金刚丸抗去卵巢大鼠骨质疏松症血清代谢组学研究

Serum metabonomics study of anti-osteoporosis effect of Jingangwan in ovariectomized rats

目的 利用代谢组学初步探讨金刚丸治疗绝经后骨质疏松症的作用机制。方法 采用去双侧卵巢手术法复制绝经后骨质疏松大鼠模型,将SD雌性大鼠随机分为空白组、假手术组、模型组、金刚丸组(3.6 g/kg)和戊酸雌二醇组(9 mg/kg),每组6只,每天灌胃1次。连续给药12周后取大鼠右侧股骨进行苏木精-伊红染色法(hematoxylin-eosin staining, H&E)及骨密度(bone mineral density, BMD)测定,取腹主动脉血采用酶联免疫吸附法(enzyme-linked immunosorbent assay, ELISE)检测骨代谢指标碱性磷酸酶(alkaline phosphatase, ALP)、骨钙素(osteocalcin, OCN)、血清抗酒石酸酸性磷酸酶5b(tartrate resistant acid phosphatase 5b, TRAP 5b)以及用生化法检测血脂(total cholesterol, TC)、甘油三酯(total triglycerides, TG)、低密度脂蛋白胆固醇(low-density lipoprotein-cholesterol, LDL-C)、高密度脂蛋白胆固醇(high-density lipoprotein-cholesterol, HDL-C)的含量;血清样品前处理后进行代谢组学检测,综合主成分分析和正交偏最小二乘法判别分析,鉴定并筛选血清代谢物,并通过代谢数据库(MetaboAnalyst)富集相关代谢通路。结果 H&E染色结果显示金刚丸组骨小梁形态较模型组明显改善。与假手术组比较,模型组BMD、HDL-C水平下降(P<0.05或P<0.01),ALP、OCN、TRAP5b、TC、TG、LDL-C水平升高(P<0.05或P<0.01);与模型组比较,金刚丸组BMD、HDL-C水平上升(P<0.05或P<0.01),ALP、OCN、TRAP5b、TC、TG、LDL-C水平下降(P<0.05或P<0.01);分析确定了与金刚丸改善绝经后骨质疏松症密切相关的13个生物标志物及5条代谢通路,分别为花生四烯酸代谢、肌醇磷酸代谢、精氨酸和脯氨酸代谢、半乳糖代谢、磷脂酰肌醇信号传导系统。结论 金刚丸改善绝经后骨质疏松症可能与调节脂代谢、氨基酸代谢与糖代谢有关。

Objective To investigate the mechanism of Jingangwan in the treatment of postmenopausal osteoporosis based on serum metabolomics by gas chromatography-mass spectrometry. Methods The rat model of postmenopausal osteoporosis was replicated by bilateral ovariectomy, and SD female rats were randomly divided into blank group(Blank), sham operation group(Sham), model group(OVX), Jingangwan group(JGW, 3.6 g/kg) and estradiol valerate group(E2, 9 mg/kg), 6 rats in each group;and the drugs were given by intragastric administration once a day. After 12 weeks of administration, the right femurs of the rats were taken for hematoxylin-eosin staining(H&E) and bone mineral density(BMD) determination, The Enzyme-Linked ImmunoSorbent Assay(ELISE) method was used to detect bone metabolism indicators alkaline phosphatase(ALP), osteocalcin(OCN), serum tartrate resistant acid phosphatase 5b(TRAP5b), and biochemical method to detect total cholesterol(TC), total triglycerides(TG), low-density lipoprotein cholesterol(LDL-C), high-density lipoprotein cholesterol(HDL-C) content. After pre-treatment, serum samples were metabolically examined, principal component analysis and orthogonal partial least square discriminant analysis were combined to identify and screen serum metabolites, and related metabolic pathways were enriched through MetaboAnalyst. Results H&E staining result showed that the morphology of bone trabecular bone in JGW group was significantly improved compared with that in model group. Compared with Sham group, BMD and HDL-C levels in OVX group were decreased(P<0.05 or P<0.01), while ALP, OCN, TRAP5b, TC, TG and LDL-C levels were increased(P<0.05 or P<0.01). Compared with OVX group, the levels of BMD and HDL-C in JGW group were increased(P<0.05 or P<0.01), while the levels of ALP, OCN, TRAP5b, TC, TG and LDL-C were decreased(P<0.05 or P<0.01);Thirteen biomarkers were determined by GC-MS analysis, and five metabolic pathways were obtained, including arachidonic acid metabolism, inositol phosphate metabolism, arginine and proline metabolism, galactose metabolism and phosphatidylinositol signal transduction system. Conclusion The effect of Jingangwan on PMOP may be related to regulating lipid metabolism, amino acid metabolism and glucose metabolism.