大鼠出生早期LPS暴露远隔抑制成年期EAE发病与巨噬细胞免疫功能抑制有关

Reduce of experimental autoimmune encephalomyelitis susceptivity after lipopolysaccharide exposure in neonatal rats and its relation with inhibition of macrophage immune function

目的探讨大鼠出生早期经脂多糖（LPS）暴露远隔抑制成年期实验性自身免疫性脑脊髓炎（EAE）发病的机制是否与外周单核细胞来源巨噬细胞免疫功能抑制有关。方法同窝雄性新生SD大鼠按随机数字表法分为3组：空白对照组（n=11）、磷酸盐缓冲液（PBS）组（n=12）、LPS组（n=12），其中LPS组于出生后第3、5天腹腔注射50此含非致死剂量LPS的PBS溶液，空白对照组及PBS组同期注射50μL无菌PBS溶液；PBS组、LPS组于12周龄用豚鼠脊髓匀浆与完全弗氏佐剂混合乳液免疫诱导制作EAE模型，空白对照组仅用完全弗氏佐剂乳液进行免疫诱导。从免疫诱导当天开始持续观察记录各组大鼠行为学评分，并在发病高峰期处死大鼠，采用HE染色观察脊髓组织病理学改变。采用流式细胞术检测外周单核细胞来源巨噬细胞中枢浸润数量及其CD86、CD80、主要组织相容性复合体（MHC）-Ⅱ表达水平，采用荧光定量PCR检测巨噬细胞CC型趋化因子受体2（CCR2）、白介素．16（IL—1β）和肿瘤坏死因子-α（TNF-α）mRNA表达。结果与PBS组比较，LPS组EAE发病率下降（11／12vs．5／12），发病潜伏期延长[（10．50±0．71）d％（12．17±1．17）d]，累积行为学评分与最高行为学评分下降（20．00±9．13vs．5．58±7．12；2．58±1．08vs1．03±0．83）．HE染色切片评分降低（2．83±0．75vs.1．17±1．17），巨噬细胞中枢浸润数量减少[（202．70±81．89）×10^3个vs．（92．58±42．65）×10^3个]，巨噬细胞CDS0、MHC-II表达水平降低，巨噬细胞CCR2、IL^-1β、TNF-α mRNA表达降低，差异均有统计学意义（P〈O．05）。结论大鼠出生早期LPS暴露可远隔抑制成年期EAE发病，其内在机制可能与巨噬细胞免疫功能抑制有关。

Objective To investigate the reduce of experimental autoimmune encephalomyelitis （EAE） susceptivity after lipopolysaccharide （LPS） exposure in neonatal rats and its relation with inhibition of macrophage immune function. Methods Litter male neonatal rats were divided into blank control group （n=11）, phosphate buffer （PBS） group （n=12） and LPS group （n=12）; rats in the LPS group were performed intraperitoneal injection of 50 μL LPS on postpartum day 3 and 5. Rats in the PBS group and LPS group were established EAE models by immune induce of spinal cord homogenate of guinea pig with complete Freund＇s adjuvant at 12 weeks old, while rats in the blank control group were given immune induce of complete Freund＇s adjuvant. Since the day of induce, the behavioral scale scores of all the rats were assessed. On the 20^th d, all rats were sacrificed and paraffin sections were cut and stained with HE to detect inflammatory cell infiltration. The number of infiltrating macrophages and the expressions of CD86, CD80, MHC-II were determined by flow cytometry. The transcriptional levels of C-C chemokine receptor type 2 （CCR2）, interleukin （IL）-1β and tumor necrosis factor （TNF）-α in macrophages were determined by real-time fluorogenic quantitative PCR. Results As compared with the PBS group, LPS group had significantly lower EAE incidence （11/12 vs. 5/12） and higher EAE latency （[ 10.50±0.71 ] d vs. [ 12.17± 1.17] d）, and statistically decreased cumulative clinic scores and peak clinic scores （20.00±9.13 vs. 5.58±7.12; 2.58±1.08 vs. 1.03±0.83, P〈0.05）. The inflammation in the spinal cord of the PBS group was severer than that in the LPS group （2.83±0.75 vs. 1.17±1.17, P〈0.05）. The number of infiltrating macrophages of the PBS group was significantly larger than that in the LPS group （[202.70±81.89] ×10^3 cells vs. [92.58 ±42.65]×10^3 cells, P〈0.05）. The expressions of CD80 and MHC-II, and mRNA levels ofCCR2, IL^-1β and TNF-α in the PBS group were significantly higher than those in the LPS group （P〈0.05）. Conclusion Neonatal LPS exposure reduces the severity of EAE, which may relate to impaired macrophage immune function.