高迁移率族蛋白B1/核因子-κB通路介导小胶质细胞极化在创伤性颅脑损伤后神经损害中的作用及机制

High mobility group protein B1/nuclear factor-κB pathway mediates the role and mechanism of microglial cell polarization in nerve damage after traumatic craniocerebral injury

目的探讨高迁移率族蛋白B1(HMGB1)/核因子-κB(NF-κB)通路介导小胶质细胞极化在创伤性颅脑损伤后神经损害中的作用及机制。方法将120只成年健康雄性SD大鼠,以随机抽签法分成假手术组、模型组、溶剂组以及拮抗剂组。除假手术组外,其余大鼠均建立创伤性颅脑损伤模型。拮抗剂组按50 mg/kg的剂量行腹腔注射甘草酸干预。溶剂组则予以等剂量的溶解剂二甲基亚砜(DMSO)注射。模型组与假手术组予以等剂量的生理盐水注射。比较各组HMGB1/NF-κB通路相关指标表达,不同时间点的改良神经功能评分(mNSS),半胱氨酰天冬氨酸特异性蛋白酶(Caspase)-3、B细胞淋巴瘤/白血病-2相关X蛋白(bax)、B细胞淋巴瘤/白血病-2(bcl-2)蛋白表达。分析大鼠HMGB1/NF-κB通路相关指标与蛋白表达的相关性。结果模型组、溶剂组、拮抗剂组大鼠的HMGB1(0.80±0.21、0.79±0.22、0.48±0.10),NF-κB(1.24±0.26、1.22±0.24、0.43±0.08),Toll样因子受体4(TLR4,0.95±0.18、0.96±0.19、0.71±0.11),Caspase-3(1.27±0.23、1.28±0.21、0.60±0.14),bax(1.43±0.25、1.45±0.26、0.87±0.17),bcl-2(0.38±0.03、0.37±0.02、0.64±0.10)蛋白表达与假手术组(0.24±0.04、0.21±0.03、0.23±0.05、0.41±0.05、0.44±0.06、0.78±0.12)比较,差异有统计学意义(t=14.348、13.472、12.205、21.555、22.872、14.103、21.110、20.351、21.758、20.013、22.074、7.000、21.091、20.732、13.064、17.712、18.459、4.909,P<0.01)。且拮抗剂组大鼠的HMGB1、NF-κB、TLR4、Caspase-3、bax、bcl-2蛋白表达与模型组、溶剂组比较,差异有统计学意义(t=7.535、7.026、16.309、17.104、6.231、6.237、13.629、14.757、10.146、10.226、13.640、14.501,P<0.01)。拮抗剂组3、7、14 d时的mNSS评分为(11.40±1.54)、(8.12±1.06)、(7.11±0.07)分,均显著低于模型组[(13.62±1.88)、(10.94±1.24)、(8.41±1.08)分]及溶剂组[(13.50±1.87)、(10.48±1.39)、(8.36±1.03)分],差异有统计学意义(t=5.003、4.748、9.468、7.395、6.579、6.632,P<0.05)。大鼠HMGB1/NF-κB通路相关指标与伤后3 d时的Caspase-3、bax蛋白表达均呈正相关(r=0.581、0.619、0.633、0.742、0.705、0.652,P<0.05),而与bcl-2蛋白表达均呈负相关(r=-0.563、-0.627、-0.646,P<0.05)。结论HMGB1/NF-κB通路介导小胶质细胞极化在创伤性颅脑损伤后神经损害中的作用机制可能和调控Caspase-3、bax、bcl-2表达水平密切相关。

Objective To investigate the role and mechanism of high mobility group protein B1(HMGB1)/nuclear factor-κB(NF-κB)pathway mediated microglia cell polarization in nerve damage after traumatic craniocerebral injury.Methods In June 2020,Animal Laboratory Center of Comparative Medicine of General Hospital of Eastern Theater of the Chinese People’s Liberation Army,120 healthy male SD rats were randomly divided into sham operation group,model group,solvent group and antagonist group.The models of traumatic brain injury were established in all the rats except the sham operation group.Rats in the antagonist group received intraperitoneal injection of glycyrrhizic acid at a dose of 50 mg/kg.The solvent group was injected with equal dose of solvent dimethylsurfoxide(DMSO).The model group and the sham group were given equal doses of normal saline.The expression levels of HMGB1/NF-κB pathway,modified neurological severity scores(mNSS)and protein expression levels of cysteinyl aspartate-specific protease(Caspase)-3,B cell lymphoma/leukemia-2 associated X protein(bax)and B cell lymphoma/leukemia-2(bcl-2)in each group were compared.The correlation between the related indexes of HMGB1/NF-κB pathway and protein expression were analyzed.Results The protein expression levels of HMGB1(0.80±0.21,0.79±0.22,0.48±0.10),NF-κB(1.24±0.26,1.22±0.24,0.43±0.08),Toll-like receptor 4(TLR4,0.95±0.18,0.96±0.19,0.71±0.11),Caspase-3(1.27±0.23,1.28±0.21,0.60±0.14),bax protein(1.43±0.25,1.45±0.26,0.87±0.17),bcl-2(0.38±0.03,0.37±0.02,0.64±0.10)in the model group,the solvent group and the antagonist group,combined with the sham group(0.24±0.04,0.21±0.03,0.23±0.05,0.41±0.05,0.44±0.06,0.78±0.12),the difference was statistically significant(t=14.348,13.472,12.205,21.555,22.872,14.103,21.110,20.351,21.758,20.013,22.074,7.000,21.091,20.732,13.064,17.712,18.459,4.909,all P<0.01).And the protein expression levels of HMGB1,NF-κB,TLR4,Caspase-3,bax,bcl-2 protein in the antagonist group combined with the model group and the solvent group,the difference was statistically significant(t=7.535,7.026,16.309,17.104,6.231,6.237,13.629,14.757,10.146,10.226,13.640,14.501,all P<0.01).And the mNSS score of the rats in the antagonist group on 3d,7d and 14d was(11.40±1.54),(8.12±1.06),(7.11±0.07)score,combined with the model group[(13.62±1.88),(10.94±1.24),(8.41±1.08)score]and the solvent group[(13.50±1.87),(10.48±1.39),(8.36±1.03)score],the difference was statistically significant(t=5.003,4.748,9.468,7.395,6.579,6.632,all P<0.05).The related indexes of HMGB1/NF-κB pathway were positively correlated with Caspase-3 and bax protein expression at 3 days after injury(r=0.581,0.619,0.633,0.742,0.705,0.652;all P<0.05),but negatively correlated with bcl-2 protein expression(r=-0.563,-0.627,-0.646;all P<0.05).Conclusion The mechanism of HMGB1/NF-κB pathway mediating microglial cell polarization in nerve damage after traumatic brain injury may be closely related to the regulation of Caspase-3,bax and bcl-2 expression levels.