高脂饮食诱导肥胖小鼠模型中氧化应激、炎症递质对骨代谢的影响及其可能机制

The effects of inflammatory mediator and oxidative stress on bone metabolism in high fat diet-induced obesity mice and the underlying mechanism

目的:探讨高脂饮食诱导肥胖小鼠模型中氧化应激和炎症递质对骨代谢的影响及其可能机制。方法:选用5周龄C57BL/6雄性小鼠16只,随机平均分为正常饮食组、高脂饮食组,分别用正常饮食和高脂饮食喂养。于实验16周末,测各组动物体质量,处死后取内脏脂肪并测量内脏脂肪/体质量比值;用ELISA法测量血清抗酒石酸酸性磷酸酶5b(TRACP-5b)、血清骨钙素(OC)、白介素-6(IL-6)和肿瘤坏死因子-α(TNF-α);分别用硫代巴比妥酸(TBA)法和羟胺法检测胫骨骨组织丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性;用骨密度仪检测右侧股骨骨密度(BMD);分别用免疫组织化学技术和实时荧光定量PCR(RT-qPCR)技术检测骨组织骨保护素(OPG)和核因子-κB受体活化因子配体(RANKL)蛋白及mRNA的表达;HE染色光镜观察左侧股骨组织形态学改变。结果:与正常饮食组相比,高脂饮食组体质量、内脏脂肪/体质量比值、骨组织RANKL mRNA/OPG mRNA比值、血清TRACP-5b、IL-6和TNF-α水平、骨组织RANKL表达及骨组织MDA均明显升高,差异均具统计学意义(P<0.05);而血清OC水平、骨组织OPG表达、骨组织SOD及BMD均明显下降,差异均具统计学意义(P<0.05)。在组织形态学上,发现高脂饮食组股骨干骺端骨小梁分布稀疏,骨小梁变细及断裂,伴骨髓腔中有大量脂肪浸润。各指标的相关分析结果显示:BMD与骨组织SOD呈正相关(r=0.627,P<0.01),与内脏脂肪/体质量比值(r=-0.858,P<0.01)、骨组织MDA均呈负相关(r=-0.538,P<0.01);SOD与OPG mRNA呈显著正相关(r=0.637,P<0.01),与TNF-α(r=-0.673,P<0.01)、IL-6(r=-0.874,P<0.01)、RANKL mRNA(r=-0.760,P<0.01)及RANKL/OPG比值(r=-0.768,P<0.01)呈显著负相关;而MDA与TNF-α、IL-6、OPG mRNA、RANKL mRNA以及RANKL/OPG无显著相关性(P>0.05)。结论:高脂饮食诱导肥胖能导致骨质疏松病理组织学改变,其机制可能与其上调炎症递质表达和氧化应激增加有关。

Objective：To investigate the effects of inlfammatory mediator and oxidative stress on bone me-tabolism in high fat diet-induced obesity mice and the underlying mechanism. MethodSixteen C57BL/6 mice aged 5 weeks were randomly divided into two groups. High fat diet group （HFD） was fed with high fat diet while normal fat diet group （NFD） was fed with normal fat diet. At the end of 16 weeks, the visceral fat was weighed and the visceral fat/body mass ratio was assessed. The levels of TRACP-5b, OC, IL-6 and TNF-αin serum were measured by ELISA. The content of malondiadehyde （MDA） and the activity of superoxide dismutase （SOD） in shin-bone were analyzed by using hydroxylamine assay and TBA colorimetry. The bone mineral density （BMD） of right femurs were detected by Hologic QDR-4500 Bone Densitometer. The expressions of OPG and RANKL in bone tissue were detected by immunohistochemical technique and RT-qPCR respectively. Histologi-cal changes in the left femurs were studied with HE stained under light microscopy. ResultCompared to the NFD group mice, the body weight,the ratio of visceral fat/body mass and the RANKL/OPG in bone, the serum levels of TRACP5b, IL-6, TNF-α, the expressions of RANKL and the content of MDA in bone were increased in HFD group mice signiifcantly （P＆lt;0.05）. But the serum level of OC, the expressions of OPG, the activity of SOD in bone and the BMD were decreased in HFD group mice signiifcantly （P＆lt;0.05）. Histologically, the volume of bones trabecular were decreased and the bones trabecular distribution were sparse, thinner and fracture and more adipocytes inifltrated in bone marrow in HFD group. The results of correlation analysis shows that BMD was positively correlated with SOD of bone （r=0.627, P＆lt;0.01） and negatively correlated with the ratio of visceral fat/body mass （r=-0.858, P＆lt;0.01）, MDA level of bone （r=-0.538, P＆lt;0.01）;SOD was positively correlated with OPG mRNA （r=0.637, P＆lt;0.01） and negatively correlated with TNF-α（r=-0.673, P＆lt;0.01）, IL-6 （r=-0.874, P＆lt;0.01）, RANKL mRNA （r=-0.760, P＆lt;0.01） and the ratio of the RANKL/OPG in bone（r=-0.768, P＆lt;0.01）, but MDA shows no correlation with TNF-α, IL-6, OPG mRNA, RANKL mRNA and the ratio of the RANKL/OPG in bone （P＆gt;0.05）. Conclusion：High fat diet induced obesity can lead to the increase of bone resorption and the patho-logical histology changes of osteoporosis and the BMD decline. The underlying mechanism may be associated with its upreguating of inlfammatory mediator and enhancing oxidative stress in bone.