程序性坏死特异性抑制剂-1对创伤失血性休克大鼠肝脏保护作用的研究

Protective effect of necrostatin-1 on the liver of rats with trauma induced hemorrhagic shock

目的探讨程序性坏死特异性抑制剂-1（Nee-1）对创伤失血性休克大鼠的肝脏保护作用。方法采用左下肢股骨、胫骨骨折及腹部软组织损伤并失血／再灌注的方法制备大鼠创伤失血性休克模型。选择雄性SD大鼠，22只按随机数字表法分为模型组和Nec-1组，每组11只，观察72h死亡率。72只同法随机分为假手术组、模型组、Nec-1组，每组24只。假手术组仅麻醉和分离、结扎血管，不进行创伤、失血、再灌注；Nee-1组于再灌注前5min经股静脉给予1mg／kgNee-1；模型组给予等体积溶剂。于再灌注后2,4、8h采集各组血清和肝组织，用全自动生化仪检测血清丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）的水平；光镜下观察肝组织病理学改变；采用逆转录-聚合酶链反应（RT—PCR）检测肝组织肿瘤坏死因子-仪（TNF—Ot）和白细胞介素-I[3（IL-1B）的mRNA表达；蛋白质免疫印迹试验（WesternBlot）检测肝组织受体相互作用蛋白酶-1／3（RIPl／RIP3）的蛋白表达。结果Nee-1组大鼠72h死亡率较模型组明显降低（18．18％（2／11）比63．64％（7／11），P=0．0403。模型组2h血清ALT、AST即较假手术组明显升高[ALT（U／L）：110．21±22．32比80．98±19．94。AST（U／L）：364．29±64．83比279．76±70．64，均P〈0．05J，8h达高峰[ALT（U／L）：387．41±47．11比82．76±22．44，AST（U／L）：973．35±77．51比261．49±52．03，均P〈0．01]。Nee-1组血清ALT、AST水平较模型组明显降低[ALT（U/L）4h：144．64±33．79比213．96±36．21，8h：159．48±43．57比387．41±47．11；AST（U／L）4h：398．78±59．48比630．61±59．93，8h：427．38±80．75比973．35±77．51，均P〈0．01]。光镜下模型组大鼠肝窦扩张、淤血，肝细胞变性、坏死，大量炎性细胞浸润；Nec-1组肝组织损伤程度明显减轻。模型组肝组织TNF—α、IL-1β的mRNA表达和RIPl、RIP3的蛋白表达随时间延长呈逐渐升高趋势；给予Nec-1后各时间点TNF—α、IL-1β的mRNA表达均较模型组明显降低，以8h最为明显（TNF—αmRNA：1．457±0．081比2．317±0．062，IL—1βmRNA：0．690±0．087比1．812±0．112，均P〈0．01），而肝组织RIP1、RIP3的蛋白表达与模型组相比差异无统计学意义（RIPl蛋白8h：0．561±0．033比0．5874-0．036，RIP3蛋白8h：0．976±0．040比1．044±0．115，均P〉O．05）。结论Nec-1对创伤失血性休克大鼠肝脏具有明显的保护作用，具体机制需进一步深入研究。

Objective To investigate the effects of necrostatin-1 （Nec-1） on the liver of rats with trauma induced hemorrhagic shock. Methods Trauma induced hemorrhagic shock model was produced by adopting the left femur, tibia fracture and soft tissue injury, bleeding and reperfusion in male Sprague-Dawley （SD） rats. A total of 22 rats were divided into model group and Nec-1 group with 11 rats in each group by randomized digital number method and the 72-hour mortality was observed. In addition, 72 rats were randomly divided into sham group, model group, Nec-1 group with 24 rats in each group. Rats in sham group were only received anesthesia, separating and ligating blood vessels, without trauma induced hemorrhagic and reperfusion, and the rats in Nec-1 group were received 1 mg/kg Nec-1 through femoral vein 5 minutes before reperfusion, while the rats in model group were received the same amount of solvent. The serum and liver tissues of each group were collected at 2, 4, 8 hours after reperfusion. Serum alanine aminotransferase （ALT） and aspartate aminotransferase （AST） were detected by automatic biochemistry analyzer. The pathology changes in liver were observed by hematoxylin-eosin （HE） staining. The mRNA expressions of tumor necrosis factor-α （TNF-α） and interleukin- 1β （IL- 1β ） in the liver were determined by reverse transcription-polymerase chain reaction （RT-PCR）. The protein expressions of receptor interaction of protease 1/3 （RIP1/RIP3） were also assessed byWestern Blot analysis. Results Compared with model group, Nec-1 significantly reduced the 72-hour mortality 18.18% （2/11 ） vs. 63.64% （7/11 ）, P= 0.0401. Two hours after trauma induced hemorrhagic shock and reperfusion, the expressions of ALT and AST in model group were significantly increased compared with those in sham group [ALT （U/L）： 110.21±22.32 vs. 80.98 ± 19.94, AST （U/L）： 364.29 ±64.83 vs. 279.76 ±70.64, both P〈0.053, and reached the peak at 8 hours [ ALT （U/L）： 387.41 ± 47.11 vs. 82.76 ± 22.44, AST （U/L） ： 973.35 ± 77.51 vs. 261.49 ± 52.03, both P〈O.O13. Levels of serum ALT and AST in Nee-1 group were significantly decreased compared with model group （ALT （U/L） 4 hours： 144.64 ± 33.79 vs. 213.96 ± 36.21, 8 hours： 159.48 ± 43.57 vs. 387.41 ±47.11 ; AST （U/L） 4 hours： 398.78 ±59.48 vs. 630.61 ± 59.93, 8 hours： 427.38 ± 80.75 vs. 973.35 ± 77.51, all P〈0.01）. Under light microscopy, it was noted that the hepatic sinus expansion, liver cells degeneration, necrosis, as well as infiltration of abundant inflammatory ceils were observed. But the pathology changes in hepatic tissues were significantly mitigated in N ec-1 group. Along with the time extension, the mRNA expressions of TNF-α and IL-1β and the protein expressions of RIP1 and RIP3 were markedly up-regulated. Compared with model group, difference in the mRNA expressions of TNF-α and IL-1β in hepatic tissues in Nec-1 group were statistically significant, and the most obvious difference was at 8 hours [TNF-α mRNA： 1.457 ± 0.081 vs. 2.317 ± 0.062, IL-1β mRNA： 0.690 ±0.087 vs. 1.812 ± 0.112, both P〈 0.013. But there was no statistically significant difference in RIP1 and RIP3 between Nee-1 group and model group [RIP1 protein 8 hours： 0.561± 0.033 vs. 0.587 ± 0.036, RIP3 protein 8 hours： 0.976± 0.040 vs. 1.044 ± 0.115, both P〉0.053. Conclusion Nec-1 may be remarkable protect effect on the liver of rats with trauma induced hemorrhage shock and reperfusion, and the intrinsic mechanisms need further investigation.