柴胡皂苷B通过Nrf2/ARE信号缓解Caco-2细胞氧化损伤  

Saikosaponin B alleviates oxidative stress and barrier damage in Caco-2 cells through activation of Nrf2/ARE signaling

作　　者：杨慧[1] 黄丽丽 YANG Hui;HUANG Lili(Department of Pharmacy,Ningbo Medical Center Lihuili Hospital,Ningbo 315040,Zhejiang,China)

机构地区：[1]宁波市医疗中心李惠利医院药学部,浙江宁波315040

出　　处：《中国现代医生》2023年第20期61-67,共7页China Modern Doctor

基　　金：国家自然科学基金项目(82003755);宁波市医学重点学科建设项目(2022-F06)。

摘　　要：目的探讨柴胡皂苷B(saikosaponin B,SSB)对棕榈酸(palmitic acid,PA)和脂多糖(lipopolysaccharide,LPS联合诱导的人结直肠腺癌细胞(Caco-2)屏障损伤的保护作用及潜在机制。方法利用Caco-2细胞构建肠道屏障模型,采用随机数字表法将细胞分为对照组(CON组,n=6),模型组(PA+LPS组,n=6)和柴胡皂苷B干预组(PA+LPS+SSB组,n=6),通过测量跨膜电阻(transepithelial electrical resistance,TEER)值和紧密连接蛋白的表达水平,评价SSB对Caco-2细胞屏障损伤的保护作用。通过检测活性氧(reactive oxygen species,ROS)和脂质过氧化物含量评估细胞氧化压力。通过定量聚合酶链反应和蛋白质免疫印迹法检测SSB对Nrf2/ARE信号通路相关基因及蛋白的表达水平的影响。通过敲降Nrf2验证SSB改善细胞屏障功能是否通过Nrf2/ARE途径。结果PA+LPS+SSB组Caco-2细胞的TEER值、ZO-1和Occludin-1的蛋白水平显著高于PA+LPS组(P<0.05)。PA+LPS组细胞内丙二醛(malondialdehyde,MDA)、4-羟基壬烯醛(4-hydroxynonenal,4-HNE)、蛋白羰基含量(protein carbonyl content,PCC)和ROS含量均显著高于对照组(P<0.05),PA+LPS+SSB组的4-HNE、MDA、ROS含量和PCC均显著低于PA+LPS组(P<0.05)。PA+LPS+SSB组细胞内Nrf2和p-Nrf2的蛋白水平、Nqo1和Ho-1的m RNA水平、过氧化氢酶(catalase,CAT)和超氧化物歧化酶(superoxide dismutase,SOD)酶活性均显著高于PA+LPS组(P<0.05)。PA+LPS+SSB^(siNrf2)组细胞中ZO-1和Occludin-1的蛋白水平和转录水平均显著低于PA+LPS+SSB组(P<0.05),ROS、4-HNE、MDA含量和PCC均显著高于PA+LPS+SSB组(P<0.05)。结论SSB通过激活Nrf2/ARE信号通路增加了肠上皮细胞的抗氧化能力,抑制了PA和LPS引起的Caco-2细胞氧化应激和屏障损伤,对肠上皮细胞具有良好保护作用。Objective To investigate the protective effect and therapeutic mechanism of saikosaponin B(SSB)on Caco-2 cell barrier damage induced by the combination of palmitic acid(PA)and lipopolysaccharide(LPS).Methods A monolayer barrier model of Caco-2 cells was constructed in vitro and the cells were divided into control(CON,n=6)group,model(PA+LPS,n=6)group and saikosaponin B(PA+LPS+SSB,n=6)group according to the random number table method.The regulatory effects of SSB on barrier injury in Caco-2 cells were observed by measuring transepithelial electrical resistance(TEER)values,gene and protein levels of tight junction molecules.Cellular oxidative stress was assessed by reactive oxygen species(ROS)and lipid peroxide levels.The effect of SSB on the expression levels of genes and proteins related to the Nrf2/ARE signaling pathway was examined by quantitative PCR and Western blot.Cell transfection was performed to construct a siNrf2 cell model to verify that SSB maintains cellular barrier function through activation of the Nrf2/ARE pathway.Results The TEER values and the protein levels of ZO-1 and Occludin-1 in the PA+LPS+SSB group were significantly higher than those in the PA+LPS group(P<0.05).The intracellular malondialdehyde(MDA),4-hydroxynonenal(4-HNE),protein carbonyl content(PCC)and ROS in the PA+LPS group were significantly higher than those in the control group(P<0.05),while the intracellular 4-HNE,MDA,PCC and ROS in the PA+LPS+SSB group were significantly lower than those in the PA+LPS group(P<0.05).The protein levels of Nrf2 and p-Nrf2,mRNA levels of Nqo1 and Ho-1,catalase(CAT)and superoxide dismutase(SOD)enzyme activities in the PA+LPS+SSB group were significantly higher than those in the PA+LPS group(P<0.05).ZO-1 and Occludin-1 in the PA+LPS+SSB^(siNrf2)group were significantly lower than those in the PA+LPS+SSB group at both protein level and transcript level(P<0.05),and the levels of ROS,4-HNE,MDA and PCC were significantly higher than those in the PA+LPS+SSB group(P<0.05).Conclusion Saikosaponin B increase t

关 键 词：柴胡皂苷B 屏障损伤 Nrf2/ARE 氧化应激 

分 类 号：R285.5[医药卫生—中药学]