【摘要】：研究背景： 急性肺損傷（acute lung injury，ALI）是多種因素引起的，以肺部微血管和肺泡上皮彌漫性損傷、肺水腫及肺間質(zhì)纖維化等為病理特征的臨床綜合癥，嚴重者可發(fā)展為急性呼吸窘迫綜合癥（acute respiratory distress syndrome，ARDS）。由于ALI/ARDS發(fā)生機制較為復雜且具有較高死亡率，目前尚無特異有效的防治方法和藥物。所以研究ALI/ARDS發(fā)生機制，研究有效治療藥物是該領域的重要研究方向。蛇床子素（Osthole，Osth）具有多種生物活性，如抗炎、抗腫瘤、抗氧化、抗凋亡等，已經(jīng)被眾多學者所關注。近年來，許多文獻已經(jīng)報道Osth具有抗氧化和抗炎的作用，但有關Osth對脂多糖引起的ALI的保護作用及其機制研究的尚鮮見報道。 脂多糖（lipopolysaccharide，LPS）是ALI/ARDS的常見致病因素之一。大量實驗證明，LPS參與激活體內(nèi)的活性氧簇（reactive oxygenspecies，ROS）從而引發(fā)體內(nèi)氧化應激（oxidative stress，OS）。在氧化抗氧化的系統(tǒng)中，核因子相關因子（Nuclearfactor erythroid-2related factor2，Nrf2）可以調(diào)節(jié)硫氧還蛋白1（thioredoxin1，Trx1）等多種抗氧化蛋白，同時Trx1起到了清除ROS及氧化還原的重要作用。有研究表明Osth具有抗氧化作用，那么Osth在LPS誘導的ALI中是否通過Nrf2和Trx1清除ROS從而發(fā)揮保護ALI的作用仍不明確。 本實驗擬觀察Osth對LPS所致小鼠ALI的預防及其治療作用和對細胞OS損傷的保護作用，并進一步研究其發(fā)揮作用機制。 實驗目的： （1）觀察Osth對LPS導致小鼠死亡率的影響。 （2）觀察Osth對LPS誘導的ALI的防治作用。 （3）觀察Osth是否通過Nrf2/Trx1通路減輕ALI。 實驗方法： 動物實驗 為了觀察Osth對小鼠死亡率的影響，將雄性小鼠（18-23g）隨機分為三組：Control組（n=20），LPS組（n=20）及Osth+LPS組（預防組，n=60）。腹腔注射50mg/kg LPS成功建立小鼠內(nèi)毒素血癥模型，在Osth+LPS組分別給予Osth（20，40或80mg/kg），，連續(xù)三天，每天一次。然后給予腹腔內(nèi)注射LPS后，每12h記錄一次小鼠的死亡情況，連續(xù)72h。 雄性BALB/c小鼠（18-23g）適應環(huán)境后，隨機分為五組：對照組（n=10），Osth組（n=10），LPS組（n=10），Osth+LPS組（n=30），LPS+Osth組（n=30）。LPS組氣管內(nèi)滴注LPS建立小鼠肺損傷模型。在對照組，Osth組與Osth+LPS組，分別以生理鹽水和Osth（40mg/kg）灌胃三天，每天一次。然后在LPS組，Osth+LPS組和LPS+Osth組，分別給予LPS（5mg/kg）氣管內(nèi)滴注。LPS+Osth組在氣管內(nèi)滴注LPS后1h給予灌胃Osth（40mg/kg）。在LPS處理6h后取小鼠肺組織進行檢測和觀察。測定肺濕重/干重比值（W/D）、肺泡灌洗液（bronchoalveolar lavage fluid，BALF）中蛋白含量，肺組織勻漿中檢測過氧化氫（hydrogenperoxide，H2O2），羥自由基（hydroxylradical， OH）和丙二醛（Methane Dicarboxylic Aldehyde，MDA）。Western blot檢測Nrf2和Trx1的蛋白含量。 細胞實驗 培養(yǎng)A549細胞系，在培養(yǎng)到對數(shù)生長期時進行傳代。用Osth（0，25，50或100μg/ml）處理A549細胞，2h后部分細胞被LPS（1μg/ml）刺激12h，取細胞上清液檢測LDH含量。將A549細胞接種到96孔板中，處理同上所述，然后使用MTT法檢測A549細胞活力。 為了證明Osth是否通過Nrf2/Trx1來清除ROS，從而發(fā)揮保護作用。將A549細胞接種于6cm細胞培養(yǎng)皿中，加入Nrf2siRNA和50μg/ml Osth，共同培養(yǎng)2h后加入1μg/ml的LPS，檢測LDH和細胞活力。RT-PCR檢測Nrf2和Trx1的mRNA和蛋白表達，并用流式細胞術(shù)檢測細胞內(nèi)ROS含量。 實驗結(jié)果： 動物實驗 Osth對LPS導致的小鼠死亡率的影響：20，40或80mg/kg Osth預處理組的小鼠72h死亡率分別為：80%，45%和40%，比LPS組的累計死亡率（85%）均有降低，并且在40，80mg/kg的Osth+LPS組72h累計死亡率明顯降低，具有統(tǒng)計學差異（P0.05）。 肺組織病理結(jié)果表明：在LPS組肺組織充血、水腫明顯，有大量炎癥細胞浸潤，并存在大面積肺不張。而在Osth+LPS組和LPS+Osth組LPS導致的肺損傷明顯減輕；LPS組W/D、BALF中蛋白含量均比對照組明顯升高（P0.05），但是在Osth+LPS組和LPS+Osth組上述兩個指標均較LPS組明顯降低（P0.05）。 LPS組的肺組織勻漿中H2O2， OH，MDA含量明顯高于對照組（P0.05），而在LPS+Osth組和Osth+LPS組，三者的含量明顯低于LPS組有統(tǒng)計學差異（P0.05）。Western Blot結(jié)果顯示：LPS組的Nrf2和Trx1的蛋白含量顯著低于對照組，而Osth+LPS組和LPS+Osth組中Nrf2和Trx1的蛋白含量顯著高于LPS組，具有統(tǒng)計學差異（P0.05）。 細胞實驗 LPS增加A549細胞上清液中的LDH含量，而Osth可濃度依賴性的抑制LPS導致的LDH含量增加。MTT分析結(jié)果顯示：單獨Osth處理組對細胞活力沒有影響，1μg/ml的LPS顯著降低A549細胞活力，Osth濃度依賴性的減輕LPS誘導的細胞活力下降。 LPS可以顯著增加LDH和細胞內(nèi)ROS的含量，并顯著降低細胞活力。然而，Nrf2和Trx1的mRNA和蛋白含量被明顯下調(diào)。給予Osth后的LPS組LDH和細胞內(nèi)ROS被抑制。與此同時，細胞活力、Nrf2和Trx1的mRNA和蛋白表達均增加。Nrf2siRNA后Nrf2和Trx1的mRNA和蛋白表達降低。Nrf2siRNA+Osth+LPS組中LDH和ROS的含量比Osth+LPS組的增加，細胞活力增加。因此，Nrf2siRNA可以下調(diào)Nrf2/Trx1的表達，同時逆轉(zhuǎn)Osth對細胞損傷的保護作用。 結(jié)論： （1） Osth明顯降低LPS導致的小鼠死亡率。 （2） Osth對LPS導致的小鼠ALI具有預防和治療作用。 （3） Osth可能通過上調(diào)Nrf2和Trx1起到防治LPS誘導的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.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R563.8

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