山柰酚对去势大鼠影响的4D-DIA蛋白质与代谢组学分析

4D-DIA protein and metabolomic analysis of the effects of kaempferol on ovariectomized rats

摘要:

[目的] 通过蛋白和代谢组学研究去势大鼠山柰酚抗骨质疏松的作用机制。[方法] 18 只雌性 SD 大鼠随机分为 3 组，每组 6 只。通过卵巢切除方法建立大鼠骨质疏松模型，山柰酚组按 40 mg/kg 标准给予山柰酚灌胃，模型组和假手术组给予蒸馏水灌胃，连续干预 12 周后，取大鼠股骨标本进行四维数据独立采集 （four-dimensional data-independent acquisition, 4DDIA）蛋白质组学及广泛靶向代谢组学，结合生物信息学探究差异蛋白及代谢物、代谢通路之间的相关性。[结果]4D-DIA 蛋白质组学结果显示 ， 3 组共鉴定出 A0A8I5Y0Y5、 A0A8I5ZMX9、 A0A8I6AI41、 D3Z9R8、 D4A7K7、 F7FLE6、 M0R6X7、 M0R7Q2、P08494、Q62740、Q68FP3、Q6AY77 等 12 个共有差异蛋白。XDH、IBSP、MGP 和 F2RL3 等蛋白的表达在山柰酚组显著上调（P＜0.05）。广泛靶向代谢组学结果显示，在 3 组中泛酸、L-精氨酸-L-丙氨酸等 25 种代谢物的表达存在差异。差异蛋白和代谢物表达相关性分析表明，山柰酚组和模型组之间有 3 个差异蛋白和代谢物呈现强相关性，XDH 与 L-异亮氨酸-L-组氨酸、 IBSP 与赖氨酰-缬氨酸、GPR183 与 L-组氨酸等代谢物呈显著负相关。差异蛋白和代谢物共同参与了维生素消化与吸收、代谢途径，其中 10 种差异代谢物和 3 个差异蛋白与代谢途径密切相关。[结论]山柰酚可能通过调节 XDH、IBSP、MGP 和 F2RL3 等蛋白的表达，调控氨基酸及其代谢物的代谢、维生素消化与吸收，起到防治骨质疏松的作用。

Abstract:

[Objective] To study the mechanism of kaempferol against osteoporosis by protein and metabolomics. [Methods] Eighteen female SD rats were randomly divided into three groups with six animals in each group. After ovariectomy or sham operation performed correspondingly, 40 mg/kg of kaempferol was given intragastrically in the kaempferol group, while distilled water was given intragastrically in the model group and sham groups. After 12 consecutive weeks of intervention, the rat femur specimens were taken for four-dimensional data-independent acquisition (4D-DIA) proteomics and broadly targeted metabolomics analysis, combined with bioinformatics to explore the correlation between differential proteins and metabolites and metabolic pathways. [Results] As results of the 4D-DIA proteomics, a total of 12 shared differential proteins were identified among the three groups, and the expression of proteins such as XDH, IBSP, MGP, and F2RL3 was up-regulated in the kaempferol group (P＜0.05). In term of extensively targeted metabolomics, differential expression of 25 metabolites, including pantothenic acid and L-arginine-L-alanine, were found among the three groups. The differential proteins and metabolites were jointly involved in vitamin digestion and absorption pathways and metabolic pathways. Specifically, 10 differential metabolites and 3 differential proteins are closely associated with metabolic pathways. [Conclusion] The kaempferol may exert its anti-osteoporotic effects by modulating the expression of proteins such as XDH, IBSP, MGP, and F2RL3, regulating the metabolism of amino acids and their metabolites, as well as vitamin digestion and absorption pathways