抑制核因子-κB对创伤休克大鼠肝脏热休克蛋白70的影响

Effects of nuclear factor kappa B （NF-κB） inhibition on heat shock protein 70 （ HSP 70） expression in a rodent model of hemorrhagic shock

目的探讨抑制核因子-κB（NF-κB）后热休克蛋白70（HSP70）在创伤失血性休克肝组织中的变化及其对肝脏结构和功能的影响。方法雄性健康Wistar大鼠66只，采用双侧股骨骨折伴失血性休克创伤模型，随机分成正常对照组6只，创伤休克组30只，NF-κB抑制伴创伤休克组30只，NF-κB抑制采用致伤前1h腹腔注射二硫代氨基甲酸吡咯烷（PDTC）200mg／kg。动态观察伤后0．5、2,4、6、8h大鼠肝组织NF-κB、HSP70、肝脏病理、肝功能、TNF-α、IL-6等变化。NF-κB采用EMSA法测定结合活性，HSP70采用免疫印迹法测定其蛋白含量，并进行计算机图像分析。数据采用SPSS12．0软件分析，两组间比较采用成组资料的t检验。结果NF-κB的活性伤后迅速升高，伤后6h达到高峰；HSP70伤后2h较正常对照相比[（10．8±1．1）vs．（4．7±0．5），P〈0．01]，伤后6h达到高峰，和正常组相比[（23．0±1．7）vs．（4．7±0．5），P〈0．01]。TNF-α、IL-6伤后逐渐升高，并于伤后6h达到高峰，和正常组相比[TNF-α（173．7±12．1）vs．（30．8±1．8）pg／ml，P〈0．01；IL-6（175．5±12．5）vs．（10．4±0．7）pg／ml，P〈0．01]；伤后8h光镜下可见肝窦内淤血明显，有大量炎性细胞浸润；血清ALT、TB伤后4h开始增高，8h达到峰值，和创伤组相比[ALT（640．6±80．2）vs．（536．8±60．0）nmol^-1·L^-1，P〈0．01；TB（4．7±1．1）vs．（1．6±0．2）mol／L，P〈0．01]。抑制NF-κB再致伤后，HSP70在肝组织中表达仍然较高，但在伤后各个时相点的表达均较未抑制NF-κB创伤性休克伤组明显回落；伤后6h和创伤组相比[（16．9±4．4）vs．（23．0±1．7），P〈0．05]。TNF-α、IL-6伤后各个时相点均迅速回落，伤后6h，和创伤组相比[TNF-α（135．2±10．2）vs．（173．7±12．1）pg／ml，P〈0．05；IL-6（113．0±10．8）vs．（175．5±12．5）pg／ml，P〈0．05]；肝脏大体淤血、肿胀明显减轻；伤后8h光镜下可见肝细胞变性明显好转，肝窦内淤血减轻，仅见少许淋巴细胞及中性粒细胞浸润；伤后4h，血清ALT、TB即明显下降，和未抑制组相比[ALT（540．8±66．2）vs．（640．6±80．2）nmol^-1·L^-1，P〈0．05；TB（2．3±0．3）vs．（4．7±1．1）mol／L，P〈0．05]。结论NF-κB、HSP70参与了严重创伤失血性休克后肝损伤与抗损伤的发生，抑制NF-κB的活性有助于减轻创伤失血性休克后肝脏的急性损害，NF-κB、HSP70可作为反映创伤休克后肝脏损害程度的重要应激指标。

Objective To study the effects of the inhibition of nuclear factor kappa-B （NF-κB） ,on the hepatic heat shock protein 70 （HSP70） expression as well as on the changes of hepatic function and uhrastructure in a rodent model of hemorrhageic shock. Method Hemorrhagic shock was produced by inducing bilateral femoral fractures in male Wistar rats. Intraperitoneal injection of pyrrolidine dithiocarbaroate（PDTC）was used to inhibit NF-κB activation 1 hour before induction of shock. A total of 66 adult male Wistar rats were randomly divided into 3 groups： control group （Control, n = 6）, trauma shock （TS, n = 30）, and NF-κB inhibition followed by trauma shock （NF-κB inhibition, n = 30）. Measurements of hepatic NF-κB and HSP70, hepatic function bio-markers, TNF-α and IL-6 were obtained 0.5, 2, 4, 6, 8 hours after trauma. Histopathological changes in liver tissues were also noted. Hepatic expression of NF-κB was determined by using electropboretic mobility shift assay, while HSP70 was assayed by＂ western blot and analyzed with computer imaging. Results In rats with trauma shock, beth hepatic NF-κB activity and HSP70 expression increased significantly compared to the control group, reaching peaks at 6 hour post injury. Serum alanine transferase （ALT） and total bilirubin （TB） also rose significantly,reaching peaks at 8 hours post trauma. Light microscopy revealed hepatic congestion with infiltration of inflammatory cells into hepatic sinusoid in the TS group at 8 hours. Inhibiting the activity of NF-κB one hour before trauma significantly decreased expression of HSP70 at 6 hours post trauma [ 16.9 ± 4.4 （NF-κB inhibition） vs. 23.0 ± 1.7 （TS）, P 〈 0.05 ]. In addition, levels TNF-α and IL-6 in the liver tissue also decreased, and hepatic congestion as well as hepatic cell degeneration were ameliorated, showing minimal inflammatory infiltrates in the hepatic sinusoids. NF-κB inhibition also significantly lowered the levels of ALT and TB at 4 hours post trauma [ ALT, 540.8 ± 66.2 nmol/L （NF-κB inhibition） vs. 640.6 ± 80.2 umol/L （TS）, P 〈 0.05 ; TB, 2.3 ± 0.3 mol/L （ NF-κB inhibition） vs. 4.7 ± 1.1 mol/L （TS）, P 〈 0. 05 ]. Conclusions NF-κB and HSP70 are involved in the pathogenesis of hepatic injury during hemorrhagic shock, and the degree of NF-κB activity and HSP70 expression may be corrsistent with the extent of hepatocellular damage. Inhibition of NF-κB helps ameliorate liver injury due to trauma shock.