单肺通气诱发肺损伤时兔肺组织核因子NF-E2相关因子2表达的变化及姜黄素的干预效应

发布时间：2018-04-23 04:01

本文选题：姜黄素 + 呼吸　；参考：《河北医科大学》2014年硕士论文

【摘要】：目的：单肺通气（OLV）是指患者开胸后只利用非手术侧肺进行通气的方式。OLV时非通气侧肺萎陷，为术者提供良好视野方便操作的同时还具有防止两肺间交叉感染等优点。但单肺通气时间过长可以诱发急性肺损伤[1]。姜黄素是自姜黄根茎中提取的活性成分，研究表明姜黄素可减轻单肺通气所诱发的肺损伤[2]，但作用机制尚不清楚。核因子NF-E2相关因子2（Nrf2）是机体内调节氧化/还原反应和炎性反应的关键性转录因子，研究发现Nrf2在急性肺损伤中发挥着重要的作用[3-4]。本研究拟评价姜黄素预先给药对单肺通气兔肺组织Nrf2蛋白表达的影响，为明确姜黄素减轻单肺通气所诱发肺损伤机制提供参考。方法：健康成年雄性新西兰大白兔24只，体重2.5~3.0kg，由河北医科大学实验动物中心提供。采用随机数字表法，将其随机分为3组（n=8）：双肺通气组（TLV组）、右肺单肺通气组（OLV组）、姜黄素预先给药组（Cur组）。取兔称重后经右耳缘静脉注射3%戊巴比妥钠30mg/kg麻醉，麻醉成功后游离右股动脉行穿刺置管术用于监测平均动脉压（MAP）及术中采集动脉血样。2%盐酸利多卡因局部浸润麻醉下切开颈部皮肤，游离右颈内静脉行穿刺置管术用于术中补液及给药。选择ID3.0mm气管导管，行气管切开术后，TLV组气管导管置入气管行双肺通气，OLV组和Cur组气管导管置入右主支气管行单肺通气。通过观察右侧胸廓运动的变化、听诊双肺呼吸音及监测气道压力的变化来判断和调整导管的位置。气管插管成功后，TLV组双肺通气3h，OLV组和Cur组右肺单肺通气3h，3组动物均采用容量通气模式，FiO2100%，调整通气参数维持SpO2＞90%。所有动物均右侧卧位，稳定10min后右颈内静脉注射维库溴铵0.1mg/kg，待自主呼吸消失后连接呼吸机行机械通气。OLV组和Cur组均先单肺通气50min，再双肺通气10min（通气参数不变，将导管退至气管内），再重复上述通气过程。同时两组均在左侧胸壁5~6肋间处开一大小约1cm小口至胸腔，用于观察左肺组织的萎陷及复张情况。实验过程中间断静脉给予维库溴铵0.1mg/kg维持肌松，必要时静脉追加3%戊巴比妥钠1ml维持麻醉，以10ml·kg-1·h-1的速率自右颈内静脉持续泵注乳酸钠林格氏液，以补充实验过程中所造成的体液丢失，维持MAP稳定。于通气开始即刻（T0）、通气1、2、3h（T1-3）采集右股动脉动脉血样进行血气分析，测定PaO2并计算氧合指数（OI），计算公式：OI=PaO2/FiO2。通气结束后采用股动脉快速放血法处死动物，取双肺下叶约1cm×1cm×2cm大小肺组织并一分为二，一半用液氮冻存24h后转80oC冰箱保存待测，另一半用4oC4%多聚甲醛溶液固定待测。将剩余肺组织称湿重后置于烤箱中（80℃，72h）烘烤，恒重后称干重，计算双肺湿/干重比（W/D比）。取4%多聚甲醛溶液中固定肺组织，常规脱水、石蜡包埋、切片及HE染色，光学显微镜下观察病理学结果并行肺组织病理学损伤评分，免疫组织化学法检测兔肺组织Nrf2蛋白表达。80oC保存肺组织采用化学比色法检测丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性及Western blot法检测兔肺组织Nrf2蛋白表达。结果：1与TLV组比较，OLV组及Cur组T2,3时OI下降（P＜0.05）；与T0时比较，OLV组及Cur组T2,3时OI下降（P＜0.05）；与OLV组比较，Cur组T3时OI升高（P＜0.05）。 2光镜下TLV组双侧肺组织肺泡结构基本完整，肺泡腔内出血少，未见明显渗出，间质轻度增厚，炎性细胞浸润较少；OLV组右侧肺组织肺泡结构破坏，肺泡腔内有少量渗出物，出血较少，炎性细胞浸润较少，间质增厚；OLV组左侧肺组织大部分肺泡结构消失，肺泡腔内有明显渗出，出血多，，间质明显增厚，炎性细胞浸润较多； Cur组右、左侧肺组织较OLV组右、左侧肺组织损伤程度轻。 3TLV组内双侧肺组织W/D比、肺组织病理学损伤评分、MDA含量、SOD活性和Nrf2蛋白表达水平差异无统计学意义（P＞0.05）；与TLV组右、左肺比较，OLV组及Cur组右、左肺W/D比、肺组织病理学损伤评分、MDA含量、Nrf2蛋白表达水平升高，SOD活性降低（P＜0.05）；与OLV组右肺比较，OLV组左肺W/D比、肺组织病理学损伤评分、MDA含量、Nrf2蛋白表达水平升高，SOD活性降低（P＜0.05）。 4与OLV组右、左肺比较，Cur组右、左肺W/D比、肺组织病理学损伤评分、MDA含量下降，SOD活性、Nrf2蛋白表达水平升高（P＜0.05）；与Cur组右肺比较，Cur组左肺W/D比、肺组织病理学损伤评分、MDA含量、Nrf2蛋白表达水平升高，SOD活性降低（P＜0.05）。结论：1单肺通气3h可导致兔动脉血氧合指数下降，双侧肺组织病理性损伤且非通气侧肺组织较通气侧损伤严重。 2Nrf2/ARE信号通路在单肺通气诱发的肺损伤中发挥肺保护作用。 3姜黄素预先给药具有减轻单肺通气所诱发的肺损伤作用，其机制与Nrf2蛋白表达上调有关。
[Abstract]:Objective: single lung ventilation (OLV) refers to the non ventilatory lung collapse in.OLV only using non operative side lung ventilation after thoracotomy, providing good visual field for operation and preventing two interpulmonary cross infection. However, the long duration of single lung ventilation can induce acute lung injury [1]. curcumin from Jiang Huanggen The active components extracted from the stem have shown that curcumin can reduce the lung injury induced by single lung ventilation, but the mechanism of action is not clear. The nuclear factor NF-E2 related factor 2 (Nrf2) is a key transcription factor for regulating oxidation / reduction reaction and inflammatory response in the body. The study found that Nrf2 plays an important role in acute lung injury, [3-4] This study is to evaluate the effect of curcumin on the expression of Nrf2 protein in lung tissue of rabbits with single lung ventilation, and to provide a reference for reducing the mechanism of lung injury induced by curcumin in single lung ventilation.
Methods: 24 healthy adult male New Zealand white rabbits, with a weight of 2.5~3.0kg, were provided by the experimental animal center of Hebei Medical University. The random digital table was used to randomly divide them into 3 groups (n=8): double lung ventilation group (group TLV), right lung single lung ventilation group (group OLV), and curcumin pre administration group (group Cur).
After the rabbit was weighed, 3% pentobarbital sodium 30mg/kg was injected into the right ear vein, and the free right femoral artery was used to monitor the average arterial pressure (MAP) and the intraoperative blood sample.2% hydrochloric acid lidocaine under local infiltration anesthesia. The ID3.0mm tracheal catheter was selected and the tracheal tube was inserted into the trachea in the TLV group after tracheotomy. The OLV group and the Cur group were placed in the right main bronchus for a single lung ventilation. By observing the changes in the right chest movement, hearing the double lung breathing sound and monitoring the airway pressure, the trachea was judged and adjusted. After the intubation was successful, the TLV group had two lung ventilation 3h, OLV group and Cur group with single lung ventilation 3H. The 3 groups all adopted the volume ventilation mode, FiO2100%, adjusted the ventilation parameters to maintain SpO2 > 90%. all the right lateral position, and the right internal jugular vein was injected with vecuronium 0.1mg/kg after the stable 10min, and the mechanical ventilation was connected to the ventilator after the spontaneous breathing disappeared. Group Cur and group Cur were first ventilated with single lung 50min, and then two lungs were ventilated by 10min (the ventilation parameters remained unchanged, the catheter was retreated to the endotracheal), and then the ventilation process was repeated. At the same time, the two groups all opened a size of about 1cm to the thoracic cavity at the left chest wall 5~6 intercostal, and used to observe the collapse and retention of the left lung tissue. The intermedium vein was given to vecuronium in the middle of the experiment. Ammonium 0.1mg/kg maintains muscle relaxation and maintains an intravenous infusion of 3% pentobarbital sodium 1ml if necessary to maintain a continuous infusion of sodium lactate solution at the rate of 10ml / kg-1 H-1 from the right internal jugular vein to supplement the fluid loss and maintain the stability of MAP during the experimental process. At the beginning of the ventilation (T0) and ventilation 1,2,3h (T1-3), the right femoral artery blood samples are collected. Blood gas analysis, determination of PaO2 and calculation of oxygenation index (OI), calculation formula: after the end of OI=PaO2/FiO2. ventilation, the animals were killed by rapid blood release of femoral artery, and the two lungs were taken to about 1cm * 1cm * 2cm size lung tissue and divided into two, and half of them were stored in 80oC refrigerator with liquid nitrogen cryopreservation, and the other half was fixed with 4oC4% polycondensation Formaldehyde Solution. The remaining lung tissue was weighed and placed in an oven (80 72h). The dry weight was calculated after constant weight and the wet / dry weight ratio (W/D ratio) of the lung was calculated.
The lung tissue was fixed in 4% polycondensation Formaldehyde Solution, routine dehydration, paraffin embedding, slice and HE staining. The pathological results were observed under optical microscope and the pathological damage score of lung tissue was observed. The expression of Nrf2 protein in lung tissue of rabbit lung tissue was detected by immunohistochemistry. The content of malondialdehyde (MDA) was detected by chemical colorimetry, and the superoxide was detected by chemical colorimetry. Dismutase (SOD) activity and Western blot method were used to detect the expression of Nrf2 protein in rabbit lung tissue.
Results: 1 compared with group TLV, OI in group OLV and Cur decreased (P < 0.05). Compared with T0, T2,3 OI decreased in OLV group and Cur group (P < 0.05).
In the 2 light microscope, the alveolar structure of bilateral lung tissues in group TLV was basically complete. There was little bleeding in the alveolus, no obvious exudation, mild thickening of interstitial tissue and less inflammatory cell infiltration. The alveolar structure in the right lung tissue of OLV group was destroyed, a small amount of exudation in the alveolus, less bleeding, less inflammatory cells infiltration, thickening of interstitial tissue, and most of the left lung tissues in group OLV. The alveolar structure disappeared, there were obvious exudation in the alveolus, more bleeding, more interstitial thickening and more inflammatory cell infiltration. Group Cur was right, left lung tissue was better than group OLV, and left lung tissue was less damaged.
There was no significant difference in the W/D ratio of bilateral lung tissues in the 3TLV group, the pathological damage score of the lung tissue, the MDA content, the SOD activity and the expression of Nrf2 protein (P > 0.05). Compared with the TLV group, the right of the TLV group, the left lung, the left lung W/D ratio, the pathological injury score of the lung tissue, the MDA content, the increase of the Nrf2 protein expression level and the decrease activity (0.05). Compared with the right lung in group OLV, the left lung W/D ratio, lung tissue pathological score, MDA content, Nrf2 protein expression level and SOD activity decreased in group OLV (P < 0.05).
4 in group OLV, right, left lung, Cur group right, left lung W/D ratio, lung tissue pathological damage score, MDA content decreased, SOD activity, Nrf2 protein expression level increased (P < 0.05); compared with Cur group right lung, Cur group left lung W/D ratio, lung tissue pathological injury score, MDA content, Nrf2 protein expression level increased and decreased activity (0.05).
Conclusion: 1 single lung ventilation 3H can lead to a decrease in arterial oxygenation index, bilateral lung tissue pathological damage and serious lung ventilation in non ventilated side.
2Nrf2/ARE signaling pathway plays a protective role in lung injury induced by one lung ventilation.
3 curcumin pretreatment can alleviate lung injury induced by one lung ventilation, and its mechanism is related to the up-regulated expression of Nrf2 protein.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R285.5

【参考文献】