The Effect Of Interleukin 8 And Its Receptor Antagonist G31P On Retinal Ganglion Cells

Objective:Glaucoma,a leading cause of irreversible blindness worldwide,is a degenerative retinal disease characterized by the structural damage to the optic nerve and progressive death of retinal ganglion cells?RGCs?.Recent experiments suggest that cytokines may play an important role in the process of glaucoma.Abnormal expression of Interleukin-8 and other cytokines were detected in aqueous humor and peripheral blood of glaucoma patients.Meanwhile,cell experiment in vitro has covered that Interleukin-8 may have negative effect on neuron.Interleukin-8?IL-8?,also called CXCL8,is a Cysteine-amino acid-Cysteine?CXC?chemokine and it is involved in regulating inflammation,proangiogenesis,proliferation and migration of tumor cells by bonding with CXCR1 and CXCR2.CXCR1/2 antagonist G31P,can block the activity of CXCL8 by binding CXCR1/2 with higher affinity,is widely used in anti-inflammation and anti-tumor research.This study investigated the effect of CXCL8 on neural RGCs and whether it can be alleviated by G31P.Method:1.Cell culture:The Retinal ganglion cell lines,RGC-5,was cultured in complete DMEM-F12mediumcontaining10%fetalbovineserumand1%penicillin/streptomycin?Gibico?at 37?in humidified incubator containing 5%CO_2,and changed the medium after 24h.2.The cells were divided into three groups:control,CXCL8,and G31P pretreatment.Treatments were administered to cultures with more than 80%confluence.The concentration of G31P used was 100ng/ml based on recommendations from previous studies.3.Methods3.1 Cell activity experiment:Cells were divided into control and CXCL8?5,10,20,25,50,100ng/ml?and the cell viability was assessed after 24h by Cell Counting Kit 8?CCK-8?under microplate reader.3.2 Cell apoptosis:Apoptosis was measured by comparing the changes of nuclear morphology by direct observation under inverted microscope and Hoechst 33258fluorescence staining,grouping as method 2.After 24h of treatment,cells were fixed,incubated with Hoechst33258 reagent,then observed under inverted fluorescence microscope.Flow cytometric analysis of the apoptosis rate,grouping as method 2,collected and then the cells were added to Annexin V-FITC and PI dye for incubation,detected by flow cytometry.3.3 Expression of CXCR1 and CXCR2:RT-PCR detected the content of CXCR1/2mRNA in the cells under the normal condition and treated with CXCL8 after 24h.3.4 Expression of apoptotic genes:The expression of apoptosis-related genes were detected by RT-PCR and Western bloting from nucleic acid and protein levels.Results:1.CCK-8 assay showed that CXCL8 significantly inhibits the proliferation of RGC-5 cells in a dose-dependent manner.The survival ratio of different CXCL8 treated groups were 0.72%±0.03%,0.62%±0.07%,0.51%±0.05%,0.38%±0.03%,0.36%±0.03%,0.33%±0.05%.2.Observation under inverted microscope showed that the morphology of cells start to change into round,cell body shorten and a small amount of them fall off.However the growth and adhesion conditions of control group were good.Hoechst33258 fluorescence staining results indicated that,compared with the control group,the morphological changes symbolize apoptosis such as condensed and fragmented fluorescent nuclei increased after exposed to CXCL8.Pre-treated with G31P can alleviate the apoptotic morphological impairment induced by CXCL8.Annexin V/PI staining assay revealed apoptosis ratio of RGC-5 in control group,CXCL8 group and CXCL8+G31P group was 2.8%±0.20%?5.62%±0.50%?2.89%±0.45%respectively.CXCL8 treatment for 24h could increase the cell apoptosis rate by about 2 times,while the apoptosis in the G31P preincubated group was similar to that of the normal control group.The difference between each group was statically significant?P<0.01?.3.RT-PCR results showed that CXCR1/2 was expressed in RGC-5 cells,and the level of those two was elevated in varying degrees after CXCL8 treatment.4.RT-PCR and Western blot results indicated that CXCL8 could promote Bax and caspase-3 expressions,but decrease the level of Bcl-2 in the aspect of nucleic acid and protein.However,pre-treatment with G31P partly attenuates these effects in RGC-5with statistical significance.Conclusion:1.CXCL8 was able to inhabit cell viability,induce apoptosis and damage RGC-5in vitro.Meanwhile,G31P could effectively protect RGC-5 against the injury caused by IL-8.2.The traumatic effect of CXCL8 may be associated with the up-regulation of classic apoptosis related genes such as Bax and Caspase-3 proteins,down-regulation of anti-apoptosis Bcl-2 expression.