Effects And Pathogenesis Of Hyperlipidemia On Murine Meibomian Gland And Corneal Endothelium

Part ?:High fat diet-induced obesity and hyperlipidemia promotes inflammation in murine meibomian gland via regulating PPAR-?signaling pathwayPurpose:To explore the link between high fat diet-induced obesity,hyperlipidemia and chronic inflammation in meibomian gland(MG)and to investigate the role of PPAR-?in it.Methods:Male C57BL/6J mice were placed on four different feeding regimens:a)standard diet(SD);b)high-fat diet(HFD);c)HFD supplemented with rosiglitazone,a PPAR-? agonist(HFD-rosi);d)HFD fed for 4 weeks followed by SD replacement for another 4 weeks(HFD-rev).Body weight,ocular sxurface integrity and eyelid changes were monitored at regular intervals.H&E or Oil Red O stained MG tissue sections.CD45,EL-1?,IL-6,TNF-? or F4/80 immunofluorescent staining evaluated immune responses.QRT?PCR evaluated PPAR-?,IL-1?,IL-6,IL-8,IL-10,TNF-?,IFN-?,MMP-3 and MMP-9 gene expression levels.Western blot determined PPAR-?,NF-?B p65,p?NF-?B p65,p38,p-p38,JNK,p-JNK,ERK1/2,and p-ERK1/2 protein expression levels.Results:HFD led to a significant increase of body weight of mice.Oil Red O staining showed accumulation of lipid in MG acini in HFD mice.H&E staining showed an increase in periglandular cell infiltrates in HFD mice.Increased infiltrations of CD45+and F4/80+ cells were observed in MG of HFD mice compared with SD mice.MGs of HFD mice exhibited downregulation of PPAR-? expression,upregulation of inflammatory cytokines and phosphorylation of MAPKs and NF-?B p65.There was significant reversal of MG inflammation in HFD-rosi and HFD-rev mice.Conclusion:HFD-induced obesity and hyperlipidemia promoted MG inflammation by inhibition of PPAR-? expression and activation of MAPK and NF-?B signaling pathways in mice.Part ?:Meibomian gland dysfunction in Apolipoprotein E-deficient micePurpose:To investigate the pathological changes of meibomian gland(MG)and ocular surface tissues in Apolipoprotein E knockout(ApoE-/-)mice,and to illustrate the association between meibomian gland dysfunction(MGD)and hyperlipidemia.Methods:The ocular surface of ApoE-/-male mice aged from 3 to 7 months,and age and sex matched wild type(WT)mice were observed under slit-lamp microscope and MG structure was clinically photographed with a stereoscopic zoom microscope.MG tissue sections were subjected to H&E staining,Oil Red O staining,TUNEL assay,and immuno-fluorescence staining for K10,Fabp5,Ki67,p63,PPAR-?,IL-6,TNF-?,NF-?B p65,p-NF-?B p65,AC-caspase 8,and immune-histochemical staining for CD45,4-HNE,NOX-4,3-NT.Quantitate RT-PCR and Western blot were performed to detect the above gene expression in MG tissues.The 5-month-old ApoE-/-mice were administered with rosiglitazone and GW9662 + rosiglitazone via oral gavage for 2 months.Results:We found eyelid abnormality,MG dropout,abnormal MG acinar morphology,dilated MG duct and plugging of MG orifice in ApoE-/-mice.MG acini in APoE-/-mice showed exaggerated lipid accumulation.Abnormal keratinization increased in MG duct and acinar cells,accompanied with decreased proliferation and increased apoptosis in MG.Inflammatory cells infiltrated into the surrounding microenvironment of MG acini,and the NF-?B signaling pathway was activated in MG acinar cells.Oxidative stress was evident in MG acinar cells of ApoE-/-mice.Further investigation showed downregulation of PPAR-? in MG acinar cells of ApoE-/-mice.PPAR-? agonist rosiglitazone treatment for 2 months reduced the morbidity of eyelid,corneal pathological changes and MG inflammation in ApoE-/-mice.Conclusion:Meibomian gland dysfunction(MGD)and hyperlipidemia are closely associated,ApoE-/-mice may be used as a model to study the pathophysiology and treatment of MGD related to lipid metabolism disorders.Part ?:Hyperlipidemia affects tight junction and pump function of corneal endotheliumPurpose:To investigate the pathological changes of corneal endothelium and related mechanism in hyperlipidemic murine models.Methods:Hyperlipidemic murine models were generated by feeding 4-week-old male wild type(WT)mice with high fat diet(HFD),and feeding Apolipoprotein E knockout(ApoE-/-)mice with standard diet(SD)or HFD.Control group was WT mice fed with SD.Body mass,cholesterol levels and fasting blood sugar concentrations were quantified after 16 weeks.Corneal endothelium density was evaluated by in vivo confocal microscopy(IVCM).Corneal endothelial tissue sections were subjected to scanning electron microscopy(SEM),transmission electron microscopy(TEM),and immunostaining for zonula occludens-1(ZO-1),N-cadherin,sodium-potassium adenosine triphosphatase(Na+-K+-ATPase),4-Hydroxynonenal(4-HNE),8hydroxy-2'-deoxyguanosine(8-OHdG)and NOX4.The gene expression of ZO-1,N-cadherin and Na+-K+-ATPase in comeal endothelium were evaluated by qRT-PCR.Corneal whole-mount tissues were subjected to Oil Red O staining to demonstrate lipid deposition.Corneal endothelium damage model was used to observe the corneal endothelium function after injuring.Aqueous humor was extracted and subjected to mass spectrometric identification.To mimic invivo hyperlipidemia,primary cultures of rabbit corneal endothelial cells(rCECs)were treated with varying concentrations of palmitate.After 24h of palmitate treatment,CCK8 viability assay and immunofluorescence staining for ZO-1,N-cadherin,Na+-K+-ATPase,translocase of the mitochondrial outer membrane 20(TOM20)and mitochondrial inner membrane 23(TIM23)were performed.The gene expression of ZO-1,N-cadherin,Na+-K+-ATPase,NADPH oxidase 4(NOX4),NFE2L2(Nrf2),superoxide dismutase 1(SOD1),catalase(CAT)and glutathione peroxidase 1(GPX1)were evaluated by qRT-PCR.Results:We found that hyperlipidemia was induced in both WT mice fed with HFD and ApoE-/-mice fed with SD or HFD.Oil Red O staining showed accumulation of lipid droplets in corneal endothelial cells(CECs)of hyperlipidemic mice.Reduced CECs density,distortion of endothelial cell shape,disruption of endothelial cell tight junction and adhesion junction,reduced CECs surface microvilli,downregulation of Na+-K--ATPase,activation of oxidative stress and damages in mitochondrial ultra-structures,and increased CECs apoptosis were observed in CECs of hyperlipidemic mice.Moreover,corneal endothelium recovery after injury was decompensated in hyperlipidemic mice.Further study showed palmitate levels in aqueous humor significantly increased in hyperlipidemia mice.Primary rabbit corneal endothelial cells(rCECs)treated with palmitate showed dose-dependent cytoxicitiy,variant cell morphology and reduced pump function in vitro hyperlipemia model.Conclusion:In conclusion,hyperlipidemia could induce oxidative stress of corneal endothelial cells,ultimately leading to pathological changes in CECs.Hyperlipidemia may become a risk factor of corneal endothelial dysfunctions.