“二次打击”脓毒症继发肺炎大鼠模型的构建与评价

Reproduction of a model of ＂two-hit＂ sepsis model with complication of pneumonia in rat

目的：模拟临床脓毒症继发感染的发病情况，构建“二次打击”动物模型，评价“二次打击”对机体损害及免疫功能的影响。方法81只雄性SD大鼠按随机数字表法分组，其中45只分为单纯脓毒症组及脓毒症继发肺炎4 d、7 d组，每组15只，用于观察生存情况；另外36只分为正常对照组，单纯脓毒症1、4、7 d组，脓毒症继发肺炎4 d、7 d组，每组6只，用于指标检测。采用盲肠结扎穿孔术（CLP）建立脓毒症模型；于CLP术后4 d或7 d分别经鼻腔注射肺炎链球菌（含菌量1×1010 cfu/mL）继发肺部感染构建“二次打击”模型。分别于相应时间点以及给菌后1 d处死大鼠，收集血液及脾脏组织，进行外周血菌落计数、脾脏细胞计数，检测血清生化指标及相关细胞因子水平，观察脾脏病理改变及细胞凋亡情况。结果①与单纯脓毒症组同期比较，脓毒症继发肺炎4 d组大鼠存活数明显减少（只：4比11，χ2＝6.533，P＝0.011），而7 d组则差异无统计学意义（只：9比11，χ2＝0.600，P＝0.439）。②单纯脓毒症组术后1 d外周血菌落计数、生化指标均明显升高，随后逐渐下降；术后4 d时脾脏免疫细胞计数已明显低于1 d〔树突细胞（DC）：（0.69±0.09）%比（0.87±0.31）%， CD4＋ T细胞：（21.05±2.89）%比（24.84±4.59）%，CD8＋ T细胞：（10.62±1.79）%比（13.40±1.31）%，均P＜0.05〕，而调节性T细胞（Treg）明显高于正常对照组〔（3.14±0.74）%比（2.87±1.08）%，P＜0.05〕；术后7 d血生化指标丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）、尿素氮（BUN）、血肌酐（SCr）较1 d明显下降〔ALT（U/L）：35.33±11.52比81.00±38.40，AST（U/L）：70.33±42.16比156.00±28.11，BUN（mmol/L）：5.30±2.27比9.13±4.04，SCr（μmol/L）：55.33±10.67比96.67±45.79，均P＜0.05〕；血清促炎因子肿瘤坏死因子-α（TNF-α）、白细胞介素（IL-6、IL-1β）高峰出现在术后1 d〔TNF-α为（18.03±2.88）ng/L，IL-6为（10.37±4.20）ng/L，IL-1β为（102.44±51.46）ng/L〕，高迁移率族蛋白B1（HMGB1）高峰出现在术后4 d〔（1.76±0.71）μg/L〕；抗炎因子转化生长因子-β1（TGF-β1）和可溶性肿瘤坏死因子受体-Ⅰ（sTNFR-Ⅰ）于脓毒症后期仍维持在较高水平〔术后7 d TGF-β1为（0.90±0.56）ng/L，sTNFR-Ⅰ为（1.56±0.39） ng/L〕。苏木素-伊红（HE）染色显示，单纯脓毒症组CLP术后脾脏组织病理改变随时间延长逐渐加重，术后4 d脾脏凋亡细胞数明显高于术后1 d（个/HP：52.99±20.79比16.05±3.28，P＜0.05）。③与单纯脓毒症组同期比较，脓毒症继发肺炎4 d组外周血菌落数明显增多（log cfu/mL：1.78±0.54比0.25±0.18，P＜0.05），而7 d组则差异无统计学意义（log cfu/mL：0.57±0.46比0.13±0.12，P＞0.05）。脓毒症继发肺炎4 d、7 d组各脾脏免疫细胞计数及生化指标变化趋势与单纯脓毒症组相似，虽较单纯脓毒症组同期有所减少，但差异均无统计学意义。与单纯脓毒症组同期比较，脓毒症继发肺炎4 d组HMGB1进一步减少（μg/L：1.17±0.74比1.76±0.71，P＜0.05），脓毒症继发肺炎7 d组IL-1β进一步升高（ng/L：105.73±25.06比61.04±31.29，P＜0.05），其余炎症因子水平则差异无统计学意义。脓毒症继发肺炎4 d组脾脏凋亡细胞较单纯脓毒症组同期明显增加（个/HP：74.48±22.47比52.99±20.79，P＜0.05），而7 d组则差异无统计学意义（个/HP：28.70±4.13比30.43±14.55， P＞0.05）。结论 CLP术后4 d继发肺炎“二次打击”模型较单纯CLP致脓毒症模型死亡大鼠明显增加，细菌清除力下降，免疫细胞数量减少且凋亡加重，这可能与病程中期机体存在的免疫麻痹有关。CLP术后7 d继发肺炎“二次打击”模型表现为宿主免疫功能重构，可能与免疫应答能力逐渐恢复有关。

ObjectiveTo reproduce a clinically relevant ＂two-hit＂ model of sepsis complicated by pneumonia and to explore the correlation between ＂two-hit＂ and immune state.Methods Eighty-one male Sprague-Dawley （ SD ） rats were divided into groups according to the random number table. Forty-five male rats were assigned respectively to sepsis-alone group, pneumonia 4 days and 7 days after sepsis groups, respectively. Survival rate of each group was observed. Another group of 36 male rats were divided into normal control group, sepsis-alone for 1, 4 and 7 days groups, and sepsis complicated by pneumonia for 4 days and 7 days after sepsis groups, each group consisted of 6 rats. Cecal ligation and puncture （CLP） was done in rats, andStreptococcus pneumoniae suspension （bacteria count 1×1010 cfu/mL） was injected via the nose on the 4th day or 7th day after CLP. Rats were sacrificed at corresponding time points, and 1 day after challenge ofStreptococcus pneumoniae on the 4 days or 7 days post CLP for the collection of blood and tissue samples to make bacterial count of the blood, splenocyte count, biochemical indices, cytokines concentration, pathological changes in spleen and apoptotic cells.Results① Compared with the rats of sepsis-alone group, the rats in pneumonia 4 days after CLP group had poor survival rate （4 vs. 11,χ2 = 6.533,P = 0.011）, while no difference was found between pneumonia 7 days after CLP group and sepsis-alone group （9 vs. 11,χ2 = 0.600,P = 0.439）.② The blood bacterial count and all the biochemical indexes were sharply increased on 1 day post-CLP in the rats of sepsis-alone group, and then they gradually lowered. Compared with the rats of 1 day post-CLP, the proportion of splenocytes were decreased on the 4th day post-CLP [dendritic cells （DC）： （0.69±0.09）% vs. （0.87±0.31）%, CD4＋T cells： （21.05±2.89）% vs. （24.84±4.59）%, CD8＋ T cells： （10.62±1.79）% vs. （13.40±1.31）%, allP〈 0.05], but T-regulatory cell （Treg） count was higher on the 4th day after CLP compared with sepsis-alone rats [（3.14±0.74 ）% vs. （2.87±1.08）%,P〈 0.05]. The biochemical indices, including alanine transaminase （ALT）, aspartate aminotransferase （AST）, blood urea nitrogen （BUN）, and serum creatinine （SCr） were obviously lowered on 7 days post-CLP compared with 1 day after CLP [ALT （U/L）： 35.33±11.52 vs. 81.00±38.40, AST （U/L）： 70.33±42.16 vs. 156.00±28.11, BUN （mmol/L）： 5.30±2.27 vs. 9.13±4.04, SCr （μmol/L）： 55.33±10.67 vs. 96.67±45.79, allP〈 0.05]. The serum levels of tumor necrosis factor-α （TNF-α） and interleukins （IL-6, IL-1β） peaked on the 1st day after CLP [TNF-α：（18.03±2.88） ng/L, IL-6： （10.37±4.20） ng/L, IL-1β： （102.44±51.46） ng/L], and high mobility group box-1 （HMGB1） peaked on the 4th day after CLP [（1.76±0.71）μg/L]. The levels of anti-inflammatory cytokines transforming growth factor-β1 （TGF-β1 ） and soluble tumor necrosis factor receptor-Ⅰ （sTNFR-Ⅰ） maintained at high levels [7 days post-CLP： TGF-β1 was （0.90±0.56） ng/L, sTNFR-Ⅰ was （1.56±0.39） ng/L]. The spleen pathology became more marked with the time in the group of sepsis-alone, meanwhile the number of apoptotic spleencytes increased 4 days post-CLP as compared with that of the 1st day post-CLP （cells/HP： 52.99±20.79 vs. 16.05±3.28,P〈 0.05）.③ Compared with the same period of sepsis-alone group, the rats with pneumonia 4 days post-CLP group showed a higher blood bacterial count （log cfu/mL： 1.78±0.54 vs. 0.25±0.18,P〈 0.05）, while no difference was found between 7-day of post-CLP pneumonia group and sepsis-alone group （log cfu/mL： 0.57±0.46 vs. 0.13±0.12,P〉 0.05）. The same trend of changes, with slight reduction in splenocytes and biochemical indices were found between the groups of sepsis followed by pneumonia and sepsis-alone, but no significant difference was found. The level of HMGB1 in the 4-day group of sepsis with complication of pneumonia was further decreased compared with sepsis-alone group （μg/L：1.17±0.74 vs. 1.76±0.71,P〈 0.05）, and IL-1β in the 7-day group of sepsis complicated pneumonia was further higher than those of sepsis-alone group in the same period （ng/L： 105.73±25.06 vs. 61.04±31.29,P〈 0.05）, while there were no differences in levels of other cytokines between ＂two-hit＂ group and sepsis-alone group. Apoptosis of spleencytes in the 4-day group of sepsis complicated pneumonia was more marked than that of sepsis-alone group at the same period （cells/HP： 74.48±22.47 vs. 52.99±20.79,P〈 0.05）, while no difference was found between the 7-day groups of sepsis complicated pneumonia and the sepsis-alone group （cells/HP： 28.70±4.13 vs. 30.43±14.55, P〉 0.05）.Conclusions The mortality of this ＂two-hit＂ model with complication of pneumonia 4 days after CLP was significantly higher than that of single sepsis model. The ability of bacteria clearance was decreased, and immunocyte apoptosis was exacerbated. These findings may be with the result of the occurrence of immunoparalysis in the mid stage of sepsis. The ＂two-hit＂ model reproduced on 7 days after CLP might suggest reconstruction of host immune function, and maybe associated with the recovery of immune response.