【作者】 许敏；

【导师】 李志超； 董明清；

【作者基本信息】 第四军医大学 ， 病理生理学， 2008， 硕士

【摘要】 研究背景:急性呼吸窘迫综合征(acute respiratory distress syndrome,ARDS)是多种因素引起的以急性肺损伤(acute lung injury,ALI)为特征,并有较高死亡率的疾病。它可导致非心源性呼吸衰竭,目前尚缺乏特异有效的治疗方法。脂多糖(lipopolysaccharide,LPS)是ARDS的常见致病因素。大量的研究证明,LPS能结合单核/巨噬细胞表面的CD14受体,并与宿主细胞相互作用后使细胞产生大量的细胞因子与炎性介质,其中,磷脂酶A2(phospholipase A2,PLA2)被认为在急性肺损伤的发生发展过程中起了重要作用。PLA2的代谢产物、血小板活化因子(platelet activating factor,PAF)和类花生酸类物质都参与ARDS的形成。丹参酮(tanshinone)具有多种生物活性,如抗肿瘤、抗血栓形成和抗炎等,常在中医中被用来治疗炎性疾病。丹参酮IIA (tanshinone IIA, TIIA)在丹参中含量最多,丹参酮IIA磺酸钠(sodium tanshinone IIA sulphonate,STS)是丹参酮IIA的衍生物。近些年来,许多文献报道丹参酮IIA具有抑制炎性介质与氧自由基释放的作用,但有关其对内毒素引起的ALI的保护作用及其机制的研究比较少。本实验拟观察STS对LPS所致小鼠ALI的预防及治疗作用和对细胞损伤的保护作用,观察STS对内毒素性小鼠死亡率的影响,并进一步研究其发挥作用的可能机制。实验目的: (1)观察STS对LPS导致的ALI的防治作用(2)观察STS对内毒素性小鼠死亡率的影响(3)观察STS是否通过抑制PLA2活力减轻LPS导致的ALI,为STS的临床应用提供理论依据实验方法:动物实验雌性KM小鼠(18~22 g)适应环境后,腹腔注射LPS建立小鼠肺损伤模型,随机分为五组:生理盐水(NS)对照组(n = 32),STS对照组(n = 32),LPS组(n = 32),STS+LPS组(n = 32)及LPS+STS组(n = 32)。在NS组、STS组与LPS组,以0.01 ml/g的体积和体重比分别腹腔注射NS、STS(10 mg/kg)和LPS(10 mg/kg)。在STS+LPS组,LPS给予前0.5小时注射STS(10 mg/kg);在LPS+STS组,LPS注射1小时后给予STS (10 mg/kg)。在NS或LPS注射后6小时取小鼠右肺进行病理观察,测定左肺湿/干重比值(W/D)、左肺/体重比值(L/B)、支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)中蛋白含量、肺组织髓过氧化物酶(MPO)活力、肺组织匀浆与BALF中PLA2活性、BALF中血栓素BB2 (TXB2)含量,以及Westernblotting检测小鼠肺核蛋白中NF-κB的含量以代表其活化程度。为观察STS对动物死亡率的影响,实验动物适应环境后,腹腔注射LPS(50 mg/kg)建立小鼠内毒素血症模型,随机将其分为三组:LPS组(n = 10),STS+LPS组(预处理组,n = 20)及LPS+STS组(治疗组,n = 20)。在STS+LPS组,LPS给予前0.5小时注射STS(30或50 mg/kg);在LPS+STS组,LPS注射1小时后给予STS(30或50 mg/kg)。小鼠注射LPS后,每12小时记录一次小鼠的死亡情况,连续记录三天。细胞实验模拟肺环境观察STS对LPS所致的肺上皮细胞损伤的保护作用,肺泡上皮细胞系A549细胞与肺泡巨噬细胞系NR8383细胞分别以1:1或5:1的比例共同培养。用0~20μg/ml的STS处理24小时,部分细胞在STS预处理2小时后被LPS(1μg/ml)刺激24小时,所有的细胞用PBS洗三次去除NR8383细胞,采用MTT法检测A549细胞的活力;将A549细胞调制浓度为1×106 /ml,并以1:1与5:1的比例与NR8383细胞共同培养,在0~20μg/ml的STS预处理2小时或未处理的情况下,用LPS(1μg/ml)刺激细胞12小时,测定上清液中LDH的含量。为了探讨STS是否通过直接作用影响PLA2的活性,将NR8383巨噬细胞调制浓度为1×106 /ml接种在24孔培养板,细胞中加入0~20μg/ml STS,2小时后部分细胞被LPS(1μg/ml)或melittin(一种PLA2激动剂)(3μg/ml)刺激6小时,测定上清中PLA2的活力。实验结果:动物实验形态学观察表明LPS组中肺组织明显充血、水肿并有大量的炎性细胞浸润,而在STS+LPS组及LPS+STS组内毒素所致的肺损伤明显减轻,肺组织结构均趋于正常;W/D、L/B、BALF中蛋白含量与肺匀浆MPO活力在LPS组均较对照组明显升高(P < 0.01),但在STS+LPS组和LPS+STS组上述指标较LPS组明显减低(P < 0.05)。LPS组小鼠肺匀浆与BALF中PLA2活性为[(49.2±4.3)U与(40.8±6.5)U],均高于正常对照组[(23.8±4.8)U与(27.2±6.9)U],而在STS+LPS组及LPS+STS组分别降至[(29.0±5.8)U,(30.0±5.8)U]与[(31.4±4.9)U,(31.0±3.8)U],与LPS组相比有统计学差异。LPS组小鼠BALF中TXB2含量高于正常对照组(P < 0.01);而在STS+LPS组及LPS+STS组降低,与LPS组相比有统计学差异(P < 0.01)。Western blotting结果显示LPS能显著增加胞核中NF-κB的含量,表明NF-κB活化;而在STS+LPS组及LPS+STS组其活化明显降低。30或50mg/kg STS预处理组小鼠3天死亡率分别为50%和40%,比LPS组(80%)显著降低(P < 0.05);STS预处理组小鼠的生存时间分别为(50.3±7.19)h与(51.62±7.89)h,比LPS组(32.80±6.30)h显著延长(P < 0.05)。但是,LPS注射后给予30或50 mg/kg的STS治疗并不能显著降低小鼠的死亡率(70%),也不能明显延长其生存时间。细胞实验MTT结果显示0~20μg/ml的STS对A549细胞的活力没有影响,1μg/ml的LPS显著降低其活力(P < 0.01),STS能浓度依赖性地逆转LPS导致的细胞活力的下降;LPS能增加上清中LDH含量,STS同样浓度依赖性地抑制LPS导致的LDH含量的升高。在NR8383细胞中,STS本身对PLA2的活力没有影响,LPS和melittin都使PLA2活力显著升高(P < 0.01)。不同浓度的STS均能抑制LPS所致的PLA2活力的增高,而且具有剂量依赖性。但是不同浓度的STS对melittin所致的PLA2活力的增高没有影响。结论: (1) STS对LPS导致的小鼠急性肺损伤有预防和治疗作用。(2) STS对LPS所致的肺泡上皮细胞损伤具有保护作用。(3) STS虽然不能逆转内毒素导致的小鼠死亡,但STS预处理能降低小鼠的死亡率并能显著延长动物的平均生存时间。(4) STS对LPS所致急性肺损伤的保护作用与其抑制肺中PLA2活性有关。(5) STS对PLA2活力的抑制不是通过直接作用,可能通过抑制NF-κB的活化间接发挥作用的。更多还原

【Abstract】 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.更多还原