摘要
为探究姜黄素通过调控miR-29-3p的表达和Toll样受体(TLR4)/核因子κB(NF-κB)信号通路发挥抗炎及抗纤维化的作用,通过转化生长因子β1(TGF-β1)诱导建立肾间质纤维化细胞模型,采用MTT法和流式细胞术分别测定姜黄素对细胞增殖活性和细胞凋亡率的影响,以酶联免疫吸附法(Elisa)和免疫印迹(WB)分别检测姜黄素和miR-29-3p mimic对人肾皮质近曲小管上皮细胞(HK-2细胞)上清液肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)分泌水平的影响以及Ⅰ型胶原(COLⅠ)、Ⅲ型胶原(COLⅢ)、α-平滑肌肌动蛋白(α-SMA)和NF-κB/TLR4信号通路相关蛋白的表达情况,用RT-PCR法检测miR-29-3、Ⅰ型胶原蛋白α1链(COL1A1)和Ⅲ型胶原蛋白α1链(COL3A1)的表达水平。研究结果表明,与模型组相比,当姜黄素浓度为10μmol/L时可显著抑制TGF-β1诱导的肾间质纤维化细胞过度增殖,提高其凋亡率(P<0.05),抑制α-SMA(P<0.05)、COLⅠ(P<0.01)、COLⅢ(P<0.05)的生成,促进miR-29-3p的表达(P<0.01),抑制IL-1β(P<0.01)、IL-6(P<0.01)和TNF-α(P<0.01)的产生和p-IκBα/IκBα(P<0.01)、TLR4(P<0.05)、p-P65/p65(P<0.05)蛋白的相对表达量;在细胞转染实验中,与模型组相比,转染miR-29-3p mimic后细胞上清液中IL-1β、IL-6和TNF-α水平显著降低(P<0.01),细胞中COL1A1和COL3A1的mRNA和α-SMA、COLⅠ、COLⅢ、TLR4、p-IκBα/IκBα、p-P65/p65蛋白表达量显著下降(P<0.01)。研究表明,姜黄素具有抗炎和抗纤维化作用,能够改善肾间质纤维化,其机制与调控miR-29-3p和TLR4/NF-κB信号通路有关。
This study was aimed to explore the anti-inflammatory and anti-fibrosis effects of curcumin by regulating the expression of miR-29-3p and the Toll-like receptor 4(TLR4)/nuclear factorκB(NF-κB)signaling pathway.Curcumin on cell apoptosis rate and cell proliferation activity were determined by flow cytometry and MTT method,respectively,on the transforming growth factor-β1(TGF-β1)induced renal interstitial fibrosis cell model.The influence of curcumin and miR-29-3p mimic on the productions of tumor necrosis factor-α(TNF-α),Interleukin-6(IL-6),Interleukin-1β(IL-1β)in the Human Kidney-2 cells(HK-2)were determined by enzyme linked immunosorbent assay(Elisa)method and the influence on the secretion ofα-smooth muscle actin(α-SMA),collagen typeⅠ(COLⅠ),collagen typeⅢ(COLⅢ),TLR4,p-IκBα/IκBα,and p-P65/p65 were detected by Western blot(WB)method.The relative expression levels of miR-29-3P,COL1A1 and COL1A3 were detected by real-time quantitative polymerase chain reaction(RT-PCR).The results showed that compared with model group,curcumin at the concentration 10μmol/L can significantly inhibit the excessive proliferation cell and improve the apoptosis rate which induced by TGF-β1(P<0.05),as well as inhibitα-SMA(P<0.05),COLⅠ(P<0.01),COLⅢ(P<0.05),IL-1β(P<0.01),IL-6(P<0.01)and of TNF-α(P<0.01)production,promote miR-29-3p expression(P<0.01),and reduce the TLR4(P<0.05),p-IκBα/IκBα(P<0.01),p-P65/p65 levels(P<0.05).In the cell transfection experiment,compared with model group,IL-6,IL-1βand TNF-αwere significantly reduced(P<0.01)as well as mRNA andα-SMA,COLⅠ,COLⅢ,TLR4,p-IκBα/IκBα,p-P65/p65 expression of COL1A1 and COL3A1 were significantly decreased(P<0.01)after the transfection miR-29-3p mimic to cells.Therefore,curcumin exerted the anti-inflammatory and anti-fibrotic effect which may be related to the regulation of miR-29-3p and TLR4/NF-κB signaling pathway.
作者
陈飞
罗惠民
谢瑜
吕琴
沈颖
CHEN Fei;LUO Huimin;XIE Yu;Lü Qin;SHEN Ying(Department of Nephrology,The First People's Hospital of Yunnan Province,The Affiliated Hospital of Kunming University of Science and Technology,Kunming 650032,China)
出处
《甘肃科学学报》
2022年第5期26-34,共9页
Journal of Gansu Sciences
基金
云南省科技厅—昆明医科大学应用基础研究联合专项(2018FE001(-289))。
