N-乙酰半胱氨酸对急性坏死性胰腺炎大鼠趋化因子MCP-1、MIP-2表达的影响

Effects of N-acetylcysteine on mRNA expression of monocyte chemotactic protein and macrophage inflammatory protein 2 in acute necrotizing pancreatits： experiment with rats

目的探讨趋化因子单核细胞趋化蛋白1（MCP-1）与巨噬细胞炎性蛋白2（MIP-2）在大鼠急性坏死性胰腺炎（ANP）早期发病机制中的作用，并观察N-乙酰半胱氨酸（NAC）对趋化因子表达的影响。方法35只SD大鼠随机分为假手术组（SO）组，ANP3h、6h、12h组和NAC干预3h、6h、12h组，每组5只。采用4％牛磺胆酸钠胰胆管逆行注射制备ANP动物模型，NAC组在诱导ANP前0．5h腹腔注射NAC500mg／kg。检测血淀粉酶、髓过氧化物酶（MPO），观察胰腺病理组织学改变，采用Rt—PCR检测胰腺组织中MCP-1与MIP-2 mRNA的表达。结果ANP各组血淀粉酶较高，浸润到胰腺组织的白细胞明显较多，胰腺病理损伤依次加重。SO组胰腺MCP-1 mRNA与MIP-2 mRNA表达阴性或弱表达（0．1492±0．0036、0．1593±0．0117），ANP各组MCP-1 mRNA（0．3653±0．0213、0．5890±0．0225、0．7164±0．0275）与MIP-2 mRNA（0．3871±0．0286、0．6040±0．0448、0．7692±0．0620）的表达随ANP病变的加重逐渐增强（均P〈0．01）。MCP-1与MIP-2 mRNA的表达与胰腺的病理损伤成正相关（r=0．76和0．82，P〈0．05）。经NAC干预后，趋化因子MCP-1、MIP-2 mRNA表达下调（0．2497±0．0168、0．4457±0．0097、0．6306±0．0423，0．2436±0．0099、0．4312±0．0221、0．6302±0．0288，均P〈0．05），白细胞浸润减少（0．63±0．03 vs 0．89±0．03，0．88±0．05VS1．42±0．14，1．31±0．09 vs 1．94±0．07，均P〈0．05），组织损伤得到改善（3．50±0．61 vs 5．10±0．42，5．60±0．65 vs 7．50±0．50，7．50±0．79VS9．90±0．96，均P〈0．05）。结论ANP早期趋化因子MCP-1、MIP-2在胰腺组织中过表达，并参与白细胞的趋化作用。NAC能显著下调胰腺组织中趋化因子MCP-1、MIP-2的表达。

Objective To explore the potential role of monocyte chemotactic protein 1 （ MCP-1 ） and macrophage inflammatory protein 2 （MIP-2） in the pathogenesis of acute necrotizing pancreatitis （ ANP）, and to study the effect of N-acetylcysteine （NAC） on the mRNA expression of MCP-1 and MIP-2. Methods Thirty-five SD rats were randomly divided into 3 groups ： sham-operation （ SO ） group （ n = 5 ）, acute necrotizing pancreatits （ANP） group （n = 15）, and NAC-pretreated group （n = 15）, ANP were induced by retrograde injection of 4% sodium taurocholate into the biliopancreatic duct. The NAC- groups underwent intraperitoneal injection of NAC 500 mg/kg 30 minutes before the induction of sodium taurocholate. The ANP and NAC groups were re-divided into 3 equal subgroups respectively： 3 h, 6 h, and 12 h subgroups. 3, 6, and 12 hours after the establishment of models heart blood samples were collected from 5 rats from each model group to detect the serum amylase. Then the rats were killed by blood letting with their pancreases taken out. HE staining and microscopy were used to observe the pathological changes of the pancreas. The wet/dry ratio of pancreas was determined. Enzyme histochemical method was used to detect the activity of myeloperoxidase （MPO） in the pancreas. RT-PCR was used to examine the mRNA expression of MCP-1 and MIP-2. Results The levels of serum amylase, wet/dry ratio of pancreas, pancreatic MPO activity, and histological score of pancreas of the ANP group increased time-dependently, all the levels at different time-points were significantly higher than those of the SO group （ all P 〈 0. 01 ）. The expression levels of MCP-1 mRNA at the time points 3, 6, and 12 h of the ANP group were 0. 3653 ±0. 0213,0. 5890 ±0. 0225,and 0. 7164±0. 0275 respectively, all significantly higher than that of the SO group （0. 1492 ± 0. 0036, all P 〈 0. 01 ）. The expression levels of MIP-2 mRNA at different time points of the ANP group were 0. 3871 ± 0. 0286,0. 6040 ± 0. 0448, and 0. 7692 ± 0. 0620 respectively, all significantly higher than that of the SO group （0. 1593 ±0. 0117, all P 〈 0. 01 ）. The intrapancreatic MPO levels at the time points 3 h, 6 h, and 12 h of the NAC group were 0. 63 ±0. 03, 0. 88 ±0. 05, and 1.31 ±0. 09 respectively, all significantly lower than those of the ANP groups （ 0. 89 ± 0. 03, 1.42 ± 0. 14, and 1.94 ± 0. 07, all P 〈 0. 05）. The MCP-1 mRNA expression levels at different time points the NAC group were 0. 2497 ±0. 0168, 0. 4457 ± 0. 0097, and 0. 6306 ± 0. 0423 respectively, and the MIP-2 mRNA expression levels at the time points 3 h, 6 h, and 12 h of the NAC group were 0. 2436 ± 0. 0099,0. 4312 ± 0. 0221, and 0. 6302 ± 0. 0288 respectively, all significantly lower than those of the ANP group （ all P 〈 0. 05 ）. The pancreatic histological scores at different time points of the NAC group were 3.50 ± 0. 61, 5.60 ± 0. 65, and 7. 50 ± 0. 79, all significantly lower than those of the ANP group （ 5. 10 ± 0. 42, 7.50 ± 0. 50, and 9. 90 ± 0. 96, all P 〈 0. 05）. The MCP-1 mRNA expression and MIP-2 mRNA expression were both correlated with the severity of pancreatic injury （ r = 0. 76 and 0. 82, both P 〈 0. 05）. Conclusion The chemokines of MCP-1 and MIP-2 are overexpressed at the early stage of acute pancreatitis. Both of them may play an important role in the pathogenesis of AP. NAC may have beneficial effects on AP through downregulation of the expression of MCP-1 and MIP-2.