蛇床子素防治脂多糖诱导的急性肺损伤的作用及其机制研究

发布时间：2018-09-05 09:00

【摘要】：研究背景： 急性肺损伤（acute lung injury，ALI）是多种因素引起的，以肺部微血管和肺泡上皮弥漫性损伤、肺水肿及肺间质纤维化等为病理特征的临床综合症，严重者可发展为急性呼吸窘迫综合症（acute respiratory distress syndrome，ARDS）。由于ALI/ARDS发生机制较为复杂且具有较高死亡率，目前尚无特异有效的防治方法和药物。所以研究ALI/ARDS发生机制，研究有效治疗药物是该领域的重要研究方向。蛇床子素（Osthole，Osth）具有多种生物活性，如抗炎、抗肿瘤、抗氧化、抗凋亡等，已经被众多学者所关注。近年来，许多文献已经报道Osth具有抗氧化和抗炎的作用，但有关Osth对脂多糖引起的ALI的保护作用及其机制研究的尚鲜见报道。 脂多糖（lipopolysaccharide，LPS）是ALI/ARDS的常见致病因素之一。大量实验证明，LPS参与激活体内的活性氧簇（reactive oxygenspecies，ROS）从而引发体内氧化应激（oxidative stress，OS）。在氧化抗氧化的系统中，核因子相关因子（Nuclearfactor erythroid-2related factor2，Nrf2）可以调节硫氧还蛋白1（thioredoxin1，Trx1）等多种抗氧化蛋白，同时Trx1起到了清除ROS及氧化还原的重要作用。有研究表明Osth具有抗氧化作用，那么Osth在LPS诱导的ALI中是否通过Nrf2和Trx1清除ROS从而发挥保护ALI的作用仍不明确。 本实验拟观察Osth对LPS所致小鼠ALI的预防及其治疗作用和对细胞OS损伤的保护作用，并进一步研究其发挥作用机制。 实验目的： （1）观察Osth对LPS导致小鼠死亡率的影响。 （2）观察Osth对LPS诱导的ALI的防治作用。 （3）观察Osth是否通过Nrf2/Trx1通路减轻ALI。 实验方法： 动物实验 为了观察Osth对小鼠死亡率的影响，将雄性小鼠（18-23g）随机分为三组：Control组（n=20），LPS组（n=20）及Osth+LPS组（预防组，n=60）。腹腔注射50mg/kg LPS成功建立小鼠内毒素血症模型，在Osth+LPS组分别给予Osth（20，40或80mg/kg），，连续三天，每天一次。然后给予腹腔内注射LPS后，每12h记录一次小鼠的死亡情况，连续72h。 雄性BALB/c小鼠（18-23g）适应环境后，随机分为五组：对照组（n=10），Osth组（n=10），LPS组（n=10），Osth+LPS组（n=30），LPS+Osth组（n=30）。LPS组气管内滴注LPS建立小鼠肺损伤模型。在对照组，Osth组与Osth+LPS组，分别以生理盐水和Osth（40mg/kg）灌胃三天，每天一次。然后在LPS组，Osth+LPS组和LPS+Osth组，分别给予LPS（5mg/kg）气管内滴注。LPS+Osth组在气管内滴注LPS后1h给予灌胃Osth（40mg/kg）。在LPS处理6h后取小鼠肺组织进行检测和观察。测定肺湿重/干重比值（W/D）、肺泡灌洗液（bronchoalveolar lavage fluid，BALF）中蛋白含量，肺组织匀浆中检测过氧化氢（hydrogenperoxide，H2O2），羟自由基（hydroxylradical， OH）和丙二醛（Methane Dicarboxylic Aldehyde，MDA）。Western blot检测Nrf2和Trx1的蛋白含量。 细胞实验 培养A549细胞系，在培养到对数生长期时进行传代。用Osth（0，25，50或100μg/ml）处理A549细胞，2h后部分细胞被LPS（1μg/ml）刺激12h，取细胞上清液检测LDH含量。将A549细胞接种到96孔板中，处理同上所述，然后使用MTT法检测A549细胞活力。 为了证明Osth是否通过Nrf2/Trx1来清除ROS，从而发挥保护作用。将A549细胞接种于6cm细胞培养皿中，加入Nrf2siRNA和50μg/ml Osth，共同培养2h后加入1μg/ml的LPS，检测LDH和细胞活力。RT-PCR检测Nrf2和Trx1的mRNA和蛋白表达，并用流式细胞术检测细胞内ROS含量。 实验结果： 动物实验 Osth对LPS导致的小鼠死亡率的影响：20，40或80mg/kg Osth预处理组的小鼠72h死亡率分别为：80%，45%和40%，比LPS组的累计死亡率（85%）均有降低，并且在40，80mg/kg的Osth+LPS组72h累计死亡率明显降低，具有统计学差异（P0.05）。 肺组织病理结果表明：在LPS组肺组织充血、水肿明显，有大量炎症细胞浸润，并存在大面积肺不张。而在Osth+LPS组和LPS+Osth组LPS导致的肺损伤明显减轻；LPS组W/D、BALF中蛋白含量均比对照组明显升高（P0.05），但是在Osth+LPS组和LPS+Osth组上述两个指标均较LPS组明显降低（P0.05）。 LPS组的肺组织匀浆中H2O2， OH，MDA含量明显高于对照组（P0.05），而在LPS+Osth组和Osth+LPS组，三者的含量明显低于LPS组有统计学差异（P0.05）。Western Blot结果显示：LPS组的Nrf2和Trx1的蛋白含量显著低于对照组，而Osth+LPS组和LPS+Osth组中Nrf2和Trx1的蛋白含量显著高于LPS组，具有统计学差异（P0.05）。 细胞实验 LPS增加A549细胞上清液中的LDH含量，而Osth可浓度依赖性的抑制LPS导致的LDH含量增加。MTT分析结果显示：单独Osth处理组对细胞活力没有影响，1μg/ml的LPS显著降低A549细胞活力，Osth浓度依赖性的减轻LPS诱导的细胞活力下降。 LPS可以显著增加LDH和细胞内ROS的含量，并显著降低细胞活力。然而，Nrf2和Trx1的mRNA和蛋白含量被明显下调。给予Osth后的LPS组LDH和细胞内ROS被抑制。与此同时，细胞活力、Nrf2和Trx1的mRNA和蛋白表达均增加。Nrf2siRNA后Nrf2和Trx1的mRNA和蛋白表达降低。Nrf2siRNA+Osth+LPS组中LDH和ROS的含量比Osth+LPS组的增加，细胞活力增加。因此，Nrf2siRNA可以下调Nrf2/Trx1的表达，同时逆转Osth对细胞损伤的保护作用。 结论： （1） Osth明显降低LPS导致的小鼠死亡率。 （2） Osth对LPS导致的小鼠ALI具有预防和治疗作用。 （3） Osth可能通过上调Nrf2和Trx1起到防治LPS诱导的ALI的作用。
[Abstract]:Research background:
Acute lung injury (ALI) is a clinical syndrome characterized by diffuse damage of pulmonary microvessels and alveolar epithelium, pulmonary edema and pulmonary interstitial fibrosis. Severe cases may develop into acute respiratory distress syndrome (ARDS). For the sake of complexity and high mortality, there are no specific and effective prevention and treatment methods and drugs. So it is an important research direction to study the pathogenesis of ALI/ARDS and effective treatment drugs. Osthole (Osth) has a variety of biological activities, such as anti-inflammation, anti-tumor, anti-oxidation, anti-apoptosis, and so on, which has been concerned by many scholars. In recent years, many literatures have reported that Osth has antioxidant and anti-inflammatory effects, but the protective effects of Osth on lipopolysaccharide-induced ALI and its mechanism are rarely reported.
Lipopolysaccharide (LPS) is one of the common pathogenic factors of ALI/ARDS. A large number of experiments have proved that LPS is involved in activating reactive oxygen species (ROS) in vivo and thus triggering oxidative stress (OS). In the oxidative-antioxidant system, nuclear factor-related factor (Nuclearfactor erythroid-2 related factor) 2, Nrf2 can regulate thioredoxin 1 (Trx1) and other antioxidant proteins, and Trx1 plays an important role in scavenging ROS and redox. Studies have shown that Osth has antioxidant effect, so it is still unclear whether Osth can protect ALI by removing ROS through Nrf2 and Trx1 in LPS-induced ALI.
In this study, we observed the preventive and therapeutic effects of Osth on LPS-induced ALI in mice and its protective effects on cell OS injury, and further studied its mechanism.
Objective:
(1) observe the effect of Osth on LPS induced mortality in mice.
(2) observe the preventive and therapeutic effects of Osth on LPS induced ALI.
(3) to observe whether Osth alleviated ALI. through the Nrf2/Trx1 pathway.
Experimental methods:
Animal experiment
To observe the effect of Osth on the mortality of mice, male mice (18-23g) were randomly divided into three groups: Control group (n=20), LPS group (n=20) and Osth+LPS group (prevention group, n=60). The model of endotoxemia in mice was successfully established by intraperitoneal injection of 50 mg/kg LPS. Osth (20, 40 or 80 mg/kg) was given to the Osth+LPS group for three consecutive days, once a day. After intraperitoneal injection of LPS, the mortality of mice was recorded every 12h, and 72h. was continuously recorded.
Male BALB/c mice (18-23g) were randomly divided into five groups: control group (n = 10), Osth group (n = 10), LPS group (n = 10), Osth + LPS group (n = 30), LPS + Osth group (n = 30). LPS group was given LPS intratracheally to establish lung injury model in mice. In LPS group, Osth+LPS group and LPS+Osth group, LPS (5mg/kg) was given intratracheal instillation respectively. LPS+Osth group was given intratracheal instillation of LPS (40mg/kg). Lung tissues of mice were taken for examination and observation after LPS treatment for 6 hours. Wet/dry weight ratio (W/D), protein content in bronchoalveolar lavage fluid (BALF), lung tissue were determined. Hydrogen peroxide (H2O2), hydroxyl radical (OH) and malondialdehyde (MDA) were detected in tissue homogenate. The protein contents of Nrf2 and Trx1 were detected by Western blot.
Cell experiment
A549 cells were treated with Osth (0,25,50 or 100 ug/ml). After 2 hours, some of the cells were stimulated by LPS (1 ug/ml) for 12 hours. LDH content was detected by cell supernatant. A549 cells were inoculated into 96-well plate and treated as described above. Then the viability of A549 cells was detected by MTT method.
In order to prove whether Osth can eliminate ROS by Nrf2/Trx1 and thus play a protective role, A549 cells were inoculated into 6 cm cell culture dishes, added Nrf2 siRNA and 50 ug/ml Osth, cultured for 2 hours and added 1 ug/ml LPS to detect LDH and cell viability. The mRNA and protein expression of Nrf2 and Trx1 were detected by RT-PCR, and the intracellular ROS was detected by flow cytometry. Content.
Experimental results:
Animal experiment
Effects of Osth on the mortality of LPS-induced mice: The 72-hour mortality rates of 20,40 or 80 mg/kg Osth pretreatment group were 80%, 45% and 40% respectively, which were lower than that of LPS group (85%), and the 72-hour mortality rate of 40,80 mg/kg Osth+LPS group was significantly lower than that of LPS group (P 0.05).
The pathological results of lung tissue showed that in LPS group, there was hyperemia, edema, infiltration of inflammatory cells, and atelectasis in a large area. In Osth+LPS group and LPS+Osth group, the lung injury caused by LPS was significantly alleviated; in LPS group, W/D, BALF protein content was significantly higher than that in control group (P 0.05), but in Osth+LPS group and LPS+Osth group, the above two groups were significantly increased (P 0.05). All indexes were significantly lower than those in group LPS (P0.05).
The contents of H2O2, OH and MDA in lung homogenate of LPS group were significantly higher than those of control group (P 0.05), while those of LPS + Osth group and Osth + LPS group were significantly lower than those of LPS group (P 0.05). Western Blot results showed that the contents of Nrf2 and Trx1 in LPS group were significantly lower than those of control group, while the contents of Nrf2 and Trx1 in Osth + LPS group and LPS + Osth group were significantly lower than those of control group. The content of white was significantly higher than that of group LPS (P0.05).
Cell experiment
LPS increased the LDH content in the supernatant of A549 cells, while Osth inhibited the increase of LDH content induced by LPS in a concentration-dependent manner. MTT analysis showed that Osth alone had no effect on the cell viability, LPS at 1 ug/ml significantly decreased the cell viability of A549 cells, and Osth concentration-dependent decreased the LPS-induced cell viability decline.
LPS significantly increased LDH and intracellular ROS levels, and significantly decreased cell viability. However, the mRNA and protein levels of Nrf2 and Trx1 were significantly down-regulated. LDH and intracellular ROS were inhibited in LPS treated with Osth. At the same time, cell viability, Nrf2 and Trx1 mRNA and protein expression were increased. Nrf2 and Trx1 mRNA and protein expression were increased after Nrf2siRNA treatment. The content of LDH and ROS in Nrf2siRNA+Osth+LPS group was higher than that in Osth+LPS group, and the cell viability was increased. Therefore, Nrf2siRNA could down-regulate the expression of Nrf2/Trx1 and reverse the protective effect of Osth on cell injury.
Conclusion:
(1) Osth significantly reduced LPS induced mortality in mice.
(2) Osth has preventive and therapeutic effects on LPS induced ALI in mice.
(3) Osth may play a role in the prevention and treatment of LPS induced ALI by up regulating Nrf2 and Trx1.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R563.8

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