Exploreing The Anti-acute Lung Injury Effects And Mechanism Of Jing-fang N-butanol And Its Isolated Fractions JFNE-A

Objective: Based on the previous discoveries that Jing-Fang n-butanol extraction(JFNE)was the effective anti-inflammatory extraction of compatible application of Schizonepeta tenuifolia Briq and Saposhnikovia divaricata Schischk.the JFNE was separated by Silica gel column chromatography and obtained JFNE-A.What’s more,LPS-induced acute lung injury mice and LPS induced RAW264.7/A549 inflammation cells was used as a carrier to observe the anti-acute lung injury and antiinflammation effect of JFNE and its isolated JFNE-A.In addition,explored the antiacute lung injury effects and mechanism of JFNE and JFNE-A based on the crosstalk mechanism of autophagy-Nrf2 signaling pathway.All these were to provide scientific basis for the in-depth study of the anti-inflammatory effect and the material basis of JFNE.Method:(1)The anti-acute lung injury effects of JFNE and JFNE-A were investigated in a mice model of LPS-induced acute lung injury.The changes of organ indices of mice were observed after 7 days of JFNE and JFNE-A administration;the lung tissues were scored for histopathological damage;the wet/dry weight ratios of lung tissues were weighed to calculate pulmonary edema;the expression of inflammatory cytokines in alveolar lavage fluid of mice was measured by Luminex kit;the MPO content of lung tissues was measured and the ultrastructure of mouse lung cells was observed by transmission electron microscopy.(2)Exploreing the anti-inflammatory response and oxidative stress mechanism of JFNE and JFNE in acute lung injury mice,Lung tissues of mice with acute lung injury were homogenized,The expression levels of NF-κB signaling pathway-related target genes IL-6,IL-1β,TNF-α,IFN-γ,i NOS,CHUK,NF-κB1,Rela m RNA were determined by RT-PCR and the expression protein levels i NOS,NF-κB p50,p65,p105,phosphorylated p65,IKKα/ β were detected by Western Blotting;Determination of Nrf2 signaling pathway-related target genes Hmox1,SOD1,SOD2,Nqo1 m RNA expression levels by RT-PCR and Nrf2,Keap1,SOD1,HO-1 protein by Western Blotting;The expression levels of Atg5,m TOR,Beclin-1,SQSTM1 m RNA were determined by RT-PCR and LC3II/I,p62,and phosphorylated p62 protein were assayed via Western Blotting.the free radical scavenging ability of JFNE and JFNE-A and the contents of SOD and GSH in lung tissues was measured according to the instructions of ABTS and DPPH kits and GSH,SOD kits.(3)exploreing the mechanism JFNE and JFNE in acute lung injury mice based on the crosstalk mechanism of autophagy-Nrf2 signaling pathway in LPSinduced RAW264.7/A549 cell model.Griess method to determine the drug effect on the content of NO in supernatant of LPS-induced RAW264.7 cell,ELISA to determine the contents of inflammatory cytokines IL-6,IL-1β,TNF-α in supernatant of LPS-induced RAW264.7/A549 cell.Determination of NF-κB signaling pathway-related target genes IL-6,IL-1β,TNF-α,IFN-γ by RT-PCR and NF-κB signaling pathway-related target proteins of i NOS,NF-κB p50,p65,p105,phosphorylated p65 expression by Western Blotting;Western Blotting method to determine the expression protein levels of Nrf2,Keap1,SOD1 and HO-1 in Nrf2 signaling pathwayand LC3II/I,p62 and phosphorylated p62 in autophagic signaling pathway in LPS-induced RAW264.7/A549 cell model.Laser confocal microscopy was used to observe the changes of autophagic flow in LC3-GFP adenovirus-infected A549 cells.The autophagy inhibitor 3-MA was used to observe the relationship between drug-regulated autophagy and inhibition of NF-κB signaling pathway,and the autophagy blocker chloroquine was used to observe the relationship between drug-regulated autophagy and activation of Nrf2 signaling pathway.(4)The effects of the active components of JFNE-A(hesperidin,cimifugin,5-O-Methylvisammioside and luteolin)on the inflammatory cytokines in the supernatant of LPS-induced RAW264.7 cells were determined by ELISA,explored the effect of the compounds in JFNE-A(hesperidin,cimifugin,5-OMethylvisammioside and luteolin)on autophagic flow in LC3-GFP adenovirusinfected A549 cells using laser confocal microscopy.Results:(1)JFNE and JFNE-A treatment significantly improved lung histopathological damage scores in mice with acute lung injury,reduced mice lung edema,improved the lung tissue cell ultrastructure,reduced MPO levels in lung tissue,and reduced IL-6,IL-1β,TNF-α,and IFN-γ contents in alveolar lavage fluid.(2)JFNE and JFNE-A treatment significantly inhibited the m RNA expression levels of IL-6,IL-1β,TNF-α,IFN-γ,i NOS,CHUK,NF-κB1,Rela genes and protein expression levels of i NOS,NF-κB p50/p105,phosphorylated p65,IKKα/β in NF-κB signaling pathwayrelated target.It also significantly increased the m RNA expression levels of Hmox1,SOD1,SOD2,Nqo1 genes and protein expression levels of Nrf2,SOD1,HO-1 and inhibited the protein expression levels of Keap1 in Nrf2 signaling pathway.It also significantly increased the m RNA expression levels of Atg5,Beclin-1 and decreased the m RNA expression levels of m TOR,SQSTM1,and also significantly decreased the protein expression levels of LC3II/I,p62 and phosphorylated p62,which are the targets related to autophagic signaling pathway.it also significantly increased the contents of GSH and SOD in lung tissues.In addition,the scavenging rate of ABTS and DPPH radicals by 5 mg JFNE-A was 95% and 89%.(3)JFNE and JFNE-A significantly reduced the release of NO in the supernatant of LPS-induced RAW264.7 cells and decreased the contents of IL-6,IL-1β,and TNF-α in the supernatant of LPS-induced RAW264.7/A549 cells.Inhibition of the m RNA expression levels of IL-6,IL-1β,TNF-α,IFN-γ in LPS-induced RAW264.7 cells and the protein expression levels of i NOS,NF-κB p50/p105 and phosphorylated p65 in NF-κB signaling pathway-related targets in LPS induced RAW264.7/A549 cells.JFNE and JFNE-A also significantly elevated the protein expression levels of Nrf2,SOD1,HO-1 and increased Keap1 protein degradation in the LPS-induced RAW264.7/A549 cells,and decreased the protein expression levels of LC3II/I,p62 and phosphorylated p62 in autophagic pathway.the results of Laser confocal microscopy of autophagic flow in LC3-GFP adenovirusinfected A549 cells showed that JFNE and JFNE-A significantly increased autophagic flow in LPS induced A549 cells.In addition,the autophagy inhibitor 3-MA reversed the inhibitory effect of JFNE-A on the NF-κB signaling pathway,and the autophagy blocker chloroquine reversed the activation of the Nrf2 signaling pathway.(4)The active compounds of JFNE-A(hesperidin,cimifugin,5-O-Methylvisammioside and luteolin)significantly inhibited the secreted IL-6,NO and TNF-α in supernatant of LPSinduced RAW264.7 cells,and the effect of luteolin was superior than other compounds.The results of Laser confocal microscopy showed that the hesperidin,cimifugin,and luteolin increased autophagic flow in LC3-GFP adenovirus-infected A549 cells significantly.Conclusion: JFNE and JFNE-A showed definite anti-inflammatory and antioxidant effects in LPS-induced acute lung injury mice.The anti-inflammatory and antioxidant effects of JFNE and JFNE-A exerted was related to its crosstalk effect through promoting autophagy-mediated activation of Nrf2 signaling pathway and inhibiting NF-κB signaling pathway activation.hesperidin,cimifugin,5-OMethylvisammioside and luteolin may be the main effective compounds in JFNE and extert anti-inflammatory and anti-acute lung injury.