The Study Of Ultraviolet B-induced Apoptosis In Rat Corneal Epithelial Cells And The Inhibition Effect Of Lycium Barbarum Polysaccharides On The Apoptosis And Its Mechanism

The animal eyes are sensitive for ultraviolet B?UVB?irradiation.The corneal epithelium,which is the first barrier when the external stimuli interact with the eyes,absorbs a large percentage of the UVB irradiation.Excess UVB irradiation induces apoptosis in the corneal epithelial cells,and it is known to be dose-dependent on UVB.However,the dynamic changes and time sequence of UVB-induced apoptosis are not systematic.It has been demonstrated that Lycium barbarum polysaccharides?LBPs?can protect lens epithelial cells,retinal pigment epithelial cells,and photoreceptor cells by inhibiting apoptosis induced by some types of damage.However,no reports have focused on the protective effect of LBPs against UVB-induced apoptosis and its mechanism in corneal epithelial cells.In the present study,the primary rat corneal epithelial?RCE?cells were isolated from adult female Wistar rats of standard body weight and cultured in vitro.Before the RCE cells used for experiments,the cell purity was detected by using cytokeratins 3and 12,which are the specific marker proteins of corneal epithelium,by immunofluorescence.We detected cell morphology,cell viability,apoptotic rate,the mitochondrial membrane potential?MMP?,and the expression levels of three apoptotic genes?Bax,caspase-8,and caspase-3?at different time points within 0-24 h after 144 mJ/cm² UVB irradiation in RCE cells to clarify the dynamic changes and time sequence of UVB-induced apoptosis in RCE cells.With or without treatment with LBPs,the RCE cells were exposed to UVB and the changes of cell viability,cell morphology,nuclear morphology,and apoptotic rate were measured to explore whether LBPs can protect RCE cells from UVB-induced apoptosis.The changes of the MMP,the expression levels of apoptotic genes?Bcl-2,Bax,and caspase-3?,phosphorylated c-Jun NH₂-terminal kinase?JNK?,and reactive oxygen species?ROS?level were detected to investigate the mechanism of this protective effect.The differentially expressed genes were analyzed after transcriptome sequencing to further explore the UVB-induced damage in RCE cells and the protective effect of LBPs.The results were as follows.The primary RCE cells were isolated and cultured successfully.The cells grew as monolayers and fibroblast-like adherence.They had well-defined bounds and rounded cell bodies after cell passage and grew confluence gradually.When cells had grown to80–90%confluence,they were digested and passaged.The purity of RCE cells was approximately80%according to the results of the cytokeratins 3 and 12 staining using immunofluorescence.After the cells were irradiated by 144 mJ/cm² UVB,the expression levels of caspase-8 and Bax were the highest at 0 h after irradiation.The MMP decreased at 0 h and remained constant for 6 h.At 6 h,caspase-3 was activated.The decrease in cell viability and the increase in apoptotic rate reached a peak at 6 h.Caspase-3 expression decreased within 12-24 h,leading to the decline in apoptotic rate and the rate of late apoptotic cells.When the RCE cells were incubated with different concentrations of LBPs,0-1 mg/mL LBPs did not affect cell viability,5 mg/mL LBPs increased cell viability,while 10 mg/mL LBPs resulted in a reduction in cell viability.0.05–1mg/mL LBPs prevented a UVB-induced decline in cell viability of RCE cells,and the optimal concentration of LBPs for this effect was 1 mg/mL.1 mg/mL LBPs significantly inhibited UVB-induced apoptotic morphology and apoptotic karyomorphology in RCE cells.It also decreased the rate of UVB-induced apoptotic nucleus from 38.06%±7.02%to 18.51%±0.94%,and decreased the apoptotic rate from 47.06%±1.83%to 13.93%±1.76%.1 mg/mL LBPs significantly prevented UVB-induced loss of MMP?P<0.05?.The Bax/Bcl-2 ratio was increased8.26±0.79-fold by UVB irradiation,and 1 mg/mL LBPs limited the increase to 2.91±0.08-fold.1 mg/mL LBPs significantly inhibited UVB-induced upregulation of caspase-3 at both the mRNA and protein levels?P<0.01?and UVB-induced JNK phosphorylation?P<0.05?.The ROS level was increased 2.41±0.15-fold by UVB irradiation,and 1 mg/mL LBPs limited the increase to1.44±0.17-fold.We found 4373 differentially expressed genes?1433 increased and 2940decreased?after UVB irradiation using transcriptome sequencing,and they were reduced to 1941?1326 increased and 615 decreased?when the cells were protected by 1 mg/mL LBPs.The differentially expressed genes were categorized by gene ontology?GO?analysis.We found that the main biological processes in the damage effect of UVB and the protective effect of LBPs included cell communication and signal transduction,cellular response to stimulus and wounding,movement of cell or subcellular component,DNA transcription,biosynthetic and metabolic processes of RNA and macromolecule,immune response and defense response,cytokine production,inflammatory response,cell motility and migration.Kyoto encyclopedia of genes and genomes?KEGG?analysis further identified the mitochondrial apoptosis pathway and JNK pathway were involved in the UVB-induced apoptosis and the inhibiton effect of LBPs for this apoptosis.In conclusion,excess UVB irradiation induced a rapid and ordered apoptotic process and the decline of cell viability in RCE cells.LBPs have a protective effect against UVB-induced apoptosis in RCE cells.The mechanism of this effect involves the upregulation of Bcl-2expression,downregulation of Bax expression,inhibition of MMP loss,and downregulation of caspase-3 expression.All factors involved in the mitochondrial apoptosis pathway.It also involves the attenuation of JNK phosphorylation and inhibition of ROS level.LBPs reduced the numbers of differentially expressed genes induced by UVB irradiation.Our work perfected the research of UVB-induced damage in corneal epithelial cells and filled the research void of the protective effect of LBPs against UVB-induced apoptosis and damage in corneal cells.It is for the first time to explore the effect of LBPs using transcriptome sequencing method.