程序性坏死特异性抑制剂-1对脓毒症大鼠肝脏单核细胞趋化蛋白-1表达的影响

The effect of necrostatin-1 on expression of liver monocyte chemotactic protein-1 in septic rats

目的：探讨程序性坏死特异性抑制剂-1（Nec-1）对脓毒症大鼠肝脏单核细胞趋化蛋白-1（MCP-1）表达的影响及其机制。方法按随机数字表法将48只雄性SD大鼠分为假手术（Sham）组、模型组、Nec-1组，每组16只。采用盲肠结扎穿孔术（CLP）制备脓毒症大鼠模型；Sham组仅麻醉、开腹翻动盲肠后关腹，不进行结扎。Nec-1组于制模前30min尾静脉注射Nec-1溶液1mg/kg〔25mgNec-1溶于2.5mL二甲基亚砜（DMSO）溶剂中〕；模型组则注射DMSO0.1mL/kg。各组分别于制模后即刻（0h）和8h取腹主动脉血及肝脏组织，采用全自动生化分析仪检测血清丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）水平；苏木素-伊红（HE）染色后光镜下观察肝组织病理学改变；酶联免疫吸附试验（ELISA）检测血清肿瘤坏死因子-α（TNF-α）和白细胞介素-6（IL-6）含量；反转录-聚合酶链反应（RT-PCR）检测肝组织MCP-1mRNA表达。结果制模后0h，3组血清ALT、AST、TNF-α、IL-6及肝脏MCP-1mRNA表达差异均无统计学意义，肝组织细胞结构正常。制模后8h，模型组和Nec-1组血清ALT、AST、TNF-α、IL-6及肝脏MCP-1mRNA表达均较Sham组明显升高〔ALT（U/L）：172.35±21.88、129.67±18.20比60.04±11.74，AST（U/L）：511.03±34.92、363.03±25.25比254.83±31.04，TNF-α（ng/L）：603.96±24.18、483.87±26.60比265.74±15.14，IL-6（ng/L）：975.62±65.37、712.09±45.47比310.42±13.88，MCP-1mRNA（2-ΔΔCt）：7.09±0.18、5.51±0.45比0.99±0.06，均P＜0.05〕；Nec-1组各指标均较模型组明显下降（均P＜0.05）。制模后8h，光镜下模型组可见肝小叶结构破坏、淤血及炎性细胞浸润；而Nec-1组肝组织病理改变较模型组明显减轻。结论 Nec-1预处理可有效减轻脓毒症大鼠肝脏损伤，减少循环中炎性因子含量及肝脏中MCP-1mRNA表达，从而减轻炎症对机体的损伤。

Objective To investigate the effect of necrostatin-1 （Nec-1） on the expression of liver monocyte chemotactic protein-1 （MCP-1） in septic rats and its mechanism. Methods Forty-eight male Sprague-Dawley （SD） rats were randomly divided into sham group, model group, and Nec-1 group by randomized digital number method, with 16 rats in each group. The model of sepsis was reproduced by cecal ligation and puncture （CLP）. Rats in sham group received anesthesia, and flipping the cecum followed by closure of the abdomen without ligation of the cecum. Rats in Nec-1 group were given 1 mg/kg Nec-1 [25 mg Nec-1 solution dissolved in 2.5 mL of dimethyl sulfoxide （DMSO）] through caudal vein 30 minutes before operation, while the rats in model group were given 0.1 mL/kg of DMSO only. Blood from abdominal aorta and liver tissue in each group were collected at 0 hour and 8 hours after operation. Serum alanine aminotransferase （ALT） and aspartate aminotransferase （AST） were determined with automatic biochemistry analyzer. The pathological changes in liver were observed under light microscope using hematoxylin-eosin （HE） staining. The serum levels of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） were determined by enzyme linked immunosorbent assay （ELISA）. The MCP-1 mRNA expression in the liver was determined by reverse transcription-polymerase chain reaction （RT-PCR）. Results There was no significant differences in the levels of serum ALT, AST, TNF-α, IL-6 and expressions of liver MCP-1 mRNA at 0 hour among three groups, and the liver cellular structure was normal. At 8 hours, compared with sham group, the expressions of serum ALT, AST, TNF-α, IL-6 and liver MCP-1 mRNA were significantly increased in model group and Nec-1 group [ALT （U/L）： 172.35±21.88, 129.67±18.20 vs. 60.04±11.74, AST （U/L）： 511.03±34.92, 363.51±25.25 vs. 254.83±31.04, TNF-α（ng/L）： 603.96±24.18, 483.87±26.60 vs. 265.74±15.14, IL-6 （ng/L）： 975.62±65.37, 712.09±45.47 vs. 310.42±13.88, MCP-1 mRNA （2-ΔΔCt）： 7.09±0.18, 5.51±0.45 vs. 0.99±0.06, all P 〈 0.05]. Levels of the above parameters in Nec-1 group at 8 hours were significantly decreased compared with those of model group （all P 〈 0.05）. Under light microscopy, it was noted that the structure of hepatic lobules was destroyed, with exacerbation of immunocyte infiltration at 8 hours in model group. At 8 hours, it was found that Nec-1 alleviated the pathological damage in Nec-1 group. Conclusion Nec-1 can protect the liver of rats with sepsis, lower the expression of serum TNF-α and serum IL-6 and liver MCP-1 mRNA, and obviously reduce the damage of inflammation.