口服IL-12重组双歧杆菌对柯萨奇B3病毒诱导BLAB/c小鼠心肌炎的影响

Bifidobacteriumas an oral delivery carrier of interleukin-12 for the treatment of coxsackievirus B3-induced myocarditis in the BALB/c mice

目的构建白细胞介素(IL)-12基因重组双歧杆菌(pBBADs-IL-12转化双歧杆菌),观察pBBADs-IL-12转化双歧杆菌是否对柯萨奇B3病毒(CVB3)诱导的小鼠心肌炎具有治疗作用。方法构建小鼠IL-12(mIL-12)基因的pBBADs-IL-12表达载体,转化双歧杆菌,体外通过酶联免疫吸附试验及Western免疫印迹验证重组双歧杆菌工程菌在L-阿拉伯糖诱导下mIL-12的表达。选取BLAB/c小鼠30只,腹腔内注射CVB3感染剂量,14 d后形成病毒性心肌炎,将感染的小鼠随机分成IL-12组、绿荧光蛋白(GFP)组及生理盐水组。IL-12组给予口服pBBADs-IL-12转化双歧杆菌;GFP组给予口服pBBADs-GFP转化双歧杆菌;生理盐水组给予腹腔注射无菌PBS(1次/d)。所有小鼠均在治疗14 d后取心脏标本观察心肌病理变化,检测心肌病毒滴度,荧光定量PCR分析Th1细胞因子水平。结果治疗14 d后,HE染色显示IL-12组小鼠心肌炎症程度较GFP组和生理盐水组明显减轻;IL-12组小鼠心脏炎症病变百分比为(18±5)%,心肌的病毒滴度为(2.89±0.18)pfu/g,显著低于GFP组[分别为(31±6)%和(4.83±0.14)pfu/g]及生理盐水组[分别为(32±9)%和(4.80±0.15)pfu/g],差异有统计学意义(均P<0.01);IL-12组小鼠心脏γ-干扰素[(2.27±0.15)pg/mL]和肿瘤坏死因子-α[(3.05±0.17)pg/mL]水平显著高于GFP组[分别为(1.32±0.11)pg/mL和(2.37±0.16)pg/mL]及生理盐水组[分别为(1.38±0.11)pg/mL和(2.37±0.12)pg/mL],差异有统计学意义(均P<0.01)。结论此次研究成功构建了一种新型表达mIL-12的双歧杆菌载体,口服IL-12转化双歧杆菌对CVB3病毒诱导的小鼠心肌炎具有较好疗效。

Objective To develop a novel oral delivery system for interleukin-12（IL-12）using genetically engineered Bifidobacterium longum（B.longum）as the carrier and further evaluate the efficacy of IL-12-expressed B.longumon the coxsackievirus B3（CVB3）-induced myocarditis in mice.Methods A mIL-12 gene expression vector pBBADs-IL-12 for B.longum was constructed and transformed into Bifidobacterium,the expression of mIL-12in the engineered B.longum was identified in vitro by Western Blot and enzyme-linked immunosorbent assay（ELISA）after L-arabinose induction.BLAB/c mice were inoculated i.p.with infectious dose of CVB3 for fourteen days and were divided randomly into three groups.The IL-12 group and green fluorescent protein group（GFP group）were orally administered with pBBADs-IL-12 and pBBADs-GFP transformed B.longumfor fourteen days respectively after the inoculation of the virus;saline group was administered i.p.with sterile PBS.All animals were killed in day14 of treatment,and the murine hearts were dissected aseptically for hematoxylin-eosin（HE）staining,viral titer and RNA extraction for Th1 cell cytokines quantification.Results After 14 days of treatment,HE staining revealed that the severity of virus-induced myocarditis in IL-12 group was reduced compared with that of GFP group and saline group;the percentage of cardiac pathological lesions and CVB3 titers in IL-12 group was（18±5）% and（2.89±0.10）pfu/g respectively,which was significantly lower than that of GFP group（[31±6]%,[4.83±0.14]pfu/g）and saline group（[32±9]%,[4.80±0.15]pfu/g）,respectively（all P0.01）;levels of interferon-γ（IFN-γ）and tumor necrosis factor-α（TNF-α）in cardiac tissue and supernatants of IL-12 group was（2.27±0.15）pg/mL and（3.05±0.17）pg/mL,respectively,which was significantly higher than that of GFP group（[1.32±0.11]pg/mL,[2.37±0.16]pg/mL）and saline group（[1.38±0.11]pg/mL,[2.37±0.12]pg/mL）,respectively（all P0.01）.Conclusion A novel oral delivery system of Bifidobacteriumfor murine IL-12 has been successfully established.Oral administration of mIL-12-transformed B.longum may play a therapeutic role in the treatment of CVB3-induced myocarditis in the mice.