[1]张麟,向楠,周广文,等.补肾化痰方对去卵巢大鼠骨形成的影响及其作用机制研究[J].中医正骨,2018,30(10):12-18.
　ZHANG Lin,XIANG Nan,ZHOU Guangwen,et al.Effect of Bushen Huatan Fang(补肾化痰方)on bone formation of ovariectomized rats and its mechanism of action[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2018,30(10):12-18.
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补肾化痰方对去卵巢大鼠骨形成的影响及其作用机制研究()
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《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第30卷
期数:
2018年10期
页码:
12-18
栏目:
基础研究
出版日期:
2018-10-20

文章信息/Info

Title:
Effect of Bushen Huatan Fang(补肾化痰方)on bone formation of ovariectomized rats and its mechanism of action
作者:
张麟1; 向楠2; 周广文2; 周亚娜3; 胡娅4; 张妍2; 李章青2
(1.湖北省武汉市第一医院,湖北 武汉 430022; 2.湖北中医药大学,湖北 武汉 430061; 3.湖北省中医院,湖北 武汉 430061; 4.长江大学,湖北 荆州 434023)
Author(s):
ZHANG Lin1; XIANG Nan2; ZHOU Guangwen2; ZHOU Yana3; HU Ya4; ZHANG Yan2; LI Zhangqing2
1.The First Hospital of Wuhan,Wuhan 430022,Hubei,China 2.Hubei University of Traditional Chinese Medicine,Wuhan 430061,Hubei,China 3.Hubei Provincial Hospital of Traditional Chinese Medicine,Wuhan 430061,Hubei,China 4.Changjiang University,Jingzhou 434023,Hubei,China
关键词:
骨质疏松;  补肾化痰方;  信号传导;  骨形态发生蛋白质2;  Runt相关转录因子2;  大鼠; Sprague-Dawley;  动物实验
Keywords:
osteoporosis;  Bushen Huatan Fang;  signal transduction;  bone morphogenetic protein 2;  Runt related transcription factor 2;  rats; Sprague-Dawley;  animal experimentation
摘要:
目的:观察补肾化痰方对去卵巢大鼠骨形成的影响及其作用机制。方法:将30只6月龄SPF级雌性SD大鼠随机分为假手术组、模型组和补肾化痰组,每组10只。模型组和补肾化痰组大鼠手术摘除双侧卵巢,假手术组不摘除卵巢,只切除卵巢周围与双侧卵巢质量相等的脂肪组织。造模术后5周开始,补肾化痰组以0.94 g·mL-1补肾化痰方药液灌胃,假手术组和模型组以等体积生理盐水灌胃,均每天1次,连续干预8周。药物干预结束后,将所有大鼠处死,摘除双侧眼球取血检测血清骨形态发生蛋白(bone morphogenetic protein,BMP)-2、Runt相关转录因子(Runt related transcription factor,RUNX)-2含量,取左侧股骨行Micro CT扫描测定骨密度、骨小梁数量、骨小梁平均厚度、骨小梁分离度,取右侧股骨上端部分骨片以蛋白免疫印迹法测定骨组织中BMP-2、RUNX-2含量,取右侧股骨下端部分骨片以免疫组织化学法检测BMP-2、RUNX-2的定位。结果:①一般情况。所有大鼠均在造模手术麻醉3～4 h后完全清醒,正常进食,1周后切口完全愈合。药物干预过程中,模型组1只大鼠死亡、补肾化痰组2只大鼠死亡。②血清BMP-2、RUNX-2含量检测结果。3组大鼠的血清BMP-2含量比较,差异有统计学意义[(245.40±10.10)pg·mL-1,(111.03±29.75)pg·mL-1,(190.35±14.00)pg·mL-1,F=109.998,P=0.000]; 模型组、补肾化痰组的血清BMP-2含量均低于假手术组(P=0.000,P=0.000),补肾化痰组的血清BMP-2含量高于模型组(P=0.000)。3组大鼠的血清RUNX-2含量比较,差异有统计学意义[(4 131.04±277.39)pg·mL-1,(1 601.09±266.10)pg·mL-1,(3 134.90±202.33)pg·mL-1,F=237.192,P=0.000]; 模型组、补肾化痰组的血清RUNX-2含量均低于假手术组(P=0.000,P=0.000),补肾化痰组的血清RUNX-2含量高于模型组(P=0.000)。③股骨Micro CT扫描结果。3组大鼠骨密度比较,差异有统计学意义[(556.90±8.86)mg HA·cm-3,(498.81±9.26)mg HA·cm-3,(530.17±10.70)mg HA·cm-3,F=90.488,P=0.000]; 模型组、补肾化痰组的骨密度均低于假手术组(P=0.000,P=0.000),补肾化痰组的骨密度高于模型组(P=0.000)。3组大鼠骨小梁数量比较,差异有统计学意义[(2.65±0.32)个·mm-1,(1.74±0.22)个·mm-1,(2.41±0.33)个·mm-1,F=23.900,P=0.000]; 模型组的骨小梁数量少于补肾化痰组和假手术组(P=0.000,P=0.000),补肾化痰组的骨小梁数量与假手术组比较,差异无统计学意义(P=0.224)。3组大鼠骨小梁平均厚度比较,差异有统计学意义[(109.33±16.12)μm,(79.27±18.17)μm,(95.73±11.38)μm,F=8.730,P=0.001]; 模型组的骨小梁平均厚度低于假手术组(P=0.001),补肾化痰组的骨小梁平均厚度分别与假手术组和模型组比较,差异无统计学意义(P=0.181,P=0.098)。3组大鼠骨小梁分离度比较,差异有统计学意义[(350.95±68.46)μm,(472.33±33.70)μm,(406.68±47.77)μm,F=12.460,P=0.000]; 模型组、补肾化痰组的骨小梁分离度均高于假手术组(P=0.000,P=0.036),补肾化痰组的骨小梁分离度低于模型组(P=0.017)。④股骨组织中BMP-2、RUNX-2蛋白含量检测结果。3组大鼠股骨组织中的BMP-2含量比较,差异有统计学意义(0.73±0.03,0.26±0.02,0.48±0.02,F=738.111,P=0.000); 模型组、补肾化痰组股骨组织中的BMP-2含量均低于假手术组(P=0.000,P=0.000),补肾化痰组股骨组织中的BMP-2含量高于模型组(P=0.000)。3组大鼠股骨组织中的RUNX-2含量比较,差异有统计学意义(0.67±0.03,0.36±0.03,0.47±0.02,F=286.493,P=0.000); 模型组、补肾化痰组股骨组织中的RUNX-2含量均低于假手术组(P=0.000,P=0.000),补肾化痰组股骨组织中的RUNX-2含量高于模型组(P=0.000)。⑤股骨组织中BMP-2、RUNX-2蛋白定位检测结果。BMP-2、RUNX-2的阳性表达主要位于骨髓基质细胞胞浆及骨小梁周边成骨细胞中,光学显微镜下阳性表达蛋白呈棕黄色颗粒。结论:补肾化痰方能通过调节BMP-2/Smads/RUNX-2信号转导通路,促进去卵巢大鼠的骨形成。
Abstract:
Objective:To observe the effects of Bushen Huatan Fang(补肾化痰方,BSHTF)on bone formation of ovariectomized rats and its mechanism of action.Methods:Thirty 6-month-old SPF-grade female SD rats were randomly divided into sham-operated group,model group and BSHTF group,10 cases in each group.The bilateral ovariectomy were performed on rats in model group and BSHTF group,and the resection of same mass of fat around bilateral ovaries were performed on rats in sham-operated group.Since the 5th week after the modeling operation,the rats in BSHTF group were intragastric administrated with BSHTF solution in dosages of 0.94 g/mL,while the rats in sham-operated group and model group were intragastric administrated with the same dose of normal saline,once a day for consecutive 8 weeks.After the end of drug intervention,all rats were executed and their bilateral eyeballs were removed and their blood were fetched out for detecting the serum contents of bone morphogenetic protein(BMP)-2 and Runt related transcription factor(RUNX)-2.Their left femurs were fetched out for determining bone mineral density(BMD),trabecular number(Tb.N),mean trabecular thickness(Tb.Th)and trabecular separation(Tb.Sp)through Micro-CT scanning.Meanwhile,the sclerites were fetched out from the upper end and the lower end of right femurs respectively for measuring the contents of BMP-2 and RUNX-2 in bone tissues by using Western blot assay and detecting the location of BMP-2 and RUNX-2 by using immunohistochemical method.Results:All rats were fully waked up from 3-4-hour anesthesia for the modeling surgery and fed normally,and their incisions healed completely 1 week later.One rat in model group and 2 rats in BSHTF group died during the drug intervention.The detection results of serum contents of BMP-2 and RUNX-2 showed that there was statistical difference in serum contents of BMP-2 between the 3 groups(245.40+/-10.10,111.03+/-29.75,190.35+/-14.00 pg/mL,F=109.998,P=0.000).The serum contents of BMP-2 were lower in model group and BSHTF group compared to sham-operated group,and were higher in BSHTF group compared to model group(P=0.000,P=0.000; P=0.000).There was statistical difference in serum contents of RUNX-2 between the 3 groups(4 131.04+/-277.39,1 601.09+/-266.10,3 134.90+/-202.33 pg/mL,F=237.192,P=0.000).The serum contents of RUNX-2 were lower in model group and BSHTF group compared to sham-operated group,and were higher in BSHTF group compared to model group(P=0.000,P=0.000; P=0.000).The results of Micro-CT scanning on femurs showed that there was statistical difference in the BMD between the 3 groups(556.90+/-8.86,498.81+/-9.26,530.17+/-10.70 mg HA/cm(-3),F=90.488,P=0.000).The BMD was lower in model group and BSHTF group compared to sham-operated group,and was higher in BSHTF group compared to model group(P=0.000,P=0.000; P=0.000).There was statistical difference in Tb.N between the 3 groups(2.65+/-0.32,1.74+/-0.22,2.41+/-0.33 /mm,F=23.900,P=0.000).The Tb.N was less in model group compared to BSHTF group and sham-operated group(P=0.000,P=0.000),and there was no statistical difference in Tb.N between BSHTF group and sham-operated group(P=0.224).There was statistical difference in mean Tb.Th between the 3 groups(109.33+/-16.12,79.27+/-18.17,95.73+/-11.38 μm,F=8.730,P=0.001).The mean Tb.N was less in model group compared to sham-operated group(P=0.001),and there was no statistical difference in mean Tb.N between BSHTF group and sham-operated group and between BSHTF group and model group(P=0.181,P=0.098).There was statistical difference in Tb.Sp between the 3 groups(350.95+/-68.46,472.33+/-33.70,406.68+/-47.77 μm,F=12.460,P=0.000).The Tb.Sp was higher in model group and BSHTF group compared to sham-operated group,and was lower in BSHTF group compared to model group(P=0.000,P=0.036; P=0.017).The detection results of contents of BMP-2 and RUNX-2 protein in femoral tissues showed that there was statistical difference in content of BMP-2 in femoral tissues between the 3 groups(0.73+/-0.03,0.26+/-0.02,0.48+/-0.02,F=738.111,P=0.000).The contents of BMP-2 in femoral tissues were lower in model group and BSHTF group compared to sham-operated group,and were higher in BSHTF group compared to model group(P=0.000,P=0.000; P=0.000).There was statistical difference in content of RUNX-2 in femoral tissues between the 3 groups(0.67+/-0.03,0.36+/-0.03,0.47+/-0.02,F=286.493,P=0.000).The contents of RUNX-2 in femoral tissues were lower in model group and BSHTF group compared to sham-operated group,and were higher in BSHTF group compared to model group(P=0.000,P=0.000; P=0.000).The detection results of location of BMP-2 and RUNX-2 protein in femoral tissues showed that the positive expressions of BMP-2 and RUNX-2 were mainly found in cytoplasms of bone marrow stromal cells and osteoblasts around bone trabecula,and the positive expressed protein presented with brown-yellow granules under the optical microscope.Conclusion:BSHTF can promote the bone formation of ovariectomized rats through regulating BMP-2/Smads/RUNX-2 signal transduction pathway.

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备注/Memo

备注/Memo:
基金项目:湖北省卫生厅中医药中西医结合科研项目(2012Z-Y31); 湖北中医药大学“青苗计划”资助项目(2016ZZX016)
通讯作者:向楠 E-mail:xiangnan61@sina.com
更新日期/Last Update: 2019-02-25