丹参、川芎提取物对高脂喂养去卵巢大鼠血脂和骨的作用及机制研究
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摘要
研究目的与意义
心血管疾病（Cardiovascular diseases，CVD）和骨质疏松症（Osteoporosis，OP）都是常见的慢性病，在老年人中发病率和死亡率非常高。越来越多的证据显示这两种疾病之间存在着很多病理生理的联系。绝经后妇女性激素（主要指雌激素和孕激素）缺乏，容易并发心血管疾病和骨质疏松症，因此常常采用以雌/孕激素为主的激素替代疗法。这种疗法对两种疾病确实表现出一定的治疗效果，但是其副作用比较大。本课题从副作用小的活血化瘀类中药入手，分别选取具有植物雌激素样的丹参和植物孕激素样的川芎为研究对象，采用高脂饮食喂养的去卵巢大鼠模拟绝经后妇女并发心血管疾病和骨质疏松症模型，首次观察两药分别对CVD和OP相关组织和器官的影响情况，旨在两病并行治疗，既可以减少病人的痛苦，又可以减轻病人和社会的经济负担，同时可以为揭示心血管疾病和骨质疏松症的内在联系提供一定的理论依据。
研究方法与结果
（1）丹参和川芎分别采用传统的水提取和80%乙醇提取的方法提取，其得率分别为13.04%和17.08%；而后HPLC方法分析显示丹参水提物中含有13.93±0.10%的丹酚酸B和0.20±0.03%的丹参素，川芎醇提物中含4.76±0.25%的阿魏酸和3.00±0.16%的川芎嗪。
（2）49只6月龄SD大鼠按体重随机分成7个组：假手术（Sham）+低脂饮食（low fat control diet, LFD）+Vehicle组、Sham+高脂喂食（high fat diet, HFD）+Vehicle组、去卵巢（OVX）+LFD+Vehicle组、OVX+HFD+Vehicle组、OVX+HFD+17β-雌二醇（17β-estradiol，E2，1mg/kg/d, PO）组、OVX+HFD+丹参水提物（600mg/kg/d, PO）组、OVX+HFD+川芎醇提物（600mg/kg/d, PO）组，每组7只，分别采用不同饮食和药物治疗12周。结果显示，HFD和OVX能明显增加大鼠的体重，降低肝脏、肾脏及脾脏的指数；E2、丹参水提物和川芎醇提物分别治疗高脂喂养的去卵巢大鼠12周后，能明显降低大鼠的体重，增加肝脏、肾脏和脾脏的指数。
（3）心血管疾病的相关指标主要检测血脂水平、肝脏的病理学变化、肝脏的脂肪含量、抗氧化活性、血管的活性。结果显示，HFD和OVX能明显诱导血脂水平异常、肝脏脂肪变性、抗氧化活性降低、血管内皮损伤。丹参水提物和川芎醇提物都能像E2一样，既能降低高脂饮食喂养的去卵巢大鼠血清TG、TC和LDL-c水平，降低血清ALT和AST活性，减少肝脏的脂肪含量，减少肝脏氧化分子MDA水平，增加抗氧化酶SOD、CAT和GSH-Px活性，上调肝脏LDLR和CYP7A1的mRNA和蛋白表达水平，下调肝脏HMGCR水平；还能增加血管对Ach诱导的内皮依赖的血管收缩活力，增加eNOS诱导的NO的产生，下调血管炎症分子TNF-α、ICAM-1和VCAM-1的mRNA水平。
（4）骨质疏松症的相关指标主要检测血清和尿液Ca和P的浓度变化，micro-CT分析松质骨（胫骨近端和第二腰椎）和皮质骨（胫骨中段和股骨中段）骨密度和骨显微结构的变化。结果显示HFD和OVX能明显诱导尿钙的渗出增加，诱导松质骨BMD降低，加剧骨显微结构改变；尽管E2、丹参水提物和川芎醇提物都能降低高脂饮食喂养的去卵巢大鼠尿Ca的渗出，增加松质骨BMD和Tb.N，降低Tb. Sp、DA和SM，诱导骨小梁由杆状向板状转变，但是它们对不同的松质骨影响不同，丹参水提物主要是影响胫骨近端的骨显微结构，川芎醇提物影响的主要是第二腰椎，而E2对两种松质骨都有明显的影响。
（5）MTS法和碱性磷酸酶活性检测法进一步检测两种提取物对大鼠类成骨细胞UMR106增殖和分化的影响，结果首次发现丹参水提物只能促进UMR106细胞分化，而不影响细胞增殖；川芎醇提物既能刺激细胞分化，又能促进细胞增殖。
结论
（1）HFD和OVX能增加大鼠体重，改变血脂水平，损伤血管和肝脏，诱导尿钙渗出、降低松质骨骨密度、改变骨显微结构，表明模拟绝经后妇女CVD和OP并发的模型建立成功。
（2）丹参水提物和川芎醇提物首次在高脂饮食喂养的去卵巢大鼠中既表现出抗CVD的效果，又表现出抗OP的作用，达到了两病同治的目的。
（3）尽管体内外实验首次证实丹参水提物和川芎醇提物对不同松质骨和类成骨细胞表现出不完全相同的影响，但它们都能促进成骨细胞分化，防止骨丢失，可能成为预防和治疗绝经后妇女骨质疏松症的新型候选药物。
OBJECTIVE AND SIGNIFICANCE
Cardiovascular diseases (CVD) and osteoporosis (OP) are common chronic diseases andaccount for significant morbidity and mortality in the aging population. Accumulatingevidence indicates a pathophysiological link between the two diseases. Due to deficiency offemale hormones (progesterone and estrogen), they frequently coexist in postmenopausalwomen. Postmenopausal women commonly take hormone replacement therapy with estrogenand/or progesterone. Such therapy is really effective in preventing CVD and OP inpostmenopausal women, but it was proven to have various adverse effects. Danshen andchuanxiong are well-known Traditional Chinese Medicinal herbs widely used for thetreatment of CVD, and their major active ingredients have been found to have estrogenic andprogestogenic effects respectively. The aim of this study was to evaluate the effect ofDanshen and Chuanxiong on cardiovascular system and bone system in an ovariectomized(OVX) rat model fed with high fat diet (HFD) for the first time. The study may not only helppostmenopausal women to relieve suffering and save financial burden, but also pave the wayto elucidate the underlying relationship between CVD and OP.
METHODS AND RESULTS
(1) Danshen herb and Chuanxiong herb were respectively ground into powder andextracted with distilled water at100°C or80%ethanol, and the yield was13.03%and17.08%. The results of HPLC analysis were shown that the Danshen aqueous extract contains3.93±0.10%salvianolic acid B and0.20±0.03%danshensu and Chuanxiong ethanolicextract contains4.76±0.25%ferulic acid and3.00±0.16%tetramethylpyrazine.
(2) Forty-nine6-month-old female Sprague-Dawley rats were randomly divided intoseven groups: sham-operated rats with low-fat control diet (LFD)+vehicle, sham-operatedrats with high-fat diet (HFD)+vehicle, OVX rats with LFD+vehicle, OVX rats with HFD+vehicle, OVX rats with HFD+17β-estradiol (1mg/kg/d, PO), and OVX rats with HFD+Danshen aqueous extract (600mg/kg/d, PO), and OVX rats with HFD+Chuanxiongethanolic extract (600mg/kg/d, PO). Rats were orally administered with vehicle, E2or herbextracts and fed with HFD or LFD respectively for12weeks. Results showed that OVX ratsfed with HFD for12weeks had highest body weight gain and lowest liver index, kidneyindex and spleen index. HFD-fed OVX rats treated with E2, Danshen aqueous extract or Chuanxiong ethanolic extract for12weeks were shown to decrease body weight and increaseliver index, kidney index and spleen index.
(3) Lipid profile levels in serum, histological examination of livers, lipid content of liver,oxidative/antioxidative activity in liver, vascular activity were detected to evaluate theprotection effect of Danshen aqueous extract or Chuanxiong ethanolic extract oncardiovascular system in HFD-fed OVX rats. The results were shown that abnormal lipidprofiles, fatty liver and impaired vascular endothelium were induced in HFD-fed OVX rats.Comsumption of Danshen aqueous extract or Chuanxiong ethanolic extract improved serumlipid profiles (lower triacylglycerols, total cholesterol and low density lipoprotein cholesterol),reduced ALT and AST activity, prevented formation of fatty liver induce by HFD and OVX,primarily via up-regulating the mRNA and protein expression of LDLR and CYP7A1anddown-regulating the mRNA and protein expression of HMGCR. In addition, they couldincrease endothelial-dependent vasorelaxation and displayed vasoprotection in OVX rats fedwith HFD, primarily by decreasing hepatic MDA levels, increasing hepatic antioxidantenzymes (SOD, CAT, GSH-Px) activities, stimulating nitric oxide (NO) production,up-regulating the mRNA expression of endothelial NO synthase, and down-regulating themRNA expression of tumor necrosis factor alpha, intercellular cell adhesion molecule-1, andvascular cell adhesion molecule-1in the isolated aortas as well as E2.
(4) Ca and P levels in serum and urine and bone properties in cancellous bone (proximaltibia and lumbar vertebra2) and cortical bone (midshaft tibia and midshaft femur) bymicro-CT were detected to evaluate the protection effect of Danshen aqueous extract orChuanxiong ethanolic extract on bone system in HFD-fed OVX rats. The results were shownthat HFD and OVX could induce rats to aggravate urinary Ca excretion, decrease bonemineral density (BMD) and change the microstructure of cancellous bone. Treatment withDanshen aqueous extract, Chuanxiong ethanolic extract or E2in HFD-fed OVX rats couldsuppress urinary Ca excretion, increase BMD, trabecular number and bone surface density,decrease trabecular separation, specific bone surface and structure model index and changetrabeculae to parallel plates instead of cylindrical rods, but cancellous bones they mainlyaffected were different. Danshen aqueous extract mainly affected proximal tibia, andChuanxiong ethanolic extract mainly affected lumbar vertebra2, and E2could affect both ofthem.
(5) MTS and alkaline phosphatase (ALP) were measured to evaluate the in vitro effect ofDanshen aqueous extract or Chuanxiong ethanolic extract on proliferation and differentiation of rat osteoblast-like UMR106cell. The results were shown that Danshen aqueous extractcould only stimulate differentiation in UMR106cells, but not affect cell proliferation. AndChuanxiong ethanolic extract exhibited to promote differentiation potential as well asproliferative activity in UMR106cells.
CONCLUSSION
(1) It was exhibited that heavy body weight, abnormal lipid profiles, impaired liver andvascular function, aggravated urinary Ca excretion, decreased BMD and changed themicrostructure of cancellous bone in HFD-fed OVX rats, indicated a successful rat modelmimicking CVD and OP in postmenopausal women.
(2) Treatment with Danshen aqueous extract or Chuanxiong ethanolic extract in HFD-fedOVX rats not only prevented CVD, but also displayed anti-osteoporosis for the first time.
(3) Effects of Danshen aqueous extract on cancellous bones and on UMR106cell were notexactly same with Chuanxiong ethanolic extract, but they both exhibited to promote celldifferentiation and prevent bone loss in vitro and in vivo, might be potential candidates forprevention and treatment of postmenopausal osteoporosis.
心血管疾病（Cardiovascular diseases，CVD）和骨质疏松症（Osteoporosis，OP）都是常见的慢性病，在老年人中发病率和死亡率非常高。越来越多的证据显示这两种疾病之间存在着很多病理生理的联系。绝经后妇女性激素（主要指雌激素和孕激素）缺乏，容易并发心血管疾病和骨质疏松症，因此常常采用以雌/孕激素为主的激素替代疗法。这种疗法对两种疾病确实表现出一定的治疗效果，但是其副作用比较大。本课题从副作用小的活血化瘀类中药入手，分别选取具有植物雌激素样的丹参和植物孕激素样的川芎为研究对象，采用高脂饮食喂养的去卵巢大鼠模拟绝经后妇女并发心血管疾病和骨质疏松症模型，首次观察两药分别对CVD和OP相关组织和器官的影响情况，旨在两病并行治疗，既可以减少病人的痛苦，又可以减轻病人和社会的经济负担，同时可以为揭示心血管疾病和骨质疏松症的内在联系提供一定的理论依据。
研究方法与结果
（1）丹参和川芎分别采用传统的水提取和80%乙醇提取的方法提取，其得率分别为13.04%和17.08%；而后HPLC方法分析显示丹参水提物中含有13.93±0.10%的丹酚酸B和0.20±0.03%的丹参素，川芎醇提物中含4.76±0.25%的阿魏酸和3.00±0.16%的川芎嗪。
（2）49只6月龄SD大鼠按体重随机分成7个组：假手术（Sham）+低脂饮食（low fat control diet, LFD）+Vehicle组、Sham+高脂喂食（high fat diet, HFD）+Vehicle组、去卵巢（OVX）+LFD+Vehicle组、OVX+HFD+Vehicle组、OVX+HFD+17β-雌二醇（17β-estradiol，E2，1mg/kg/d, PO）组、OVX+HFD+丹参水提物（600mg/kg/d, PO）组、OVX+HFD+川芎醇提物（600mg/kg/d, PO）组，每组7只，分别采用不同饮食和药物治疗12周。结果显示，HFD和OVX能明显增加大鼠的体重，降低肝脏、肾脏及脾脏的指数；E2、丹参水提物和川芎醇提物分别治疗高脂喂养的去卵巢大鼠12周后，能明显降低大鼠的体重，增加肝脏、肾脏和脾脏的指数。
（3）心血管疾病的相关指标主要检测血脂水平、肝脏的病理学变化、肝脏的脂肪含量、抗氧化活性、血管的活性。结果显示，HFD和OVX能明显诱导血脂水平异常、肝脏脂肪变性、抗氧化活性降低、血管内皮损伤。丹参水提物和川芎醇提物都能像E2一样，既能降低高脂饮食喂养的去卵巢大鼠血清TG、TC和LDL-c水平，降低血清ALT和AST活性，减少肝脏的脂肪含量，减少肝脏氧化分子MDA水平，增加抗氧化酶SOD、CAT和GSH-Px活性，上调肝脏LDLR和CYP7A1的mRNA和蛋白表达水平，下调肝脏HMGCR水平；还能增加血管对Ach诱导的内皮依赖的血管收缩活力，增加eNOS诱导的NO的产生，下调血管炎症分子TNF-α、ICAM-1和VCAM-1的mRNA水平。
（4）骨质疏松症的相关指标主要检测血清和尿液Ca和P的浓度变化，micro-CT分析松质骨（胫骨近端和第二腰椎）和皮质骨（胫骨中段和股骨中段）骨密度和骨显微结构的变化。结果显示HFD和OVX能明显诱导尿钙的渗出增加，诱导松质骨BMD降低，加剧骨显微结构改变；尽管E2、丹参水提物和川芎醇提物都能降低高脂饮食喂养的去卵巢大鼠尿Ca的渗出，增加松质骨BMD和Tb.N，降低Tb. Sp、DA和SM，诱导骨小梁由杆状向板状转变，但是它们对不同的松质骨影响不同，丹参水提物主要是影响胫骨近端的骨显微结构，川芎醇提物影响的主要是第二腰椎，而E2对两种松质骨都有明显的影响。
（5）MTS法和碱性磷酸酶活性检测法进一步检测两种提取物对大鼠类成骨细胞UMR106增殖和分化的影响，结果首次发现丹参水提物只能促进UMR106细胞分化，而不影响细胞增殖；川芎醇提物既能刺激细胞分化，又能促进细胞增殖。
结论
（1）HFD和OVX能增加大鼠体重，改变血脂水平，损伤血管和肝脏，诱导尿钙渗出、降低松质骨骨密度、改变骨显微结构，表明模拟绝经后妇女CVD和OP并发的模型建立成功。
（2）丹参水提物和川芎醇提物首次在高脂饮食喂养的去卵巢大鼠中既表现出抗CVD的效果，又表现出抗OP的作用，达到了两病同治的目的。
（3）尽管体内外实验首次证实丹参水提物和川芎醇提物对不同松质骨和类成骨细胞表现出不完全相同的影响，但它们都能促进成骨细胞分化，防止骨丢失，可能成为预防和治疗绝经后妇女骨质疏松症的新型候选药物。
OBJECTIVE AND SIGNIFICANCE
Cardiovascular diseases (CVD) and osteoporosis (OP) are common chronic diseases andaccount for significant morbidity and mortality in the aging population. Accumulatingevidence indicates a pathophysiological link between the two diseases. Due to deficiency offemale hormones (progesterone and estrogen), they frequently coexist in postmenopausalwomen. Postmenopausal women commonly take hormone replacement therapy with estrogenand/or progesterone. Such therapy is really effective in preventing CVD and OP inpostmenopausal women, but it was proven to have various adverse effects. Danshen andchuanxiong are well-known Traditional Chinese Medicinal herbs widely used for thetreatment of CVD, and their major active ingredients have been found to have estrogenic andprogestogenic effects respectively. The aim of this study was to evaluate the effect ofDanshen and Chuanxiong on cardiovascular system and bone system in an ovariectomized(OVX) rat model fed with high fat diet (HFD) for the first time. The study may not only helppostmenopausal women to relieve suffering and save financial burden, but also pave the wayto elucidate the underlying relationship between CVD and OP.
METHODS AND RESULTS
(1) Danshen herb and Chuanxiong herb were respectively ground into powder andextracted with distilled water at100°C or80%ethanol, and the yield was13.03%and17.08%. The results of HPLC analysis were shown that the Danshen aqueous extract contains3.93±0.10%salvianolic acid B and0.20±0.03%danshensu and Chuanxiong ethanolicextract contains4.76±0.25%ferulic acid and3.00±0.16%tetramethylpyrazine.
(2) Forty-nine6-month-old female Sprague-Dawley rats were randomly divided intoseven groups: sham-operated rats with low-fat control diet (LFD)+vehicle, sham-operatedrats with high-fat diet (HFD)+vehicle, OVX rats with LFD+vehicle, OVX rats with HFD+vehicle, OVX rats with HFD+17β-estradiol (1mg/kg/d, PO), and OVX rats with HFD+Danshen aqueous extract (600mg/kg/d, PO), and OVX rats with HFD+Chuanxiongethanolic extract (600mg/kg/d, PO). Rats were orally administered with vehicle, E2or herbextracts and fed with HFD or LFD respectively for12weeks. Results showed that OVX ratsfed with HFD for12weeks had highest body weight gain and lowest liver index, kidneyindex and spleen index. HFD-fed OVX rats treated with E2, Danshen aqueous extract or Chuanxiong ethanolic extract for12weeks were shown to decrease body weight and increaseliver index, kidney index and spleen index.
(3) Lipid profile levels in serum, histological examination of livers, lipid content of liver,oxidative/antioxidative activity in liver, vascular activity were detected to evaluate theprotection effect of Danshen aqueous extract or Chuanxiong ethanolic extract oncardiovascular system in HFD-fed OVX rats. The results were shown that abnormal lipidprofiles, fatty liver and impaired vascular endothelium were induced in HFD-fed OVX rats.Comsumption of Danshen aqueous extract or Chuanxiong ethanolic extract improved serumlipid profiles (lower triacylglycerols, total cholesterol and low density lipoprotein cholesterol),reduced ALT and AST activity, prevented formation of fatty liver induce by HFD and OVX,primarily via up-regulating the mRNA and protein expression of LDLR and CYP7A1anddown-regulating the mRNA and protein expression of HMGCR. In addition, they couldincrease endothelial-dependent vasorelaxation and displayed vasoprotection in OVX rats fedwith HFD, primarily by decreasing hepatic MDA levels, increasing hepatic antioxidantenzymes (SOD, CAT, GSH-Px) activities, stimulating nitric oxide (NO) production,up-regulating the mRNA expression of endothelial NO synthase, and down-regulating themRNA expression of tumor necrosis factor alpha, intercellular cell adhesion molecule-1, andvascular cell adhesion molecule-1in the isolated aortas as well as E2.
(4) Ca and P levels in serum and urine and bone properties in cancellous bone (proximaltibia and lumbar vertebra2) and cortical bone (midshaft tibia and midshaft femur) bymicro-CT were detected to evaluate the protection effect of Danshen aqueous extract orChuanxiong ethanolic extract on bone system in HFD-fed OVX rats. The results were shownthat HFD and OVX could induce rats to aggravate urinary Ca excretion, decrease bonemineral density (BMD) and change the microstructure of cancellous bone. Treatment withDanshen aqueous extract, Chuanxiong ethanolic extract or E2in HFD-fed OVX rats couldsuppress urinary Ca excretion, increase BMD, trabecular number and bone surface density,decrease trabecular separation, specific bone surface and structure model index and changetrabeculae to parallel plates instead of cylindrical rods, but cancellous bones they mainlyaffected were different. Danshen aqueous extract mainly affected proximal tibia, andChuanxiong ethanolic extract mainly affected lumbar vertebra2, and E2could affect both ofthem.
(5) MTS and alkaline phosphatase (ALP) were measured to evaluate the in vitro effect ofDanshen aqueous extract or Chuanxiong ethanolic extract on proliferation and differentiation of rat osteoblast-like UMR106cell. The results were shown that Danshen aqueous extractcould only stimulate differentiation in UMR106cells, but not affect cell proliferation. AndChuanxiong ethanolic extract exhibited to promote differentiation potential as well asproliferative activity in UMR106cells.
CONCLUSSION
(1) It was exhibited that heavy body weight, abnormal lipid profiles, impaired liver andvascular function, aggravated urinary Ca excretion, decreased BMD and changed themicrostructure of cancellous bone in HFD-fed OVX rats, indicated a successful rat modelmimicking CVD and OP in postmenopausal women.
(2) Treatment with Danshen aqueous extract or Chuanxiong ethanolic extract in HFD-fedOVX rats not only prevented CVD, but also displayed anti-osteoporosis for the first time.
(3) Effects of Danshen aqueous extract on cancellous bones and on UMR106cell were notexactly same with Chuanxiong ethanolic extract, but they both exhibited to promote celldifferentiation and prevent bone loss in vitro and in vivo, might be potential candidates forprevention and treatment of postmenopausal osteoporosis.
引文
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