N-苄基十六碳酰胺促小鼠Leydig细胞增殖和分泌睾酮的 1H NMR代谢组学研究

doi:10.7503/cjcu20190256

摘要/Abstract

摘要：

采用基于核磁共振氢谱(¹H NMR)的细胞代谢组学技术探讨了玛咖有效成分N-苄基十六碳酰胺(NBH)促进小鼠Leydig细胞增殖和分泌睾酮的作用机制. 测定了小鼠Leydig细胞在给药前后的细胞增殖率和细胞培养液中的睾酮含量, 采用主成分分析和正交偏最小二乘判别分析, 研究了小鼠Leydig细胞在给药前后细胞破碎液中的代谢物差异, 并进行了代谢通路分析. 实验结果表明, NBH能提高小鼠Leydig细胞增殖率和睾酮分泌量, 给药后小鼠Leydig细胞破碎液中的代谢轮廓明显改变, 共鉴定亮氨酸、赖氨酸、鲨肌醇、缬氨酸和丙氨酸等24种差异代谢物. 经Metaboanalyst分析发现, 差异代谢物主要涉及丙氨酸/天冬氨酸/谷氨酸代谢、甘氨酸/丝氨酸/苏氨酸代谢、谷胱甘肽代谢及丙酮酸代谢等10条新陈代谢和遗传信息处理代谢通路, 初步阐明了NBH通过调节上述代谢通路的相关节点发挥促进小鼠Leydig细胞增殖和分泌睾酮作用.

关键词: 细胞代谢组学, 核磁共振氢谱, N-苄基十六碳酰胺, 小鼠Leydig细胞

Abstract:

A metabolomic mechanism was evaluated for inducing proliferation and testosterone secretion of mouse testicular Leydig cells by N-benzylhexadecanamide(NBH) from maca based on ¹H NMR metabolism approach. The cell proliferation rate of mouse testicular Leydig cell sbefore and after administration and the testosterone content in the cell culture medium was determined experimentally. Based on principal component analysis(PCA) and orthogonal partial least squares discriminant analysis(OPLS-DA), metabolite differences in mouse testicular Leydig cells before and after administration were studied, and metabolic pathway analysis was performed. The results showed that NBH could increase the proliferation rate and testosterone secretion of mouse testicular Leydig cells. After administration, the profile of the faulty liquid metabolic in mouse testicular Leydig cells was changed significantly, which identified 24distinct metabolites such as leucine, lysine, scyllo-inositol, valine and alanine. Metaboanalyst showed that distinct metabolites mainly involves 10 pathways including alanine, aspartate and glutamate metabolism, glycine, serine and threonine metabolism, glutathione metabolism, pyruvate metabolism, pantothenate and CoA biosynthesis, aminoacyl-tRNA biosynthesis, lysine degradation, Inositol phosphate metabolism, D-glutamine and D-glutamate metabolism as well as glycerophospholipid metabolism. The result indicated that NBH promotes proliferation and secretion of testosterone in mouse Leydig cells by organizing the relevant nodes of the above metabolic pathway.

Key words: Cell metabonomics, ¹H NMR, N-Benzylhexadecanamide, Mouse testicular Leydig cells

中图分类号:

O657.2

TrendMD:

张楠茜, 吕经纬, 金平, 李晶峰, 边学峰, 张辉, 孙佳明. N-苄基十六碳酰胺促小鼠Leydig细胞增殖和分泌睾酮的 ¹H NMR代谢组学研究. 高等学校化学学报, 2019, 40(9): 1832.

ZHANG Nanxi, LV Jingwei, JIN Ping, LI Jingfeng, BIAN Xuefeng, ZHANG Hui, SUN Jiaming. ¹H NMR Metabonomic Investigations of N-Benzylhexadecanamide Induced Proliferation and Iestosterone Secretion of Mouse Testicular Leydig Cells^†. Chem. J. Chinese Universities, 2019, 40(9): 1832.

图/表 6

参考文献 31

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Group code	Concentration of NBH/(μg·mL_-1)	Cell viablility rate(%)	Testosteron secretion/(nmol·L_-1)
Control	0	100±1.73_**	15.70±0.85_**
NBH	62.5	121.49±1.59_**	24.50±1.77_**
NBH	125.0	125.82±2.12_*	28.71±2.08_**
NBH	250.0	130.54±1.93_**	31.10±1.93_**

Group code	Concentration of NBH/(μg·mL_-1)	Cell viablility rate(%)	Testosteron secretion/(nmol·L_-1)
Control	0	100±1.73_**	15.70±0.85_**
NBH	62.5	121.49±1.59_**	24.50±1.77_**
NBH	125.0	125.82±2.12_*	28.71±2.08_**
NBH	250.0	130.54±1.93_**	31.10±1.93_**

No.	Name	δ
1	Pantothenic acid	0.90(s), 0.94(s), 3.96(s)
2	Leucine	0.96(d), 0.97(d), 1.68(m), 3.74(m)
3	Valine	0.99(d, 7.0), 1.01(d, 7.0), 1.04(d)
4	3-Hydroxybutyrate	1.15(d, 6.0)
5	Ethanol	1.19(t, 7.2), 3.65(q)
6	Lipid	1.27(m), 2.0(m), 4.24(m)
7	Lactate	1.33(d), 4.11(q)
8	Alanine	1.48(d, 7.2), 3.78(m)
9	Lysine	1.72(m), 1.89(m), 3.04(t, J=7.57 Hz), 3.76(t, J=6.11 Hz)
10	Acetic acid	1.92(s)
11	Pyroglutamate	2.06(m), 2.35(m)
12	Glutamate	2.09(m), 2.13(m), 2.36(m), 2.46(m)
13	Methionine	2.14(s), 2.65(t)
14	Citrate	2.53(d), 2.69(d)
15	Dimethylamine	2.73(s)
16	Sarcosine	2.76(s), 3.60(s)
17	Aspartate	2.85(dd, 17.3, 3.7), 3.91(m)
No.	Name	δ
18	Glutathione	2.16(m), 2.56(m), 2.94(m)
19	Creatine	3.03s, 3.23(s), 3.93s
20	Ethanolamine	3.12(t, 5.5 Hz), 3.15(t, J=7.2 Hz), 3.86(t)
21	Malonate	3.15(s)
22	O-Phosphocholine	3.21(s)
23	Phosphocholine	3.22(s), 3.59(m), 4.17(m)
24	Histidine	3.25(dd), 7.08(s), 7.82(s)
25	Betaine	3.27(s)
26	Inositol	3.28(t, J=9.0 Hz), 3.63(t, J=9.6 Hz), 4.07(t, J=3.0 Hz)
27	Scyllo-inositol	3.37(s)
28	Taurine	3.44(t)
29	Methyl phosphate	3.47(d, 10.2)
30	Glycero	3.55(dd, 4.2, 9.6), 3.66(dd, 4.2, 11.4)
31	Glycine	3.57(s), 3.68(s)
32	Threonine	1.33(d), 3.58(d), 4.25(m)
33	Hippurate	3.97(d, 6.0), 7.54(t)
34	Adenosine monophosphate	4.01(m), 4.37(m), 4.51(m), 6.14(d), 8.27(s), 8.61(s)
35	Adenosine	4.30(dd), 8.24(s), 8.35(s)
36	Inosine	4.28(m), 4.44(m), 6.11(d), 8.23(s), 8.34(s)
37	Lactose	4.46(d), 4.66(d, J=7.8 Hz)
38	β-Glucose	4.50(d, 7.9)
39	α-Glucose	5.24(d, 3.6)
40	Urea	5.81(s)
41	Uracil	5.81(d, J=9.0 Hz)
42	Guanosine monophosphate	5.94(d), 8.20(s)
43	Uridine monophosphate	6.00(m), 8.11(d)
44	Cytidine	6.08(d), 7.85(d)
45	Fumaric acid	6.52(s)
46	Tyrosine	6.89(d), 7.19(d)
47	Tryptophan	7.33(d)
48	Phenylacetylglycine	3.68(s), 7.37(m), 7.43(m)
49	Uridine	7.92(d, 8.2)
50	Inosine monophosphate	8.25(s), 8.58(s)
51	Formate	8.46(s)

No.	Name	δ
1	Pantothenic acid	0.90(s), 0.94(s), 3.96(s)
2	Leucine	0.96(d), 0.97(d), 1.68(m), 3.74(m)
3	Valine	0.99(d, 7.0), 1.01(d, 7.0), 1.04(d)
4	3-Hydroxybutyrate	1.15(d, 6.0)
5	Ethanol	1.19(t, 7.2), 3.65(q)
6	Lipid	1.27(m), 2.0(m), 4.24(m)
7	Lactate	1.33(d), 4.11(q)
8	Alanine	1.48(d, 7.2), 3.78(m)
9	Lysine	1.72(m), 1.89(m), 3.04(t, J=7.57 Hz), 3.76(t, J=6.11 Hz)
10	Acetic acid	1.92(s)
11	Pyroglutamate	2.06(m), 2.35(m)
12	Glutamate	2.09(m), 2.13(m), 2.36(m), 2.46(m)
13	Methionine	2.14(s), 2.65(t)
14	Citrate	2.53(d), 2.69(d)
15	Dimethylamine	2.73(s)
16	Sarcosine	2.76(s), 3.60(s)
17	Aspartate	2.85(dd, 17.3, 3.7), 3.91(m)
No.	Name	δ
18	Glutathione	2.16(m), 2.56(m), 2.94(m)
19	Creatine	3.03s, 3.23(s), 3.93s
20	Ethanolamine	3.12(t, 5.5 Hz), 3.15(t, J=7.2 Hz), 3.86(t)
21	Malonate	3.15(s)
22	O-Phosphocholine	3.21(s)
23	Phosphocholine	3.22(s), 3.59(m), 4.17(m)
24	Histidine	3.25(dd), 7.08(s), 7.82(s)
25	Betaine	3.27(s)
26	Inositol	3.28(t, J=9.0 Hz), 3.63(t, J=9.6 Hz), 4.07(t, J=3.0 Hz)
27	Scyllo-inositol	3.37(s)
28	Taurine	3.44(t)
29	Methyl phosphate	3.47(d, 10.2)
30	Glycero	3.55(dd, 4.2, 9.6), 3.66(dd, 4.2, 11.4)
31	Glycine	3.57(s), 3.68(s)
32	Threonine	1.33(d), 3.58(d), 4.25(m)
33	Hippurate	3.97(d, 6.0), 7.54(t)
34	Adenosine monophosphate	4.01(m), 4.37(m), 4.51(m), 6.14(d), 8.27(s), 8.61(s)
35	Adenosine	4.30(dd), 8.24(s), 8.35(s)
36	Inosine	4.28(m), 4.44(m), 6.11(d), 8.23(s), 8.34(s)
37	Lactose	4.46(d), 4.66(d, J=7.8 Hz)
38	β-Glucose	4.50(d, 7.9)
39	α-Glucose	5.24(d, 3.6)
40	Urea	5.81(s)
41	Uracil	5.81(d, J=9.0 Hz)
42	Guanosine monophosphate	5.94(d), 8.20(s)
43	Uridine monophosphate	6.00(m), 8.11(d)
44	Cytidine	6.08(d), 7.85(d)
45	Fumaric acid	6.52(s)
46	Tyrosine	6.89(d), 7.19(d)
47	Tryptophan	7.33(d)
48	Phenylacetylglycine	3.68(s), 7.37(m), 7.43(m)
49	Uridine	7.92(d, 8.2)
50	Inosine monophosphate	8.25(s), 8.58(s)
51	Formate	8.46(s)

No.	Metabolites	VIP	FC	P-value	Trend_a	Related pathway
1	Pantothenic acid	1.70	4.20	3.65×10_-5	↑_**	Pantothenate and CoA biosynthesis
2	Leucine	3.36	9.11	3.57×10_-4	↑_**	Aminoacyl-tRNA biosynthesis
3	Valine	2.27	9.52	1.89×10_-3	↑_**	Aminoacyl-tRNA biosynthesis, pantothenate
						and CoA biosynthesis
6	Lipid	1.67	9.06	3.10×10_-5	↑_**	-
7	Lactate	1.42	4.06	3.86×10_-3	↑_**	Pyruvate metabolism
8	Alanine	2.25	9.68	1.14×10_-3	↑_**	Aminoacyl-tRNA biosynthesis, alanine, aspar-
						tate and glutamate metabolism
9	Lysine	2.46	11.12	2.61×10_-5	↑_**	Aminoacyl-tRNA biosynthesis
						Lysine degradation
10	Acetic acid	2.21	12.07	2.37×10_-4	↑_**	Pyruvate metabolism
11	Pyroglutamate	1.97	5.44	1.63×10_-5	↑_**	Glutathione metabolism
12	Glutamate	1.78	6.95	1.04×10_-5	↑_**	Aminoacyl-tRNA biosynthesis, glutathione metabolism, alanine, aspartate and glutamate metabolism, D-glutamine and D-glutamate metabolism
13	Methionine	1.68	6.89	2.25×10_-4	↑_**	Aminoacyl-tRNA biosynthesis
17	Aspartate	1.42	4.01	3.64×10_-6	↑_**	Aminoacyl-tRNA biosynthesis, alanine, aspartate and glutamate metabolism, pantothenate and CoA biosynthesis, glycine, serine and threonine metabolism
18	Glutathione	1.08	18.44	1.87×10_-5	↑_**	Glutathione metabolism
20	Ethanolamine	1.09	4.45	1.08×10_-5	↑_**	Glycerophospholipid metabolism
21	Malonate	1.64	3.84	1.21×10_-3	↑_**	-
23	Phosphocholine	2.02	2.86	3.41×10_-5	↑_**	Glycerophospholipid metabolism
26	Inositol	1.66	6.59	2.38×10_-4	↑_**	Inositol phosphate metabolism
27	Scyllo-inositol	2.36	0.17	4.76×10_-4	↓_**	-
31	Glycine	1.14	0.67	4.67×10_-2	↓_*	Aminoacyl-tRNA biosynthesis, glutathione metabolism, glycine, serine and threonine metabolism, lysine degradation
No.	Metabolites	VIP	FC	P-value	Trend_a	Related pathway
32	Threonine	1.42	4.06	3.86×10_-3	↑_**	Aminoacyl-tRNA biosynthesis, glycine, serine
						and threonine metabolism
34	Adenosine monophosphate	1.16	4.30	2.00×10_-5	↑_**
35	Adenosine	1.23	4.37	2.27×10_-5	↑_**
36	Inosine	1.06	4.62	4.67×10_-5	↑_**
48	Phenylacetylglycine	1.14	0.67	4.67×10_-2	↓_*

N-苄基十六碳酰胺促小鼠Leydig细胞增殖和分泌睾酮的 ¹H NMR代谢组学研究

¹H NMR Metabonomic Investigations of N-Benzylhexadecanamide Induced Proliferation and Iestosterone Secretion of Mouse Testicular Leydig Cells^†

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