拮抗高迁移率族蛋白B1对烫伤小鼠白细胞介素-35表达及T淋巴细胞免疫功能的影响

Effects of antagonist of high mobility group box-1 protein on interleukin-35 expression and T cell- mediated immunity in mice after thermal injury

目的观察特异性拮抗高迁移率族蛋白B1（HMGB1）对烫伤小鼠白细胞介素-35（IL-35）表达及T淋巴细胞免疫功能的影响。方法选择成年雄性BALB／e小鼠30只，按随机数字表法分为正常组、假伤组、烫伤组、Abox干预组，正常组6只，其余各组每组8只。将小鼠背部皮肤置于97℃恒温热水中6s造成约15％体表面积烫伤模型，Abox干预组小鼠烫伤后2h和12h腹腔注射HMGBl特异性拮抗剂Abo×300ug。烫伤后24h处死小鼠，取心、肝、脾、肺等组织，用酶联免疫吸附试验（ELISA）测定各组织中HMGBl和IL-35含量；用实时荧光定量反转录一聚合酶链反应（qRT-PCR）分析IL-35亚基p35和EB病毒诱导基因3（EBB）的mRNA表达水平；由脾脏提取单个核细胞检测HMGBl和IL-35分泌水平，分离脾脏淋巴细胞检测效应T细胞增殖活性及IL-2、γ-干扰素（IFN-γ）、IL-4分泌水平。结果与烫伤组比较，Abox干预后，各器官HMGBl含量和脾脏单个核细胞HMGB1分泌水平显著降低[心脏（mg／g）：65．78±4．42比110．49±11．19，肝脏（mgCg）：50．90±3．72比117．77±8．10，脾脏（mg／g）：83．34±3．90比149．84±11．93，肺脏（mg／g）：125．15±8．75比236．20±17．51，脾脏单个核细胞（ug／L）：34．57±0．69比73．66±19．03]；各器官组织p35和EBl3的mRNA表达[相对定量（RQ）值]及IL-35蛋白水平均有不同程度下降[p35mRNA（r{Q值）：心脏为0．91±0．62比19．74±18．05，肝脏为0．42±0．19比14．85±8．39，脾脏为1．99±1．12比21．94±6．90，肺脏为0．50±0．21比5．15±3．46；EB13mRNA（RQ值）心脏为0．63±0．61比74．25±15．19，肝脏为0．64±0．09比56．59±19．99，脾脏为1．24±0．48比32．72±3．34，肺脏为0．30±0．06比2．41±1．43；IL-35蛋白（ug，g：心脏为142．40±19．95比241．64±12．40，肝脏为272．68±14．90比409．76±21．60。脾脏为66．94±9．60比92．28±8．82，肺脏为235．88±15．65比193．50±27．06；均P〈0．05），脾脏T淋巴细胞增殖活性增强（A值：1．26±0．26比1．03±0．05）及IL-2分泌水平也显著升高（ng／L：153．48±26．64比101．77±34．55），IFN-γ／IL-4比值显著增加（7．44±1．70比0．33±0．07），差异均有统计学意义（均P〈0．01）。结论特异性拮抗HMGB1可显著下调烧伤小鼠重要组织IL-35表达水平，有效促进脾脏T细胞增殖及Th1功能极化，从而有助于改善严重烧伤后免疫抑制状态。

Objective To investigate the effects of specific antagonist of high mobility group box-1 protein （HMGB1） on interleukin-35 （IL-35） expression and T cell-mediated immunity in mice after major burns. Methods Thirty male adult BALB/c mice were randomly divided into normal control group （n = 6）, sham group （n = 8）, burn group （n = 8） and Abox （specific antagonist of HMGB1） treatment group （n = 8）. The dorsal and lateral surfaces of the mice were shaved for 15% total body surface area （TBSA） under ether anesthesia, and the shaved areas in burn group and Abox treatment group were exposed and immersed in 97 ~C hot water for 6 seconds creating burn models. Abox interference group was given intraperitoneal injection of the specific antagonist of HMGB1 300ug per mouse at 2 hours and 12 hours after burn injury. After modeling for 24 hours, the mice were sacrificed and their hearts, livers, spleens and lungs were harvested and homogenized. Enzyme linked immunosorbent assay （ELISA） was applied to measure the contents of HMGB1 and IL-35. The real time fluorescence quantitative polymerase chain reaction （q-PCR） was used to analyze the mRNA expression levels of the sub-gene p35 of IL-35 and EB virus induced gene 3 （EBI3）. Lymphocytes collected from the spleen were used to detect the effective T cell proliferation activity and secretion levels of IL-2, 3＇ -interferon （IFN- γ ） and IL-4. Results Compared with burn group, the levels of HMGB 1 in different organ tissues and spleen mononuclear cells were significantly decreased after interference of Abox [hearts （mg/g）： 65.78 ± 4.42 vs. 110.49 ± 11.19, livers （rag/g）： 50.90 ± 3.72 vs. 117.77 ± 8.10, spleens （mg/g）： 83.34 ± 3.90 vs. 149.84 ± 11.93, lungs （rag/g）： 125.15 ± 8.75 vs. 236.20 ± 17.51, spleen mononuelear cells （txg/L）： 34.57 ± 0.69 vs. 73.66 ± 19.03, all P 〈 0.01]; the mRNA expressions of p35 and EBI3 and protein levels of IL-35 in various organ tissues were decreased to certain different degrees [hearts （p35 mRNA）： 0.91 ± 0.62 vs. 19.74 ± 18.05, livers （p35 mRNA）： 0.42 ± 0.19 vs. 14.85 ± 8.39, spleens （p35 mRNA）： 1.99 ± 1.12 vs. 21.94 ± 6.90, lungs （p35 mRNA）： 0.50 ± 0.21 vs. 5.15 ± 3.46; hearts （EBI3 mRNA）： 0.63 ± 0.61 vs. 74.25 ± 15.19, livers （EBI3 mRNA）： 0.64 ± 0.09 vs. 56.59 ± 19.99, spleens （EBI3 mRNA）： 1.24 ± 0.48 vs. 32.72 ± 3.34, lungs （EBI3 mRNA）： 0.30 ± 0.06 vs. 2.41 ± 1.43 ; hearts （IL-35 protein, p^g/g）： 142.40± 19.95 vs. 241.64 ± 12.40, livers （IL-35 protein, ug/g）： 272.68± 14.90 vs. 409.76± 21.6, spleens （IL-35 protein, txg/g）： 66.94±9.60 vs. 92.28±8.82, lungs （IL-35 protein, Ixg/g）： 235.88± 15.65 vs. 193.50 ± 27.06, all P 〈 0.05], the T cell proliferate activity was enhanced （A value： 1.26 ± 0.26 vs. 1.03 ± 0.05）, the secretion level of IL-2 was signifieantly elevated （ng/L ： 153.48 ± 26.64 vs. 101.77 ± 34.55） and the IFN- γ/IL-4 ratio was obviously increased （7.44± 1.70 vs. 0.33 ± 0.07）, all the differenees being statistically significant （all P 〈 0.01）. Conclusions The specific antagonist of HMGB1 can obviously down-regulate the IL-35 expression in vital organs and effectively promote the T eell proliferation and the Thl cell function polarization in spleen in mice with thermal injury, in turn eontributing to improving the immune inhibitory status after severe burn.