酪氨酸激酶Src和酪氨酸磷酸酶SHP-2在脓毒症相关性脑病中的变化及其意义

Changes of tyrosine kinase Src and tyrosine phosphatase SHP-2 and their significance in sepsisassociated encephalopathy

目的研究酪氨酸激酶Src和酪氨酸磷酸酶SHP．2在脓毒症相关性脑病中的变化及其意义。方法Wistar大鼠88只按照随机数字表法分为脓毒症组m=80）和对照组（n=8）。脓毒症组大鼠通过腹腔注射革兰阴性菌脂多糖（LPS）10mg／kg建立脓毒症相关性脑病模型（32只大鼠成功建模），并分别在建模后6h、12h、24h和48h处死大鼠并取脑组织。对照组大鼠给予腹腔注射10mg／kg0．9％生理盐水后即刻处死，取脑组织作为对照组标本。通过Westernblotting、实时定量PCR（RT．PCRl和免疫荧光染色法观察Src和SHP．2蛋白及mRNA的变化。比较给予活性Src和SHP-2蛋白与线粒体蛋白反应前后呼吸链复合物酶活性的变化情况。结果Westernblotting、RT-PCR结果显示脓毒症组大鼠Src在6h出现明显的下降，持续至48h；同时间段SHP-2则出现明显的上升，与对照组比较差异均具有统计学意义（P〈0．05）。免疫荧光染色结果也显示48h脓毒症组大鼠Src表达较对照组明显下降，而SHP-2表达则明显升高，差异均有统计学意义（P〈0．05）。与活性Src蛋白反应后．48h脓毒症组大鼠呼吸链复合物Ⅰ、Ⅱ和Ⅲ酶活性明显升高，与活性SHP-2蛋白反应后呼吸链复合物Ⅰ、Ⅱ和Ⅲ酶活性明显下降，差异均具有统计学意义（P〈0．05）。结论脓毒症相关性脑病模型中，酪氨酸激酶Src和酪氨酸磷酸酶SHP-2可能是通过下调线粒体蛋白磷酸化降低了线粒体呼吸锛复合物酶活性．从而导致线粒体功能障碍。

Objective To investigate the changes of tyrosine kinase Src and tyrosine phosphatase SHP-2 and their significance in sepsis-associated encephalopathy. Methods Eighty-eight Wistar rats were randomly divided into septic group （n=80） and control group （n=60）; intrapefitoneal injection of I 0 mg/kg of lipopolysaccharide （LPS） was given to induce sepsis-associated encephalopathy models in the septic group; and 10 mg/kg 0.9% normal saline was given to the rats in the control group. Animals were sacrificed and brain tissues were quickly removed at the indicated time points （0 h for control group and 6, 12, 24 and 48 h after injections for septic group）. Western blotting, real time quantitative-PCR and immunofluorescence were used to analyze the protein and mRNA alterations of Src and SHP-2. Activities of mitochondrial oxidative phosphorylation complexes I-V were measured by enzyme assay kits, and enzyme activities with or without pretreatment of mitochondrial proteins with active Src or SHP-2 were analyzed. Results Western blotting and real time-PCR indicated that Src levels in the septic group were obviously decreased at the 6 h and till the 48 h, with significant differences as compared with those in the control group （/9〈0.05）; however, the opposite trend was noted in the SHP-2 levels. Immunofluorescence showed the same results of Western blotting. Pretreatment of mitochondrial proteins with active Src significantly enhanced complex I, II and III activities in vitro,while pretreatment of active SHP-2 produced opposite effects; significant difference was noted between the two （P〈0.05）. Conclusion Brain mitochondrial dysfunction of septic rat is partly affected due to a decrease in mitochondria proteins tyrosine phosphorylation by Src and SHP-2.