表面活性蛋白D缓解脓毒症诱发的急性肾损伤的机制

The mechanism of SP-D in alleviating acute renal injury induced by sepsis

目的探讨表面活性蛋白D(SP-D)缓解脓毒症通过调节细胞凋亡和核转录因子-κB(NF-κB)介导的炎症诱发急性肾损伤的机制。方法行盲肠结扎穿刺术建立脓毒症动物模型,C57BL/6小鼠144只,分4组(n=36):SP-D敲除组(KO)、SP-D野生组(WT),两组均设置手术组(Sepsis)和假手术组(Sham),即WT sham组、KO sham组、WT sepsis组、KO sepsis组,并建立假手术组作为对照组。术后6及24 h取材。用酶联免疫吸附法(ELISA)测定血清肿瘤坏死因子-α(TNF-α)和单核细胞趋化蛋白1(MCP-1)含量;用钙结合蛋白V/碘化丙啶(Annexin V/PI)双染色法和流式细胞仪测定凋亡细胞;用ELISA方法测定肾脏组织中NF-κB水平,用Western blot方法测定NF-κB的肾脏组织入核水平;TUNEL法检测凋亡。结果术后6及24 h WT sepsis组和KO sepsis组血清TNF-α水平高于对照组(P <0.05),且KO sepsis组TNF-α水平高于WTsepsis组(P <0.05);术后24 h WT sepsis组及KO sepsis组的TNF-α水平均高于术后6 h(P <0.05)。在术后6及24 h WT sepsis组和KO sepsis组血清MCP-1水平均高于对照组(P <0.05),KO sepsis组MCP-1水平高于WT sepsis组(P <0.05);术后24 h WT sepsis组及KO sepsis组的MCP-1水平均高于术后6 h(P <0.05)。WT小鼠肾脏中,术后6和24 h SP-D表达降低(P <0.05)。脓毒性SP-D KO小鼠的肾脏中TUNEL阳性细胞的数目高(P <0.05)。与假手术组相比,在脓毒症造模后6和24 h,脓毒症小鼠中NF-κB增加,来自脓毒性SP-D KO小鼠的肾脏中的NF-κB比WT小鼠高(P <0.05)。结论 SP-D降低小鼠血清炎症因子水平,通过抑制NF-κB的活性和细胞凋亡减轻脓毒症诱导的急性肾损伤。

Objective To investigate the mechanism by which innate immune molecule surfactant protein D(SP-D) attenuates sepsis-induced acute kidney injury through modulating apoptosis and NF-KB-mediated inflammation. Methods In the present study, animal models of sepsis were established by cecal ligation and puncture with SP-D knockout group and wild-type mice, and a sham-operated group was established as a control group. The experimental materials were extracted 6 and 24 hours after the operation. The levels of TNF-α and MCP-1 in plasma were determined by enzyme-linked immunosorbent assay(ELISA). Apoptosis was measured by double staining with Annexin V/PI and flow cytometry the levels of NF-κB in renal tissues were measured by ELISA. The levels of NF-κB in the kidney were measured by Western Blot. The apoptosis was detected by TUNEL. Results The levels of TNF-α in WT sepsis and KO sepsis groups were higher than those in control at 6 and 24 hours after operation(P < 0.05). The level of TNF-α in KO sepsis group was higher than that in WT sepsis group(P < 0.05). TNF-αlevels in WT sepsis group and KO sepsis group at 24 hours after operation were higher than those at 6 hours after operation( P < 0.05).The levels of MCP-1 in WT sepsis group and KO sepsis group at 6 and 24 hours after operation were higher than those in control group(P < 0.05), and MCP-1 levels in KO sepsis group were higher than those in WT sepsis group(P < 0.05). MCP-1 levels in WT sepsis group and KO sepsis group at 24 hours after operation were higher than those at 6 hours after operation(P < 0.05). The expression of SP-D in WT kidneys was lower at 6 and 24 hours after operation(P < 0.05). Quantitative analysis of apoptotic cells showed that the number of TUNEL-positive cells in the kidneys from septic SP-D KO mice was higher(P < 0.05). The levels of NF-κB in septic mice were increased at 6 hours and 24 hours after sepsis modeling compared with the sham-operated group,and the septic SP-D KO mice. The level of NF-κB was higher than WT mice(P < 0.05). Conclusion Surfactant protein D decreases plasma levels of inflammatory cytokines in mice and attenuates sepsis-induced acute kidney injury by inhibiting NF-κB activity and apoptosis.