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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 effect and mechanism of B7 homologue 3 (B7-H3) on the inflammatory response and the integrity of the blood-brain barrier in Streptococcus pneumoniae meningitis rats. Methods The SP suspension was injected into the lateral ventricle of mice to prepare meningitis model. Wild-type mice were divided into 5 groups: PBS group, SP group, SP + B7-H3 group, SP + isotype group and SP + B7-H3 group. Toll-like receptor 2 gene knockout (TLR2-KO) rats were divided into 3 groups: PBS group, SP group, SP + B7-H3 protein group. After 6 h, 18 h, 30 h, the orbital blood was collected for anesthesia, and the brain tissue was harvested to prepare brain tissue homogenate. The level of serum TNF-α, IL-6, IL-1β and MCP-1 in serum were detected by ELISA. The levels of albumin and IgG in brain homogenate were measured. The expression of TNF-α, IL-6 and MCP-1 in serum SP group and SP + B7-H3 group after 18 h of injection were detected by ELISA and TNF-α, IL-6 and IL- (P <0.05). Compared with SP group, there was no significant difference in the expression of inflammatory cytokines at each time point between SP + The expression of MCP-1 at 18 h after injection of SP + B7-H3 and the expression of TNF-α and IL-6 at 30 h were significantly higher than those in SP group [(42.010 ± 3.883) ng.L-1 vs (29.620 ± 3.830) (66.560 ± 5.766) ng.L-1 vs (48.630 ± 4.418) ng.L-1, (37.550 ± 3.232) ng.L-1 vs (24.570 ± 2.377) ng.L- Pa <0.05]. The levels of TNF-α and IL-6 in SP + B7-H3 blocking mAb group were significantly lower than those in SP group [(18.680 ± 1.798) ng.L-1 vs ± 2.377) ng.L-1; (37.180 ± 3.150) ng.L-1 vs (48.630 ± 4.418) ng.L-1, Pa <0.05]. ELISA detection of brain tissue homogenate: SP group, SP + B7-H3 group 18 h, 30 h after injection of Albumin, IgG were significantly higher than the PBS group (Pa; 0.05); SP + (P> 0.05). In the SP + B7-H3 group, the expression of Albumin in brain homogenate after 18 h and 30 h and the expression of IgG in brain homogenate after 30 h (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]. The expression of Albumin and the expression of 30-h IgG at 18 h and 30 h after SP + B7-H3 blocking mAb injection were significantly lower than those in SP group [(20.590 ± 1.720) μg.g-1 vs (35.450 ± 4.256 (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]. ELISA detection of TLR2-KO serum and brain tissue homogenate showed that there was no significant difference in the expression of each monitoring index between SP + B7-H3 group and SP group at any time point (P> 0.05). Conclusion B7-H3 promotes the inflammatory response and the destruction of the blood-brain barrier in the SP-induced meningitis mice via a TLR2-dependent mechanism.