Impact of PTEN inhibition on inflammation induced by lipopolysaccharide in corneal fibroblasts
Xiang Yang,Li Xuan,Tang Xin
Tianjin Eye Hospital, Tianjin Key Laboratory of Ophthalmology and Vision Science, Clinical College of Ophthalmology, Tianjin Medical Univesiity, Tianjin Eye Institute, Tianjin 300020, China
Objective To investigate the impact of BPV, the inhibitor of phosphatase and tensin homologue deleted on chromosome ten (PTEN), on lipopolysaccharide (LPS)-induced expression of inflammatory cytokines in rabbit corneal fibroblasts and to study its possible mechanism. Methods Ten healthy, mature New Zealand white rabbits bought in the Animal Lab Center of Tianjin Medical University were used in this experimental study. Primary cultures of rabbit corneal fibroblasts were isolated and cultured from corneal tissue explants in vitro. In the preliminary experiment, second-generation cells were divided into groups based on different doses of LPS (0.1, 1, 10 µg/ml) and BPV(250, 500, 1 000 nmol/L), and the survival rate of the cells was determined by MTT assay to determine proper concentrations. In the next stage of the experiment, corneal fibroblasts were randomly categorized into three groups based on different stimuli: a control group, LPS group and LPS+BPV group. The gene expression of inflammatory 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 and phospho-AKT were evaluated by Western blot. ELISA was conducted to detect the levels of IL-6 and IL-8 in the supernatant of each group. Data were analyzed by ANOVA and an LSD-t test. Results The results of the MTT assay in the preliminary experiment showed that different doses of LPS and BPV could reduce the survival rate of cells and the differences were statistically significant (χ²=22.32, 17.82, P<0.01). After multiple comparisons among groups, 1 µg/ml LPS and 500 nmol/L BPV were chosen as the proper concentrations. The results of real-time PCR indicated that the relative expression levels of IL-6 mRNA and IL-8 mRNA in all groups showed significant differences (F=46.65, 42.59, P<0.01). And IL-6 mRNA and IL-8 mRNA in the LPS group were 1.29±0.19 and 1.11±0.04, which were significantly higher than expression levels in the control group (P<0.01), but much lower than 1.17±0.08 and 0.92±0.15 in the LPS+BPV group (P<0.01). Western blot analysis showed that the relative expression of PTEN in the BPV group was significantly lower than expression in the control group (t=29.39, P<0.01). The relative expression levels of phospho-AKT in all groups showed statistically significant differences (F=62.83, P<0.01). And the relative expression of phospho-AKT in the LPS+BPV group was 0.82±0.07, which was significantly higher than 0.63±0.05 in the LPS group. All differences were statistically significant (P<0.01). The results of ELISA indicated that the relative mRNA expression levels of IL-6 and IL-8 in all groups were statistically different (F=32.41, 27.13, P<0.01). The relative expression levels of IL-6 and IL-8 in the supernatant of the LPS group were significantly higher than those in the control group (P<0.01) but much lower than levels in the LPS+BPV group (P<0.01). Conclusion BPV, the inhibitor of PTEN, may decrease LPS-induced gene expression and release of IL-6 and IL-8, inhibiting the inflammation reaction in rabbit corneal fibroblasts. The PI3K/AKT signal pathway may be involved in the process.
向阳,李轩,汤欣. PTEN抑制剂对脂多糖诱导兔角膜基质细胞炎性反应的影响[J]. 中华眼视光学与视觉科学杂志, 2016, 18(12): 729-735.
Xiang Yang,Li Xuan,Tang Xin. Impact of PTEN inhibition on inflammation induced by lipopolysaccharide in corneal fibroblasts. Chinese Journal of Optometry Ophthalmology and Visual science, 2016, 18(12): 729-735. DOI: DOI:10.3760/cma.j.issn.1674-845X.2016.12.006
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