摘要
研究背景及目的：激素性股骨头坏死是临床常见的难治性疾病之一。抗凝、降脂、增加成骨、促进纤溶等方法仅仅针对股骨头坏死发病机制中的某一方面,治疗效果并不确切、并发症较多,而且股骨头坏死发生后再进行治疗只能延缓病程的发展,并不能使病情逆转,因此预防股骨头坏死意义更大。白藜芦醇是多酚类化合物的一种,具有抗氧化、改善脂质代谢和血管内皮功能、调节炎症因子及抑制血小板的聚集和黏附等多种功能,而这些在激素性股骨头坏死的发病机制中起重要作用,因此,我们采用白藜芦醇对兔激素性骨坏死模型进行干预,明确白藜芦醇是否降低骨坏死发生率并探讨其作用机制。
方法：选取新西兰大白兔72只,随机分为四组。白藜芦醇预防组(Pro组)：共30只,静脉注射1次LPS(脂多糖内毒素,10μg/kg)和3次MPS(甲基强的松龙,20mg/kg),每次注射时间间隔24h。注射LPS当日开始腹腔注射白藜芦醇4mg/kg/d，共2周。骨坏死模型组(AVN组)：共30只,与Pro组相同时间点静脉注射等量LPS及MPS,同时腹腔注射等量稀释酒精,共2周。白藜芦醇对照组(Res组)：共6只,与Pro组相同时间点静脉注射等量生理盐水,同时腹腔注射白藜芦醇4mg/kg/d，共2周。空白对照组(Con组)：共6只,与Pro组相同时间点静脉注射等量生理盐水,同时每日腹腔注射等量稀释酒精,共2周。静脉注射MPS 2周和3个月后处死动物并留取双侧股骨及肱骨标本。ELISA法检测不同时间点血浆组织纤溶酶原激活剂(t-PA)、纤溶酶原激活剂抑制物-1(PAI-Ⅰ)、血栓调节蛋白(TM)和血管内皮生长因子(VEGF)水平。动态增强MRI观察不同时间股骨头血流灌注情况。HE染色及免疫组化评估各组骨坏死发生率、组织因子(TF)和VEGF表达情况。
结果：2周时骨坏死模型组骨坏死率为80.0%,白藜芦醇预防组为43.8%,两组间具有明显差异(P=0.043)。3个月时两组骨坏死率分别为50%和36.4%,无统计学差异。白藜芦醇对照组和空白对照组无骨坏死发生。MPS注射后1周、2周和4周时骨坏死模型组血浆TM和VEGF浓度明显高于白藜芦醇预防组,但3个月时两组无明显差异。骨坏死模型组和白藜芦醇预防组t-PA和PAI-1浓度呈波浪状改变,两组间无明显差异。MPS注射1周时骨坏死模型组ME明显降低,2周时明显升高,3个月时下降,而白藜芦醇预防组ME变化不明显。骨坏死模型组TF及VEGF表达阳性(++)或强阳性(+++),而白藜芦醇预防组TF及VEGF表达弱阳性(+)或阳性(++)。
结论：经静脉单次小剂量内毒素和3次大剂量甲基强的松龙可成功诱导出兔激素性骨坏死模型。动态增强MRI能有效判断股骨头灌注情况,与病理学表现具有一致性,是早期诊断骨坏死的一个可靠方法。白藜芦醇可通过多种机制改善骨组织局部血供、降低骨坏死的发生率。
Background:Steroid-associated osteonecrosis (ON) is an intractable and disabling disease in orthopedics. Anticoagulant,lipid-lowering agent,ossification agent,and fibrinolytic agent may exert some indefinite effect on ON and have many complications.Drug treatment for ON can only delay the progess of the disease,but not reverse it,so it is more efficient to prevent the development of ON. The etiopathogenesis of steroid-associated ON is not very clear,but intravascular thrombosis and extravascular lipid-deposit-induced compression are the two generally accepted mechanism. Resveratrol,one kind of polyphenol, has the potential of anti-oxidation,regulation of lipid metabolism,protection of vascular endothelium cell,modification of inflammatory factor and inhibition of platelet aggregation and secretion. We will establish a steroid-associated ON model in rabbits and evaluate the effect of resveratrol on preventing steroid-associated ON development in rabbits.
Methods:Seventy-two New-Zealand white rabbits were randomly divided into four grous.30 rabbits were included in resveratrol preventing group (Pro) and intravenously injected with 10μg/kg body weight of Lippolysaccharide (LPS) on day 0 (week 0),following with three times intravenous injections of 20 mg/kg body weight of Methylprednisolone (MPS) at a time interval of 24 h. Intraperitoneal injection of 4 mg/kg body weight of resveratrol was given on day 0 and lasted for two weeks at a time interval of 24 h.30 rabbits were involved in Osteonecrosis group(AVN) and intravenous injected with LPS and MPS as that in Pro group.but intraperitoneal injected 2 weeks with diluted alcohol.6 rabbits of resveratrol control group (Res) were intravenous injected four times with physiological saline and intraperitoneal injected two weeks with 4 mg/kg body weight of resveratrol.6 rabbits of control group were intravenous injected four times with physiological saline and intraperitoneal injected 2 weeks with diluted alcohol.36 rabbits were separately killed week 2 and month 3 after the last MPS injection,and bilateral femoral and humeral samples were harvested. Plasma t-PA (Tissue-type plasminogen activator), PAI-I (plasminogen activator inhibitor type I), TM(thrombomodulin) and VEGF(Vascular endothelial growth factor) were detected by ELISA(Enzyme-linked immunosorbent assay). Dynamic contrast-enhanced MRI was perfomed for local intraosseous perfusion. The incidence of osteonecrosis was assessed by HE staining and local TF and VEGF expression were graded by immunohistochemistry.
Results:The ON incidence was 80.0%in AVN group and 43.8%in Pro group at week 2,and the difference is significant (P=0.043).The ON incidence was 50%and 36.4%respectively at month 3 and without significant difference.There were no ON in the Res group and Con group.TM and VEGF concentration were significant higher in the AVN group than that in Pro group at week 1, week 2 and week 4,but there was no significance at month 3.The level of t-PA and PAI-1 showed a wavy change in both AVN and Pro group,and no significant difference occurred between the two groups.ME in the AVN group decreased significantly at week 1 postinduction (P<0.05), then significantly increased over baseline at week 2 and decreased at month 3.The variance of ME in the Pro gourp is not significant. Moderate (++) and strong immunoreactivity (+++) of local TF and VEGF expression were found in AVN group and weak (+)and moderate (++) immunoreactivity were found in Pro group.
Conclusions:Single intravenous injection of low dose LPS,combined with three intravenous injection of high dose MPS can successfully establish steroid-associated ON in rabbits. Dynamic contrast-enhanced MRI can reflect the perfusion of femoral head,consist with the pathological features and it is a reliable method to predict the ON development at early stage. Resveratrol can reduce the ON incidence by different mechanisms.
引文
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