Study On Ocular Surface Energy Metabolism In Dry Eye And Its Mechanism

Part Ⅰ:The changes of energy metabolism of corneal epithelial cells in dry eyeBACKGROUND:The continuous self-renewal ability of corneal epithelial cells requires stable energy supply,in which glucose metabolism plays a major role.However,the changes of energy metabolism in dry eye development are poorly understood.PURPOSE:To investigate the changes of energy metabolism in corneal epithelial cells in dry eyeMETHODS:Female C57BL/6 mice aged 8-11 weeks were randomly divided into two groups.The dry eye group was induced by "dry environment(relative humidity 20±3%)combined with scopolamine subcutaneous injection",and the normal control group was fed in the normal environment without any treatment.Hypertonic stimulation(hypertonic medium containing 69 mM sodium chloride)of human corneal epithelial cell lines(HCECs)was used to induce in vitro cell drying model,normal control group was cultured on complete medium.The changes of energy rich phosphate compounds(AMP,ADP,ATP)and the activation of adenosine monophosphate-activated protein kinase(AMPK)in HCECs and murine corneal epithelial cells were detected to evaluate the changes of energy homeostasis.Then,the changes of mitochondrial morphology and structure,mitochondrial biogenesis including the expression of fusion proteins and mitotic proteins,mitochondrial membrane potential(MMP)level in corneal epithelial cells were detected to assess mitochondrial quality and Mitochondrial energy metabolism.The respiratory function and oxidative phosphorylation level of corneal epithelial cells were evaluated by measuring oxygen consumption rate,combined with the changes of glucose metabolites,the changes in the metabolic pathways during dry eye would be determined.To further evaluate the glucose uptake and utilization ability during dry eye,the expression of GLUT1 and key enzymes of glucose metabolism(HK,PFK,PKM and LDH)in cornea were detected by immunohistochemical staining,qRT PCR and WB,the glycogen content of corneal epithelial cells was detected by PAS staining.RESULTS:Compared with the normal control group,the relative level of AMP increased,ATP decreased,and AMPK was activated in HCECs after hypertonic stimulation and murine corneal epithelium in dry eye.Mitochondrial morphology and mitochondrial biogenesis of murine corneal epithelium impaired in dry eye,the level of mitochondrial membrane potential and Cellular oxidative phosphorylation decreased.The level of lactic acid,the expression of GLUT1 and key enzymes in glucose metabolism increased significantly in murine corneal epithelium of dry eye,and the content of glycogen decreased significantly.CONCLUSIONS:Energy dynamic equilibrium of corneal epithelium was disrupted in dry eye.Mitochondrial oxidative phosphorylation and mitochondrial biogenesis impaired,glycolysis increased,glucose uptake ability increased,and the glycogen synthesis decreased.Part Ⅱ:The regulation of AMPK on corneal epithelial metabolism and clinical phenotype during dry eyeBACKGROUND:AMPK is a key energy regulator that plays an important role in regulating cellular glucose homeostasis,mitochondrial homeostasis and metabolic reprogramming.However,the relationship between AMPK and the changes of corneal epithelial energy metabolism in dry eye remains unclear.PURPOSE:To investigate the regulation of AMPK on corneal epithelial metabolism and clinical phenotype during dry eyeMETHODS:AMPK-siRNA was used to transfect HCECs,the metabolic and clinical phenotypes of HCECs with and without hypertonic stimulation were observed.The experiment was divided into four groups:normal control group(NS),siRNA transfection group(NS-si),hypertonic group(HOsM)and hypertonic combined siRNA transfection group(HOSM-si).The changes of respiratory function and metabolic pathways of corneal epithelial cells after AMPK knockdown were evaluated by detecting the relative levels of ATP and lactic acid in cells and measuring the oxygen consumption rate of cells.The energy metabolism of HCECs after AMPK knockdown was further evaluated by assessing mitochondrial biogenesis and MMP.Then immunofluorescence staining and qRT PCR were used to detect the barrier function,apoptosis and inflammatory state of HCECs after AMPK knockdown.Finally,AMPKαlf/fα2f/f transgenic mice were hybridized with K12-Cre transgenic mice to obtain AMPKα1-/-αa2-/--K 12Cre conditional knockout mice.The corneal barrier function after AMPK deletion were observed by slit lamp,OCT and in vivo confocal microscopy.RESULTS:The relative level of ATP and lactate decreased in HCECs after AMPK knockdown,which suggesting the glycolysis pathway is inhibited.In AMPK knockdown HCECs,Cellular OCR decreased,mitochondrial biogenesis impaired and MMP decreased significantly,and more serious damage was more obvious in the HOSM-si group.In addition,the expressions of MMP3,IL1β and Caspase8 in HCECs were significantly increased after AMPK knockdown.And corneal epithelial AMPK conditional knockout mice had impaired ocular surface barrier function.CONCLUSIONS:The enhancement of glycolysis in dry eyes is AMPK dependent,and the activation of AMPK plays a protective role on the ocular surface.Part Ⅲ:Therapeutic effect of regulating energy metabolism in dry eyeBACKGROUND:Currently,various pharmacological treatments have been developed according to the existing pathological mechanisms,but none of them can completely relieve the discomfort of patients.It has been shown above that the energy dynamic equilibrium of cornea epithelium disrupted in dry eye,but the therapeutic effect of regulating corneal epithelial energy metabolism on dry eye is still unclear.PURPOSE:To investigate the therapeutic effect of regulating energy metabolism in dry eyeMETHODS:Murine tear production and OGD staining was to detect after topical application of A769662 in mice dry eye model.To investigate therapeutic effect of amino acid supplementation,HCECs was cultured in hypertonic medium to establish in vitro desiccating model.The cell viability was detected by CCK8 assasy,and the expression of AC-caspaseC3 and AC-caspase8 was detected by immunofluorescence staining.Finally,the relative level of ATP in HCECs after amino acid supplementation were detected by LCMS,and the activation of mTOR signaling pathway was detected by WB to verify the regulation of amino acid on energy metabolism of cornealepithelial cells.RESULTS:The tear production and OGD staining was not improved in A769662 group mice.Compared with hypertonic group,amino acid treatment can significantly improve the cell viability and apoptosis level of HCECs,and restore the intracellular ATP level to a certain extent.In addition,mTOR signaling pathway was significantly activated in HCECs after amino acid supplementation.CONCLUSIONS:Further activation of AMPK has no obvious therapeutic effect on dry eye,and replenishing energy substrates(amino acids)can restore ocular surface energy homeostasis to a certain extent,effectively treating dry eye.