二至丸及其组方药物防治骨质疏松与作用机制研究
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摘要
研究背景
骨质疏松症(Osteoporosis, OP)属于一种全身性骨骼系统疾病,主要表现为骨量减少、骨组织微观结构退化,最终导致骨脆性增加和骨折危险性增加。目前,全世界患有OP的病人数已经超过2亿,占总人口的3%,在全世界常见病、多发病中,OP位置已经上升到第7位。随着老龄化社会的到来,在我国乃至全球,这种与增龄有关的OP疾病将大幅上升。OP已成为严重威胁老年人健康的全世界共同关注的公共问题,这也是全球函待探索和解决的老年性、慢性疾病之一。当前,由于现有防治OP药物特性的限制,研制开发安全、长效、经济的抗OP药物仍然是医药学界的主攻目标。
由于中药及其复方含有大量不同的化学成分,可以通过多途径、多靶点、多程序发挥整体作用,因此在防治骨质疏松症方面具有较大的潜力与优势,被很多国内外研究抗骨质疏松症药物的学者视为重点开发对象。现代研究证实,中医药中所说的“肾”涉及到人体多种系统功能,例如内分泌系统、神经系统、免疫系统、代谢系统等功能,而中药及其复方在此方面又具有西药无法比拟的整体疗效。因此,在防治骨质疏松症的研究工作中,继承和发扬中医药特长,研发出显著减少西药副作用、提高疗效、标本兼治的中药及其复方,并运用现代先进科技手段探明其机理,既是符合中医药现代化的客观要求,又是推动中医药发展、服务于全人类健康的必然举措。
研究目的
二至丸最早记录于明代王三才的《医便》中,由女贞子和墨旱莲二味中药等份配伍而得,是中医临床应用的经典名方。二至丸药味精少而药性平和,补而不滞,滋而不腻,防治与OP相类似的临床骨症已有近七百年的历史。本课题在复制OVX大鼠骨质疏松模型及维甲酸所致急性OP模型的基础上,系统地研究了二至丸、女贞子、墨旱莲防治OP的药理效应；采用体外培养的大鼠原代成骨细胞为研究工具,从细胞水平上对二至丸、女贞子、墨旱莲含药血清对大鼠成骨细胞的增殖、分化及矿化整个过程中的促进作用进行研究。同时,采用分子生物学检测方法,研究二至丸、女贞子、墨旱莲对大鼠股骨组织中OPG蛋白表达、RANKL蛋白表达和OPG/RANKL蛋白表达比率的影响,从而揭示二至丸、女贞子、墨旱莲防治OP的作用机制。本研究为二至丸、女贞子、墨旱莲进一步研究开发提供理论依据,为中药及其复方防治骨质疏松症提供思路和方法学的借鉴。
研究方法
1.选取3月龄未经产雌性Sprague--Dawley(SD)大鼠共80只,适应环境3天后随机分为假手术(Sham, n=10)组和去卵巢(OVX, n=70)组。将去卵巢后的大鼠分为以下组别：模型组(OVX, n=10),戊酸雌二醇组(EV,0.4mg/kg/day, n=10),二至丸大剂量组(EZW-H,9g/kg/day, n=10),二至丸中剂量组(EZW-M,4.5g/kg/day,n=10),二至丸小剂量组(EZW-L,2.25g/kg/day, n=10),女贞子组(FLL,4.5g/kg/day, n=10),墨旱莲组(HE,4.5g/kg/day, n=10).进行卵巢摘除手术后第5周,各组开始给药,共给药26周,每周称量各组大鼠体重一次。动物处死前,收集大鼠24h尿液,麻醉动物采集血清。全部动物处死并解剖,采集大鼠子宫及重要脏器。检测脏器体质量及子宫重量和病理变化,分析长期用药对子宫和重要脏器的影响；大鼠血清和尿液中的Ca、P和ALP等骨代谢指标及肝肾功能指标采用全自动生化分析仪进行分析检测；对尿液中DPD和血清中BALP、OCN等骨转换标记性指标成分采用ELISA免疫学方法进行测定；采用三点弯曲材料力学实验和骨压碎力实验检测分析大鼠股骨生物力学性能；采用DEXA及Micro-CT对大鼠股骨、第四椎骨、胫骨的BMD及股骨干骺端骨小梁微结构特性进行检测。
2.选取3月龄未经产雌性二级Sprague--Dawley(SD)大鼠共80只,适应环境2天后,随机分为8组,每组10只。即,正常组(Sham, n=10),模型组(VA,75mg/kg/day, n=10),仙灵骨葆组(XLGB,1.5g/kg/day, n=10),二至丸大剂量组(EZW-H,9.0g/kg/day, n=10),二至丸中剂量组(EZW-M,4.5g/kg/day, n=10),二至丸小剂量组(EZW-L,2.25g/kg/day, n=10),女贞子组(FLL,4.5g/kg/day, n=10),墨旱莲组(HE,4.5g/kg/day, n=10).除正常组外,其余各组每天上午维甲酸灌胃给药1次,连续2周,制备急性骨质疏松模型。造模同时,下午各组按规定剂量灌胃给予相应药物,连续给药6周,正常组和模型组给予蒸馏水,各组动物每天称重一次。动物处死前,收集大鼠24h尿液,麻醉动物采集血清。全部动物处死并解剖,采集以下标本并进行测定分析：观察大鼠重要脏器的体质量变化,分析药物用药对脏器的影响；采用全自动生化分析仪对大鼠血清和尿液中的Ca、P和ALP等骨代谢指标及肝肾功能指标进行分析；采用ELISA等免疫学方法对骨转换指标尿DPD和血清OCN进行测定；采用三点弯曲实验对大鼠股骨生物力学性能进行分析；采用DEXA及Micro-CT对大鼠股骨、第四椎骨、胫骨的骨密度(bone mineral density, BMD)及股骨干骺端骨小梁微结构特性进行分析。
3.采用多次酶消化法获得大鼠原代成骨细胞,采用MTT、碱磷酶染色、矿化结节茜素红染色等方法,分析二至丸、女贞子、墨旱莲含药血清对大鼠原代成骨细胞的增殖、分化及矿化整个过程的影响。
4.采用western-blot技术分析二至丸、女贞子、墨旱莲对大鼠股骨组织中OPG蛋白表达、RANKL蛋白表达和OPG/RANKL蛋白表达比率的影响。
结果
1.二至丸、女贞子、墨旱莲连续治疗26周对大鼠去卵巢所致的骨质疏松症有良好的治疗效果,可以防止雌激素减少引起的体重增加,降低骨转换率,增加大鼠股骨、第四椎骨、胫骨的骨密度,提高大鼠股骨生物力学性能、改善股骨干骺端骨小梁微结构,从而降低绝经后骨折的发生率。与雌激素相比较具有无子宫刺激的优点,并且在防治OP的同时,长期用药无明显的毒副作用。
2.二至丸、女贞子、墨旱莲连续治疗6周能显著对抗维甲酸所致的大鼠血钙减少、血液及尿液中ALP和DPD活性增强、股骨、第四椎骨、胫骨的骨密度降低、股骨生物力学性能下降、骨小梁微结构退化等现象,从而降低骨折的发生率,起到防治药物所致急性骨质疏松症的作用。
3.二至丸、女贞子、墨旱莲能显著促进大鼠原代培养成骨细胞的增殖、分化及成熟,从而促进成骨作用。
4.二至丸中剂量组、女贞子、墨旱莲可以上调去卵巢大鼠股骨组织中OPG蛋白的表达,同时降低RANKL蛋白的表达,提高OPG/RANKL蛋白表达比率,表明三者可能通过调节OPG/RANKL表达从而达到抑制破骨细胞的活性,使骨吸收降低。
结论
二至丸、女贞子、墨旱莲治疗26周对大鼠去卵巢所致的骨质疏松症有良好的治疗效果,可以防止雌激素减少引起的体重增加,降低骨转换率,增加大鼠股骨、第四椎骨、胫骨的骨密度,提高股骨生物力学性能、改善骨小梁微结构,从而降低绝经后骨折的发生率。与雌激素相比较具有无子宫刺激的优点,并且在防治OP的同时,长期用药无明显的毒副作用。二至丸、女贞子、墨旱莲连续治疗6周能显著对抗维甲酸所致的大鼠血钙减少、ALP和DPD活性增强、骨密度降低、骨生物力学性能下降、骨小梁微结构退化等现象,从而降低骨折率,起到防治急性骨质疏松症的作用。二至丸、女贞子、墨旱莲既能防治去卵巢所致的原发性骨质疏松症又能防治维甲酸所致的继发性骨质疏松症,作用机制与其能显著促进成骨细胞活性,调节OPG/RANKL表达从而抑制破骨细胞活性,降低骨吸收密切相关。
Background
Osteoporosis is a systemic skeletal disease characterized by a reduction in bone mass and microarchitectural deterioration of bone tissue, leading to bone fragility and an increased susceptibility to fractures. Among of common and frequently-occurring diseases, the location of the OP has risen to the seventh, and now200million people have infected OP.In China and even in the world, the ratio of osteoporosis associated with the increasing of age will rise considerably. With the arrival of aging society, OP has become a serious threat to the elderly health all over the world. Thus, it needs to explore and solve this global public health problem. As the present cures for OP have their limitation, there is great demand for the exploration of safe, effective and economical medicine for the prevention and treatment of OP.
It is verified by modern researches of traditional Chinese medicine (TCM) that "kidney" involves series of functions such as endocrine, nerve, immune and metabolism. TCM contains a variety of ingredients, which functions wholy through multiple aspects, more ways and more targets. Therefore, it has more advantages and potentions in curing OP, which has attracted attention of specialists worldwide and has been considered as an important means to vaccinate and cure OP. Inheriting and carrying forward the superiority of TCM, preparing TCM and its formulations which can decrease side effects of western medicine and enhance curative effect, studying its mechanism by using modern science and technology methods, are the request of promoting the modernization of TCM, fostering the development of TCM, and serving human health.
Objective
Er-Zhi-Wan (EZW) is a famous traditional Chinese formulation firstly recorded in "Yi Bian" written in Ming Dynasty, contains two herbs viz. Fructus Ligustri Lucidi(FLL) and Herba Ecliptae (HE). EZW is simple and mild, widely used to prevent and treat various kidney diseases for its actions of nourishing the kidney yin and strengthening tendon and bone, with a long history of safe use for treatment of bone fractures and joint diseases in China. An equal weight ratio (1:1) of Fructus Ligustri Lucidi to Herba Eclipta is widely used to prevent and treatvarious kidney diseases for its actions of nourishing the kidneyand strengthening tendon and bone. Current knowledge whether EZW, FLL and HE have potential antiosteoporotic activity in rats should be evaluated. Therefore, the aim of this study was to systematically evaluate whether EZW,FLL and HE have the functions of treatment for osteoporosis induced by ovariectomy (OVX) and by retinoic acid in rats; isolate and purify the primary osteoblasts of rats and observe the EZW, FLL and HE effects of on primary osteoblasts;investigate the effects of EZW, FLL and HE on the expression of OPG and RANKL protein in rat femoral tissue by western-blot, discuss the mechanism of the antiosteoporotic effects of EZW, FLL and HE on osteoporosis; provide the theoretic witness for the advanced research of EZW, FLL and HE, provide methodology reference for studies of TCM and its compound to prevent and treat osteoporosis.
Methods
1. Eigthty3-month-old female Sprague-Dawley rats were randomly assigned into sham-operated group (Sham,n=10) and seven OVX subgroups, OVX with vehicle (OVX,n=10); OVX with Estradiol Valerate (EV,0.4mg/kg body weight/day,n=10); OVX with EZW of graded doses (9.0,4.5, or2.25g/kg/day,n=10); Fructus Ligustri Lucidi (FLL,4.5g/kg/day, n=10), Herba Ecliptae (HE,4.5g/kg/day, n=10). Daily oral administration of EV, EZW, FLL and HE on5th week for26weeks. The body weight of the animals was recorded weekly during the experimental period. Bone turnover markers (Serum alka-line phosphatase (ALP), bone-specific alkaline phosphatase (BALP), osteocalcin (OCN), deoxypyridinoline(DPD)), other parameters, including serum calcium (S-Ca), serum phosphorus (S-P), urine calcium (U-Ca), phosphorus (U-P), and bone mineral density (BMD) of the femur,4th lumbar vertebra and tibia, bone biomechanical properties and trabecular microarchitecture parameters were measured.
2. Eigthty3-month-old female Sprague-Dawley rats were randomly assigned into8groups, i.e. sham-operated group (Sham,n=10), retinoic acid vehicle (VA,75mg/kg/day,n=10); VA with XLGB (XLGB,1.5g/kg/day, n=10); OVX with EZW of graded doses (9.0,4.5, or2.25g/kg/day,n=10); OVX with Fructus Ligustri Lucidi(FLL,4.5g/kg/day, n=10), OVX with Herba Ecliptae(HE,4.5g/kg/day, n=10). In the morning all the rats were given retinoic acid through intragastric administration for2weeks, at the same time in the atfernoon with corresponding drugs for6weeks. The body weight of the animals was recorded daily during the experimental period. Bone turnover markers (Serum alka-line phosphatase (ALP), osteocalcin (OCN), deoxypyridinoline (DPD)), other parameters, including serum calcium (S-Ca), serum phosphorus (S-P), urine calcium (U-Ca), phosphorus (U-P), and bone mineral density (BMD) of the femur,4th lumbar vertebra and tibia, bone biomechanical properties and trabecular microarchitecture parameters were measured.
3. Primary cultures of rats osteoblasts were obtained by method of enzyme digestion and identified by morphological features, the alkaline phosphatase staining and Alizarin Red S staining of calcified nodules. The effects of EZW, FLL and HE on the proliferation and differentiation of rat osteoblast were evaluated by the MTT method, measuring the activity of alkaline phosphatase activity and Alizarin red staining.
4. The effects of EZW, FLL and HE on the expression of OPG and RANKL protein in rat femoral tissue by western-blot, discuss the mechanism of the antiosteoporotic effects of EZW, FLL and HE on osteoporosis.
Results
1.Treatment with EZW three graded doses,(9.0,4.5, or2.25g/kg/day), FLL(4.5g/kg/day) and HE(4.5g/kg/day) over a26-week period in the adult female OVX rats was found to be able to significantly inhibited estrogen deficiency-induced BMD decrease in the femur,4th lumbar vertebra and tibia. Biochemical markers were measured to show an increase in S-Ca, S-P levels, decreased levelof bone turnover markers and U-Ca, U-P levels. The preventionin the deterioration of trabecular microarchitecture and maintaining biomechanical competence of bone without an increase in the weight of the uterus were also exhibited during EZW, FLL and HE treatment. Long-term using of EZW, FLL and HE had not influcence on other organs and the liver and kidney functions.
2. The osteoprotective effects of EZW, FLL and HE in the retinoic acid-induced OP modelas acute OP model was studied. The results showed that daily oral administration of EZW, FLL and HE over a6-week period was able to prevent the decreased S-Ca, lower the active energy of bone turnover markers ALPand DPD, increase the BMD of the femur,4th lumbar vertebra and tibia, strengthen the biomechanical properties and mainten the trabecular bone structure compared to VA treatments.
3. Medicine containing serum of EZW, FLL and HE could promote the proliferation, differentiationand of rat primary cultured osteoblasts, which may indicate the potential protective action of EZW, FLL and HE can promote bone formation.
4. Our investigation showed treatment with EZW-M, FLL and HE for26weeks was able to prevent the decreased OPG protein, lower the decreased of RANKL protein induced by the OVX and increase the ratio of OPG/RANKL.EZW-M, FLL and HE can prevents bone resorption may be due to regulation the ratio of OPG/RANKL and slow down the proliferation, differentiation, maturity of osteoblasts.
Conclusions
Our data shows that daily oral administration of EZW, FLL and HE over a26-week period in the adult female OVX rats prevented the estrogen deficiency-induced bone loss. Biochemical markers were measured to show an increase in S-Ca, S-P levels, decreased levelof bone turnover markers and U-Ca, U-P levels. The preventionin the deterioration of trabecular microarchitecture and maintaining biomechanical competence of bone without an increase in the weight of the uterus were also exhibited during EZW, FLL and HE treatment. Long-term using of EZW, FLL and HE had not influcence on other organs and the liver and kidney functions; The administration of EZW, FLL and HE over a6-week period was able to prevent the decreased S-Ca, lower the active energy of bone turnover markers ALP and DPD, increase the BMD of the femur,4th lumbar vertebra and tibia, strengthen the biomechanical properties and mainten the trabecular bone structure in the retinoic acid-induced OP rats; EZW, FLL and HE might be potential medicines for treatment of postmenopausal osteoporosis and acid-induced acute osteoporosis, the effects of these three medicines can promote bone formation, increase osteoblastic activity, regulate the ratio of OPG/RANKL, slow down osteoclastic activity.
骨质疏松症(Osteoporosis, OP)属于一种全身性骨骼系统疾病,主要表现为骨量减少、骨组织微观结构退化,最终导致骨脆性增加和骨折危险性增加。目前,全世界患有OP的病人数已经超过2亿,占总人口的3%,在全世界常见病、多发病中,OP位置已经上升到第7位。随着老龄化社会的到来,在我国乃至全球,这种与增龄有关的OP疾病将大幅上升。OP已成为严重威胁老年人健康的全世界共同关注的公共问题,这也是全球函待探索和解决的老年性、慢性疾病之一。当前,由于现有防治OP药物特性的限制,研制开发安全、长效、经济的抗OP药物仍然是医药学界的主攻目标。
由于中药及其复方含有大量不同的化学成分,可以通过多途径、多靶点、多程序发挥整体作用,因此在防治骨质疏松症方面具有较大的潜力与优势,被很多国内外研究抗骨质疏松症药物的学者视为重点开发对象。现代研究证实,中医药中所说的“肾”涉及到人体多种系统功能,例如内分泌系统、神经系统、免疫系统、代谢系统等功能,而中药及其复方在此方面又具有西药无法比拟的整体疗效。因此,在防治骨质疏松症的研究工作中,继承和发扬中医药特长,研发出显著减少西药副作用、提高疗效、标本兼治的中药及其复方,并运用现代先进科技手段探明其机理,既是符合中医药现代化的客观要求,又是推动中医药发展、服务于全人类健康的必然举措。
研究目的
二至丸最早记录于明代王三才的《医便》中,由女贞子和墨旱莲二味中药等份配伍而得,是中医临床应用的经典名方。二至丸药味精少而药性平和,补而不滞,滋而不腻,防治与OP相类似的临床骨症已有近七百年的历史。本课题在复制OVX大鼠骨质疏松模型及维甲酸所致急性OP模型的基础上,系统地研究了二至丸、女贞子、墨旱莲防治OP的药理效应；采用体外培养的大鼠原代成骨细胞为研究工具,从细胞水平上对二至丸、女贞子、墨旱莲含药血清对大鼠成骨细胞的增殖、分化及矿化整个过程中的促进作用进行研究。同时,采用分子生物学检测方法,研究二至丸、女贞子、墨旱莲对大鼠股骨组织中OPG蛋白表达、RANKL蛋白表达和OPG/RANKL蛋白表达比率的影响,从而揭示二至丸、女贞子、墨旱莲防治OP的作用机制。本研究为二至丸、女贞子、墨旱莲进一步研究开发提供理论依据,为中药及其复方防治骨质疏松症提供思路和方法学的借鉴。
研究方法
1.选取3月龄未经产雌性Sprague--Dawley(SD)大鼠共80只,适应环境3天后随机分为假手术(Sham, n=10)组和去卵巢(OVX, n=70)组。将去卵巢后的大鼠分为以下组别：模型组(OVX, n=10),戊酸雌二醇组(EV,0.4mg/kg/day, n=10),二至丸大剂量组(EZW-H,9g/kg/day, n=10),二至丸中剂量组(EZW-M,4.5g/kg/day,n=10),二至丸小剂量组(EZW-L,2.25g/kg/day, n=10),女贞子组(FLL,4.5g/kg/day, n=10),墨旱莲组(HE,4.5g/kg/day, n=10).进行卵巢摘除手术后第5周,各组开始给药,共给药26周,每周称量各组大鼠体重一次。动物处死前,收集大鼠24h尿液,麻醉动物采集血清。全部动物处死并解剖,采集大鼠子宫及重要脏器。检测脏器体质量及子宫重量和病理变化,分析长期用药对子宫和重要脏器的影响；大鼠血清和尿液中的Ca、P和ALP等骨代谢指标及肝肾功能指标采用全自动生化分析仪进行分析检测；对尿液中DPD和血清中BALP、OCN等骨转换标记性指标成分采用ELISA免疫学方法进行测定；采用三点弯曲材料力学实验和骨压碎力实验检测分析大鼠股骨生物力学性能；采用DEXA及Micro-CT对大鼠股骨、第四椎骨、胫骨的BMD及股骨干骺端骨小梁微结构特性进行检测。
2.选取3月龄未经产雌性二级Sprague--Dawley(SD)大鼠共80只,适应环境2天后,随机分为8组,每组10只。即,正常组(Sham, n=10),模型组(VA,75mg/kg/day, n=10),仙灵骨葆组(XLGB,1.5g/kg/day, n=10),二至丸大剂量组(EZW-H,9.0g/kg/day, n=10),二至丸中剂量组(EZW-M,4.5g/kg/day, n=10),二至丸小剂量组(EZW-L,2.25g/kg/day, n=10),女贞子组(FLL,4.5g/kg/day, n=10),墨旱莲组(HE,4.5g/kg/day, n=10).除正常组外,其余各组每天上午维甲酸灌胃给药1次,连续2周,制备急性骨质疏松模型。造模同时,下午各组按规定剂量灌胃给予相应药物,连续给药6周,正常组和模型组给予蒸馏水,各组动物每天称重一次。动物处死前,收集大鼠24h尿液,麻醉动物采集血清。全部动物处死并解剖,采集以下标本并进行测定分析：观察大鼠重要脏器的体质量变化,分析药物用药对脏器的影响；采用全自动生化分析仪对大鼠血清和尿液中的Ca、P和ALP等骨代谢指标及肝肾功能指标进行分析；采用ELISA等免疫学方法对骨转换指标尿DPD和血清OCN进行测定；采用三点弯曲实验对大鼠股骨生物力学性能进行分析；采用DEXA及Micro-CT对大鼠股骨、第四椎骨、胫骨的骨密度(bone mineral density, BMD)及股骨干骺端骨小梁微结构特性进行分析。
3.采用多次酶消化法获得大鼠原代成骨细胞,采用MTT、碱磷酶染色、矿化结节茜素红染色等方法,分析二至丸、女贞子、墨旱莲含药血清对大鼠原代成骨细胞的增殖、分化及矿化整个过程的影响。
4.采用western-blot技术分析二至丸、女贞子、墨旱莲对大鼠股骨组织中OPG蛋白表达、RANKL蛋白表达和OPG/RANKL蛋白表达比率的影响。
结果
1.二至丸、女贞子、墨旱莲连续治疗26周对大鼠去卵巢所致的骨质疏松症有良好的治疗效果,可以防止雌激素减少引起的体重增加,降低骨转换率,增加大鼠股骨、第四椎骨、胫骨的骨密度,提高大鼠股骨生物力学性能、改善股骨干骺端骨小梁微结构,从而降低绝经后骨折的发生率。与雌激素相比较具有无子宫刺激的优点,并且在防治OP的同时,长期用药无明显的毒副作用。
2.二至丸、女贞子、墨旱莲连续治疗6周能显著对抗维甲酸所致的大鼠血钙减少、血液及尿液中ALP和DPD活性增强、股骨、第四椎骨、胫骨的骨密度降低、股骨生物力学性能下降、骨小梁微结构退化等现象,从而降低骨折的发生率,起到防治药物所致急性骨质疏松症的作用。
3.二至丸、女贞子、墨旱莲能显著促进大鼠原代培养成骨细胞的增殖、分化及成熟,从而促进成骨作用。
4.二至丸中剂量组、女贞子、墨旱莲可以上调去卵巢大鼠股骨组织中OPG蛋白的表达,同时降低RANKL蛋白的表达,提高OPG/RANKL蛋白表达比率,表明三者可能通过调节OPG/RANKL表达从而达到抑制破骨细胞的活性,使骨吸收降低。
结论
二至丸、女贞子、墨旱莲治疗26周对大鼠去卵巢所致的骨质疏松症有良好的治疗效果,可以防止雌激素减少引起的体重增加,降低骨转换率,增加大鼠股骨、第四椎骨、胫骨的骨密度,提高股骨生物力学性能、改善骨小梁微结构,从而降低绝经后骨折的发生率。与雌激素相比较具有无子宫刺激的优点,并且在防治OP的同时,长期用药无明显的毒副作用。二至丸、女贞子、墨旱莲连续治疗6周能显著对抗维甲酸所致的大鼠血钙减少、ALP和DPD活性增强、骨密度降低、骨生物力学性能下降、骨小梁微结构退化等现象,从而降低骨折率,起到防治急性骨质疏松症的作用。二至丸、女贞子、墨旱莲既能防治去卵巢所致的原发性骨质疏松症又能防治维甲酸所致的继发性骨质疏松症,作用机制与其能显著促进成骨细胞活性,调节OPG/RANKL表达从而抑制破骨细胞活性,降低骨吸收密切相关。
Background
Osteoporosis is a systemic skeletal disease characterized by a reduction in bone mass and microarchitectural deterioration of bone tissue, leading to bone fragility and an increased susceptibility to fractures. Among of common and frequently-occurring diseases, the location of the OP has risen to the seventh, and now200million people have infected OP.In China and even in the world, the ratio of osteoporosis associated with the increasing of age will rise considerably. With the arrival of aging society, OP has become a serious threat to the elderly health all over the world. Thus, it needs to explore and solve this global public health problem. As the present cures for OP have their limitation, there is great demand for the exploration of safe, effective and economical medicine for the prevention and treatment of OP.
It is verified by modern researches of traditional Chinese medicine (TCM) that "kidney" involves series of functions such as endocrine, nerve, immune and metabolism. TCM contains a variety of ingredients, which functions wholy through multiple aspects, more ways and more targets. Therefore, it has more advantages and potentions in curing OP, which has attracted attention of specialists worldwide and has been considered as an important means to vaccinate and cure OP. Inheriting and carrying forward the superiority of TCM, preparing TCM and its formulations which can decrease side effects of western medicine and enhance curative effect, studying its mechanism by using modern science and technology methods, are the request of promoting the modernization of TCM, fostering the development of TCM, and serving human health.
Objective
Er-Zhi-Wan (EZW) is a famous traditional Chinese formulation firstly recorded in "Yi Bian" written in Ming Dynasty, contains two herbs viz. Fructus Ligustri Lucidi(FLL) and Herba Ecliptae (HE). EZW is simple and mild, widely used to prevent and treat various kidney diseases for its actions of nourishing the kidney yin and strengthening tendon and bone, with a long history of safe use for treatment of bone fractures and joint diseases in China. An equal weight ratio (1:1) of Fructus Ligustri Lucidi to Herba Eclipta is widely used to prevent and treatvarious kidney diseases for its actions of nourishing the kidneyand strengthening tendon and bone. Current knowledge whether EZW, FLL and HE have potential antiosteoporotic activity in rats should be evaluated. Therefore, the aim of this study was to systematically evaluate whether EZW,FLL and HE have the functions of treatment for osteoporosis induced by ovariectomy (OVX) and by retinoic acid in rats; isolate and purify the primary osteoblasts of rats and observe the EZW, FLL and HE effects of on primary osteoblasts;investigate the effects of EZW, FLL and HE on the expression of OPG and RANKL protein in rat femoral tissue by western-blot, discuss the mechanism of the antiosteoporotic effects of EZW, FLL and HE on osteoporosis; provide the theoretic witness for the advanced research of EZW, FLL and HE, provide methodology reference for studies of TCM and its compound to prevent and treat osteoporosis.
Methods
1. Eigthty3-month-old female Sprague-Dawley rats were randomly assigned into sham-operated group (Sham,n=10) and seven OVX subgroups, OVX with vehicle (OVX,n=10); OVX with Estradiol Valerate (EV,0.4mg/kg body weight/day,n=10); OVX with EZW of graded doses (9.0,4.5, or2.25g/kg/day,n=10); Fructus Ligustri Lucidi (FLL,4.5g/kg/day, n=10), Herba Ecliptae (HE,4.5g/kg/day, n=10). Daily oral administration of EV, EZW, FLL and HE on5th week for26weeks. The body weight of the animals was recorded weekly during the experimental period. Bone turnover markers (Serum alka-line phosphatase (ALP), bone-specific alkaline phosphatase (BALP), osteocalcin (OCN), deoxypyridinoline(DPD)), other parameters, including serum calcium (S-Ca), serum phosphorus (S-P), urine calcium (U-Ca), phosphorus (U-P), and bone mineral density (BMD) of the femur,4th lumbar vertebra and tibia, bone biomechanical properties and trabecular microarchitecture parameters were measured.
2. Eigthty3-month-old female Sprague-Dawley rats were randomly assigned into8groups, i.e. sham-operated group (Sham,n=10), retinoic acid vehicle (VA,75mg/kg/day,n=10); VA with XLGB (XLGB,1.5g/kg/day, n=10); OVX with EZW of graded doses (9.0,4.5, or2.25g/kg/day,n=10); OVX with Fructus Ligustri Lucidi(FLL,4.5g/kg/day, n=10), OVX with Herba Ecliptae(HE,4.5g/kg/day, n=10). In the morning all the rats were given retinoic acid through intragastric administration for2weeks, at the same time in the atfernoon with corresponding drugs for6weeks. The body weight of the animals was recorded daily during the experimental period. Bone turnover markers (Serum alka-line phosphatase (ALP), osteocalcin (OCN), deoxypyridinoline (DPD)), other parameters, including serum calcium (S-Ca), serum phosphorus (S-P), urine calcium (U-Ca), phosphorus (U-P), and bone mineral density (BMD) of the femur,4th lumbar vertebra and tibia, bone biomechanical properties and trabecular microarchitecture parameters were measured.
3. Primary cultures of rats osteoblasts were obtained by method of enzyme digestion and identified by morphological features, the alkaline phosphatase staining and Alizarin Red S staining of calcified nodules. The effects of EZW, FLL and HE on the proliferation and differentiation of rat osteoblast were evaluated by the MTT method, measuring the activity of alkaline phosphatase activity and Alizarin red staining.
4. The effects of EZW, FLL and HE on the expression of OPG and RANKL protein in rat femoral tissue by western-blot, discuss the mechanism of the antiosteoporotic effects of EZW, FLL and HE on osteoporosis.
Results
1.Treatment with EZW three graded doses,(9.0,4.5, or2.25g/kg/day), FLL(4.5g/kg/day) and HE(4.5g/kg/day) over a26-week period in the adult female OVX rats was found to be able to significantly inhibited estrogen deficiency-induced BMD decrease in the femur,4th lumbar vertebra and tibia. Biochemical markers were measured to show an increase in S-Ca, S-P levels, decreased levelof bone turnover markers and U-Ca, U-P levels. The preventionin the deterioration of trabecular microarchitecture and maintaining biomechanical competence of bone without an increase in the weight of the uterus were also exhibited during EZW, FLL and HE treatment. Long-term using of EZW, FLL and HE had not influcence on other organs and the liver and kidney functions.
2. The osteoprotective effects of EZW, FLL and HE in the retinoic acid-induced OP modelas acute OP model was studied. The results showed that daily oral administration of EZW, FLL and HE over a6-week period was able to prevent the decreased S-Ca, lower the active energy of bone turnover markers ALPand DPD, increase the BMD of the femur,4th lumbar vertebra and tibia, strengthen the biomechanical properties and mainten the trabecular bone structure compared to VA treatments.
3. Medicine containing serum of EZW, FLL and HE could promote the proliferation, differentiationand of rat primary cultured osteoblasts, which may indicate the potential protective action of EZW, FLL and HE can promote bone formation.
4. Our investigation showed treatment with EZW-M, FLL and HE for26weeks was able to prevent the decreased OPG protein, lower the decreased of RANKL protein induced by the OVX and increase the ratio of OPG/RANKL.EZW-M, FLL and HE can prevents bone resorption may be due to regulation the ratio of OPG/RANKL and slow down the proliferation, differentiation, maturity of osteoblasts.
Conclusions
Our data shows that daily oral administration of EZW, FLL and HE over a26-week period in the adult female OVX rats prevented the estrogen deficiency-induced bone loss. Biochemical markers were measured to show an increase in S-Ca, S-P levels, decreased levelof bone turnover markers and U-Ca, U-P levels. The preventionin the deterioration of trabecular microarchitecture and maintaining biomechanical competence of bone without an increase in the weight of the uterus were also exhibited during EZW, FLL and HE treatment. Long-term using of EZW, FLL and HE had not influcence on other organs and the liver and kidney functions; The administration of EZW, FLL and HE over a6-week period was able to prevent the decreased S-Ca, lower the active energy of bone turnover markers ALP and DPD, increase the BMD of the femur,4th lumbar vertebra and tibia, strengthen the biomechanical properties and mainten the trabecular bone structure in the retinoic acid-induced OP rats; EZW, FLL and HE might be potential medicines for treatment of postmenopausal osteoporosis and acid-induced acute osteoporosis, the effects of these three medicines can promote bone formation, increase osteoblastic activity, regulate the ratio of OPG/RANKL, slow down osteoclastic activity.
引文
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