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目的 应用不同的抗体蛋白研究小鼠肠道缺血-再灌注致远隔组织损伤时Toll样受体4(TLR4)-高迁移率族蛋白1(HMGB1)及其下游信号的传导途径.方法 SPF级雄性C57BL/6小鼠40只,采用随机数字表法分为5组:假手术组、对照组、抗HMGB1组、抗髓样细胞分化基因88 (MyD88)组和抗含诱导干扰素β的衔接蛋白(TRIF)组,每组8只.对照、抗-HMGB1、抗-MyD88和抗-TRIF组在行肠道缺血-再灌注手术前30 min分别经尾静脉注射对照IgG、HMGB1、MyD88和TRIF抗体(剂量为1 mg/kg,浓度为0.025％).所有小鼠麻醉、开腹后,除假手术组外的4组用无创血管夹夹闭肠系膜上动脉60 min后再灌注60 min,假手术组只开腹120 min,不进行缺血-再灌注.检测血浆核因子-κB(NF-κB) p65、白细胞介素6(IL-6)及肿瘤坏死因子α(TNF-α)浓度;肺和肠道组织形态学变化和HMGB1与NF-κB的mRNA和蛋白表达.结果 小鼠肠道缺血-再灌注损伤时,与对照组[(228.534-24.85)、(104.91±31.18)及(70.81±46.97) ng/L]比较,缺血前通过注射抗体抑制HMGB1[(145.00±33.63)、(62.28±6.73)及(52.76±5.71) ng/L]、MyD88[(191.12± 13.22)、(85.90± 17.37)及(63.19±5.47) ng/L]和TRIF[(183.73±10.81)、(78.14±7.38)及(59.70±4.63) ng/L]的表达能明显降低血浆炎症因子NF-κB(P=0.000、0.005、0.001)、IL-6 (P=0.000、0.004、0.000)及TNF-α(P=0.000、0.024、0.002)的水平,同时减轻了肺脏和小肠组织的损伤.抗-HMGB1组、抗-MyD88组和抗-TRIF组小鼠肺脏和回肠的HMGB1 mRNA(肺脏:1.89±0.18、2.35±0.31和2.29±0.28,回肠:4.93±0.55、5.96±0.73和5.76±0.51)、NF-κB mRNA(肺脏:1.42±0.23、1.77±0.18和1.70±0.13,回肠:2.23±0.55、3.11±0.38和2.99±0.24)和蛋白表达较假手术组[肺脏HMGB1mRNA (1.04±0.19) (P=0.000、0.000、0.000)、NF-κB mRNA (1.03±0.21) (P=0.004、0.000、0.000),回肠HMGB1 mRNA (1.14±0.54) (P=0.000、0.000、0.000)、NF-κB mRNA (1.03±0.23)(P=0.000、0.000、0.000)]升高,差异有统计学意义,但与对照组[肺脏HMGB1 mRNA(2.67±0.30) (P=0.000、0.035、0.016)、NF-κB mRNA (2.04±0.29) (P=0.000、0.039、0.012),回肠HMGB1 mRNA (6.70±0.66) (P=0.001、0.038、0.015)、NF-κB mRNA(3.71±0.53) (P=0.000、0.018、0.006)]相比其余3组的升高程度明显降低,其中抗-HMGB1组降低的程度最为显著.抗-HMGB1抗体、抗-MyD88抗体和抗-TRIF抗体的应用能明显减轻缺血-再灌注引起的组织损伤,其中抗-HMGB1抗体的作用最为显著.结论 HMGB1及其下游信号途径在小鼠肠道缺血-再灌注损伤中具有重要作用,在两条下游信号途径中,TRIF依赖途径发挥的作用比MyD88依赖途径更大些.“,”Objective To investigate the effect of different antibodies on Toll-like Receptor 4-High Mobility Group Box 1 and its downstream signal transductions in distant organ injuries caused by intestinal ischemia/reperfusion in mice.Methods A total of 40 mice (C57BL/6,SPF level) were by random number table method assigned into five groups:sham,control,anti-HMGB1,anti-Myeloid differentitation gene,and antiTIR domain containing adaptor inducing IFN-β (n=8).In the control,anti-HMGB1,anti-MyD88,and antiTRIF groups,the IgG,HMGB1,MyD88,and TRIF antibodies were injected,respectively,via the tail vein 30 minutes before ischemia (1 mg/kg body weight,0.025％).After anesthesia and abdomen incision,all mice,except the sham group,underwent intestinal ischemia by clamping the superior mesenteric artery for 60 minutes followed by 60 minutes of reperfusion.Sham group underwent the same surgical procedures except for clamping the artery.Serum nuclear factor-κB p65,Interleukin-6 and Tumor Necrosis Factor-α were measured.Morphological changes in the lung and intestine were evaluated.mRNA and protein expressions of HMGB1 and NF-κB in lung and intestinal tissues were assayed.Results Compared with the control group [(228.53± 24.85),(104.91±31.18),and (70.81±46.97) ng/L],HMGB1 [(145.00±33.63),(62.28±6.73),and (52.76± 5.71) ng/L],MyD88 [(191.12± 13.22),(85.90± 17.37),and (63.19 ± 5.47) ng/L],and TRIF [(183.73±10.81),(78.14±7.38),and (59.70±4.63) ng/L] significantly decreased the serum level of NF-κB (P=0.000,0.005,0.001),IL-6 (P=0.000,0.004,0.000) and TNF-α (P=0.000,0.024,0.002) after ischemia reperfusion.Tissue injuries in the lung and intestine were also alleviated by HMGB1,MyD88,and TRIF.The anti-HMGB1,anti-MyD88,and anti-TRIF groups displayed significant elevations of HMGB1 mRNA [lung (1.89±0.18),(2.35±0.31),and (2.29±0.28),ileum (4.93±0.55),(5.96± 0.73),and (5.76±0.51)],NF-κB mRNA [lung (1.42±0.23),(1.77±0.18) and (1.70±0.13),ileum (2.23±0.55),(3.11±0.38) and (2.99±0.24)] and NF-κB protein expressions in lung and ileum tissues compared to the sham group [lung HMGB1 mRNA (1.04±0.19) (P=0.000,0.000,0.000),NF-κBmRNA (1.03±0.21) (P=0.004,0.000,0.000),ileum HMGB1 mRNA (1.14±0.54) (P=0.000,0.000,0.000),NF-κB mRNA (1.03±0.23) (P=0.000,0.000,0.000)].However,incornparison with the control group [lung HMGB1 mRNA (2.67±0.23) (P=0.000,0.035,0.016),NF-κB mRNA (2.04±0.29) (P=0.000,0.039,0.012),ileum HMGB1 mRNA (6.70±0.66) (P=0.001,0.038,0.015),NF-κBmRNA (3.71±0.53) (P=0.000,0.018,0.006)],the other three groups showed a significant down-regulation,with the most remarkable decrement in the anti-HMGB1 group.Application of anti-HMGB1,anti-MyD88,and anti-TRIF could drastically attenuate the tissue injuries in ischemia reperfusion.anti-HMGB1 exhibited the most significant effect.Conclusions HMGB1 and its downstream signals play an important role in intestinal ischemia reperfusion injuries in mice.Of two downstream signals,the TRIF-dependent pathway exerts a more important effect than that of the MyD88-dependent pathway.