大黄附子汤对重症急性胰腺炎肺损伤大鼠肺组织Toll样受体4／核转录因子-κB的影响

A study on effect of Dahuang Fuzi decoction （大黄附子汤） on toll-like receptor 4/nuclear factor-κB in acute lung injury with severe acute pancreatitis in rats

目的探讨大黄附子汤治疗重症急性胰腺炎-急性肺损伤（SAP—ALI）的作用机制。方法将128只SD大鼠随机分为假手术组、SAP—ALI组、大黄附子汤组和血必净组，每组再按处死时间点分为术后3、6、12、24h4个亚组，每个亚组8只。假手术组开腹后翻动胰腺数次，行空肋造瘘；SAP—ALI组采用胰胆管逆行注入牛磺胆酸钠1ml／kg制模，行空肠造瘘；大黄附子汤组和血必净组于制模后0．5h经空肠造瘘管分别注入大黄附子汤或血必净注射液2ml，其他两组注入等量生理盐水。各组在处死大鼠前从心脏取血，用全自动生化仪检测血清淀粉酶水平，动态比浊法测定血清内毒素含量。处死大鼠后取肺脏，观察肺组织病理学变化、计算肺损伤评分和湿／干重（W／D）比值；用蛋白质免疫印迹法（Western blotting）检测肺Toll样受体4（TLR4）／核转录因子-κB p65（NF—κB p65）的表达。结果SAP—ALI组各时间点血清淀粉酶、内毒素，肺损伤评分及W／D比值较假手术组均明显升高（P均〈0．05）；大黄附子汤组各指标均显著下降（P均〈0．05）。Western blotting结果显示，SAP—ALI组TLR4、NF—κBp65表达于术后3h开始较假手术组升高，12h达高峰（P均〈0．05）；大黄附子汤组TLR4于术后3h、NF—κB p65于术后6h表达较SAP—ALI组显著降低，12h达峰值（P均〈0．05）。结论TLR4／NF-κB信号转导通路在SAP—ALI早期炎症级联反应中发挥了重要作用；大黄附子汤通过降低血清内毒素水平、降低肺组织TLR4的活化、抑制下游NF-κB p65转导路径，从而减轻SAP—ALI的进程。

Objective To approach the therapeutic mechanism of Dahuang Fuzi decoction （大黄附子汤） in acute lung injury with severe acute pancreatitis （SAP-ALI） in rats. Methods One hundred and twenty-eight Sprague-Dawley （SD） rats were randomly divided into sham operation group, SAP-ALI group, Dahuang Fuzi decoction group, and Xuebijing injection （血必净注射液） group, and then according to the time point of sacrifice after operation, each group was subdivided into 3, 6, 12 and 24 hour-subsets （each, n=8）. After the belly of a rat in the sham operation group was cut open, the pancreas was flipped several times, and then a stoma was made in the jejunum to form its fistula. In the SAP-ALI group, 1 ml/kg sodium taurocholate was reversely injected into the pancreatobile duct to establish the model of SAP, and then the jejunum fistula was performed. The SAP-ALI model in Dahuang Fuzi decoction group or Xuebijing injection group was treated by injection of 2 ml of Dahuang Fuzi decoction or Xuebijing injection into the fistula respectively, and 2 ml of normal saline （NS） was injected similarly to the other two groups. Blood was collected from heart to detect serum amylase and lipopolysaccharide （LPS） levels before the rat being executed. The lung histopathologie changes, pulmonary injury scores and wet/dry weight （W/D） ratios were observed after the rats were executed. The expression of toil-like receptor 4/nuclear factor-κB p65 （TLR4/NF-κB p65） of the lung was detected by Western blotting. Results The levels of serum amylase, LPS, pulmonary injury score and W/D ratio of SAP-ALI group were significantly higher than those of sham operation group at each time point （all P〈0. 05）. The Western blotting showed that the expressions of TLR4 and NF-κB p65 in SAP-ALI group were significantly higher than those of sham operation group, and the elevation of expressions began from 3 hours and reached the peak at 12 hours during the course （all P〈0. 05）. The expression of TLR4 decreased from 3 hours and of NF-κB p65 descended from 6 hours in Dahuang Fuzi decoction group, and reached their lowest points at 12 hours, demonstrating that they were lower than those in the SAP-ALI group obviously （all P〈0. 05）. Conclusion TLR4/NF-κB signal transduetion pathway plays an important role during the early inflammation cascade reaction of SAP-ALI. Dahuang Fuzi decoction can significantly reduce the level of LPS, diminish the activation of TLR4 and inhibit NF-κB p65 downstream transduction pathway in the lung tissue, thereby the progress of SAP-ALI is ameliorated.