B7同源体3经Toll样受体2依赖性机制增强肺炎链球菌脑膜炎小鼠的炎性反应和病理损伤

B7 Homolog 3 Promote Inflammatory Response and Pathological Injury of Streptococcus Pneumoniae Meningitis via A Toll-Like Receptor 2 Dependent Mechanism in Mice

目的探讨B7同源体3(B7-H3)对肺炎链球菌(SP)脑膜炎大鼠的炎性反应和血脑屏障完整性的影响及其机制。方法经小鼠侧脑室穿刺注入SP悬液,制备脑膜炎模型。野生型鼠分5组,分别为PBS组、SP组、SP+B7-H3组、SP+同型单抗组、SP+B7-H3阻断性单抗组。Toll样受体2基因剔除(TLR2-KO)鼠分3组:PBS组、SP组、SP+B7-H3蛋白组。术后6 h、18 h、30 h,麻醉其下眼眶采血法收集血清,取脑组织,制备脑组织匀浆。ELISA检测其血清TNF-α、IL-6、IL-1β和单核细胞趋化因子蛋白-1(MCP-1)水平以及脑组织匀浆中血清蛋白(Albumin)和IgG水平。结果 1.ELISA检测野生型鼠血清SP组、SP+B7-H3组注射18 h后TNF-α、IL-6和MCP-1的表达及30 h后TNF-α、IL-6、IL-1β和MCP-1的表达均较PBS组显著升高(Pa<0.05);SP+同型单抗组与SP组比较,各时间点各炎性因子的表达差异均无统计学意义(Pa>0.05);SP+B7-H3组注射18 h后MCP-1的表达及30 h后TNF-α和IL-6表达均显著高于SP组[(42.010±3.883)ng.L-1 vs(29.620±3.830)ng.L-1;(37.550±3.232)ng.L-1 vs(24.570±2.377)ng.L-1;(66.160±5.766)ng.L-1 vs(48.630±4.418)ng.L-1,Pa<0.05];SP+B7-H3阻断性单抗组注射30 h后,TNF-α和IL-6的表达均显著低于SP组[(18.680±1.798)ng.L-1 vs(24.570±2.377)ng.L-1;(37.180±3.150)ng.L-1 vs(48.630±4.418)ng.L-1,Pa<0.05]。2.ELISA检测脑组织匀浆:SP组、SP+B7-H3组注射18 h、30 h后Albumin、IgG的表达较PBS组均显著升高(Pa<0.05);SP+同型单抗组与SP组比较,各时间点Albumin、IgG的表达差异均无统计学意义(Pa>0.05);SP+B7-H3组注射18 h、30 h后脑组织匀浆Albumin的表达及30 h后脑组织匀浆IgG的表达均显著高于SP组[(59.090±4.184)μg.g-1vs(35.450±4.256)μg.g-1;(59.890±4.701)μg.g-1 vs(43.790±3.508)μg.g-1;(36.220±2.775)μg.g-1 vs(25.440±2.620)μg.g-1,Pa<0.05]。3.SP+B7-H3阻断性单抗组注射后18 h、30 h Albumin的表达及30 h IgG的表达显著低于SP组[(20.590±1.720)μg.g-1 vs(35.450±4.256)μg.g-1;(28.650±3.063)μg.g-1 vs(43.790±3.508)μg.g-1;(17.380±1.595)μg.g-1 vs(25.440±2.620)μg.g-1,Pa<0.05]。3.TLR2-KO鼠血清和脑组织匀浆的ELISA检测显示:SP+B7-H3组较SP组各监测指标的表达在任何时间点的差异均无统计学意义(Pa>0.05)。结论 B7-H3通过TLR2依赖性机制促进SP诱导的脑膜炎小鼠的炎性反应和血脑屏障的破坏。

Objective To investigate the impact of B7 homolog 3（B7-H3） on inflammatory response and blood-brain barrier integrity during streptococcus pneumoniae（SP）meningitis,and explore its mechanism. Methods SP meningitis was established by intracerebral ventricular injection of SP suspension.Wild-type mice were randomly divided into 5 groups and received following injections：PBS,SP alone,SP plus recombinant murine B7-H3,SP plus control IgG mAb,or SP plus anti-B7-H3 blocking mAb.Toll-like receptor 2（TLR2）-deficient mice were divided into 3 groups and received PBS,SP alone,or SP plus recombinant murine B7-H3 injection.At 6 h,18 h,and 30 h post infection,mice anesthetized,blood sample in mice eyes and brains were collected,and brain homogenates were prepared.Tumor necrosis factor-alpha（TNF-α）,interleukin-6（IL-6）,interleukin-1 beta（IL-1β）,and monocyte chemoattractant protein-1（MCP-1） le-vels in serum,and albumin and IgG levels in brain homogenates were assessed by enzyme-linked immunosorbent assay（ELISA）. Results 1.In wild-type mice,injection with SP alone and SP plus recombinant murine B7-H3 led to higher TNF-α,IL-6 and MCP-1 levels in serum at 18 h,and higher TNF-α,IL-6,IL-1β and MCP-1 levels in serum at 30 h,as compared with PBS injection group（Pa〈0.05）.Compared with SP injection group,levels of any cytokines in serum at each time point were not changed after SP plus control IgG mAb group（Pa〉0.05）.SP plus recombinant murine B7-H3 injection induced higher MCP-1 level at 18 h,and higher TNF-α and IL-6 levels at 30 h in serum,as compared with SP injection group[MCP-1：（42.010±3.880） ng·L-1 vs（29.620±3.830） ng·L-1;TNF-α：（37.550±3.232） ng·L-1 vs（24.570±2.377） ng·L-1;IL-6：（66.160±5.766） ng·L-1 vs（48.630±4.418） ng·L-1,Pa〈0.05].2.In SP plus anti-B7-H3 blocking mAb group,at 30 h,TNF-α and IL-6 levels in serum were lower than those in SP injection group[（18.680±1.798） ng·L-1 vs（24.570±2.377） ng·L-1;（37.180±3.150） ng·L-1vs（48.630±4.418） ng·L-1,Pa〈0.05];injection with SP alone and SP plus recombinant murine B7-H3 led to higher levels of albumin and IgG in serum at 18,30 h,as compared with PBS injection group（Pa〈0.05）.Compared with SP injection group,levels of albumin and IgG in brain at each time point were not changed after SP plus control IgG mAb injection group（Pa〉0.05）.SP plus recombinant murine B7-H3 injection increased albumin level at 18,30 h,and IgG le-vel at 30 h in brain,as compared with SP injection group[albumin：（59.090±4.184） μg·g-1 vs（35.450±4.256） μg·g-1;（59.890±4.701） μg·g-1 vs（43.790±3.508） μg·g-1;IgG：（36.220±2.775） μg·g-1 vs（25.440±2.620） μg·g-1,Pa〈0.05].In SP plus anti-B7-H3 blocking mAb group,levels of albumin at 18,30 h,and IgG at 30 h in brain were lower than those in SP injection group [Albumin：（20.590±1.720） μg·g-1 vs（35.450±4.256） μg·g-1;（28.650±3.063） μg·g-1 vs（43.790±3.508） μg·g-1;IgG：（17.380±1.595） μg·g-1 vs（25.440±2.620） μg·g-1,Pa〈0.05].3.In TLR2-deficient mice,SP plus recombinant murine B7-H3 injection failed to further enhance proinflammatory cytokines further,albumin and IgG release in serum and brain homogenates compared with SP injection alone group（Pa〉0.05）. Conclusions B7-H3 enhances inflammatory response and exacerbates blood-brain barrier injury via a TLR2-dependent mechanism during SP meningitis in mice.