细胞因子信号转导抑制因子在内毒素耐受大鼠肝组织中的作用

The study on the role of SOCS1 and SOCS3 in the livers of endotoxin tolerance rats

目的研究内毒素耐受（endotoxin tolerance，ETT）及正常大鼠接受D-胺基半乳糖（D—gaiactosamine，D—GaiN）／脂多糖（lipopolysacharide，LPS）刺激后肝组织细胞因子信号转导抑制因子（SOCSs）基因表达的异同，探讨内毒素耐受的可能机制。方法雄性SD大鼠随机分为急性肝功能衰竭模型组（acute liver failure，ALF组）和内毒素耐受组（ETT组）。ETT组及ALF组先分别以0．1mg／kg LPS和生理盐水腹腔注射，每日1次，连续5次，于LPS或生理盐水第5次注射24h后同时腹腔注射D—GaIN 800mg／kg和LPS 8μg/只，分别在注射D—Gain／LPS前（0h）和注射后2、6、12、24和48h 6个时间点留取大鼠血及肝脏标本。HE染色及透射电镜下观察肝组织病理及超微结构变化；RT—PCR法检测动物肝组织中SOCS1和SOCS3 mRNA表达；采用鲎试剂基质显色法测定血清内毒素水平，ELISA法检测TNF-α水平。结果ETT组大鼠肝组织病理改变及超微结构变化明显轻于ALF组，其血清内毒素、TNF-α水平明显低于ALF组。内毒素：6h组分别为1．11±0．38和0．74±0．22，24h组分别为1．12±0．24和0．86±0．21，均P〈0．05，12h组分别为1．88±0．35和0．62±0．16，48h组分别为1．10±0．13和0．84±0．19，均P〈0．01。TNF—α：6h组分别为86．9±12．6和70．0±12．8，P〈0．05，12h组分别为77．0±18．1和48．8±12．8，24h组分别为63．8±9．2和39．1±5．7，48h组分别为53．2±8．3和38．2±9．9，均P〈0．01。ALF组大鼠肝组织SOCS1和SOCS3 mRNA表达均明显上调，造模后2h即明显高于对照组，分别于12h、6h达峰值，ETT组的SOCS1和SOCS3 mRNA表达较模型组明显上升。SOCS1：6h组分别为0．955±0．186和1．349±0．390，48h组分别为0．766±0．145和0．970±0．205，均P〈0．05，2h组分别为0．554±0．164和0．841±0．175，12h组分别为1．130±0．181和1．888±0．573，24h组分别为0．990±0．212和1．550±0．439，均P〈0．01。SOCS3：6h组分别为0．914±0．054和1．039±0．109，12h组分别为0．±0．044和0．863±0．063，均P〈0．05，2h组分别为0．681±0．139和0．898±0．058，24h组分别为0．700±0．065和0．811±0．055，均P〈0．01。结论内毒素耐受时，大剂量的D—GaIN和LPS腹腔注射引起的肝脏损害减轻，内毒素、TNF-α释放减少，可能与肝组织SOCS1、SOCS3的高表达有关；

Objective To explore the mechanism of endotoxin tolerance （ETT） through observing the gene expression of SOCS1 and SOCS3 in the livers of ETF rats. Methods SD male rats were divided randomly into acute liver failure model group（ALF group） and ETF group. LPS 0. 1 mg/kg（ETT group） or normal saline （ALF group）was administered five consecutive intraperitoneal injections at 24 h intervals, then, the two groups were treated with intraperitoneal injection of D-GaIN 800 mg/Kg and LPS 8 μg/rat 24 h later. Liver histopathology and fine structure of rats were observed by HE staining and electron microscope. The TNF-α level were estimated by ELISA, the concentrations of endotoxin were determined by tachypleus amebocyte lysate and the gene expressions of SOCSI and SOCS3 in the liver were measured by RT-PCR at 0,2, 6 ,12 ,24 and 48 h after the injection of D-GalN/LPS. Results D-GalN/LPS induced acute liver injury was attenuated significantly in ETT group. The concentrations of endotoxin and the TNF-α level were evidently lower in the ETT group than those in the ALF group （ endotoxin： 6 h ： 1.11 ± 0. 38 vs 0. 74 ±0. 22,24 h：1. 12 ±0. 24vs0. 86 ±0. 21 ,all P 〈0. 05,12 h：1. 88 ±0. 35vs 0. 62 ±0. 16,48 h：1. 10 ± 0.13vs0. 84 ± 0.19, all P 〈0.01;TNF-α：6 h：86.9 ± 12.6 vs70.0 ± 12.8, P 〈0.05,12 h：77.0 ± 18.1vs48.8±12.8,24 h：63.8 ±9.2vs39. 1 ±5.7, 48 h：53.2±8.3vs38.2 ±9.9,all P〈0.01）. In the ALF group, the expressions of SOCS-1 and SOCS-3 were obviously higher than those in the controls and reached peak at 12th hours and 6th hours respectively. The gene expression of SOCS1 and SOCS3 in the liver in ETT group were increased significantly and much higher than those in ALF groups. （SOCS1：6 h ：0. 955 ± 0.186vs 1.349±0.390,48 h：0.766±0.145 vs0.970 ±0.205,all P〈0.05,2 h： ：0.554±0.164 vs 0.841±0.175,12h：1.130±0.181 vs 1.888 ± 0.573,24 h：0.990±0.212 vs 1. 550 ± 0. 439, all P 〈 0. 01 ; SOCS3 -6 h：0. 914 ±0. 054 vs 1. 039 ±0. 109,12 h：0. 781 ± 0. 044 vs 0. 863 ±0. 063 ,all P 〈0. 05, 2 h,0. 681 ± 0. 139 vs 0. 898 ± 0. 058,24 h .0. 700 ±0. 065 vs 0. 811 ±0. 055, all P 〈 0. 01 ）. Conclusions LPS pretreatment can induce endotoxin tolerance of rats, inhibited the level of TNF-α and endotoxin . The up-regulation of SOCS1 and SOCS3 gene expression may be one of the possible mechanisms responsible for endotoxin tolerance.