五味子乙素对大鼠创伤性急性肺损伤的保护作用及机制研究

发布时间：2018-07-08 19:23

  本文选题：创伤性急性肺损伤 + 五味子乙素　； 参考：《陕西中医药大学》2016年硕士论文

【摘要】：目的采用重物高处自由坠落的方法复制大鼠创伤性急性肺损伤(ALI)模型,观察肺损伤过程中氧化应激反应通路的变化,及五味子乙素对大鼠外伤性急性肺损伤干预的作用,并通过脂质过氧化物丙二醛(MDA)含量的改变、抗氧化活性酶超氧化物歧化酶(SOD)活性的改变及iNOS蛋白表达的变化,来初步解释作用的机制。为中药五味子在治疗创伤性急性肺损伤的临床应用上提供理论支持。方法选取健康SD大鼠50只,使用数字随机法,随机分为对照组(Control group)(n=10)、模型组(Model group, n=10)、五味子乙素低剂量组(SchB-LD group, n=10)、五味子乙素高剂量组(SchB-HD group, n=10)和地塞米松阳性对照组(Dex-Group, n=10),共5组。模型组和各实验组大鼠水合氯醛腹腔麻醉后,使用自制钝性胸外伤实验台,以500g的钢制撞击锤从40.8cm高度落下,达到2.0J的撞击能量,建立创伤性肺损伤模型。SchB-LD组、SchB-HD组造模结束后,立即腹腔注射准备好低、高浓度的五味子乙素注射剂(五味子乙素溶于PBS),每2h一次,共3次。地塞米松对照组同时分次注射地塞米松注射液。对照组不做处理,正常饲养。各组鼠分笼饲养,自由饮食,观察生存状态。在0-48h中,从活动状况、呼吸状态、皮毛色泽、觅食觅水频率等方面观察记录各组大鼠的生存情况,每4h为一个时间节点统计大鼠死亡数量。使用Kaplan-Meier法计算绘制各组大鼠和对照与治疗(高、低五味子乙素)组的生存曲线,并进行Log Rank分析。所有大鼠观察48h后处死,解剖取材。固定后的肺组织常规脱水、石蜡包埋,2μm层厚切片,HE染色观察各组鼠肺的组织病理学改变；按试剂盒方法检测肺组织MDA浓度、超氧化物歧化酶SOD活性；免疫组织化学染色检测诱导性一氧化氮合酶iNOS表达。结果与对照组比较,模型组大鼠肺的水含量(湿重／干重比)显著升高；肺组织的ALI病理评分明显增高；模型组大鼠肺组织MDA水平明显增高,SOD活性下降,iNOS蛋白的表达率显著上升。提示氧自由基显著增多,抗氧化能力减弱,各项指标均出现有统计学意义的显著改变,表明本研究制备的外伤致ALI模型成功可靠。治疗组(高、低剂量五味子乙素组)大鼠与模型组大鼠比较,48h生存曲线具有显著差异,生存率明显提高。(x2= 7.16,p＜0.01, HR (Mantel-Haenszel)=5.33)。与模型组比较,高剂量五味子乙素组的生存曲线具有显著差异：(x2=6.03,p＜0.01, HR (Mantel-Haenszel)=5.03);高、低剂量五味子乙素组和地塞米松对照组间生存率无明显差异(x2=0.79,p=0.67)。对各组数据进行比较,模型组肺组织含水量较对照组显著增多(p0.01),高、低剂量五味子乙素处理组较模型组肺的含水量显著降低(p0.01)；高、低剂量的五味子乙素处理组之间比较：高剂量组的肺含水量有所降低,但无统计学意义(p=0.07)。高、低剂量五味子乙素处理组肺组织的ALI病理评分显著低于模型组(p0.01),具体表现为炎性细胞浸润明显减轻,透明膜明显减少,高剂量五味子乙素组与低剂量五味子乙素组肺组织的ALI病理评分之间差异无统计学意义(p=0.6)。阳性对照组的病理评分与五味子乙素组之间比较无明显差异。模型组和高、低量的五味子乙素组的组织匀浆中MDA的水平均显著高于对照组,差异有统计学意义(p0.01)。高、低剂量的五味子乙素组组织匀浆中MDA水平显著低于模型组,差异具有统计学意义p0.05)。与低剂量五味子乙素组相比,高剂量五味子乙素组MDA水平降低幅度更大,具有统计学意义p0.05)。模型组匀浆中SOD活性显著低于对照组(p0.01)；高、低剂量的五味子乙素组的组织匀浆中SOD活性均显著低于模型组,差异有统计学意义。经One-Way ANOVA分析,在高、低剂量五味子乙素组和地塞米松组之间,组织匀浆中SOD活性无统计学意义的差异(F=2.549,p=0.09)。iNOS蛋白表达评分：模型组的评分显著高于对照组；高、低剂量的五味子乙素治疗组评分显著低于模型组,差异有统计学意义(p0.01)。经One-Way ANOVA分析,高、低剂量五味子乙素组和地塞米松组间的iNOS蛋白表达无明显差异(F=2.35,p=0.11)。与模型组比较各项指标均的到显著改善：肺水含量(湿重/干重比)降低,提示肺泡上皮细胞通透性降低；肺组织的ALI病理评分显著降低,MDA水平显著降低,SOD活性提高,iNOS蛋白的表达率显著降低,提示五味子乙素干预能抑制肺部氧化应激反应,减轻炎性损伤。结论采用重物自由落体外伤实验台可制造稳定的大鼠创伤性肺损伤模型,氧化应激反应通路参与肺损伤过程,是治疗靶点之一；五味子乙素能有效缓解创伤性肺损伤的病理学改变,减轻肺水肿,减少炎性细胞的浸润,起到创伤后肺保护的作用,增加存活率；五味子乙素对于创伤性肺损伤的这种保护作用的潜在机制可能与五味子乙素减少氧自由基含量,减轻氧化应激反应程度有关。
[Abstract]:Objective to reproduce the rat model of traumatic acute lung injury (ALI) with heavy weight and free fall, and to observe the changes of oxidative stress pathway in the process of lung injury, and the effect of schisandrin on acute lung injury in rats, and the changes of lipid peroxides (MDA) and antioxidant enzyme superoxide. The changes in the activity of SOD and the change of iNOS protein expression were used to provide a preliminary explanation for the clinical application of Schisandra chinensis in the treatment of traumatic acute lung injury. Methods 50 healthy SD rats were selected and randomly divided into the control group (Control group) (n=10), and the model group (Model GRO). Up, n=10), the low dose group of schisandin (SchB-LD group, n=10), the high dose group of schisandin (SchB-HD group, n=10) and the positive control group of dexamethasone (Dex-Group, n=10), 5 groups. The model group and the rats in the experimental group were anesthetized with chloral hydrate in the abdominal cavity, and made the homemade blunt chest trauma test bench and the 500g steel impact hammer from the 40.8cm height. Falling, reaching the impact energy of 2.0J, establishing the model.SchB-LD group of traumatic lung injury. After the end of the model of group SchB-HD, the low and high concentration of schisandrin injection (Fructus schisandrin dissolved in PBS) was immediately prepared, 3 times per 2H. The dexamethasone injection was injected into the control group at the same time. The control group was not treated with the control group. In 0-48h, the survival status of rats in each group was recorded from activity status, breathing state, fur color, foraging and foraging frequency, and the number of rats died per 4H for a time node. Kaplan-Meier method was used to calculate and draw the rats and control and treatment of each group. The survival curve of the treatment group (high, low schisandin) group and Log Rank analysis. All rats were killed and dissected after observation of 48h. After the fixation, the lung tissue was dehydrated, paraffin embedded, 2 m thick slices, and HE staining was used to observe the histopathological changes of lung tissue in each group; the MDA concentration of lung tissue and superoxide dismutase SOD were detected by the test kit method. The expression of inducible nitric oxide synthase iNOS was detected by immunohistochemical staining. Compared with the control group, the water content (wet weight / dry weight ratio) of lung in the model group increased significantly, and the pathological score of ALI in lung tissue was significantly higher; the MDA level of lung tissue in the model group was significantly higher, the activity of SOD decreased and the expression rate of iNOS protein was significant. The increase of oxygen free radicals showed a significant increase in oxygen free radicals, the weakening of antioxidant capacity, and significant changes in all indexes, which showed that the ALI model produced by this study was successful and reliable. The 48h survival curve of the treatment group (high and low dose schisandrin group) was significantly different from that of the model group, and the survival rate was obviously improved. (x2= 7.16, P < 0.01, HR (Mantel-Haenszel) =5.33). Compared with the model group, the survival curves of the high dose schisandrin group were significantly different: (x2=6.03, P < 0.01, HR (Mantel-Haenszel) =5.03); high, low dose of schisandrin and dexamethasone had no significant difference between groups (x2=0.79, p=0.67). Compared with the control group, the water content of the lung tissue in the model group was significantly increased (P0.01), and the low dose of schisandrin treated group was significantly lower than that of the model group (P0.01). The high, low dose of schisandrin treatment group was compared with the high dose group, but the lung water content of the high dose group decreased, but there was no statistical significance (p=0.07). The ALI pathological score of lung tissue in the treated group was significantly lower than that in the model group (P0.01). The specific expression was that the infiltration of inflammatory cells was significantly reduced and the transparent membrane was significantly reduced. There was no significant difference between the high dose schisandrin group and the low dose schisandrin group (p=0.6) in the ALI pathological score of lung tissue (p=0.6). The pathological score of the positive control group and the five flavour of the positive control group were not statistically significant. The level of MDA in the tissue homogenate of the model group and the high and low quantity of schisandin group was significantly higher than that of the control group, and the difference was statistically significant (P0.01). The level of MDA in the tissue homogenate of the low dose of schisandrin was significantly lower than that of the model group, the difference was statistically significant P0.05) and the low dose of five. The level of MDA in the high dose group of schisandin was significantly lower than that in the high dose group. The activity of SOD in the homogenate of the model group was significantly lower than that of the control group (P0.01), and the SOD activity in the tissue homogenate of the low dose of schisandrin was significantly lower than that in the model group, and the difference was statistically significant. The One-Way ANOVA analysis, In the high, low dose schisandin group and dexamethasone group, the SOD activity of the tissue homogenate was not statistically significant (F=2.549, p=0.09).INOS protein expression score: the score of the model group was significantly higher than that of the control group; the high, low dose of schisandrin treatment group was significantly lower than the model group, the difference was statistically significant (P0.01). After On, the difference was statistically significant (P0.01). E-Way ANOVA analysis showed that there was no significant difference in iNOS protein expression between high and low dose schisandrin group and dexamethasone group (F=2.35, p=0.11). Compared with the model group, the indexes of the lung water content (wet weight / dry weight ratio) decreased, the alveolar epithelial cell permeability decreased, and the pathological score of ALI in lung tissue decreased significantly, MDA water was significantly reduced. The level of SOD and the expression rate of iNOS protein decreased significantly, suggesting that the intervention of schisandrin can inhibit the oxidative stress of the lungs and reduce the inflammatory damage. Conclusion the free falling body trauma test bed can produce a stable model of traumatic lung injury in rats, and the oxygen stress reaction pathway participates in the process of lung injury, and it is the target of treatment. One point: schisandrin can effectively alleviate the pathological changes of the traumatic lung injury, reduce the pulmonary edema, reduce the infiltration of inflammatory cells, play a role in the protection of the lung after trauma, and increase the survival rate; the potential mechanism of schisandrin's protective effect on traumatic lung injury may be associated with the reduction of oxygen free radical content with schisandrin, It is related to reducing the degree of oxidative stress.
【学位授予单位】：陕西中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R641
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本文编号：2108503