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.
Kai NiuChenguang LiSong YuanLei ZhangQi ShiYongjun WangWeichao Zheng
目的观察不同浓度的苦杏仁苷对IL-1β诱导的大鼠椎间盘软骨终板细胞的影响,并进一步探讨其作用的可能机制。方法从1月龄SD大鼠椎间盘中分离软骨终板并培养,经鉴定后,随机分组为正常组、诱导组、苦杏仁苷10-2、10-3、10-4、10-5mol·L-1给药组,CCK-8法检测各组对大鼠椎间盘软骨终板细胞增殖的影响。Real-time PCR(RTPCR)检测聚集蛋白聚糖(aggrecan-1)、Ⅱ型胶原(type II collagen,Col2a1)、Ⅹ型胶原(type X collagen,Col10al)、基质金属蛋白酶-13(matrix metalloproteinase 13,MMP-13)基因的表达情况。细胞免疫荧光检测分析Col2a1、Col10al的表达,流式细胞仪检测其对软骨终板细胞凋亡的影响。结果苦杏仁苷10-2mol·L-1给药组能够抑制椎间盘软骨终板细胞的增殖,与正常组比较差异有统计学意义(P<0.05)。流式分析,其细胞凋亡比例较诱导组及正常组均较高。RT-PCR检测结果显示一定浓度的苦杏仁苷能够上调Aggrecan、Col2a1mRNA的表达,下调Col10al、MMP-13 mRNA的表达,与诱导组比较,差异有统计学意义(P<0.05)。一定浓度的苦杏仁苷能够上调Col2a1蛋白的表达,下调Col10al蛋白的表达。结论一定浓度的苦杏仁苷能够抑制IL-1β诱导大鼠椎间盘软骨终板细胞发生退变,起到延缓椎间盘退变的作用。
