基于蛋白质组学方法研究左归丸治疗去卵巢所致骨质疏松症大鼠的作用机制

Action mechanism of Zuogui Pill in ovariectomized-induced osteoporotic rats based on proteomics techniques

目的基于蛋白质组学方法研究左归丸治疗去卵巢所致骨质疏松症大鼠的作用机制,进而为阐明"肾主骨"理论的科学内涵寻找实验依据。方法将60只雌性Wistar大鼠随机分为2组,正常对照组20只和造模组40只。造模组大鼠通过摘除双侧卵巢建立绝经后骨质疏松症(PMOP)模型,正常对照组大鼠只摘取卵巢周围的少许脂肪组织。手术2周后,再将40只造模组大鼠随机分为模型组及左归丸组,各20只。左归丸组予左归丸水煎剂灌胃,正常对照组及模型组予等容积蒸馏水灌胃。给药3个月后,比较3组大鼠胫骨骨小梁体积百分比(TBV%)水平,取大鼠双侧股骨骨髓进行蛋白质双向电泳并对电泳图谱进行分析,筛选出左归丸干预的相关蛋白,并进行质谱分析鉴定。结果模型组、左归丸组大鼠胫骨TBV%水平低于正常对照组(P<0.05),左归丸组大鼠胫骨TBV%水平高于模型组(P<0.05)。3组大鼠共筛选出23个差异蛋白质点,其中12个蛋白质点由于蛋白质分数不具有统计学意义而未得到鉴定,11个蛋白质点得到鉴定。检索数据库显示10号差异蛋白质点alpha-enolase为α-烯醇化酶,其免疫组化法检测显示模型组α-烯醇化酶阳性表达密度高于正常对照组(P<0.05),而左归丸组α-烯醇化酶阳性表达密度低于模型组(P<0.05),这与双向凝胶电泳图谱分析和质谱鉴定结果相一致。结论左归丸可能通过调节骨髓内与细胞增殖、分化、代谢、凋亡及氧化反应等相关蛋白质来实现对PMOP的防治作用,这些差异蛋白质可能与PMOP的发生发展具有一定的相关性。

Objective To study the action mechanism of Zuogui Pill in ovariectomized - induced osteoporotic rats based on proteomics techniques, and to provide the experimental evidence of the theory of ＂Kidney dominating bone＂. Methods 60 female Wistar rats were randomly divided into two groups, 20 in normal control group and 40 in molding group. Postmenopausal osteoporosis （ PM OP） model was established by removing the bilateral ovaries in molding group, while only a little fatty tissue nearby the ovaries in normal control group were extirpated. Two weeks after operation, 40 rats in molding group were randomly divided into model and Zuogui pill group, 20 in each group. The Zuogui pill group received decoction of Zuogui pills by gavage, the normal control and model group received same dose distilled water by gavage. After three months administration, the percentage of trabecular bone volume （ TBV % ） of tibia were compared among three groups. The related proteins of Zuogui Pill intervention were selected from the femur bone by means of 2 - DE and electrophoretogram, then use for mass spectrometry analysis. Results The TBV % of tibia in model and Zuogui pill groups were lower than that in normal control group （P 〈 0. 05 ） , and its in Zuogui pill groups were higher than that in model group （P 〈 0 .05 ） . 12 protein spots, which selected from total 23 differential protein spots of three groups, were unrecognized by reason of the protein scores without statistical difference, and other 11 protein spots obtain correct identification. Search databases showed that the NO. 10 differential protein spot was alpha - enolase. The positive expression density of alpha - enolase which measured by mmunohistochemistry in model group was higher than that in normal control group （ P 〈 0. 05 ） , and its in Zuogui pill group was lower than that in model （ P 〈 0. 05 ） , and the result was in agreement with 2 - DE map and mass spectrometry analysis. Conclusion Zuogui pill has preventive and therapeutic effects on PMOP which may be through the regulation of bone marrows proteins relate to cell proliferation, differentiation, metabolism, apoptosis and oxidation reaction, such differential proteins may be have a positive correlation with occurrence and development of PMOP.