本文選題：Nrf-2 + 膿毒癥��； 參考：《第二軍醫(yī)大學(xué)》2017年碩士論文

【摘要】：目的通過盲腸結(jié)扎穿孔法(CLP)建立大鼠膿毒癥急性肝損傷(acute liver injury,ALI)模型,以核因子E2相關(guān)因子2(Nrf-2)激動劑萊菔硫烷(SFN)、抑制劑全反式維甲酸(ATRA)干預(yù)CLP模型,驗證Nrf-2轉(zhuǎn)錄活性,明確Nrf-2信號通路在膿毒癥急性肝損傷時是否具有肝功能保護作用。方法SPF級SD大鼠共計128只,設(shè)置成假手術(shù)組、盲腸結(jié)扎穿孔組、萊菔硫烷組以及全反式維甲酸組。四組大鼠分別按時間點再次平均分成6h、12h、18h及生存分析組四個亞組。假手術(shù)組大鼠麻醉后,用1ml注射器根據(jù)體重按5ml/kg劑量給予腹腔注射溶劑玉米油后關(guān)閉大鼠腹腔。盲腸結(jié)扎穿孔組大鼠,在距離盲腸盲端1/4盲腸全長處用絲線結(jié)扎,然后用16G留置針在結(jié)扎段盲腸腸系膜對側(cè)總長度的1/2和遠端1/4點處各扎一孔,擠出部分腸內(nèi)容物,按5ml/kg劑量腹腔注射玉米油后關(guān)腹。萊菔硫烷組和全反式維甲酸組分別在CLP建模后腹腔注射SFN(5mg/kg)、ATRA(lmg/kg)后關(guān)腹。各時間點經(jīng)心臟采血,離心獲取血清行轉(zhuǎn)氨酶(ALT、AST)、總膽紅素水平檢測,處死大鼠后留取肝臟行組織形態(tài)學(xué)、炎癥因子、抗氧化酶水平測定及Western-Blot法檢測Nrf-2蛋白水平,RT-PCR法檢測目的基因Nrf-2以及抗氧化酶基因表達水平。結(jié)果(1)大鼠血清AST、ALT、總膽紅素值的變化:建模后6h,盲腸結(jié)扎穿孔組、萊菔硫烷組及全反式維甲酸組大鼠AST、ALT和總膽紅素值均較假手術(shù)組升高;萊菔硫烷組大鼠總膽紅素值較盲腸結(jié)扎穿孔組降低,而全反式維甲酸組大鼠總膽紅素值較盲腸結(jié)扎穿孔組升高;全反式維甲酸組大鼠轉(zhuǎn)氨酶以及總膽紅素水平均較萊菔硫烷組升高。建模后12h及18h,盲腸結(jié)扎穿孔組、萊菔硫烷組及全反式維甲酸組轉(zhuǎn)氨酶、總膽紅素值均較假手術(shù)組顯著升高;萊菔硫烷組數(shù)值較盲腸結(jié)扎穿孔組均有所降低,全反式維甲酸組數(shù)值較盲腸結(jié)扎穿孔組均有所升高;全反式維甲酸組數(shù)值較萊菔硫烷組則明顯升高。(2)肝組織外觀及病理HE染色:各時間點假手術(shù)組大鼠肝臟大體形態(tài)正常,鏡下觀其結(jié)構(gòu)輪廓清晰,未見明確病理改變。盲腸結(jié)扎穿孔組、萊菔硫烷組和全反式維甲酸組大鼠肝臟鏡下可見不同程度損傷,且隨時間延長呈加重趨勢,在同一時間點萊菔硫烷組病理改變范圍及嚴重程度較盲腸結(jié)扎穿孔組輕,全反式維甲酸組較盲腸結(jié)扎穿孔組重。(3)肝組織勻漿炎癥因子值的變化:建模后各時間點,盲腸結(jié)扎穿孔組、萊菔硫烷組、全反式維甲酸組大鼠肝臟組織勻漿炎癥因子數(shù)值均較假手術(shù)組升高;萊菔硫烷組數(shù)值較盲腸結(jié)扎穿孔組下降,而全反式維甲酸組數(shù)值較盲腸結(jié)扎穿孔組有所升高,全反式維甲酸組數(shù)值較萊菔硫烷組則明顯升高。(4)肝組織勻漿抗氧化酶水平檢測:SOD活力檢測結(jié)果:建模后6h時間點盲腸結(jié)扎穿孔組比假手術(shù)組SOD活力水平增高,18h時間點較其下降;各時間點萊菔硫烷組、全反式維甲酸組SOD值分別比假手術(shù)組以和盲腸結(jié)扎穿孔組升高、下降,全反式維甲酸組SOD值比萊菔硫烷組明顯降低。GSH-Px活力檢測結(jié)果:盲腸結(jié)扎穿孔組與假手術(shù)組相比,建模后6h及12h時間點GSH-Px活力水平增高,18h時間點較其下降;各時間點萊菔硫烷組、全反式維甲酸組GSH-Px值分別比假手術(shù)組以和盲腸結(jié)扎穿孔組升高、下降,全反式維甲酸組GSH-Px值比萊菔硫烷組明顯降低。(5)肝組織中Nrf-2蛋白表達情況:各時間點盲腸結(jié)扎穿孔組、萊菔硫烷組肝臟Nrf-2蛋白表達水平均較假手術(shù)組增高;萊菔硫烷組較盲腸結(jié)扎穿孔組也有所升高;全反式維甲酸組在18h時間點較假手術(shù)組有所降低,同時各時間點全反式維甲酸組Nrf-2蛋白表達水平均較盲腸結(jié)扎穿孔組有所下降,較萊菔硫烷組明顯降低。(6)肝組織中Nrf-2 m RNA、GSH-PX mRNA、SODmRNA表達水平:Nrf-2 mRNA:各時間點盲腸結(jié)扎穿孔組、萊菔硫烷組均較假手術(shù)組增高,同時萊菔硫烷組、全反式維甲酸組分別較盲腸結(jié)扎穿孔組升高、下降,全反式維甲酸組較萊菔硫烷組明顯降低。GSH-Px mRNA:盲腸結(jié)扎穿孔組在6h、12h較假手術(shù)組增高;萊菔硫烷組在各時間點較假手術(shù)組、盲腸結(jié)扎穿孔組增高;全反式維甲酸組表達水平在18h時間點較假手術(shù)組降低,在各時間點較盲腸結(jié)扎穿孔組降低,較萊菔硫烷組明顯降低。SOD mRNA:盲腸結(jié)扎穿孔組在6h、12h時間點均較假手術(shù)組增高,萊菔硫烷組在各時間點較假手術(shù)組與盲腸結(jié)扎穿孔組增高;全反式維甲酸組在12h、18h時時間點較假手術(shù)組降低,在各時間點較盲腸結(jié)扎穿孔組都有所下降,較萊菔硫烷組也有所降低。結(jié)論Nrf-2信號通路激活后能通過提高抗氧化酶如GSH-Px、SOD的表達,從而發(fā)揮抗炎、抗氧化效應(yīng),改善肝功能,對膿毒癥急性肝損傷起保護作用。
[Abstract]:Objective to establish a rat acute liver injury (acute liver injury, ALI) model of sepsis by cecal ligation and perforation (CLP), using the nuclear factor E2 related factor 2 (Nrf-2) agonist sulforaphane (SFN) and all trans retinoic acid (ATRA) as the inhibitor of CLP model to verify the Nrf-2 transcriptional activity and to clarify whether the Nrf-2 signal pathway is in the acute liver injury of sepsis. Methods a total of 128 SPF grade SD rats were set up into sham operation group, cecum ligation and perforation group, sulforaphane group and all trans retinoic acid group. The four rats were divided into 6h, 12h, 18h and four subgroups of survival analysis group at the time point respectively. The rats in the artificial hand group were anesthetized with 1ml syringe according to weight 5ml/ according to weight. The rat abdominal cavity was closed after the kg dose of corn oil was injected into the abdominal cavity. The rats in the cecum ligation group were ligated with silk thread at the distance from the blind 1/4 cecum in the blind end of the cecum, and then used the 16G indwelling needle in the ligation section of the cecum mesentery at the total length of the 1/2 and the distal 1/4 point each hole, squeezing out part of the contents of the intestines, and intraperitoneally injected at the dose of 5ml/kg. After CLP modeling, the group of sulforaphane and all trans retinoic acid group were injected with SFN (5mg/kg) and ATRA (lmg/kg) after the modeling of the abdominal cavity. The serum transaminase (ALT, AST), the total bilirubin level was measured by the centrifugation at each time point, and the liver was left to take the liver histomorphology, inflammatory factors and antioxidant enzyme levels. The level of Nrf-2 protein was determined by Western-Blot method, and the expression level of Nrf-2 and antioxidant enzyme gene was detected by RT-PCR. Results (1) the changes of serum AST, ALT, total bilirubin value in rat serum: 6h, cecum ligation and perforation group after modeling, AST in sulforaphane group and all trans retinoic acid group, ALT and total bilirubin value were higher than those in sham operation group. The total bilirubin value in the sulforaphane group was lower than that in the cecal ligation and perforation group, while the total bilirubin value in the total trans retinoic acid group was higher than that in the cecal ligation group. The total trans retinoic acid group and the total bilirubin level were higher than the sulforaphane group. After modeling, the 12h and 18h, the cecal ligation and perforation group, the sulforaphane group and all trans form of the group of sulforaphane and the total trans retinoic acid group. The value of transaminase and total bilirubin in the retinoic acid group was significantly higher than that in the sham group; the value of sulforaphane group was lower than that of the cecal ligation group, and all trans retinoic acid group was higher than that of the cecal ligation group; the total trans retinoic acid group was significantly higher than the sulforaphane group. (2) the appearance of liver tissue and pathological HE staining: each time The liver of the rats in the pseudo operation group was generally normal, and the structure of the rat liver was clear and the pathological changes were clear. The cecum ligation and perforation group, the sulforaphane group and the total trans retinoic acid group showed different degrees of injury in the liver microscope, and the trend was prolonged with the time. The pathological changes of the sulforaphane group at the same time and the same time point, the pathological changes of the group and the pathological changes of the sulforaphane group were found at the same time. Compared with the cecal ligation and perforation group, the total trans retinoic acid group was heavier than the cecal ligation and perforation group. (3) the changes in the inflammatory factor value of the liver tissue homogenate: all time points, the cecal ligation and perforation group, the sulforaphane group, the total trans retinoic acid group were all higher than the sham operation group, and the number of sulforaphane groups in the total trans retinoic acid group was higher than that of the sham group. Compared with the cecal ligation and perforation group, the total trans retinoic acid group was higher than the cecal ligation and perforation group, and the total trans retinoic acid group was significantly higher than the sulforaphane group. (4) the detection of antioxidant enzyme levels in the liver tissue homogenate: SOD activity test results: the SOD activity level of the cecum ligation and perforation group at the time of modeling 6h was more than that of the sham operation group. The time point of 18h was higher than that of it, and the SOD value of sulforaphane group and all trans retinoic acid group was higher than that of the sham group and the cecum ligation and perforation group, and the SOD value of all trans retinoic acid group was significantly lower than that of the sulforaphane group. Compared with the sham group, the 6h and 12h time after the modeling was compared with the sham group. The activity level of point GSH-Px was higher and the time point of 18h decreased. The GSH-Px value of sulforaphane group and all trans retinoic acid group was higher than that of the sham group and the cecum ligation and perforation group, and the GSH-Px value of all trans retinoic acid group was significantly lower than that of the sulforaphane group. (5) the expression of Nrf-2 protein in the liver tissue: the cecum ligation at each time point In the perforation group, the expression level of Nrf-2 protein in the liver of sulforaphane group was higher than that of the sham group, and the group of sulforaphane group was also higher than that of the cecal ligation group, and the total trans retinoic acid group decreased at the time point of 18h compared with the sham group. At the same time, the expression level of Nrf-2 egg white in all trans retinoic acid group was lower than that of the cecal ligation group. Compared with the sulforaphane group, (6) the expression of Nrf-2 m RNA, GSH-PX mRNA, SODmRNA in the liver tissue: the cecum ligation and perforation group at every time point of Nrf-2 mRNA:, the sulforaphane group was higher than the sham group, and the sulforaphane group and all trans retinoic acid group were higher than the blind intestinal perforation group, and the total trans retinoic acid group was compared with sulforaphane. Group.GSH-Px mRNA: cecum ligation group was significantly lower in 6h and 12h than in sham operation group, and sulforaphane group was higher in each time point than in sham operation group and cecum ligation group. The expression level of all trans retinoic acid group was lower than that of sham group at 18h time point, lower than the blind intestinal ligation group at all time points, and significantly lower than the sulforaphane group. The time point of low.SOD mRNA: cecum ligation was higher than that of sham operation group at 6h, 12h time point was higher than that of sham operation group and cecum ligation group at all time points, and all trans retinoic acid group was lower than that of sham group at 12h and 18h at all time points, and compared with the blind intestinal perforation group at each time point, compared with the sulforaphane group, the group also had a lower sulforaphane group than the sulforaphane group. Conclusion the activation of Nrf-2 signal pathway can improve the expression of antioxidant enzymes such as GSH-Px and SOD, thus exerting anti-inflammatory, antioxidant effect, improving liver function and protecting the acute liver injury of sepsis.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R459.7

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