抗氧化剂对急性胰腺炎大鼠核因子-B和一氧化氮合酶的影响

摘要

目的: 探讨抗氧化剂N-乙酰半胱氨酸(N-acetylcysteine, NAC)对急性胰腺炎大鼠胰腺核因子-B(nuclear factor-kappa B, NF-κB)和诱导型一氧化氮合酶 (inducible nitric oxide synthase, iNOS)表达的影响.

方法: ♂SD大鼠95只, 随机分成正常对照组(C组, 25只)、急性胰腺炎组(A组, 35只)和NAC干预组(N组, 35只). A组分2次腹腔内注射8 g/L的L-精氨酸(L-arginine, L-Arg) 1.2 mg/g诱导急性坏死性胰腺炎(acute necrotizing pancreatitis, ANP)模型; C组同法腹腔内注射等量生理盐水; N组先提前1h腹腔内注射0.5 mol/L的NAC 0.05 mg/g, 然后同A组方法诱导ANP. 在首次注射L-Arg后于6, 12, 24, 36, 48 h 5个时点分批处死大鼠, 检测胰腺NF-κB及 iNOS活性.

结果: N组NF-κB浓度在ANP早期比A组的明显降低(10.4±2.3 vs 89.7±6.4, 6.8±3.2 vs 21.5±3.5, 7.9±3.4 vs 32.5±4.5, 5.4±2.7vs 14.7±5.2, 5.0±3.7vs 11.1±2.3, P<0.05 及 P<0.01). A组各时点iNOS明显升高, N组各时点iNOS活性均较A组的明显下降(15.2±4.0 vs 24.2±3.8, 28.3±8.0 vs 36.8±6.0, 25.2±3.8 vs 30.5±3.5, 21.2±3.7 vs 28.7±7.2, 18.8±5.5 vs 28.2±4.2, P<0.05及P<0.01).

结论: 抗氧化剂可通过抑制NF-κB的激活而抑制iNOS的表达.

关键词: N/A

Effects of antioxidant on NF-κB and iNOS in rats with acute necrotizing pancreatitis

Zhi-Rou Tan, Guo-Du Tang, Hai-Xing Jiang, De-Hai Deng, Hai-Feng Yuan

Zhi-Rou Tan, Guo-Du Tang, Hai-Xing Jiang, De-Hai Deng, Department of Gastroenterology, the First Affiliated Hospital, Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China

Hai-Feng Yuan, Ruikang Hospital, Guangxi Traditional Chinese Medical University, Nanning 530011, Guangxi Zhuang Autonomous Region, China

Correspondence to: Zhi-Rou Tan, Department of Gastroenterology, the First Affiliated Hospital, Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China. zhirout@yahoo.com.cn

Received: October 21, 2003
Revised: November 5, 2003
Accepted: November 13, 2003
Published online: March 15, 2004

Abstract

AIM: To observe the influence of antioxidant N-acetylcysteine (NAC) on nuclear factor-kappa B (NF-κB) and inducible nitric oxide synthase (iNOS) in pancreatic tissue of rats with acute necrotizing pancreatitis (ANP).

METHODS: A total of 95 Spraque-Dawley (SD) male rats were randomly divided into control group (group C, n = 25), acute pancreatitis group (group A, n = 35) and NAC intervention group (group N, n = 35). In group A, SD rats were injected twice intraperitoneally with 8 g/L L-arginine (2×1.2 mg/g) in an interval of 1 hour for ANP. In group C, SD rats received the same amount of saline at the same time. In group N, 0.5 mol/L NAC (0.05 mg/g) was administered intraperitoneally 1 hour before the start of L-arginine injection. Animals were killed at 6, 12, 24, 36, and 48 hours after the first L-arginine injection. The concentration of NF-κB and the activity of iNOS in rat's pancreatic tissue of each group were assayed.

RESULTS: The concentration of NF-κB in pancreatic tissue in group N significantly decreased in earlier period than that in group A (10.4±2.3 vs 89.7±6.4, 6.8±3.2 vs 21.5±3.5, 7.9±3.4 vs 32.5±4.5, 5.4±2.7 vs 14.7±5.2, and 5.0±3.7 vs 11.1±2.3, P < 0.05 or P < 0.01). iNOS activity increased in group A, whereas it significantly decreased in group N (15.2± 4.0 vs 24.2±3.8, 28.3±8.0 vs 36.8±6.0, 25.2±3.8 vs 30.5±3.5 , 21.2±3.7 vs 28.7±7.2, and 18.8±5.5 vs 28.2±4.2, P < 0.05 or P < 0.01).

CONCLUSION: Antioxidants may decrease the activity of iNOS through the inhibition of NF-κB activation.

Key Words: N/A

0 引言

急性胰腺炎(acute pancreatitis, AP)是临床常见急症之一[1-8], 尤其是急性出血坏死性胰腺炎(acute necrotizing pancreatitis, ANP)临床表现危重, 病死率高[9-10], 其发病机制极为复杂且尚未完全阐明[11-13]. 最近的研究表明, 前炎症因子iNOS的过度表达参与加重AP时对胰腺组织的损害[14-15]. iNOS在正常生理条件下并不表达, 只有在炎症等病理条件下细胞受到刺激后方表达, 但iNOS的转录合成还受到转录因子复合体NF-κB的调控[16]; 抗氧化剂N-乙酰半胱氨酸(NAC)对NF-κB活性有抑制作用[17]. 但NAC在ANP的发展中对大鼠胰腺组织NF-κB、iNOS活性的影响, 国内外尚未见报道. 我们采用分2次腹腔内注射大剂量左旋精氨酸(L-arginine, L-Arg)法制备大鼠ANP模型, 观察NAC对大鼠胰腺组织NF-κB, iNOS活性的影响及其意义.

1 材料和方法

1.1 材料

健康♂SD大鼠95只, 2-3月龄, 体质量250-300 g (广西医科大学动物中心提供). iNOS检测试剂盒购自南京建成生物工程研究所, 大鼠转录因子检测试剂盒购自美国Clontech公司. L-Arg, NAC, 蛋白酶抑制物混合液, 胰蛋白酶抑制剂, 羟乙基哌嗪乙磺酸(HEPES), 二硫苏糖醇(DTT)均购自美国Sigma公司.

1.2 方法

大鼠随机分成3组: 生理盐水对照组(C组, 25只), 急性胰腺炎组(A组, 35只), NAC干预组(N组, 35只). 所有大鼠实验前均禁食16 h, 自由饮水. A组大鼠分2次腹腔内共注射80 g/L L-Arg溶液2×1.2 mg/g, 中间间隔1 h; C组同法腹腔内注射等量的生理盐水. N组则于L-Arg注射前1 h, 预先腹腔内注射0.5 moI/L的NAC溶液0.05 mg/g, 1 h后按照A组同法腹腔内注射等量的80 g/L L-Arg溶液. 各组实验大鼠在注射80 g/L L-Arg或等量生理盐水后分别于6, 12, 24, 36, 48 h 5个时点分批处死; C组每个时点处死5只大鼠, A组和N组每个时点处死7只大鼠. 解剖大鼠, 取胰头部胰腺组织行组织病理学检查, A组、N组大鼠经大体病理及组织病理检查证实为ANP. 将胰腺组织用滤纸吸干水分, 电子天平称取 200 mg胰腺组织, 剪碎后加入PBS溶液至2 mL, 匀浆机制成100 g/L的胰腺组织匀浆, 4 ℃低温离心, 取上清-20 ℃冰箱保存, 采用L-精氨酸比色法测定iNOS活性. 另切取胰腺组织250-300 mg以同等量冷PBS液中冲洗, 于5 mL冷PBS 中充分剪碎, 经150目的滤网冰上研磨, 收集中层悬液、离心, 弃上清取细胞沉淀. 细胞沉淀于溶解缓冲液(HEPES 100 mmol/L, MgCl₂ 15 mmol/L, KCl 100 mmol/L, DTT 0.1 mol/L, 蛋白酶抑制物)中冰冻孵育15 min, 离心、再悬浮, 用小27号针头反复吸喷以破碎细胞, 离心破碎的细胞液, 收集核沉淀物. 再于提取缓冲液(HEPES 20 mmol/L, MgCl₂ 1.5 mmol/L, NaC1 0.42 mol/L, EDTA 0.2 mmol/LM, 甘油, DTT 0.1 mol/L, 蛋白酶抑制物)中悬浮粗制核沉淀物, 注射器反复吸喷以破碎细胞核, 离心核悬浮液, 收集上清液(细胞核提取物), 测量核提取物的蛋白浓度, 小整量分装、快速冷冻(-70 ℃), 采用ELISA法测定 NF-κB浓度.

统计学处理 采用SPSS10.0软件包作统计学处理, 数据以mean±SD表示, 进行方差分析, P<0.05为差异有统计学意义.

2 结果

2.1 NAC对NF-κB的影响

经NAC预处理的大鼠胰腺腺泡细胞核内NF-κB浓度显著下降(表1).

表1 大鼠胰腺腺泡细胞核内NF-κB的变化 (mg/L, mean±SD).

分组	n	t/h
		6	12	24	36	48
C	5	3.3±3.1	4.3±2.9	4.3±2.9	3.3±3.1	3.3±3.1
ANP	7	89.7±6.4b	21.5±3.5b	32.5±4.5b	14.7±5.2b	11.1±2.3b
NAC	7	10.4±2.3ad	6.8±3.2d	7.9±3.4d	5.4±2.7d	5.0±3.7c

^aP<0.05,

^bP<0.01 vs C;

^cP<0.05,

^dP<0.01 vs ANP.

2.2 NAC对iNOS活性的影响

C组各时点胰腺组织匀浆iNOS活性均未能检出. 以NAC预处理, 使大鼠胰腺组织匀浆iNOS活性显著下降(表2).

表2 大鼠胰腺组织iNOS活性(kat/g, mean±SD, n = 7).

分组	t/h
	6	12	24	36	48
ANP	24.2±3.8	36.8±6.0	30.5±3.5	28.7±7.2	28.2±4.2
NAC	15.2±4.0b	28.3±8.0a	25.2±3.8a	21.2±3.7a	18.8±5.5b

^aP<0.05,

^bP<0.01 vs ANP.

3 讨论

在实验性AP的发生早期有NF-κB的激活, 在实验性AP模型的发生中起着重要的作用[18-19]. NF-κB是最近发现的极重要的基因转录调控因子, 他是一种具有多向转录调节作用的核蛋白. NF-κB几乎存在于所有细胞中; 但通常情况下, NF-κB与抑制性抗核因子-B (IB)共价结合成复合物, 以无活性形式存在于细胞质内. NF-κB的活化可诱导前炎症因子iNOS的过度表达, iNOS的大量表达参与AP的发生、发展, 加重了AP时对胰腺组织的损害[14-15]. 我们发现ANP大鼠细胞核内NF-κB浓度在6 h即有明显升高, 6 h后呈现急剧下降, 12 h降至较低水平, 24 h有所回升并呈现第二峰值, 以后时点呈缓慢下降趋势, 支持AP早期有NF-κB的激活. 使用NAC后NF-κB的活性明显降低, 提示NAC可抑制NF-κB的活性. 其作用机制尚不十分明确, 推测可能是: NAC是一含巯基(-SH)物质, 是体内游离巯基的重要组成部分之一. NAC进入体内迅速脱去乙酰基变为左旋半胱氨酸, 捕捉未配对电子, 阻止急性胰腺炎胰腺缺血再灌注时中性粒细胞氧自由基的瞬间爆发. 同时NAC可直接作用于过氧化氢(H₂O₂)生成H₂O和O₂, 还可清除超氧阴离子、羟自由基[20]. 此外, NAC为小分子物质, 进入细胞后可为谷胱甘肽(GSH)的合成提供原料. GSH 对稳定细胞膜及细胞器膜, 稳定细胞内酶和蛋白质的功能具有重要作用; 能解除病理状态下蛋白质分子二硫键交联, 利用巯基灭活活性氧. NAC可能通过其抗氧化和细胞保护作用, 抑制NF-κB的激活[21].

我们发现经NAC预处理的大鼠胰腺组织iNOS的含量显著下降, 可能是NAC通过抑制NF-κB激活, 从而抑制了胰腺组织iNOS表达的结果. iNOS在正常生理条件下并不表达, 只有在炎症等病理条件下细胞受到多种刺激物如脂多糖(LPS)、肿瘤坏死因子-(TNF-)、干扰素、白介素-1(IL-1)等刺激诱导下, 启动定位于第17对常染色体上的II型iNOS基因转录合成[22-23]; 但iNOS的转录合成还受到NF-κB 的调控[24-25]; iNOS表达的调控主要发生在转录水平, iNOS基因的启动子区域含有多个转录因子的结合位点包括NF-κB[26-27], 要诱导iNOS的表达, 必须有NF-κB的激活[28-29]. 本研究发现, C组各时点胰腺腺泡核内NF-κB浓度无明显升高, 各时点胰腺组织匀浆iNOS活性均未能检出; A组NF-κB浓度急剧升高后, iNOS活性也明显升高. 应用抗氧化剂NAC后, NF-κB浓度明显下降, iNOS的活性也降低, 与文献[17,30]报道基本一致; 提示NAC通过抑制NF-κB的激活而有效抑制iNOS的表达, 因此, 抗氧化剂可能成为急性胰腺炎治疗中具有应用前景的一类药物.

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备注

编辑: N/A