Efforts Of PTEN Inhibition On Inflammation Induced By Lipopolysaccharide In Corneal Fibroblasts

Purpose To investigate the efforts of biperoxovanadate(BPV), the inhibitor of phosphatase and tensin homologue deleted on chromosome ten(PTEN), on lipopolysaccharide(LPS)-induced expression of inflammatory cytokins in rabbit corneal fibroblasts and to explore its possible mechanism.Methods Experimental study.Primary cultures of rabbit corneal fibroblasts were isolated and cultured from cornea tissue explants in vitro.The cells of fouth generation in exponential phase were collected and included in the study. In the preliminary experiment, the cells were divided into groups based on different doses of LPS and BPV, and the survival rate of the cells was detected by MTT assay to determine the proper concentration in the following experiment. Then, corneal fibroblasts were randomly categorized into four groups according to different stimulus: control group, LPS group, BPV group and LPS+BPV group. The gene expression of inflammation cytokines, for example interleukelin-6(IL-6) and Interleukelin-8(IL-8), were examined by Real-time quantitative polymerase chain reaction(PCR). Expressions of associated proteins, like PTEN, phospho-AKT were evaluated by Western blotting. Enzyme-linked immunosorbent assay(Elisa)was conducted to detect the level of IL-6 and IL-8 in the supernatant of each group. Data were analyzed statistically using ANOVA and LSD-t test.Results Primary rabbit corneal fibroblasts were isolated successfully and under regular culture procedure. The cells of fouth generation in exponential phase were collected and included in the study. The results of MTT assay in the preliminary experiment showed that the survival rate of cells in 0.1?g/ml LPS group, 1?g/ml LPS group, 10?g/ml LPS group was(98.9±3.56)%,(94.1±3.48)%,(86.6±2.80)% respectively, with the survival rate in control group set 100%. The differences were statistically significant(F=73.29,P<0.01). After the multiple comparison among groups, 1?g/ml LPS was chosen to build the inflamation models. Meanwhile, the survival rate of cells in 250nmol/L BPV group, 500nmol/L BPV group, 1000nmol/L BPV group was(96.0±2.23)%,(90.4±2.01)%,(86.0±2.47)% respectively, with the survival rate in control group set 100%. The differences were also statistically significant(F=41.86,P<0.01). After the multiple comparison among groups, 500nmol/L BPV was chosen to build the inflamation models. The results of realtime PCR indicated that the relative expression levels of IL-6 m RNA and IL-8 m RNA in LPS group were 1.79±0.19 and 1.37±0.18, significantly higher than these of control group(P<0.01) but much lower than 1.23±0.15 and 1.05±0.14 in LPS+BPV group( P<0.01). Western blot analysis showed that the relative expression of PTEN in BPV group were significantly lower than these of control group while the relative expression in LPS+BPV group were significantly lower than that of LPS group; the relative expression of phospho-AKT in LPS+BPV group were significantly higher than these of LPS group. All the differences were statistically significant(P<0.01). The results of Elisa indicated that the relative expression levels of IL-6 and IL-8 in the supernatant of LPS group were 1.21±0.03 and 1.37±0.04, significantly higher than these of control group(P<0.01) but much lower than 1.09±0.05 and 1.18±0.12 in LPS+BPV group( P<0.01).Conclusions: The inflammation of corneal fibroblasts can be successfully induced by LPS, with the upregulation of IL-6 and IL-8 both in the gene level and in the secretion level. As expected, the presence of BPV could inhibit the expression of PTEN in corneal fibroblasts. LPS could upregulate the expression of PTEN, as well as reduce the activation of AKT, which indicates that PTEN and AKT might be involved in the occurrence and development of inflammation reaction induced by LPS. BPV could inhibit the expression and secretion of IL-6 and IL-8, reduce the expression of PTEN and upregulate the activation of AKT after the LPS stimulus. In conclusion, BPV, the inhibitor of PTEN, may decrease the LPS-induced gene expression of IL-6 and IL-8 in corneal fibroblasts via the activition of AKT and up-regulation of PI3K/AKT signal pathway, resulting in the reduction of inflammatory mediators release and inhibition of inflammation reaction in corneal fibroblasts.