升高脑脊液瘦素水平预防兔骨质疏松的实验研究

Experimental study on precaution osteoporosis by the increase of cerebrospinal fluid leptin level on rabbit model

目的经枕骨大孔向小脑延髓池注射给药提高脑脊液瘦素水平，探索脑脊液瘦素水平的升高对兔双侧卵巢切除联合甲泼尼龙琥珀酸钠诱导的骨质疏松的影响。方法选取30只体重相近的5月龄健康新西兰雌兔，按随机数字表法分为对照组、生理盐水组、低剂量瘦素组、中剂量瘦素组和高剂量瘦素组。5组均行双侧卵巢切除术，并于术后起每日肌注甲泼尼龙琥珀酸钠（1mg／kg）持续8周。术后第0、3、7天，对照组仅经枕骨大孔小脑延髓池穿刺，生理盐水组小脑延髓池内注射生理盐水0．05ml，低、中、高剂量组小脑延髓池内注射质量浓度为0．0l、0．1、1．0μg/μl的瘦素蛋白溶液0．05ml。于术前和术后4、8周双能x线仪扫描测定双侧股骨骨密度，酶联免疫吸附法检测脑脊液瘦素浓度和血骨钙素、生长激素和胰岛素生长因子-1，并检测血钙、磷、碱性磷酸酶浓度。第8周处死所有动物后，PCR测定脑组织内瘦素受体和骨组织内骨形成蛋白-2、I型胶原的表达情况。结果脑脊液瘦素水平的改变，给药后第4、8周，低、中、高剂量组脑脊液瘦素浓度较对照组和生理盐水组保持较高水平，且与给药浓度呈正相关。骨密度的变化，瘦素给药组骨密度较非瘦素给药组下降趋势不明显，且高剂量组呈持平趋势。血骨钙素、生长激素、胰岛素生长因子-1的变化，第4周各组血骨钙素明显降低，且瘦素给药组较非瘦素给药组下降更加明显，第8周各组血骨钙素均明显回升，瘦素给药组较非瘦素给药组回升明显，低剂量组较高剂量组回升明显；第4、8周血生长激素呈下降趋势，但非瘦素给药组较瘦素给药组下降不明显；第4周各组血胰岛素生长因子-1均明显下降，第8周对照组、低剂量组较生理盐水组、中、高剂量组明显升高。血钙、磷、碱性磷酸酶的变化，第8周瘦素给药组血钙较非瘦素给药组明显升高，且与给药浓度呈正相关；第4周各组血磷呈下降趋势，各组间无明显差异，第8周各组均升高，各组问无差异；各组血碱性磷酸酶在第4、8周呈持续下降趋势，且中剂量组下降较其他组明显。PCR表达量的变化，第8周脑瘦素受体mRNA表达量各组间差异无统计学意义；瘦素给药组骨骨形成蛋白-2和I型胶原mRNA较非瘦素给药组表达明显增高。结论升高脑脊液瘦素水平可促进成骨代谢，减少骨质流失和预防双侧卵巢切除联合甲强龙导致的兔骨质疏松发生，并呈剂量依赖性，为兔骨质疏松的防治提供了可能的新方法。

Objective To explore effect of increasing cerebrospinal fluid （CSF） leptin level by administration from the fo- ramen magnum to the cerebellum medulla oblongata pool in osteoporosis in rabbit model established by ovariectomy （OVX） and methylprednisolones. Methods According to certain inclusion and exclusion criteria, 30 adult rabbits which were healthy 5- month-old female New Zealand white rabbits with the same batch and weight were divided into 5 groups （n=6） with random num- ber table, including control group, normal saline group, Low dose group, middle dose group and high dose group. The animal mod- el of osteoporosis （OP） was established 8 weeks after ovariectomy （bilateral ovarian resection） and intramuscular injection of meth- ylprednisolone daily for 8 consecutive weeks in all groups. The control group were only simulated puncture and didn＇ t inject any drugs or normal saline, the normal saline group were intracerebroventricular （ICV） injection with NS and Low, Middle and High dose group injected with low, middle and high dose of leptin respectively at 0,3,7 days after OVX. Before （0 week） and 4,8 weeksafter the operation, the measure of dual energy X-ray absorptiometry was done on all the rabbits femur and bone mineral density （BMD） was delivered. Before （0 week） and 4, 8 weeks after the first administration, leptin in cerebrospinal fluid （CSF） and serum growth hormone （GH）, insulin growth factor 1 （IGF-1） and osteocalcin （OT/BGP） were tested with Enzyme linked immunosorbent assay and serum calcium, phosphorus and alkaline phosphatase （ALP） with automatic biochemical analyzer （ACA）. The rabbits were sacrificed and the thalamus and femoral neck were harvested to test the expression of leptin receptor （LEPR） in thalamus and bone morphogenetic protein-2 （BMP-2） and type I collagen （Col I ） at 8 weeks after the first administration. Results Compared with the control group and NS group, the cerebrospinal fluid （CSF） leptin concentration of leptin administration groups kept in a higher level significantly at 4 and 8 weeks after leptin administration, it was positively correlated with the concentration of adminis- tration. After administration 4 and 8 weeks, the bone mineral density （BMD） of leptin administration groups were not significantly lower than that of non-administration groups, and the high dose group was unchanged. The serum osteocalcin/bone glad protein （OT/BGP） concentration of leptin administration groups decreased more significantly compared with the non-administration groups at 4 weeks after leptin administration; and it rebounded more significantly than the non-administration groups and low dose group rebounded more significantly than other non-administration groups at 8 weeks. The concentration of serum growth hormone （GH） of 5 groups showed a downward trend at 4 and 8 weeks, the serum GH concentration of leptin administration groups decreased more significantly than the non-administration groups. The concentration of serum insulin-like growth IGF- 1 of 5 groups were de- creased significantly at 4 weeks, and the serum IGF- 1 concentration of control and low dose groups were significantly higher than other 3 groups at 8weeks. Compared with the non-administration groups, the concentration of serum Calcium of leptin administra- tion groups were increased more obvious, and it was positively correlated with the concentration of administration. Serum phospho- rus of 5 groups were decreased at 4 weeks and elevated at 8 weeks; there was no difference among the groups. Serum ALP showed a continuous downward trend, and the middle dose group decreased significantly compared with other groups at 4 and 8 weeks. RT- PCR results showed that the BMP-2 and Col I mRNA expression increased and positively correlated with leptin administration concentration at 8weeks. Conclusion The increase of cerebrospinai fluid leptin level can promote the bone metabolism, reduce bone loss and precaution the occurrence of osteoporosis in rabbit model caused by OVX and methylprednisolone, and it was posi- tively correlated with the concentration of administration. That provides a new method for the prevention and treatment of osteopo- rosis in rabbit model.