The Effect Of Plumbagin On Rats Traumatic Tracheal Stenosis By Regulating NOX4,TGF-β1/Smad And AKT/mTOR Signaling Pathway

Traumatic tracheal stenosis is usually caused by too much granulation hyperplasia and scar formation after tracheal injury,and the common causes are tracheal cannula compression injury,incision injury and even chemical and physical burns.With the development of intensive care medicine,the number of patients receiving endotracheal intubation increases,and the incidence of tracheal stenosis tends to increase.Surgical methods or bronchoscopic interventional therapy can quickly remove narrow tissues,but also lead to recurrent granulation hyperplasia and restenosis.At present,there is no specific drug treatment,and its pathogenesis is unclear.TGF-β1 is an important factor in tissue injury repair,which involves many signal pathways,and it is difficult to target therapy.Recent studies have shown that NOX4 regulates fibroblast differentiation and promotes fibrosis through oxidative stress,and it can interact with TGF-β1/Smad signal.Up-regulation of NOX4 can also activate AKT/mTOR signaling pathway,and the inhibition of mTOR can significantly inhibit the expression of NOX4.however,there are few reports about NOX4 in traumatic tracheal stenosis.Is there an oxidative stress response with increasing NOX4 in traumatic tracheal stenosis? Is it involved in the mechanism of abnormal repair by interacting with TGF-β1/Smad and AKT/mTOR pathway?Traditional Chinese medicine components can be obtained from nature,with wide active components and lower side effects.It has unique and important research value for tracheal stenosis that may be required long-term medication intervention.Plumbagin(PLB)is a kind of traditional Chinese medicine with anti-fibrosis,anti-oxidation and anti-inflammatory activities.Does it play a therapeutic role in the pathogenesis of traumatic tracheal stenosis? Is it related to inhibiting oxidative stress with increasing NOX4 and regulating TGF-β1/Smad,AKT/mTOR signaling pathway? These problems are still unknown.The purpose of this study was to explore the pathogenesis of traumatic tracheal stenosis.By observing the expression of NOX4,TGF-β1/Smad and AKT/mTOR pathway in animals and cells by the intervention effect of plumbagin,the treatment effect and mechanism of plumbagin on traumatic tracheal stenosis were discussed.Therefore,this study is carried out as follows:PART Ⅰ: Establishment of Rats Traumatic Tracheal Stenosis Model and the Effective Mechanism of Plumbagin Objective: To investigate the effect of plumbagin on traumatic tracheal stenosis in rats.Methods: Twenty-four SD male rats were randomly divided into four groups(n=6 in each group),in which Group a was no template control group(NC),Group b was tracheal stenosis group(Model),Group c was tracheal stenosis model+ normal saline group(TS+NS),and Group d was tracheal stenosis model+plumbagin group(TS+PLB).Groups b to d underwent tracheotomy,then by scraping airway mucosa with nylon brush.Group d was intraperitoneally injected with plumbagin at a dose of 4mg/kg/d for 5 days from the first day of operation,while group c was treated with the same dose of normal saline instead of plumbagin,and the other methods were the same as those of group d.The pathological changes and stenosis degree of tracheal tissue were observed by HE staining.The results of IL-6,COL1 and α-SMA were detected by qPCR.Results: Except group a,in groups b ～ d,there were different degrees of granulation tissue hyperplasia causing tracheal stenosis,increased inflammatory cells and fibroblasts,obvious collagen staining and lumen stenosis.Among them,the lumen stenosis rate in group a was(12.04± 1.8)%,and the tracheal stenosis rates in group b ～ d were(61.08 ±2.4)%,(58.52± 1.9)% and(35.21± 4.1)%,respectively.The tracheal stenosis rate in group b was significantly higher than that in group a(P < 0.05).The rate of tracheal stenosis in group d was significantly lower than that in group c(p < 0.05).Compared with group a,the mRNA expressions of IL-6,COL1 and α-SMA in group b increased(p < 0.05),while the mRNA expressions of COL1,α-SMA and IL-6 in group d decreased(p< 0.05).Conclusion: Tracheal stenosis model was established in rats by scraping airway mucosa with nylon brush after tracheotomy.After using plumbagin,the degree of tracheal stenosis in rats can be reduced,and the inflammatory reaction and fibrogenic reaction of tracheal stenosis in rats can be alleviated.PART Ⅱ: The Effect of Plumbagin on the Expression of NOX4,TGF-β1/Smad and AKT/mTOR Pathway in Rat Tracheal StenosisObjective: To investigate the role of plumbagin on TGF-β1/Smad,AKT/mTOR signal pathway and oxidative stress characterized by NOX4 changes in the rats traumatic tracheal stenosis.Methods: The methods of establishment rat model and experimental grouping are the same as the first part.The expressions of NOX4,TGF-β1,COL1 andα-SMA were detected by immunohistochemistry.The expressions of NOX4 and TGF-β1 were detected by qPCR and Wb.The expressions of 4HNE,Smad2/3,p-Smad2/3,mTOR,p-mTOR,AKT,p-AKT,COL1 and α-SMA were detected by WB.Results: Compared with group a,immunohistochemistry results showed that the expressions of NOX4,TGF-β1,COL1 and α-SMA in group b were higher,while those in group d were significantly lower than those in group c(all p < 0.05).Compared with group a,the expressions of NOX4,TGF-β1 protein and mRNA in tracheal stenosis tissue of rats in group b were higher,and the expressions of4 HNE,Smad2/3,p-Smad2/3,mTOR,p-mTOR,AKT,p-AKT,COL1 andα-SMA protein were also higher(all p < 0.05).Compared to group c,the expressions of NOX4,TGF-β1 protein and mRNA were decreased,and the expressions of 4HNE,Smad2/3,p-Smad2/3,mTOR,p-mTOR,AKT,p-AKT,COL1 and α-SMA protein were decreased in group d(all p < 0.05).Conclusion:1.The expressions of NOX4,TGF-β1 protein and mRNA were increased in model rats,and the expressions of 4HNE,Smad2/3,p-Smad2/3,mTOR,p-mTOR,AKT,p-AKT,COL1 and α-SMA protein were also increased in model rats.2.plumbagin can down-regulate the expressions of NOX4,4HNE,TGF-β1/Smad and AKT/mTOR signaling pathway in rats with tracheal stenosis,and alleviate the oxidative stress response in rats with tracheal stenosis.PART Ⅲ: The Effect of Plumbagin in Inhibiting Proliferation and Differentiation of Fibroblasts by Regulating NOX4,TGF-β1/Smad and AKT/mTOR PathwayObjective: To explore the effective mechanism of plumbagin in inhibiting proliferation and differentiation of fibroblasts.Methods: Human lung fibroblasts(IMR-90)were cultured in vitro.The first part was divided into four groups: normal control group,TGF-β1-induced group,plumbagin treatment group and TGF-β1-induced + plumbagin group.The second part was divided into six groups: normal control group,TGF-β1-induced group,TGF-β1-induced + plumbagin group,TGF-β1-induced+LY294002+ plumbagin group,TGF-β1-induced +SB-431542+ plumbagin group,respectively The third part is divided into eight groups: blank control group;TGF-β1-induced +blank transfection group;C: plumbagin+blank transfection group;D: blank transfection group +plumbagin +TGF-β1-induced;E: NOX4 gene silencing group;F: TGF-β1-induced +NOX4 gene silencing group;G: plumbagin +NOX4 gene silencing group;H: plumbagin +TGF-β1-induced +NOX4 gene silencing group.The above groups were detected NOX4,4HNE,Smad2/3,p-Smad2/3,mTOR,p-mTOR,AKT,p-AKT,COL1 and α-SMA by WB method.COL1 and α-SMA of the first part were detected by qPCR.CCK-8+Ed U method was used to detect the proliferation of lung fibroblasts.Immunofluorescence method was used to detect the expression ofα-SMA.Results: Plumbagin could significantly down-regulate NOX4,4HNE,Smad2/3,p-Smad2/3,mTOR,p-mTOR,AKT,p-AKT,COL1 and α-SMA in IMR-90 induced by TGF-β1(all P < 0.05).Immunofluorescence showed that the expression of α-SMA decreased further with the increasing of PLB concentration.In TGF-β1-induced IMR-90,compared with PLB treatment alone,COL1,α-SMA decreased more significantly and the cells proliferation were further inhibited detected by Ed U after PLB combined with LY294002,SB-431542 and Rapamycin treatment respectively(all P < 0.05).Combined PLB with LY294002 or SB-431542,NOX4 and 4HNE decreased further(all p <0.05),but combined with Rapamycin had little effect(P>0.05).Compared with PLB treatment group,the expression of COL1,α-SMA,p-Smad2/Smad2,mTOR,p-mTOR,AKT and p-AKT in IMR-90 induced by TGF-β1 decreased further in the NOX4 gene silencing plus PLB treatment group(all p < 0.05),except p-Smad3/Smad3(P>0.05).Conclusion:1.Plumbagin may affect the proliferation and differentiation of lung fibroblasts by regulating NOX4,TGF-β1/Smad and AKT/mTOR signaling pathway.2.Plumbagin may affect the expression of NOX4 and 4HNE in lung fibroblasts by regulating TGF-β1/Smad and AKT signaling pathway.3.Plumbagin can alleviate oxidative stress by decreasing NOX4 to down-regulate AKT/mTOR and TGF-β1/Smad2 signal pathway activity.