丹参酮ⅡA对去卵巢大鼠骨密度及骨形态计量学的影响

Effect of tanshinone ⅡA on bone mineral density and bone histomorphometry in ovariectomized rat

目的研究丹参酮ⅡA对去卵巢大鼠骨密度(bone mineral density,BMD)和骨形态计量学的影响,探讨丹参酮ⅡA对雌激素缺乏引起的骨质疏松症(osteoporosis,OP)的治疗作用。方法 3月龄SD雌性大鼠40只,采用数字表法随机分为假手术组(Sham)、单纯卵巢切除组(Ovx)、雌激素治疗组和丹参酮ⅡA治疗组,每组10只。手术后1周,雌激素组给予炔雌醇200μg/kg;丹参酮ⅡA治疗组给予丹参酮ⅡA 11mg/kg;Ovx组与Sham组每日给予与治疗组同等体积的0.9%氯化钠注射液,给药方式均为口服,1次/d。每2周称1次体质量,每个月检测全身BMD,3个月后处死所有实验动物并取材。测定椎骨和右侧股骨的离体BMD和生物力学性能;品红-苦味酸染色(Van-Gieson,VG)进行骨形态分析;大鼠处死前按时间依次皮下注射四环素、茜素红和钙黄绿素,镜下观察各组3种荧光之间的距离变化;计算子宫系数,并在光镜显微镜下观察子宫病理切片中各组子宫内膜厚度的变化。结果 Ovx组大鼠体质量(282.38±16.92)明显增加,给予丹参酮ⅡA治疗的大鼠体质量增长与Sham组差异无统计学意义(P>0.05),雌激素组大鼠体质量(248.33±15.57)明显低于其他各组,差异有统计学意义(P<0.01);丹参酮ⅡA组大鼠的子宫系数(0.925±0.553)与Ovx组(0.900±0.389)比较略高,差异无统计学意义(P>0.05),但明显低于雌激素组(2.386±0.495),差异有统计学意义(P<0.01);与Ovx组相比,丹参酮ⅡA组的全身BMD(0.149±0.008)、股骨三点弯曲试验和椎骨压缩试验的最大载荷(159.86±12.37、267.58±33.4)均有所提高,差异有统计学意义(P<0.05),而丹参酮ⅡA组和雌激素组组间差异无统计学意义(P>0.05);丹参酮ⅡA组的弹性模量与Ovx组之间差异无统计学意义(P>0.05),但丹参酮ⅡA组均值高于Ovx组;骨形态计量分析结果显示丹参酮ⅡA组和雌激素组较Ovx组骨小梁明显变多变密,改善了松质骨的微结构;荧光标记观察表明口服丹参酮ⅡA和炔雌醇可加快骨的生长;子宫病理切片观察到Ovx组子宫内膜组织萎缩变薄,丹参酮ⅡA组与假手术组相差不大,雌激素组子宫内膜明显增厚,与子宫系数呈现相同的趋势。结论丹参酮ⅡA可通过增加BMD,提高骨质量,改善骨微结构预防与治疗雌激素缺乏引起的OP,而刺激子宫组织增生的不良反应又远比雌激素小,对探索抗OP中药单体新药有非常重要的意义。

Objective To study effects of tanshinone Ⅱ A on bone mineral density and bone histomorphometry in ovariectomized rat, and to explore the therapeutic effect of tanshinone Ⅱ A on osteoporosis induced by estrogen deficiency.Methods Forty female SD rats only 3 months old, randomly divided into control group （Sham）, ovariotomized group （Ovx）, estrogen therapy group and tanshinone ⅡA treatment group, 10 in each group. 1 week after the operation, estrogen group were administered as estrogen, 200 Ixg/kg; Tanshinone IlA treatment group were given tanshinone ⅡA, 11 mg/kg; The Ovx group and sham group were given the same volume of saline to the treatment group daily, orally once. Rats were weighed once every 2 weeks, whole body bone density was detected monthly, All experiment animals were executed after 3 months and got a sample. The bone density and biomechanical properties of the vertebrae and the right femur were determined. The bone morphological analysis was carried out by the dyeing of magenta-bitter acid （ Van-Gieson, VG）. Before the rats were executed, they were injected tetracycline, alizarin red and calculitin subcutaneously, and observed the changes of distance between the three fluorescence of each group under the microscope. The uterine coefficient was calculated and the changes of endometrial thickness were observed under the microscope. Results The body quality of Ovx group （282. 38±16. 92） was obviously increased, there was no significant difference between the tanshinone HA group and sham group; estrogen group （248. 33± 15.57） was obviously lower than other groups, and has a very significant difference （P〈0. 01）. The rat uterus coefficient （0. 925±0. 553） of tanshinone IlA group is slightly higher than that of Ovx group （0. 900±0. 389）, but without significant difference （P〉0. 05）, which was significantly lower than the estrogen group （2. 386±0. 495）, the difference was statistically significant （P〈0. 01） ; Compared with the Ovx group, whole body BMD （0. 149±0. 008）, the maximum load of three point bending test and compression test of vertebrae （ 159.86±12. 37, 267. 58±33.40） of tanshinone HA group were improved, the difference was significantly （P〈0. 05）, while there was no significant difference between tanshinone ⅡA group and estrogen group; the modulus of elasticity has no significant difference between tanshinone ⅡA group and Ovx group （P〉0. 05）, but the average of tanshinone ⅡA group is higher than that of Ovx group; Bone morphometric analysis showed that the bone trabeculas of tanshinone ⅡA group and estrogen group were more variable density than that of Ovx group, suggested that tanshinone ⅡA could improve bone microstructure ; Fluorescent tags observation showed that oral tanshinone IIA and ethinylestradiol could accelerate the growth of bone; Endometrial tissue atrophy and thinning were ob- served in Ovx group, the difference between tanshinone ⅡA group and sham group was not significant, and endometrium thickening was observed in estrogen group, and those trend was the same as that in uterine coefficient. Conclusion Tanshinone ⅡA could prevent and treat the osteoporosis caused by lack of estrogen by increasing bone mineral density, bone quality and improve bone microstructure, however, the adverse reaction of stimulating uterine hyperplasia is much smaller than that of estrogen, which is of great significance in exploring new anti-osteoporosis traditional Chinese medicine monomer.