金荞麦总黄酮对脂多糖诱导小鼠急性肺损伤的影响

Effects of total flavonoids of Fagopyrum dibotrys against lipopolysaccharide-induced acute lung injury

该研究旨在探讨金荞麦总黄酮对脂多糖(lipopolysaccharide,LPS)诱导小鼠急性肺损伤的影响。应用金荞麦根以乙醇为提取溶剂获得金荞麦总黄酮,采用昆明小鼠连续12 d灌胃金荞麦总黄酮(25、50、100 mg/kg),1 mg/kg LPS滴鼻12 h诱导小鼠急性肺损伤;通过20μg/mL LPS和RAW 264.7细胞共孵育建立细胞损伤模型,应用10、20、30μg/mL金荞麦总黄酮处理RAW 264.7细胞。采用CCK-8法和乳酸脱氢酶(dehydrogenase,LDH)释放量对LPS和金荞麦总黄酮进行RAW 264.7细胞毒性评价;采用ELISA检测炎性细胞因子[白细胞介素1β(interleukin-1β,IL-1β)、白细胞介素6(interleukin-6,IL-6)、肿瘤坏死因子(tumor necrosis factor,TNF-α)和白细胞介素18(interleukin-18,IL-18))]含量;分光光度计法检测氧化生物标志物[活性氧(reactive oxygen,ROS)、丙二醛(malonaldehyde,MDA)、超氧化物歧化酶(superoxide dismutase,SOD)、过氧化氢酶(catalase,CAT)、还原型谷胱甘肽(glutathione,GSH)和H_(2)O_(2)]水平;Hoechst 33342/PI双染色检测RAW 264.7细胞凋亡水平。结果表明,金荞麦总黄酮可减轻小鼠肺病理损伤及肺组织水肿程度,显著降低LPS染毒小鼠的血清中IL-1β、IL-6、TNF-α的分泌和氧化应激产物的水平,并增强CAT活力;金荞麦总黄酮能抑制RAW 264.7细胞向M1极化进而减少促炎介质分泌;降低LDH的释放量,清除积累的ROS和MDA,升高SOD及CAT活性,提高GSH的含量,并抵御LPS诱导细胞凋亡,且呈现浓度依赖性。金荞麦总黄酮能有效抑制促炎介质的产生,逆转氧化生物标志物的表达,抵御细胞凋亡,抑制巨噬细胞向M1极化,缓解LPS诱导的小鼠急性肺损伤。

This study aimed to investigate the effects of total flavonoids of Fagopyrum dibotrys on acute lung injury induced by lipopolysaccharide(LPS)in mice.In this study,the total flavonoid extract of Fagopyrum dibotrys root was obtained with ethanol as an extraction solvent.Kunming mice were given intragastric administration of total flavonoid extract of buckwheat(25,50,and 100 mg/kg)for 12 days,and acute lung injury was induced by intranasal 1 mg/kg LPS.The cell injury model was established by co-incubation of 20μg/mL LPS and RAW 264.7 cells,and RAW 264.7 cells were treated with 10,20,and 30μg/mL of total flavonoids.The cytotoxicity of LPS and total flavonoids of Fagopyrum dibotrys was evaluated by the CCK-8 method and dehydrogenase(LDH)release.The contents of inflammatory cytokines,including interleukin-1β(IL-1β),interleukin-6(IL-6),tumor necrosis factor(TNF-α),and interleukin-18(IL-18)were detected by ELISA.The levels of oxidative biomarkers,including reactive oxygen(ROS),malonaldehyde(MDA),superoxide dismutase(SOD),catalase(CAT),glutathione(GSH),and H_(2)O_(2) were detected by spectrophotometer and the apoptosis of RAW 264.7 cells was detected by Hoechst33342/PI double staining.Results showed that total flavonoids of Fagopyrum dibotrys could effectively inhibit the polarization of RAW 264.7 cells to M1 and reduce the secretion of pro-inflammatory mediators such as IL-1β,IL-6,IL-18,and TNF-α,reduce the release of LDH,eliminate accumulated ROS and MDA,increase the activity of SOD and CAT,increase the content of GSH,and resist apoptosis induced by LPS in a concentration-dependent manner.The effect of total flavonoid extract of Fagopyrum dibotrys was verified in mice with acute lung injury induced by LPS.It significantly decreased the secretion of IL-1β,IL-6,and TNF-αin serum and the levels of oxidative stress products MDA and H_(2)O_(2),increased the activity of antioxidant enzyme CAT,and prevented the accumulation of edematous fluid in lung tissue.To sum up,total flavonoids of Fagopyrum dibotrys can effectively inhibit the production of pro-inflammatory mediators,reverse the expression of oxidative biomarkers,and resist apoptosis,thus inhibiting the polarization of macrophages to M1 and alleviating LPS-induced acute lung injury in mice by anti-inflammatory,antioxidant,and anti-apoptosis effects.