缺锌对内毒素／脂多糖所致脓毒症大鼠相关免疫功能的影响

Effects of zinc deficiency on the relevant immune function in rats with sepsis induced by endotoxin/lipopolysaccharide

目的观察缺锌对LPS所致脓毒症大鼠相关免疫功能的影响。方法将60只大鼠按随机数字表法分为低锌组、常锌配饲组、常锌对照组，每组20只。低锌组大鼠进食低锌饲料；常锌配饲组大鼠进食正常含锌饲料，并通过人工控制使其进食量与低锌组大鼠相同；常锌对照组大鼠自由进食正常含锌饲料。饲养7d后禁食，按随机数字表法将前述3组大鼠组内依次分为低锌LPS亚组、低锌生理盐水（NS）亚组，常锌配饲LPS亚组、常锌配饲NS亚组，常锌对照LPS亚组、常锌对照NS亚组，每组10只。各LPS亚组大鼠腹腔注射1mg／mLLPS（5mg／kg），各NS亚组大鼠腹腔注射等量NS。注射6h后各组大鼠断头取血并留取脾脏、胸腺。采用电感耦合等离子体质谱仪检测血清锌含量，采用血生化自动分析仪检测血清碱性磷酸酶（ALP）活性，称量各亚组大鼠体质量、胸腺及脾脏的质量并计算脾脏指数和胸腺指数，采用全自动血细胞分析仪检测6项血常规指标，ELISA法检测血清γ干扰素、IL-4、TNF—α、IL-10含量并计算γ干扰素／IL-4比值。对数据行单因素方差分析、SNK检验。结果（1）血清锌含量、ALP活性：各LPS亚组大鼠均显著低于相应的NS亚组，常锌配饲NS亚组、常锌对照NS亚组大鼠均显著高于低锌NS亚组，常锌配饲LPS亚组、常锌对照LPS亚组大鼠均显著高于低锌LPS亚组，P值均小于0．05。（2）体质量、脾脏质量、脾脏指数、胸腺质量、胸腺指数：各LPS亚组大鼠与相应的NS亚组相近（P值均大于0．05）；除体质量外，常锌配饲NS亚组、常锌对照NS亚组大鼠余指标均显著高于低锌NS亚组，常锌配饲LPS亚组、常锌对照LPS亚组余指标均显著高于低锌LPS亚组，P值均小于0．05。（3）白细胞计数：低锌LPS亚组、常锌配饲LPS亚组大鼠显著高于相应的NS亚组（P值均小于0．05），常锌对照NS亚组大鼠显著高于低锌NS亚组（P〈0．05），常锌对照LPS亚组大鼠显著低于低锌LPS亚组（P〈0．05）。中性粒细胞计数及中性粒细胞：各LPS亚组大鼠均显著高于相应的NS亚组（P值均小于0．05），常锌对照LPS亚组大鼠显著低于低锌LPS亚组（P值均小于0．05）；常锌对照NS亚组大鼠中性粒细胞显著低于低锌NS亚组（P〈0．05）。淋巴细胞计数及淋巴细胞：低锌NS亚组、低锌LPS亚组、常锌配饲NS亚组、常锌配饲LPS亚组、常锌对照NS亚组、常锌对照LPS亚组大鼠淋巴细胞计数分别为（1．8±0．4）×10^9／L、（1．0±0．3）×10^9／L、（2．6±0．7）×10^9／L、（1．4±0．4）×10^9／L、（3．3±0．6）×10^9／L、（1．5±0．5）×10^9／L，淋巴细胞分别为0．39±0．10、0．11±0．03、0．47±0．12、0．14±0．04、0．50±0．09、0．24±0．07，各LPS亚组大鼠均显著低于相应的NS亚组，常锌对照NS亚组大鼠均显著高于低锌NS亚组，常锌配饲LPS亚组、常锌对照LPS亚组大鼠显著高于低锌LPS亚组，P值均小于0．05；常锌配饲NS亚组大鼠淋巴细胞计数显著高于低锌NS亚组（P〈0．05）。血小板计数：常锌配饲LPS亚组、常锌对照LPS亚组大鼠显著低于相应的NS亚组（P值均小于0．05），常锌配饲NS亚组、常锌对照NS亚组大鼠显著高于低锌NS亚组（P值均小于0．05），常锌对照LPS亚组大鼠显著高于低锌LPS亚组（P〈0．05）。（4）各组大鼠血清TNF-α、IL-4、IL-10含量相近（P值均大于0．05）。血清γ干扰素含量及γ干扰素／IL4比值：低锌Ns亚组、低锌LPS亚组、常锌配饲NS亚组、常锌配饲LPS亚组、常锌对照NS亚组、常锌对照LPS亚组血清γ干扰素含量分别为（75±21）、（233±40）、（80±14）、（345±74）、（66±7）、（821±189）pg／mL，γ干扰素／IL-4比值分别为3．1±1．0、6．6±1．7、3．9±1．7、20．2±8．3、3．4±1．5、45．7±7．6，各LPS亚组大鼠均显著高于相应的NS亚组（P值均小于0．05），常锌配饲NS亚组、常锌对照NS亚组大鼠与低锌NS亚组相近（P值均大于0．05），常锌配饲LPS亚组、常锌对照LPS亚组大鼠均显著高于低锌LPS亚组（P值均小于0．05）。结论锌缺乏可造成月龄大鼠胸腺、脾脏萎缩，外周血淋巴细胞减少，在脓毒症状态下，γ干扰素产生进一步减少，从而使Th1／Th2细胞因子发生了向Th2漂移，加剧免疫功能失衡。

Objective To investigate the effects of zinc deficiency on the relevant immune function in rats with LPS-induced sepsis. Methods Sixty rats were divided into low zinc group （LZ） , normal zinc pair-fed group （NP）, and normal zinc control group （NC） according to the random number table, with 20 rats in each group. The rats in group LZ were fed with low zinc diet, and the rats in group NP were fed with normal zinc diet, with the same intake as that of group LZ by manual control, and the rats in group NC were fed with normal zinc diet freely. After being fed for 7 d, the rats all fasted and were further divide into the below subgroups named LZ-LPS, LZ-normal saline （NS）, NP-LPS, NP-NS, NC-LPS, and NC-NS according to the random number table, with 10 rats in each subgroup. Rats in the LPS subgroups were intraperitoneally injected with 1 mg/mL LPS solution with the dosage of 5 mg/kg, rats in the corresponding NS subgroups were intraperitoneally injected with equivalent NS. The rats were sacrificed at post injection hour 6 to collect blood, spleen, and thymus. The serum level of zinc was detected by inductively coupled plasma mass spectrometry, and the serum alkaline phosphatase （ALP） activity was detected by automatic blood biochemical analyzer. The body weight and weight of spleen and thymus of rats were weighed, and the indices of spleen and thymus were calculated. Six routine blood indices were examined by automatic blood cell analyzer. The serum levels of interferon gamma （IFN-γ） , TNF-α, IL-4, and IL-10 were determined with ELISA, and the ratio of IFN-γ to IL-4 was calculated. Data were processed with one-way analysis of variance and SNK test. Results （ 1 ） Serum levels of zinc and ALP activity in the LPS subgroups were significantly lower than those in the corresponding NS subgroups （ with P values below 0. 05）. The two former indices in subgroups NP-NS and NC-NS were significantly higher than those in subgroup LZ-NS （ with P values below 0.05）. The two former indices in subgroups NP-LPS and NC-LPS were significantly higher than those in subgroup LZ-LPS （ with P values below 0.05 ）. （2） Body weight, spleen and thymus weight, indices of spleen and thymus in the LPS subgroups were similar with those in the corresponding NS subgroups （with P values above 0. 05）. The 4 former indices, except for body weight, in subgroups NP-NS and NC-NS were significantly higher than those in subgroup LZ-NS （ with P values below 0. 05）. The 4 former indices, except for body weight, in subgroups NP-LPS and NC-LPS were significantly higher than those in subgroup LZ-LPS （with P values below 0.05）. （3） Levels of leucocyte count in subgroups LZ-LPS and NP-LPS were signifi- cantly higher than those in the corresponding NS subgroups （with P values below 0.05）. Level of leucocyte count in subgroup NC-NS was significantly higher than that in subgroup LZ-NS （ P 〈 0.05）. Level of leucocyte count in subgroup NC-LPS was significantly lower than that in subgroup LZ-LPS （ P 〈 0. 05 ）. Levels of neutrophilic granulocyte count （NGC） and NG in the LPS subgroups were significantly higher than those in the corresponding NS subgroups （with P values below 0.05）. The two former indices in subgroup NC-LPS were significantly lower than those in subgroup LZ-LPS （ with P values below 0.05 ）. Level of NG in subgroup NC-NS was significantly lower than that in subgroup LZ-NS （ P 〈 0.05）. Levels of lymphocyte count and lymphocyte in subgroups LZ-NS, LZ-LPS, NP-NS, NP-LPS, NC-NS, and NC-LPS were respectively （ 1.8 ± 0.4） ×10^9/L, （ 1.0 ± 0.3 ） ×10^9/L, （ 2.6 ± 0.7 ）×10^9/L, （ 1.4 ±0.4 ）×10^9/L, （ 3.3 ± 0.6）×10^9/L, （1.5 ±0.5） ×10^9/L, and 0.39 ±0.10, 0.11 ±0.03, 0.47 ±0.12, 0.14 ±0.04, 0.50 ± 0.09, 0.24 ±0.07. The two former indices in the LPS subgroups were significantly lower than those in the corresponding NS subgroups （ with P values below 0.05 ）. The two former indices in subgroup NC-NS were significantly higher than those in subgroup LZ-NS （with P values below 0.05）. The two former indices in subgroups NP-LPS and NC-LPS were significantly higher than those in subgroup LZ-LPS （with P values below 0.05）. Level of lymphocyte count in subgroup NP-NS was significantly higher than that in subgroup LZ-NS（ P 〈 0.05）. Levels of platelet count （PC） in subgroups NP-LPS and NC-LPS were significantly lower than those in the corresponding NS subgroups （with P values below 0.05）. Levels of PC in subgroups NP-NS and NC-NS were significantly higher than those in subgroup LZ-NS （ with P values below 0.05 ）. Level of PC in subgroup NC-LPS was significantly higher than that in subgroup LZ-LPS （ P 〈0.05）. （4） Serum levels of TNF-α, IL-4, and IL-10 in each subgroup showed no significant differences （with P values above 0.05）. Serum levels of IFN-γ and ratios of IFN-γ to IL--4 in subgroups LZ-NS, LZ-LPS, NP-NS, NP-LPS, NC-NS, and NC-LPS were respectively （75 ±21）, （233 ±40）, （80 ± 14）, （345 ±74）, （66 ± 7）, （821 ±189） pg/mL, and3.1 ±1.0, 6.6 ±1.7, 3.9 ±1.7, 20.2 ±8.3, 3.4 ±1.5, 45.7 ±7.6. The two former indices in the LPS subgroups were significantly higher than those in the corresponding NS subgroups （ with P values below 0.05 ）. The two former indices in subgroups NP-NS and NC-NS were similar with those in subgroup LZ-NS （ with P values above 0.05 ）. The two former indices in subgroups NP-LPS and NC-LPS were significantly higher than those in subgroup LZ-LPS （with P values below 0. 05 ）. Conclusions Zinc deficiency can induce the atrophy of spleen and thymus, and reduction of peripheral blood lymphocyte. In sepsis, zinc deficiency can further decrease the production of IFN-γ, thus making the cytokines of Th1/Th2 shift to Th2 and the immune imbalance worse.