Effects of different concentrations of tetramethylpyrazine,an active constituent of Chinese herb, on human corneal epithelial cell damaged by hydrogen peroxide

AIM: To discuss the effects of different concentrations of tetramethylpyrazine(TMP), an active constituent of Chinese herb, on damaged Shandong human corneal epithelial cell(SDHCEC) induced by hydrogen peroxide.METHODS: We detected the combined effects of TMP with concentrations ranging from 4 mg/m L to 0.03 mg/m L and 800 μM hydrogen peroxide on SDHCEC. The methyl thiazolyl tetrazolium(MTT) assay was processed at 3, 6and 12 h separately while the detection of cell apoptosis at 6h only by flow cytometry.RESULTS: The viability of SDHCEC with 0.5 mg/m L,0.25 mg/m L, 0.125 mg/m L and 0.06 mg/m L TMP joint with800 μM hydrogen peroxide at 3h and 6h was significantly higher than that with 800 μM hydrogen peroxide only, P <0.05. However, except 0.25 mg/m L, TMP with other concentrations joint with 800 μM hydrogen peroxide at12 h could not significantly inhibit decreased SDHCEC viability induced by 800 μM hydrogen peroxide. At 12 h,TMP of 0.5 mg/m L, 0.25 mg/m L, 0.125 mg/m L and 0.06 mg/m L could significantly inhibit SDHCEC early apoptosis induced by 800 μM hydrogen peroxide, most remarkable at 0.25 mg/m L TMP, P <0.05.CONCLUSION: Our results suggested that hydrogen peroxide can induce apoptosis related damage to SDHCEC. TMP can protect SDHCEC from the damage,and the protective effects may be associated with its anti-apoptosis mechanism.

The role of Dectin-1/Raf-1 signal cascade in innate immune of human corneal epithelial cells against Aspergillus fumigatus infection

AIM: To investigate the expression of the v-raf-1murine leukemia viral oncogene homolog 1(Raf-1) and its role in the innate immune response of human corneal epithelial cells(HCECs) infected by Aspergillus fumigatus.METHODS: HCECs were cultured in vitro.They were randomly divided into 4 groups,including control group,Aspergillus fumigatus group,GW5074(an inhibitor of Raf-1) group and Laminarin [an inhibitor of Dendriti-cell-associated C-type lectin 1(Dectin-1)] group.The protein expression level of total Raf-1 and p-Raf-1 was measured by Western blot.The expression of IL-6 and IL-8 m RNA in each group was detected by real-time polymerase chain reaction.RESULTS: In Aspergillus fumigatus group,total Raf-1 protein levels in HCECs remained unchanged at 5,15,30 and 45min after infection,while p-Raf-1 expression was significantly enhanced at 30 min after infection compared with control group.However,the expression of p-Raf-1 was apparently declined after treated with GW5074 or Laminarin compared with Aspergillus fumigatus group.The expression levels of IL-6,IL-8 m RNA were significantly increased after stimulation with fumigatus compared with control group.Pre-treated with GW5074 significantly inhibited Aspergillus fumigatus-induced upregulation of IL-8 and IL-6.CONCLUSION: Aspergillus fumigatus stimulation can elevate the expression of p-Raf-1 in HCECs in vitro.Dectin-1/Raf-1 signal pathway may play a role on regulating the expression of inflammatory cytokines,including IL-6 and IL-8.

Cytoprotective effect of amniotic membrane extracts on human corneal epithelial cells exposed to benzalkonium chloride in vitro

Background:The goal was to explore the protective effect and potential mechanism of amniotic membrane extracts(AME)on the ocular surface exposed to benzalkonium chloride(BAC).Methods:The human corneal epithelial cell(HCEC)line SD-HCEC1s was cultured in 5 groups:normal control(NC),NC+AME,BAC,BAC+NC,and BAC+AME.Cell viability analysis,flow cytometry analysis,real-time polymerase chain reaction(PCR),and western blot were employed to measure changes in cell function.Matrix metalloproteinases(MMPs)and inflammatory cytokines were assayed by enzyme-linked immunosorbent assay(ELISA)and activity assays.Results:Real-time PCR and western blot analysis demonstrated that the expressional level of caspase-8 was increased while the levels of Muc1,Muc4,and Muc16 were decreased after treatment with 0.02%BAC for 1 h.When the SD-HCEC1s were withdrawn from the BAC and switched to media containing 10%AME for 2 days,the expression level of capsase-8 was decreased while the levels of Muc1,Muc4,and Muc16 were increased.Real-time PCR and ELISA demonstrated that the mRNA and protein levels of MMP-1,MMP-3,MMP-13,CXCL1,interleukin(IL)-1β,IL-6,and tumor necrosis factor-alpha(TNF-α)were significantly increased after treatment with 0.02%BAC,whereas those of MMP-8 were decreased.When the 0.02%BAC was withdrawn and the SD-HCEC1s were cultured in 10%AME,the mRNA and protein levels of MMP-1,MMP-3,MMP-13,CXCL1,IL-1β,IL-6,and TNF-αwere decreased,while those of MMP-8 were increased.MMP-8 activity assays confirmed that IL-1βand TNF-αdownregulated the protein levels of MMP-8.Conclusions:AME protects SD-HCEC1s when stressed in BAC via upregulation of MMP-8 and downregulation of IL-1βand TNF-α.AME may have the potential functions to be employed as a topical adjunctive therapy in eyes chronically exposed to BAC.