The Role Of NF-κB And Renin-angiotensin System In Acute Lung Injury Induced By Hemorrhage-LPS Two Hits And The Effects Of PDS

Acute Lung Injury (ALI) is a kind of acute progressive respiratory failure caused by various pathogenic factors such as trauma, infection and ischemia-resuscitation and burn. ALI will develop into acute respiratory distress syndrome (ARDS) and even multiple organ disfunction syndrome (MODS) if it is not control in time. So ALI has a very high fatality rate. It is not known clearly about the molecular mechanism of ALI, and"two-hits"theory has been accepted by most of the researchers. In this theory, it is thought that activation of effective cells by over-reaction of inflammation play important roles in progress of ALI. In this study, the two-hits model was built up by hemorrhage-LPS in rats to study the mechanism of two-hit and observe the effect of PDS on it, Dex as positive control.In hemorrhagic shock, the Gram-negative bacteria in intestine will enter blood circulation. The major pathogenic component LPS will bond to circulating LPS-binding protein (LBP),and formed LPS-LBP complex which subsequently combind with cell membrane receptor CD14 ,and formed LPS-LBP-CD14,then activate TLR4-MD2, in turn affecte a series of subsequent protein. After phosphorylation of IκB which induced the translocation of NF-κB to the nucleus, induces the expression of mRNA of a variety of inflammatory mediators .This process finally becomes the major cause to the acute lung injury.RAS is the central modulator of cardiovascular system, and it play a key role in pathophysiology of cardiovascular and kidney. The angiotensinogen, AT1, AT2 and AngⅡare expressed in rat and human lung tissue. The evidence shows that RAS take part in the pathophysiology of different kinds of lung injury, including permeability increasing, vascular tensity and fibroblast activating, survive rate of alveolar epithelium decreasing. The newcomer,new receptor,new medicine of RAS is emerging with the technical developing. The research pay attention to the relationship of RAS and prognosis of lung disease.So, we observe the changes of RAS related metabolic enzyme and its receptor,the cytocine and it regulating gene and nuclear transcription factor NF-κB by seting up two hits shock model. The aim is to study the mechanism of ALI and the protective effect of PDS.The results of this experimental research are as follows:1. The acute lung injury model induced by hemorrhage-LPS two hitsForty Wistar rats were divided randomly into 4 groups: the sham operational group(S group) ; the two-hits groups with hemorrhage- lipopolysaccharides ( hemorrhagic shock/ resuscitation with shed blood and lipopolysaccharides administered group,HL group) ; the Dexamethasone preventive therapy group( the hemorrhagic shock/ resuscitation / Dexamethasone and lipopolysaccharides administered group,HLD group);the panaxadiol saponins preventive therapy group( the hemorrhagic shock/ resuscitation / panaxadiol saponins and lipopolysaccharides administered group ,HLP group). The rats were made first-hit by hemorrhagic shock, and glucose solution, Dex and PDS were administered intraperitoneally separately in HL, HLD and HLP groups. Ten minutes later, LPS were administered intraperitoneally to make endotoxic shock and induce two-hit in rats. The mean arterial blood pressure (MABP) was monitored dynamically by the RM6000 4-lead physiologic recording apparatus, serum enzymes (AST, ALT, ALP, LDH and CK), TNF-αand IL-6 levels were detected.1.1 MABP dynamic observation: When blood pressure reduced to 40mmHg, it was regarded as the start of the hemorrhagic shock. After one hour, blood and saline were refused until blood pressure come up to 110mmHg, followed by LPS injection. Although MABP in all groups were decreased again just after LPS injection, 60 min later, only rats in HL groups had progressing decreasing MABP, and the rats in HLDand HLP groups both had steady MABP. This implied that PDS has the effect to increase the lowered MABP by two-hits, which is similar to Dex, and this way be much effective for prevention and therapy of vital shock.1.2 Changes of serum enzymesSerum enzymes including AST, ALT, ALP, LDH and CK in rats of HL groups were significantly increased compared to that of S groups, implying that two-hits have damaged several important organs such as liver and heart and several metabolic enzymes have been secreted to blood because of cell damage . Injection of PDS could decrease the levels of serum enzymes, showing that PDS has the effect of stabilizing cell membrane in most tissues.1.3 Contents of TNF-αand IL-6Serum TNF-αand IL-6 in rats of HL groups were much higher than that of S groups, which could be significantly decreased by PDS therapy, implying that PDS had the effect to decrease inflammatory factors and prevent SIRS.To sum up, two-hits of hemorrhage-LPS could decrease MABP and increase serum enzymes and TNF-αand IL-6 levels significantly, implied that important organs such as heart and liver were vitally damaged and the model of two-hits have been built up very successfully, which made up a substantial backbone for next experiments. PDS could increase MABP, decrease serum enzymes and TNF-αand IL-6 levels, thus to prevent cells in most tissue from seriously damage.2. Oxidative stress in two-hit rats and the effect of PDSLPS can bind to the receptor on the surface of endothelial cells and macrophages, to induce the synthesis and secretion of NO as a result of NOS expression. As a kind of free radical, NO plays a quite important role in endotoxic shock, and it can induce the injury of LPS to various tissues directly or indirectly. NOS is the restriction enzyme in NO synthesis. In this study, MDA content, SOD, NOS, iNOS activities and NO2-/NO3- index in lung tissue were detected to observe the change of oxidative stress and the effect of PDS on it.2.1 MDA content and SOD activity in lung tissueMDA content and NO2―/NO3― index in lung tissues from rats of HL group were significantly higher than that of S group, while SOD activities were much lower. This implied that hemorrhage-LPS two-hits could elevate significantly oxygen free radical in lung tissue, and cause structural and functional injury in cellular and subcellular levels, which will decrease the body's ability to eliminate oxygen free radical. It was probably the cause of morphologic changes of lung tissue in rats of HL group. PDS could decrease MDA content and NO2―/NO3― index, and increase SOD activity, proving the anti-lipid peroxidation effect of PDS.2.2 NOS and iNOS activities and content of NO2-/NO3- in lung tissueNOS and iNOS activities of lung tissues in rats of HL group were significantly increased compared to that of S group, and PDS injection could decrease their activities thus to decrease the NO synthesis. PDS could prevent lung tissue from LPS harm via decreasing content of lung oxygen free radical.To summarize, PDS could decrease MDA level, NOS and iNOS activity and NO2-/NO3- index, and increase SOD activity in hemorrhage-LPS two-hit rats. This may be caused by blockage of LPS binding to its receptor by PDS, thus to inhibit NOS activity and then decrease the synthesis and secretion of NO. Further studies are needed to clarify the mechanism of PDS blockage of LPS binding to its receptor.3. NF-κB signal transduction pathway of lung in two-hit ratsCD14 and Toll-like receptors (TLRs) are two very important model-recognizing receptors of LPS. Once LPS enters human body, it will bind to LPS-binding protein (LBP) first, and secondly CD14 to make LPS-LBP-CD14 complex, which will interact with TLR4-MD2. The intracellular signal pathway of LPS will be initiated and nuclear factor-κB (NF-κB) is activated to nuclear translocation. After phosphorylation of IκB, NF-κB will associate with specific DNA binding sites to initiate gene transcription. It induces the expression of mRNA of a variety of pro-inflammatory mediators including TNF-α, adhesive factor, iNOS/NO and IL-18 et al. This process finally becomes the major cause to the lung tissue injury. In this study, lung CD14, TLR4, IκB , nuclear NF-κB, TNF-α, IL-6 and IL-18 expression were detected to clarify the changes of LPS signal transduction in two-hit rats and the effect of PDS on it.3.1 CD14 and TLR4 content in lung tissueIn rats of hemorrhage-LPS two-hit group, the CD14 mRNA and protein expression were significantly higher than that of S group, and PDS injection could decrease the CD14 expression. Immunohistochemical staining also showed that CD14 expression in cytoplasm were much higher in rats of HL group. TLR4 mRNA expression in rats of HL group was also markedly increased compared to that of S group, while PDS injection could decrease it. The results implied that PDS could block LPS signal transduction by inhibiting LPS binding to its receptors. PDS could play important roles in upstream of LPS signal transduction, avoiding the successive harm to lung tissue.3.2 NF-κB expression in lung tissueProtein expressions of P65 and P50 in cell nucleus of HL group were significantly increased compared to S group, while PDS injection could decrease the NF-κB protein level. Immunohistochemical staining showed that dark-yellow staining can only be seen in cytoplasm in S group, but can been seen in both cytoplasm and nucleus in HL group. PDS and Dex injection both could decrease protein expression level. This means that PDS could decrease nuclear translocation of NF-κB to decrease cytokines transcription induced by NF-κB, and avoid vital harm to lung tissue.3.3 IκB expression in lung tissue IκB mRNA level in lung tissues of HL group were significantly lower than that in S group, implying LPS stimulation could activate NF-κB to translocate to nucleus by releasing and degrading the binding IκB. Nuclear NF-κB would activate various cytokines, to induce broad harm to most of the important organs of human body. PDS therapy could increase IκB expression obviously, and inhibit too much activation of NF-κB.3.4 Expression of effective cytokines in lung tissueExpression of TNF-α,IL-6 and IL-18 mRNA were much higher in lung tissues of HL group than that of S group, while PDS injection could decrease the expression levels of these cytokines, which may be the results of decreased nuclear NF-κB.In conclusion, LPS could increase the expressions of its receptors, CD14 and TLR4, decrease IκB content, increase nuclear translocation of NF-κB, and increase expressions of various cytokine such as TNF-α, IL-6 and IL-18. This would induce acute damage to lung tissue. PDS could inhibit the binding of LPS to its receptors and block the signal transduction of LPS, thus to decrease the levels of subsequent cytokines and reduce lung injury in two-hit.It was observed in this study that AQP1 mRNA and protein expression in lung tissues of HL groups were significantly decreased compared to S group, and immunohistochemistry analysis got the similar result. PDS could increase AQP1 expression in lung tissues. Because AQP1 plays important roles in water transportation through cell membrane, its decrease will undoubtly destroy the pulmonary alveoli- blood capillary barrier, and block water transport in lung stroma which will cause lung edema. PDS could increase AQP1 expression in lung tissue, lighten the edema, and maintain the intact structure of lung tissue. PDS may be a potential reagent for lung edema therapy.4.The change of Renin-angiotensin system in two hit ratsThe renin-anigotensin system was also activated by two hits, then the content of Angiotensin is increasing by the expression of ACE and AGT increasing. AngⅡis the key mediator in inflammation, and NF-κB is the key role in inflammation injury mediated by AngⅡ. So NF-κB and other transcription factor synergy to form a complex regulation crossing in transcription level. It participate in the regulation of gene transcription of inflammation mediators by different mechanism, and this determinate the onset and development of ALI. As an inflammation mediator, AngⅡcan activate NF-κB, so decreasing produce of AngⅡor down regulation of NF-κB activity can prevent the onset of ALI. PDS can inhibit directly the NF-κB activity by decreasing the expression of CD14 and TLR4, or inhibit indirectly the NF-κB activity by decreasing the produce of AngⅡ, This will lessen the lung damage.To conclude, LPS, which is the major component of endotoxin of Gram negative bacteria will enter body circulation during two-hit, to form LPS-LBP-CD14 complex. The complex will activate NF-κB through LPS signal transduction to secret large quantities of cytokine such as TNF-α, IL-1, IL-6 and iNOS, which are also strong inducer of NF-κB and will enhance the activation of NF-κB. The activated NF-κB could alone or combined with other transcript factors to induce the expression of various inflammatory mediators, whose over-expression are the major cause of ALI. Furtherly, the activated iNOS could increase the formation of oxygen free radical, which aggravate the lung injury. The synergetic effect of the above factors will induce obvious inflammation in lung tissues of hemorrhage-LPS rats as well as lung atrophy. AngⅡis the key mediator in inflammation, and NF-κB is the key role in inflammation injury mediated by AngⅡ. AngⅡcan also mediate related gene expression of NF-κB by AT1 and AT2. PDS injection could block the LPS induced signal transduction pathway by inhibiting the activity of NF-κB via different mechanism. PDS can also decrease levels of oxygen free radical, thus to prevent structure and function of lung tissue. It was also observed that lung tissues of rats in PDS group had no obvious inflammation, which had no significant difference compared to S group. The results implied that PDS could prevent effectively the ALI caused by two-hit.