参附注射液对失血性休克大鼠肺损伤的影响

Effect of Shenfu injection on acute lung injury in hemorrhagic shock rats

中文关键词: 参附注射液  失血性休克  肺损伤  缺氧诱导因子1α  视黄酸相关孤核受体γt  叉头翼状螺旋转录因子3

英文关键词: Shenfu injection  Hemorrhagic shock  Lung injury  Hypoxia-inducible factor-1α  Retinoic acid-related orphan nuclear receptor γt  Transcription factor forkhead box protein 3

中文摘要:

      
目的：观察参附注射液对大鼠失血性休克（HS）致肺损伤的影响并探讨潜在机制。
方法：选择SPF级健康雄性SD大鼠36只，16～17周龄，体重400～600 g。采用随机数字表法将大鼠分为三组：假手术组（SH组）、失血性休克组（HS组）和参附注射液组（SF组），每组12只。SH组麻醉后仅分离出右股静脉和股动脉，不行动静脉置管，HS组和SF组制备HS大鼠模型。HS组经静脉导管进行液体复苏，复苏液为所失自体血加1.5倍失血量的复方氯化钠注射液与生理盐水10 ml/kg，SF组复苏液为所失自体血加1.5倍失血量的复方氯化钠注射液与参附注射液10 ml/kg，灌注时间约60 min。于术后24、48 h随机处死6只大鼠，取肺组织检测湿重与干重比（W/D），采用ELISA法检测白细胞介素-6（IL-6）、IL-17、IL-10、转化生长因子-β（TGF-β）浓度，采用PCR法检测肺组织视黄酸相关孤核受体γt（RORγt）、叉头翼状螺旋转录因子3（Foxp3）、缺氧诱导因子1α（HIF-1α） mRNA表达量，采用Western blot法检测RORγt、Foxp3、HIF-1α、水通道蛋白1（AQP1）和AQP5蛋白含量，肺组织行HE染色后在光镜下观察病理学改变并行肺损伤评分。
结果：与术后24 h比较，术后48 h SF组肺组织W/D、IL-6和IL-17浓度、RORγt和HIF-1α mRNA表达量及蛋白含量、肺损伤评分均明显降低（P<0.05），IL-10和TGF-β浓度、Foxp3 mRNA表达量及蛋白含量、AQP1蛋白含量均明显升高（P<0.05）。与SH组比较，术后24、48 h HS组和SF组W/D、IL-6、IL-17、IL-10和TGF-β浓度、RORγt 、Foxp3和HIF-1α mRNA表达量及蛋白含量、肺损伤评分均明显升高（P<0.05），AQP1、AQP5蛋白含量明显降低（P<0.05），光镜下可见肺泡结构破坏，肺泡间质充满大量水肿液，其间浸润着大量炎性细胞。与HS组比较，术后24、48 h SF组W/D、IL-6和IL-17浓度、RORγt和HIF-1α mRNA表达量及蛋白含量、肺损伤评分明显降低（P<0.05），IL-10和TGF-β浓度、Foxp3 mRNA表达量及蛋白含量、AQP1和AQP5蛋白含量均明显升高（P<0.05），光镜下可见肺泡结构紊乱改善，水肿程度减轻，炎性细胞数量减少。
结论：参附注射液可调节促炎因子IL-6、IL-17与抗炎因子IL-10、TGF-β平衡，升高肺组织AQP1、AQP5蛋白含量，降低肺组织W/D和肺损伤评分，减轻HS大鼠肺损伤，机制可能与调节HIF-1α-RORγt/Foxp3平衡有关。

英文摘要:

      
Objective: To observe the effect of Shenfu injection on lung injury caused by hemorrhagic shock (HS) in rats and explore the related potential mechanism.
Methods: Thirty-six SPF healthy male SD rats, aged 16-17 weeks, weighing 400-600 g, were randomly divided into three groups: sham operation group (group SH), HS group (group HS), and Shenfu injection group (group SF), 12 rats in each group. In group SH, only the right femoral vein and femoral artery were separated after anesthesia , and venous catheterization was not performed. HS model was established in groups SF and HS. In group HS, liquid resuscitation was performed through an intravenous catheter, and the resuscitation fluid consisted of the autoblood lost and the compound sodium chloride injection equivalent to 1.5 times the blood loss and 10 ml/kg normal saline. In group SF, the resuscitation fluid consisted of the lost autoblood and the compound sodium chloride injection equivalent to 1.5 times the blood loss and Shenfu injection 10 ml/kg. The whole perfusion time was about 60 minutes. Six rats in the three groups were randomly anesthetized 24 and 48 hours after operation. The wet/dry weight ratio (W/D) of lung tissues was detected. The concentrations of interleukin-6 (IL-6), IL-17, IL-10, and transforming growth factor-β (TGF-β) were detected by ELISA, the mRNA expression of retinoic acid-related orphan nuclear receptor γt (RORγt), transcription factor forkhead box protein 3 (Foxp3), and hypoxia-inducible factor-1α (HIF-1α) in lung tissues were detected by PCR. The protein contents of RORγt, Foxp3, HIF-1α, aquaporin 1 (AQP1), and AQP5 in lung tissue were detected by Western blot. Pathological changesunder HE staining light microscope and lung injury scores were observed.
Results: Compared with 24 hours after operation, W/D, the concentrations of IL-6 and IL-17, mRNA expression and protein content of RORγt and HIF-1α, and lung injury score were significantly decreased (P < 0.05), the concentrations of IL-10, and TGF-β, Foxp3 mRNA expression and protein content, and AQP1 protein content were significantly increased in group SF 48 hours after operation (P < 0.05). Compared with group SH, W/D, the concentrations of IL-6, IL-17, IL-10, and TGF-β, mRNA expression and protein content of RORγt, Foxp 3, and HIF-1α, and lung injury score were significantly increased (P < 0.05), AQP1 and AQP5 protein contents were significantly decreased in groups HS and SF 24 and 48 hours after operation (P < 0.05), and alveolar structure was damaged under light microscope and alveolar interstitium was filled with a large amount of edematous fluid, during which a large number of inflammatory cells infiltrated. Compared with group HS, W/D, the concentrations of IL-6 and IL-17, mRNA expression and protein content of RORγt and HIF-1α, and lung injury score were significantly decreased (P < 0.05), the concentrations of IL-10 and TGF-β, Foxp3 mRNA expression and protein content, AQP1 and AQP5 protein contents were significantly increased in group SF 24 and 48 hours after surgery(P < 0.05), and the alveolar structure was improved under light microscope, and edema was reduced, and the number of inflammatory cells was reduced.
Conclusion: Shenfu injection can regulate the balance between pro-inflammatory factors IL-6 and IL-17, and anti-inflammatory factors IL-10 and TGF-β, increase the protein content of AQP1 and AQP5 in lung tissue, and decrease the W/D and injury score in lung tissue, thus alleviating lung injury in HS rats. The mechanism may be related to the regulation of HIF-1α-RORγt/Foxp3 balance.