The Effects Of Niu-po-zhi-bao Pellet On Transforming Growth Factor Beta 1/Smad2 Pathway On Acute Lung Injury Induced By Endotoxin In Rats

Background and ObjectiveIn the cytokine network which regulates lung injury and pulmonary fibrosis,transforming growth factor beta(TGF-β1)has been proved as one of the key cytokines.Previous studies demonstrated that the expression of TGF-β1 was obviously up-regulation at the early period of ALI/ ARDS and was closely related with levels of pulmonary fibrosis and mortality.Recent studies confirmed that Chinese medicine Niu-po-zhi-bao pellet took protective effects in rats of acute lung injury induced by endotoxin shock,especially in inhibiting the early formation of collagen fibers.But so far,the possible mechanism of this effect is still unknown.Whether Niu-po-zhi-bao pellet can reduce levels of acute lung injury and pulmonary fibrosis by the regulation of TGF-β/Smads pathway has not yet been reported in any literature.In this study,we used an acute lung injury model induced by lipopolysaccharide in rats and to investigate the effects of Niu-po-zhi-bao pellet on transforming growth factor beta 1 and the positive regulatory proteins-Smads2 at its downstream,exploring the possible protective mechanism of this pellet on acute lung injury and fibrosis.MethodsForty eight Sprague Dawley rats,weight 250～280g,were randomly assigned to the following four groups(n=12 per group):the saline control group(Control group),the endotoxin model group(LPS alone group),low-dose Niu-po-zhi-bao pellet pretreatment group(Low-dose group),high-dose Niu-po-zhi-bao pellet pretreatment group(High-dose group).Rats were pretreatment with 5 mg/100g Suspended solution of Niu-po-zhi-bao pellet in low-dose group,with 10mg/100g in high-dose group,twice a day,five consecutive garage Days.The other two groups were treated with equivalent saline.Experiment started after drug pretreatment.Model of acute lung injury was set by tracheal joint intraperitoneal injection of endotoxin.In day 1,3 and 5,a dose of LPS 1mg/kg was intraperitoneally injected,with the tracheal injection of LPS 2mg/kg at the same time.Corresponding saline was injected in the control group,In day 6,the survivals of each group were killed and specimens were collected.Lung tissues were sampled to measure values of wet-to-dry lung weight ratio(W/D), pulmonary microvascular(EB value),HE stain used for detection of levels of lung injury by microscopy;Van Gieson stain used for detection of levels of lung collagen fibers;Immunohistochemical and Westernblot methods used for detection of expression of TGF-β1 and Western Blot used for detection of expression of Smad2.Results1.Changes of pulmonary vascular permeability Compared with the control group,values of EB and W/D were significantly higher in the LPS alone group and the two Chinese medicine groups(P＜0.01);Compared with the LPS alone group, the two Chinese medicine groups significantly decreased the values of EB and W/D(P＜0.01);Compared with the low-dose group,the high-dose Chinese medicine group significantly decreased the values of EB and W/D(P＜0.01).2.Histopathological examination The lung tissues were histopathologically studied.Control group:No swelling,inflammation,or other special changes were observed in the interstitium.Lung tissue Structures were integrity,and alveolar spaces were clear.LPS alone group:Significant numbers of inflammatory cells migrated into the alveolar spaces and a large number of effusion,transparent membrane formation,alveolar wall damage,pulmonary edema,pulmonary capillary obstruction and bleeding could be seen in the alveolar spaces.The original structures were severely damaged.Low-dose group: Some neutrophils accumulated in the alveolar spaces.There was still a small amount of effusion and transparent membrane formation.High-dose group:Only a small amount of neutrophil accumulated in the lung tissue with local distribution.There was still a certain number of alveolar wall thicken or broken.Compared with the LPS alone group,pathological changes in the two Chinese medicine gourps were significantly reduced,such as the Board of neutrophils accumulated,the alveolar wall damaged and pulmonary edema, particularly in the High-dose group.3.Morphology of Pulmonary fibrosis Control group:A small amount of collagen fibers was only seen around the blood vessels and bronchial wall. LPS alone group:A large amount of red collagen fibers was seen around muscular layer and endothelium in the blood vessels,bronchus and interalveolar septum. Low-dose group:Some of the red collagen fibers expressed,mainly in the bronchial muscular wall and vascular endothelium.High-dose group:Only a small amount of red collagen fibers was expression,mainly distributed in the bronchial muscular wall and vascular endothelium.Compared with the LPS alone group,the Board of pulmonary fibrosis in the two Chinese medicine groups was significantly reduced,especially in high-dose group.4.Immunohistochemical staining and location of TGF-β1 Immunostaining of lung samples with anti-TGF-β1 antibody was shown.In the control group,weak TGF-β1 staining was detected in the alveolar epithelial cells.However, intense positive immunostaining for TGF-β1 was observed in alveolar epithelial cells,pulmonary interstitial inflammatory cell as well as macrophage cells of alveolar space of the LPS alone group.Similar levels of staining were seen in the two pretreatment group.There was a marked reduction in the immunostaining in the lungs pre-treated with low dose medicine.A more profound reduction in TGF-β1 staining was seen pretreated with high dose medicine.5.Expression of TGF-β1 in the lung tissue Western blot analysis demonstrated that the LPS alone group caused significantly increase of TGF-β1 protein when compared with the control group.In contrast,the two Chinese medicine pretreatment groups,especially the high-dose group obviously inhibited levels of endotoxin-enhanced TGF-β1 protein,that yielded statistical significance when compared to the LPS alone group(P＜0.01).6.Expression of p-Smad2 in the lung tissue Western blot analysis demonstrated that the LPS alone group caused significantly increase of p-Smad2 protein when compared with the control group.In contrast,the two Chinese medicine pretreatment groups,especially the high-dose group obviously inhibited levels of endotoxin-enhanced p-Smad2 protein,that yielded statistical significance when compared to the LPS alone group(P＜0.01).Conclusion Our present findings showed that:1.Rats receiving lipopolysaccharide not only up-regulated TGF-β1 expression but also induced a striking increase in Smad2 expression.The activation of TGF-β1/Smad2 signaling pathway may play an important role in regulation of pulmonary permeability and severity of pulmonary fibrosis.2.Pre-treatment with the Chinese medicine Niu-po-zhi-bao pellet, especially with the high dose medicine can reduce lung tissue damage and the severity of pulmonary fibrosis after ALI induced by endotoxin which caused, in part by down-regulation the expression of TGF-β1/Smad2 signaling pathway.