Coptisine Ameliorates LPS-Induced Acute Lung Injury Through Its Anti-inflammatory Activity By Inhibition Of The PI3K/Akt/NF-?B Pathway

Objective : The aim of the present study was to explore the effect of coptisine on lipopolysaccharide(LPS)-induced acute lung injury(ALI)in a mouse model,as well as the underlying mechanism.Methods:Animals were randomly assigned to four groups: PBS-treated control group,LPS-induced ALI group,hight dose coptisine-treated group and low dose Coptisine-treated group.Coptisine(20 mg/kg)or Coptisine(10 mg/kg)was administered orally for 7 consecutive days,starting 7 days prior to induction of ALI to hight dose or low dose Coptisine-treated mice,respectively.PBS-treated control group and LPS-induced mice received distilled water alone on the same schedule.The survival rates of mice,lung wet/dry weight ratios,lung injury score,protein levels and lung myeloperoxidase(MPO)activity,the number of total cells,infiltrated neutrophils and leukocytes in bronchoalveolar lavage fluid(BALF).In addition,the BALF levels of inflammatory cytokines such as TNF-?,IL-6,interleukin(IL)-1?,were determined,along with the levels of phosphorylated NF-?B P65,phosphorylated PI3 K,and phosphorylated Akt in lung tissues.Results:1.Effect of Coptisine pretreatment on suvival rate of LPS-treated mice After the LPS challenge 4h,mice manifested lethargy,piloerection,diarrhea,huddling,and malaise,clear signs of endotoxic shock.At 108 h after LPS injection,the mortality rates were 0%,100%,and 50% and 70% for the LPS,respectively.The hight dose Coptisine pretreatment group had a significantly lower mortality than the LPS groups(P<0.05,n=10 animals per group).This observation suggests that Coptisinat partially decreases the mortality rate caused by LPS injection.2.Effect of Coptisine pretreatment on lung W/D ratioLung W/D ratio was significantly higher in the LPS group than in the control group.Coptisine pretreatment significantly decreased the W/D ratio(P <0.05,P < 0.01).MPO,concentration of proteins and total cells,neutrophi,leukocyte in BALF were significantly higher in the LPS group than in the control group.Coptisine pretreatment significantly decreased MPO,concentration of proteins and total cells,neutrophi,leukocyte in BALF(P <0.05,P < 0.01).3.Effect of Coptisine pretreatment on LPS-induced pathological results of acute lung injuryThe structure of lung tissue of normal control mice was clear,the alveolar structure was intact,no obvious infiltration of inflammatory cells and no thickening of alveolar wall.LPS group mice lung tissue showed alveolar congestion,bleeding,alveolar wall thickening,fracture or even collapsed,a large number of inflammatory cells gathered in the alveolar cavity and interstitial lung tissue.Coptisine significantly inhibited the histoligical changes of LPS-induced acute lung injury and the pathological changes of LPS-induced acute injury in rats treated with pretreatment of Coptisine(20mg/kg)and Coptisine(10mg/kg).4.Measurement of lung myeloperoxidase(MPO)activity: Frozen tissue samples of the right lower lung lobe were homogenized with RIPA buffer,as a protease and phosphatase inhibitor and sonicated.The suspensions were centrifuged at 2000 x g for 10 min at 4?C.The MPO activity in the supernatant was measured as the absorbance changes at 460 nm.5.Measurement of total protein concentration: To assess the effect of Coptisine on lung permeability,BALF total protein was measured by using a Bradford assay.6.Effect of Coptisine pretreatment on TNF-?,IL-6 and IL-1?The concentration of TNF-?,IL-6 and IL-1? in bronchoalveolar lavage fluid(BALF).was significantly higher in the LPS group than in the control group.Coptisine pretreatment significantly decreased the concentrations of TNF-?,IL-6 and IL-1? in bronchoalveolar lavage fluid(BALF).(P <0.05,P < 0.01)?7.Effect of Coptisine pretreatment on protein expression of NF-?BPhosphoinositide-NF-?B p65 protein in mice lung tissue significantly higher in the LPS group than in the control group.Coptisine pretreatment significantly decreased Phosphoinositide-NF-?B p65 protein in mice lung tissue(P <0.05,P < 0.01)?8.Effect of Coptisine pretreatment on protein expression of PI3 K /Akt in lung tissues of LPS-treated miceThe lvels of phosphoinositide-3-kinase(PI3K)and p-Akt protein.in mice lung tissue were significantly higher in the LPS group than in the control group.Coptisine pretreatment significantly decreased Phosphoinositide-3-kinase(PI3K)and p-Akt protein(P <0.05,P < 0.01).Conclusion: The results showed that The levels of Coptisine pretreatment significantly increased the survival rate,improved histopathologic changes,alleviated pulmonary edema and lung vascular leak,reduced neutrophil infiltration as well as downregulated the levels of inflammatory cytokines in the BALF.Moreover,Coptisine pretreatment reduced the protein lvels of phosphorylated NF-?B p65 and phosphorylated protein levels of p-PI3 K and p-Akt.