N-乙酰半胱氨酸治疗大鼠急性坏死性胰腺炎作用机制的实验研究

An experimental study about the treatment mechanism for using N-acety-L-cysteine to cure acute necrotizing pancreatitis in rats

目的:探讨N乙酰半胱氨酸(NAC)对急性坏死性胰腺炎(ANP)胰腺腺泡细胞核转录因子κB(NFκB)、κB抑制蛋白(IκB)活性以及血液中炎症细胞因子IL6、TNFα及ICAM1含量的影响,揭示NAC治疗ANP的作用机制。方法:采用5%牛磺胆酸钠胰管逆行注射制备ANP动物模型。SD雄性大鼠60只,分为A、B、C三组,每组分4个时间点,每组每个时间点大鼠5只,采用随机分配原则分组。A组:假手术组,给予0.9%NS0.3ml/100g;B组:ANP空白对照组,给予0.9%NS0.3ml/100g;C组:NAC治疗组,给予0.075%NAC液0.3ml/100g。ANP后再继续分别观察1、3、5及7h后采用颈椎脱位法处死大鼠,并立即取血、腹水、胰腺组织及肺组织供实验用。实验中采用免疫组化法(SABC法)观察胰腺腺泡细胞NFκB活性和IκB表达及酶联免疫法(ELISA)检测血液IL6、TNFα及ICAM1含量。结果:NAC能够减轻ANP胰腺和肺组织损伤。SABC法显示抗NFκB与NFκB结合活性在3h时B组较C组显著增强;而抗IκB与IκB结合活性强度在3h时B组较C组显著减弱。ELISA法提示,呈点状出血坏死病理学改变的ANP血液中炎症细胞因子IL6、TNFα及ICAM1在3h即显著升高,5h即达高峰,7h又呈逐渐下降趋势,应用NAC治疗ANP后血液中炎症细胞因子IL6、TNFα及ICAM1在3、5及7h亦较ANP模型组显著降低。结论:(1)大鼠ANP胰腺腺泡细胞NFκB活性增高,胰腺腺泡细胞胞质IκB活性受到抑制,启动或促进控制炎症细胞因子IL6、TNFα及ICAM1的基因转录,从而加重全身炎症反应。(2)NAC能抑制大鼠ANP胰腺腺泡细胞NFκB活性,增强胰腺腺泡细胞胞质IκB活性,抑制大鼠ANP血液中促炎因子的产生,减轻全身炎症反应。(3)NAC能明显改善大鼠ANP胰腺和肺组织损伤。

Objective： This study was to investigate the effects of N-acety-L-cysteine on transcription factor nuclear-κB（NF-κB）,I-κB activity of pancreatic cells and inflammatory cytokines （IL-6, TNF-α and ICAM-1 ）of blood in acute necrotizing pancreatitis（ANP）of rats. Methods： ANP of rats was induced by retrograde injection of 5% sodium taurocholate into the pancreatic duct. Sixty male-rats were randomly divided into three groups, namely group A,group B and group C. There were four time points for every group（namely at 1 hr, 3hr, 5hr, and 7hr）and there were five rats at every time point. Group A：ANP（ - ）contrast group; Group B： ANP （ ＋ ） contrast group; Group C： ANP （ ＋ ） NAC treatment group. Rats of ANP were killed by the way of cervical spine dislocation after observed at 1 hr, 3hr, 5hr, 7hr and blood, hydropericardium,lung tissue, pancreas tissue were measured. NF-κB activity in the cell nuclear of pancreatic canard cells and IκBa activity in the cell spasm of pancreatic acinar cells were measured by immunohistochemistry. The concentrations of inflammatory cytokines（IL-6, TNF-α and ICAM-1 ）of the blood were measured by ELISA. Results： N acety-L-cysteine can decrease the damage of preacreas and lung tissue. NAC can significantly inhibit the NF-κB activity and stabIize I-κB activity. EIASA method indicated that the concentrations of inflammatory cytokines （IL-6, TNF-α and ICAM-1）in the blood of ANP（ ＋ ）rats significantly increased in 3hr and at the top of it in 5hr and decreased in 7hr. The concentrations of inflammatory cytokines（IL-6,TNF-α and ICAM- 1）in the blood of NAC treatment group rats remarkably decreased compared with the ANP contrast group respectively（in 3hr, 5hr and 7hr）. Conclusions： （1）The results indicate that the NF-κB activity of pancreatic acinar cells in experimental A NP rats significantly increases and leads to the production of inflammatory cytokines,irritating or aggravating the systemic inflammatory response. （2）NAC can inhibit the NF-κB activity of pancreatic acinar cells of ANP Rats, decrease the concentrations of inflammatory cytokines （IL-6, TNF-α and ICAM-1）, and limit the systemic inflammatory response. （3）N-acety-L-cysteine could significantly improve the damage of preacreas and lung in ANP rats.