狭鳕鱼皮胶原多肽对去势大鼠骨微结构影响的研究

Effect of collagen peptides from walleye pollock skin on bone microstructure of ovariectomized rats

目的通过观察狭鳕鱼皮胶原多肽对去势大鼠骨质疏松模型骨微结构的影响,探讨其防治骨质疏松的可行性。方法成年Wistar雌性大鼠60只,体质量(250±10)g,随机分为5组(n=12),分别为正常对照组(A组)、骨质疏松模型组(B组)、骨质疏松模型+狭鳕鱼皮胶原多肽预防组(C组)、骨质疏松模型+狭鳕鱼皮胶原多肽低剂量治疗组(D组)、骨质疏松模型+狭鳕鱼皮胶原多肽高剂量治疗组(E组),每组12只。B、C、D、E组采用摘除双侧卵巢法制备大鼠骨质疏松模型。C组从术前4周开始、D、E组从术后6周开始,每天按照1.0、0.5、1.0 g/kg行狭鳕鱼皮胶原多肽灌胃,连续6周;A、B组于术后同时间点给予等体积生理盐水灌胃。末次给药24 h后,A、B组大鼠摄股骨X线片并测定灰度值;然后颈椎脱臼法处死各组大鼠,取左侧胫骨近端骨组织行HE染色,观察骨组织病理学改变,测量骨小梁数量(trabecular number,TN)、平均骨小梁厚度(mean trabecular plate thickness,MTPT)、平均骨小梁间距(mean trabecular plate spacing,MTPS)、骨小梁体积百分比(trabecular bone volume,TBV)、平均骨皮质厚度(mean bone cortical thickness,MBCT);免疫组织化学染色观测降钙素受体(caltitonin receptor,CTR)及IL-1表达水平。结果 B组大鼠股骨灰度值显著低于A组(t=45.130,P=0.000),表明去势大鼠骨质疏松模型制备成功。组织学观察示,A、C、E组TN、MTPS、TBV、MBCT与B组比较,差异有统计学意义(P<0.05);C组各骨组织形态计量学参数与D、E组比较,差异均有统计学意义(P<0.05);D组TN、MTPS、TBV、MBCT与A组比较差异有统计学意义(P<0.05);E组仅MTPS与A组比较差异有统计学意义(P<0.05);E组MTPS、TBV、MBCT与D组比较,差异有统计学意义(P<0.05)。免疫组织化学染色观察示,A、C、D、E组CTR、IL-1表达水平较B组降低,C、E组低于D组,差异有统计学意义(P<0.05);其余组间比较差异均无统计学意义(P>0.05)。结论狭鳕鱼皮胶原多肽能够改善骨质疏松大鼠的骨微结构,其机制可能与抑制骨组织中CTR、IL-1表达有关,但尚未发现其对骨质疏松症有预防作用。

Objective To investigate the effect of collagen peptides from walleye pollock skin on the microstructure of osteoporosis model in ovariectomized rats, and to explore the feasibility of preventing and treating oste- oporosis. Methods Sixty adult Wistar female rats, weighing （250±10） g, were randomly divided into 5 groups （12 rats each group）： normal group （group A）, osteoporosis model group （group B）, osteoporosis model＋collagen peptides from walleye pollock skin prevention group （group C）, osteoporosis model＋low concentration of collagen peptides from walleye pollock skin treatment group （group D）, and osteoporosis model＋high concentration of collagen peptides from walleye pollock skin treatment group （group E）. The rats in groups B, C, D, and E were removed bilateral ovarian to establish osteoporosis model. The rats in group C were treated with stomach perfusion of the collagen peptides from walleye pollock skin （1.0 g/kg） from 4 weeks after operation for 6 weeks; and the rats in groups D and E were treated with stomach perfusion of the collagen peptides from walleye pollock skin （0.5, 1.0 g/kg respectively） at 6 weeks after operation for 6 weeks. The rats in groups A and B were given equal volume of normal saline at the same time after operation. At 24 hours after the last administration, the femoral gray value of rats in groups A and B were measured by X-ray film; HE staining was performed on the proximal tibial bone of the left side in 4 groups; the histopathological changes of the bone were observed and the trabecular number （TN）, mean trabecular plate thickness （MTPT）, mean trabecular plate spacing （MTPS）, trabecular bone volume （TBV）, mean bone cortical thickness （MBCT） were measured; immunohistochemical staining was performed to observe the expression levels of caltitonin receptor （CTR） and interleukin 1 （IL-1）. Results The femoral gray value of group B was significantly lower than that of group A （t=45.130, P=0.000）, which indicated that the ovariectomized rat model was successfully prepared. Histological observation showed that TN, MTPS, TBV, and MBCT in groups A, C, and E were significantly different from those in group B （P〈0.05）. The histological parameters of bone tissue in group C were significantly different from those in groups D and E （P〈0.05）. TN, MTPS, TBV, and MBCT in group D were significantly different from those in group A （P〈0.05）; only MTPS in group E was significantly different from that in group A （P〈0.05）. MTPS, TBV, and MBCT in group E were significantly different from those in group D （P〈0.05）. The immunohistochemical staining showed that the levels of CTR and IL-1 in groups A, C, D, and E were lower than those in group B, in groups C and E were lower than in group D, showing significant differences （P〈0.05）. Conclusion Collagen peptides from walleye pollock skin can improve the bone microstructure of osteoporotic rats, and its mechanism may be related to the inhibition of CTR and IL-1 expression in bone tissue, but it has not been found to prevent osteoporosis.