摘要
本试验通过建立细菌脂多糖(LPS)诱导的小鼠巨噬细胞RAW264.7炎症模型,旨在探讨中国杨属型蜂胶乙醇提取物(EECP)对巨噬细胞能量代谢及抗炎活性的影响。试验设正常组、LPS组(1.00μg/mL)、低浓度EECP组(1.00μg/mL LPS+5.00μg/mL EECP)、高浓度EECP组(1.00μg/mL LPS+10.00μg/mL EECP),每组至少3次重复。试验检测一氧化氮(NO)的生成、活性氧(ROS)含量和线粒体膜电位。通过实时荧光定量PCR分析炎症相关基因、抗氧化相关基因、糖代谢相关基因和M1/M2型巨噬细胞标志物相关基因的表达。通过蛋白免疫印迹分析Toll样受体4/核因子-κB(TLR4/NF-κB)信号通路、糖代谢相关蛋白表达水平。结果表明:1)EECP中总黄酮含量为(265.88±3.98)mg芦丁当量/g,总酚酸含量为(278.57±2.69)mg没食子酸当量/g。2)与LPS组相比,低、高浓度EECP均极显著抑制了细胞NO的生成(P<0.001),且分别显著和极显著降低了细胞中ROS含量(P<0.05和P<0.001),并分别显著和极显著逆转了线粒体膜电位水平下降(P<0.05和P<0.001)。3)实时荧光定量PCR结果显示,与LPS组相比,高浓度EECP显著下调了促炎因子肿瘤坏死因子-α(TNF-α)和环氧合酶-2(Cox-2)的基因相对表达量(P<0.05),并极显著下调了白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)、单核细胞趋化蛋白-1(Mcp-1)、诱导型一氧化氮合酶(iNOS)的基因相对表达量(P<0.01或P<0.001)。此外,低、高浓度EECP均极显著上调了抗氧化相关基因血红素氧合酶-1(HO-1)的相对表达量(P<0.01和P<0.001),且高浓度EECP分别显著和极显著上调了核因子E2相关因子2(Nrf2)和硫氧还蛋白还原酶(TXNRD)的基因相对表达量(P<0.05和P<0.001)。4)与LPS组相比,低、高浓度EECP均显著抑制了核因子κB抑制蛋白α(IκBα)的活化(P<0.05),而高浓度EECP极显著抑制了TLR4基因相对表达量和蛋白表达水平(P<0.01)。此外,低浓度EECP极显著抑制了NF-κB p65蛋白的活化(P<0.001)。5)与LPS组相比,高浓度EECP显著抑制了己糖激酶2(HK2)和葡萄糖转运体3(GLUT3)的基因相对表达量(P<0.05),并极显著下调了葡萄糖转运体1(GLUT1)的基因相对表达量(P<0.001)。在蛋白水平上,与LPS组相比,高浓度EECP显著抑制了HK2和乳酸脱氢酶A(LDHA)的蛋白表达水平(P<0.05),并极显著降低了GLUT3和丙酮酸激酶M2型(PKM2)的蛋白表达水平(P<0.01)。6)与LPS组相比,高浓度EECP显著降低了M1型巨噬细胞标志物分化簇86(CD86)的基因相对表达量(P<0.05),并分别显著和极显著上调了M2型标志物转化生长因子-β(TGF-β,P<0.05)和分化簇206(CD206,P<0.01)基因相对表达量。综上所述,EECP富含多酚类物质,通过降低能量代谢和抑制TLR4/NF-κB炎症信号通路,在LPS诱导的小鼠巨噬细胞RAW264.7中具有抗炎和抗氧化活性,从而抑制促炎极化。
The aim of this experiment was to investigate the effects of ethanol extracts of Chinese poplar propolis(EECP)on the energy metabolism and anti-inflammatory activity of macrophages by establishing a bacterial lipopolysaccharide(LPS)-induced inflammation model of mouse macrophage RAW264.7.Four groups were established:a normal group,a LPS group(1.00μg/mL),a low-concentration EECP group(1.00μg/mL LPS+5.00μg/mL EECP),and a high-concentration EECP group(1.00μg/mL LPS+10.00μg/mL EECP),with at least three replicates per group.Nitric oxide(NO)production,reactive oxygen species(ROS)levels,and mitochondrial membrane potential were measured.The expressions of inflammation-related genes,antioxidant-related genes,glucose metabolism-related genes,and genes related to M1/M2 macrophage markers were analyzed by real-time fluorescence quantitative PCR.The expression levels of proteins related to the Toll-like receptor 4/nuclear factor kappa-B(TLR4/NF-κB)signaling pathway and glucose metabolism were analyzed by Western blot.The results showed as follows:1)the total flavonoid content of EECP was(265.88±3.98)mg rutin equivalents/g,the total phenolic acid content was(278.57±2.69)mg gallic acid equivalents/g.2)Compared with the LPS group,both low and high concentrations of EECP significantly inhibited macrophage NO production(P<0.001).Additionally,low and high concentrations of EECP significantly and highly significantly reduced cellular ROS content(P<0.05 and P<0.001,respectively)and significantly and highly significantly reversed the mitochondrial membrane potential(P<0.05 and P<0.001,respectively).3)Real-time fluorescence quantitative PCR(RT-qPCR)results demonstrated that,compared with the LPS group,high concentrations of EECP significantly down-regulated the gene relative expression levels of pro-inflammatory factors tumor necrosis factor-α(TNF-α)and cyclooxygenase-2(Cox-2)(P<0.05)and highly significantly(P<0.01 or P<0.001)down-regulated the gene relative expression level of interleukin-6(IL-6),interleukin-1β(IL-1β),monocyte chemotactic protein-1(Mcp-1),and inducible nitric oxide synthase(iNOS).Furthermore,both low and high concentrations of EECP highly significantly upregulated the relative expression level of the antioxidant-related gene heme oxygenase-1(HO-1)(P<0.01 or P<0.001).Moreover,the high concentration of EECP significantly and highly significantly upregulated the gene relative expressions of nuclear factor E2-related factor 2(Nrf2)and thioredoxin reductase(TXNRD),respectively(P<0.05 and P<0.001).4)Compared with the LPS group,both low and high concentrations of EECP significantly inhibited the activation of inhibitor of nuclear factor-kappa B alpha(IκBα)(P<0.05),while the high concentration of EECP highly significantly inhibited the relative expression level of TLR4 gene and protein expression level(P<0.01).Moreover,the low concentration of EECP highly significantly inhibited the activation of nuclear factor-kappa B p65(NF-κB p65)protein(P<0.001).5)Compared with the LPS group,the high concentration EECP significantly inhibited the gene expressions of hexokinase 2(HK2)and glucose transporter 3(GLUT3)(P<0.05)and highly significantly downregulated the gene relative expression of glucose transporter 1(GLUT1)(P<0.001).At the protein level,compared with the LPS group,the high concentration EECP significantly inhibited the protein expression levels of HK2 and lactate dehydrogenase A(LDHA)(P<0.05)and highly significantly reduced the protein expressions of GLUT3 and pyruvate kinase M2(PKM2)(P<0.01).6)Compared with the LPS group,high concentrations of EECP significantly decreased the gene expression of the M1-type macrophage marker CD86(P<0.05)and significantly and highly significantly upregulated the relative expression levels of the M2-type markers TGF-β(P<0.05)and CD206(P<0.001),respectively.In conclusion,EECP,which is rich in polyphenols,exhibits anti-inflammatory and antioxidant activities in LPS-induced mouse macrophage RAW264.7 by reducing energy metabolism and inhibiting the TLR4/NF-κB inflammatory signaling pathway,thereby suppressing pro-inflammatory polarization.
作者
刘正鑫
袁洁
玄红专
LIU Zhengxin;YUAN Jie;XUAN Hongzhuan(College of Agriculture and Biology,Liaocheng University,Liaocheng 252059,China)
出处
《动物营养学报》
北大核心
2025年第6期4073-4089,共17页
CHINESE JOURNAL OF ANIMAL NUTRITION
基金
山东省蜂产业技术体系产品加工与质量控制岗位(SDAIT-24-05)
山东省自然科学基金(ZR2021MC110)
泰山学者工程专项经费(tsqn202211172)
聊城大学畜牧学学科开放课题(319312105-28)。
