抗氧化应激对脂多糖介导的肝脏和枯否细胞MAPK和STAT信号转导的影响

发布时间：2018-03-27 14:14

本文选题：枯否细胞　切入点：STAT　出处：《山西医科大学》2006年硕士论文

【摘要】： 目的:探讨抗氧化剂N-乙酰半胱氨酸(NAC)对脂多糖介导的急性肝损伤发生过程中肝脏和枯否细胞MAPK和STAT信号转导的影响。方法:实验1.雄性昆明种小鼠54只随机分为3组:(1)对照组(n=6):腹腔注射0.9 % NaCl 0.2 ml;(2)Galn /LPS组(n=24):(Galn 520 mg + LPS 120μg)/kg,i.p.;(3)NAC+ Galn /LPS组(n=24):NAC 150 mg/kg,连续灌胃3天;第3天灌胃后1h腹腔注射GalN/LPS,剂量、分组和处理与Galn /LPS组相同。以上各组动物分别于注射Galn /LPS或生理盐水后0.5h、1h、2h和6h后,在戊巴比妥钠麻醉下经眼内眦采血和开腹取肝,用于血浆ALT、肝MDA和GSH含量测定及肝脏病理组织学检查。实验2.动物和分组同实验1。(1)对照组(n=6):0.9 % NaCl 0.2 ml,i.p.;(2)LPS组(n=24):LPS 5 mg/kg,i.p.;(3)NAC+LPS组(n=24):NAC 150mg/kg,连续灌胃3天,第3天灌胃后1h,腹腔注射LPS 5 mg/kg,i.p.。分别于注射LPS或生理盐水后0.5h、1h、2h和6h后,在戊巴比妥钠麻醉下开腹取肝脏,用Western blotting方法检测肝脏MEK1/2、ERK1/2、p38MAPK、STAT1、STAT3和HSP 70表达,用放免法测定肝TNF-α含量。实验3.雄性昆明种小鼠40只分为3组,无菌分离枯否细胞(kupffer cells, KCs),培养24h后,分为:(1)对照组(n=8):直接收集KCs和培养基上清液;(2)LPS组(n=16):DMEM培养基中加入100 ng/ml LPS剌激0.5h或1h,收集KCs和培养基上清液;(3)NAC+LPS组(n=16):用含NAC(10 mmol/L)的DMEM培养基培养2h后,再加入含LPS(100 ng/ml)的DMEM基培养刺激0.5h或1h,收集KCs和培养基上清液。KCs MEK1/2、ERK1/2、p38MAPK、STAT1、STAT3和HSP70表达及培养上清液中TNF-α含量检测同实验二。结果:NAC预处理可明显减轻GalN/LPS所致急性肝损伤,经NAC预处理动物血浆ALT活性和肝脏MDA含量明显下降,而GSH含量则升高。LPS剌激可使肝脏和枯否细胞的MEK1/2、ERK1/2、p38MAPK的磷酸化表达显著增强,p-MEK1/2、p-ERK1/2在LPS剌激0.5h达到高峰,2h后恢复至基础水平,而p38MAPK则在LPS刺激0.5h后持续保持高磷酸化水平。肝匀浆和KCs培养基的TNF-α水平亦明显增高。STAT1和STAT3在LPS剌激0.5h后发生磷酸化,1h时达高峰并持续保持较高的磷酸化水平。NAC预处理可显著抑制LPS所致肝脏或KCs MEK1/2、ERK1/2、p38MAPK、STAT1和STAT3的磷酸化,与此同时,伴有肝脏或KCs培养基上清中TNF-α水平显著降低。HSP 70在LPS剌激0.5h时表达明显高于对照组,1h后逐渐趋于正常水平。结论:LPS介导KCs激活产生的ROS,在激活MAPK和STAT的信号转导中起了重要作用,并导致TNF-α的释放增加。NAC通过其抗氧化应激作用可部分抑制LPS介导的细胞信号转导,使TNF-α释放减少,从而发挥其抗损伤作用。在LPS作用下HSP 70的表达增强可能是机体拮抗LPS诱导肝损伤发生的重要自我保护机制。
[Abstract]:Aim: to investigate the effects of antioxidant N-acetylcysteine (NAC-) on MAPK and STAT signal transduction in liver and Kupffer cells during lipopolysaccharide mediated acute liver injury. Methods: experiment 1. Fifty-four male Kunming mice were randomly divided into 3 groups: control group (n = 6): intraperitoneal injection of 0.9% NaCl 0.2 ml / L ~ (2 +) Gal ~ (2 +) LPS group (n ~ (24): n ~ (24): n ~ (20) mg / LPS ~ (120) 渭 g 路kg ~ (-1) 路kg ~ (-1) 路kg ~ (-1) 路kg ~ (-1) NaCl Galn / LPS group, n = 40% NAC 150 mg 路kg ~ (-1) 路kg ~ (-1)) intraperitoneal injection of Galnr-LPS at 1 hour after intragastric administration for 3 days. The animals were divided into groups and treated in the same way as those in the Galn / LPS group. The animals in the above groups were collected blood from the canthus under pentobarbital sodium anesthesia for 2 hours and 6 hours after the injection of Galn / LPS or normal saline, and the liver was obtained by laparotomy. For the determination of plasma alt, liver MDA and GSH contents and liver histopathology. Experiment 2. Animals and groups are the same as experiment 1. 1) in the control group, control group (6: 0.9% NaCl 0.2 ml, i.p.) and lipopolysaccharide group (LPS-5 mg 路kg-1 路kg ~ (-1) 路kg ~ (-1)) n24% LPS-5 mg 路kg ~ (-1) 路L ~ (-1) 路L ~ (-1) + n24% NAC 150 mg / kg 路L ~ (-1) 路L ~ (-1). 1 h after intragastric administration of LPS 5 mg / kg i.p., LPS 5 mg / kg i.p. was injected intraperitoneally at 0.5 h after injection of LPS or normal saline for 2 h and 6 h, respectively. The liver was anesthetized with pentobarbital sodium. The expression of MEK1 / 2mmpMAPKSTAT1STAT3 and HSP 70 were detected by Western blotting. The content of TNF- 伪 in liver was measured by radioimmunoassay. Experiment 3. Forty male Kunming mice were divided into 3 groups. Kupffer cells (KCs1) were isolated from Kupffer cells and cultured for 24 h. The control group was divided into two groups: the control group was divided into two groups: direct collection of KCs and culture medium supernatant / lipopolysaccharide added 100 ng/ml LPS for 0.5 h or 1 h, and KCs and the supernatant of KCs and the supernatant of NAC LPS for 2 h, then cultured in DMEM medium containing NAC(10 mmol / L for 2 h. The DMEM base containing LPS(100 / ml was added to the culture medium for 0.5 h or 1 h. The supernatants of KCs and culture medium were collected. The expression of STAT3 and HSP70 in supernatant of KCs and culture medium was detected by the same method as experiment 2. The expression of STAT3 and HSP70 in the supernatant of KCs and the supernatant of culture medium was detected by the same method as that of the supernatant. Results the acute liver injury induced by GalN/LPS was significantly alleviated by the pretreatment of GalN/LPS. The plasma ALT activity and hepatic MDA content decreased significantly after NAC preconditioning. The phosphorylation of MEK1 / 2ERK1 / 2p38MAPK in liver and Kupffer cells was significantly enhanced by GSH. The phosphorylation of p-MEK1 / 2pERK1 / 2 was restored to the basic level at 0.5 h after LPS stimulation. The levels of TNF- 伪 in liver homogenate and KCs medium also increased significantly. STAT1 and STAT3 reached the peak at 1 h after LPS stimulation and maintained a high level of phosphorylation. NAC. Pretreatment significantly inhibited phosphorylation of LPS induced liver or KCs MEK1 / 2 ERK1 / 2 p38 MAPKP STAT1 and STAT3. At the same time, the expression of TNF- 伪 in the supernatant of liver or KCs medium decreased significantly, and the expression of HSP70 was significantly higher than that of the control group at 0.5 h after stimulation with LPS. Conclusion ROSmediated by KCs may play an important role in signal transduction of MAPK and STAT, and increase the release of TNF- 伪. NAC can partly inhibit the cellular signal transduction mediated by LPS and decrease the release of TNF- 伪 through its antioxidant stress. The increased expression of HSP 70 under the action of LPS may be an important self-protective mechanism against LPS induced liver injury.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R363

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