脂氧素A4对内毒素攻击的肺泡Ⅱ型上皮细胞Na＋-K＋-ATP酶的影响

Effect of lipoxin A4 on the Na＋-K＋-ATPase in alveolar type Ⅱ epithelial cells of rats treated with endotoxin

目的 探讨促炎症消退介质脂氧素A4（LipoxinA4,LXA4）对内毒素（endotoxin）攻击的大鼠肺泡Ⅱ型上皮细胞（Alveolar type Ⅱ epithelial cells,ATⅡ）Na＋-K＋-ATP酶的影响.方法 AT Ⅱ成功分离纯化后随机（随机数字法）分为5组：①空白组（PBS）;②溶剂对照组（乙醇,0.7μL/mL）;③脂多糖（lipopolysaccharide,LPS）（1μg/mL）组;④LXA4（1×10-7mol/mL）组;⑤LPS（1μg/mL）＋LXA4（1×10-7mol/mL）组.药物干预4 h后用RT-PCR法检测ATⅡ上Na＋-K＋-ATP酶α1,β1亚基的mRNA的表达,用高效液相法测定细胞ATP,ADP,AMP和腺嘌呤核苷酸总量（TAN）,间接测得Na＋-K＋-ATP酶的活性和AT Ⅱ能荷.结果 RT-PCR结果显示：LPS组α1,β1亚基的mRNA表达较空白组明显降低（P＜0.05）,而LPS＋LXA4组的α1,β1亚基的mRNA表达较LPS模型组有明显增高（P＜0.05）.酶活性检测结果显示：LPS组Na＋-K＋-ATP酶活性较空白组明显增高（P＜0.05）.与LPS刺激组比较,LPS＋LXA4组酶活性明显增高（P＜0.05）.能荷结果显示：LPS组较空白组明显增高（P＜0.05）,其余各组间差异无统计学意义（P＞0.05）.结论 LXA4能上调内毒素攻击的大鼠肺泡Ⅱ型上皮细胞Na＋-K＋-ATP酶α1,β1亚基mRNA表达,增强Na＋-K＋-ATP酶的活性,并能有效维持细胞的能量代谢平衡,提示LXA4可能通过上调Na＋-K＋-ATP酶的基因表达和功能活性,维持细胞代谢稳定,从而起到促进肺泡水肿液清除的作用.

Objective To study the protective role of pre-resolving mediator lipoxin A4（LXA4） in the NA＋ -K＋-ATPase in alveolar type Ⅱ （AT Ⅱ ） epithelial cells of rats exposed to lipopolysaccharide （LPS）. Method The AT Ⅱ cells were isolated and purified, and divided randomly into control group （PBS）, vehiculum （alcohol 0.7 μL/mL） group, LPS （1 μg/mL） group, LXA4（1/10 mol/mL） group and LPS （1 μg/mL LPS） ＋ LXA4（1/10 mol/mL） group. After exposure to LPS and/or LXA4 for4 hours, NA＋-K＋ -ATPase and β1-subunits mRNA in AT Ⅱ epithelial cells were detected by using RT-PCR, and ATP, ADP, AMP, total adenine nucleotides （TAN） and energy charge （EC） were measured by using high performance liquid chromatography （HPLC）, and then the activities of Na＋-K＋-ATPase were calculated accordingly. Results The NA＋-K＋-ATPase α-subunit and β-subunit mRNA were significantly decreased in LPS group （ P 〈 0.05 vs. control group）. However, the expressions of NA＋ -K＋-ATPase mRNA were significantly enhanced by application of LXA4 to AT Ⅱ epithelial cells exposed to LPS （P 〈0.05 vs. LPS group）. The activities of NA＋ -K＋ -ATPase were enhanced in LPS group （P 〈0.05 vs. control group）. Compared with control group and LPS group, the activities of NA＋-K＋-ATpase in LPS ＋ LXA4 group were significantly increased （P 〈0.01 vs. control group; P 〈0.05 vs. LPS group）. The EC of AT Ⅱ epithelial cells were higher in LPS group （ P 〈 0.01 vs. control group）. There were no significant differences in EC between control group and LPS ＋ LXA4group（P 〉0.05）. Conclusions The pro-resolving mediator LXA4 can enhance the expressions of NA ＋ -K ＋ -ATPase α-subunit and β-subunit mRNA, and the activities of NA ＋ -K ＋ -ATPase in AT Ⅱ epithelial cells or rats exposed to LPS, and ca also balance the metabolism of AT Ⅱ epithelial cells. These findings suggest that LXA4 plays an important role in lung edema clearance in lung injury induced by endotoxin, and the role is likely associated with the enhancement of the expressions of Na＋ -K＋ -AT-Pase α-subunit and β-subunit, and the activities of Na＋ -K＊ -ATPase, maintaining the balance of metabolism of AT Ⅱ epithelial cells.