The Study On Caveolin-1 And Cytokine Of Acute Lung Injury With Qidonghuoxue Decoction

Objective To observe the protection of Qidonghuoxue decoction on acute lung injury mice and human pulmonary microvascular endothelial cells (HPMEC), and to investigate the possible effects of caveolin-1 and cytokine on ALI, to provide theoretical basis for clinical application of Qidonghuoxue decoction.Methods This study is divided into two parts:1. In vivo study of intervention and mechanism of Qidonghuoxue decoction on acute lung injury mice:60 healthy female mice were randomly divided into control group, model group, Qidonghuoxue decoction low and high dose groups, dexamethasone group. The five groups were given intragastric administration with different medicines and concentrations for 5 days before modeling by LPS endotracheal instillation. The serum, bronchoalveolar lavage fluid and lung tissue were collected 24h later and were detected by Milliplex map Kit, ELISA, RT-PCR, immunohistochemical assay and pathological detection on the levels of cytokines, caveolin-1, and so on.2. The effect of Qidonghuoxue decoction medicated serum on expression of E-selectin and IP-10 by LPS-induced human pulmonary microvascular endothelial cells:The HPMEC cells were randomly divided into five groups:blank group; LPS group; Normal rat serum and LPS group; 5% QidongHuoxue decoction medicated serum and LPS group; 10% QidongHuoxue decoction medicated serum and LPS group. All group cells were pretreated with medicated serum for 4h, then followed by 1μg/ml LPS. The mRNA level of E-selectin and IP-10 were measured by RT-PCR, its protein level were detected by ELISA.Results 1.Pathological result showed that model group mice had severe pulmonary congestion, edema, alveolar structure damage, widely alveolus effusion, alveolar decreasing with bleeding, thickening of alveolar interval, and a large number of inflammatory cells infiltration in interstitial lung parenchyma. And all the lung injury of treatment groups were alleviated. What’s more, dexamethasone group was lower than Qidonghuoxue decoction high dose group, while high dose group was lower than low dose group.2. Milliplex map Kit and ELISA assay showed that the anti-inflammatory cytokines(G-CSF, GM-CSF, IFN-γ, IL-1β, IL-5, IL-6, IL-7, IL-15, IL-17, IP-10, KC, MCP-1, MIP-1α, MIP-1β, RANTES, TNF-α, IL-12p70, IL-12, IL-la) and pro-inflammatory cytokines(IL-10, IL-13) in BALF and serum of mice were higher in model group than control group(P<0.05). The three groups’ treatment had inhibiting effects on cytokines, and the effect in dexamethasone group was stronger than in Qidonghuoxue decoction high dose group, while the effect in high dose group was stronger than in low dose group(P<0.05).3. RT-PCR detection showed that the expression of IL-6, TNF-α, IL-1β and caveolin-1 mRNA in model group were higher than in control group(P< 0.05). All of the three groups’treatment had inhibiting effects on there expression, and the effect on IL-6 and TNF-α which were the strongest in dexamethasone group, were the weakest in Qidonghuoxue decoction low dose group, while the effect on IL-1β which was the strongest in dexamethasone group was the weakest in Qidonghuoxue decoction high dose group(P<0.05).4. Immunohistochemical assay result and mean optical density showed that MyD88 and caveolin-1 were strong in lung tissue which were stronger in model group than control group(P<0.05). While they were weaker in the three treatment groups with or without difference between each other(P>0.05). eNOS was also strong in lung tissue which had no difference among all the groups(P>0.05).5.The expression and secretion of E-selectin and IP-10 in LPS group were elevated than blank group (P<0.01), while Qidonghuoxue decoction medicated serum suppressed their mRNA and protein level (P<0.05).Conclusion 1. Lipoplolysaccharide(LPS) endotracheal instillation can induce the acute lung injury mice model, and generate the imbalance bewteen pro-inflammatory cytokines and anti-inflammatory cytokines, as well as activate the TLR4/Caveolin-1 signal pathway.2. Qidonghuoxue decoction can alleviate the lung injury of ALI mice model induced by LPS.3. Qidonghuoxue decoction can inhibit cytokines in serum, BALF and lung tissue of ALI mice, such as G-CSF, GM-CSF, IFN-γ, IL-1β, IL-5, IL-6, IL-7, IL-15, IL-17, IP-10, KC, MCP-1, MIP-1α, MIP-1β, RANTES, TNF-α, IL-12p70, IL-12, IL-la, IL-10 and IL-13.4. Qidonghuoxue decoction can block TLR4/Caveolin-1 signal transduction pathway of ALI by inhibiting caveolin-1 and MyD88 depression.5.Qidonghuoxue decoction medicated serum can inhibit the HPMEC activation, reduce the cytokine release and alleviate the inflammatory injury.