The inflammatory cytokine interleukin-1 beta （IL-1β） plays a key role in the process of intervertebral disc degenera- tion （IVDD）. In the present study, we aimed to evaluate the effect of pharmaco-serum of ＂Taor...The inflammatory cytokine interleukin-1 beta （IL-1β） plays a key role in the process of intervertebral disc degenera- tion （IVDD）. In the present study, we aimed to evaluate the effect of pharmaco-serum of ＂Taoren-Honghua-herb pair＂ on IL-1β- induced chondrocyte degeneration in vitro. Taoren （Semen persicae） and Honghua （Safflower carthamus） were administered to the rats, and the pharmaco-serum was collected and prepared. Chondrocytes of the third passage, isolated from the rat＇s vertebral endplates, were treated by standard medium only （Group NC）, IL-1β （Group IL） or combination of IL-1β and pharmaco-serum （Group TRHH）. Cell proliferation and apoptosis were determined, and the expression of aggrecan, Col2ul, Coll0ul, IL-6 and SOX9 at the mRNA level in chondrocytes was quantified by real-time PCR. Immunohistochemistry staining of type II and X collagen and Safranine O staining were also used to evaluate the chondrocytes. Compared with the Group NC, IL-1β treatment inhibited the cell proliferation and induced the cell apoptosis （P〈0.05）, and the expression of aggrecan, Col2αl and SOX9 at the mRNA level was down-regulated. In contrast, the expression of Coll0ul and IL-6 was up-regulated after IL-1β treatment （P〈0.05）. Meanwhile, the immune-staining of type II collagen and Safranine O staining were decreased, while the staining of type X collagen was increased. Compared with the Group IL, cell proliferation was increased, and apoptosis of chondrocytes was decreased when cells were treated with the pharmaco-semm of TRHH-herb pair （P〈0.05）. The expression of aggrecan, Col2cd and SOX9 at the mRNA level was up-regulated, while that of Coll0cd and IL-6 was down-regulated （P〈0.05）. Saffanine O staining also showed increased positive staining （P〈0.05）. Taken together, the treatment of pharmaco-serum of TRHH-herb pair could prevent endplate chondrocyte degeneration induced by IL-1β.展开更多
文摘目的探究独活寄生汤水提物对经IL-1β诱导退变的大鼠椎间盘软骨细胞中Wnt4、GSK-3β、β-catenin、DKK-1的影响。方法采用机械-酶消化法分离大鼠椎间盘软骨组织,进行软骨细胞体外培养、镜下观察与鉴定,分为正常组、模型组(经10 ng/m L浓度IL-1β造模)、实验1组(独活寄生汤水提物组200μg/m L干预24 h)、实验2组(独活寄生汤水提物组200μg/m L干预48 h)。观察4组大鼠椎间盘软骨细胞中Wnt4、GSK-3β、β-catenin、DKK-1 m RNA与蛋白的表达及上清液中Sox 9表达。结果 (1)第2代大鼠椎间盘软骨细胞增殖速度快,呈现多边形,胞核清晰,且含有1~2个核仁,融合后出现"铺路石"状,经Ⅱ型胶原法染色后,阳性对照组胞浆区域浸染为棕黄色;(2)与正常组比较,模型组中软骨细胞Wnt 4、GSK-3β、β-catenin m RNA与蛋白表达及上清液Sox 9含量明显提高(P<0.05),DKK-1 m RNA与蛋白表达明显降低(P<0.05);与模型组比较,实验1组、实验2组中软骨细胞Wnt 4、GSK-3β、β-catenin m RNA与蛋白表达及上清液Sox 9含量明显降低(P<0.05),DKK-1m RNA与蛋白表达明显提高(P<0.05),以实验2组变化最为显著(P<0.05)。结论独活寄生汤水提物组可调控退变椎间盘软骨细胞的功能,下调Wnt 4、GSK-3β、β-catenin和上调DKK-1 m RNA与蛋白表达,进而延缓大鼠椎间盘软骨细胞的退变。
基金National Natural Science Foundation of China(Grant No.81273777,81102606,81072831)the National Basic Re search Program of China(Grant No.2010CB530400)the Program for Changjiang Scholars and Innovative Research Team in University(PCSIRT,IRT1270)
文摘The inflammatory cytokine interleukin-1 beta （IL-1β） plays a key role in the process of intervertebral disc degenera- tion （IVDD）. In the present study, we aimed to evaluate the effect of pharmaco-serum of ＂Taoren-Honghua-herb pair＂ on IL-1β- induced chondrocyte degeneration in vitro. Taoren （Semen persicae） and Honghua （Safflower carthamus） were administered to the rats, and the pharmaco-serum was collected and prepared. Chondrocytes of the third passage, isolated from the rat＇s vertebral endplates, were treated by standard medium only （Group NC）, IL-1β （Group IL） or combination of IL-1β and pharmaco-serum （Group TRHH）. Cell proliferation and apoptosis were determined, and the expression of aggrecan, Col2ul, Coll0ul, IL-6 and SOX9 at the mRNA level in chondrocytes was quantified by real-time PCR. Immunohistochemistry staining of type II and X collagen and Safranine O staining were also used to evaluate the chondrocytes. Compared with the Group NC, IL-1β treatment inhibited the cell proliferation and induced the cell apoptosis （P〈0.05）, and the expression of aggrecan, Col2αl and SOX9 at the mRNA level was down-regulated. In contrast, the expression of Coll0ul and IL-6 was up-regulated after IL-1β treatment （P〈0.05）. Meanwhile, the immune-staining of type II collagen and Safranine O staining were decreased, while the staining of type X collagen was increased. Compared with the Group IL, cell proliferation was increased, and apoptosis of chondrocytes was decreased when cells were treated with the pharmaco-semm of TRHH-herb pair （P〈0.05）. The expression of aggrecan, Col2cd and SOX9 at the mRNA level was up-regulated, while that of Coll0cd and IL-6 was down-regulated （P〈0.05）. Saffanine O staining also showed increased positive staining （P〈0.05）. Taken together, the treatment of pharmaco-serum of TRHH-herb pair could prevent endplate chondrocyte degeneration induced by IL-1β.
