脂多糖诱导大鼠急性肺损伤时核因子-κB信号转导及盐酸氨溴索的作用

Effects of ambroxoi on nuclear factor-kappa B signaling transducfion in lipopolysaccaride-induced acute lung injury in rats

目的探讨脂多糖（LPS）诱导的大鼠急性肺损伤（acute lung injury，ALI）时核因子κB（NF-κB）的信号转导，以及盐酸氨溴索（AMB）对其的作用。方法雄性SD大鼠108只，实验分为两部分：第一部分选择其中90只，采用随机排列表法分为6组（n=15）：A组无菌生理盐水（NS）0．5ml腹腔注射1h后，再腹腔注射NS0．5ml；B组腹腔注射AMB10mg／kg 1h后腹腔注射NS0．5ml；C组腹腔注射NS0．5ml 1h后腹腔注射LPS5mg／kg；D、E、F组分别腹腔注射AMB5、10、20mg／kg后1h腹腔注射LPS5mg／kg。各组分别按1、2、4h再分为3个亚组，每组5只。采用酶联吸附免疫法（ELISA）测定肺泡灌洗液（BALF）中肿瘤坏死因子-α（TNF-α）、白介素-1β（IL-1β）和巨噬细胞炎性蛋白-2（MIP-2）水平，并测定BALF中乳酸脱氢酶（LDH）与总蛋白量的变化。采用蛋白印迹法（Western blot）测定肺组织细胞胞核中NF-κBp65、胞浆中NF-κB抑制蛋白-α（IκBβ）及C—Jun氨基末端激酶（JNK）的表达。第二部分选择18只大鼠按上述分组法随机分为6组（n=3），A、B、C、D、E、F组，给药方法同第一部分，腹腔注射NS（A组、B组）或腹腔注射LPS（C、D、E、F组）后4h观察大鼠肺组织病理形态学变化。结果第一部分：与A组比较，C、D、E、F组BALF中LDH、总蛋白量、总蛋白量、TNF-α、IL-1β和MIP-2均明显升高（P〈0．05或P〈0．01）：与C组比较，E、F组上述指标均降低（P〈0．05或P〈0．01）。与A组比较，C、D、E、F组肺组织细胞中的NF-κB p65与JNK表达明显增多（P〈0．01），而IκBα的表达量减少（P〈0．01）；与C组比较，E、F组NF-κB p65与磷酸化JNK的表达减少（P〈0．05或P〈0．01），而IκBα的表达量增多（P〈0．05或P〈0．01）。第二部分：A、B组肺组织形态学正常，C组呈明显的肺损伤病理形态学改变，D组肺损伤程度与C组相似，E、F组肺损伤程度较C组减轻。结论AMB可以减轻LPS诱导大鼠ALI．其机制可能与抑制肺组织细胞NF-κB065与JNK的活化，并促进IκBα的表达，以及下调TNF-α、IL-1β和MIP-2的表达有关，且呈剂量依赖性。

Objective To study the effects of ambroxol （AMB） on lipopolysaccaride （LPS） -induced acute lung injury （ALI） in rats, and to study the possible mechanism. Method One hundred and eight mail Sprague- Dawley rats were randomly divided into two parts. In the first part, 90 rats were randomly divided into 6 groups （ n = 15, each group ） ： group A received normal saline （NS） by intraperitoneal injection （ i. p. ） ; group B received AMB 10 mg·kg^-1 （i. p. ） ; group C received LPS 5 mg·kg^-1 （i. p. ） ; group D, group E, group F received AMB5, 10, 20 mg·kg^-1 （i. p.）, respectively, 1 hours before LPS 5mg·kg^-1（i. p.）. The animals were killed at 1, 2, 4 hours after NS （group A and group B） or LPS （group C, group D, group E and group F） treatment and every group was divided into three subgroups according to the 1, 2, 4 hours. The total protein, lactic dehydrogenase （LDH）, tumor necrosis factor-α （TNF-α）, interleukin-1β （IL-1β）, and macrophage inflammatory protein-2 （MIP-2） concentration were determined by ELISA in broncho-αlveolar lavage fluid （BALF）, and activity of nuclear factor-κB p65 （NF-κB p65）, inhibitor of NF-κBα （IκBα） and c-Jun-N terminal kinase （JNK） in lung tissue were measured by western blot. In the second part, the rest 18 rats were divided into group A, B, C, D, E, F, and the treatment was the same as in the first part. After 4 hours of Ns injection （group A and group B） or LPS injection （group C, group D, group E, group F）, the rats were killed and the lung tissue was collected for pathological observation. Results In the first part, LDH, total protein, TNF-α, IL- 1β and MIP-2 concentrations in BALF and the NF-κB p65, JNK activity in the lung tissue were significantly increased in group C, D, E, F when compared with group A, and were significantly decreased in group E and F when compared with group C. In the second part, lung injury didn＇t occur in group A and B, lung injury was serious in group C and D, but it was slight in group E and F. Conclusions Pretreatment with AMB has protective effect against LPS-induced ALI in rats by inhibiting NF-κBp65 and JNK activity, promoting IκBα activity, and down-regulating the expression of TNF-α, IL-1β and MIP-2 in a dose-dependent manner.