本文選題：紫草素 + 脂多糖�。� 參考：《第四軍醫(yī)大學(xué)》2013年博士論文

【摘要】：目的與背景 急性肺損傷（ALI）是常見的重癥疾病，其病死率高，臨床尚缺乏特效的治療方法。中藥紫草用于治療感染炎癥性的疾病已有上千年記載，而且其提取物紫草素在多項體內(nèi)外實驗中已初顯其抗炎癥作用。本課題旨在研究紫草素在ALI中的作用與機制，為ALI的藥物治療提供新的思路。 方法 1．采集健康人外周血，分離培養(yǎng)其中外周血單個核細胞（PBMC），向培養(yǎng)基中加入3μM的紫草素進行預(yù)處理，1h后加入100ng/ml的脂多糖（LPS）誘導(dǎo)PBMC產(chǎn)生炎癥反應(yīng)，24h后收集細胞上清液，通過酶聯(lián)免疫吸附試驗（ELISA）測定其中TNF-α、IL-1β及IL-8的濃度，對結(jié)果進行統(tǒng)計學(xué)分析，驗證紫草素是否抑制LPS刺激的PBMC炎性因子的產(chǎn)生。 2．通過向BALB/C小鼠氣管內(nèi)滴入5mg/kg的LPS構(gòu)建ALI模型，分別于3h、6h、12h、24h采集支氣管肺泡灌洗液（BALF），采用ELISA法檢測其中TNF-α及IL-1β的濃度；取肺組織，行組織病理學(xué)切片觀察，測定肺濕干重比，比色法檢測組織勻漿液中髓過氧化物酶（MPO）活性及一氧化氮（NO）濃度。對上述數(shù)據(jù)進行統(tǒng)計學(xué)分析，驗證ALI小鼠模型構(gòu)建成功與否。 3．將BALB/C小鼠分成空白對照組、紫草素組(50mg/kg）、LPS組和LPS+紫草素3個劑量組（12.5mg/kg、25mg/kg、50mg/kg），小鼠氣管內(nèi)滴入LPS之前1h，預(yù)先通過灌胃法給予小鼠相應(yīng)劑量的紫草素或其溶劑。LPS給藥6h后采集BALF及肺組織，用ELISA法檢測BALF中TNF-α和IL-1β的濃度，BCA法測定BALF中蛋白濃度；肺組織行病理組織切片檢查，測定肺濕干重比，比色法檢測組織勻漿液中MPO活性及NO濃度，Western Blot法檢測肺組織中誘生型一氧化氮合成酶（iNOS）和環(huán)氧化酶-2（COX-2）的表達，，電泳遷移實驗（EMSA）檢測肺組織核蛋白內(nèi)核轉(zhuǎn)錄因子-κB（NF-κB）水平。對上述結(jié)果進行分析，研究紫草素在ALI小鼠體內(nèi)的抗炎作用與機制。 結(jié)果 1．體外紫草素的抗炎作用：LPS刺激PBMC后產(chǎn)生的TNF-α、IL-1β和IL-8均顯著增加，分別為199.3±21pg/ml、100.3±11.24pg/ml和101.0±11.6pg/ml，均較單純紫草素處理組和空白對照組顯著增高，組間差異有統(tǒng)計學(xué)意義（P0.05）。在LPS之前給予紫草素預(yù)處理能夠明顯抑制TNF-α、IL-1β和IL-8的產(chǎn)生，其濃度分別為126.3±10.0pg/ml、55.7±9.1pg/ml和60.7±12.1pg/ml，與LPS組差異具有統(tǒng)計學(xué)意義（P0.05）。 2．成功構(gòu)建小鼠ALI模型：經(jīng)小鼠氣管滴入LPS后3h，BALF中TNF-α和IL-1β濃度（3038±344.1pg/ml，1841.0±320.0pg/ml）均較對照組（109.7±46.06pg/ml，104.7±33.25pg/ml）明顯升高，達到最高值，其他各時間點TNF-α及IL-1β濃度較對照組明顯升高，組間差異有統(tǒng)計學(xué)意義（P0.05）。氣管滴入LPS3h后肺組織中MPO活性（1.49±0.39U/g）較空白對照組（0.39±0.09U/g）升高，后逐漸增高至24h達到最高值（5.09±1.04U/g），各時間點均較空白對照組明顯升高，組間差異有統(tǒng)計學(xué)意義（P0.05）。氣管內(nèi)滴入LPS3h后，肺組織中NO濃度（697.9±63.18μmol/g）較空白對照組（210.6±19.16μmol/g）顯著升高，各時間點NO濃度較空白對照組明顯升高，差異有統(tǒng)計學(xué)意義（P0.05）。氣管內(nèi)滴入LPS3h后，肺組織濕干重比（5.03±0.46）較對照組（4.02±0.27）即升高，到24h達到峰值，差異有統(tǒng)計學(xué)意義（P0.05）。LPS給藥24h后肺組織病理學(xué)表現(xiàn)為大量炎細胞向肺間質(zhì)及肺泡腔內(nèi)浸潤，肺泡內(nèi)出血，肺泡塌陷及廣泛的肺泡壁增厚、水腫。 3．紫草素對小鼠ALI的保護作用：檢測BALF中TNF-α和IL-1β的濃度以及BALF中的蛋白含量發(fā)現(xiàn)，3個劑量紫草素+LPS組的濃度均較LPS組顯著降低，差異有統(tǒng)計學(xué)意義（P0.05）。比色法測定肺組織中MPO活性和NO濃度發(fā)現(xiàn)，紫草素+LPS組也明顯低于LPS組，統(tǒng)計學(xué)差異顯著（P0.05）。紫草素預(yù)處理組的肺濕干重比也較LPS組明顯降低，差異有統(tǒng)計學(xué)意義（P0.05）。此外，紫草素預(yù)處理組的肺組織病理學(xué)改變也較LPS組有所減輕，尤其體現(xiàn)在肺組織炎性細胞的浸潤明顯減少。Western Blot示3個劑量紫草素+LPS組的COX-2和iNOS表達量均較LPS組明顯下降，存在顯著性差異（P0.05）。EMSA則顯示小鼠肺組織核蛋白內(nèi)NF-κB水平在紫草素預(yù)處理組較LPS組明顯降低，存在顯著性差異（P0.05）。 結(jié)論 1．紫草素可顯著降低LPS誘導(dǎo)的PBMC促炎癥因子TNF-α、IL-1β和細胞趨化因子IL-8的產(chǎn)生，提示紫草素在體外具有抗炎癥作用。 2．通過檢測ALI小鼠模型BALF中TNF-α及IL-1β的濃度、肺組織勻漿液中MPO活性及NO濃度，觀察肺組織濕干重比和病理學(xué)結(jié)果，多項指標結(jié)果表明氣管內(nèi)給予LPS成功誘導(dǎo)小鼠建立ALI模型，為體內(nèi)試驗提供了模擬活體病理生理改變理想的平臺。 3．在ALI小鼠體內(nèi)，紫草素可減輕病理學(xué)改變，減輕肺水腫，減少炎癥因子TNF-α、IL-1β的釋放和中性粒細胞的浸潤，降低肺組織勻漿中的MPO活性和NO濃度，使肺組織中iNOS、COX-2和核蛋白中NF-κB水平下降，表明紫草素對LPS誘導(dǎo)的ALI的保護作用，而且這種保護作用的潛在機制可能與紫草素抑制NF-κB、iNOS和COX-2有關(guān)。 本研究從體外到體內(nèi)兩個層面研究了紫草素的抗炎作用，表明紫草素對ALI具有一定的保護作用，并初步探討了其潛在機制，為紫草素的臨床抗炎治療的應(yīng)用奠定了一定基礎(chǔ)。
[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.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R563.8

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