乌司他丁对脑出血大鼠局部黏着斑激酶 /细胞外信号调节激酶信号通路及神经元凋亡的影响

目的探讨乌司他丁( UTI)对脑出血( ICH)大鼠局部黏着斑激酶( FAK)/细胞外信号调节激酶( ERK)信号通路及神经元凋亡的影响。方法建立 ICH大鼠模型，使用随机数字表法将造模成功的大鼠随机分为模型组(尾静脉注射 0.9%氯化钠溶液)、抑制剂组( FAK抑制剂 PF562271，50 mg/kg灌胃)、 UTI组(尾静脉注射 10万 U/kg UTI)、 UTI+抑制剂组( FAK抑制剂 PF562271，50 mg/kg灌胃同时尾静脉注射 10万 U/kg UTI)每组 20只，另设 20只为假手术组(尾静脉注射 0.9%氯化钠溶液)作为对照，所有处理均每天 1次，连续 7d。干湿比重法测定各，组大鼠脑组织含水率；酶联免疫吸附(ELISA)法各组大鼠血清中炎性因子含量；原位末端标记( TUNEL)法检测各组大鼠海马组织神经元凋亡；免疫组织化学分析法检测大鼠海马组织抗凋亡因子 B细胞淋巴瘤 -2(Bcl-2)和促凋亡因子 Bcl相关 X(Bax)、胱天蛋白酶 3(caspase-3)表达；尼氏染色法检测各组大鼠海马组织中神经元存活情况；蛋白质印迹法( Western blotting)法检测大鼠海马组织中 FAK蛋白表达和 ERK1/2磷酸化水平。结果与假手术组相比，模型组大鼠脑组织含水率( 84.51±7.42)%、炎性因子含量、神经元凋亡指数( 44.51±3.77)%以及促凋亡因子 Bax(3.05±0.41)、 caspase-3(3.27±0.46)表达和 ERK1/2磷酸化水平升高，神经元存活数目、抑凋亡因子 Bcl-2(1.23±0.21)表达以及 FAK(0.29±0.07)蛋白表达水平降低( P<0.05)。与模型组相比， UTI组大鼠脑组织含水率(71.43±6.11)%、炎性因子含量、神经元凋亡指数( 15.34±0.76)%以及促凋亡因子 Bax(2.03±0.15)、 caspcase-3(2.27±0.61)表达和 ERK1/2磷酸化水平降低，神经元存活数目、抑凋亡因子 Bcl-2(2.67±0.27)表达以及 FAK(0.58±0.09)蛋白表达升高( P<0.05)；抑制剂组大鼠脑组织含水率( 92.83±7.56)%、炎性因子含量、神经元凋亡指数( 51.99±5.65)%以及促凋亡因子 Bax(3.73±0.37)、 caspcase-3(3.99±0.26)表达和 ERK1/2磷酸化水平升高，神经元存活数目、抑凋亡因子 Bcl-2(0.73±0.06)表达以及 FAK(0.12±0.02)蛋白表达水平降低( P<0.05)。与 UTI组相比， UTI+抑制剂组大鼠脑组织含水率( 84.16±6.76)%、炎性因子含量、神经元凋亡指数( 43.22±4.07)%以及促凋亡因子 Bax(2.98±0.35)、 caspcase-3(3.26±0.31)表达和 ERK1/2磷酸化水平升高，神经元存活数目、抑凋亡因子 Bcl-2(1.27±0.12)表达以 及 FAK(0.28±0.03)蛋白表达水平降低( P<0.05)。结论 UTI可通过促进 FAK蛋白表达，抑制 ERK磷酸化，进而抑制 ICH大鼠神经元凋亡。

Effects of ulinastatin on FAK/ERK signaling pathway and neuronal apoptosis in rats with intracerebral hemorrhage

Objective To investigate the effects of ulinastatin (UTI) on focal adhesion kinase (FAK)/extracellular signal regulated ki.nase (ERK) signal pathway and neuronal apoptosis in rats with intracerebral hemorrhage.Methods ICH rat models were established and randomly divided into model group (tail vein injection of 0.9% sodium chloride solution), inhibitor group (FAK inhibitor PF562271,50 mg/kg by gavage), UTI group (tail vein injection of 100 000 U/kg UTI), UTI+inhibitor group (gavage of FAK inhibitor pf562271 and50 mg/kg, meanwhile, tail vein injection of 100 000 U/kg UTI), with 20 rats in each group, another 20 rats were divided into sham opera.tion group (tail vein injection of 0.9% sodium chloride solution) as control, all treatments were once a day for 7 consecutive days. Thewater content of brain tissue was measured by dry wet specific gravity method; the content of inflammatory factors in serum of rats ineach group was detected by enzyme-linked immunosorbent assay (ELISA); TUNEL method was used to detect neuronal apoptosis in hip.pocampus;the expressions of anti apoptotic factor Bcl-2 and pro apoptotic factor Bax, caspase-3 were detected by immunohistochemistry;the survival of neurons in hippocampal tissues of rats were detected by Nysch staining; and Western blotting was used to detect FAKprotein expression and ERK1/2 phosphorylation level in hippocampus.Results Compared with those in the sham operation group, thewater content of brain tissue(84.51±7.42)%, inflammatory factor content, neuronal apoptosis index(44.51±3.77)%, Bax(3.05±0.41) andcaspase-3(3.27±0.46) expressions, ERK1/2 phosphorylation level in model group were higher, while the expressions of Bcl-2(1.23± 0.21) and FAK(0.29±0.07) protein was lower (P< 0.05). Compared with those in the model group, the water content of brain tissue(71.43±6.11)%, inflammatory factor content, neuronal apoptosis index(15.34±0.76)%, Bax(2.03±0.15) and caspase-3(2.27±0.61) expres.sions, ERK1/2 phosphorylation level in UTI group were lower, while the number of neurons alive, the expressions of Bcl-2(2.67±0.27) and FAK(0.58±0.09) protein was higher (P<0.05); the water content of brain tissue(92.83±7.56)%, inflammatory factor content, neuro.nal apoptosis index(51.99±5.65)%, Bax(3.73±0.37) and caspase-3(3.99±0.26) expressions, ERK1/2 phosphorylation level in inhibitorgroup were higher, while the number of neurons alive, the expressions of Bcl-2(0.73±0.06) and FAK(0.12±0.02) protein was lower (P< 0.05). Compared with those in UTI group, the water content of brain tissue(84.16±6.76)%, inflammatory factor content(43.22±4.07)%,neuronal apoptosis index, Bax(2.98±0.35) and caspase-3(3.26±0.31) expressions, ERK1/2 phosphorylation level in UTI+inhibitor groupwere higher, while the number of neurons alive, the expressions of Bcl-2(1.27±0.12) and FAK(0.28±0.03) protein was lower (P<0.05). Conclusion UTI can inhibit ERK phosphorylation by promoting FAK protein expression, then inhibit neuronal apoptosis in ICH rats.