本文選題：創(chuàng)傷性急性肺損傷 + 五味子乙素��； 參考：《陜西中醫(yī)藥大學》2016年碩士論文

【摘要】：目的采用重物高處自由墜落的方法復制大鼠創(chuàng)傷性急性肺損傷(ALI)模型,觀察肺損傷過程中氧化應激反應通路的變化,及五味子乙素對大鼠外傷性急性肺損傷干預的作用,并通過脂質過氧化物丙二醛(MDA)含量的改變、抗氧化活性酶超氧化物歧化酶(SOD)活性的改變及iNOS蛋白表達的變化,來初步解釋作用的機制。為中藥五味子在治療創(chuàng)傷性急性肺損傷的臨床應用上提供理論支持。方法選取健康SD大鼠50只,使用數字隨機法,隨機分為對照組(Control group)(n=10)、模型組(Model group, n=10)、五味子乙素低劑量組(SchB-LD group, n=10)、五味子乙素高劑量組(SchB-HD group, n=10)和地塞米松陽性對照組(Dex-Group, n=10),共5組。模型組和各實驗組大鼠水合氯醛腹腔麻醉后,使用自制鈍性胸外傷實驗臺,以500g的鋼制撞擊錘從40.8cm高度落下,達到2.0J的撞擊能量,建立創(chuàng)傷性肺損傷模型。SchB-LD組、SchB-HD組造模結束后,立即腹腔注射準備好低、高濃度的五味子乙素注射劑(五味子乙素溶于PBS),每2h一次,共3次。地塞米松對照組同時分次注射地塞米松注射液。對照組不做處理,正常飼養(yǎng)。各組鼠分籠飼養(yǎng),自由飲食,觀察生存狀態(tài)。在0-48h中,從活動狀況、呼吸狀態(tài)、皮毛色澤、覓食覓水頻率等方面觀察記錄各組大鼠的生存情況,每4h為一個時間節(jié)點統(tǒng)計大鼠死亡數量。使用Kaplan-Meier法計算繪制各組大鼠和對照與治療(高、低五味子乙素)組的生存曲線,并進行Log Rank分析。所有大鼠觀察48h后處死,解剖取材。固定后的肺組織常規(guī)脫水、石蠟包埋,2μm層厚切片,HE染色觀察各組鼠肺的組織病理學改變；按試劑盒方法檢測肺組織MDA濃度、超氧化物歧化酶SOD活性；免疫組織化學染色檢測誘導性一氧化氮合酶iNOS表達。結果與對照組比較,模型組大鼠肺的水含量(濕重／干重比)顯著升高；肺組織的ALI病理評分明顯增高；模型組大鼠肺組織MDA水平明顯增高,SOD活性下降,iNOS蛋白的表達率顯著上升。提示氧自由基顯著增多,抗氧化能力減弱,各項指標均出現(xiàn)有統(tǒng)計學意義的顯著改變,表明本研究制備的外傷致ALI模型成功可靠。治療組(高、低劑量五味子乙素組)大鼠與模型組大鼠比較,48h生存曲線具有顯著差異,生存率明顯提高。(x2= 7.16,p＜0.01, HR (Mantel-Haenszel)=5.33)。與模型組比較,高劑量五味子乙素組的生存曲線具有顯著差異：(x2=6.03,p＜0.01, HR (Mantel-Haenszel)=5.03);高、低劑量五味子乙素組和地塞米松對照組間生存率無明顯差異(x2=0.79,p=0.67)。對各組數據進行比較,模型組肺組織含水量較對照組顯著增多(p0.01),高、低劑量五味子乙素處理組較模型組肺的含水量顯著降低(p0.01)；高、低劑量的五味子乙素處理組之間比較：高劑量組的肺含水量有所降低,但無統(tǒng)計學意義(p=0.07)。高、低劑量五味子乙素處理組肺組織的ALI病理評分顯著低于模型組(p0.01),具體表現(xiàn)為炎性細胞浸潤明顯減輕,透明膜明顯減少,高劑量五味子乙素組與低劑量五味子乙素組肺組織的ALI病理評分之間差異無統(tǒng)計學意義(p=0.6)。陽性對照組的病理評分與五味子乙素組之間比較無明顯差異。模型組和高、低量的五味子乙素組的組織勻漿中MDA的水平均顯著高于對照組,差異有統(tǒng)計學意義(p0.01)。高、低劑量的五味子乙素組組織勻漿中MDA水平顯著低于模型組,差異具有統(tǒng)計學意義p0.05)。與低劑量五味子乙素組相比,高劑量五味子乙素組MDA水平降低幅度更大,具有統(tǒng)計學意義p0.05)。模型組勻漿中SOD活性顯著低于對照組(p0.01)；高、低劑量的五味子乙素組的組織勻漿中SOD活性均顯著低于模型組,差異有統(tǒng)計學意義。經One-Way ANOVA分析,在高、低劑量五味子乙素組和地塞米松組之間,組織勻漿中SOD活性無統(tǒng)計學意義的差異(F=2.549,p=0.09)。iNOS蛋白表達評分：模型組的評分顯著高于對照組；高、低劑量的五味子乙素治療組評分顯著低于模型組,差異有統(tǒng)計學意義(p0.01)。經One-Way ANOVA分析,高、低劑量五味子乙素組和地塞米松組間的iNOS蛋白表達無明顯差異(F=2.35,p=0.11)。與模型組比較各項指標均的到顯著改善：肺水含量(濕重/干重比)降低,提示肺泡上皮細胞通透性降低；肺組織的ALI病理評分顯著降低,MDA水平顯著降低,SOD活性提高,iNOS蛋白的表達率顯著降低,提示五味子乙素干預能抑制肺部氧化應激反應,減輕炎性損傷。結論采用重物自由落體外傷實驗臺可制造穩(wěn)定的大鼠創(chuàng)傷性肺損傷模型,氧化應激反應通路參與肺損傷過程,是治療靶點之一；五味子乙素能有效緩解創(chuàng)傷性肺損傷的病理學改變,減輕肺水腫,減少炎性細胞的浸潤,起到創(chuàng)傷后肺保護的作用,增加存活率；五味子乙素對于創(chuàng)傷性肺損傷的這種保護作用的潛在機制可能與五味子乙素減少氧自由基含量,減輕氧化應激反應程度有關。
[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.
【學位授予單位】：陜西中醫(yī)藥大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R641
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本文編號：2108503