甲基莲心碱对腰椎间盘退变大鼠的影响

Effects of neferine on lumbar disc degeneration in rats

目的研究甲基莲心碱对髓核细胞生长、髓核组织胶原蛋白变性和免疫反应对椎间盘退变的修复作用。方法(1)细胞实验:将髓核细胞分为4组:空白对照组、加药组、诱导损伤组和损伤加药组。空白对照组细胞正常培养,诱导损伤组和损伤加药组细胞加10μg·mL^-1白细胞介素-1β(IL-1β),加药组和损伤加药组细胞用20μg·mL^-1甲基莲心碱处理。以细胞计数试剂盒(CCK-8)检测髓核细胞存活率,以蛋白质印迹检测细胞增殖标记物(Ki67)和半胱氨酸天冬氨酸蛋白酶3(Caspase-3)的表达。(2)动物实验:按照体重将大鼠随机分成4组:正常组、健康给药组、模型组和模型给药组,每组10只;健康给药组和模型给药组大鼠腹腔注射甲基莲心碱20 mg·kg^-1,每天1次;另外2组大鼠腹腔注射等量0. 9%Na Cl,连续6周。用纤维环穿刺法建立椎间盘退变模型,以酶联免疫吸附法检测IL-1β、肿瘤坏死因子(TNF-α)和诱导性一氧化氮合酶(i NOS)的表达。结果(1)细胞结果:空白对照组、加药组、诱导损伤组和损伤加药组的细胞存活率分别为(100. 00±0. 00)%,(100. 00±0. 00)%,(32. 69±9. 09)%和(84. 35±8. 37)%;上述这4组Ki67的表达量分别为0. 38±0. 05,0. 40±0. 06,0. 07±0. 03和0. 30±0. 05;上述这4组Caspase-3的表达水平分别为0. 05±0. 01,0. 04±0. 01,0. 33±0. 06和0. 10±0. 02;诱导损伤组与空白对照组相比,细胞存活率、Ki67表达量显著降低,Caspase-3的表达水平显著升高,差异均有统计学意义(均P <0. 01);损伤加药组的上述指标与诱导损伤组相比,差异均有统计学意义(均P <0. 01)。(2)动物结果:正常组、健康给药组、模型组和模型给药组IL-1β的水平分别为(17. 92±8. 27),(14. 78±6. 12),(89. 21±17. 22)和(32. 37±12. 34) pg·mg^-1;上述这4组TNF-α的水平分别为(30. 24±5. 11),(27. 93±5. 20),(108. 67±9. 89)和(45. 93±7. 19)pg·mg^-1;上述这4组i NOS的水平分别为(54. 19±5. 44),(49. 06±5. 77),(175. 97±22. 13)和(78. 93±9. 19) pg·mg^-1。模型组的上述指标与正常组比较,差异均有统计学意义(均P <0. 01);模型给药组的上述指标与模型组比较,差异均有统计学意义(均P <0. 01)。结论甲基莲心碱诱导髓核细胞生长,抑制髓核组织胶原蛋白变性和免疫紊乱,对腰椎间盘退变具有修复作用。

Objective To investigate the repairing effect of neferine on the growth of nucleus pulposus cells,collagen degeneration of nucleus pulposus tissue and immune response to degeneration of intervertebral disc. Methods ( 1) Cell experiment: The nucleus pulposus cells were divided into 4 groups: blank control group( were cultured normally),do- sing group( 20 μg·mL^-1 neferine),induced injury group[interleukin - 1β( IL -1β) 10 μg·mL^-1 induced],and injury dosing group( IL - 1β+ neferine). Cell counting kit ( CCK -8) was used to detect the survival rate of nucleus pulposus cells,and protein imprinting was used to detect the expression of cell proliferation marker ( Ki67) and cysteine aspartate protease 3( caspase -3).( 2) Animal experiment: Rats were randomly divided into four groups: normal group,healthy administration group,model group,model administration group,each group had ten rats. The rats in the healthy and model administration groups were intraperitoneally injected with 20 mg·kg^-1 of neferine once a day;the rats in the other two groups were intraperitoneally injected with the same amount of physiological liquid, for 6 weeks. The model of intervertebral disc degeneration was established by using fiber ring puncture method. The ex- pressions of the IL -1β,nitric oxide synthase ( iNOS),tumor necrosis factor -α( TNF -α) were detected by enzyme linked immunosorbent assay. Results ( 1) Cell results: The cell survival rates of blank control group,dosing group,in- duced injury group,and injury dosing group were ( 100. 00 ± 0. 00)%,( 100. 00 ±0. 00)%,( 32. 69 ± 9. 09)% and ( 84. 35 ±8. 37)%. The expression levels of Ki67 in these four groups were 0. 38 ±0. 05,0. 40 ±0. 06,0. 07 ±0. 03, and 0. 30 ± 0. 05,respectively. The expression levels of Caspase - 3 in these four groups were 0. 05 ± 0. 01, 0. 04 ±0. 01,0. 33 ±0. 06,and 0. 10 ±0. 02,respectively. Comparing between induced injury group and blank control group,the cell viability and Ki67 expression decreased significantly and Caspase -3 expression s increased significantly ( all P < 0. 01);comparing between injury dosing group with the induced injury group,the cell viability and Ki67 expression increased significantly and the Caspase -3 expression decreased significantly ( all P <0. 01).( 2) Animal results: The levels of IL - 1 β in normal group,experimental - healthy group,model group and experimental - model group were ( 17. 92 ±8. 27),( 14. 78 ±6. 12),( 89. 21 ±17. 22),and ( 32. 37 ±12. 34) pg·mg^-1;and the levels of TNF -α in these 4 groups were ( 30. 24 ±5. 11),( 27. 93 ±5. 20),( 108. 67 ±9. 89),and ( 45. 93 ±7. 19) pg·mg^-1 , respectively;the levels of iNOS in these 4 groups were ( 54.19 ±5.44),( 49.06 ± 5.77),( 175.97 ± 22.13),and ( 78.93 ±9.19) pg·mg^-1 . Comparing between model group with normal group,the differences of the factors were significant ( all P <0.01);comparison between experimental - model group with model group,the differences of the factors were signifi- cant( all P <0.01). Conclusion Neferine can repair the degeneration of lumbar intervertebral disc by inducing the growth of nucleus pulposus cells and inhibiting the degeneration of collagen and immune disorders in the nucleus pulposus.