目的 探讨丁香苷调节核苷酸结合寡聚化结构域样受体蛋白3(nucleotide-binding oligomerization domain-like receptor protein 3,NLRP3)/半胱氨酸天冬氨酸酶1(Caspase-1)信号通路对脓毒症肺损伤大鼠细胞焦亡的影响。方法 取Wistar大鼠腹腔注射脂多糖建立脓毒症模型,随机分为模型组、丁香苷低剂量组、丁香苷高剂量组、尼日利亚菌素(Nigericin,NLRP3激活剂)组、丁香苷高剂量+Nigericin组,每组12只,另取12只大鼠腹腔注射等剂量生理盐水设为对照组,以丁香苷和Nigericin分组干预后,检测各组大鼠肺组织含水量和肺指数;以苏木精-伊红(HE)和醋酸铀-枸橼酸铅双重染色分别检测各组大鼠肺组织病理变化与超微结构改变;以酶联免疫吸附测定(ELISA)试剂盒测定各组大鼠肺组织及血清促炎因子白细胞介素(interleukin,IL)-6、IL-17水平;以免疫荧光染色检测各组大鼠肺组织细胞焦亡标志蛋白NLRP3表达;以免疫印迹检测各组大鼠肺组织NLRP3/Caspase-1通路蛋白表达。结果 与对照组相比,模型组大鼠肺组织病理形态和超微结构呈现严重损伤改变,肺组织含水量和肺指数、肺组织与血清IL-6、IL-17水平、肺组织NLRP3相对荧光强度、NLRP3与Caspase-1蛋白表达升高(P＜0.05)。与模型组相比,丁香苷给药组大鼠肺组织病理损伤和超微结构受损均减轻,肺组织含水量和肺指数、肺组织与血清IL-6、IL-17水平、肺组织NLRP3相对荧光强度、NLRP3与Caspase-1蛋白表达均降低(P＜0.05);丁香苷高剂量组大鼠肺组织病理损伤和超微结构受损相比丁香苷低剂量组进一步减轻,肺组织含水量和肺指数、肺组织与血清IL-6、IL-17水平、肺组织NLRP3相对荧光强度、NLRP3与Caspase-1蛋白表达进一步降低(P＜0.05);Nigericin组大鼠肺组织病理损伤和超微结构受损加重,肺组织含水量和肺指数、肺组织与血清IL-6、IL-17水平、肺组织NLRP3相对荧光强度、NLRP3与Caspase-1蛋白表达升高(P＜0.05)。与丁香苷高剂量组相比,丁香苷高剂量+Nigericin组大鼠肺组织病理损伤和超微结构受损加重,肺组织含水量和肺指数、肺组织与血清IL-6、IL-17水平、肺组织NLRP3相对荧光强度、NLRP3与Caspase-1蛋白表达升高(P＜0.05)。结论 丁香苷可通过下调NLRP3/Caspase-1信号通路而抑制炎症水平和细胞焦亡发生,减轻脓毒症大鼠肺水肿及肺组织损伤。
Abstract
OBJECTIVE To investigate the effect of syringin on the pyroptosis of alveolar epithelial cells in septic rats by regulating the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3)/Caspase-1 signal pathway. METHODS The sepsis model of Wistar rats was established by intraperitoneal injection of lipopolysaccharide. The rats were randomly divided into model group, low-dose syringin group, high-dose syringin group, nigericin (NLRP3 activator) group, high-dose syringin+nigericin group, with 12 rats in each group, and another 12 rats were intraperitoneally injected with equal dose of normal saline as the control group. After intervention with syringin and nigericin, the water content and lung index of lung tissue of rats in each group were measured; hematoxylin-eosin (HE) and uranium acetate lead citrate double staining were used to detect the pathological changes and ultrastructural changes of lung tissues of rats in each group; the levels of interleukin (IL)-6 and IL-17 in lung tissue and serum of rats in each group were measured by enzyme linked immunosorbent assay (ELISA) kit; immunofluorescence staining was used to detect the expression of NLRP3 in lung tissue of rats in each group; and the expression of NLRP3/Caspase-1 pathway protein in lung tissue of rats in each group was detected by Western blot. RESULTS Compared with the control group, the pathological morphology and ultrastructure of lung tissue in the model group showed severe damage, the water content and lung index of lung tissue, levels of IL-6 and IL-17 in lung tissue and serum, relative fluorescence intensity of NLRP3 in lung tissue, and expression of NLRP3 and Caspase-1 proteins increased (P<0.05). Compared with the model group, the pathological damage and ultrastructural damage of lung tissue in syringin groups were alleviated, the water content and lung index of lung tissue, levels of IL-6 and IL-17 in lung tissue and serum, relative fluorescence intensity of NLRP3 in lung tissue, and expression of NLRP3 and Caspase-1 proteins decreased (P<0.05). Compared with the low-dose syringin group, the pathological damage and ultrastructural damage of lung tissue in the high-dose syringin group were further alleviated, the water content and lung index of lung tissue, levels of IL-6 and IL-17 in lung tissue and serum, relative fluorescence intensity of NLRP3 in lung tissue, and expression of NLRP3 and Caspase-1 proteins further decreased (P<0.05); the pathological damage and ultrastructural damage of lung tissue in nigericin group were aggravated, the water content and lung index of lung tissue, levels of IL-6 and IL-17 in lung tissue and serum, relative fluorescence intensity of NLRP3 in lung tissue, and expression of NLRP3 and Caspase-1 proteins increased (P<0.05). Compared with the high-dose syringin group, the pathological damage and ultrastructural damage of lung tissue in the high-dose syringin+nigericin group were aggravated, the water content and lung index of lung tissue, levels of IL-6 and IL-17 in lung tissue and serum, relative fluorescence intensity of NLRP3 in lung tissue, and expression of NLRP3 and Caspase-1 proteins increased (P<0.05). CONCLUSION Syringin can inhibit the occurrence and progression of inflammation by down-regulation of NLRP3/Caspase-1 signal pathway, reduce pulmonary edema and lung tissue damage in septic rats, and inhibit the pyroptosis of alveolar epithelial cells.
关键词
丁香苷 /
核苷酸结合寡聚化结构域样受体蛋白3/半胱氨酸天冬氨酸酶1 /
脓毒症 /
肺损伤 /
焦亡
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Key words
syringin /
NLRP3/Caspase-1 /
sepsis /
lung injury /
pyroptosis
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中图分类号：
R966
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参考文献
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脚注
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基金
海南省卫生健康委员会科研项目资助(21A200316)
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