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目的观察热射病(heat stroke,HS)大鼠枯否细胞(kupffer cells,KCs)吞噬及分泌功能的变化并探讨其可能机制。方法用动物体温维持仪建HS大鼠动物模型。对照组及HS组大鼠肝脏苏木精-伊红(hematoxylin-eosin,HE)染色,观察KCs吞噬印度墨水的情况。留取各组大鼠实验前后外周血,鲎试剂盒检测内毒素浓度,酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)试剂盒检测巨噬细胞炎症蛋白1α(macrophage inflammatory protein-1 alpha,MIP-1α)、肿瘤坏死因子α(tumor necrosis factor-alpha,TNF-α)、白介素1β(interleukin-1 beta,IL-1β)及干扰素γ(interferongamma,INF-γ)浓度。制备石蜡切片,免疫荧光技术检测大鼠KCs内磷酸化c-Jun氨基端激酶(phosphorylation c-Jun Nterminal kinase,p-JNK)及MIP-1α的表达。用细胞裂解液匀浆提取肝组织总蛋白,蛋白免疫印迹(Western blot)检测各组大鼠KCs内p-JNK及MIP-1α蛋白的表达。结果大鼠肝脏HE染色显示HS组与对照组相比,KCs吞噬印度墨水的能力明显下降(P<0.05)。HS组大鼠外周血内毒素浓度明显高于对照组(P<0.05);MIP-1α、TNF-α、IL-1β及INF-γ浓度明显高于对照组(P<0.05)。大鼠肝脏免疫荧光结果显示p-JNK和MIP-1α主要表达在KCs,与对照组相比,HS组KCs内p-JNK和MIP-1α的表达明显增多,HS组大鼠肝脏p-JNK和MIP-1α蛋白表达均明显上调(P<0.05)。结论 HS组大鼠KCs吞噬功能减弱,分泌TNF-α、IL-1β及INF-γ等炎症因子的能力增强,其机制可能与c-Jun氨基端基酶信号通路激活及MIP-1α表达增多有关。
Objective To observe the changes of phagocytic and secretory functions of kupffer cells (KCs) in heat stroke (HS) rats and to explore its possible mechanism. Methods The animal model of HS rat was established by keeping body temperature of animals. The hematoxylin-eosin (HE) staining of the liver of rats in the control group and the HS group was performed to observe the engulfment of Indian inks by KCs. Peripheral blood samples were taken from peripheral blood of rats in each group. The concentration of endotoxin was detected by 鲎 kit, and macrophage inflammatory protein-1 alpha (MIP) was detected by enzyme linked immunosorbent assay (ELISA) -1α), TNF-α, IL-1β and INF-γ. Paraffin sections were prepared and the expression of phosphorylated c-Jun Nterminal kinase (p-JNK) and MIP-1α in KCs were detected by immunofluorescence staining. The total protein of liver tissue was homogenized with cell lysate, and the protein expression of p-JNK and MIP-1α in KCs of each group were detected by Western blot. Results HE staining showed that the ability of KCs to swallow Indian ink was significantly lower than that of the control group (P <0.05). The concentration of endotoxin in HS group was significantly higher than that in control group (P <0.05). The concentrations of MIP-1α, TNF-α, IL-1β and INF-γ in HS group were significantly higher than those in control group (P <0.05). Immunofluorescence results of rat liver revealed that p-JNK and MIP-1α were mainly expressed in KCs. Compared with the control group, the expression of p-JNK and MIP-1α in KCs of HS group was significantly increased. The expressions of p-JNK and MIP-1α protein expression were significantly increased (P <0.05). Conclusions The ability of phagocytosis of KCs in HS group is weakened and the secretion of TNF-α, IL-1β and INF-γ and other inflammatory factors is enhanced. The mechanism may be related to the activation of c-Jun N-terminal kinase signaling pathway and the increase of MIP-1α expression .