Protective Effects And Mechanisms Of Salvianolic Acid B In Rat With Acute Lung Injury

BackgroundAcute lung injury(ALI)is a common clinical critical illness.Its rapid onset and development lead to poor prognosis and high mortality.The pathogenesis of ALI is a complex process associated with cell injury and apoptosis of alveolar epithelial cells and vascular endothelial cells,inflammation and oxidative stress in the lung tissue.Currenly,there are no effective treatments for the disease.Therefore,searching for new treatment strategies for ALI is an important issue in clinical research and practice.Salvianolic acid B(SAB),one of Salvia Finland acids,has been reported to have antioxidant role by scavenging oxygen free radicals,effectively inhibit platelet aggregation and thrombosis formation,and thus improve the blood flow.In addition,SAB has a regulatory role on lipid metabolism and other biological activities such as anti-apoptotic and anti-fibrotic effects.These results pin-point the pharmacological functions of SAB in the prevention and treatment of cardiovascular disease,liver and lung fibrosis,and disorders of other vital organs.While its clinical value in the treatment of cardiovascular diseases has been widespread approved,therapeutic roles of SAB on ALI is still elusive.ObjectiveIn the present study,we observed protective effects of SAB in a rat ALI model by comparing pathological changes and biochemical and inflammatory parameters in the lung tissue.We further investigated the cellular and molecular mechanisms underlying these effects by using type ? alveolar epithelial cells in vitro.Hopefully,we expect thatthese results will provide new ideas for ALI treatment with SAB.Method(1)Establishment of rat ALI model.The SD rat ALI model was established by intratracheal instillation of 1mg/kg lipopolysaccharide(Lipopolysaccharides,LPS).1,2,4,6 and10 hours after the treatment,wet / dry quality ratio of the lung tissue and serum TNF-? and IL-6 levels were determined to reflect pulmonary edema and inflammation in the lung tissue,and lactate dehydrogenase(LDH)level in the lavage fluid was detected to observe cell injury in the lung.Pathological changes in the lung tissue,along with these parameters,were used for evaluation of the model.(2)Protective effects of SAB in the rat ALI model.SD rats were divided into 3 groups: control group,ALI model group and SAB treatment group.In the control group,the rats were intratracheally instilled with saline,and 1 h later intravenously injected with saline,and continued the injection 1 time/day for 4 weeks;In the ALI model group,the rats were intratracheally instilled with 1mg/kg LPS to induce acute lung injury,and 1 h later intravenously injected with saline,and continued the injection 1 time/day for 4 weeks;In the SAB treatment group,the rats were intratracheally instilled with 1mg/kg LPS,and 1 h later intravenously administrated with 20mg/kg of SAB,and continued the SAB administration 1 time/day for 4 weeks.4 weeks later the rats in each group were sacrificed and the lung tissue was collected for lung wet / dry weight ratio determination and for pathological observation;parameters associated with metabolism of reactive oxygen species including activities of Caspase-3,superoxide dismutase(SOD),catalase(CAT)and glutathione peroxidase(GPX)and levels of malondialdehyde(MDA),and acute inflammation-related cytokine IL-6 in the lung tissue homogenate were detected by ELISA;Expression of Col-I,TGF-?1 and ?-SMA proteins in the lung tissue,which are associated with lung fibrosis, as detected by Western Blotting.(3)Protective effects of SAB in vitro.As in the rat ALI model,A549 human alveolar epithelial cells were divided into control group,LPS injury group and SAB treatment group.Acute injury of the A549 cells was induced by addition of LPS with a final concentration of 1 ?g / m L,then the cells was treated with 100? mol / L SAB to observe its protective effects.Apoptotic cells,expression of the apoptosis-associated proteins Caspase-3,Bcl-2and Bax and cell viability were compared between each group by flow cytometry,Western Blotting and MTT assay,respectively.Results1.The rat lung tissue wet / dry weight ratio,serum TNF-?,IL-6 and LDH levels were significantly increased at 1,2,4,6 and 10 h after administration of LPS(P<0.05,0.05 and 0.01,respectively),compared with those in the control group.Histopathological observation indicated that intratracheal instillation of LPS resulted in severe inflammatory cell infiltration and vascular congestion.2.Salvia acid B can improve inflammatory infiltration and vascular congestion induced by the LPS in the lung tissue.Also,lung tissue wet / dry weight ratio,Caspase-3 activity,levels of MDA,TNF-?,IL-6,Col-I,TGF-?1 and ?-SMA in the lung tissue,and serum LDH levels were significantly decreased by administration of SAB(all P values <0.01),while activities of SOD,CAT and GPX were significantly increased(P <0.01).3.Flow cytometry results showed that SAB significantly reduced the rate apoptotic A549 cells(P <0.01),compared with that of the cells treated with LPS alone.Cell viability detected by MTT assay confirmed the protective role of SAB.Further analysis by Western Blotting indicated that treatment with SAB decreased expression of pro-apoptotic Caspase-3 and Bax protein and increased xpression of the anti-apoptotic protein Bcl-2(P <0.01).Conclusion By using a rat ALI model,we demonstrated that SAB suppressed inflammation,reduce oxidative stress and apoptosis in the lung tissue induced by LPS.In vitro studies on A549 cells confirmed that SAB protected the cells from apoptosis caused by LPS.These results suggest that SAB is a potential choice for ALI in clinical practice.