摘要
目的
探讨具有温补肾阳作用的单味中药-沙苑子、狗脊、肉苁蓉和韭菜子对去卵巢所致大鼠骨质疏松症的治疗作用,并从破骨细胞分化调控因子、与骨代谢密切相关的激素和细胞因子等角度,揭示它们治疗的作用机理以及作用特点,为中医药防治骨质疏松症提供进一步实验依据。
方法
选用雌性Wistar大鼠94只,随机分为3组：空白对照组12只,假手术组12只,造模组70只。在术后1个月,将造模组大鼠随机分为6组,即模型组、阳性对照组、沙苑子组和狗脊组,每组各12只,肉苁蓉组和韭菜子组,每组各11只。然后,开始灌胃给药：各中药组给予浓度为1g生药／mL的水煎剂,给药体积为5.6mL/kg体重,即给药剂量均为5.6 g生药／kg体重；阳性对照组给予0.008 mg/mL的己烯雌酚,给药体积为5.6 mL/kg体重,即给药剂量为0.0448mg/kg体重。以上给药1d 1次,连续6 d,休息1 d后,如此给药3个月。空白对照组、假手术组、模型组按同法灌服等体积的蒸馏水。
各组大鼠于处死前15 d和前3 d分别腹腔注射盐酸四环素30 mg/kg体重,对骨进行荧光标记。给药结束后,将大鼠以45mg/kg剂量的戊巴比妥钠腹腔注射麻醉,股动脉取血,用于检测外周血清中IL-1、IL-6、BGP、CT、PTH的含量。然后,将各组大鼠处死,取右侧胫骨制作不脱钙骨切片,用于骨组织形态计量学指标的检测；取左侧胫骨制作冰冻切片,用于成骨细胞和骨髓基质细胞OPG和RANKL蛋白及其mRNA表达的检测。
统计学处理方法：实验结果皆以均数±标准差(x±s)表示,采用单因素方差分析(ANOVA)进行处理,组间两两比较,采用q检验。
结果
1沙苑子、狗脊、肉苁蓉和韭菜子对去卵巢大鼠胫骨骨组织形态计量学指标的影响
1.1沙苑子、狗脊、肉苁蓉和韭菜子对去卵巢大鼠胫骨TBV%的影响
模型组大鼠胫骨TBV%较假手术组明显降低。与模型组比较,沙苑子组、狗脊组和阳性对照组的大鼠胫骨TBV%显著增加：而肉苁蓉组和韭菜子组则无显著性差异。狗脊组、肉苁蓉组和韭菜子组大鼠胫骨TBV%明显低于假手术组；沙苑子组、阳性对照组较假手术组则无显著性差异。见表1。注：与假手术组比较：*P<0.05,**P<0.01：与模型组比较：△P<0.05,△△P<0.01。下同。
1.2沙苑子、狗脊、肉苁蓉和韭菜子对去卵巢大鼠胫骨TRS%的影响
与假手术组相比,模型组大鼠胫骨TRS%明显增高。沙苑子组、狗脊组和阳性对照组较模型组大鼠胫骨TRS%明显降低；肉苁蓉组和韭菜子组较模型组无显著性变化。狗脊组、肉苁蓉组和韭菜子组大鼠胫骨TRS%较假手术组显著增高；沙苑子组和阳性对照组较假手术组则无显著性差异。见表2。
1.3沙苑子、狗脊、肉苁蓉和韭菜子对去卵巢大鼠胫骨TFS%和MAR的影响
模型组大鼠胫骨TFS%和MAR均明显高于假手术组。与模型组相比,沙苑子组和阳性对照组大鼠胫骨TFS%和MAR显著降低；狗脊组、肉苁蓉组和韭菜子组无显著性差异。与假手术组相比,沙苑子组、狗脊组、肉苁蓉组和韭菜子组大鼠胫骨TFS%均显著增高；阳性对照组无明显差异。狗脊组、肉苁蓉组和韭菜子组大鼠胫骨MAR高于假手术组；沙苑子组和阳性对照组则无明显差异。见表3。
1.4沙苑子、狗脊、肉苁蓉和韭菜子对去卵巢大鼠胫骨mAR和OSW的影响
与假手术组相比,模型组大鼠胫骨mAR显著升高。与模型组相比,沙苑子：组和阳性对照组大鼠胫骨mAR明显降低：狗脊组、肉苁蓉组和韭菜子组无明显差异。狗脊组、肉苁蓉组和韭菜子组大鼠胫骨mAR明显高于假手术组；沙苑子组和阳性对照组较假手术组无显著性差异。
模型组大鼠胫骨OSW较假手术组明显升高。沙苑子组、狗脊组、肉苁蓉组和韭菜了组大鼠胫骨OSW较模型组和假手术组均无显著性差异。阳性对照组大鼠胫骨OSW低于模型组,与假手术组无显著性差异。见表4。
2沙苑子、狗脊、肉苁蓉和韭菜子对去卵巢大鼠胫骨成骨细胞和骨髓基质细胞oPG和RANKL蛋白及其mRNA表达的影响
2.1沙苑子、狗脊、肉苁蓉和韭菜子对去卵巢大鼠胫骨成骨细胞和骨髓基质细胞OPG和RANKL蛋白表达的影响
2.1.1沙苑子、狗脊、肉苁蓉和韭菜了对去卵巢大鼠胫骨成骨细胞和骨髓基质细胞OPG蛋白表达的影响
与假手术组相比,模型组大鼠胫骨成骨细胞和骨髓基质细胞OPG蛋白表达均明显下调。与模型组相比,沙苑子组、狗脊组和阳性对照组大鼠胫骨成骨细胞和骨髓基质细胞OPG蛋白表达明显上调；肉苁蓉组和韭菜了组无显著差异。狗脊组、肉苁蓉组和韭菜了组较假手术组明显下调；沙苑子组和阳性对照组与假手术组相比,无明显差异。见表5。
2.1.2沙苑了、狗脊、肉苁蓉和韭菜子对去卵巢大鼠胫骨成骨细胞和骨髓基质细胞RANKL蛋白表达的影响
模型组大鼠胫骨成骨细胞和骨髓基质细胞RANKL蛋白表达明显高于假手术组。沙苑子组、阳性对照组大鼠胫骨成骨细胞和骨髓基质细胞RANKL蛋白表达较模型组显著下调；狗脊组、肉苁蓉组和韭菜子组无显著性变化。与假手术组相比,狗脊组、肉苁蓉组和韭菜子组大鼠胫骨成骨细胞RANKL蛋白表达上调；沙苑了组、狗脊组、肉苁蓉组和韭菜子组大鼠胫骨骨髓基质细胞RANKL蛋白表达上调；沙苑子组大鼠胫骨成骨细胞RANKL蛋白表达较假手术组无显著性差异。阳性对照组大鼠胫骨成骨细胞和骨髓基质细胞RANKL蛋白表达与假手术相比,均无明显差异。见表6。
2.2沙苑子、狗脊、肉苁蓉和韭菜子对去卵巢大鼠胫骨成骨细胞和骨髓基质细胞oPG和RANKL mRNA表达的影响
2.2.1沙苑子、狗脊、肉苁蓉和韭菜子对去卵巢大鼠胫骨成骨细胞和骨髓基质细胞OPG mRNA表达的影响
与假手术组相比,模型组大鼠胫骨成骨细胞和骨髓基质细胞OPG mRNA表达均明显下调。与模型组相比,沙苑子组、狗脊组和阳性对照组大鼠胫骨成骨细胞和骨髓基质细胞OPG mRNA表达明显上调；肉苁蓉组和韭菜了组无显著差异。狗脊组、肉苁蓉组和韭菜子组较假手术组明显下调；沙苑子组和阳性对照组与假手术组相比,无明显差异。见表7。
2.2.2沙苑子、狗脊、肉苁蓉和韭菜子对去卵巢大鼠胫骨成骨细胞和骨髓基质细胞RANKL mRNA表达的影响
模型组大鼠胫骨成骨细胞和骨髓基质细胞RANKL mRNA表达明显高于假手术组。沙苑了组以及阳性对照组大鼠胫骨成骨细胞和骨髓基质细胞RANKL mRNA较模型组显著下调；狗脊组、肉苁蓉组和韭菜子组无显著性变化。与假手术组相比,狗脊组、肉苁蓉组和韭菜子组大鼠胫骨成骨细胞和骨髓基质细胞RANKL mRNA表达上调;沙苑子组、阳性对照组无明显差异。见表8。
3沙苑子、狗脊、肉苁蓉和韭菜子对去卵巢大鼠外周血清中IL-1、IL-6含量的影响
与假手术组相比,模型组大鼠外周血清中IL-1含量显著增高。与模型组相比,沙苑子组及阳性对照组大鼠外周血清中IL-1含量明显降低;其它各给药组则无显著性变化。沙苑子组、狗脊组、肉苁蓉组以及韭菜子组大鼠外周血清中1L-1含量较假手术组均显著升高；阳性对照组无明显差异。
模型组大鼠外周血清中IL-6含量高于假手术组。与模型组相比,沙苑子组、狗脊组及阳性对照组大鼠外周血清中IL-6含量较模型组明显降低；肉苁蓉组和韭菜子组则无显著性差异。与假手术组相比,狗脊组、肉苁蓉组以及韭菜子组大鼠外周血清中IL-6含量明显增高；沙苑了组和阳性对照组无明显差异。见表9。
4沙苑子、狗脊、肉苁蓉和韭菜子对去卵巢大鼠外周血清中BGP、CT和PTH含量的影响
模型组大鼠外周血清中BGP含量较假手术组明显增高。沙苑子组和阳性对照组大鼠外周血清中BGP含量显著低于模型组；其余各给药组较模型组无显著性的变化。沙苑子组、狗脊组、肉苁蓉组以及韭菜子组较假手术组均显著升高；阳性对照组无明显差异。
模型组大鼠外周血清中CT含量较假手术组明显降低。与模型组相比,沙苑子组及阳性对照组CT含量明显增加；其余各组药组则无显著性变化。与假手术组相比,狗脊组、肉苁蓉组以及韭菜子组大鼠外周血清中CT含量显著降低；沙苑了组和阳性对照组无明显差异。
与假手术组相比,模型组及各给药组大鼠外周血清中PTH含量均无显著性变化；各给药组大鼠外周血清中PTH含量较模型组亦无显著性变化。见表10。
假手术组与空白对照组相比,以上各项指标均未发生显著性变化,从而排除了手术本身对实验的影响。
结论
沙苑子和狗脊能在一定程度上使去卵巢所致的大鼠高转换型骨质疏松症恢复到一种骨形成大于骨吸收的相对平衡的状态,从而防止骨量的丢失,达到治疗作用；而肉苁蓉、韭菜子则无明显的治疗作用。沙苑子和狗脊的具体作用环节和作用特点如下：
(1)沙苑子不仅能提高成骨细胞和骨髓基质细胞OPG的蛋白及其mRNA表达,抑制RANKL的蛋白及其mRNA表达,而且还能降低外周血中IL-1、IL-6的水平,并且提高CT的水平,从而达到对骨质疏松症的治疗作用；
(2)狗脊能提高成骨细胞和骨髓基质细胞OPG的蛋白及其mRNA表达,使之与RANKL结合增多,进而抑制破骨细胞的分化、成熟及活性,并且对IL-6的也有一定地抑制作用,从而防止骨的过度吸收。
Objective
This study is to estimate the therapeutic effect of kidney yang-supplementing Chinese medicines, including Semen Astragali Complanati decoction (SACD), Rhizoma Cibotii decoction (RCD), Herba Cistanches decoction (HCD), and Semen Allii Tuberosi decoction (SATD), on ovariectomized (OVX) rats. The effective mechanism of the four Chinese medicine on OVX rats is revealed by focusing on the osteoclast differentiation and regulatory factors and hormones and cytokines related to bone metabolism closely, which provide further experimental evidence for osteoporosis and the traditional Chinese medicine theory of" The kidney dominating bone"
Material and Methods
Ninety-four virgin Wistar clean grade female rats (weight 250±20.0g) were randomly divided into three groups, including a normal control group (n=12), a sham control group (n=12), and a bilaterally ovariectomized (OVX, n=70) group. OVX rats were randomly divided into six groups, including OVX group (n=12), positive control group (n=12), SACD group (n=12), RCD group (n=12), HCD group (n=11), and SATD group (n=11). Diethylstilbestrol 0.008mg/mL dissolved in distilled water was administered intragastrically to the rats in the positive control group. The rats of the SACD group, the RCD group, the HCD group, and the SATD group were treated with SACD 5.6mL/(kg body weight day), RCD 5.6mL/(kg body weight day), HCD 5.6mL/(kg body weight day), and SATD 5.6mL/(kg body weight day), respectively. At the same time, the rats of the normal control group, the sham control group, and the OVX group were administered with the same volume of distilled water. The treatment started four weeks after the surgery and continued for 12 weeks.
On the fifteenth and third days before sacrifice all the rats received tetracyclin 30mg/kg by intraperitoneal injection in order to mark the bone with fluorescence. Then all of them were taken blood for testing the serum contents of IL-1,1L-6, BGP, CT, and PTH by means of enzyme linked immunosorbent. After sacrifice, we estimated the proximal right tibiae by measuring TBV%, TRS%, TFS%, MAR, mAR, and OSW of the undecalcified tibia slides. Proximal left tibiae were sliced into 5μm sections by freezing microtome. Frozen sections were prepared for immunohistochemistry and in situ hybridization.
All values were expressed as means±standard(x±s) deviations. The difference between the groups regarding the evaluated parameters was tested using the ANOVA test followed by the q test.
Results
1. In contrast to the OVX group, TBV% in both SACD and RCD groups increased significantly, while TRS%, TFS%, MAR, and mAR decreased significantly in the SACD group, and only TRS% decreased significantly in the RCD group. No changes of these parameters above had been observed in either HCD or SATD group.
2. Compared with the OVX group, the protein expression of OPG in OB and MSC of the SACD group and the RCD group significantly increased, but that of the HCD group and the SATD group was not significantly different compared to the OVX group. The protein expression of OPG in the RCD group, the HCD group, and the SATD group was below the sham control group, but there were no significant changes in the SACD group.
The protein expression of RANKL in the SACD group was significantly lower than that of the OVX group. However, there were no significant changes in the RCD group, the HCD group, the SATD group, and the OVX group. Compared with the sham control group, the protein expression of RANKL in OB of the RCD group, the HCD group, and the SATD group significantly increased, and the protein expression of RANKL in MSC of the SACD group, the RCD group, the HCD group, and the SATD group also significantly increased, but there was no significant difference in the protein expression of RANKL in OB of the SACD group.
3. Compared with the OVX group, OPG mRNA expression in OB and MSC of the SACD group and the RCD group significantly increased, but that of the HCD group and the SATD group was not significantly different compared to the OVX group. OPG mRNA expression in OB and MSC of the RCD group, the HCD group, and the SATD group was below the sham control group, but there were no significant changes in the SACD group.
RANKL mRNA expression in OB and MSC of the SACD group was significantly lower than that of the OVX group. However, there were no significant changes in the RCD group, the HCD group, the SATD group, and the OVX group. Compared with the sham control group, RANKL mRNA expression in OB and MSC of the RCD group, the HCD group, and the SATD group was significantly increased. There were no significant changes in the SACD group and sham control group.
4. In contrast to the OVX group, the level of IL-1 in the peripheral blood of the SACD group decreased significantly, but that of RCD group, the HCD group, and the SATD group was not significantly different compared to the OVX group. However, IL-1 in the SACD group, the RCD group, the HCD group, and the SATD group was higher than that in the sham control group.
The level of IL-6 in the SACD group, and the RCD group was significantly lower than that of the OVX group. However, there were no significant changes in the HCD group, the SATD group, and the OVX group. Compared with the sham control group, IL-6 in the RCD group, the HCD group, and the SATD group was significantly increased. There were no significant changes in the SACD group and sham control group.
5. The level of BGP in the peripheral blood of the SACD group was below the OVX group, but there were no significant changes in the RCD group, the HCD group, and the SATD group. However, BGP of the SACD group, the RCD group, the HCD group, and the SATD group was higher than that in the sham control group.
Compared with the OVX group, CT of the SACD group significantly increased, but that of the RCD group, the HCD group and the SATD group was not significantly different compared to the OVX group. CT of the RCD group, the HCD group, and the SATD group was below the sham control group, but there were no significant changes in the SACD group.
No changes of PTH had been observed in the SACD group, the RCD group, the HCD group, the SATD group, the OVX group, or the sham control group.
Conclusions
The effective mechanism of the four kidney yang-supplementing Chinese medicines on OVX rats is revealed by focusing on the osteoclast differentiation and regulatory factors and hormones and cytokines related to bone metabolism closely. We found that the OVX rat was a high-transformational model of osteoporosis, and SACD and RCD enhanced overall bone quality of OVX rats significantly
Our experiments implied that the protein expression of OPG in OB and MSC was promoted by SACD, and the protein expression of RANKL in OB and MSC was inhibited by SACD. At the same time, SACD could reduce the level of IL-1, IL-6 and enhance the level of CT in the peripheral blood. We also found that the protein expression of OPG in OB and MSC was promoted by RCD, and the level of IL-6 was inhibited by RCD. However, no change of RANKL had been observed in the RCD group.
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