Aim: To study the effects of tanshinone IIA (TIIA) about lipopolysaccharide (LPS)-induced acute lung injury in mice and the underlying mechanisms. inflammatory responses and apoptosis, which is definitely mediated via inhibition of the NF-B and HIF-1 pathways. Bunge, is definitely widely used in China for the treatment of many diseases. TIIA may exert a series of biochemical effects, such as anti-oxidant and anti-inflammatory effects. Our previous work has shown that TIIA was able to alleviate ALI induced by lipopolysaccharide (LPS)8,9 and seawater exposure10,11,12,13, indicating that TIIA may be a potential agent to treat ALI. Although we have previously found that TIIA was able to prevent the event of ALI to a certain GSK1070916 extent having a pretreatment method, little is known about its restorative effect. The development of ALI is definitely a cascade reaction, which is definitely involved in many mechanisms; as a result, although pretreatment with TIIA GSK1070916 may attenuate lung injury, it is unclear whether it has restorative effects when lung injury has already occurred. However, as most patients in private hospitals have been diagnosed GSK1070916 with ALI rather than are at risk of developing ALI, there is also a tremendous need to explore the restorative effect of TIIA on lung injury. In the present study, to accelerate its medical use, we examined whether TIIA was able to therapeutically reduce LPS-induced ALI and explored the underlying molecular mechanisms in mice. Our results demonstrate that TIIA alleviated LPS-induced lung injury, attenuated lung inflammatory reactions, and reduced lung cell apoptosis, which was via the inhibition of NF-B and HIF-1 signaling pathways. Materials and methods Chemicals Tanshinone IIA (sulfonate, purity is definitely 99%) was purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The structure of TIIA is definitely shown in Number 1. The kit for determining myeloperoxidase (MPO) activity was from Jiancheng Bioengineering Institute (Nanjing, China). Enzyme-linked immunosorbent assay (ELISA) packages for TNF- and IL-1 were from R&D Systems (Minneapolis, MN, USA). cell death detection kits and proteinase were from Roche Molecular Biochemicals (Indianapolis, IN, USA). Bcl-2 and caspase-3 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies specific for total and phosphorylated NF-B, and HIF-1 were purchased form Millipore (Bedford, MA, USA). Monoclonal -actin antibody, endotoxin LPS (O55:B5), Evans blue dye and all the other reagents were from Sigma-Aldrich Inc (St Louis, MO, USA). The purity of all chemical reagents was at least in Mouse Monoclonal to E2 tag. analytical grade. Number 1 The chemical structure of TIIA. Animal preparation Male BALB/c mice, which weighed 18C22 g, were from the Animal Center (the 88th Hospital of PLA, Taian, China). Mice were kept inside a temperature-controlled house with 12-h light-dark cycles and were fed standard laboratory diet and water for 10 min, total and differential cell counts and protein concentration in the BALF were identified from your cell portion16,17. The supernatant was utilized for measurements of LDH and inflammatory cytokines (TNF- and IL-1) by ELISA according to the related manufacturer’s instructions. TUNEL staining Paraffin-embedded cells slices were dewaxed, washed with PBS, and digested with proteinase K in the damp package for 30 min at 37 C. After becoming washed with PBS, the slides were dipped in TUNEL reaction mixture, and then incubated for 1 h at 37 C in the damp box. After washing, the sections were incubated with converter-AP for 30 min at 37 C in the damp box, and then washed with PBS. Subsequently, the sections were stained with NBT/BCIP substrate remedy for 1 h, and signals were observed having a microscope. All cells with purple nuclei were considered to be dead. Protein extraction and Western blotting Cells from the right lungs (saline control group. eLPS group. TIIA alleviated LPS-induced lung injury To evaluate the ALI model, numerous parameters related to the acute phase response, such as cell injury, lung edema, vascular and protein leakage were identified. First, we used BALF LDH content to assess the degree of cell injury in ALI model. Results showed that there were GSK1070916 no variations in BALF LDH content material between saline and TIIA control organizations, but BALF LDH content material was significantly improved in the LPS group, indicating that LPS induced ALI in the treated mice (Number 3A, Bunge, offers.