CYP3A酶在激素性股骨头坏死发病中的作用及健骨方对其影响的实验研究
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摘要
目的
股骨头坏死(Osteonecrosis of the femoral head, ONFH)是骨科领域难治性疾病之一,临床上ONFH分为创伤性与非创伤性两大类,前者主要因髋部外伤(股骨颈骨折、髋关节脱位等)引起,后者主要由于皮质类固醇应用及长期大量饮酒引起；激素性股骨头坏死占非创伤性ONFH的首位,约占50%,ONFH为渐进性疾病,未经有效治疗及预防,约80%会在发病后1-4年内进展至股骨头塌陷；一旦出现股骨头塌陷,多数患者将发展成为严重骨性关节炎而需行人工关节置换术。由于非创伤性ONFH好发于中青年,中青年的人工关节置换术后远期疗效仍难以预测。因此,寻求有效的保髋治疗方法尤为重要,而取得优良治疗效果的前提是获得激素性股骨头坏死的早期诊断及早期干预。探索激素性股骨头坏死的确切发病机理、寻找敏感的早期诊断方法及有效的早期治疗方法或预防途径,具有非常重要意义。
激素性股骨头坏死是全身疾病的局部表现,中医药治疗的优势在于全身用药整体治疗以减少激素应用的副作用、同时可以进行高危人群的预防、亚临床期与塌陷前的个体化辨证治疗等。由于本病发病机制及遗传背景特征的复杂性,准确确定高危人群十分困难。亚临床期及塌陷前因缺少临床症状与X线表现,医患常不加注意,更不可能采用比较敏感但昂贵的核磁共振(MRI)进行普查。由于本病早期诊断困难,导致无法运用中医药进行有效预防以及在塌陷前针对发病机制进行治疗；因此,有必要对激素性ONFH的发病机制进行深入研究。
临床研究发现大剂量激素的使用是导致激素性股骨头坏死有意义的危险因素,对于激素性股骨头坏死,同样的激素用量,不同患者常出现不同的结果,有些患者会引起ONFH,有些患者不会,说明个体对于激素的敏感性是不同的。而糖皮质激素在体内代谢关键酶是细胞色素P4503A酶(CYP3A),研究者发现CYP3A的活性范围较大,低活性的CYP3A,可能是引起血清中皮质类固醇清除率下降,导致游离的糖皮质激素水平升高,副作用增大的主要原因,因此,提出“CYP3A的活性水平与激素性ONFH相关”的假说,本研究旨在初步探讨CYP3A活性在激素性ONFH发病中的作用及临床上用于治疗激素性ONFH的经验方—健骨方对CYP3A活性的影响,通过观察其对股骨头局部PPARr的影响探讨其对局部成脂分化影响；通过观察其对局部脂联素、Runx2、Osterix的影响探讨其对局部成骨分化的影响,为其治疗与预防激素性ONFH提供依据。
方法
以30只健康SPF级新西兰家兔为研究对象,雌雄各半,按体重均衡原则随机分成5组即空白对照组、模型组、诱导剂组、抑制剂组、健骨方组,每组6只,分笼标准饲料饲养,自由摄食水,室温控制在23℃-25℃,湿度50%,通风良好,适应喂养1周后给药；造模方法参照Yamamoto等、参照Toshiaki Masada的实验研究方法及相关药物用量,分组后的实验家兔,根据不同的施加因素,诱导剂组予每天肌肉注射苯巴比妥注射液,25mg/kg体重；抑制剂组予隔天灌胃伊曲康唑,50mg/kg体重,实验前用生理盐水将伊曲康唑胶囊内容物配制成5mg/ml的混悬液；健骨方组予每天灌胃健骨方混悬液,10ml/kg体重,实验前用生理盐水将健骨方煎煮的药液配制成含生药0.719g/ml的混悬液；空白对照组、模型组予灌胃等剂量生理盐水,10ml/kg体重；每周称重一次,连续用药3周后,实验动物均选择在左大腿内侧肌肉注射注射用甲泼尼龙琥珀酸钠,40mg/kg体重,连续用药3天,继续常规饲养3周。
实验开始前以及3周后,动物禁食不禁水8小时,次日凌晨家兔耳缘静脉取血4ml,用肝素锂抗凝,立即放入离心机中,3000RPM,离心10分钟,小心移取上层黄色液体到新的离心管中,放入-70℃冰箱里保存备测,采用的是GENMED比色法定量检测试剂盒,该方法是通过红霉素去甲基酶反应系统中红霉素A去甲基后的产物甲醛与纳什试剂作用后吸光峰值的变化来测定样品中肝CYP3A酶活性；采用双抗体一步夹心法酶联免疫吸附试验(ELISA)测定血浆糖皮质激素水平,实验开始6周后空气栓塞法处死家兔,剖取左侧股骨头,置于液氮罐中,用ELISA法测定局部股骨头组织内糖皮质激素、脂联素PPARγ、Runx2、Osterix含量,取右后侧股骨头,剔除周围软组织,分组标记好标本袋,置于10%福尔马林溶液中固定,进行病理组织学检测；数据用SPSS 13.0 For Windows统计分析软件处理,计量资料用均数((?))±标准差(S)表示,采用t检验；对坏死率进行单因素方差分析(One-Way ANOVA)；相关分析采用Spearman等级相关分析。
结果
参照Matsui等对成年兔激素诱导股骨头坏死标本的组织病理改变分级方法,本研究选择使用病理结果评价激素性股骨头坏死模型复制的成功性。肉眼观察可见股骨头外观完整,未见软骨下塌陷或碎裂,但股骨头关节软骨颜色瘀暗；HE染色切片置于光学显微镜下观察结果显示除空白对照组5只家兔病理改变为0级外,其余19只使用激素造模的家兔出现2级病理改变的实验动物为9只(47.4%)、1级病理改变为10只(52.6%)。
在用药物干预之前,与空白对照组比较,模型组、诱导剂组、抑制剂组、健骨方组肝CYP3A酶活性差异均无统计学意义(P值分别为0.253、0.647、0.242、0.262>0.05),用药干预3周后与空白对照组比较,模型组差异无统计学意义(t=1.848,P=0.118>0.05)、诱导剂组用苯巴比妥注射液诱导后肝CYP3A酶活性明显提高,差异具有统计学意义(t=3.678,P=0.004<0.05)、抑制剂组经伊曲康唑胶囊抑制后肝CYP3A酶活性则明显降低,差异具有统计学意义(t=6.268,P=0.001<0.05)、健骨方组也可以提高该酶活性,差异具有统计学意义(t=3.84,P=0.004<0.05)；与模型组比较,诱导剂组、健骨方组酶活性增高、抑制剂组酶活性降低,差异均具有统计学意义(t=3.82,3.93,2.57,P=0.013、0.09、0.021<0.05)与诱导剂组比较,抑制剂组肝CYP3A酶活性明显降低,差异具有统计学意义(t=3.714,P=0.013<0.05)、健骨方组CYP3A酶活性有所下降,但差异无统计学意义(t=0.105,P=-0.322>0.05)；与抑制剂组比较,健骨方组CYP3A酶活性明显提高,差异具有统计学意义(t=5.60,P=-0.02<0.05)。
各组用药前后自身比较,空白对照组用药前后比较,肝CYP3A酶活性差异无统计学意义(t=1.859,P=0.122>0.05)；模型组用药后肝CYP3A酶活性较用药前也有所升高,差异无统计学意义(t=1.92,P=0.097>0.05)；诱导剂组用药后肝CYP3A酶活性较用药前明显升高,差异具有统计学意义(t=5.06,P=0.003<0.05)；抑制剂组用药后肝CYP3A酶活性较用药前明显降低,差异具有统计学意义(t=7.482,P=-0.002<0.05)；健骨方组用药后肝CYP3A酶活性较用药前明显升高,差异具有统计学意义(t=5.059,P=0.003<0.05)。
在用糖皮质激素造模之前,与空白对照组比较,模型组、诱导剂组、抑制剂组、健骨方组血浆糖皮质激素水平差异不显著(P值分别为0.952、0.18、0.099、0.351>0.05),组间采用单因素方差分析结果显示F=1.289,P=0.301>0.05说明组间差异无统计学意义；用药干预3周后,组间采用单因素方差分析结果显示F=26.72,P=0.001<0.05说明组间差异显著,经方差齐性检验,P=-0.076>0.05,表明方差齐,采用LSD方法进行两两比较,结果显示与空白对照组比较,血浆糖皮质激素水平模型组升高,差异具有统计学意义(P=0.001<0.05)、诱导剂组有所降低,差异无统计学意义(P=-0.527>0.05)、抑制剂组明显升高,差异具有统计学意义(P=0.0001<0.05)、健骨方组也明显降低,但差异无统计学意义(P=0.124>0.05)；与模型组比较,血浆糖皮质激素水平诱导剂组、健骨方组均降低、抑制剂组升高,差异均具有统计学意义(P=0.0001、0.0001、0.003<0.05)；与诱导剂组比较,抑制剂组血浆糖皮质激素水平升高明显,差异具有统计学意义(P=0.0001<0.05),健骨方组有所下降,但差异无统计学意义(P=0.346>0.05)；与抑制剂组比较,健骨方组明显降低,差异具有统计学意义(P=0.001<0.05)。
各组股骨头局部糖皮质激素含量的数据,组间采用单因素方差分析结果显示F=143.8,P=0.001<0.05说明组间差异显著,经方差齐性检验,P=0.494>0.05,表明方差齐,采用LSD方法进行两两比较,与空白对照组比较,局部糖皮质激素水平模型组明显升高,差异具有统计学意义(P=0.001<0.05)、诱导剂组有所降低,差异无统计学意义(P=0.059>0.05)、抑制剂组明显升高,差异具有统计学意义(P=0.0001<0.05)、健骨方组也明显降低,但差异无统计学意义(P=0.324>0.05)；与模型组比较,局部糖皮质激素水平诱导剂组、健骨方组均降低、抑制剂组升高,差异均具有统计学意义(P=0.0001、0.0001、0.001<0.05)；与诱导剂组比较,抑制剂组股骨头局部糖皮质激素水平升高明显,差异具有统计学意义(P=0.0001<0.05),健骨方组有所下降,但差异无统计学意义(P=-0.331>0.05)；与抑制剂组比较,健骨方组明显降低,差异具有统计学意义(P=0.001<0.05)。肝CYP3A酶活性与股骨头局部糖皮质激素水平呈显著的负相关(RR值为-0.657,P=0.001)。
各组股骨头局部脂联素含量数据,组间采用单因素方差分析结果显示F=15.78,P=0.001<0.05说明组间差异显著,经方差齐性检验,P=0.102>0.05,表明方差齐,采用LSD方法进行两两比较,与空白对照组比较,局部脂联素水平模型组明显降低,差异具有统计学意义(P=0.005<0.05)、诱导剂组有所升高,差异无统计学意义(P=0.163>0.05)、抑制剂组明显降低,差异具有统计学意义(P=0.0001<0.05)、健骨方组也有所降低,但差异无统计学意义(P=0.464>0.05)；与模型组比较,局部脂联素水平诱导剂组、健骨方组均升高、抑制剂组明显降低,差异均具有统计学意义(P=0.0001、0.013、0.023<0.05)；与诱导剂组比较,抑制剂组股骨头局部脂联素水平降低明显,差异具有统计学意义(P=0.0001<0.05),健骨方组有所降低,差异有统计学意义(P=0.041<0.05)；与抑制剂组比较,健骨方组明显升高,差异具有统计学意义(P=0.001<0.05)。
各组股骨头局部PPARγ含量的数据,组间采用单因素方差分析结果显示F=91.79,P=0.001<0.05说明组间差异显著,经方差齐性检验,P=0.001<0.05,表明方差不齐,采用Tamhane方法进行两两比较,与空白对照组比较,局部PPARγ水平模型组明显升高,差异具有统计学意义(P=0.018<0.05)、诱导剂组有所升高,差异无统计学意义(P=1.00>0.05)、抑制剂组明显升高,差异具有统计学意义(P=0.0001<0.05)、健骨方组也略有所升高,但差异无统计学意义(P=1.00>0.05)；与模型组比较,局部PPARγ水平诱导剂组、健骨方组均降低、抑制剂组明显升高,差异均具有统计学意义(P=0.045、0.006、0.033<0.05)；与诱导剂组比较,抑制剂组股骨头局部PPARγ水平明显升高,差异具有统计学意义(P=0.002<0.05),健骨方组有所降低,差异无统计学意义(P=0.891>0.05)；与抑制剂组比较,健骨方组明显降低高,差异具有统计学意义(P=0.001<0.05)。
各组股骨头局部Runx2含量数据,组间采用单因素方差分析结果显示F=88.4,P=0.001<0.05说明组间差异显著,经方差齐性检验,P=0.003<0.05,表明方差不齐,采用Tamhane方法进行两两比较,与空白对照组比较,股骨头局部Runx2含量模型组明显降低,差异具有统计学意义(P=0.0031<0.05)、诱导剂组明显升高,差异有统计学意义(P=0.013<0.05)、抑制剂组明显降低,差异具有统计学意义(P=0.0001<0.05)、健骨方组也明显升高,差异具有统计学意义(P=0.002<0.05)；与模型组比较,股骨头局部Runx2含量诱导剂组、健骨方组均升高、抑制剂组明显降低,差异均具有统计学意义(P=0.003、0.001、0.001<0.05)；与诱导剂组比较,抑制剂组股骨头局部Runx2含量降低明显,差异具有统计学意义(P=0.001<0.05),健骨方组有所降低,差异无统计学意义(P=0.288>0.05)；与抑制剂组比较,健骨方组明显升高,差异具有统计学意义(P=0.001<0.05)。
股骨头局部Osterix含量组间采用单因素方差分析结果显示F=38.07,P=0.001<0.05说明组间差异显著,经方差齐性检验,P=0.002<0.05,方差不齐,采用Tamhane方法进行两两比较,与空白对照组比较,股骨头局部Osterix含量模型组明显降低,差异具有统计学意义(P=0.037<0.05)、诱导剂组明显升高,差异有统计学意义(P=0.006<0.05)、抑制剂组明显降低,差异具有统计学意义(P=0.004<0.05)、健骨方组也明显升高,但差异无统计学意义(P=0.066>0.05)；与模型组比较,股骨头局部Osterix含量诱导剂组、健骨方组均升高、抑制剂组明显降低,差异均具有统计学意义(P=0.001、0.025、0.022<0.05)；与诱导剂组比较,抑制剂组股骨头局部Osterix含量降低明显,差异具有统计学意义(P=O.001<0.05),健骨方组有所升高,差异无统计学意义(P=0.76>0.05)；与抑制剂组比较,健骨方组明显升高,差异具有统计学意义(P=-0.012<0.05)。
各组坏死面积与坏死率数据,使用单因素方差分析(One-Way ANOV A),统计显示：经Levene方差齐性检验,方差齐(P=0.091及P=0.151)；在方差齐性条件下,采用LSD检测方法,坏死面积结果显示：模型组与诱导剂组比较,诱导剂组坏死面积较模型组降低,两者有显著性差异P<0.05(P=0.037)；模型组与抑制剂组比较,两者坏死面积间显著性差异P<0.05(P=0.023)；模型组与健骨方组比较,健骨方组组坏死面积较模型组降低,两者有显著性差异P<0.05(P=0.015)；与诱导剂组比较,抑制剂组坏死面积明显升高,差异具有统计学意义P<0.05(P=0.012),健骨方组坏死面积有所升高,但差异不显著P>0.05(P=0.423)；与抑制剂组,健骨方组坏死面积明显降低,P<0.05(P=0.034)；坏死率结果显示：模型组与诱导剂组比较,诱导剂组坏死率较模型组降低,两者有显著性差异P<0.05(P=0.023)；模型组与抑制剂组比较,两者坏死率间显著性差异P<0.05(P=0.044)；模型组与健骨方组比较,健骨方组坏死率较模型组降低,两者有显著性差异P<0.05(P=0.024)；与诱导剂组比较,抑制剂组坏死率明显升高,差异具有统计学意义P<0.05(P=0.016),健骨方组坏死率有所升高,但差异不显著P>0.05(P=0.623)；与抑制剂组,健骨方组坏死率明显降低,P<0.05(P=0.025)。肝CYP3A酶活性与股骨头坏死面积、坏死率均成显著的负相关；肝CYP3A酶活性与股骨头局部PPARγ含量、脂联素、RUNX2的相关性,经Pearson相关性分析,RR值分别为-0.269,0.097,0.211,P值分别为0.203,0.652,0.321均>0.05,无明显相关性。
结论
本次研究结果表明肝CYP3A酶的活性与激素性股骨头坏死的发生呈显著负相关①肝CYP3A酶活性增高可使股骨头局部糖皮质激素含量减少。②肝CYP3A酶活性增高可降低激素性股骨头坏死率,减少坏死面积,减轻病理分级。这可能是肝CYP3A酶能对外源性糖皮质激素起清除作用,使糖皮质激素在股骨头局部的蓄积时间缩短、浓度降低,从而降低了股骨头坏死的发生率。中药健骨方对肝CYP3A酶的影响与激素性ONFH的实验研究结果表明①健骨方能够增强肝CYP3A酶的活性。②健骨方能够预防激素性股骨头坏死的发生,减轻坏死病理程度。③健骨方防治激素性股骨头坏死的发生可能是通过降低股骨头局部PPARγ和提高Runx2、Osterix以及脂联素的水平,达到抑制成脂、促进成骨的作用。④为中医药“治未病”理论防治激素性股骨头坏死提供实验依据。
Objective
Osteonecrosis of the femoral head (ONFH) is regard as one of the difficult to treat disease in the field of Orthopedics. ONFH can be divided into traumatic and non-traumatic causes. ONFH which is caused by the traumatic mainly because of injury of hip such as neck fracture and hip dislocation, etc.. The non-traumatic ONFH is caused by a long-term corticosteroids use and Alcoholism. A long-term corticosteroids use is the most causes ONFH in the non-traumatic part, it is about 50%. ONFH is a progressive disease, without effective treatment and prevention, about 80% people pathogenesis femoral head collapse in 1 to 4 years, the event of a femoral head collapse will develop into a serious arthritis in the majority of patients, finally the arthroplasty is needed. non-traumatic ONFH is regularity occurs in middle-aged, middle-aged in the long term effect of arthroplasty surgery is still difficult to predict. Therefore, the search for effective treatment of hip protection is especially important, early diagnosis and early intervention is the premise to have achieved excellent therapeutic effect. Explore the ONFH pathogenesis, find the sensitive methods for early diagnosis and effective early treatment, it has has very important significance.
Clinical data indicate that the ONFH caused by glucocorticoids is systemic disease but performance in the part of the body. Advantages of Chinese medicine treatment of systemic drug therapy to the whole to reduce the side effects of hormone application, prevention the high-risk groups, sub-clinical phase and before the collapse to use individual differentiation therapy. As the complexity of disease pathogenesis and genetic background characteristics, it was quite difficult to determine high-risk groups accurately. Because of lack of clinical symptoms and X-ray performance within the sub-clinical phase and before the collapse, patients often less liked to notice even not to adopt sensitive but expensive magnetic resonance imaging (MRI) performance. As the early diagnosis was difficult, it couldn't use Chinese medicine to prevention effectively and treat before the collapse. Therefore, it was necessary to research the ONFH caused by glucocorticoids in-depth.
Clinical study found that the use of high-dose hormone therapy is significant risk factor for ONFH. But the same hormone dosage in different patients are often comes out different results, some patients will lead to ONFH, some patients not. that means the individual to hormones is different of sensitivity. The metabolism of glucocorticoids in the body is the key enzyme cytochrome P4503A enzymes (CYP3A), researchers found that the activity of CYP3A large range of low activity of CYP3A, may cause the serum clearance rate of corticosteroids due to the decreasing level of free glucocorticoids increased side effects increased, therefore, "CYP3A activity levels and associated femoral head necrosis" hypothesis was made, this study aims to initially explore the CYP3A activity in steroid ONFH pathogenesis and clinical for the treatment of the experience of the femoral head necrosis side-bone with effects on CYP3A activity.
Observe the influence of PPARr in partial femoral head to investigate the locality fat differentiation. Observe the influence of adiponectin、Osterix and Runx2 to investigate the influence of locality osteoblast, its treatment and prevention of steroid-induced femoral head necrosis provide a theoretical basis.
Methods
Thirty adult SPF healthy New Zealand rabbits were used, male and female half and half. According to the principle of weight balance, the rabbits were devided into five groups:Control group, Model group, Group inducer, Inhibitor group, JIANGUFANG group. Each group was feed under control and was administrate after one week. The molding method was refer to Yamanoto and the research method and dosage was Toshiaki Masada. In group inducer, the six animals were received daily intramuscular Phenobarbital 25mg/kg, Inhibitor group received oral itraconazole 50 mg/kg every other day to inhibit CYP3A activity; JIANGUFANG group received oral JIANGUFANG Suspension 10ml/kg, Drug content was 0.719g/ml every day and the other six animals were given with saline 10ml/kg as a control.Weight each of the animal for three consecutive weeks, then intramuscular injection of methylprednisolone sodium succinate 40mg/kg for 3 days in the left thight of the rabbits, continue to feed 3 weeks.
Before the Experiment or three weeks later, collection 4ml vein blood from the rabbit ear after 8-hour fasting, mixed with Lithium heparin, then put it into the Centrifuge,3000RPM,10 minute. Collect the upper yellow liquid into the Centrifuge Tube,-70℃Storage. Use the GENMED quantitative colorimetric detection kit detect the sample. This method is by erythromycin demethylation enzyme reaction system to methyl erythromycin A product of formaldehyde and Nash after the reagents after the change of absorption peak to determine CYP3A activity in liver samples. Adopt double antibody step clip neo-confucianism, enzyme-linked immunosorbent adsorption test (ELISA) to determinate the glucocorticoid levels in plasma. Air embolism execute the rabbit after six weeks,take the left femoral in liquid nitrogen tank, determinate the glucocorticoid, adiponectin PPARγ, Runx2, Osterix in locality femoral head with ELISA, take the right posterior femoral head, remove soft tissue, marked the specimen bag, fix the sample with 10% Formalin, prosecution pathology testing. Analyzed the data with SPSS 13.0 For Windows Statistical software. Representing measurement data by T test with arithmetic mean(x)and Standard Deviation (S). Analyzed necrosis rate by One-Way ANOVA.Abdopted Spearman rank correlation analysis to progress Analysis of Correlation.
Results
Refer to Matsui, the stage division of histopathology in adult rabbit hormone induced ONFH, this study used the pathologic result to evaluate the success of copying the model. Macroscopic observation showed that visible appearance complete, did not see the femoral subchondral collapse or chip, but the color of femoral articular cartilage was dark; HE dyed slice which was observed by optical microscope showed that in addition to 5 in control group only for 0 level of rabbit pathological changes, there were 9 (47.4%) of grade 2 pathological changes and 10(52.6%) of grade 1 in the rest of the 19 used hormone.
Before to use of the drug intervention, hepatic CYP3A activity had no statistically significant difference (P=0.253,0.647,0.242,0.262> 0.05)in compared with the control group, model group, Group inducer, inhibitor group and JIANGUFANG group. After 3 weeks, Compared with the control group, the model group difference was statistically significant (t=1.848, P=> 0.05), the enzymatic enzyme activity of CYP3A was obviously improved after phenobarbital injection and differences are statistically significant (t= 3.678, P=0.004 < 0.05), itraconazole capsule inhibited the CYP3A enzyme activity in the inhibitor group,the difference was statistically significant (t=6.268, P=0.001<0.05), JIANGUFANG can also raise the enzyme's activity, the difference has a statistics meaning; (t=3.84, P=0.004<0.05) And the comparison with model set, inducement set, JIANGUFANGset the enzyme activity increases, and the depressant set enzyme activity lower, and the difference all has a statistics meaning; (t=3.82,3.93,2.57, P=0.013,0.09,0.021<0.05)And comparison with inducement set, the activity obviously lowers of the liver CYP3 A enzyme in depressant set, the difference has a statistics meaning (t=3.714, P=0.013<0.05), JIANGUFANG set the CYP3 A Enzyme the activity has to descend, but the difference has no statistics meaning; (t=0.105, P=0.322>0.05)And with the comparison depressant set, the CYP3 A Enzyme the activity in JIANGUFANG set obviously raises, the difference has a statistics meaning. (t=5.60, P=0.02<0.05)
Each comparison used self-comparison of medicine, the blank matched control is used a medicine self-comparison, the liver CYP3 A Enzyme live difference has no statistics meaning; (t=1.859, P=0.122>0.05) The model set is used the medicine the liver CYP3 A Enzyme activity more than before, the difference has no statistics meaning; (t=1.92, P=0.097>0.05)The inducement set is used the medicine the liver CYP3 A Enzyme activity obviously higher than before, the difference has a statistics meaning; (t=5.06, P=0.003<0.05)The depressant set is used the medicine the liver CYP3 A Enzyme activity more lower than before, the difference has a statistics meaning;(t=7.482, P=0.002<0.05)The JIANGUFANG set is used the medicine the liver CYP3 A Enzyme activity more obviously higher than before, the difference has a statistics meaning. (t=5.059, P=0.003<0.05)
Just before making a mold with glucocorticoid, Blank control group, model group, induced dose group, inhibitor group, JIANGUFANG plasma glucocorticoid levels not significantly different (the P value distinguishes to 0.952,0.18, 0.099,0.351>0.05), the set adopts a single factor variance to analyze to show F=1.289, P=0.301>0.05 difference have no the statistics;With the medicine interfere after 3 weeks, the set adopts a single factor variance analytical show F=26.72,P=0.001<0.05 shows significant differences between groups, The test of homogeneity of variance, P=0.076>0.05, Show that the homogeneity of variance. Use the using LSD pairwise comparison method, The results showed that compared with the control group, blood plasma sugar skin quality hormone level model set goes up, the difference has a statistics meaning(P=0.001<0.05) and inducement the set has to lower, difference have no the statistics meaning(P=0.527>0.05), depressant set obviously goes up, the difference has a statistics meaning(P=0.0001<0.05), JIANGUFANG set also obviously lower, but the difference have no the statistics meaning;(P=0.124>0.05)And model set comparison, the blood plasma level glucocorticoid in inducement set, JIANGUFANG set all lower, the depressant set raise, the difference all has a statistics meaning;(P=0.0001,0.0001,0.003<0.05)And comparison in inducement set, depressant set blood plasma glucocorticoid level goes up obviously, the difference has a statistics meaning(P=0.0001<0.05), and the JIANGUFANG set has to descend, but the difference has no statistics meaning;(P=0.346>0.05)And depressant set comparison, the JIANGUFANG set obviously lower, the difference has a statistics to learn meaning. (P=0.001<0.05)
The data of partial s glucocorticoid content of each bone, the set adopts a single factor variance analytical show F=143.8, P=0.001<0.05 difference shows obvious, through data examination, P=0.494>0.05, express that the data equal. Use the LSD method carries to compare with blank matched control comparison, partial glucocorticoid level model set obviously goes up, the difference has a statistics meaning(P=0.001<0.05) and inducement the set has to lower, difference have no the statistics learn the meaning(P=0.059>0.05), depressant set obviously goes up, the difference has a statistics to learn the meaning(P=0.0001<0.05), JIANGUFANG set also obviously lowers, but difference have no the statistics learn meaning;(P=0.324>0.05)And model set comparison, partial set, JIANGUFANG set of sugar skin quality hormone level inducement all lowers, the depressant set goes up, the difference all has a statistics-to learn meaning; (P=0.0001,0.0001,0.001<0.05) And inducement set comparison, the depressant set the bone partial sugar the skin quality hormone level goes up obviously, the difference has a statistics to learn meaning (p=0.0001<0.05), and the JIANGUFANG set has to descend, but the difference has no statistics meaning;(P=0.331>0. 05)And depressant set comparison, the JIANGUFANG set obviously lowers, the difference has a statistics meaning. (P=0.001<0.05)The liver CYP3 A enzyme activity and the bone partial glucocorticoid level presents to show negative related. (RR value is-0.657, P=0.001)
The partial fat allied plain content data of each bone, the set adopts a single factor variance analytical show F=15.78, P=0.001<0.05 difference shows Obvious, through homogeneity of variance test, P=0.102>0.05, express that the variance adopts together the LSD method carries to compare, and blank matched control comparison, the partial fat unites plain level model, the set obviously lowers, the difference has a statistics to learn a meaning(P=0.005<0.05) and inducement the set has to go up, difference have no the statistics learn the meaning(P=0.163>0.05), depressant set obviously lowers, the difference has a statistics meaning(P=0.0001<0.05), JIANGUFANG set also has to lower, but difference have no the statistics learn meaning;(P=0.464>0.05)And model set comparison, partial fat allied plain level inducement the set, JIANGUFANG set all goes up, the depressant set obviously lowers, the difference all has a statistics meaning; (P=0.0001,0.013, 0.023<0.05) And inducement set comparison, the depressant set the bone partial fat allied plain level lowers obviously, the difference has a statistics meaning(P=0.0001<0.05), the JIANGUFANG set has to lower, and the difference includes a statistics meaning; (P=0.041m<0.05)And depressant set comparison, the JIANGUFANG set obviously goes up, the difference has a statistics meaning. (P=0.001<0.05)
The data of partial content of PPARγof each bone, the set adopts a single factor variance analytical show F=91.79, P=0.001<0.05 difference shows Obvious, the test of homogeneity of variance, P=0.001<0.05, express that the variance adopts not and together the Tamhane method carries on compare, and blank matched control comparison, the partial PPAR theγlevel model set obviously goes up, the difference has a statistics to learn a meaning(P=0.018<0.05) and inducement the set has to go up, difference have no the statistics learn the meaning(P=1.00>0.05), depressant set obviously goes up, the difference has a statistics meaning(P=0.0001<0.05), JIANGUFANG set also slightly has to go up, but difference have no the statistics learn meaning; (P=1.00>0.05)And model set comparison, partial PPAR the set, JIANGUFANG set of theγlevel inducement all lowers, the depressant set obviously goes up, the difference all has a statistics meaning; (P=0.045,0.006, 0.033<0.05)And inducement set comparison, depressant set the bone partial level of PPARγobviously goes up, the difference has a statistics meaning (P=0.002<0.05), the JIANGUFANG set has to lower, and the difference has no statistics meaning;(P=0.891>0.05)And depressant set comparison, the JIANGUFANG set obviously lowers Gao, the difference has a statistics meaning. (P=0.001<0.05)
The partial Runx2 content data of each bone, the set adopts a single factor variance analytical show F=88.4, P=0.001<0.05 difference shows obvious, the test of homogeneity of variance, P=0.003<0.05, express that the variance adopts not and together the Tamhane method carries to compare, and blank matched control comparison, bone partial Runx2 content model the set obviously lowers, the difference has a statistics to learn a meaning(P=0.0031<0.05) and inducement the set obviously goes up, the difference includes a statistics meaning(P=0.013<0.05), depressant set obviously lowers, the difference has a statistics meaning (P=0.0001<0.05), JIANGUFANG set also obviously goes up, the difference has a statistics meaning;(P=0.002<0.05)And model set comparison, bone partial Runx2 content inducement the set, JIANGUFANG set all goes up, the depressant set obviously lowers, the difference all has a statistics meaning; (P=0.003,0.001,0.001<0.05) And inducement set comparison, depressant set the bone partial Runx2 contents lower obviously, the difference has a statistics meaning(P=0.001<0.05), the JIANGUFANG set has to lower, and the difference has no statistics meaning;(P=0.288>0.05)And depressant set comparison, the JIANGUFANG set obviously goes up, the difference has a statistics meaning. (P=0.001<0.05)
Bone partial Osterix content set adopts a single factor variance analytical show F=38.07, P=0.001<0.05 difference shows Obvious, the test of homogeneity of variance, P=0.002<0.05, the variance adopts not and together the Tamhane method carries to compare, and blank matched control comparison, bone partial Osterix content model the set obviously lowers, the difference has a statistics to learn a meaning(P=0.037<0.05) and inducement the set obviously goes up, the difference includes a statistics meaning(P=0.006<0.05), depressant set obviously lowers, the difference has a statistics meaning(P=0.004<0.05), JIANGUFANG set also obviously goes up, but difference have no the statistics learn meaning; (P=0.066>0.05) And model set comparison, bone partial Osterix content inducement the set, JIANGUFANG set all goes up, the depressant set obviously lowers, the difference all has a statistics meaning;(P=0.001,0.025,0.022<0.05)And inducement set comparison, the depressant set the bone partial Osterix content lowers obviously, the difference has a statistics meaning(P=0.001<0.05), the JIANGUFANG set has to go up, and the difference has no statistics meaning; (P=0.76>0.05) And depressant set comparison, the JIANGUFANG set obviously goes up, the difference has a statistics meaning. (P=0.012<0.05)
Each bad dead area with bad dead rate data, use a single factor variance analysis(One-Way ANOVA), the statistics shows:Through Levene variance examines, the variance is together;(P=0.091 and P=0.151)Together adopt a LSD examination method under the variance, the bad dead area result shows:Model set and inducement set comparison, to compare with model set, the inducement the set bad dead area lowers, both have already shown the Obvious difference P<0.05;(P=0.037)Model set and depressant set comparison, both the bad dead area shows the Obvious difference P<0.05;(P=0.023)Model set and JIANGUFANG set comparison, to compare with model set, the JIANGUFANG set set bad dead area lowers, both have already shown the Obvious difference P<0.05;(P=0.015)And inducement set comparison, the depressant set bad dead area obviously goes up, the difference has a statistics meaning P<0.05(P=0.012), the JIANGUFANG set bad dead area has to go up, but the difference don't show Obvious P>0.05;(P=0.423)And depressant set, the JIANGUFANG set bad dead area obviously lowers, P<0.05; (P=0.034) The bad dead rate shows as a result:Model set and inducement set comparison, to compare with model set, the inducement the set bad dead rate lowers, both have already shown the Obvious difference P<0.05;(P=0.023)Model set and depressant set comparison, both the bad dead rate shows the Obvious difference P<0.05; (P=0.044)Model set and JIANGUFANG set comparison, to compare with model set, the JIANGUFANG set bad dead rate lowers, both have already shown the Obvious difference P<0.05;(P=0.024)And inducement set comparison, the depressant set bad dead rate obviously goes up, the difference has a statistics meaning P<0.05(P=0.016), the JIANGUFANG set bad dead rate has to go up, but the difference don't show Obvious P>0.05;(P=0.623)And depressant set, the JIANGUFANG set bad dead rate obviously lowers, P<0.05. (P=0.025)The liver CYP3 enzyme activity and the bone bad dead area, bad dead rate all become to show Obvious of negative related.
Conclusions
This research result expresses the activity of The liver CYP3A enzyme presents with the occurrence of the hormone femoral head necrosis to show Obvious negative the related①liver CYP3 A Mao activity increase Gao to make a bone partial glucocorticoid content reduced.②The liver CYP3 enzyme activity increases Gao to lower hormone the bone bad dead lead, reduce bad dead area and ease pathologic ratings. This may be The liver CYP3 enzyme can outward the source glucocorticoid contain clearance function and make glucocorticoid shortenned in time of storing of bone part, the density lower and lowered the incidence rate of the bone bad dead thus. The experiment research of the influence of Chinese herbal medicine JIANGUFANG upon The liver CYP3 enzyme and hormone ONFH expresses that①JIANGUFANG can strengthen the activity of The liver CYP3 enzyme as a result.②JIANGUFANG can prevent°fro the occurrence of the hormone femoral head necrosis, ease bad dead pathologic degree.③JIANGUFANG prevents and cures the occurrence of the hormone femoral head necrosis to be the level that passes to lower a bone partial PPARγand exaltation Runx2, Osterix and the fat allied vegetable and attains to repress into a fat and promotes the function of ossification.④Provide an experiment for the Chinese medicine medicine"cure don't disease" theories prevention and cure hormone femoral head necrosis basis.
股骨头坏死(Osteonecrosis of the femoral head, ONFH)是骨科领域难治性疾病之一,临床上ONFH分为创伤性与非创伤性两大类,前者主要因髋部外伤(股骨颈骨折、髋关节脱位等)引起,后者主要由于皮质类固醇应用及长期大量饮酒引起；激素性股骨头坏死占非创伤性ONFH的首位,约占50%,ONFH为渐进性疾病,未经有效治疗及预防,约80%会在发病后1-4年内进展至股骨头塌陷；一旦出现股骨头塌陷,多数患者将发展成为严重骨性关节炎而需行人工关节置换术。由于非创伤性ONFH好发于中青年,中青年的人工关节置换术后远期疗效仍难以预测。因此,寻求有效的保髋治疗方法尤为重要,而取得优良治疗效果的前提是获得激素性股骨头坏死的早期诊断及早期干预。探索激素性股骨头坏死的确切发病机理、寻找敏感的早期诊断方法及有效的早期治疗方法或预防途径,具有非常重要意义。
激素性股骨头坏死是全身疾病的局部表现,中医药治疗的优势在于全身用药整体治疗以减少激素应用的副作用、同时可以进行高危人群的预防、亚临床期与塌陷前的个体化辨证治疗等。由于本病发病机制及遗传背景特征的复杂性,准确确定高危人群十分困难。亚临床期及塌陷前因缺少临床症状与X线表现,医患常不加注意,更不可能采用比较敏感但昂贵的核磁共振(MRI)进行普查。由于本病早期诊断困难,导致无法运用中医药进行有效预防以及在塌陷前针对发病机制进行治疗；因此,有必要对激素性ONFH的发病机制进行深入研究。
临床研究发现大剂量激素的使用是导致激素性股骨头坏死有意义的危险因素,对于激素性股骨头坏死,同样的激素用量,不同患者常出现不同的结果,有些患者会引起ONFH,有些患者不会,说明个体对于激素的敏感性是不同的。而糖皮质激素在体内代谢关键酶是细胞色素P4503A酶(CYP3A),研究者发现CYP3A的活性范围较大,低活性的CYP3A,可能是引起血清中皮质类固醇清除率下降,导致游离的糖皮质激素水平升高,副作用增大的主要原因,因此,提出“CYP3A的活性水平与激素性ONFH相关”的假说,本研究旨在初步探讨CYP3A活性在激素性ONFH发病中的作用及临床上用于治疗激素性ONFH的经验方—健骨方对CYP3A活性的影响,通过观察其对股骨头局部PPARr的影响探讨其对局部成脂分化影响；通过观察其对局部脂联素、Runx2、Osterix的影响探讨其对局部成骨分化的影响,为其治疗与预防激素性ONFH提供依据。
方法
以30只健康SPF级新西兰家兔为研究对象,雌雄各半,按体重均衡原则随机分成5组即空白对照组、模型组、诱导剂组、抑制剂组、健骨方组,每组6只,分笼标准饲料饲养,自由摄食水,室温控制在23℃-25℃,湿度50%,通风良好,适应喂养1周后给药；造模方法参照Yamamoto等、参照Toshiaki Masada的实验研究方法及相关药物用量,分组后的实验家兔,根据不同的施加因素,诱导剂组予每天肌肉注射苯巴比妥注射液,25mg/kg体重；抑制剂组予隔天灌胃伊曲康唑,50mg/kg体重,实验前用生理盐水将伊曲康唑胶囊内容物配制成5mg/ml的混悬液；健骨方组予每天灌胃健骨方混悬液,10ml/kg体重,实验前用生理盐水将健骨方煎煮的药液配制成含生药0.719g/ml的混悬液；空白对照组、模型组予灌胃等剂量生理盐水,10ml/kg体重；每周称重一次,连续用药3周后,实验动物均选择在左大腿内侧肌肉注射注射用甲泼尼龙琥珀酸钠,40mg/kg体重,连续用药3天,继续常规饲养3周。
实验开始前以及3周后,动物禁食不禁水8小时,次日凌晨家兔耳缘静脉取血4ml,用肝素锂抗凝,立即放入离心机中,3000RPM,离心10分钟,小心移取上层黄色液体到新的离心管中,放入-70℃冰箱里保存备测,采用的是GENMED比色法定量检测试剂盒,该方法是通过红霉素去甲基酶反应系统中红霉素A去甲基后的产物甲醛与纳什试剂作用后吸光峰值的变化来测定样品中肝CYP3A酶活性；采用双抗体一步夹心法酶联免疫吸附试验(ELISA)测定血浆糖皮质激素水平,实验开始6周后空气栓塞法处死家兔,剖取左侧股骨头,置于液氮罐中,用ELISA法测定局部股骨头组织内糖皮质激素、脂联素PPARγ、Runx2、Osterix含量,取右后侧股骨头,剔除周围软组织,分组标记好标本袋,置于10%福尔马林溶液中固定,进行病理组织学检测；数据用SPSS 13.0 For Windows统计分析软件处理,计量资料用均数((?))±标准差(S)表示,采用t检验；对坏死率进行单因素方差分析(One-Way ANOVA)；相关分析采用Spearman等级相关分析。
结果
参照Matsui等对成年兔激素诱导股骨头坏死标本的组织病理改变分级方法,本研究选择使用病理结果评价激素性股骨头坏死模型复制的成功性。肉眼观察可见股骨头外观完整,未见软骨下塌陷或碎裂,但股骨头关节软骨颜色瘀暗；HE染色切片置于光学显微镜下观察结果显示除空白对照组5只家兔病理改变为0级外,其余19只使用激素造模的家兔出现2级病理改变的实验动物为9只(47.4%)、1级病理改变为10只(52.6%)。
在用药物干预之前,与空白对照组比较,模型组、诱导剂组、抑制剂组、健骨方组肝CYP3A酶活性差异均无统计学意义(P值分别为0.253、0.647、0.242、0.262>0.05),用药干预3周后与空白对照组比较,模型组差异无统计学意义(t=1.848,P=0.118>0.05)、诱导剂组用苯巴比妥注射液诱导后肝CYP3A酶活性明显提高,差异具有统计学意义(t=3.678,P=0.004<0.05)、抑制剂组经伊曲康唑胶囊抑制后肝CYP3A酶活性则明显降低,差异具有统计学意义(t=6.268,P=0.001<0.05)、健骨方组也可以提高该酶活性,差异具有统计学意义(t=3.84,P=0.004<0.05)；与模型组比较,诱导剂组、健骨方组酶活性增高、抑制剂组酶活性降低,差异均具有统计学意义(t=3.82,3.93,2.57,P=0.013、0.09、0.021<0.05)与诱导剂组比较,抑制剂组肝CYP3A酶活性明显降低,差异具有统计学意义(t=3.714,P=0.013<0.05)、健骨方组CYP3A酶活性有所下降,但差异无统计学意义(t=0.105,P=-0.322>0.05)；与抑制剂组比较,健骨方组CYP3A酶活性明显提高,差异具有统计学意义(t=5.60,P=-0.02<0.05)。
各组用药前后自身比较,空白对照组用药前后比较,肝CYP3A酶活性差异无统计学意义(t=1.859,P=0.122>0.05)；模型组用药后肝CYP3A酶活性较用药前也有所升高,差异无统计学意义(t=1.92,P=0.097>0.05)；诱导剂组用药后肝CYP3A酶活性较用药前明显升高,差异具有统计学意义(t=5.06,P=0.003<0.05)；抑制剂组用药后肝CYP3A酶活性较用药前明显降低,差异具有统计学意义(t=7.482,P=-0.002<0.05)；健骨方组用药后肝CYP3A酶活性较用药前明显升高,差异具有统计学意义(t=5.059,P=0.003<0.05)。
在用糖皮质激素造模之前,与空白对照组比较,模型组、诱导剂组、抑制剂组、健骨方组血浆糖皮质激素水平差异不显著(P值分别为0.952、0.18、0.099、0.351>0.05),组间采用单因素方差分析结果显示F=1.289,P=0.301>0.05说明组间差异无统计学意义；用药干预3周后,组间采用单因素方差分析结果显示F=26.72,P=0.001<0.05说明组间差异显著,经方差齐性检验,P=-0.076>0.05,表明方差齐,采用LSD方法进行两两比较,结果显示与空白对照组比较,血浆糖皮质激素水平模型组升高,差异具有统计学意义(P=0.001<0.05)、诱导剂组有所降低,差异无统计学意义(P=-0.527>0.05)、抑制剂组明显升高,差异具有统计学意义(P=0.0001<0.05)、健骨方组也明显降低,但差异无统计学意义(P=0.124>0.05)；与模型组比较,血浆糖皮质激素水平诱导剂组、健骨方组均降低、抑制剂组升高,差异均具有统计学意义(P=0.0001、0.0001、0.003<0.05)；与诱导剂组比较,抑制剂组血浆糖皮质激素水平升高明显,差异具有统计学意义(P=0.0001<0.05),健骨方组有所下降,但差异无统计学意义(P=0.346>0.05)；与抑制剂组比较,健骨方组明显降低,差异具有统计学意义(P=0.001<0.05)。
各组股骨头局部糖皮质激素含量的数据,组间采用单因素方差分析结果显示F=143.8,P=0.001<0.05说明组间差异显著,经方差齐性检验,P=0.494>0.05,表明方差齐,采用LSD方法进行两两比较,与空白对照组比较,局部糖皮质激素水平模型组明显升高,差异具有统计学意义(P=0.001<0.05)、诱导剂组有所降低,差异无统计学意义(P=0.059>0.05)、抑制剂组明显升高,差异具有统计学意义(P=0.0001<0.05)、健骨方组也明显降低,但差异无统计学意义(P=0.324>0.05)；与模型组比较,局部糖皮质激素水平诱导剂组、健骨方组均降低、抑制剂组升高,差异均具有统计学意义(P=0.0001、0.0001、0.001<0.05)；与诱导剂组比较,抑制剂组股骨头局部糖皮质激素水平升高明显,差异具有统计学意义(P=0.0001<0.05),健骨方组有所下降,但差异无统计学意义(P=-0.331>0.05)；与抑制剂组比较,健骨方组明显降低,差异具有统计学意义(P=0.001<0.05)。肝CYP3A酶活性与股骨头局部糖皮质激素水平呈显著的负相关(RR值为-0.657,P=0.001)。
各组股骨头局部脂联素含量数据,组间采用单因素方差分析结果显示F=15.78,P=0.001<0.05说明组间差异显著,经方差齐性检验,P=0.102>0.05,表明方差齐,采用LSD方法进行两两比较,与空白对照组比较,局部脂联素水平模型组明显降低,差异具有统计学意义(P=0.005<0.05)、诱导剂组有所升高,差异无统计学意义(P=0.163>0.05)、抑制剂组明显降低,差异具有统计学意义(P=0.0001<0.05)、健骨方组也有所降低,但差异无统计学意义(P=0.464>0.05)；与模型组比较,局部脂联素水平诱导剂组、健骨方组均升高、抑制剂组明显降低,差异均具有统计学意义(P=0.0001、0.013、0.023<0.05)；与诱导剂组比较,抑制剂组股骨头局部脂联素水平降低明显,差异具有统计学意义(P=0.0001<0.05),健骨方组有所降低,差异有统计学意义(P=0.041<0.05)；与抑制剂组比较,健骨方组明显升高,差异具有统计学意义(P=0.001<0.05)。
各组股骨头局部PPARγ含量的数据,组间采用单因素方差分析结果显示F=91.79,P=0.001<0.05说明组间差异显著,经方差齐性检验,P=0.001<0.05,表明方差不齐,采用Tamhane方法进行两两比较,与空白对照组比较,局部PPARγ水平模型组明显升高,差异具有统计学意义(P=0.018<0.05)、诱导剂组有所升高,差异无统计学意义(P=1.00>0.05)、抑制剂组明显升高,差异具有统计学意义(P=0.0001<0.05)、健骨方组也略有所升高,但差异无统计学意义(P=1.00>0.05)；与模型组比较,局部PPARγ水平诱导剂组、健骨方组均降低、抑制剂组明显升高,差异均具有统计学意义(P=0.045、0.006、0.033<0.05)；与诱导剂组比较,抑制剂组股骨头局部PPARγ水平明显升高,差异具有统计学意义(P=0.002<0.05),健骨方组有所降低,差异无统计学意义(P=0.891>0.05)；与抑制剂组比较,健骨方组明显降低高,差异具有统计学意义(P=0.001<0.05)。
各组股骨头局部Runx2含量数据,组间采用单因素方差分析结果显示F=88.4,P=0.001<0.05说明组间差异显著,经方差齐性检验,P=0.003<0.05,表明方差不齐,采用Tamhane方法进行两两比较,与空白对照组比较,股骨头局部Runx2含量模型组明显降低,差异具有统计学意义(P=0.0031<0.05)、诱导剂组明显升高,差异有统计学意义(P=0.013<0.05)、抑制剂组明显降低,差异具有统计学意义(P=0.0001<0.05)、健骨方组也明显升高,差异具有统计学意义(P=0.002<0.05)；与模型组比较,股骨头局部Runx2含量诱导剂组、健骨方组均升高、抑制剂组明显降低,差异均具有统计学意义(P=0.003、0.001、0.001<0.05)；与诱导剂组比较,抑制剂组股骨头局部Runx2含量降低明显,差异具有统计学意义(P=0.001<0.05),健骨方组有所降低,差异无统计学意义(P=0.288>0.05)；与抑制剂组比较,健骨方组明显升高,差异具有统计学意义(P=0.001<0.05)。
股骨头局部Osterix含量组间采用单因素方差分析结果显示F=38.07,P=0.001<0.05说明组间差异显著,经方差齐性检验,P=0.002<0.05,方差不齐,采用Tamhane方法进行两两比较,与空白对照组比较,股骨头局部Osterix含量模型组明显降低,差异具有统计学意义(P=0.037<0.05)、诱导剂组明显升高,差异有统计学意义(P=0.006<0.05)、抑制剂组明显降低,差异具有统计学意义(P=0.004<0.05)、健骨方组也明显升高,但差异无统计学意义(P=0.066>0.05)；与模型组比较,股骨头局部Osterix含量诱导剂组、健骨方组均升高、抑制剂组明显降低,差异均具有统计学意义(P=0.001、0.025、0.022<0.05)；与诱导剂组比较,抑制剂组股骨头局部Osterix含量降低明显,差异具有统计学意义(P=O.001<0.05),健骨方组有所升高,差异无统计学意义(P=0.76>0.05)；与抑制剂组比较,健骨方组明显升高,差异具有统计学意义(P=-0.012<0.05)。
各组坏死面积与坏死率数据,使用单因素方差分析(One-Way ANOV A),统计显示：经Levene方差齐性检验,方差齐(P=0.091及P=0.151)；在方差齐性条件下,采用LSD检测方法,坏死面积结果显示：模型组与诱导剂组比较,诱导剂组坏死面积较模型组降低,两者有显著性差异P<0.05(P=0.037)；模型组与抑制剂组比较,两者坏死面积间显著性差异P<0.05(P=0.023)；模型组与健骨方组比较,健骨方组组坏死面积较模型组降低,两者有显著性差异P<0.05(P=0.015)；与诱导剂组比较,抑制剂组坏死面积明显升高,差异具有统计学意义P<0.05(P=0.012),健骨方组坏死面积有所升高,但差异不显著P>0.05(P=0.423)；与抑制剂组,健骨方组坏死面积明显降低,P<0.05(P=0.034)；坏死率结果显示：模型组与诱导剂组比较,诱导剂组坏死率较模型组降低,两者有显著性差异P<0.05(P=0.023)；模型组与抑制剂组比较,两者坏死率间显著性差异P<0.05(P=0.044)；模型组与健骨方组比较,健骨方组坏死率较模型组降低,两者有显著性差异P<0.05(P=0.024)；与诱导剂组比较,抑制剂组坏死率明显升高,差异具有统计学意义P<0.05(P=0.016),健骨方组坏死率有所升高,但差异不显著P>0.05(P=0.623)；与抑制剂组,健骨方组坏死率明显降低,P<0.05(P=0.025)。肝CYP3A酶活性与股骨头坏死面积、坏死率均成显著的负相关；肝CYP3A酶活性与股骨头局部PPARγ含量、脂联素、RUNX2的相关性,经Pearson相关性分析,RR值分别为-0.269,0.097,0.211,P值分别为0.203,0.652,0.321均>0.05,无明显相关性。
结论
本次研究结果表明肝CYP3A酶的活性与激素性股骨头坏死的发生呈显著负相关①肝CYP3A酶活性增高可使股骨头局部糖皮质激素含量减少。②肝CYP3A酶活性增高可降低激素性股骨头坏死率,减少坏死面积,减轻病理分级。这可能是肝CYP3A酶能对外源性糖皮质激素起清除作用,使糖皮质激素在股骨头局部的蓄积时间缩短、浓度降低,从而降低了股骨头坏死的发生率。中药健骨方对肝CYP3A酶的影响与激素性ONFH的实验研究结果表明①健骨方能够增强肝CYP3A酶的活性。②健骨方能够预防激素性股骨头坏死的发生,减轻坏死病理程度。③健骨方防治激素性股骨头坏死的发生可能是通过降低股骨头局部PPARγ和提高Runx2、Osterix以及脂联素的水平,达到抑制成脂、促进成骨的作用。④为中医药“治未病”理论防治激素性股骨头坏死提供实验依据。
Objective
Osteonecrosis of the femoral head (ONFH) is regard as one of the difficult to treat disease in the field of Orthopedics. ONFH can be divided into traumatic and non-traumatic causes. ONFH which is caused by the traumatic mainly because of injury of hip such as neck fracture and hip dislocation, etc.. The non-traumatic ONFH is caused by a long-term corticosteroids use and Alcoholism. A long-term corticosteroids use is the most causes ONFH in the non-traumatic part, it is about 50%. ONFH is a progressive disease, without effective treatment and prevention, about 80% people pathogenesis femoral head collapse in 1 to 4 years, the event of a femoral head collapse will develop into a serious arthritis in the majority of patients, finally the arthroplasty is needed. non-traumatic ONFH is regularity occurs in middle-aged, middle-aged in the long term effect of arthroplasty surgery is still difficult to predict. Therefore, the search for effective treatment of hip protection is especially important, early diagnosis and early intervention is the premise to have achieved excellent therapeutic effect. Explore the ONFH pathogenesis, find the sensitive methods for early diagnosis and effective early treatment, it has has very important significance.
Clinical data indicate that the ONFH caused by glucocorticoids is systemic disease but performance in the part of the body. Advantages of Chinese medicine treatment of systemic drug therapy to the whole to reduce the side effects of hormone application, prevention the high-risk groups, sub-clinical phase and before the collapse to use individual differentiation therapy. As the complexity of disease pathogenesis and genetic background characteristics, it was quite difficult to determine high-risk groups accurately. Because of lack of clinical symptoms and X-ray performance within the sub-clinical phase and before the collapse, patients often less liked to notice even not to adopt sensitive but expensive magnetic resonance imaging (MRI) performance. As the early diagnosis was difficult, it couldn't use Chinese medicine to prevention effectively and treat before the collapse. Therefore, it was necessary to research the ONFH caused by glucocorticoids in-depth.
Clinical study found that the use of high-dose hormone therapy is significant risk factor for ONFH. But the same hormone dosage in different patients are often comes out different results, some patients will lead to ONFH, some patients not. that means the individual to hormones is different of sensitivity. The metabolism of glucocorticoids in the body is the key enzyme cytochrome P4503A enzymes (CYP3A), researchers found that the activity of CYP3A large range of low activity of CYP3A, may cause the serum clearance rate of corticosteroids due to the decreasing level of free glucocorticoids increased side effects increased, therefore, "CYP3A activity levels and associated femoral head necrosis" hypothesis was made, this study aims to initially explore the CYP3A activity in steroid ONFH pathogenesis and clinical for the treatment of the experience of the femoral head necrosis side-bone with effects on CYP3A activity.
Observe the influence of PPARr in partial femoral head to investigate the locality fat differentiation. Observe the influence of adiponectin、Osterix and Runx2 to investigate the influence of locality osteoblast, its treatment and prevention of steroid-induced femoral head necrosis provide a theoretical basis.
Methods
Thirty adult SPF healthy New Zealand rabbits were used, male and female half and half. According to the principle of weight balance, the rabbits were devided into five groups:Control group, Model group, Group inducer, Inhibitor group, JIANGUFANG group. Each group was feed under control and was administrate after one week. The molding method was refer to Yamanoto and the research method and dosage was Toshiaki Masada. In group inducer, the six animals were received daily intramuscular Phenobarbital 25mg/kg, Inhibitor group received oral itraconazole 50 mg/kg every other day to inhibit CYP3A activity; JIANGUFANG group received oral JIANGUFANG Suspension 10ml/kg, Drug content was 0.719g/ml every day and the other six animals were given with saline 10ml/kg as a control.Weight each of the animal for three consecutive weeks, then intramuscular injection of methylprednisolone sodium succinate 40mg/kg for 3 days in the left thight of the rabbits, continue to feed 3 weeks.
Before the Experiment or three weeks later, collection 4ml vein blood from the rabbit ear after 8-hour fasting, mixed with Lithium heparin, then put it into the Centrifuge,3000RPM,10 minute. Collect the upper yellow liquid into the Centrifuge Tube,-70℃Storage. Use the GENMED quantitative colorimetric detection kit detect the sample. This method is by erythromycin demethylation enzyme reaction system to methyl erythromycin A product of formaldehyde and Nash after the reagents after the change of absorption peak to determine CYP3A activity in liver samples. Adopt double antibody step clip neo-confucianism, enzyme-linked immunosorbent adsorption test (ELISA) to determinate the glucocorticoid levels in plasma. Air embolism execute the rabbit after six weeks,take the left femoral in liquid nitrogen tank, determinate the glucocorticoid, adiponectin PPARγ, Runx2, Osterix in locality femoral head with ELISA, take the right posterior femoral head, remove soft tissue, marked the specimen bag, fix the sample with 10% Formalin, prosecution pathology testing. Analyzed the data with SPSS 13.0 For Windows Statistical software. Representing measurement data by T test with arithmetic mean(x)and Standard Deviation (S). Analyzed necrosis rate by One-Way ANOVA.Abdopted Spearman rank correlation analysis to progress Analysis of Correlation.
Results
Refer to Matsui, the stage division of histopathology in adult rabbit hormone induced ONFH, this study used the pathologic result to evaluate the success of copying the model. Macroscopic observation showed that visible appearance complete, did not see the femoral subchondral collapse or chip, but the color of femoral articular cartilage was dark; HE dyed slice which was observed by optical microscope showed that in addition to 5 in control group only for 0 level of rabbit pathological changes, there were 9 (47.4%) of grade 2 pathological changes and 10(52.6%) of grade 1 in the rest of the 19 used hormone.
Before to use of the drug intervention, hepatic CYP3A activity had no statistically significant difference (P=0.253,0.647,0.242,0.262> 0.05)in compared with the control group, model group, Group inducer, inhibitor group and JIANGUFANG group. After 3 weeks, Compared with the control group, the model group difference was statistically significant (t=1.848, P=> 0.05), the enzymatic enzyme activity of CYP3A was obviously improved after phenobarbital injection and differences are statistically significant (t= 3.678, P=0.004 < 0.05), itraconazole capsule inhibited the CYP3A enzyme activity in the inhibitor group,the difference was statistically significant (t=6.268, P=0.001<0.05), JIANGUFANG can also raise the enzyme's activity, the difference has a statistics meaning; (t=3.84, P=0.004<0.05) And the comparison with model set, inducement set, JIANGUFANGset the enzyme activity increases, and the depressant set enzyme activity lower, and the difference all has a statistics meaning; (t=3.82,3.93,2.57, P=0.013,0.09,0.021<0.05)And comparison with inducement set, the activity obviously lowers of the liver CYP3 A enzyme in depressant set, the difference has a statistics meaning (t=3.714, P=0.013<0.05), JIANGUFANG set the CYP3 A Enzyme the activity has to descend, but the difference has no statistics meaning; (t=0.105, P=0.322>0.05)And with the comparison depressant set, the CYP3 A Enzyme the activity in JIANGUFANG set obviously raises, the difference has a statistics meaning. (t=5.60, P=0.02<0.05)
Each comparison used self-comparison of medicine, the blank matched control is used a medicine self-comparison, the liver CYP3 A Enzyme live difference has no statistics meaning; (t=1.859, P=0.122>0.05) The model set is used the medicine the liver CYP3 A Enzyme activity more than before, the difference has no statistics meaning; (t=1.92, P=0.097>0.05)The inducement set is used the medicine the liver CYP3 A Enzyme activity obviously higher than before, the difference has a statistics meaning; (t=5.06, P=0.003<0.05)The depressant set is used the medicine the liver CYP3 A Enzyme activity more lower than before, the difference has a statistics meaning;(t=7.482, P=0.002<0.05)The JIANGUFANG set is used the medicine the liver CYP3 A Enzyme activity more obviously higher than before, the difference has a statistics meaning. (t=5.059, P=0.003<0.05)
Just before making a mold with glucocorticoid, Blank control group, model group, induced dose group, inhibitor group, JIANGUFANG plasma glucocorticoid levels not significantly different (the P value distinguishes to 0.952,0.18, 0.099,0.351>0.05), the set adopts a single factor variance to analyze to show F=1.289, P=0.301>0.05 difference have no the statistics;With the medicine interfere after 3 weeks, the set adopts a single factor variance analytical show F=26.72,P=0.001<0.05 shows significant differences between groups, The test of homogeneity of variance, P=0.076>0.05, Show that the homogeneity of variance. Use the using LSD pairwise comparison method, The results showed that compared with the control group, blood plasma sugar skin quality hormone level model set goes up, the difference has a statistics meaning(P=0.001<0.05) and inducement the set has to lower, difference have no the statistics meaning(P=0.527>0.05), depressant set obviously goes up, the difference has a statistics meaning(P=0.0001<0.05), JIANGUFANG set also obviously lower, but the difference have no the statistics meaning;(P=0.124>0.05)And model set comparison, the blood plasma level glucocorticoid in inducement set, JIANGUFANG set all lower, the depressant set raise, the difference all has a statistics meaning;(P=0.0001,0.0001,0.003<0.05)And comparison in inducement set, depressant set blood plasma glucocorticoid level goes up obviously, the difference has a statistics meaning(P=0.0001<0.05), and the JIANGUFANG set has to descend, but the difference has no statistics meaning;(P=0.346>0.05)And depressant set comparison, the JIANGUFANG set obviously lower, the difference has a statistics to learn meaning. (P=0.001<0.05)
The data of partial s glucocorticoid content of each bone, the set adopts a single factor variance analytical show F=143.8, P=0.001<0.05 difference shows obvious, through data examination, P=0.494>0.05, express that the data equal. Use the LSD method carries to compare with blank matched control comparison, partial glucocorticoid level model set obviously goes up, the difference has a statistics meaning(P=0.001<0.05) and inducement the set has to lower, difference have no the statistics learn the meaning(P=0.059>0.05), depressant set obviously goes up, the difference has a statistics to learn the meaning(P=0.0001<0.05), JIANGUFANG set also obviously lowers, but difference have no the statistics learn meaning;(P=0.324>0.05)And model set comparison, partial set, JIANGUFANG set of sugar skin quality hormone level inducement all lowers, the depressant set goes up, the difference all has a statistics-to learn meaning; (P=0.0001,0.0001,0.001<0.05) And inducement set comparison, the depressant set the bone partial sugar the skin quality hormone level goes up obviously, the difference has a statistics to learn meaning (p=0.0001<0.05), and the JIANGUFANG set has to descend, but the difference has no statistics meaning;(P=0.331>0. 05)And depressant set comparison, the JIANGUFANG set obviously lowers, the difference has a statistics meaning. (P=0.001<0.05)The liver CYP3 A enzyme activity and the bone partial glucocorticoid level presents to show negative related. (RR value is-0.657, P=0.001)
The partial fat allied plain content data of each bone, the set adopts a single factor variance analytical show F=15.78, P=0.001<0.05 difference shows Obvious, through homogeneity of variance test, P=0.102>0.05, express that the variance adopts together the LSD method carries to compare, and blank matched control comparison, the partial fat unites plain level model, the set obviously lowers, the difference has a statistics to learn a meaning(P=0.005<0.05) and inducement the set has to go up, difference have no the statistics learn the meaning(P=0.163>0.05), depressant set obviously lowers, the difference has a statistics meaning(P=0.0001<0.05), JIANGUFANG set also has to lower, but difference have no the statistics learn meaning;(P=0.464>0.05)And model set comparison, partial fat allied plain level inducement the set, JIANGUFANG set all goes up, the depressant set obviously lowers, the difference all has a statistics meaning; (P=0.0001,0.013, 0.023<0.05) And inducement set comparison, the depressant set the bone partial fat allied plain level lowers obviously, the difference has a statistics meaning(P=0.0001<0.05), the JIANGUFANG set has to lower, and the difference includes a statistics meaning; (P=0.041m<0.05)And depressant set comparison, the JIANGUFANG set obviously goes up, the difference has a statistics meaning. (P=0.001<0.05)
The data of partial content of PPARγof each bone, the set adopts a single factor variance analytical show F=91.79, P=0.001<0.05 difference shows Obvious, the test of homogeneity of variance, P=0.001<0.05, express that the variance adopts not and together the Tamhane method carries on compare, and blank matched control comparison, the partial PPAR theγlevel model set obviously goes up, the difference has a statistics to learn a meaning(P=0.018<0.05) and inducement the set has to go up, difference have no the statistics learn the meaning(P=1.00>0.05), depressant set obviously goes up, the difference has a statistics meaning(P=0.0001<0.05), JIANGUFANG set also slightly has to go up, but difference have no the statistics learn meaning; (P=1.00>0.05)And model set comparison, partial PPAR the set, JIANGUFANG set of theγlevel inducement all lowers, the depressant set obviously goes up, the difference all has a statistics meaning; (P=0.045,0.006, 0.033<0.05)And inducement set comparison, depressant set the bone partial level of PPARγobviously goes up, the difference has a statistics meaning (P=0.002<0.05), the JIANGUFANG set has to lower, and the difference has no statistics meaning;(P=0.891>0.05)And depressant set comparison, the JIANGUFANG set obviously lowers Gao, the difference has a statistics meaning. (P=0.001<0.05)
The partial Runx2 content data of each bone, the set adopts a single factor variance analytical show F=88.4, P=0.001<0.05 difference shows obvious, the test of homogeneity of variance, P=0.003<0.05, express that the variance adopts not and together the Tamhane method carries to compare, and blank matched control comparison, bone partial Runx2 content model the set obviously lowers, the difference has a statistics to learn a meaning(P=0.0031<0.05) and inducement the set obviously goes up, the difference includes a statistics meaning(P=0.013<0.05), depressant set obviously lowers, the difference has a statistics meaning (P=0.0001<0.05), JIANGUFANG set also obviously goes up, the difference has a statistics meaning;(P=0.002<0.05)And model set comparison, bone partial Runx2 content inducement the set, JIANGUFANG set all goes up, the depressant set obviously lowers, the difference all has a statistics meaning; (P=0.003,0.001,0.001<0.05) And inducement set comparison, depressant set the bone partial Runx2 contents lower obviously, the difference has a statistics meaning(P=0.001<0.05), the JIANGUFANG set has to lower, and the difference has no statistics meaning;(P=0.288>0.05)And depressant set comparison, the JIANGUFANG set obviously goes up, the difference has a statistics meaning. (P=0.001<0.05)
Bone partial Osterix content set adopts a single factor variance analytical show F=38.07, P=0.001<0.05 difference shows Obvious, the test of homogeneity of variance, P=0.002<0.05, the variance adopts not and together the Tamhane method carries to compare, and blank matched control comparison, bone partial Osterix content model the set obviously lowers, the difference has a statistics to learn a meaning(P=0.037<0.05) and inducement the set obviously goes up, the difference includes a statistics meaning(P=0.006<0.05), depressant set obviously lowers, the difference has a statistics meaning(P=0.004<0.05), JIANGUFANG set also obviously goes up, but difference have no the statistics learn meaning; (P=0.066>0.05) And model set comparison, bone partial Osterix content inducement the set, JIANGUFANG set all goes up, the depressant set obviously lowers, the difference all has a statistics meaning;(P=0.001,0.025,0.022<0.05)And inducement set comparison, the depressant set the bone partial Osterix content lowers obviously, the difference has a statistics meaning(P=0.001<0.05), the JIANGUFANG set has to go up, and the difference has no statistics meaning; (P=0.76>0.05) And depressant set comparison, the JIANGUFANG set obviously goes up, the difference has a statistics meaning. (P=0.012<0.05)
Each bad dead area with bad dead rate data, use a single factor variance analysis(One-Way ANOVA), the statistics shows:Through Levene variance examines, the variance is together;(P=0.091 and P=0.151)Together adopt a LSD examination method under the variance, the bad dead area result shows:Model set and inducement set comparison, to compare with model set, the inducement the set bad dead area lowers, both have already shown the Obvious difference P<0.05;(P=0.037)Model set and depressant set comparison, both the bad dead area shows the Obvious difference P<0.05;(P=0.023)Model set and JIANGUFANG set comparison, to compare with model set, the JIANGUFANG set set bad dead area lowers, both have already shown the Obvious difference P<0.05;(P=0.015)And inducement set comparison, the depressant set bad dead area obviously goes up, the difference has a statistics meaning P<0.05(P=0.012), the JIANGUFANG set bad dead area has to go up, but the difference don't show Obvious P>0.05;(P=0.423)And depressant set, the JIANGUFANG set bad dead area obviously lowers, P<0.05; (P=0.034) The bad dead rate shows as a result:Model set and inducement set comparison, to compare with model set, the inducement the set bad dead rate lowers, both have already shown the Obvious difference P<0.05;(P=0.023)Model set and depressant set comparison, both the bad dead rate shows the Obvious difference P<0.05; (P=0.044)Model set and JIANGUFANG set comparison, to compare with model set, the JIANGUFANG set bad dead rate lowers, both have already shown the Obvious difference P<0.05;(P=0.024)And inducement set comparison, the depressant set bad dead rate obviously goes up, the difference has a statistics meaning P<0.05(P=0.016), the JIANGUFANG set bad dead rate has to go up, but the difference don't show Obvious P>0.05;(P=0.623)And depressant set, the JIANGUFANG set bad dead rate obviously lowers, P<0.05. (P=0.025)The liver CYP3 enzyme activity and the bone bad dead area, bad dead rate all become to show Obvious of negative related.
Conclusions
This research result expresses the activity of The liver CYP3A enzyme presents with the occurrence of the hormone femoral head necrosis to show Obvious negative the related①liver CYP3 A Mao activity increase Gao to make a bone partial glucocorticoid content reduced.②The liver CYP3 enzyme activity increases Gao to lower hormone the bone bad dead lead, reduce bad dead area and ease pathologic ratings. This may be The liver CYP3 enzyme can outward the source glucocorticoid contain clearance function and make glucocorticoid shortenned in time of storing of bone part, the density lower and lowered the incidence rate of the bone bad dead thus. The experiment research of the influence of Chinese herbal medicine JIANGUFANG upon The liver CYP3 enzyme and hormone ONFH expresses that①JIANGUFANG can strengthen the activity of The liver CYP3 enzyme as a result.②JIANGUFANG can prevent°fro the occurrence of the hormone femoral head necrosis, ease bad dead pathologic degree.③JIANGUFANG prevents and cures the occurrence of the hormone femoral head necrosis to be the level that passes to lower a bone partial PPARγand exaltation Runx2, Osterix and the fat allied vegetable and attains to repress into a fat and promotes the function of ossification.④Provide an experiment for the Chinese medicine medicine"cure don't disease" theories prevention and cure hormone femoral head necrosis basis.
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