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目的探讨六味地黄丸含药血清对兔退变椎间盘软骨终板细胞的保护作用。方法选择8月龄成年新西兰兔利用“异常应力负荷及椎间失稳法”建立新西兰兔腰椎椎间盘退变模型。病理切片确认模型成功后,取椎间盘分离下位软骨终板进行细胞培养,将生长良好的细胞随机分为含药组、损伤组与对照组。含药组体积分数为10%的六味地黄丸含药血清加体积分数为10%的胎牛血清与浓度为5μg·L-1的TNF-α混合的DMEM培养基;损伤组给予体积分数为10%的胎牛血清与浓度为5μg·L-1的TNF-α混合的DMEM培养基;对照组细胞培养用胎牛血清体积分数为10%的DMEM培养基。使用细胞增殖-毒性检测试剂盒(CCK-8)检测各组兔退变椎间盘软骨终板细胞增殖的变化。TUNEL法计数软骨终板活细胞与凋亡细胞数量,RT-PCR测定其Fas/Fas L的表达水平。结果对照组软骨终板细胞初期正常生长,增殖良好,随着时间延长,软骨终板细胞数量减少,凋亡细胞数量增多,细胞外基质胶原减少,细胞形态变化快。损伤组能够明显抑制椎间盘软骨终板细胞的增殖,软骨细胞及细胞外基质胶原数量减少明显,同一时间点比较,软骨细胞凋亡数量增多,与对照组比较差异有统计学意义(P<0.05)。而含药组软骨终板细胞增殖缓慢,软骨细胞下降少,细胞凋亡数量减少,细胞外基质胶原成分下降不明显,3组间比较差异有统计学意义(P<0.05)。对照组软骨终板内Fas/Fas L的表达水平随时间逐渐增强,同期比较,损伤组Fas/Fas L表达水平高于对照组,而含药组软骨终板水平Fas/Fas L较损伤组表达明显下降,差异有统计学意义(P<0.05)。结论六味地黄丸含药血清能够抑制TNF-α诱导的兔退变椎间盘软骨终板细胞损伤,减少软骨细胞凋亡,起到延缓椎间盘损伤的作用,其机制可能与抑制Fas/Fas L基因在退变椎间盘软骨终板中的过度表达有关。
Objective To investigate the protective effect of Liuwei Dihuang Pills containing serum on the degeneration of intervertebral disc cartilage end-plate cells in rabbits. Methods Eight-month-old New Zealand white rabbits were selected to establish a model of lumbar disc degeneration in New Zealand rabbits by using “abnormal stress load and intervertebral instability method”. After the pathological section confirmed the model was successful, the lower cartilage endplate was isolated from the disc for cell culture, and the well-growing cells were randomly divided into drug-containing group, injury group and control group. Drug-containing group volume fraction of 10% of Liuweidihuangwan drug-containing serum plus 10% fetal bovine serum concentration of 5μg · L-1 of TNF-α mixed with DMEM medium; injury group given volume fraction of 10 % Fetal bovine serum and TNF-α at a concentration of 5 μg · L-1. The control group was cultured in DMEM with 10% fetal bovine serum. The proliferation of degenerative disc cartilage end-plate cells in each group was detected by CCK-8. The number of viable cells and apoptotic cells were counted by TUNEL method. The expression of Fas / Fas L was determined by RT-PCR. Results In the control group, the cartilage end-plate cells grew normally and proliferated well. With the extension of time, the number of cartilage end-plate cells decreased, the number of apoptotic cells increased, the extracellular matrix collagen decreased, and the cell morphology changed rapidly. Compared with control group, the number of chondrocytes apoptosis in the injured group was significantly decreased (P <0.05), and the number of chondrocytes and extracellular matrix collagen in the injury group was significantly decreased (P <0.05) . The drug-containing group of cartilage end-plate cells proliferation slow, chondrocyte decreased, the number of apoptotic cells decreased, the extracellular matrix collagen composition was not significantly reduced, the difference between the three groups was statistically significant (P <0.05). The expression level of Fas / Fas L in cartilaginous endplate in control group increased gradually with time, and the expression level of Fas / Fas L in injured group was higher than that in control group at the same period Significantly decreased, the difference was statistically significant (P <0.05). Conclusion Liuweidihuangwan serum can inhibit TNF-α-induced degeneration of intervertebral disc cartilage end-plate cells, reduce chondrocyte apoptosis and play a role in delaying the injury of the intervertebral disc. The mechanism may be related to inhibition of Fas / Fas L gene in retreat Variable disc cartilage endplate overexpression.