紫草素在急性肺损伤中保护作用与机制的研究

发布时间：2018-04-25 05:33

本文选题：紫草素 + 脂多糖　；参考：《第四军医大学》2013年博士论文

【摘要】：目的与背景急性肺损伤（ALI）是常见的重症疾病，其病死率高，临床尚缺乏特效的治疗方法。中药紫草用于治疗感染炎症性的疾病已有上千年记载，而且其提取物紫草素在多项体内外实验中已初显其抗炎症作用。本课题旨在研究紫草素在ALI中的作用与机制，为ALI的药物治疗提供新的思路。方法 1．采集健康人外周血，分离培养其中外周血单个核细胞（PBMC），向培养基中加入3μM的紫草素进行预处理，1h后加入100ng/ml的脂多糖（LPS）诱导PBMC产生炎症反应，24h后收集细胞上清液，通过酶联免疫吸附试验（ELISA）测定其中TNF-α、IL-1β及IL-8的浓度，对结果进行统计学分析，验证紫草素是否抑制LPS刺激的PBMC炎性因子的产生。 2．通过向BALB/C小鼠气管内滴入5mg/kg的LPS构建ALI模型，分别于3h、6h、12h、24h采集支气管肺泡灌洗液（BALF），采用ELISA法检测其中TNF-α及IL-1β的浓度；取肺组织，行组织病理学切片观察，测定肺湿干重比，比色法检测组织匀浆液中髓过氧化物酶（MPO）活性及一氧化氮（NO）浓度。对上述数据进行统计学分析，验证ALI小鼠模型构建成功与否。 3．将BALB/C小鼠分成空白对照组、紫草素组(50mg/kg）、LPS组和LPS+紫草素3个剂量组（12.5mg/kg、25mg/kg、50mg/kg），小鼠气管内滴入LPS之前1h，预先通过灌胃法给予小鼠相应剂量的紫草素或其溶剂。LPS给药6h后采集BALF及肺组织，用ELISA法检测BALF中TNF-α和IL-1β的浓度，BCA法测定BALF中蛋白浓度；肺组织行病理组织切片检查，测定肺湿干重比，比色法检测组织匀浆液中MPO活性及NO浓度，Western Blot法检测肺组织中诱生型一氧化氮合成酶（iNOS）和环氧化酶-2（COX-2）的表达，，电泳迁移实验（EMSA）检测肺组织核蛋白内核转录因子-κB（NF-κB）水平。对上述结果进行分析，研究紫草素在ALI小鼠体内的抗炎作用与机制。结果 1．体外紫草素的抗炎作用：LPS刺激PBMC后产生的TNF-α、IL-1β和IL-8均显著增加，分别为199.3±21pg/ml、100.3±11.24pg/ml和101.0±11.6pg/ml，均较单纯紫草素处理组和空白对照组显著增高，组间差异有统计学意义（P0.05）。在LPS之前给予紫草素预处理能够明显抑制TNF-α、IL-1β和IL-8的产生，其浓度分别为126.3±10.0pg/ml、55.7±9.1pg/ml和60.7±12.1pg/ml，与LPS组差异具有统计学意义（P0.05）。 2．成功构建小鼠ALI模型：经小鼠气管滴入LPS后3h，BALF中TNF-α和IL-1β浓度（3038±344.1pg/ml，1841.0±320.0pg/ml）均较对照组（109.7±46.06pg/ml，104.7±33.25pg/ml）明显升高，达到最高值，其他各时间点TNF-α及IL-1β浓度较对照组明显升高，组间差异有统计学意义（P0.05）。气管滴入LPS3h后肺组织中MPO活性（1.49±0.39U/g）较空白对照组（0.39±0.09U/g）升高，后逐渐增高至24h达到最高值（5.09±1.04U/g），各时间点均较空白对照组明显升高，组间差异有统计学意义（P0.05）。气管内滴入LPS3h后，肺组织中NO浓度（697.9±63.18μmol/g）较空白对照组（210.6±19.16μmol/g）显著升高，各时间点NO浓度较空白对照组明显升高，差异有统计学意义（P0.05）。气管内滴入LPS3h后，肺组织湿干重比（5.03±0.46）较对照组（4.02±0.27）即升高，到24h达到峰值，差异有统计学意义（P0.05）。LPS给药24h后肺组织病理学表现为大量炎细胞向肺间质及肺泡腔内浸润，肺泡内出血，肺泡塌陷及广泛的肺泡壁增厚、水肿。 3．紫草素对小鼠ALI的保护作用：检测BALF中TNF-α和IL-1β的浓度以及BALF中的蛋白含量发现，3个剂量紫草素+LPS组的浓度均较LPS组显著降低，差异有统计学意义（P0.05）。比色法测定肺组织中MPO活性和NO浓度发现，紫草素+LPS组也明显低于LPS组，统计学差异显著（P0.05）。紫草素预处理组的肺湿干重比也较LPS组明显降低，差异有统计学意义（P0.05）。此外，紫草素预处理组的肺组织病理学改变也较LPS组有所减轻，尤其体现在肺组织炎性细胞的浸润明显减少。Western Blot示3个剂量紫草素+LPS组的COX-2和iNOS表达量均较LPS组明显下降，存在显著性差异（P0.05）。EMSA则显示小鼠肺组织核蛋白内NF-κB水平在紫草素预处理组较LPS组明显降低，存在显著性差异（P0.05）。结论 1．紫草素可显著降低LPS诱导的PBMC促炎症因子TNF-α、IL-1β和细胞趋化因子IL-8的产生，提示紫草素在体外具有抗炎症作用。 2．通过检测ALI小鼠模型BALF中TNF-α及IL-1β的浓度、肺组织匀浆液中MPO活性及NO浓度，观察肺组织湿干重比和病理学结果，多项指标结果表明气管内给予LPS成功诱导小鼠建立ALI模型，为体内试验提供了模拟活体病理生理改变理想的平台。 3．在ALI小鼠体内，紫草素可减轻病理学改变，减轻肺水肿，减少炎症因子TNF-α、IL-1β的释放和中性粒细胞的浸润，降低肺组织匀浆中的MPO活性和NO浓度，使肺组织中iNOS、COX-2和核蛋白中NF-κB水平下降，表明紫草素对LPS诱导的ALI的保护作用，而且这种保护作用的潜在机制可能与紫草素抑制NF-κB、iNOS和COX-2有关。本研究从体外到体内两个层面研究了紫草素的抗炎作用，表明紫草素对ALI具有一定的保护作用，并初步探讨了其潜在机制，为紫草素的临床抗炎治疗的应用奠定了一定基础。
[Abstract]:Purpose and background
Acute lung injury (ALI) is a common severe disease with high mortality and lack of special therapeutic methods. The Chinese herbal medicine herb has been used for the treatment of inflammatory diseases for thousands of years, and the extract of the herb has shown its anti inflammatory effect in a number of experiments in and out of the body. The purpose of this study is to study the role of shikonin in ALI. And the mechanism provides new ideas for the drug treatment of ALI.
Method
1. the peripheral blood of healthy people was collected, and the peripheral blood mononuclear cells (PBMC) were isolated and cultured, and 3 mu M was added to the medium for preconditioning. 1H was added to 100ng/ml to induce PBMC to induce the inflammatory reaction. The cell supernatant was collected after 24h, and the concentration of TNF- alpha, IL-1 beta and IL-8 was determined by the enzyme linked immunosorbent assay (ELISA). The results were statistically analyzed to verify whether shikonin inhibited the production of PBMC inflammatory factors stimulated by LPS.
2. the ALI model was constructed by dropping 5mg/kg LPS into the trachea of BALB/C mice. The bronchoalveolar lavage fluid (BALF) was collected in 3h, 6h, 12h, 24h, and TNF- and IL-1 beta concentration was detected by ELISA. The lung tissue was observed by histopathology, and the ratio of dry weight of lung wet was measured and myeloperoxidase in tissue homogenate was detected. PO) activity and nitric oxide (NO) concentration. Statistical analysis was performed to verify the success of the ALI mouse model.
3. the BALB/C mice were divided into blank control group, 50mg/kg group (50mg/kg), LPS group and LPS+ purple herb (12.5mg/kg, 25mg/kg, 50mg/kg). The mice were injected into the trachea before LPS, and the mice were given the corresponding dose of lachycin or its solvent.LPS for 6h after the dosing of the BALF and lung tissue. The concentration of IL-1 beta and the concentration of protein in BALF were measured by BCA; the lung tissue was examined by pathological tissue section, the ratio of lung wet dry weight was measured, MPO activity and NO concentration in tissue homogenate were detected by colorimetry. Western Blot method was used to detect the expression of inducible nitric oxide synthase (iNOS) and -2 (COX-2) in lung tissue, and the electrophoretic migration test (EMSA) was used to detect lung. Tissue nuclear protein nuclear factor kappa B (NF- B) level was analyzed. The anti-inflammatory effect and mechanism of shikonin were studied in ALI mice.
Result
1. the anti-inflammatory effect of LPS in vitro: TNF- alpha, IL-1 beta and IL-8 were increased significantly after PBMC stimulation, respectively, 199.3 + 21pg/ml, 100.3 + 11.24pg/ml and 101 + 11.6pg/ml, which were significantly higher than those in the pure purple grass treatment group and the blank control group. The difference between the groups was statistically significant (P0.05). The production of TNF- alpha, IL-1 beta and IL-8 was significantly inhibited, the concentration was 126.3 + 10.0pg/ml, 55.7 + 9.1pg/ml and 60.7 + 12.1pg/ml, respectively, and had statistical significance with the LPS group (P0.05).
2. the mouse ALI model was successfully constructed: the concentrations of TNF- alpha and IL-1 beta in BALF (3038 + 344.1pg/ml, 1841 + 320.0pg/ml) in BALF were significantly higher than those of the control group (109.7 + 46.06pg/ml, 104.7 + 33.25pg/ml), reaching the highest value. The concentration of TNF- alpha and IL-1 beta in the other time points was significantly higher than that in the control group. There was a statistical difference between the groups. Significance (P0.05). The activity of MPO (1.49 + 0.39U/g) in the lung tissue was higher than that in the blank control group (0.39 + 0.09U/g), and then gradually increased to the highest value of 24h (5.09 + 1.04U/g), and all time points were significantly higher than that in the blank control group. The difference between the groups was statistically significant (P0.05). The concentration of NO in the lung tissue was (697.9 + 6) (697.9 + 6). 3.18 mol/g) was significantly higher than that in the blank control group (210.6 + 19.16 mu mol/g), and the concentration of NO in each time point was significantly higher than that in the blank control group. The difference was statistically significant (P0.05). After the intratracheal drop into LPS3h, the ratio of wet dry weight to the lung tissue was (5.03 + 0.46), which was higher than the control group (4.02 + 0.27), to the peak of 24h, and the difference was statistically significant (P0.05).LPS administration 2. After 4h, the pathological features of lung tissue were massive infiltration of inflammatory cells into pulmonary interstitium and alveolar cavity, alveolar hemorrhage, alveolar collapse and extensive alveolar wall thickening and edema.
3. the protection of ALI in mice: the concentration of TNF- alpha and IL-1 beta in the BALF and the protein content in BALF found that the concentration of 3 doses of the +LPS group was significantly lower than that in the LPS group. The difference was statistically significant (P0.05). The determination of MPO activity and NO concentration in lung tissue by colorimetric method was also found to be significantly lower than that of LPS group. The statistical difference was significant (P0.05). The lung wet dry weight ratio in the pretreated group was also significantly lower than that in the LPS group (P0.05). In addition, the pulmonary histopathological changes in the pretreated group were also less than those in the LPS group, especially the infiltration of inflammatory cells in the lung tissue, which significantly reduced.Western Blot to show 3 doses of LPS +L. The expression of COX-2 and iNOS in group PS was significantly lower than that in group LPS, and there was a significant difference (P0.05).EMSA showed that the level of NF- kappa B within the lung tissue of mice was significantly lower than that in the group LPS (P0.05).
conclusion
1. LPS can significantly reduce the production of PBMC - induced TNF- - alpha, IL-1 - beta and cell chemokines IL-8, suggesting that it has an anti - inflammatory effect in vitro.
2. by detecting the concentration of TNF- alpha and IL-1 beta in the ALI mouse model BALF, the activity of MPO and the concentration of NO in the homogenate of lung tissue, the wet dry weight ratio of the lung tissue and the pathological results were observed. The results showed that the intratracheal LPS was successfully induced to induce the mice to establish the ALI model, which provided an ideal platform to simulate the pathophysiological changes of the living body in vivo.
3. in ALI mice, shikonin alleviates pathological changes, reduces pulmonary edema, reduces the release of inflammatory factors TNF- a, IL-1 beta release and neutrophil infiltration, reduces MPO activity and NO concentration in lung tissue homogenate, and reduces the level of NF- kappa B in iNOS, COX-2 and nucleoprotein in lung tissue, indicating the protective effect of ALI on LPS induced by LPS. Moreover, the potential mechanism of this protective effect may be related to inhibition of NF- kappa B, iNOS and COX-2 by shikonin.
In this study, the anti-inflammatory effect of purple grass was studied from two levels in vitro and in vivo. It showed that the protective effect of purple grass on ALI and its potential mechanism were preliminarily discussed, which laid a foundation for the application of the clinical anti-inflammatory treatment of purple herb.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R563.8

【参考文献】