[1]黄晨,施杞,王拥军,等.基于代谢组学技术探讨肾阳方治疗骨质疏松症肾阳虚证的作用机制[J].中医正骨,2023,35(09):1-7,29.
　HUANG Chen,SHI Qi,WANG Yongjun,et al.A study of mechanism of Shenyang Fang(肾阳方)in treatment of osteoporosis with syndrome of kidney-yang deficiency based on metabonomics technology[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2023,35(09):1-7,29.
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基于代谢组学技术探讨肾阳方治疗骨质疏松症肾阳虚证的作用机制()
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《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第35卷
期数:
2023年09期
页码:
1-7,29
栏目:
基础研究
出版日期:
2023-09-20

文章信息/Info

Title:
A study of mechanism of Shenyang Fang(肾阳方)in treatment of osteoporosis with syndrome of kidney-yang deficiency based on metabonomics technology
作者:
黄晨; 施杞; 王拥军; 唐德志
上海中医药大学附属龙华医院,上海 200032
Author(s):
HUANG Chen; SHI Qi; WANG Yongjun; TANG Dezhi
Longhua Hospital Shanghai University of Traditional Chinese Medicine,Shanghai 200032,China
关键词:
骨质疏松;  肾阳虚;  代谢组学
Keywords:
osteoporosis;  syndrome of deficiency of kidney yang;  metabonomics
摘要:
目的:探讨肾阳方治疗骨质疏松症(osteoporosis,OP)肾阳虚证的作用机制。方法:将30只2月龄雌性SD大鼠随机分为3组,每组10只。对照组大鼠从双侧卵巢附近切除少量脂肪组织,模型组和肾阳方组大鼠摘除双侧卵巢,造模手术1个月后模型组和肾阳方组大鼠皮下注射氢化可的松注射液进行肾阳虚证造模。造模结束后,肾阳方组以肾阳方药液灌胃,其余2组均以等量生理盐水灌胃,连续干预60 d。药物干预结束后,观察各组大鼠一般情况,采用ELISA法进行血清指标检测,以Micro-CT检测大鼠L4骨密度及骨组织微结构,采用超高效液相色谱-质谱法进行血清代谢组学分析。结果:①一般情况观察结果。模型组大鼠出现肾阳虚证表现,肾阳方组大鼠肾阳虚证表现较模型组明显改善。②血清指标检测结果。3组大鼠雌二醇(estradiol,E2)血清含量、环磷酸腺苷(cyclic adenosine monophosphate,cAMP)血清含量、cAMP/环磷酸鸟苷(cyclic guanosine monophosphate,cGMP)、骨钙素(osteocalcin,OCN)血清含量、Ⅰ型胶原C末端肽(C-telopeptide of typeⅠcollagen,CTX-Ⅰ)血清含量比较,组间差异均有统计学意义[E2:(99.74±9.53)ng·L-1,(42.84±10.84)ng·L-1,(60.53±13.03)ng·L-1,F=48.850,P=0.000; cAMP:(206.00±13.30)nmol·L-1,(184.70±13.01)nmol·L-1,(198.80±10.64)nmol·L-1,F=7.182,P=0.003; cAMP/cGMP:3.98±0.34,3.11±0.48,3.58±0.35,F=10.374,P=0.001; OCN:(4.62±1.04)ng·L-1,(2.36±0.87)ng·L-1,(4.24±1.63)ng·L-1,F=6.366,P=0.007; CTX-Ⅰ:(756.16±74.82)ng·L-1,(949.36±86.23)ng·L-1,(856.69±55.44)ng·L-1,F=13.776,P=0.000]; 3组大鼠cGMP血清含量的差异无统计学意义[(52.28±7.69)nmol·L-1,(61.39±14.26)nmol·L-1,(56.15±8.06)nmol·L-1,F=1.572,P=0.228]。对照组和肾阳方组大鼠的E2血清含量、cAMP血清含量、cAMP/cGMP、OCN血清含量均高于模型组[E2:P=0.000,P=0.002; cAMP:P=0.001,P=0.014; cAMP/cGMP:P=0.000,P=0.012; OCN:P=0.004,P=0.008],CTX-Ⅰ血清含量均低于模型组(P=0.000,P=0.013)。③骨密度和骨组织微结构检测结果。3组大鼠骨密度、骨体积分数、骨小梁数目、骨小梁厚度、骨小梁分离度比较,组间差异均有统计学意义[骨密度:(385.630±46.664)mg·cm-3,(246.648±21.253)mg·cm-3,(283.183±24.332)mg·cm-3,F=47.533,P=0.000; 骨体积分数:(0.441±0.055)%,(0.286±0.022)%,(0.325±0.027)%,F=45.608,P=0.000; 骨小梁数目:(4.525±0.497)个·mm-1,(3.206±0.289)个·mm-1,(3.536±0.232)个·mm-1,F=35.684,P=0.000; 骨小梁厚度:(0.094±0.005)mm,(0.086±0.007)mm,(0.090±0.005)mm,F=4.596,P=0.019; 骨小梁分离度:(0.199±0.028)mm,(0.300±0.039)mm,(0.262±0.021)mm,F=23.218,P=0.000]。对照组和肾阳方组的骨密度、骨体积分数、骨小梁数目均高于模型组(骨密度:P=0.000,P=0.007; 骨体积分数:P=0.000,P=0.012; 骨小梁数目:P=0.000,P=0.023),骨小梁分离度均低于模型组(P=0.000,P=0.006); 对照组的骨小梁厚度大于模型组(P=0.006),肾阳方组和模型组骨小梁厚度的差异无统计学意义(P=0.068)。④血清代谢组学分析结果。对照组和模型组、肾阳方组和模型组的代谢物均能明显分离。模型组较对照组有21个代谢物上调、7个代谢物下调,肾阳方组较模型组有89个代谢物上调、9个代谢物下调。这些差异代谢物中13个为氨基酸或与氨基酸有关,93个为胆固醇酯、鞘磷脂、甘油二酯、甘油三酯等脂质代谢物。模型组较对照组CE(15:0)浓度上升、TG(16:0_37:3)浓度下降,肾阳方组较模型组CE(15:0)浓度下降、TG(16:0_37:3)浓度上升。KEGG通路富集分析结果显示,对照组和模型组差异代谢物的信号通路有氨酰tRNA生物合成、谷胱甘肽代谢、氨基乙酸-丝氨酸-苏氨酸代谢、苯丙氨酸-酪氨酸-色氨酸生物合成、缬氨酸-亮氨酸-异亮氨酸生物合成、泛醌和其它萜类-醌生物合成、苯丙氨酸代谢、组氨酸代谢、β-丙氨酸代谢、卟啉和叶绿素代谢、二羧酸代谢、精氨酸和脯氨酸代谢、酪氨酸代谢、初级胆汁酸生物合成,肾阳方组和模型组差异代谢物的信号通路有氨酰tRNA生物合成、生物素代谢、精氨酸生物合成、硒代谢、赖氨酸降解、丙氨酸-天冬氨酸-谷氨酸代谢、半胱氨酸和蛋氨酸代谢。结论:OP肾阳虚证的发生可能与氨基酸、脂质代谢紊乱有关,肾阳方治疗可以纠正部分氨基酸、脂质代谢紊乱,从而治疗OP肾阳虚证。
Abstract:
Objective:To explore the mechanism of Shenyang Fang(肾阳方,SYF)in treatment of osteoporosis(OP)with kidney-yang deficiency syndrome(KYDS).Methods:Thirty 2-month-old female Sprague-Dawley(SD)rats were randomly assigned into control group,model group and SYF group,10 cases in each group.The rats in control group were subjected to removal of a small amount adipose tissues near the ovaries,with ovaries retained; while the ones in model group and SYF group underwent bilateral ovariectomy for inducing OP,followed by subcutaneous injection of hydrocortisone injection at 1 month after the modeling surgery for inducing KYDS.After the end of molding,the mice in SYF group were intragastric administrated with SYF solution,while the ones in control group and model group with the same dose of normal saline.All mice in the 3 groups were intragastric administrated for consecutive 60 days.After the end of drug intervention,the general condition of the rats was observed,the serum indicators were detected by using ELISA method,and the bone mineral density(BMD)and bone microstructure of L4 lumbar vertebra were evaluated by using Micro-CT.Furthermore,the serum metabolomic analysis was performed by using ultra-high-performance liquid chromatography-mass spectrometry(UHPLC-MS).Results:①The KYDS was found in rats of model group,and it was significantly improved in rats of SYF group in contrast to that of model group.②There was statistical difference in cAMP/cGMP and serum levels of estradiol(E2),cAMP,osteocalcin(OCN)and CTX-Ⅰamong the 3 groups(cAMP/cGMP:3.98±0.34,3.11±0.48,3.58±0.35,F=10.374,P=0.001; E2:99.74±9.53,42.84±10.84,60.53±13.03 ng/L,F=48.850,P=0.000; cAMP:206.00±13.30,184.70±13.01,198.80±10.64 nmol/L,F=7.182,P=0.003; OCN:4.62±1.04,2.36±0.87,4.24±1.63 ng/L,F=6.366,P=0.007; CTX-Ⅰ:756.16±74.82,949.36±86.23,856.69±55.44 ng/L,F=13.776,P=0.000); while there was no statistical difference in the serum level of cGMP among the 3 groups(52.28±7.69,61.39±14.26,56.15±8.06 nmol/L,F=1.572,P=0.228).The cAMP/cGMP and serum levels of E2,cAMP and OCN were higher,while the serum level of CTX-Ⅰ was lower in control group and SYF group compared to model group(cAMP/cGMP:P=0.000,P=0.012; E2:P=0.000,P=0.002; cAMP:P=0.001,P=0.014; OCN:P=0.004,P=0.008; CTX-Ⅰ:P=0.000,P=0.013).③The differences in BMD,bone volume fraction(BVF),trabecular number(Tb.N),trabecular thickness(Tb.Th)and trabecular separation(Tb.Sp)were statistically significant among the 3 groups(BMD:385.630±46.664,246.648±21.253,283.183±24.332 mg/cm(3),F=47.533,P=0.000; BVF:0.441±0.055,0.286±0.022,0.325±0.027%,F=45.608,P=0.000; Tb.N:4.525±0.497,3.206±0.289,3.536±0.232 trabeculae/mm,F=35.684,P=0.000; Tb.Th:0.094±0.005,0.086±0.007,0.090±0.005 mm,F=4.596,P=0.019; Tb.Sp:0.199±0.028,0.300±0.039,0.262±0.021 mm,F=23.218,P=0.000).The control group and SYF group had higher BMD,BVF and Tb.N,but lower Tb.Sp compared to model group(BMD:P=0.000,P=0.007; BVF:P=0.000,P=0.012; Tb.N:P=0.000,P=0.023; Tb.Sp:P=0.000,P=0.006).The Tb.Th was greater in control group compared to model group,while there was no statistical difference between SYF group and model group(P=0.006; P=0.068).④The metabolites could be distinctly separated between control group and model group as well as between SYF group and model group.Twenty-one metabolites were up-regulated and 7 ones were down-regulated in model group compared with that of control group; while 89 metabolites were up-regulated and 9 ones were down-regulated in SYF group compared with that of model group.Among these differential metabolites,13 ones were amino acids or related to amino acids,and 93 ones were lipid metabolites,including cholesterol esters,sphingomyelin,diglycerides and triglycerides.The concentration of CE(15:0)increased,and TG(16:0_37:3)decreased in model group compared to control group; while the concentration of CE(15:0)decreased,and TG(16:0_37:3)increased in SYF group compared with that of model group.The results of KEGG signaling pathway enrichment analysis indicated that the signaling pathways of differential metabolites between control group and model group included aminoacyl-tRNA biosynthesis,glutathione metabolism,glycine-serine-threonine metabolism,phenylalanine-tyrosine-tryptophan biosynthesis,valine-leucine-isoleucine biosynthesis,ubiquinone and other terpenoid-quinone biosynthesis,phenylalanine metabolism,histidine metabolism,β-alanine metabolism,porphyrin and chlorophyll metabolism,dicarboxylic acid metabolism,arginine and proline metabolism,tyrosine metabolism and primary bile acid biosynthesis; the signaling pathways of differential metabolites between SYF group and model group included aminoacyl-tRNA biosynthesis,biotin metabolism,arginine biosynthesis,selenium metabolism,lysine degradation,alanine-aspartate-glutamate metabolism,and cysteine and methionine metabolism.Conclusion:The occurrence of OP with KYDS may be related to the disorder of amino acid and lipid metabolism.SYF can correct some of these metabolism disorders so as to treat KYDS of OP.

参考文献/References:

[1] 中华医学会骨质疏松和骨矿盐疾病分会.原发性骨质疏松症诊疗指南(2017)[J].中华骨质疏松和骨矿盐疾病杂志,2017,10(5):413-444.
[2] 游利.骨质疏松症的现状、筛查和预防[J].中国全科医学,2016,19(14):1616-1619.
[3] 中华人民共和国国家统计局.中国统计年鉴2021[M].北京:中国统计出版社,2021:54.
[4] MESSINGER-RAPPORT B J,THACKER H L.Prevention for the older woman.A practical guide to prevention and treatment of osteoporosis[J].Geriatrics,2002,57(4):16-18.
[5] 梅润宏.基于脂质代谢组学技术探讨中药骨蚀灵胶囊干预激素性股骨头坏死的作用机制[D].南昌:南昌大学,2022.
[6] 韩艳,温利平,刘娜,等.补肾活血方对去卵巢大鼠骨代谢及骨密度的影响[J].中医正骨,2015,27(12):7-11.
[7] 李媛,许红涛,李华强,等.肾阳虚骨质疏松大鼠动物模型的建立[J].药物评价研究,2015,38(2):135-139.
[8] 陈剑磨,张胜军,夏炳江.骨质疏松肾阳虚病证结合模型构建的实验研究[J].中国中医急症,2015,24(2):201-203.
[9] 招文华,任辉,沈耿杨,等.骨质疏松症肾阳虚病证结合模型的研究进展[J].中华中医药杂志,2018,33(1):219-223.
[10] 张海洋.辛甘温阳法对肺阳虚反复感冒者cGMP、cAMP/cGMP相关性的验证性试验研究[D].长春:长春中医药大学,2011.
[11] 张子怡,陈宝军,张英杰,等.肾阳虚、肾阴虚证大鼠模型的建立与稳定性观察[J].福建中医药,2015,46(1):51-54.
[12] 郭胜男,戚晓楠,姚啸生,等.UP LC-MS/MS对肾阳虚原发性骨质疏松临床代谢标志物筛查研究[J].中国骨质疏松杂志,2022,28(10):1410-1415.
[13] 朱庭辰.基于阴/阳虚证的原发性骨质疏松症临床代谢组学研究[D].南京:南京中医药大学,2020.
[14] MOUTIEZ M,BELIN P,GONDRY M.Aminoacyl-tRNA-Utilizing enzymes in natural product biosynthesis[J].Chem Rev,2017,117(8):5578-5618.
[15] 张琳琳,张静莹,曲哲,等.还原型谷胱甘肽促进高糖成骨细胞的增殖和分化[J].中国组织工程研究,2015,19(7):985-990.
[16] 杨剑锋,孙春汉.成骨细胞增殖分化中还原型谷胱甘肽的作用研究[J].中国卫生工程学,2017,16(3):401-402.
[17] 代志鹏.谷胱甘肽对激素性股骨头坏死骨髓间充质干细胞分化的影响[D].武汉:华中科技大学,2015.
[18] JIANG M Y,CAI D P.Oral arginine improves linear growth of long bones and the neuroendocrine mechanism[J].Neurosci Bull,2011,27(3):156-162.
[19] 张世旭,路考生,石昌浩,等.左旋精氨酸对骨质疏松骨折骨愈合及血生化的影响[J].中国组织工程研究,2013,17(28):5120-5125.
[20] 镐英杰,王珍,李秀群,等.左旋精氨酸对大鼠成骨细胞的影响[J].中华实验外科杂志,2007,24(9):1148.
[21] 雍家成,朱菲,陈昊,等.自然人群甘油三酯葡萄糖指数与骨质疏松关系的横断面研究[J].实用临床医药杂志,2023,27(6):29-32.
[22] YE M,ZHANG C,JIA W,et al.Metabolomics strategy reveals the osteogenic mechanism of yak(Bos grunniens)bone collagen peptides on ovariectomy-induced osteoporosis in rats[J].Food Funct,2020,11(2):1498-1512.
[23] 朱玉杰.罗非鱼鱼头脂质改善大鼠骨质疏松症的作用及其机制研究[D].海口:海南大学,2022.
[24] 唐翠松,汤光宇.骨髓脂肪酸与骨质疏松相关性的研究进展[J].中国骨质疏松杂志,2023,29(3):420-425.

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[11]曹俊青,郑剑南,张麟.右归丸联合阿仑膦酸钠口服治疗绝经后骨质疏松症肾阳虚证的临床研究[J].中医正骨,2018,30(05):20.
　CAO Junqing,ZHENG Jiannan,ZHANG Lin.A clinical study of oral application of Yougui Wan(右归丸)and alendronate sodium for treatment of postmenopausal osteoporosis with kidney-yang deficiency syndrome[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2018,30(09):20.
[12]李中万,徐绍俊,杨广钢,等.健肾方联合碳酸钙D3咀嚼片(Ⅱ) 治疗绝经后骨质疏松症肾阳虚证[J].中医正骨,2018,30(08):11.
　LI Zhongwan,XU Shaojun,YANG Guanggang,et al.Oral application of Jianshen Fang(健肾方)and calcium carbonate and Vitamin D3 chewable tablets(Ⅱ)for treatment of postmenopausal osteoporosis with kidney-yang deficiency syndrome[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2018,30(09):11.
[13]刘魏,童培建,肖鲁伟,等.益骨汤口服联合太极拳锻炼治疗老年性骨质疏松症肾阳虚证[J].中医正骨,2018,30(11):6.
　LIU Wei,TONG Peijian,XIAO Luwei,et al.Oral application of Yigu Tang(益骨汤)combined with shadow boxing exercises for treatment of osteoporosis with kidney-yang deficiency syndrome in the aged[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2018,30(09):6.
[14]汪青,黄昊强,陈勇,等.二仙汤在绝经后骨质疏松症肾阳虚证治疗中的应用价值及作用机制研究[J].中医正骨,2022,34(03):8.
　WANG Qing,HUANG Haoqiang,CHEN Yong,et al.Applied values and mechanism of action of oral application of Erxian Tang(二仙汤)in treatment of postmenopausal osteoporosis with syndrome of kidney-yang deficiency:a clinical study[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2022,34(09):8.

备注/Memo

备注/Memo:
基金项目:国家重点研发计划项目(2018YFC1704300); 国家中医药传承创新团队项目(ZYYCXTD-C-202202)
通讯作者:唐德志 E-mail:dztang702@126.com
更新日期/Last Update: 1900-01-01