睡莲总多酚对脂多糖诱导的RAW 264.7细胞炎症的保护机制OA
Preventive effect of the total polyphenols from Nymphaea candida in lipopolysaccharide-induced inflammation in RAW 264.7 cells
采用脂多糖(lipopolysaccharide,LPS)诱导的RAW 264.7细胞炎症模型探究睡莲总多酚(total polyphenols from Nymphaea candida,NCTP)的抗炎机制.通过LPS诱导构建RAW 264.7细胞炎症模型,采用CCK-8法检测细胞活力;利用Griess试剂法检测细胞上清液中一氧化氮(NO)含量;ELISA法检测细胞上清液中环氧合酶-2(cyclooxygenase-2,COX-2)、前列腺素 E2(prostaglandin E2,PGE2)、白细胞介素-1β(interleukin-1β,IL-1β)、IL-6、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)含量;采用免疫荧光和流式细胞仪检测细胞线粒体膜电位(mitochondrial membrane potential,MMP)和活性氧(reactive oxygen species,ROS)水平;流式细胞术检测细胞M1极化;Western blot测定细胞中Toll样受体4(Toll-like receptor 4,TLR4)、髓样分化因子 88(myeloid differentiation factor 88,MyD88)、转化生长因子激酶 1(transforming growth factor β-activated kinase 1,TAK1)、κB 抑制因子激酶(inhibitor of κB kinase,IKK)、核转录因子 κB抑制蛋白(inhibitor of NF-κB,IκB)、核转录因子-κB(nuclear factor-κB,NF-κB)、细胞外调节蛋白激酶(extracellular reg-ulated protein kinases,ERK)、c-Jun 氨基末端激酶(c-Jun N-terminal kinase,JNK)、P38 丝裂原活化蛋白激酶(P38 mito-gen-activated protein kinase,P38 MAPK)、NOD 样受体蛋白 3(NOD-like receptor protein 3,NLRP3)、凋亡相关颗粒样蛋白(apoptosis-associated speck-like protein containing a CARD,ASC)、半胱氨酸天冬氨酸特异蛋白酶-1(cysteinyl aspar-tate specific proteinase-1,Caspase-1)、血红素氧合酶-1(heme oxygenase-1,HO-1)、Kelch 样 ECH 相关蛋白 1(Kelch-like ECH-associated protein 1,KEAP1)和核因子 E2 相关因子 2(nuclear factor erythroid 2-related factor 2,NRF2)的蛋白表达.结果显示,NCTP抑制了 PGE2、COX-2、NO、TNF-α、IL-1β和IL-6的水平(P＜0.01),并防止LPS诱导的RAW 264.7 巨噬细胞 M1 极化(P＜0.01);NCTP 可抑制 TLR4、MyD88、TAK1、IKK、p-IκB/IκB、p-NF-κB/NF-κB、p-JNK/JNK、p-P38 MAPK/P38 MAPK、NLRP3、ASC、Caspase-1、KEAP1 水平,上调 NRF2 和 HO-1 蛋白的表达(P＜0.05,P＜0.01);同时保护MMP、减少ROS的产生(P＜0.05,P＜0.01).以上结果表明,NCTP对LPS诱导的RAW 264.7巨噬细胞炎症损伤具有较好的防治作用,其机制与抗氧化,改善线粒体功能,调节TLR4/MAPK/NF-κB、NLRP3、KEAP1-NRF2/HO-1途径密切相关.
This study aims to investigate the anti-inflammatory mechanism of total polyphenols from Nymphaea candida(NCTP)by using lipopolysaccharide(LPS)-induced RAW 264.7 cell inflammation model.In vitro,CCK-8 assay was used to test cell viability;the Griess reagent method was used to detect the content of nitric oxide(NO)in cell supernatant;ELISA methods were used to measure the levels of cyclooxygenase-2(COX-2),prostaglandin E2(PGE2),interleukin-1β(IL-1β),IL-6,and tumor necrosis factor-α(TNF-α)in the cell supernatant;immunofluorescence and flow cytometry were used to measure mitochondrial membrane potential(MMP)and reactive oxygen species(ROS)levels;flow cytometry was used to test M1 polarization;Western blot was utilized to determine the protein expression levels of Toll-like receptor 4(TLR4),mye-loid differentiation factor 88(MyD88),transforming growth factor β-activated kinase 1(TAK1),inhibitor of κB kinase(IKK),inhibitor of NF-κB(IκB),nuclear factor-κB(NF-κB),extracellular regulated protein kinases(ERK),c-Jun N-ter-minal kinase(JNK),P38 mitogen-activated protein kinase(P38 MAPK),NOD-like receptor protein 3(NLRP3),apoptosis-associated speck-like protein containing a CARD(ASC),cysteinyl aspartate specific proteinase-1(Caspase-1),heme oxyge-nase-1(HO-1),Kelch-like ECH-associated protein 1(KEAP1)and nuclear factor erythroid 2-related factor 2(NRF2).The results showed that NCTP could inhibit the levels of NO,PGE2,COX-2,IL-1β,IL-6 and TNF-α(P＜0.01);and prevented LPS-induced RAW 264.7 macrophages M1 polarization(P＜0.01).NCTP also downregulated the expression of TLR4,MyD88,TAK1,IKK,p-IκB/IκB,p-NF-κB/NF-κB,p-JNK/JNK,p-P38 MAPK/P38 MAPK,NLRP3,ASC,Caspase-1,KEAP1 proteins,and upregulated the expression of NRF2 and HO-1 in RAW 264.7 cell(P＜0.05,P＜0.01).Meanwhile,NCTP el-evated MMP and reduced ROS production(P＜0.05,P＜0.01).These results demonstrated that anti-inflammatory mecha-nism of NCTP might be closely related to antioxidation,ameliorating mitochondrial function,and regulating TLR4/MAPK/NF-κB,NLRP3,KEAP1/NRF2/HO-1 pathways.
李晨阳;姚雨含;艾迪达尔·托力肯;陈燕;赵军
新疆医科大学公共卫生学院,乌鲁木齐 830011||新疆维吾尔自治区药物研究院维吾尔药重点实验室,乌鲁木齐 830004新疆维吾尔自治区药物研究院维吾尔药重点实验室,乌鲁木齐 830004新疆维吾尔自治区药物研究院维吾尔药重点实验室,乌鲁木齐 830004新疆维吾尔自治区药物研究院维吾尔药重点实验室,乌鲁木齐 830004新疆医科大学公共卫生学院,乌鲁木齐 830011||新疆维吾尔自治区药物研究院维吾尔药重点实验室,乌鲁木齐 830004
睡莲花睡莲总多酚炎症TLR4/MAPK/NF-κBNLRP3RAW 264.7细胞
Nymphaea candida Presltotal polyphenols from Nymphaea candidainflammationTLR4/MAPK/NF-κBNL-RP3RAW 264.7 cells
《天然产物研究与开发》 2026 (8)
1629-1639,11
新疆维吾尔自治区自然科学基金重点项目(2021D01D14)新疆维吾尔自治区公益性科研院所基本科研业务费项目(KY2025083)
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