PTEN抑制剂对脂多糖诱导兔角膜基质细胞炎性反应的影响

Impact of PTEN inhibition on inflammation induced by lipopolysaccharide in corneal fibroblasts

目的探讨第10号染色体丢失的张力蛋白同源的磷酸酶基因（PTEN）抑制剂双过氧化矾（BPV）对细菌脂多糖（LPS）诱导的兔角膜基质细胞炎症因子释放的影响及可能机制。方法实验研究。选取10只健康清洁级成年新西兰大白兔，采用组织块酶消化法获得原代兔角膜基质细胞并常规培养。取第2代细胞随机分为不同浓度梯度的LPS组（0．1、1、10μg／ml）及BPV组（250、500、1000nmol／L）进行预实验，MTT比色法检测各组细胞存活率以确定合适的LPS及BPV浓度。实验组分为正常对照组、LPS组、LPS＋BPV组，运用实时荧光定量PCR技术检测以上各组中白细胞介素．6（IL．6）及白细胞介素一8（IL广8）的mRNA表达水平；Westernblot法分别检测各实验组PTEN及磷酸化蛋白激酶B（p-AKT）的蛋白相对表达量。ELISA法检测各实验组上清液中IL-6及IL-8的分泌量。数据分析采用单因素方差分析，独立样本t检验、线性回归分析。结果在预实验的MTT比色法检测中，不同浓度梯度的LPS、BPV均可降低角膜基质细胞存活率，总体差异均有统计学意义（χ2=22．32、17．82，P〈0．01），选取1μg／ml、500nmol／L分别作为LPS、BPV的实验浓度。实时荧光定量PCR检测结果显示，正常对照组、LPS组、LPS＋BPV组IL-6、IL-8mRNA的表达量总体差异均有统计学意义（F=46．65、42．59，P〈0．01），两两比较显示LPS组IL-6、IL-8mRNA的表达量（分别为1．29±0．19、1．11±0．04），明显高于正常对照组（均为1．00±0．00）（P〈0．01），而LPS＋BPV组IL-6、IL-8mRNA的表达量（分别为1．17±0．08、0．92±0．15）明显低于LPS组（P〈0．01）。Western blot结果显示相对于正常对照组，LPS＋BPV组细胞PTEN蛋白表达水平较低（t=29．39，P〈0．01）。3组P-AKT的蛋白相对表达量总体差异均有统计学意义（F=62．83，P〈0．01），两两比较显示LPS＋BPV组p-AKT的蛋白表达量（0．82±0．07）明显高于LPS组（0．63±0．05）（P〈0．01）。ELISA法检测结果显示，3组上清液中IL-6、IL-8相对浓度的差异均有统计学意义（F=32．41、27．13，P〈0．01），与LPS组相比．正常对照组和LPS＋BPV组的IL-6、IL-8的相对浓度均明显较低．差异有统计学意义（P〈0．01）。结论PTEN的抑制剂BPV可下调由LPS诱导的兔角膜基质细胞炎症因子IL-6、IL-8基因表达及分泌，抑制角膜基质细胞炎症反应，PI3K／AKT信号传导通路可能在其中发挥重要调节作用。

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 cuhured 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 bloL 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 MTr 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 muhiple 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 1L-8, inhibiting the inflammation reaction in rabbit corneal fibroblasts. The PI3K/AKT signal pathway may be involved in the process.