| Background:Acute respiratory distress syndrome (ARDS), which is characterized by acute lung injury (ALI) caused by a variety of factors, is a disease of high mortality. ARDS can lead to noncardiogenic respiratory failure and there is no specific and effective treatment for it at present. Lipopolysaccharide (LPS) is one of common reasons of ARDS. A large number of researches prove that LPS can bind to the CD14 receptor on the surface of the monocytes/macrophages lineage, and a variety of cytokines and other inflammatory mediators are released after the interaction of LPS with host cells. Among the mediators, PLA2 is one of important mediators contributing to ALI. Metabolites of PLA2, platelet activating factor (PAF) and eicosanoids, are potentially involved in the development of ARDS.Tanshinone posseses lots of biological features such as anti-cancer, anti- thrombosis and anti-inflammation activities, and has been commonly used in traditional oriental herbal medicine to treat inflammatory diseases. Tanshinone IIA (TIIA) is one of the key components of tanshinone, and sodium tanshinone IIA sulphonate (STS) is a derivative of TIIA. STS can inhibit the release of inflammatory mediators and oxygen free radicals. However, there is little study of the protective effects of STS on LPS-induced ALI and the underlying mechanism. This experiment is to investigate the roles of STS in the prevention and treatment of ALI in mice and in the protection of cell injury induced by LPS, to observe the effects of STS in LPS-induced mortality of mice, and to further study the possible mechanism.Objective: (1) To study the preventive and therapeutic effects of STS on LPS-induced ALI (2) To investigate the effects of STS on LPS-induced mortality in mice (3) To investigate whether STS ameliorate LPS-induced ALI through inhibiting PLA2 activityMethods:Animal experimentFemale KM mice (18~22 g) were randomly divided into five groups: saline control group (n = 32), STS control group (n = 32), LPS group (n = 32), STS+LPS group (n = 32) and LPS+STS group (n = 32); the animal model of ALI was established by intraperitoneal administration of LPS. In the saline, STS and LPS groups, mice received saline, STS (10 mg/kg) and LPS (10 mg/kg) intraperitoneally by the volume to body weight ratio at 0.01 ml/g, respectively. In the STS+LPS group, STS (10 mg/kg) was administered half an hour before LPS administration, and in the LPS+STS group, mice received STS (10 mg/kg) one hour after LPS. Six hour after LPS or saline administration, the right lungs of each group were used for histological study, the left lung wet-to-dry (W/D) and lung-to-body (L/B) weight ratios, protein content in bronchoalveolar lavage fluid (BALF), myeloperoxidase (MPO) activity in lung homogenate, PLA2 activities in both lung homogenate and BALF and thromboxane B2 (TXB2) content in BALF were measured. Meanwhile, the content of NF-κB in nuclear protein was detected by Western blotting, representing the extent of its activation.To investigate the effects of STS on LPS-induced mortality of mice, female KM mice (18~22 g) were randomly divided into three groups: LPS group (n = 10), STS+LPS group (n = 20) and LPS+STS group (n = 20); the animal model of ALI was established by intraperitoneal administration of LPS (50 mg/kg). In STS+LPS and LPS+STS group, 30 or 50 mg/kg STS was administrated 0.5 h before or 1 h after LPS challenge. The mortality of mice was recorded every 12 h for 3 days after LPS challenge in each treated group.Cell experimentTo closely model the lung environment to survey the effects of STS on LPS-induced pulmonary epithelial cell injury,A549 cells (a human lung adenocarcinoma cell line) were co-cultured with NR8383 cells (the AMs cell line) at the ratios of 1:1 or 5:1. Cells were pretreated with 0~20μg/ml of STS for 24 h and some cells were activated for 24 h with LPS (1μg/ml) after preincubation of STS for 2 h. All the cells were washed three times with PBS in order to remove the NR8383 cells, and MTT assay was used for cell viability of A549 cells. A549 cells were adjusted to 1×106 cells/ml and co-cultured with NR8383 cells at the ratios of 1:1 and 5:1; After cells were stimulated with LPS (1μg/ml) for 12 h in the presence or absence of 0~20μg/ml STS, supernatant was used for determination of LDH activity at the end of the experiments. In order to investigate whether STS influence PLA2 activity directly, AM cells were adjusted to 1×106 cells/ml in 24 well cell culture cluster. Cells were pretreated with 0~20μg/ml of STS and some cells were activated with 1μg/ml LPS or 3μg/ml melittin (a PLA2 activator) for 6 h after preincubation of STS for 2 h. Supernatant was used for determination of PLA2 activity at the end of the experiments.Results:Animal experimentHistological studied showed that there were congestion, edema and the sequestration of inflammatory cells in lung tissues in LPS group, but lung injury was significantly alleviated in STS+LPS and LPS+STS groups; the W/D, L/B, protein content in BALF and MPO activity in lung homogenate were highly increased in LPS group (P < 0.01), and both prevention and therapy of STS can inhibit the increased parameters (P < 0.05).The PLA2 activities in lung homogenate and BALF in LPS group were [(49.2±4.3) U and (40.8±6.5) U], which were higher than that in the control group [(23.8±4.8) U and (27.2±6.9) U]; the PLA2 activities in STS+LPS and LPS+STS groups were reduced to [(29.0±5.8)U, (30.0±5.8)U] and [(31.4±4.9)U, (31.0±3.8)U] respectively, which had significant difference compared with LPS group. The BALF TXB2 content in LPS group was higher than that in the control group (P < 0.01), but it was reduced in both STS+LPS and LPS+STS groups, which was significantly lower than that in LPS group (P < 0.01). Western blotting data showed that LPS significantly increased the nuclear content of NF-κB, indicating the activation of NF-κB; but its activation was inhibited to some extent in both STS+LPS and LPS+STS groups.Mortalities during 3 days in 30 or 50 mg/kg STS pretreatment group were 50% and 40% respectively, which were significantly lower than that in LPS group (80%) (P < 0.05), and the survival time in STS pretreatment groups, (50.3±7.19) h and (51.62±7.89) h respectively, were significantly longer than that in LPS group (32.80±6.30) h (P < 0.05). However, the mortality of mice with treatment of STS (30, 50 mg/kg) after LPS challenge was 70%, and STS could not prolong the survival time compared to LPS group.Cell experimentMTT assay indicated that the concentrations (0~20μg/ml) of STS used had no effect on the viability of A549 cells. LPS at 1μg/ml reduced cell viability (P < 0.01), but STS concentration-dependently reversed LPS-induced reduction of cell viability. Furthermore, LPS increased LDH content in supernatant. STS also concentration-dependently reduced LPS-induced LDH content.In NR8383 cells, STS per se had no effect on PLA2 activity, and both LPS and melittin significantly increased its activity (P < 0.01). STS of different concentrations could reduce LPS-induced PLA2 activity and this effect was concentration-dependent, but it had no influence on melittin-induced activity.Conclusion: (1) STS could play an important role in the prevention and treatment of LPS-induced ALI in mice. (2) STS could exert protective effects on pulmonary epithelial cell injury induced by LPS. (3) Although STS could not reverse LPS-induced mortality of mice, pretreatment with STS could reduce mortality of mice and prolong their survival time. (4) The protective effect of STS on LPS-induced ALI was correlated with inhibition of lung PLA2 activity. (5) The inhibitive effect of STS on PLA2 activity was not through a direct way, probably through inhibiting the activation of NF-κB indirectly.
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