Nrf-2信号通路在大鼠脓毒症急性肝损伤中的作用研究

发布时间：2018-06-03 18:11

本文选题：Nrf-2 + 脓毒症　；参考：《第二军医大学》2017年硕士论文

【摘要】：目的通过盲肠结扎穿孔法(CLP)建立大鼠脓毒症急性肝损伤(acute liver injury,ALI)模型,以核因子E2相关因子2(Nrf-2)激动剂莱菔硫烷(SFN)、抑制剂全反式维甲酸(ATRA)干预CLP模型,验证Nrf-2转录活性,明确Nrf-2信号通路在脓毒症急性肝损伤时是否具有肝功能保护作用。方法SPF级SD大鼠共计128只,设置成假手术组、盲肠结扎穿孔组、莱菔硫烷组以及全反式维甲酸组。四组大鼠分别按时间点再次平均分成6h、12h、18h及生存分析组四个亚组。假手术组大鼠麻醉后,用1ml注射器根据体重按5ml/kg剂量给予腹腔注射溶剂玉米油后关闭大鼠腹腔。盲肠结扎穿孔组大鼠,在距离盲肠盲端1/4盲肠全长处用丝线结扎,然后用16G留置针在结扎段盲肠肠系膜对侧总长度的1/2和远端1/4点处各扎一孔,挤出部分肠内容物,按5ml/kg剂量腹腔注射玉米油后关腹。莱菔硫烷组和全反式维甲酸组分别在CLP建模后腹腔注射SFN(5mg/kg)、ATRA(lmg/kg)后关腹。各时间点经心脏采血,离心获取血清行转氨酶(ALT、AST)、总胆红素水平检测,处死大鼠后留取肝脏行组织形态学、炎症因子、抗氧化酶水平测定及Western-Blot法检测Nrf-2蛋白水平,RT-PCR法检测目的基因Nrf-2以及抗氧化酶基因表达水平。结果(1)大鼠血清AST、ALT、总胆红素值的变化:建模后6h,盲肠结扎穿孔组、莱菔硫烷组及全反式维甲酸组大鼠AST、ALT和总胆红素值均较假手术组升高;莱菔硫烷组大鼠总胆红素值较盲肠结扎穿孔组降低,而全反式维甲酸组大鼠总胆红素值较盲肠结扎穿孔组升高;全反式维甲酸组大鼠转氨酶以及总胆红素水平均较莱菔硫烷组升高。建模后12h及18h,盲肠结扎穿孔组、莱菔硫烷组及全反式维甲酸组转氨酶、总胆红素值均较假手术组显著升高;莱菔硫烷组数值较盲肠结扎穿孔组均有所降低,全反式维甲酸组数值较盲肠结扎穿孔组均有所升高;全反式维甲酸组数值较莱菔硫烷组则明显升高。(2)肝组织外观及病理HE染色:各时间点假手术组大鼠肝脏大体形态正常,镜下观其结构轮廓清晰,未见明确病理改变。盲肠结扎穿孔组、莱菔硫烷组和全反式维甲酸组大鼠肝脏镜下可见不同程度损伤,且随时间延长呈加重趋势,在同一时间点莱菔硫烷组病理改变范围及严重程度较盲肠结扎穿孔组轻,全反式维甲酸组较盲肠结扎穿孔组重。(3)肝组织匀浆炎症因子值的变化:建模后各时间点,盲肠结扎穿孔组、莱菔硫烷组、全反式维甲酸组大鼠肝脏组织匀浆炎症因子数值均较假手术组升高;莱菔硫烷组数值较盲肠结扎穿孔组下降,而全反式维甲酸组数值较盲肠结扎穿孔组有所升高,全反式维甲酸组数值较莱菔硫烷组则明显升高。(4)肝组织匀浆抗氧化酶水平检测:SOD活力检测结果:建模后6h时间点盲肠结扎穿孔组比假手术组SOD活力水平增高,18h时间点较其下降;各时间点莱菔硫烷组、全反式维甲酸组SOD值分别比假手术组以和盲肠结扎穿孔组升高、下降,全反式维甲酸组SOD值比莱菔硫烷组明显降低。GSH-Px活力检测结果:盲肠结扎穿孔组与假手术组相比,建模后6h及12h时间点GSH-Px活力水平增高,18h时间点较其下降;各时间点莱菔硫烷组、全反式维甲酸组GSH-Px值分别比假手术组以和盲肠结扎穿孔组升高、下降,全反式维甲酸组GSH-Px值比莱菔硫烷组明显降低。(5)肝组织中Nrf-2蛋白表达情况:各时间点盲肠结扎穿孔组、莱菔硫烷组肝脏Nrf-2蛋白表达水平均较假手术组增高;莱菔硫烷组较盲肠结扎穿孔组也有所升高;全反式维甲酸组在18h时间点较假手术组有所降低,同时各时间点全反式维甲酸组Nrf-2蛋白表达水平均较盲肠结扎穿孔组有所下降,较莱菔硫烷组明显降低。(6)肝组织中Nrf-2 m RNA、GSH-PX mRNA、SODmRNA表达水平:Nrf-2 mRNA:各时间点盲肠结扎穿孔组、莱菔硫烷组均较假手术组增高,同时莱菔硫烷组、全反式维甲酸组分别较盲肠结扎穿孔组升高、下降,全反式维甲酸组较莱菔硫烷组明显降低。GSH-Px mRNA:盲肠结扎穿孔组在6h、12h较假手术组增高;莱菔硫烷组在各时间点较假手术组、盲肠结扎穿孔组增高;全反式维甲酸组表达水平在18h时间点较假手术组降低,在各时间点较盲肠结扎穿孔组降低,较莱菔硫烷组明显降低。SOD mRNA:盲肠结扎穿孔组在6h、12h时间点均较假手术组增高,莱菔硫烷组在各时间点较假手术组与盲肠结扎穿孔组增高;全反式维甲酸组在12h、18h时时间点较假手术组降低,在各时间点较盲肠结扎穿孔组都有所下降,较莱菔硫烷组也有所降低。结论Nrf-2信号通路激活后能通过提高抗氧化酶如GSH-Px、SOD的表达,从而发挥抗炎、抗氧化效应,改善肝功能,对脓毒症急性肝损伤起保护作用。
[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.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R459.7

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