes of 130 patients aged≥18y with Ocular Surface Disease Index(OSDI)scores≥13,total meibomian gland scores(MGS)of<15 in the lower eyelid of each eye,and non-invasive tear break-up time(NI-TBUT)<10s,who were randomized 1:1 to ILux®or MMGE.RESULTS:The mean age was 58±17.49y.Baseline total MGS scores in both treatment groups were comparable.During follow-up,there were significant differences in total MGS and per sector with P<0.001.Multivariate analysis was performed using generalized estimating equations corresponding to the generalized linear model for repeated means to determine the treatment relationship with total MGS,NIBUT,and OSDI.There was a significant difference between ILux®and MMGE(P<0.001)at follow-up from the first to the twelfth month in MGS,NI-BUT,and OSDI scores.No adverse events were reported.CONCLUSION:ILux®treatment compared to MMGE significantly improves symptoms and signs in patients with moderate to severe MGD for one year without adverse events.

Standardization of meibomian gland dysfunction in an Egyptian population sample using a non-contact meibography technique

AIM:To develop normative data for meibomian gland dysfunction(MGD)parameters,using non-contact meibography technique of Sirius Costruzione Strumenti Oftalmici(CSO)machine,in an Egyptian population sample.METHODS:Observational,cross-sectional,analytic study,in which 104 Egyptian volunteers were included.Both upper lids were examined,using“Sirius CSO”machine.Each eyelid was given a degree of meibomian gland loss(MGL),which was calculated by the software of the machine.RESULTS:Mean percentage MGL in right upper lid was of 30.9%±12.6%,and that of left upper lid was 32.6%±11.8%.Thirty-four volunteers(32.7%)had first-degree MGL in their right upper lid,and 67.3%had second-degree loss.One volunteer(1%)had zero-degree MGL in left upper lid,28(26.9%)had first-degree loss,and 75(72.1%)had second-degree loss.Degree of MGL in right upper eyelid was not related to age,but degree of MGL in left upper eyelid increased with age.There was statistically significant difference between both genders for degree of MGL in right eye(P=0.036)and in left eye(P=0.027).CONCLUSION:Noncontact meibography is a useful non-invasive tool for diagnosing MGL.MGL is diagnosed in 100%of apparently normal individuals;26.9%-32.7%of which have first-degree MGL,and 67.3%-72.1%have second-degree MGL.

Interleukin 1βreceptor and synaptic dysfunction in recurrent brain infection with Herpes simplex virus type-1

Several experimental evidence suggests a link between brain Herpes simplex virus type-1 infection and the occurrence of Alzheimer’s disease.However,the molecular mechanisms underlying this association are not completely understood.Among the molecular mediators of synaptic and cognitive dysfunction occurring after Herpes simplex virus type-1 infection and reactivation in the brain neuroinflammatory cytokines seem to occupy a central role.Here,we specifically reviewed literature reports dealing with the impact of neuroinflammation on synaptic dysfunction observed after recurrent Herpes simplex virus type-1 reactivation in the brain,highlighting the role of interleukins and,in particular,interleukin 1βas a possible target against Herpes simplex virus type-1-induced neuronal dysfunctions.

Frequency and associated factors of accommodation and non-strabismic binocular vision dysfunction among medical university students

AIM:To investigate the frequency and associated factors of accommodation and non-strabismic binocular vision dysfunction among medical university students.METHODS:Totally 158 student volunteers underwent routine vision examination in the optometry clinic of Guangxi Medical University.Their data were used to identify the different types of accommodation and nonstrabismic binocular vision dysfunction and to determine their frequency.Correlation analysis and logistic regression were used to examine the factors associated with these abnormalities.RESULTS:The results showed that 36.71%of the subjects had accommodation and non-strabismic binocular vision issues,with 8.86%being attributed to accommodation dysfunction and 27.85%to binocular abnormalities.Convergence insufficiency(CI)was the most common abnormality,accounting for 13.29%.Those with these abnormalities experienced higher levels of eyestrain(χ2=69.518,P<0.001).The linear correlations were observed between the difference of binocular spherical equivalent(SE)and the index of horizontal esotropia at a distance(r=0.231,P=0.004)and the asthenopia survey scale(ASS)score(r=0.346,P<0.001).Furthermore,the right eye's SE was inversely correlated with the convergence of positive and negative fusion images at close range(r=-0.321,P<0.001),the convergence of negative fusion images at close range(r=-0.294,P<0.001),the vergence facility(VF;r=-0.234,P=0.003),and the set of negative fusion images at far range(r=-0.237,P=0.003).Logistic regression analysis indicated that gender,age,and the difference in right and binocular SE did not influence the emergence of these abnormalities.CONCLUSION:Binocular vision abnormalities are more prevalent than accommodation dysfunction,with CI being the most frequent type.Greater binocular refractive disparity leads to more severe eyestrain symptoms.

Interplay between metabolic dysfunction-associated fatty liver disease and renal function: An intriguing pediatric perspective

Over recent years,the nomenclature of non-alcoholic fatty liver disease has undergone significant changes.Indeed,in 2020,an expert consensus panel proposed the term“Metabolic(dysfunction)associated fatty liver disease”(MAFLD)to underscore the close association of fatty liver with metabolic abnormalities,thereby highlighting the cardiometabolic risks(such as metabolic syndrome,type 2 diabetes,insulin resistance,and cardiovascular disease)faced by these patients since childhood.More recently,this term has been further replaced with metabolic associated steatotic liver disease.It is worth noting that emerging evidence not only supports a close and independent association of MAFLD with chronic kidney disease in adults but also indicates its interplay with metabolic impairments.However,comparable pediatric data remain limited.Given the progressive and chronic nature of both diseases and their prognostic cardiometabolic implications,this editorial aims to provide a pediatric perspective on the intriguing relationship between MAFLD and renal function in childhood.

Punicalagin prevents obesity-related cardiac dysfunction through promoting DNA demethylation in mice

The aim of this study was to investigate whether punicalagin(PU)could prevent obesity-related cardiac dysfunction by promoting DNA demethy lation,and to explore its possible mechanism.C57BL/6J mice were fed with standard diet,high-fat diet(HFD),HFD supplemented with resveratrol,low-dose PU(LPU)and high-dose PU(HPU)for 8 weeks.Compared with HFD group,body weight was significantly lower in PU treatment groups,number of cardionwocytes and the protein level of myosin heavy chain 7B were significantly higher in PU treatment groups.Levels of 5-hydroxymethylcytosine and 5-formylcytosine were significantly lower in HFD group than in other groups.Compared with the HFD group,the protein level of ten-eleven translocation enzyme(TET)2 was significantly higher in PU treatment groups,p-AMP-activated protein kinase(AMPK)was significantly higher in LPU group.Levels of total antioxidant capacity and the protein levels of complexesⅡ/Ⅲ/Ⅴ,oxoglutarate dehydrogenase,succinate dehydrogenase B and fumarate hdrolase were significantly lower in HFD group than PU treatment group.The ratio of(succinic acid+fumaric acid)/a-ketoglutarate was significantly higher in HFD group than other groups.In conclusion,PU up-regulated TETs enzyme activities and TET2 protein stability through alleviating mitochondrial dysfunction and activating AMPK,so as to promote DNA demethylation,thus preventing obesity-related cardiac dysfunction.

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.