[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.

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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