Prevention Of Lipopolysaccharide Induced Aute Lung Injury In Mice By Tibetan Medicine Duo Xue Kang Capsule And Its Mechanism

The Duo Xue Kang capsule(DXK)comes from the experience of old Tibetan medicine master Trroru Tsenam,consist of Rhodiola crenulata,Hippophae rhamnoides,Phyllanthus emblica and Zingiberoffcinale,commonly used in the clinical treatment ofhigh altitude polycythemia(HAPC).Rhodiola has the functions of anti-inflammation,anti-fatigue,anti-lipid peroxidation,anti-tumor,hypoxia resistance and stasis removement.Recent studies shown that Rhodiola extracts can fight against chemicals,biological toxins and chronic ischemia-induced lung injury,and is able to combat allergic asthma induced airway inflammation.The extract of Hippophae has protective effect of vascular endothelial cells,and it can inhibit the inflammatory reaction in the lung injury induced bylipopolysaccharide in mice.Therefore,this study put forward the hypothesis that "DXK has lung protective effects",and to explore its mechanism in preventing acute lung injury(ALI)in mice.Objective:To study the prevention of lipopolysaccharide(LPS)induced acute lung injury in mice by DXK and the possible underling mechanism.Methods: 1.The replication of ALI model in mice.The 60 male KM mice were randomly assigned to control group,trachea group and ip group according to body weight.Trachea group was injected with LPS(2 mg/kg)and ip group was intraperitoneally injected with LPS(10 mg/kg).Mice were sacrificed after modeling 10 h.Lung tissues were taken for measurement of water content,protein concentration in bronchoalveolar lavage fluid(BALF)and pathological analysis.2.Protective effect of DXK on LPS induced ALI in mice.The 100 male KM mice were randomly assigned to 6 groups: control group(nomal and nomal sailine),model group(LPS and nomal sailine),dex group(LPS and 1 mg/kg dexamethasone),DXK high does group(LPS and 3.6 g/kg DXK),DXK middle does group(LPS and 1.8 g/kg DXK)and DXK low does group(LPS and 0.9 g/kg DXK).Mice were sacrificed after modeling 10 h.Blood and lung samples were collected for measurement of water content,protein and neutrophilconcentration in BALF,blood count and pathological analysis.Evans blue solution was used to test pulmonary vascular permeability.3.Effect of DXK on Nr-f2 signaling pathway in ALI mice.The 40 male KM mice were randomly assigned to 4 groups: Control group,model group,dex group,DXK low does group.The animal experimental and treatment handle the same as part 2.The concentrations of SOD,MDA and GSH-PX in lung tissue were detected by test kits.GCLC,NQO1 and HO-1 expression levels of mRNA were detected by Rral-time PCR technique,andfluorescence and Western blot were used to detect the nuclear transfer of Nr-f2 protein.4.Effect of DXK on NF-?B signaling pathway in ALI mice.Grouping,modeling and treating the same as part 3.ELISA method was used to detect the levels of IL-6 and TNF-?in serum,and the expression of CD62 E and ICAM-1 protein in lung tissue was determined by Western blot.TNF-?,IL-6,CD62 E and ICAM-1 mRNA expression levels were detected by Rral-time PCR technique,and Western blot was used to detect the concentration of IKK and P65 protein in NF-?B pathway.Results:1.The replication of ALI model in mice.The survival rate of control group was 95%,and the rate of trachea and ip group was 35% and 100% separately.The water content,total protein in BALF,pathological score were higher in trachea and ip groups compared with Control group.Moreover,there was no statistical difference between trachea and ip group.2.Protective effect of DXK on LPS induced ALI in mice.At the end of experimental,the pulmonary water content,total protein and neutrophils in BALF,vascular permeability and inflammatory cell infiltration was significantly higher in model group compared wih control group(P<0.05).The pathological grading shown the lung in model group has obvious pathological damage.Afer oral administration of DXK for 7 days,each indicator has improved.Date showed that the DXK had a does-dependent effect on decresing pulmonary edema and declining vascular permeability.The best effect is low dose.3.Effect of DXK on Nr-f2 signaling pathway in ALI mice.In model group,the SOD,GSH-Px was decreased by LPS and DXK-L could up-regulatedSOD,GSH-Px concentration.The expression of HO-1 mRNA in model group was higher than of control group,and DXK-L could further promoteHO-1 mRNA level.Nr-f2 was raised at the same time.4.Effect of DXK on NF-?B signaling pathway in ALI mice.At the end of experiment,in ALI model group,the expression of TNF-?,IL-6,ICAM-1 are higher than control group,DXK-L could inhibition the expression.The RT PCR results shown DXK-L had remarkable down-regulation effect on TNF-?,ICAM-1mRNA compared with model group.The protein of IKK in cytoplasm and p65 in nucleus were rose by LPS.DXK-L can effective relieve IKK and p65 protein levels.Conclusions: 1.Obvious lung damage was found in Trachea group and ip group.Conserdering the stabiility of model and mortality,we decided intraperitoneal injection as the flowing study condition.2.DXK decreased ALI and vascular permeability.At the same time,the lung pathology damage was also improves.DXK-L does showd the better effect.3.DXK-L may effect ALI by decreasing inflammatory reaction in pulmonary via improving ability of resistance to oxidative stress injury,promoting activating of Nrf2 and suppressing activating of NF-?B,and decresing inflammatory gene transcription and protein expression in ALI.