The Protective Effect And Mechanisms Of Nervilifordin F On Acute Lung Injury

BACKGROUND: Acute lung injury(ALI)and its most severe manifestation,the acute respiratory distress syndrome(ARDS),occurs in the process of non-cardiac diseases such as severe infection,shock,trauma and aspiration,manifested as diffuse alveolar parenchymal injury and acute,progressive respiratory distress and refractory hypoxemia.It is generally believed that ALI/ARDS is a combination of multiple inflammatory mediators and effector cells,and cascade-amplified waterfall-like inflammation mediated by multiple pathogenesis.If get no effective treatment,it will develop into acute hypoxic respiratory function insufficiency,and even caused multiple organ dysfunction syndrome(MODS)and death.The essence of ALI/ARDS is the uncontrolled inflammatory response,but the pathogenesis has not yet been fully elucidated.There is still no effective treatment.The treatment strategy only reduces the bacterial load and reduces the organ damage caused by excessive inflammation,and the mortality rate is still high.Nervilia fordii(Hance)Schltr is a commonly used heat-clearing and detoxifying medicinal material in Guangdong,Guangxi and Southeast Asia.It is often used for the treatment of pulmonary inflammatory diseases with good results.During the outbreak of SARS,several anti-SARS prescriptions with Nervilia fordii as a drug have achieved good results.Nervilifordin F(NF),particularly,a preliminary cell experiment has demonstrated its outstanding antiinflammatory activity in cells and is substantially non-cytotoxic.This study intends to further study the protective effect of Nervilifordin F on acute lung injury and its related mechanism.OBJECTIVE: 1.To verify the effects of lipopolysaccharide(LPS)and intestinal ischemia/reperfusion(II/R)induced ALI animal models;2.To investigate the effect of NF on LPS and intestinal ischemia/reperfusion-induced ALI;To elucidate the protective mechanism of NF on LPS-induced ALI;4.To elucidate the protective mechanism of NF on intestinal ischemia/reperfusioninduced ALI;5.Based on network pharmacology to explore the the mechanism and its target of Nervilifordin Fon.METHODS: 1.SD rats were divided into normal control group(normal saline),model group,positive control group(dexamethasone 3 mg/kg)and NF high,medium and low dose groups(10.0,5.0,2.5 mg/kg),The NF group were intragastrically administered for 5 days,and the positive control group was intraperitoneally administered once.The normal control group and the model group were intragastrically administered with physiological saline of the corresponding administration volume.In addition to the normal control group,tracheal instillation of LPS(10mg/kg)or II/R(intestinal ischemia for 1h and then reperfusion for 2h)induced ALI in rats(the normal control group only performed sham operation),1 h after the last administration,peripheral blood was collected from the abdominal aorta after anesthetized animals,and the lung tissue were collected after the animals were sacrificed.Hematoxylin-Eosin stain(H&E)was used to observe the pathological changes of lung tissue in each group.TNF-?,IL-1? and IL-6 in the peripheral blood and tissue of mouse lung were detected by enzyme-linked immunosorbent assay(ELISA);The mRNA expression FKBP25,mTOR,p70S6 K,LPS,ATG5,Beclin 1,TLR4,NLRP3,Caspase1,p65 were detected by Real-time quantitative PCR(RT-QPCR)in ALI rats induced by LPS;The protein expression FKBP25,mTOR,p70S6 K,LPS,ATG5,Beclin 1,TLR4,NLRP3,Caspase1,p65 were detected by western blot in ALI rats induced by LPS,the Expression of FKBP25,LC3 B,and TLR4 were also detected by immunohistochemistry in ALI rats induced by LPS.3.The mRNA expression FKBP25,mTOR,p70S6 K,LPS,ATG5,Beclin 1,TLR4,NLRP3,Caspase1,p65 were detected by Real-time quantitative PCR(RTQPCR)in ALI rats induced by intestinal ischemia/reperfusion;The protein expression FKBP25,mTOR,p70S6 K,LPS,ATG5,Beclin 1,TLR4,NLRP3,Caspase1,p65 were detected by western blot in ALI rats induced by intestinal ischemia/reperfusion,the Expression of FKBP25,LC3 B,and TLR4 were also detected by immunohistochemistry in ALI rats induced by intestinal ischemia/reperfusion;2.The possible targets of NF were collected through ctd database and Genecards database;The related targets of acute lung injury were collected through Genecards database and DPDR-CPI database;Enrichment analysis was performed on the predicted targets predicted by the David database and GeneMANIA database;The network visualization and topology were analyzed by cytoscape.RESULTS: 1.H&E staining showed that the structure of alveolar tissue in the normal control group remained intact,and no congestion and neutrophil adhesion,no tissue fluid was observed and the structure of lung tissue was normal.In the LPS group or the intestinal ischemia/reperfusion group,the pathological changes occurred in the lung tissue,mainly in the severity of lung tissue congestion and alveolar expansion.At the same time,neutrophils were attached to the alveolar capillary wall.Pathological changes in acute lung injury such as thickening and destruction of alveolar walls,exudation of tissue fluids,and incomplete alveolar structure.The levels of TNF-?,IL-1? and IL-6 in serum and lung tissues of LPS group or intestinal ischemia/reperfusion group were significantly higher than those of normal control group(p<0.01).2.H&E staining results showed that NF can significantly reduce the pathological damage of rat ALI induced by LPS or intestinal ischemia/reperfusion,and inhibit neutrophil infiltration.In addition,NF can significantly reduce the levels of inflammatory factors TNF-?,IL-1? and IL-6 in lung tissue of rats ALI model induced by LPS or intestinal ischemia/reperfusion-induced;3.RT-QPCR and Western blot results showed that NF can activate the expression of FKBP25 and down-regulated the expression of mTOR and p70S6 K in lung tissue of rat ALI model induced by LPS or intestinal ischemia/reperfusion.At the same time,after NF intervention,the expression levels of autophagy-related genes ATG5,Beclin 1 and LC3 B were significantly increased in ALI group.4.RT-QPCR and Western blot showed NF could significantly inhibit the expression of TLR4 receptor,NF-?B,NLRP3 and Caspase 1 in lung tissue of rat ALI model induced by LPS or intestinal ischemia/reperfusion;5.Obtaining 98 possible target of NF in acute lung injury,15 of them were selected as interest targets for further verification.CONCLUSION: 1.The rats models induced by LPS or intestinal ischemia/reperfusion were constructed successfully;2.NF can attenuate the degree of pathological damage and the expression level of inflammatory factors in the lung tissue of the ALI rat model induced by LPS or intestinal ischemia/reperfusion,thereby exerting a protective effect on ALI;3.NF can protecting LPS or intestinal ischemia/reperfusion-induced ALI rats by activating the FKBP25 and inhibiting the mTOR pathway,which activate the level of autophagy in tissue cells;4.NF can inhibit the TLR4 receptor in the lung tissue of the rat ALI model induced by LPS or intestinal ischemia/reperfusion,inhibit NF-?B expression,thereby down-regulating the expression of precursors such as IL-1?,while the reduction of NF-?B down-regulates the assembly of NLRP3/ASC/pro-caspase-1 protein complexes,resulting in inflammatory factors such as IL-1? significantly reduced,thereby inhibiting the level of inflammatory factors in the body and protecting rat ALI;5.Network pharmacology studies have shown that NF affects acute lung injury through immune,inflammation,apoptosis,autophagy and other related pathways.NF is a potential drug for the treatment of acute lung injury.