Protective Effects And Mechanisms Of Ginsenoside Rg3 On LPS-induced Acute Lung Injury In A Murine Model

Part? The anti-inflammatory and antioxidant effect of ginsenosides in vitroObjectives: To assess the anti-inflammatory and antioxidant effect of ginsenoside Rh2, Rg1 and Rg3 in vitro, and screen out the most effective one.Methods: PBMCs(peripheral blood mononuclear cells) were isolated from peripheral blood which were obtaind from healthy volunteer. 3?M ginsenoside Rh2, Rg1 and Rg3 were added into the culture media of 96-well plate respectively before lipopolysaccharide(100ng/ml) stimulation, and 24 hours later the supernatants were collected. The concentration of inflammatory cytokines tumor necrosis factor(TNF)-?, interleutin(IL)-1? and nitric oxide(NO) were measured respectively by enzymelinked immunosorbent assay(ELISA). The effect of ginsenoside Rh2, Rg1 and Rg3 in LPS- stimulated PBMCs was analyzed statistiacally.Results:Compared with control group, the concentration of TNF-?, IL-1? and NO in supernatants in LPS group were significantly increased(P <0.05). After the pretreatment of ginsenoside Rh2, Rg1 and Rg3, the concentration of TNF-?, IL-1? and NO in supernatants were significantly reduced(P <0.05). For the concentration of TNF-?, the effect of decrease is GRg3>GRg1>GRh2; as for the IL-1?, the downward trend is GRg3>GRh2 and GRg1; as for the NO, the downward trend is GRg3 and GRh2>GRg1Conclusions: 1. The pretreatment of ginsenoside Rh2, Rg1 and Rg3 can reduce the concentration of TNF-?, IL-1? and NO induced by LPS in PBMC.2. The combined anti-inflammatory and antioxidant effect of ginsenoside Rg3 is better than ginsenoside Rh2, Rg1 in vitro.Part The establishment of lipopolysaccharide induced ?self-regression acute lung injury model and the expression of cyclooxygenase-2Objectives: To establish the acute lung injury mice model by intra-tracheal injection of LPS, and then inspect the dynamic process of the disease. Detect the expression of cyclooxygenase-2, and provide a theoretical basis for the subsequent experiment.Methods: 78 Male BALB/c mice, 6-8 weeks, 16-20 g, were randomly divided into three groups: zero point group, control group and ALI group. The mice model of acute lung injury was established through intra-tracheal instillation of 10?g/50?l LPS. The mice in zero point group were sacrificed immediately at 0 point. After administration, the mice in ALI and control groups were sacrificed at 6h, 12 h, 1d, 2d, 4d, 7d point. To evaluate the pathological condition, wet-to-dry weight ratio and MPO score of the lung tissues. The bronchoalveolar lavage fluid(BALF) was collected for cell count, and the expression of TNF-?, IL-1? in BALF was detected by ELISA. The expression of COX-2 at m RNA level in lung tissue was detected by reverse transcription polymerase chain reaction(RT-PCR).Results: 1. There were no significant changes in pathological condition of the lung tissues in zero point group and control group. The damage of alveolar structure, the thickness of alveolar septum, and the infiltration of inflammatory cells were most obvious at 12 hour in ALI group, and these phenomena gradually attenuated. The degree of acute lung injury showed the similar phenomena as the pathological changes of the lung.2. Compared with zero point group and control group, the wet/dry weight ratio of lung tissues in ALI group was increased significantly. The ratio peaked at 12 hour and then decreased gradually.3. The MPO activity of lung tissues in ALI group increased from 6 hour to 1 day, and then the number decreased.4. The total cell number in BALF of ALI group peaked at 1 day, and then decreased as time went by.5. Compared with zero point group and control group, the concentration of TNF-? and IL-1? in BALF in ALI group reached the peak at 1 day, and afterward decreased gradually.6. The expression of COX-2 at m RNA level in lung tissue of ALI group was an increasing statement from 6 hour to 12 hour, and decreased as time went by.Conclusions: 1. Intra-tracheal instillation of LPS can induce self- regression acute lung injury in the murine model.2. The peak of pathological changes, wet-to-dry weight ratio and COX-2 mRNA of lung tissue of the murine acute lung injury model was 12 hour. The lung tissue MPO score, total cell number in BALF, TNF-? and IL-1? levels in BALF peaked at 1day in the ALI model.Part Protective effects and mechanisms of ginsenoside Rg3 on ?LPS-induced acute lung injury in a murine modelObjectives: To investigate the effects of ginsenoside Rg3(GRg3) on LPS-induced ALI mice, and to analyze the expression of genes and proteins related with inflammation and oxidative stress. In order to unravel the protective effects of ginsenoside Rg3, and further explore the molecular mechanisms of ginsenoside Rg3 in acute lung injury.Methods: Male BALB/c mice, 6-8 weeks, 16-20 g, were randomly divided into six groups: control group, GRg3(30mg/Kg) group, LPS group, LPS + GRg3(10mg/Kg) group, LPS + GRg3(20mg/Kg) group, LPS + GRg3(30mg/Kg) group. ALI model was set up in mice by intra-tracheal injection of LPS as previously described. The mice in the GRg3 and GRg3+LPS groups were treated with GRg3 for 2 days via tail vein injection prior to LPS challenge.To assess the pathological condition, wet-to-dry weight ratio and MPO score of the lung tissues in different groups. The BALF was collected for cell count, and the expression of TNF-?, IL-1?, IL-6 and NO in BALF was detected by ELISA. The expression of NF-?B p65, i NOS, COX-2 in lung tissues was detected by Western-Blotting. The expression of i NOS, COX-2 at m RNA level in lung tissue was detected by real-time PCR.To evaluate NF-?B p65 DNA binding activity of the lung tissues.Results: 1.The degree of histopathology injury, wet / dry weight ratio and MPO score of lung tissues in LPS group was significantly higher as compared with the control and GRg3 groups(P <0.05). GRg3 could reduce the degree of pathological injury, wet / dry weight ratio and MPO score in lung tissues(P <0.05), and showed a dose-dependent manner.2. LPS-induced increase of the total cells, neutrophils and macrophages in the BALF were significantly inhibited by GRg3 pretreatment in a dose dependent fashion(P <0.05).3. The levels of pro-inflammatory cytokines including TNF-?, IL-1?, IL-6 and NO in BALF increased significantly after LPS administration(P <0.05). And the pretreatment of GRg3 can reduce the expression of these cytokines in BALF as compared with LPS group in a dose-dependent manner(P <0.05).4. Compared with the control group and GRg3 group, the expression of iNOS and COX-2 at m RNA level and the NF-?B p65 DNA binding ability in lung were increased significantly in LPS group(P <0.05); but they were dose-dependently reduced by GRg3 pretreatment( P <0.05)5. The ratio of NF-?B p65 phosphorylation and the expression of i NOS, COX-2 protein in lung in LPS group were significantly increased as compared with the control group and GRg3 group(P <0.05). After pretreatment of GRg3, these were significantly decreased in a dose-dependent manner(P <0.05).Conclusions: 1. In acute lung injury, LPS can induce inflammation and oxidative stress through the up-regulation of NF-?B p65 phosphorylation, COX-2 and i NOS in lung.2. GRg3 could play an anti-inflammatory and antioxidant effect on LPS-induced acute lung injury though inhibiting NF-?B p65 phosphorylation, and the expression of COX-2 and i NOS in lung.