幼鼠脓毒症模型中Rankl调控胸腺自然调节性T细胞免疫平衡的作用机制研究

Mechanism of Rankl regulating immune balance of thymus natural regulatory T cells in the rat model of sepsis

目的探讨核因子κB受体激活因子配体(receptor activator of nuclear factor kappa-B ligand,Rankl)对脓毒症幼鼠胸腺自然调节性T细胞(regulatory T cell,Treg)的免疫调控机制。方法采用脂多糖(lipopolysaccharide,LPS)腹腔注射构建SD雄性幼鼠脓毒症模型。48只幼鼠按LPS浓度梯度分为4组,分别注射生理盐水(对照组)和5、8、10 mg/kg LPS,每组12只。24只幼鼠按给药分组,随机分为对照组、脓毒症组、野黄芩苷(Rankl抑制剂)组、白桦脂酸(NF-κB激动剂)组共4组,每组6只。取胸腺组织行免疫组化染色,计算Rankl、自身免疫调节因子(autoimmunity regulator,Aire)、叉头翼状螺旋转录因子(forkhead box P3,Foxp3)阳性细胞的平均光密度。胸腺组织匀浆免疫印迹法测定Rankl、核因子κB(nuclear factor kappa-B,NF-κB)p65、Aire、Foxp3的蛋白表达水平。统计学分析采用单因素方差分析和t检验。结果不同浓度梯度模型中,对照组与腹腔注射LPS 5、8、10 mg/kg组,48 h时胸腺组织阳性细胞平均光密度比较[Rankl:(0.209±0.021)%、(0.256±0.008)%、(0.302±0.015)%、(0.361±0.023)%;Aire:(0.279±0.017)%、(0.350±0.021)%、(0.402±0.013)%、(0.459±0.024)%;Foxp3:(0.207±0.014)%、(0.237±0.010)%、(0.277±0.011)%、(0.310±0.016)%],蛋白表达水平比较(Rankl:0.577±0.079、0.740±0.079、0.935±0.043、1.240±0.109;NF-κBp 65:0.150±0.064、0.306±0.055、0.534±0.077、0.949±0.077;Aire:0.324±0.039、0.571±0.062、0.737±0.091、1.019±0.120;Foxp3:0.226±0.098、0.475±0.035、0.824±0.070、1.148±0.087),LPS造模组均高于对照组(P值均<0.05);且随LPS剂量增加而增加,10 mg/kg组增加最明显。不同给药模型中,脓毒症组、野黄芩苷组、白桦脂酸组的胸腺组织阳性细胞平均光密度比较[Rankl:(0.343±0.022)%、(0.262±0.014)%、(0.299±0.020)%;Foxp3:(0.380±0.016)%、(0.340±0.013)%、(0.426±0.012)%;Aire:(0.671±0.079)%、(0.437±0.109)%、(0.893±0.034)%],蛋白表达水平比较(Rankl:0.945±0.059、0.626±0.072、0.801±0.052;NF-κB p65:0.671±0.079、0.633±0.191、1.229±0.106;Aire:0.815±0.144、0.437±0.109、1.219±0.114;Foxp3:0.773±0.093、0.453±0.143、1.256±0.086),野黄芩苷组均低于脓毒症组,白桦脂酸组除了Rankl,其余指标均高于脓毒症组(P值均<0.05)。结论在幼鼠脓毒症模型中,Rankl通过激活NF-κB/Aire/Foxp3信号通路使胸腺Treg大量增殖,发生免疫失调。Rankl抑制剂野黄芩苷可调节此失衡状态,对脓毒症幼鼠产生保护效应。

Objective To investigate the immune regulation mechanism of receptor activator of nuclear factor kappa-B ligand(Rankl)on thymus natural regulatory T cells(Treg)in juvenile rats with sepsis.Method The sepsis model of SD male juvenile rats was established by means of lipopolysaccharide(LPS)intra-abdominal injection.48 rats were divided into 4 groups according to the concentration gradient of LPS,12 rats in each group were injected with normal saline(control group)and LPS(5,8,10 mg/kg).24 rats were randomly divided into control group,sepsis group,scutellarin(Rankl inhibitor)group and belobulinic acid(NF-κB agonist)group,with 6 rats in each group.Immunohistochemical staining was performed on thymus tissues,and the average optical density of Rankl,autoimmunity regulator(Aire),forkhead box P3(Foxp3)positive cells was calculated.The protein expression levels of Rankl,nuclear factor kappa-B(NF-κB)p65,Aire and Foxp3 were determined by Western blotting in thymus tissues.One-way analysis of variance and t test were used for statistical analysis.Result In different concentration gradient models,the average optical density of thymus positive cells at 48 hours was compared between the control group and the intraperitoneal injection group of LPS(5,8,10 mg/kg)[Rankl:(0.209±0.021)%,(0.256±0.008)%,(0.302±0.015)%,(0.361±0.023)%;Aire:(0.279±0.017)%,(0.350±0.021)%,(0.402±0.013)%,(0.459±0.024)%;Foxp3:(0.207±0.014)%,(0.237±0.010)%,(0.277±0.011)%,(0.310±0.016)%];so was protein expression(Rankl:0.577±0.079,0.740±0.079,0.935±0.043,1.240±0.109;NF-κB p65:0.150±0.064,0.306±0.055,0.534±0.077,0.949±0.077;Aire:0.324±0.039,0.571±0.062,0.737±0.091,1.019±0.120;Foxp3:0.226±0.098,0.475±0.035,0.824±0.070,1.148±0.087);LPS modeling groups were higher than control group(all P<0.05).It increased with the increase of LPS dosage,and the increase was most obvious in 10 mg/kg group.Comparison of average optical density of thymus positive cells in sepsis group,scutellarin group and betulinic acid group in different administration models[Rankl:(0.343±0.022)%,(0.262±0.014)%,(0.299±0.020)%;Foxp3:(0.380±0.016)%,(0.340±0.013)%,(0.426±0.012)%;Aire:(0.671±0.079)%,(0.437±0.109)%,(0.893±0.034)%],comparison of protein expression levels(Rankl:0.945±0.059,0.626±0.072,0.801±0.052;NF-κB p65:0.671±0.079,0.633±0.191,1.229±0.106;Aire:0.815±0.144,0.437±0.109,1.219±0.114;Foxp3:0.773±0.093,0.453±0.143,1.256±0.086),scutellarin group was lower than sepsis group,betulinic acid group was higher than sepsis group except Rankl(all P<0.05).Conclusion In sepsis model,Rankl activates the NF-κB/Aire/Foxp3 signaling pathway to induce the proliferation of thymus Treg,resulting in immune dysregulation.Rankl inhibitor scutellarin can regulate the imbalance and produce protective effect on juvenile rats with sepsis.