柚皮苷对大鼠激素性股骨头坏死组织修复的影响

Protective effects of naringin on steroid-induced femoral head necrosis in rats

摘要:

[目的]探究柚皮苷（naringin, Nar）预防大鼠激素性股骨头坏死（osteonecrosis, ON）的作用机制。[方法]SpragueDawley （SD） 雄性大鼠 73 只 （SPF 级），随机分为 4 组，包括空白组 （n=16）、模型组 （n=19）、低量组 （n=19）、高量组 （n= 19）。模型组、低量组和高量组的 57 只 SD 大鼠，按 20 μg/kg 腹腔注射 LPS 2 次后，每隔 24 h 按 40 mg/kg 肌注 MPS 3 次。低量组、高量组分别在初次注射 MPS 后每日按 300 mg/kg 和 600 mg/kg 分别给予 Nar 灌胃给药 6 周，空白组和模型组则予生理盐水。观测血清指标、股骨骨密度和病理学观察。分离培养大鼠 BMSCs，测定 ALP 活性、钙沉积、RT-qPCR 和 Western-blot。 [结果]ON 发病率模型组＞低量组＞高量组 （89.5% vs 63.2% vs 26.3%, P＜0.001），坏死灶集中于干骺端，各组 ON 严重度差异无统计学意义（P＞0.05）。四组骨小梁面积比率 [(0.41±0.04) vs (0.37±0.03) vs (0.35±0.02) vs (0.30±0.03), P＜0.001 ]、微血管密度 [(2.90± 1.20) vs (1.90±1.30) vs (1.50±1.10) vs (1.00±0.60), P＜0.001 ] 比较均为空白组＞高量组＞低量组＞模型组，而骨髓脂肪细胞直径 [(136.60± 9.60) μm vs (158.40±5.50) μm vs (184.40±8.00) μm vs (223.60±11.60) μm, P＜0.001 ]、脂肪细胞密度 [(29.70±2.70) /mm2 vs (33.70±1.60) /mm2 vs (36.60±2.10) /mm2 vs (40.70±2.00) /mm2 , P＜0.001] 均为空白组＜高量组＜低量组＜模型组。Nar 以剂量依赖方式恢复激素诱发的异常血清部分凝血酶原时间（activated partial thromboplastin time, APTT）、血栓调节蛋白（thrombomodulin, TM）、组织型纤溶酶原激活物（tissue plasminogen activator, t-PA）、纤溶酶原激活抑制剂（plasminogen activator inhibitor-1, PAI-1）、低密度脂蛋白（low-den- sity lipoprotein, LDL）、高密度脂蛋白（high-density lipoprotein, HDL）、谷胱甘肽（glutathione, GSH）、过氧化脂质（lipid peroxide, LPO），高剂量组接近空白组水平（P＜0.05）。Nar 以剂量依赖方式抑制 PPARγ2 诱导的 BMSCs 脂向分化，增强成骨 mRNA 和蛋白表达（P＜0.05）。[结论]天然配体 Nar 将 PPARγ 作为关键靶点促进骨修复并抑制脂肪增生，有助于多重机制参与的激素性股骨头坏死的早期干预。

Abstract:

[Objective] To explore the mechanism of naringin (Nar) in preventing steroid-induced osteonecrosis (ON) in rats. [Methods] Seventy-three Sprague-Dawley (SD) male rats (SPF grade) were randomly divided into four groups, including blank group (n=16), model group (n=19), low-dose group (n=19) and high-dose group (n=19). Fifty-seven SD rats in model group, low-dose group and high-dose group were intraperitoneally injected 20 μg/kg LPS twice, and intramuscularly injected 40 mg/kg MPS three times every 24 h. After the initial injection of MPS, the low-dose group and the high-dose group were given Nar at 300 mg/kg and 600 mg/kg daily for 6 weeks, respectively, while the blank group and the model group were given normal saline. Serum marks, bone mineral density of femur and histological observation were assayed. The rat BMSCs were isolated and cultured, and ALP activity, calcium deposition, RT-qPCR and Western-blot were tested. [Results] The incidence of ON was ranked as model group ＞ lowdose group ＞ highdose group (89.5% vs 63.2% vs 26.3%, P 0.001), while the necrotic focus was concentrated in metaphysis, and there was no a significant difference in the severity of ON among all the groups (P＞0.05). The trabecular bone area ratio [(0.41±0.04) vs (0.37±0.03) vs (0.35±0.02) vs (0.30± 0.03), P 0.001] and microvascular density [(2.90±1.20) vs (1.90±1.30) vs (1.50±1.10) vs (1.00±0.60), P 0.001] were ranked up-down as blank group ＞ high-dose group ＞ low-dose group ＞ model group. However, bone marrow fat cells diameter [(136.60±9.60) μm vs (158.40±5.50) μm vs (184.40±8.00) μm vs (223.60± 11.60) μm, P 0.001] and fat cell density [(29.70±2.70) /mm2 vs (33.70±1.60) /mm2 vs (36.60±2.10) /mm2 vs (40.70±2.00) /mm2 , P 0.001] were of blank group ＜ highdose group ＜ lowdose group ＜ model group. The Nar restored abnormal thrombomodulin, activated partial thromboplastin time (APTT) and thrombomodulin, tissue plasminogen activator (t-PA), tissue plasminogen activator inhibitor-1 (plasminogen activator inhibitor-1, PAI-1), lowdensity lipoprotein (LDL), highdensity lipoprotein (HDL), glutathione (GSH), and lipid peroxide (LPO) all in the dose-dependent manner, with high-dose group close to the blank group (P＜0.05). Nar inhibited PPARγ2-induced lipotropic differentiation of BMSCs and enhanced osteogenic mRNA and protein expression in the dose-dependent manner (P＜0.05). [Conclusion] Natural ligand Nar uses PPARγ as a key target to promote bone repair and inhibit fat hyperplasia, which is helpful for early intervention of steroid-induced femoral head necrosis involving multiple mechanisms.