左归丸对去卵巢所致骨质疏松症大鼠股骨骨髓中相关蛋白质的影响
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摘要
目的
以卵巢切除大鼠作为绝经后骨质疏松症动物模型,用左归丸进行干预,在检测肾虚状态和补肾后骨量以及骨形成与骨吸收变化情况的基础上,采用蛋白质组学方法,从骨髓中探索左归丸对去卵巢所致大鼠骨质疏松症产生调节作用的相关蛋白质,从而在分子水平上揭示补肾方剂左归丸对去卵巢所致大鼠骨质疏松症的作用机理,进而为阐明“肾主骨”理论的科学内涵提供进一步的实验依据。
方法
1动物分组和取材
将100只雌性Wistar大鼠随机分为3组：正常对照组20只、假手术组20只和造模组60只。造模组大鼠用戊巴比妥钠腹腔注射麻醉,摘除双侧卵巢；假手术组只摘取卵巢周围的少许脂肪组织。手术2周后,将造模组大鼠随机分为3组：模型组20只、己烯雌酚组20只、左归丸组20只。分组后开始给大鼠灌胃给药：己烯雌酚组灌胃给予己烯雌酚0.046mg/kg,给药浓度为0.0046mg/ml;左归丸组灌胃给予左归丸9.68g生药/kg,给药浓度为0.968g生药/ml。给药体积均为1ml/100g体重。以上给药一天一次,连续6天,休息一天后,再连续给药6天,如此给药12周。正常对照组、假手术组、模型组则按同法灌服等体积的蒸馏水。每周称重一次,据此调整给药量。各组大鼠在处死前15天和前3天分别腹腔注射盐酸四环素30mg/kg体重,以对骨进行荧光标记。给药结束后,将各组大鼠处死,取右侧胫骨近端1/3,制作不脱钙骨切片,其中一张甲苯胺蓝染色,另一张切片则直接用于荧光观察；左侧胫骨近端1/3,制作冰冻切片,以作蛋白质的免疫组化法验证；取双侧股骨,用作蛋白质双向凝胶电泳测定和免疫印迹验证。
2骨组织形态计量学指标测定
采用Qwin V3图像分析系统检测各组大鼠胫骨骨小梁体积百分比(TBV%)、骨小梁吸收表面百分比(TRS%)、骨小梁形成表面百分比(TFS%)、骨小梁矿化率(MAR)、类骨质平均宽度(OSW)、骨皮质矿化率(mAR),观察各组大鼠骨形成及骨吸收等骨代谢情况。
3骨髓蛋白质分析鉴定
分别提取正常组、模型组和左归丸组三组大鼠股骨骨髓蛋白质,应用蛋白质双向凝胶电泳技术分析比较三组大鼠股骨骨髓之间蛋白质的表达,找出左归丸调节纠正的去卵巢所致骨质疏松症大鼠骨髓中的差异蛋白质点。通过质谱方法对这些差异蛋白质进行进一步鉴定,获取差异蛋白质的肽质量指纹图谱和相关数据库信息。对差异蛋白质的质谱鉴定结果应用免疫印迹法和免疫组化方法予以验证。
结果
1左归丸对去卵巢所致骨质疏松症大鼠骨组织形态计量学指标的影响
1.1左归丸对去卵巢所致骨质疏松症大鼠胫骨TBV%的影响
模型组大鼠胫骨TBV%显著低于假手术组。与模型组相比,左归丸组、己烯雌酚组的TBV%均显著升高。左归丸组、已烯雌酚组的TBV%明显低于假手术组。
1.2左归丸对去卵巢所致骨质疏松症大鼠胫骨TRS%的影响
模型组大鼠胫骨TRS%较假手术组显著增高。与模型组相比,左归丸组和已烯雌酚组的TRS%均显著降低。与假手术组相比,已烯雌酚组和左归丸组的TRS%显著升高。
1.3左归丸对去卵巢所致骨质疏松症大鼠胫骨TFS%和MAR的影响
模型组大鼠胫骨TFS%和MAR较假手术组皆显著增高。与模型组相比,左归丸组和己烯雌酚组的TFS%和MAR均显著降低；而与假手术组相比,左归丸组明显增高,己烯雌酚组则无显著性差异。
1.4左归丸对去卵巢所致骨质疏松症大鼠胫骨mAR和OSW的影响
模型组大鼠胫骨mAR和OSW皆显著高于假手术组。左归丸组和己烯雌酚组的mAR和OSW均明显低于模型组；与假手术组相比,左归丸组的mAR和OSW显著增高,而己烯雌酚组无显著性差异。
假手术组与空白对照组比较,以上指标均无显著性差异,从而排除手术因素对实验的影响。
2左归丸对去卵巢所致骨质疏松症大鼠股骨骨髓中相关蛋白质的影响
2.1双向凝胶电泳结果
双向凝胶电泳图像结果经Image Master2D Platinum5.0软件分析,所识别的蛋白质点个数为(826±35),以3倍差异表达做为标准,配合人工校正,在正常组、模型组和左归丸组三组之间共找到差异蛋白质点为23个。
2.2质谱分析结果
对23个差异蛋白质点进行酶解、质谱分析得到PMF图谱后,查询NCBInr20111014数据库,其中个12点由于数据搜索的蛋白的分数不具有统计学意义而未得到鉴定定(≥61分具有统计意义),11个蛋白质点得到鉴定,其中两个蛋白为未命名蛋白。在模型中骨髓高表达的蛋白包括白果糖醛缩酶A、抗氧化蛋白6、α烯醇化酶、烯醇化酶3、白蛋白、钙网蛋白、蛋白二硫化物异构酶A3；在模型组骨髓中低表达蛋白质为碳酸酐酶1、S100-A8蛋白。2.3蛋白质验证结果2.3.1Western-blot验证骨髓中果糖醛缩酶A蛋白表达的结果
果糖醛缩酶A (ALDOA)蛋白在各组大鼠中均有表达,在模型组中表达水平较高,明显高于正常组和左归丸组,说明ALDOA蛋白在去卵巢骨质疏松症大鼠发病过程中为异常高表达蛋白,而具有补肾作用的左归丸对ALDOA蛋白的高表达有一定的调节作用,这与凝胶分析和质谱分析结果相符。
2.3.2免疫组化法验证骨髓中碳酸酐酶1和抗氧化蛋白6表达的结果
2.3.2.1免疫组化法验证骨髓中碳酸酐酶1表达的结果
碳酸酐酶1(CA1)在各组大鼠中均有表达,模型组大鼠骨髓基质细胞CA1蛋白表达阳性密度较正常对照组显著降低。左归丸组CA1蛋白表达阳性密度明显高于模型组,但与正常对照组相比,无显著差异。说明左归丸对CA1蛋白的低表达有调节作用,结果与凝胶分析和质谱分析结果相符。
2.3.2.2免疫组化验证骨髓中抗氧化蛋白6表达的结果
抗氧化蛋白6(PRDX6)在各组大鼠中均有表达,模型组大鼠骨髓基质细胞PRDX6蛋白表达阳性密度较正常对照组显著升高。左归丸组PRDX6蛋白表达阳性密度显著低于模型组,但与正常对照组相比,无显著差异。说明左归丸刘PRDX6蛋白的高表达有调节作用,结果凝胶分析和质谱分析结果相符。
结论
1.切除大鼠卵巢14周后,作为骨量主要标志的胫骨TBV%显著降低,而代表骨吸收参数的TRS%,以及代表骨形成参数的TFS%、MAR、OSW和mAR均显著增高,说明卵巢切除所造成的是一种骨吸收大于骨形成的高转换型骨质疏松症；而左归丸能在一定程度上使上述指标发生逆转,表明其对卵巢切除所致的大鼠骨质疏松症具有一定的预防作用。
2.应用蛋白组学方法从大鼠股骨骨髓中分析鉴定出11个差异蛋白质。其中,模型组骨髓中高表达而被左归丸下调至正常组水平的蛋白质有果糖醛缩酶A、抗氧化蛋白6、α烯醇化酶、烯醇化酶3、白蛋白、钙网蛋白、蛋白二硫化物异构酶A3；模型组骨髓中低表达而被左归丸上调至正常组水平的蛋白质有碳酸酐酶1、S100-A8蛋白。这些差异蛋白质主要涉及能量代谢、抗氧化、信号传导、细胞增殖、分化和凋亡等多方面的功能。说明在分子水平上,左归丸可通过调节骨髓内细胞增殖、分化、凋亡和能量代谢以及氧化反应等来实现对去卵巢所致大鼠骨质疏松症的预防作用。同时基于中医理论对绝经后骨质疏松症的认识,这些差异蛋白质是与绝经后骨质疏松症发生密切相关的蛋白质,也是与“肾主骨”密切相关的蛋白质。
Objective
To explore the changed proteins which is related with Zuoguiwan as a regulatory role by the methods of proteomics in the bone marrow of the model rats which have osteoporosis caused by bilateral ovariectomy.And then reveal the mechanism of Zuoguiwan during the treatment of the rats'osteoporosis induced by bilateral ovariectomy at the protein level which will also to provide further experimental evidence for exploring the scientific connotation of the mechanism of "The kidney dominating bone"
Methods
1Rats grouping and drawn
Firstly,one hundred female Wistar rats were randomly divided into three groups:twenty in normal control group,twenty in the pseudo-operation group,twenty in model group,twenty in stilbestrol group, twenty in Zuoguiwan group.Both ovaries of all rats in the operation group were extirpated,while only a little fatty tissue nearby the ovaries in the pseudo-operation group were extirpated.Distilled water was administrated orally to normal control group,pseudo-operation group and model group at the same time. The rats in Stilbestrol group and Zuoguiwan group were treated with stilbestrol tablets and decoction of Zuoguiwan.The Stilbestrol tablets was crushed,used distilled water as the the suspension concentration0.0046mg/ml,and the dose was0.046g/kg weight.The concentration of Zuoguiwan was0.968g/ml, and the dose was9.68/kg weight. The drugs were given once a day and last six days. There is one day paused every six days. The whole period last twelve weeks.
At the third day and the fifteenth day before the day all the rats were killed,hydrochloric acheomycin were respectively injected into the abdomen of the rats according to30mg/kg to mark the bone with fluorescence.After being killed the right tibia of the rats were retained for bone tissue section. At the same time the left tibia of the rats were used for Frozen tissue section after being decalcified to observe the protein expression by means of immunohistochemistry comfirm the result of mass spectrographic analysis, and the bilateral femur for2-DE and Western-blot.
2Bone histomorphometry indicators analysis
Detected the rats'tibia TBV%,TRS%,TFS%,MAR.OSW and mAR by Qwin V3image analysis system.
3Bone marrow protein analysis and identification
Get the femur bone marrow from the rats of normal group.model group and Zuoguiwan group, and extract the proteins. With2-D electrophoresis technology to analyze and compare the expressing proteins in the marrow of the three groups, and find thedeferent proteins adjusted by Zuoguiwan which are considered to be the related protein with postmenopausal osteoporosis.Then these diferent proteins were identified by mass spectrographic analysis and get their relateded informations.Chose protein ADLOA to be verified by Western-blot and protein CA1and PRDX6to be verified by immunohistochemical methods.
Results
1Effects of Zuoguiwan on the bone histomorphometry indicators in ovariectomized rats suffered osteoporosis
After the bilateral ovaries of the rats were extiroated,TBV%of tibia significantly decreased, while TRS%、TFS%、MAR、OSW and mAR significantly increased,which indicated that the model established by extirpating bilateral ovaries is a kind of high transformed osteoporosis model which has higher resorption than formation in bone.Compared with model group, TBV%significantly increased while TRS%、 TFS%、MAR、OSW and mAR significantly decreased after being treated by zuoguiwan. So Zuoguiwan can treat osteoporosis by reducing the high transformation of the bone.
2Result of2-DE
Through Image Master2D Platinum5.0analysis,the numbers of protein expression in the femur bone marrow showed two-dimensional gel electrophoresis profiles is(826±35).Three-fold expression is used as the standard, and twenty-three differentially expressed protein spots were detected which were regulated and corrected by zuoguiwan in the model group.
3Results of Mass spectrometry
Eleven differentially expressed protein spots were detected by MALDI-TOP masss pectra,and11proteins spots obtain correct identification results. Among these proteins, the proteins which were highly expressed in model group and corrected by Zuoguiwan are carbonic anhydrase1, calreticulin, fructose-bisphosphate, aldolaseA, peroxiredoxin-6, enolase3, albumin, disulfide-isomerase A3, enolasel; the proteins which were lowly expressed in model group and corrected by Zuoguiwan are carbonic anhydrase1and protein S100-A8.
4Results of verifications
4.1Results of western-blot
Aldolase A was verified by western-blot.The map of western-blot shows: Aldolase A is expressed in the3groups.The level of expression is highest in the model group. The level of expression in zuoguiwan group is significantly decreased and is similar to that in stilbestrol group This result is in accorded with gel analysis and mass identification.
4.2Results of immunohistochemistry
CA1and PRDX6were verified by immunohistochemical methods.The results show:The expression of CA1is lowest in the model group and significantly increased in zuoguiwan group.The expression of PRDX6is highest in the model group and significantly decreased in zuoguiwan group.
Conclusion
1. After bilateral ovariectomy, Rats'bone resorption and bone formation were significantly enhanced, but bone resorption is more than bone formation, which resulted in a significant reduction in bone mass. This is a kind of high conversion of osteoporosis. Zuoguiwan can improve the abnormal changes in bone resorption and bone formation, reduce bone loss,and prevent the occurrence of ovariectomized induced osteoporosis disease.
2. Eleven differentially expressed proteins in the rats' femur bone marrow are screened and identified by proteomic methods. Among these proteins, the proteins which were highly expressed in model group but reduced by Zuoguiwan include carbonic anhydrase1, calreticulin, fructose-bisphosphate, aldolaseA, peroxiredoxin6, enolase3, albumin, disulfide-isomerase A3, alpha-enolase. The proteins which were lowly expressed in model group and increased by Zuoguiwan are carbonic anhydrase1and protein S100-A8. The functions of these proteins are related to energy metabolism, antioxidant, signal transduction, cell differentiation, proliferation and apoptosis, etc. Then we conclude that Zuoguiwan' treatment effect on the osteoporosis of the ovariectomized rat is achieved by regulating cell proliferation, differentiation, metabolism, apoptosis and oxidative reactions in the bone marrow. These proteins are also closely related with Postmenopausal osteoporosis and "The kidney dominating bone".
以卵巢切除大鼠作为绝经后骨质疏松症动物模型,用左归丸进行干预,在检测肾虚状态和补肾后骨量以及骨形成与骨吸收变化情况的基础上,采用蛋白质组学方法,从骨髓中探索左归丸对去卵巢所致大鼠骨质疏松症产生调节作用的相关蛋白质,从而在分子水平上揭示补肾方剂左归丸对去卵巢所致大鼠骨质疏松症的作用机理,进而为阐明“肾主骨”理论的科学内涵提供进一步的实验依据。
方法
1动物分组和取材
将100只雌性Wistar大鼠随机分为3组：正常对照组20只、假手术组20只和造模组60只。造模组大鼠用戊巴比妥钠腹腔注射麻醉,摘除双侧卵巢；假手术组只摘取卵巢周围的少许脂肪组织。手术2周后,将造模组大鼠随机分为3组：模型组20只、己烯雌酚组20只、左归丸组20只。分组后开始给大鼠灌胃给药：己烯雌酚组灌胃给予己烯雌酚0.046mg/kg,给药浓度为0.0046mg/ml;左归丸组灌胃给予左归丸9.68g生药/kg,给药浓度为0.968g生药/ml。给药体积均为1ml/100g体重。以上给药一天一次,连续6天,休息一天后,再连续给药6天,如此给药12周。正常对照组、假手术组、模型组则按同法灌服等体积的蒸馏水。每周称重一次,据此调整给药量。各组大鼠在处死前15天和前3天分别腹腔注射盐酸四环素30mg/kg体重,以对骨进行荧光标记。给药结束后,将各组大鼠处死,取右侧胫骨近端1/3,制作不脱钙骨切片,其中一张甲苯胺蓝染色,另一张切片则直接用于荧光观察；左侧胫骨近端1/3,制作冰冻切片,以作蛋白质的免疫组化法验证；取双侧股骨,用作蛋白质双向凝胶电泳测定和免疫印迹验证。
2骨组织形态计量学指标测定
采用Qwin V3图像分析系统检测各组大鼠胫骨骨小梁体积百分比(TBV%)、骨小梁吸收表面百分比(TRS%)、骨小梁形成表面百分比(TFS%)、骨小梁矿化率(MAR)、类骨质平均宽度(OSW)、骨皮质矿化率(mAR),观察各组大鼠骨形成及骨吸收等骨代谢情况。
3骨髓蛋白质分析鉴定
分别提取正常组、模型组和左归丸组三组大鼠股骨骨髓蛋白质,应用蛋白质双向凝胶电泳技术分析比较三组大鼠股骨骨髓之间蛋白质的表达,找出左归丸调节纠正的去卵巢所致骨质疏松症大鼠骨髓中的差异蛋白质点。通过质谱方法对这些差异蛋白质进行进一步鉴定,获取差异蛋白质的肽质量指纹图谱和相关数据库信息。对差异蛋白质的质谱鉴定结果应用免疫印迹法和免疫组化方法予以验证。
结果
1左归丸对去卵巢所致骨质疏松症大鼠骨组织形态计量学指标的影响
1.1左归丸对去卵巢所致骨质疏松症大鼠胫骨TBV%的影响
模型组大鼠胫骨TBV%显著低于假手术组。与模型组相比,左归丸组、己烯雌酚组的TBV%均显著升高。左归丸组、已烯雌酚组的TBV%明显低于假手术组。
1.2左归丸对去卵巢所致骨质疏松症大鼠胫骨TRS%的影响
模型组大鼠胫骨TRS%较假手术组显著增高。与模型组相比,左归丸组和已烯雌酚组的TRS%均显著降低。与假手术组相比,已烯雌酚组和左归丸组的TRS%显著升高。
1.3左归丸对去卵巢所致骨质疏松症大鼠胫骨TFS%和MAR的影响
模型组大鼠胫骨TFS%和MAR较假手术组皆显著增高。与模型组相比,左归丸组和己烯雌酚组的TFS%和MAR均显著降低；而与假手术组相比,左归丸组明显增高,己烯雌酚组则无显著性差异。
1.4左归丸对去卵巢所致骨质疏松症大鼠胫骨mAR和OSW的影响
模型组大鼠胫骨mAR和OSW皆显著高于假手术组。左归丸组和己烯雌酚组的mAR和OSW均明显低于模型组；与假手术组相比,左归丸组的mAR和OSW显著增高,而己烯雌酚组无显著性差异。
假手术组与空白对照组比较,以上指标均无显著性差异,从而排除手术因素对实验的影响。
2左归丸对去卵巢所致骨质疏松症大鼠股骨骨髓中相关蛋白质的影响
2.1双向凝胶电泳结果
双向凝胶电泳图像结果经Image Master2D Platinum5.0软件分析,所识别的蛋白质点个数为(826±35),以3倍差异表达做为标准,配合人工校正,在正常组、模型组和左归丸组三组之间共找到差异蛋白质点为23个。
2.2质谱分析结果
对23个差异蛋白质点进行酶解、质谱分析得到PMF图谱后,查询NCBInr20111014数据库,其中个12点由于数据搜索的蛋白的分数不具有统计学意义而未得到鉴定定(≥61分具有统计意义),11个蛋白质点得到鉴定,其中两个蛋白为未命名蛋白。在模型中骨髓高表达的蛋白包括白果糖醛缩酶A、抗氧化蛋白6、α烯醇化酶、烯醇化酶3、白蛋白、钙网蛋白、蛋白二硫化物异构酶A3；在模型组骨髓中低表达蛋白质为碳酸酐酶1、S100-A8蛋白。2.3蛋白质验证结果2.3.1Western-blot验证骨髓中果糖醛缩酶A蛋白表达的结果
果糖醛缩酶A (ALDOA)蛋白在各组大鼠中均有表达,在模型组中表达水平较高,明显高于正常组和左归丸组,说明ALDOA蛋白在去卵巢骨质疏松症大鼠发病过程中为异常高表达蛋白,而具有补肾作用的左归丸对ALDOA蛋白的高表达有一定的调节作用,这与凝胶分析和质谱分析结果相符。
2.3.2免疫组化法验证骨髓中碳酸酐酶1和抗氧化蛋白6表达的结果
2.3.2.1免疫组化法验证骨髓中碳酸酐酶1表达的结果
碳酸酐酶1(CA1)在各组大鼠中均有表达,模型组大鼠骨髓基质细胞CA1蛋白表达阳性密度较正常对照组显著降低。左归丸组CA1蛋白表达阳性密度明显高于模型组,但与正常对照组相比,无显著差异。说明左归丸对CA1蛋白的低表达有调节作用,结果与凝胶分析和质谱分析结果相符。
2.3.2.2免疫组化验证骨髓中抗氧化蛋白6表达的结果
抗氧化蛋白6(PRDX6)在各组大鼠中均有表达,模型组大鼠骨髓基质细胞PRDX6蛋白表达阳性密度较正常对照组显著升高。左归丸组PRDX6蛋白表达阳性密度显著低于模型组,但与正常对照组相比,无显著差异。说明左归丸刘PRDX6蛋白的高表达有调节作用,结果凝胶分析和质谱分析结果相符。
结论
1.切除大鼠卵巢14周后,作为骨量主要标志的胫骨TBV%显著降低,而代表骨吸收参数的TRS%,以及代表骨形成参数的TFS%、MAR、OSW和mAR均显著增高,说明卵巢切除所造成的是一种骨吸收大于骨形成的高转换型骨质疏松症；而左归丸能在一定程度上使上述指标发生逆转,表明其对卵巢切除所致的大鼠骨质疏松症具有一定的预防作用。
2.应用蛋白组学方法从大鼠股骨骨髓中分析鉴定出11个差异蛋白质。其中,模型组骨髓中高表达而被左归丸下调至正常组水平的蛋白质有果糖醛缩酶A、抗氧化蛋白6、α烯醇化酶、烯醇化酶3、白蛋白、钙网蛋白、蛋白二硫化物异构酶A3；模型组骨髓中低表达而被左归丸上调至正常组水平的蛋白质有碳酸酐酶1、S100-A8蛋白。这些差异蛋白质主要涉及能量代谢、抗氧化、信号传导、细胞增殖、分化和凋亡等多方面的功能。说明在分子水平上,左归丸可通过调节骨髓内细胞增殖、分化、凋亡和能量代谢以及氧化反应等来实现对去卵巢所致大鼠骨质疏松症的预防作用。同时基于中医理论对绝经后骨质疏松症的认识,这些差异蛋白质是与绝经后骨质疏松症发生密切相关的蛋白质,也是与“肾主骨”密切相关的蛋白质。
Objective
To explore the changed proteins which is related with Zuoguiwan as a regulatory role by the methods of proteomics in the bone marrow of the model rats which have osteoporosis caused by bilateral ovariectomy.And then reveal the mechanism of Zuoguiwan during the treatment of the rats'osteoporosis induced by bilateral ovariectomy at the protein level which will also to provide further experimental evidence for exploring the scientific connotation of the mechanism of "The kidney dominating bone"
Methods
1Rats grouping and drawn
Firstly,one hundred female Wistar rats were randomly divided into three groups:twenty in normal control group,twenty in the pseudo-operation group,twenty in model group,twenty in stilbestrol group, twenty in Zuoguiwan group.Both ovaries of all rats in the operation group were extirpated,while only a little fatty tissue nearby the ovaries in the pseudo-operation group were extirpated.Distilled water was administrated orally to normal control group,pseudo-operation group and model group at the same time. The rats in Stilbestrol group and Zuoguiwan group were treated with stilbestrol tablets and decoction of Zuoguiwan.The Stilbestrol tablets was crushed,used distilled water as the the suspension concentration0.0046mg/ml,and the dose was0.046g/kg weight.The concentration of Zuoguiwan was0.968g/ml, and the dose was9.68/kg weight. The drugs were given once a day and last six days. There is one day paused every six days. The whole period last twelve weeks.
At the third day and the fifteenth day before the day all the rats were killed,hydrochloric acheomycin were respectively injected into the abdomen of the rats according to30mg/kg to mark the bone with fluorescence.After being killed the right tibia of the rats were retained for bone tissue section. At the same time the left tibia of the rats were used for Frozen tissue section after being decalcified to observe the protein expression by means of immunohistochemistry comfirm the result of mass spectrographic analysis, and the bilateral femur for2-DE and Western-blot.
2Bone histomorphometry indicators analysis
Detected the rats'tibia TBV%,TRS%,TFS%,MAR.OSW and mAR by Qwin V3image analysis system.
3Bone marrow protein analysis and identification
Get the femur bone marrow from the rats of normal group.model group and Zuoguiwan group, and extract the proteins. With2-D electrophoresis technology to analyze and compare the expressing proteins in the marrow of the three groups, and find thedeferent proteins adjusted by Zuoguiwan which are considered to be the related protein with postmenopausal osteoporosis.Then these diferent proteins were identified by mass spectrographic analysis and get their relateded informations.Chose protein ADLOA to be verified by Western-blot and protein CA1and PRDX6to be verified by immunohistochemical methods.
Results
1Effects of Zuoguiwan on the bone histomorphometry indicators in ovariectomized rats suffered osteoporosis
After the bilateral ovaries of the rats were extiroated,TBV%of tibia significantly decreased, while TRS%、TFS%、MAR、OSW and mAR significantly increased,which indicated that the model established by extirpating bilateral ovaries is a kind of high transformed osteoporosis model which has higher resorption than formation in bone.Compared with model group, TBV%significantly increased while TRS%、 TFS%、MAR、OSW and mAR significantly decreased after being treated by zuoguiwan. So Zuoguiwan can treat osteoporosis by reducing the high transformation of the bone.
2Result of2-DE
Through Image Master2D Platinum5.0analysis,the numbers of protein expression in the femur bone marrow showed two-dimensional gel electrophoresis profiles is(826±35).Three-fold expression is used as the standard, and twenty-three differentially expressed protein spots were detected which were regulated and corrected by zuoguiwan in the model group.
3Results of Mass spectrometry
Eleven differentially expressed protein spots were detected by MALDI-TOP masss pectra,and11proteins spots obtain correct identification results. Among these proteins, the proteins which were highly expressed in model group and corrected by Zuoguiwan are carbonic anhydrase1, calreticulin, fructose-bisphosphate, aldolaseA, peroxiredoxin-6, enolase3, albumin, disulfide-isomerase A3, enolasel; the proteins which were lowly expressed in model group and corrected by Zuoguiwan are carbonic anhydrase1and protein S100-A8.
4Results of verifications
4.1Results of western-blot
Aldolase A was verified by western-blot.The map of western-blot shows: Aldolase A is expressed in the3groups.The level of expression is highest in the model group. The level of expression in zuoguiwan group is significantly decreased and is similar to that in stilbestrol group This result is in accorded with gel analysis and mass identification.
4.2Results of immunohistochemistry
CA1and PRDX6were verified by immunohistochemical methods.The results show:The expression of CA1is lowest in the model group and significantly increased in zuoguiwan group.The expression of PRDX6is highest in the model group and significantly decreased in zuoguiwan group.
Conclusion
1. After bilateral ovariectomy, Rats'bone resorption and bone formation were significantly enhanced, but bone resorption is more than bone formation, which resulted in a significant reduction in bone mass. This is a kind of high conversion of osteoporosis. Zuoguiwan can improve the abnormal changes in bone resorption and bone formation, reduce bone loss,and prevent the occurrence of ovariectomized induced osteoporosis disease.
2. Eleven differentially expressed proteins in the rats' femur bone marrow are screened and identified by proteomic methods. Among these proteins, the proteins which were highly expressed in model group but reduced by Zuoguiwan include carbonic anhydrase1, calreticulin, fructose-bisphosphate, aldolaseA, peroxiredoxin6, enolase3, albumin, disulfide-isomerase A3, alpha-enolase. The proteins which were lowly expressed in model group and increased by Zuoguiwan are carbonic anhydrase1and protein S100-A8. The functions of these proteins are related to energy metabolism, antioxidant, signal transduction, cell differentiation, proliferation and apoptosis, etc. Then we conclude that Zuoguiwan' treatment effect on the osteoporosis of the ovariectomized rat is achieved by regulating cell proliferation, differentiation, metabolism, apoptosis and oxidative reactions in the bone marrow. These proteins are also closely related with Postmenopausal osteoporosis and "The kidney dominating bone".
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