Exploring The Protective Effect Of Hinokitol Against PolyI:C And Hydrogen Peroxide Induced Damage In Human Corneal Epithelial Cell And The Underlying Mechanisms

Part Ⅰ PolyI:C and hydrogen peroxide induce inflammation and oxidative stress in human corneal epithelial cellsPurposeDry eye disease(DED)is reported as a multifactor-related disfunction of tear with potential damage to the ocular surface.Although people haven’t drawn a unanimous conclusion of the pathogenesis of DED,there is increasing envidence pointing to ocular surface inflammation and oxidative stress.This part of experiment was aiming to use PolyI:C and H2O2 to induce inflammation response and oxidative stress of human corneal epithelial cells(HCECs),as cellular models which are foundaments of intervening DED.MethodsIn vitro cultured HCECs were divided into 3 groups:normal control group,25μg/ml PolyI:C group and 500 μM H2O2.The transcriptional levels of IL-1β,IL-6 and IL-8 were measured by Real time quantitative polymerase chain reaction(RT-qPCR)and translational levels of these three cytokines were measured by Enzyme linked immunosorbent assay(ELISA),as assessments of inflammation status of HCECs.Cell counting kit-8(CCK-8)was used to test cell viablity.2’,7’-Dichlorodihydrofluorescein diacetate(DCFH-DA)fluorescent probe was used to measure reactive oxygen species(ROS).Terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)method was applied to detect apoptotic cells.And we used Western blot to observe the change of cleaved caspase-3,phosphorylated yH2AX and cytochrome c located inside and outside of mitochondrion.ResultsThe mRNA and protein levels of IL-1β,IL-6 and IL-8 were all significantly elevated by 25 μg/ml PolyI:C,indicating that it can effectivly cause inflammation response of HCECs.Cell viability was significantly suppressed by 500 μM H2O2.Besides,the intensity of DCFH-DA increased and the number of TUNEL positive cell rose after incubation of H2O2.Western blot showed that cleaved caspase-3 and phosphorylated yH2AX significantly increased and cytochrome c translocated from mitochondrion to cytoplasm.Conclusions25 μg/ml PolyI:C was able to upregulate proinflammatory cytokines so as to induce inflammation response of HCECs.500 μM H2O2 could cause ROS imbalance of HCECs,suppress cell viability and activate cell apoptotic pathway.Part Ⅱ Protective effect of Hinokitol against PolyI:C and hydrogen peroxide induced injury on HCECsPurposeThe part I experiment addressed that 25 μg/ml PolyI:C and 500μM H2O2 were capable of inducing inflammation response and oxidative stress on HCECs,respectively.Hinokitiol(HK),a natural tropolone-related compound isolated from the heartwood of Chymacyparis taiwanensis,has a wide variety of biochemical and pharmacological activities.Part II experiment was aiming to explore the effect of HK against those two cellular pathological models mentioned above.MethodsAfter incubation with HCECs with 0 μM、25 μM、50 μM、100 μM HK,we used CCK-8 to test the cellular toxicity of HK.HCECs were incubated with different concentrations of HK for 2 h before stimulated with 25 μg/ml PolyI:C for 24 h,then the mRNA and protein levels of IL-1β、IL-6 and IL-8 were measured by RT-qPCR and ELISA,respectively.HCECs were:incubated with different concentrations of HK for 2 h before stimulated with 500 μM H2O2 for 24 h,then CCK-8 was applied to value cell viability at different time points.DCFH-DA probe and TUNEL were adopted to detect ROS and cell apoptosis,respectively.Western blot was used to view changes of yH2AX,cleaved caspase-3 and cytochrome c.Results25-100 μM HK didn’t display significant cellular toxicity,campared with normal control.HK also decreased the mRNA and protein levels of IL-1β,IL-6 and IL-8 in a dose-dependent manner.Different concentrations of HK revived cell viability at different time points in different degrees.Cellular ROS and apoptosis were relieved by 100 μM HK.Phosphorylated γH2AX,cleaved caspase-3 and translocation of cytochome c were also suppressed by 100 μM HK.ConclusionHK was able to efficiently intervene the inflammation response and oxidative stress caused by PolyI:C and H2O2 by downregulating proinflammatory cytokines,suppressing ROS accumulation,attenuating the activation of apoptotic pathway,which may suggest a potential action of protecting HCECs and preventing DED.Part Ⅲ Mechanisms of protective effect of Hinokitol against PolyI:C and hydrogen peroxide induced injury on HCECsPurposeThe above experiment addressed that we successfully established inflammation response and oxidative stress models of HCECs and HK was able to efficiently intervene the inflammation response and oxidative stress caused by PolyI:C and H2O2.This part experiment was aiming to explore the mechanisms of protective effect of HK.MethodHCECs were incubated with different concentrations of HK for 2 h before stimulated with 25 μg/ml PolyI:C for 24 h,then Western blot was used to measure the protein levels of I IκBα and NFκB subunit p65 located inside and outside of nucleus.HCECs were incubated with 100μM of HK for 2 h before stimulated with 500 μM H2O2 for 24 h,then the level of malondialdehyde(MDA),the activities of superoxide dismutase(SOD),catalase(CAT)and total antioxidant capacity(T-AOC)were measured.Besides,protein levels of Bcl-2 and Bax were assessed by Western blot.Results100μM HK could maintain the cellular level of IκBα,restrain translocation of p65 from cytoplasm to nucleus,suppress generation of MDA,revive activities of SOD,CAT and T-AOC,rebalance the relative levels of Bcl-2/Bax,attenuate the activation of apoptotic pathway.ConclusionHK might protect HCECs through suppressing NFκB,attenuating oxidative stress and maintaining the relative relationship of Bcl-2/Bax.