摘要
目的探讨苯甲酰芍药苷对脂多糖(LPS)诱导小鼠急性肺损伤(ALI)的保护作用及可能分子机制。方法将75只SPF级C57BL/6J小鼠按照随机数字表法分成五组,每组15只:空白对照组(生理盐水200μL)、ALI组(2mg/kg LPS 100μL+生理盐水100μL)、阳性对照组(2mg/kg LPS100μL+血必净注射液100μL)、苯甲酰芍药苷低剂量组(2mg/kg LPS 100μL+2.5mg/kg苯甲酰芍药苷100μL)、苯甲酰芍药苷高剂量组(2mg/kg LPS 100μL+5.0mg/kg苯甲酰芍药苷100μL),通过腹腔注射LPS构建ALI模型并给予药物干预。称重检测各组肺组织湿/干重比;苏木素-伊红(HE)染色观察肺组织病理形态变化;酶联免疫吸附试验(ELISA)法检测ALI小鼠肺组织肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)水平;蛋白免疫印迹(WB)法检测肺组织IκB激酶(IKKβ)/核因子κB(NF-κB)表达及其磷酸化激活水平;荧光定量PRC(RT-PCR)检测ALI小鼠肺组织mi R-520c-3p表达;Targetscan 7.0预测mi R-520c-3p靶基因;A549转染mi R-520c-3p inhibitor验证苯甲酰芍药苷对炎症的调控作用。结果与ALI组比较,低、高剂量苯甲酰芍药苷均明显降低ALI小鼠肺组织湿/干重比[(5.64±0.14)、(4.75±0.11)比(6.21±0.64),P均<0.05];ALI损伤的肺组织结构得到缓解;ELISA结果显示,与ALI组比较,低、高剂量苯甲酰芍药苷能明显降低ALI小鼠肺组织TNF-α、IL-6水平[TNF-α:(20.68±1.06)pg/m L、(7.34±0.37)pg/m L比(32.27±1.89)pg/m L,P均<0.05;IL-6:(29.16±2.36)pg/m L、(14.26±1.06)pg/m L比(48.26±3.19)pg/m L,P均<0.05];WB结果显示,与ALI组比较,苯甲酰芍药苷低、高剂量组IKKβ、NF-κB p65磷酸化水平以及总NF-κB p65水平下降;RTPCR结果显示,与ALI组比较,低、高剂量苯甲酰芍药苷有效促进mi R-520c-3p在ALI中的表达[(0.51±0.13)、(0.74±0.09)比(0.34±0.11),P均<0.05];Targetscan预测结果显示,mi R-506-3p直接靶向NF-k B p65亚基RELA基因3'UTR序列;补救实验显示,与苯甲酰芍药苷组比较,苯甲酰芍药苷+mi R-520c-3p inhibitor组TNF-α、IL-6 m RNA水平明显升高[TNF-α:(3.94±0.49)比(1.55±0.36),P<0.05;IL-6:(6.95±1.21)比(2.11±0.42),P<0.05]。结论苯甲酰芍药苷能有效缓解LPS引起的小鼠ALI病理进程中炎症的发生,其作用机制可能是通过上调mi R-520c-3p表达,从而抑制其靶基因NF-κB p65生成,抑制IKKβ/NF-κB信号通路的活化,导致炎症因子TNF-α、IL-6的合成受到抑制。
Objective To investigate the protective effect of benzoyl paeoniflorin on lipopolysaccharide(LPS)-induced acute lung injury(ALI)in mice and its molecular mechanism.Methods Seventy-five SPF C57BL/6J mice were randomly divided into five groups,with 15 animals in each group:blank control group(normal saline 200μL),ALI group(2mg/kg LPS 100μL+normal saline 100μL),positive control group(2mg/kg LPS 100μL+Xuebijing injection 100μL),low-dose benzoyl paeoniflorin group(2mg/kg LPS 100μL+2.5mg/kg benzoyl paeoniflorin 100μL),and high-dose benzoyl paeoniflorin group(2mg/kg LPS 100μL+5.0mg/kg benzoyl paeoniflorin 100μL).The ALI model was constructed by intraperitoneal injection of LPS,and drug intervention was performed.The wet/dry weight ratio of lung tissue in each group was measured by weighing.Hematoxylin-eosin(HE)staining was used to observe the pathological changes of lung tissue.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)in lung tissue of ALI mice.Western blot(WB)was performed to check the expression of inhibitor of nuclear factor kappa B kinase subunit beta(IKKβ)/nuclear factor kappa B subunit 2(NF-κB)and its phosphorylation activation level in lung tissue.The expression of miR-520c-3p in lung tissue of ALI mice was tested by fluorescence quantitative PCR(RT-PCR).miR-520c-3p target gene was predicted by Targetscan 7.0.A549 was transfected with miR-520c-3p inhibitor to verify the regulatory effect of benzoyl paeoniflorin on inflammation.Results Compared to the ALI group,both low and high doses of benzoyl paeoniflorin significantly reduced the wet/dry weight ratio of lung tissue in ALI mice[(5.64±0.14)and(4.75±0.11)vs(6.21±0.64),P<0.05];The injury lung tissue structure of ALI was relieved;ELISA results showed that benzoyl paeoniflorin at both low and high dose significantly decreased the levels of TNF-αand IL-6 in the lung tissue of ALI mice,i.e.,TNF-α:[(20.68±1.06)and(7.34±0.37)vs(32.27±1.89)pg/mL,P<0.05],IL-6:[(29.16±2.36)and(14.26±1.06)vs(48.26±3.19)pg/mL,P<0.05];WB results indicated that the phosphorylation levels of IKKβ,NF-κB p65 and total NF-κB p65 decreased in both low and high-dose benzoyl paeoniflorin groups;RT-PCR results revealed that both low and high-dose benzoyl paeoniflorin effectively promoted the expression of miR-520c-3p in ALI[(0.51±0.13)and(0.74±0.09)vs(0.34±0.11),P<0.05].The results of Targetscan showed that miR-506-3p directly targeted the 3'-UTR sequence of RELA gene of NF KB p65 subunit.Rescue experiments showed that compared with the benzoyl paeoniflorin group,the levels of TNF-αand IL-6 mRNA of the benzoyl paeoniflorin+MiR-520c-3p inhibitor group were significantly increased,i.e.,TNF-α:[(3.94±0.49)vs(1.55±0.36),P<0.05],IL-6:[(6.95±1.21)vs(2.11±0.42),P<0.05].Conclusion Benzoyl paeoniflorin can effectively alleviate the inflammation in the pathological process of ALI induced by LPS.Its mechanism may be through up-regulating the expression of miR-520c-3p,thus inhibiting the production of its target gene NF-κB p65,inhibiting the activation of IKKβ/NF-κB signaling pathway,resulting in the inhibition of the synthesis of inflammatory factors TNF-αand IL-6.
作者
周垂杨
杨明
高仁贤
ZHOU Chuiyang;YANG Ming;GAO Renxian(Department of Emergency Medicine,Wenzhou People's Hospital,Wenzhou,Zhejiang province,325000,China;The Second Clinical College,Zhejiang Chinese Medical University,Hangzhou,Zhejiang province,310053,China)
出处
《浙江中西医结合杂志》
2020年第9期698-702,708,I0001,共7页
Zhejiang Journal of Integrated Traditional Chinese and Western Medicine
