阿伦磷酸钠联合肾骨安对去势小鼠终板结构和椎间盘退变的影响

Effects of alendronate combined with "Shen gu an" on the endplate structure and intervertebral disc degeneration in ovariectomized mice

中文关键词:  椎间盘退变  卵巢切除  终板  阿伦膦酸钠  肾骨安  小鼠

英文关键词:Intervertebral disc degeneration  Ovariectomy  Endplate  Alendronate  Herb  Mice

中文摘要:

  【摘要】 目的：观察阿伦磷酸钠（alendronate，ALN）联合补肾中药———肾骨安（herbs，HB）对去势小鼠椎体终板、椎间盘退变（intervertebral disc degeneration，IVDD）的影响，初步探讨ALN+HB干预IVDD的作用机制。方法：32只12周龄健康雌性C57BL/6J小鼠，随机分为正常对照组（对照组，n=8）、去卵巢组（OVX组，n=8）、ALN组（n=8）及ALN+HB组（n=8）。对照组行假手术，其余各组行双侧卵巢摘除。术后3d开始给药，连续给药2个月后取材，HE及番红O-固绿染色观察椎间盘组织形态学改变并进行病理评分，显微计算机断层扫描（u-CT）检测终板表面孔隙变化及附近骨赘增生情况，免疫组织化学法检测椎间盘中Ⅱ型胶原（Collagen-type Ⅱ，Col2）的表达。结果：实验过程中无小鼠死亡。HE及番红O-固绿染色对照组椎间盘结构和形态基本正常；OVX组椎间盘终板发生严重骨化重塑，以下位终板更为明显，同时出现终板与髓核的边界结构紊乱，椎间盘基质的丢失及裂隙形成；ALN组终板骨化重塑较OVX组轻，髓核结构形态改善，软骨终板内仍可见一些小的骨化区；ALN+HB组终板骨化重塑不明显，番红O固绿染色染色可见软骨终板着色加深。IVDD病理评分、终板表面孔隙率（%）和Col2积分光密度（IOD）值分别为1.20±0.84、28.60±4.04和27764.7±1958.2，OVX组为4.00±1.58、56.80±9.39和17213.6±1021.3，ALN组为2.40±0.89、36.20±3.42和18921.4±888.1，ALN+HB组为2.00±0.71、29.60±6.19和23420.1±2439.6。OVX组与对照组比较IVDD病理评分、终板孔隙率均显著性升高（P<0.05）；ALN组、ALN+HB组与OVX组比较二者均显著性降低（P<0.05）；ALN组、ALN+HB组与对照组比较，及ALN+HB组与ALN组比较二者均无统计学差异（P>0.05）。OVX组、ALN组和ALN+HB组与对照组比较Col2 IOD值均显著性降低（P<0.05），ALN组与OVX组比较无统计学差异（P>0.05），ALN+HB组与OVX组、ALN组比较均显著性升高（P<0.05）。结论：OVX小鼠雌激素缺乏可引起椎体终板的骨化重塑及孔隙率增加，导致IVDD。ALN及ALN+HB均可以抑制椎体终板的重塑，降低终板孔隙率，保证终板结构和功能完整，从而能延缓雌激素缺乏相关IVDD，ALN+HB效果更为明显。

英文摘要:

  【Abstract】 Objectives: To observe the effects of alendronate(ALN) combined with tonifying-kidney herbs (herbs, HB) on the endplate leision and intervertebral disc degeneration(IVDD) in ovariectomized mice, and to investigate the mechanism of ALN+HB in the treatment of IVDD. Methods: Thirty-two 12-week-old healthy female C57BL/6J mice were randomly divided into four groups as follows: control group(CT, n=8), the ovariectomized group(OVX, n=8), the alendronate treated group(ALN, n=8) and the group treated with alendronate combining with tonifying-kidney herbs(ALN+HB, n=8). The mice in CT group underwent sham operation, and the mice in the other groups were bilateral ovariectomized. Animals were dosed with medicine three days after surgery and sacrificed at two months later. HE and safranine O-fast green staining were used to detect the morphological changes of intervertebral disc, and the pathological score was recorded. The u-CT scan was used to examine the surface porosity of endplate and the osteophyte formation nearby. The expression of collagen-type Ⅱ(Col2) in the intervertebral disc was detected by immunohistochemistry. Results: No mice died during the experiment. The structure and morphology of intervertebral disc in CT group showed normal in Hematoxylin-Eosin and Safranine O-Fast green staining. In OVX group, severe ossification occurred on the endplate which was more obvious in the caudal side. Besides, the border between the endplate and the nucleus pulposus was disordered with matrix degradation and crack formation. However, the ossification remodeling of the endplate and structural morphology of the nucleus pulposus in ALN group were obviously improved when compared with OVX group, although small ossification lesions in endplates were still existed. In ALN+HB group, there was hardly no ossification remodeling in endplates, and safranin O staining showed deep dying in cartilagous endplate. The IVDD score, endplate surface porosity(%) and Col2 integral optical density(IOD) were 1.20±0.84, 28.60±4.04 and 27764.7±1958.2 in CT group, 4.00±1.58, 56.80±9.39 and 17213.6±1021.3 in OVX group, 2.40±0.89, 36.20±3.42 and 18921.4±888.1 in ALN group, and 2.00±0.71, 29.60±6.19 and 23420.1±2439.6 in ALN+HB group, respectively. Both the pathological score of IVDD and the porosity of the endplate were significantly higher in OVX group than in tCT group, ALN group and ALN+HB group(P<0.05). There was no significant difference among ALN group, ALN+HB group and CT group(P>0.05). The Col2 IOD in OVX, ALN and ALN+HB groups was significantly lower than that in CT group(P<0.05), but had no significant difference between ALN group and OVX group(P>0.05). However, the Col2 IOD in ALN+HB group was significantly higher than that in OVX group and ALN group(P<0.05). Conclusions: Estrogen deficiency in OVX mice can induce ossification remodeling and increasing permeability of endplate, which affects the disc metabolism and results in IVDD. Meanwhile, ALN and ALN+HB can inhibit the ossification remodeling, reduce the porosity, maintain the structural and the functional integrity of endplate, which delays the menopausal related IVDD. Futhermore, ALN＋HB has a better effect on IVDD.