Protective Effect And Mechanism Of Pulsatilla Saponin B4 On Acute Lung Injury Induced By LPS

Objective:The purpose of this study was to investigate the effects of Pulsatilla saponin B4 on mice with acute lung injury and its mechanism of action,and provide a theoretical basis for the in-depth study of Pulsatilla saponins,and provide new clues for the prevention and treatment of acute lung injury.Part Ⅰthe effect of Pulsatilla saponin B4 on acute lung injury in mice and its mechanism.Method:Sixty male Balb/c mice were randomly divided into control group,model group,B4 high dose group(8 mg/kg),B4 medium dose group(4 mg/kg),B4 low dose group(2 mg/kg),dexamethasone.Groups(5 mg/kg),10 in each group,were intranasally instilled into LPS to establish a mouse model of acute lung injury.B4 high-dose group(8 mg/kg),B4 middle-dose group(4 mg/kg),B4 low-dose group(2 mg/kg),dexamethasone group(5 mg/kg)were intraperitoneally injected 1h before model establishment.Groups and model groups were given saline.After establishing the model for 24 h,lung tissue and bronchoalveolar lavage fluid(BALF)were taken.The classification and quantity of inflammatory cells in BALF were observed.HE staining was used to observe the pathological changes of lung tissue.Elisa method was used to detect tumor necrosis factor(TNF-α)in BALF.The levels of interleukin-1β(IL-1β)and interleukin-6(IL-6);Western blot analysis of NF-κB expression.Result:The number of neutrophils in leukocytes and BALF of model group mice increased significantly(P<0.01),and Pulsatilla saponin B4(2mg/kg)significantly decreased the number of neutrophils in BALF of ALI mice(P<0.05).The number of white blood cells also decreased.HE staining observed the lung tissue of mice.The model group showed obvious alveolar space widening,alveolar shape change,inflammatory cell infiltration and so on.The inflammatory cells and erythrocyte infiltration of lung tissue of Pulsatilla saponin B4(2 mg/kg)were significantly relieved compared with the model group,and the lung injury was also significantly reduced.At the same time,the levels of TNF-α,IL-1β and IL-6 in the BALF of the model group were significantly increased(P<0.01),and the IL-6 content of the Pulsatilla saponin group(2 mg/kg)was significantly decreased(P<0.05);In addition,the Pulsatilla saponin group(2 mg/kg)can reduce the expression level of NF-κB in the lung tissue of mice with acute lung injury.Conclusion:Pulsatilla saponin B4(2 mg/kg)has a protective effect on acute lung injury induced by LPS in mice.The mechanism may inhibit the release of inflammatory factors by inhibiting the expression of NF-κB protein,thereby improving LPS.Acute lung injury.Part Ⅱthe effect of Pulsatilla saponin B4 on the differentiation of RAW264.7 macrophages.Method:RAW264.7 cells were cultured in vitro,and cells in logarithmic growth phase were plated.Randomly grouped into: normal group,LPS group,Pulsatilla saponin B4 group(10 μg/mL),Pulsatilla saponin B4 group(1 μg/mL),added with different concentrations of B4 for 24 h and then added LPS(1 μg /mL),the cell supernatant was collected after 24 h,the NO content in the cell supernatant was detected by Griess method;the content of TNF-α,IL-6 and IL-1β in the supernatant was detected by Elisa method;Western Blot method The expression of p-IκB,IκB,p-NF-κB,NF-κB,p-p38,p38,p-JNK,JNK,p-ERK,and ERK was detected in the cells.Result:After LPS treatment,the contents of NO,TNF-α,IL-6 and IL-1β in the cell culture supernatant increased significantly(P<0.01),and each dose of Pulsatilla saponin B4 could significantly reduce NO,TNF-α,IL.-6,IL-1β content(P <0.01),and is dependent;at the same time,compared with the model group,Pulsatilla saponin B4 can effectively down-regulate MD2,MyD88,p-IKK,p-IκB,p-NF-κB,expression of p-p38,p-JNK,p-ERK.Conclusion: Pulsatilla saponin B4 has a good anti-inflammatory protective effect on LPS-induced RAW264.7 macrophages,and its mechanism may be related to the regulation of MD2/MyD88/NF-κB/MAPK.