大鼠脑创伤后MKP-1表达上调、NO的生成减少

Increased expression of MKP-1 and reduced production of NO in brain tissue of rat after traumatic injury

目的研究创伤性脑损伤(TBI)周围皮质中丝裂原活化蛋白激酶磷酸酶-1(MKP-1)的表达变化,探讨其对一氧化氮(NO)含量的影响及机制。方法用自由落体法制作大鼠TBI模型;酶化学法检测周围皮质中NO的含量;免疫组织化学和免疫印迹等方法检测脑皮质MKP-1、e NOS及MAPKs的表达。结果对照组脑皮质NO水平为34.4±3.2μmol/L,TBI损伤后3、6、24及72 h均显著下降,分别为20.8±2.5、23.9±3.8、24.0±1.6及26.8±2.6μmol/L(均P<0.05)。脑损伤周围皮质中胞质和突起呈棕黄色的MKP-1免疫阳性细胞数量增加。脑损伤后3和6 h MKP-1蛋白表达水平明显增加(P<0.05),随后降低至正常水平;e NOS蛋白表达水平在脑损伤后3和6h则明显降低(均P<0.05),至24 h接近正常水平;p ERK和p-P38 MAPK蛋白表达水平在脑损伤后3和6 h也分别下降(均P<0.05)。结论大鼠TBI后,皮质中MKP-1表达上调可能负向调控MAPK信号而降低e NOS水平,引起NO生成减少造成脑血流灌注不足。

Objective To detect expressive changes of MKP-1 and measure NO content in pericontusional cerebral cortex of rat after traumatic brain injury （TBI）, to explore regulatory role and molecular mechanism of MKP-1 on NO generation after TBI. Methods We constructed TBI model by Feeney＇s method. Nitric oxide detection kits were used to measure NO content in pericontusional cerebral cortex between control group and TBI group. Distribu- tion of MKP-1 was detected by immunohistochemistry. Western blot analysis was performed to detect expression lev- el of MKP-1, endothelial NOS and MAPKs in the rats of pericontusional cerebral cortex after TBI. Results NO level significantly decreased at 3, 6, 24 and 72 h with concentration as （20. 8 ±2. 5）μmol/L, （23.9 ±3.8）μmol/L, （24.0 ± 1.6） μmol/L, （26.8 ± 2.6 ）μmo]/L （ P 〈 0. 05 ） respectively, compared with the control as （ 34. 4 ± 3.2） μmoL/L after TBI. The positive staining MKP-1 cells were scattered and distributed in pericontusional cerebral cortex with brown cytoplasma and processes and increased significantly at 3 h and 6 h after TBI. Compared to the control group, the protein expressions of MKP-1 were increased at 3 h and 6 h respectively with normal level at 24 hafter TBI （ P 〈 0. 05 ）. While the protein level of eNOS decreased at 3 h and 6 h respectively after TBI （ P 〈 0.05 ）. Both protein expression of pERK and p-P38 decreased at 3 h and 6 h respectively after TBI compared with control group （P 〈 0. 05 ）. Conclusions Increased MKP-1 expression in pericontusional cerebral cortex after TBI specific- ally inhibits the expression level of pERK and p-P38 MAPK with the pJNK intact to down-regulated the expression of endothelial NOS, which decrease NO content and attribute to the defect of cerebral blood flow.