慢性华支睾吸虫感染对脂多糖刺激巨噬细胞反应的影响

 目的：了解慢性华支睾吸虫（Cs）感染的免疫状态及其对巨噬细胞表型和炎症反应的影响。方法：选择Sprague-Dawley雄性大鼠，按随机数字表法分组进行2部分实验。(1) 经口华支睾吸虫虫卵灌胃感染70 d建立慢性Cs感染模型，分为正常对照组和慢性Cs感染组，每组10只，用酶联免疫吸附法（ELISA）检测大鼠血清白细胞介素 4（IL-4)、IL-10、肿瘤坏死因子α（TNF-α）和干扰素γ（IFN-γ）水平，腹腔灌洗获取巨噬细胞，流式细胞术检测腹腔巨噬细胞亚型；(2)腹腔灌洗获取巨噬细胞，按照腹腔巨噬细胞的来源，分为空白对照组、正常组和慢性Cs感染组，以脂多糖（LPS,10 μg/L）刺激孵育，用ELISA法动态监测LPS刺激后0、2、12和24 h细胞培养上清液的TNF-α和IL-10的变化。结果：(1) 慢性Cs感染组大鼠血清促炎因子TNF-α［(77.64±3.25) ng/L］和IFN-γ［(212.69±12.60)  ng/L］水平较正常组TNF-α［(55.13±3.25) ng/L］和IFN-γ［(108.15±10.49)  ng/L］升高（均P＜0.05），而抗炎因子IL-4［(248.24±8.99) ng/L］和IL-10［(223.56±5.60) ng/L］水平较正常组血清IL-4［(52.40±3.97) ng/L］和IL-10［(60.18±5.36) ng/L］也显著升高（均P＜0.01）,并可使腹腔巨噬细胞向替代活化型（M2型）巨噬细胞分化；(2) 2组腹腔巨噬细胞培养上清液TNF-α水平在LPS刺激后2 h即开始上升，至24 h达到高峰，慢性Cs感染组巨噬细胞在LPS刺激后2、12和24 h上清液的TNF-α水平均显著低于正常大鼠组［2 h： （88.20±1.91） ng/L vs（123.45±2.98） ng/L；12 h：（123.66±4.31） ng/L vs（161.11±7.38） ng/L；24 h：（154.52±4.83） ng/L vs（227.85±10.67） ng/L，均P＜0.05］，而IL-10的水平较正常组升高［2 h：（105.46±12.28） ng/L vs（80.86±2.28） ng/L；12 h：（194.19±8.62） ng/L vs（117.56±8.02） ng/L；24 h：（280.02±11.31） ng/L vs（168.14±8.97） ng/L，均P＜0.05］。结论：慢性Cs感染时宿主血清的促炎因子升高，而抗炎因子升高得更明显，使巨噬细胞向M2型极化；体外实验表明慢性Cs感染宿主的巨噬细胞对LPS刺激产生耐受。

Effects of chronic Clonorchis sinensis infestation on inflammatory reaction of macrophages

AIM：To evaluate the immune state in rats with chronic Clonorchis sinesis (Cs) infestation by investigating the effects of Cs on macrophage polarization and inflammatory reactions. METHODS：Sprague-Dawley rats were used in the study. Chronic Cs infestation model was reproduced by intragastric perfusion with Cs eggs. Twenty rats were randomly divided into normal group (n=10) and Cs infestation group (n=10). The serum levels of interleukin (IL-4) and IL-10, tumor necrosis factor α(TNF-α) and interferon γ (IFN-γ) were detected by ELISA. The macrophages were harvested by peritoneal lavage. The differentiation proportion of M1 and M2 macrophages were detected by flow cytometry. The macrophages were divided into control group, normal group and chronic Cs infestation group according to the sources of macrophages. The levels of TNF-α and IL-10 in the culture supernatants were detected by ELISA at 0, 2, 12 and 24 h after lipopolysaccharide (LPS, 10 μg/L) stimulation in vitro. RESULTS：Compared with normal group, chronic Cs infestation increased the serum levels of TNF-α, IFN-γ, IL-4 and IL-10. The differentiation proportion of M1 detected by flow cytometry was 92.1% in normal group and that of M2 macrophages was 93.8% in Cs infestation group. The levels TNF-α and IL-10 in culture supernatants were increased at 2~24 h after LPS stimulation both in normal group and Cs infestation group, but the levels of TNF-α were lower in chronic Cs infestation group than that in normal group at 2 h,12 h and 24 h after LPS stimulation. The level of anti-inflammatory cytokine IL-10 was higher in Cs infestation group than that in normal group at 2 h, 12 h and 24 h after LPS stimulation. CONCLUSION：Chronic Cs infestation increases the serum levels of both pro-inflammatory cytokines and anti-inflammatory cytokines, thus inducing the polarization of M2 macrophages. The macrophages derived from chronic Cs-infected rats produce tolerance in the inflammatory process against LPS in vitro.