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Gene symbol	Gene bank	Primer sequence
Cpt1b	ENSRNOG00000010438	CATGTATCGCCGCAAACTGG	CCTGGGATGCGTGTAGTGTT
Pcsk9	ENSRNOG00000006280	AGGGCCAGAGAAGCAATGTC	GCACAGCCTGTCCTCTCATT
Abca1	ENSRNOG00000018126	CTGAAGCCACCACAACGTTG	AGAAACATCACCTCCTGCCG
Ascm5	ENSRNOG00000031211	GTGAAAGCAGCCTGGACTCT	TGTACAAGCCGCCGGAAGAG
Gng7	ENSRNOG00000019857	CAGAGAGGGCCAGGACTTTG	TCCCGGTCCCCAGAATGTAT
Col6a6	ENSRNOG00000023007	GACCAGCTGCTCAGGGAAAT	ACTGCTAAAGAACGTGGCGA
Sgk1	ENSRNOG00000011815	GCAGTGACGAGCATCCAGAT	ACGGCTCTGACTGACAACTG
Nr4a1	ENSRNOG00000007607	TGCCAAGTACATCTGCCTGG	GGCTGCTTGGGTTTTGAAGG
Gapdh	ENSRNOG00000018630	AAAGGGTCATCATCTCCGCC	AGTGATGGCATGGACTGTGG

Gene symbol	Gene bank	Primer sequence
Cpt1b	ENSRNOG00000010438	CATGTATCGCCGCAAACTGG	CCTGGGATGCGTGTAGTGTT
Pcsk9	ENSRNOG00000006280	AGGGCCAGAGAAGCAATGTC	GCACAGCCTGTCCTCTCATT
Abca1	ENSRNOG00000018126	CTGAAGCCACCACAACGTTG	AGAAACATCACCTCCTGCCG
Ascm5	ENSRNOG00000031211	GTGAAAGCAGCCTGGACTCT	TGTACAAGCCGCCGGAAGAG
Gng7	ENSRNOG00000019857	CAGAGAGGGCCAGGACTTTG	TCCCGGTCCCCAGAATGTAT
Col6a6	ENSRNOG00000023007	GACCAGCTGCTCAGGGAAAT	ACTGCTAAAGAACGTGGCGA
Sgk1	ENSRNOG00000011815	GCAGTGACGAGCATCCAGAT	ACGGCTCTGACTGACAACTG
Nr4a1	ENSRNOG00000007607	TGCCAAGTACATCTGCCTGG	GGCTGCTTGGGTTTTGAAGG
Gapdh	ENSRNOG00000018630	AAAGGGTCATCATCTCCGCC	AGTGATGGCATGGACTGTGG

Group	ALB/g·L^-1	BUN/mmol·L^-1	Scr/μmol·L^-1	TG/mmol·L^-1	TP/g·L^-1	TCHO/mmol·L^-1
C	33.06 ±1.73	5.06 ± 0.91	46.09 ± 11.32	0.36 ± 0.052	54.46 ± 1.79	1.42 ± 0.32
M	30.36 ± 2.19^*	5.69 ± 1.10^*	46.95 ± 9.43	0.512 ± 0.17^*	52.26 ± 2.90^**	2.49 ± 0.55^**
HQ	31.96 ± 0.97^#	5.07 ± 0.91^#	34.06 ± 2.85^##	0.37 ± 0.089^#	55.46 ± 2.02^##	1.66 ± 0.38^#

Group	ALB/g·L^-1	BUN/mmol·L^-1	Scr/μmol·L^-1	TG/mmol·L^-1	TP/g·L^-1	TCHO/mmol·L^-1
C	33.06 ±1.73	5.06 ± 0.91	46.09 ± 11.32	0.36 ± 0.052	54.46 ± 1.79	1.42 ± 0.32
M	30.36 ± 2.19^*	5.69 ± 1.10^*	46.95 ± 9.43	0.512 ± 0.17^*	52.26 ± 2.90^**	2.49 ± 0.55^**
HQ	31.96 ± 0.97^#	5.07 ± 0.91^#	34.06 ± 2.85^##	0.37 ± 0.089^#	55.46 ± 2.02^##	1.66 ± 0.38^#

Gene bank	Gene symbol	P-value	Fold change	Description	Trend
ENSRNOG00000032508	Acot5	0.047	0.423 797	Acyl-CoA thioesterase 5	↑↓
ENSRNOG00000005780	Adamts13	0.047	0.271 354	ADAM metallopeptidase with thrombospondin type 1 motif, 13	↑↓
ENSRNOG00000029844	Akr1c2	0.031	0.520 374	Aldo-keto reductase family 1, member C2	↑↓
ENSRNOG00000007980	Anxa13	0.004	0.479 046	Annexin A13	↑↓
ENSRNOG00000006365	Asb15	0.001	0.393 735	Ankyrin repeat and SOCS box containing 15	↑↓
ENSRNOG00000020685	Atp12a	0.042	0.547 411	ATPase H⁺/K⁺ transporting non-gastric alpha 2 subunit	↑↓
ENSRNOG00000002381	Bmp3	0.001	0.382 219	Bone morphogenetic protein 3	↑↓
ENSRNOG00000010438	Cpt1b	0.016	0.543 593	Carnitine palmitoyltransferase 1B	↑↓
ENSRNOG00000006873	Dnase1	0.000 07	0.408 402	Deoxyribonuclease 1	↑↓
ENSRNOG00000013770	Folh1	0.021	0.513 944	Folate hydrolase 1	↑↓
ENSRNOG00000011599	Gldc	0.001	0.450 592	Glycine decarboxylase CRA_a	↑↓
ENSRNOG00000003616	Grem2	0.041	0.364 554	Gremlin 2, DAN family BMP antagonist	↑↓
ENSRNOG00000029237	Klk1c2	0.001	0.306 455	Kallikrein 1-related peptidase C2	↑↓
ENSRNOG00000056076	Mif	0.001	0	Macrophage migration inhibitory factor	↑↓
ENSRNOG00000034078	Mxi1	0.006	0.537 201	MAX interactor 1, dimerization protein	↑↓
ENSRNOG00000007607	Nr4a1	0.004	0.414 562	Nuclear receptor subfamily 4, group A, member 1	↑↓
ENSRNOG00000051837	Nwd2	0.001	0.034 365	NACHT and WD repeat domain containing 2	↑↓
ENSRNOG00000006280	Pcsk9	0.001	0.040 151	Proprotein convertase subtilisin/kexin type 9	↑↓
ENSRNOG00000007387	Per1	0.012	0.541 879	Period circadian clock 1	↑↓
ENSRNOG00000014761	Rasd2	0.008	0.459 304	RASD family, member 2	↑↓
ENSRNOG00000029651	Rdh16	0.001	0.439 728	Retinol dehydrogenase 16 (all-trans)	↑↓
ENSRNOG00000014524	S1pr3	0.011	0.460 415	Sphingosine-1-phosphate receptor 3	↑↓
ENSRNOG00000011815	Shisa3	0.001	0.230 381	Shisa family member 3	↑↓
ENSRNOG00000025670	Sgk1	0.002	0.506 214	Serum/glucocorticoid regulated kinase 1	↑↓
ENSRNOG00000017072	Slc16a14	0.021	0.583 361	Solute carrier family 16, member 14	↑↓
ENSRNOG00000018131	Slc16a4	0.001	0.463 205	Solute carrier family 16, member 4	↑↓
ENSRNOG00000014338	Slc25a25	0.002	0.504 666	Solute carrier family 25, member 25	↑↓
ENSRNOG00000007591	Slc5a8	0.007	0.541 283	Solute carrier family 5, member 8	↑↓
ENSRNOG00000006367	Slc45a3	0.037	0.586 863	Solute carrier family 45, member 3	↑↓
ENSRNOG00000033830	Slc7a12	0.004	0.514 671	Solute carrier family 7, member 12	↑↓
ENSRNOG00000036984	Slco1a1	0.003	0.518 432	Solute carrier organic anion transporter family, member 1a1	↑↓
ENSRNOG00000021726	Tlr3	0.046	0.611 419	Toll-like receptor 3	↑↓
ENSRNOG00000039464	Tmem229a	0.016	0.438 692	Transmembrane protein 229A	↑↓
ENSRNOG00000019486	Trpv1	0.023	0.483 102	Transient receptor potential cation, subfamily Ⅴ, member 1	↑↓
ENSRNOG00000003832	Vash2	0.001	0.153 739	Vasohibin 2	↑↓
ENSRNOG00000018126	Abca1	0.034	1.803 634	ATP binding cassette subfamily A, member 1	↓↑
ENSRNOG00000031211	Acsm5	0.001	3.260 038	Acyl-CoA synthetase medium-chain family member 5	↓↑
ENSRNOG00000006460	Amdhd2	0.037	1.783 825	Amidohydrolase domain containing 2	↓↑
ENSRNOG00000029543	Cish	0.021	1.879 914	Cytokine inducible SH2-containing protein	↓↑
ENSRNOG00000023007	Col6a6	0.001	2.460 307	Collagen type Ⅵ alpha 6 chain	↓↑
ENSRNOG00000008534	Dusp15	0.043	2.175 131	Dual specificity phosphatase 15	↓↑
ENSRNOG00000009660	Enpp6	0.011	1.909 883	Ectonucleotide pyrophosphatase/phosphodiesterase 6	↓↑
ENSRNOG00000001806	Fetub	0.001	4.472 346	Fetuin B	↓↑
ENSRNOG00000000168	Gatm	0.001	2.314 874	Glycine amidinotransferase	↓↑
ENSRNOG00000019857	Gng7	0.045	1.784 171	Acyl-CoA thioesterase 5	↓↑
ENSRNOG00000014233	Krt19	0.009	2.704 678	ADAM metallopeptidase with thrombospondin type 1 motif, 13	↓↑
ENSRNOG00000006857	Ndnf	0.003	7.001 253	Aldo-keto reductase family 1, member C2	↓↑
ENSRNOG00000015353	Prss12	0.007	2.502 539	Annexin A13	↓↑
ENSRNOG00000014861	Rnf183	0.001	3.599 888	Ankyrin repeat and SOCS box containing 15	↓↑
ENSRNOG00000057620	Slc6a8	0.042	1.584 699	ATPase H⁺/K⁺ transporting non-gastric alpha 2 subunit	↓↑
ENSRNOG00000010296	Slc7a7	0.001	3.960 724	Bone morphogenetic protein 3	↓↑
ENSRNOG00000019924	Thop1	0.048	1.757 756	Carnitine palmitoyltransferase 1B	↓↑

Gene bank	Gene symbol	P-value	Fold change	Description	Trend
ENSRNOG00000032508	Acot5	0.047	0.423 797	Acyl-CoA thioesterase 5	↑↓
ENSRNOG00000005780	Adamts13	0.047	0.271 354	ADAM metallopeptidase with thrombospondin type 1 motif, 13	↑↓
ENSRNOG00000029844	Akr1c2	0.031	0.520 374	Aldo-keto reductase family 1, member C2	↑↓
ENSRNOG00000007980	Anxa13	0.004	0.479 046	Annexin A13	↑↓
ENSRNOG00000006365	Asb15	0.001	0.393 735	Ankyrin repeat and SOCS box containing 15	↑↓
ENSRNOG00000020685	Atp12a	0.042	0.547 411	ATPase H⁺/K⁺ transporting non-gastric alpha 2 subunit	↑↓
ENSRNOG00000002381	Bmp3	0.001	0.382 219	Bone morphogenetic protein 3	↑↓
ENSRNOG00000010438	Cpt1b	0.016	0.543 593	Carnitine palmitoyltransferase 1B	↑↓
ENSRNOG00000006873	Dnase1	0.000 07	0.408 402	Deoxyribonuclease 1	↑↓
ENSRNOG00000013770	Folh1	0.021	0.513 944	Folate hydrolase 1	↑↓
ENSRNOG00000011599	Gldc	0.001	0.450 592	Glycine decarboxylase CRA_a	↑↓
ENSRNOG00000003616	Grem2	0.041	0.364 554	Gremlin 2, DAN family BMP antagonist	↑↓
ENSRNOG00000029237	Klk1c2	0.001	0.306 455	Kallikrein 1-related peptidase C2	↑↓
ENSRNOG00000056076	Mif	0.001	0	Macrophage migration inhibitory factor	↑↓
ENSRNOG00000034078	Mxi1	0.006	0.537 201	MAX interactor 1, dimerization protein	↑↓
ENSRNOG00000007607	Nr4a1	0.004	0.414 562	Nuclear receptor subfamily 4, group A, member 1	↑↓
ENSRNOG00000051837	Nwd2	0.001	0.034 365	NACHT and WD repeat domain containing 2	↑↓
ENSRNOG00000006280	Pcsk9	0.001	0.040 151	Proprotein convertase subtilisin/kexin type 9	↑↓
ENSRNOG00000007387	Per1	0.012	0.541 879	Period circadian clock 1	↑↓
ENSRNOG00000014761	Rasd2	0.008	0.459 304	RASD family, member 2	↑↓
ENSRNOG00000029651	Rdh16	0.001	0.439 728	Retinol dehydrogenase 16 (all-trans)	↑↓
ENSRNOG00000014524	S1pr3	0.011	0.460 415	Sphingosine-1-phosphate receptor 3	↑↓
ENSRNOG00000011815	Shisa3	0.001	0.230 381	Shisa family member 3	↑↓
ENSRNOG00000025670	Sgk1	0.002	0.506 214	Serum/glucocorticoid regulated kinase 1	↑↓
ENSRNOG00000017072	Slc16a14	0.021	0.583 361	Solute carrier family 16, member 14	↑↓
ENSRNOG00000018131	Slc16a4	0.001	0.463 205	Solute carrier family 16, member 4	↑↓
ENSRNOG00000014338	Slc25a25	0.002	0.504 666	Solute carrier family 25, member 25	↑↓
ENSRNOG00000007591	Slc5a8	0.007	0.541 283	Solute carrier family 5, member 8	↑↓
ENSRNOG00000006367	Slc45a3	0.037	0.586 863	Solute carrier family 45, member 3	↑↓
ENSRNOG00000033830	Slc7a12	0.004	0.514 671	Solute carrier family 7, member 12	↑↓
ENSRNOG00000036984	Slco1a1	0.003	0.518 432	Solute carrier organic anion transporter family, member 1a1	↑↓
ENSRNOG00000021726	Tlr3	0.046	0.611 419	Toll-like receptor 3	↑↓
ENSRNOG00000039464	Tmem229a	0.016	0.438 692	Transmembrane protein 229A	↑↓
ENSRNOG00000019486	Trpv1	0.023	0.483 102	Transient receptor potential cation, subfamily Ⅴ, member 1	↑↓
ENSRNOG00000003832	Vash2	0.001	0.153 739	Vasohibin 2	↑↓
ENSRNOG00000018126	Abca1	0.034	1.803 634	ATP binding cassette subfamily A, member 1	↓↑
ENSRNOG00000031211	Acsm5	0.001	3.260 038	Acyl-CoA synthetase medium-chain family member 5	↓↑
ENSRNOG00000006460	Amdhd2	0.037	1.783 825	Amidohydrolase domain containing 2	↓↑
ENSRNOG00000029543	Cish	0.021	1.879 914	Cytokine inducible SH2-containing protein	↓↑
ENSRNOG00000023007	Col6a6	0.001	2.460 307	Collagen type Ⅵ alpha 6 chain	↓↑
ENSRNOG00000008534	Dusp15	0.043	2.175 131	Dual specificity phosphatase 15	↓↑
ENSRNOG00000009660	Enpp6	0.011	1.909 883	Ectonucleotide pyrophosphatase/phosphodiesterase 6	↓↑
ENSRNOG00000001806	Fetub	0.001	4.472 346	Fetuin B	↓↑
ENSRNOG00000000168	Gatm	0.001	2.314 874	Glycine amidinotransferase	↓↑
ENSRNOG00000019857	Gng7	0.045	1.784 171	Acyl-CoA thioesterase 5	↓↑
ENSRNOG00000014233	Krt19	0.009	2.704 678	ADAM metallopeptidase with thrombospondin type 1 motif, 13	↓↑
ENSRNOG00000006857	Ndnf	0.003	7.001 253	Aldo-keto reductase family 1, member C2	↓↑
ENSRNOG00000015353	Prss12	0.007	2.502 539	Annexin A13	↓↑
ENSRNOG00000014861	Rnf183	0.001	3.599 888	Ankyrin repeat and SOCS box containing 15	↓↑
ENSRNOG00000057620	Slc6a8	0.042	1.584 699	ATPase H⁺/K⁺ transporting non-gastric alpha 2 subunit	↓↑
ENSRNOG00000010296	Slc7a7	0.001	3.960 724	Bone morphogenetic protein 3	↓↑
ENSRNOG00000019924	Thop1	0.048	1.757 756	Carnitine palmitoyltransferase 1B	↓↑

Pathway	Pathway ID	Gene	Description	Fold change
PI3K-AKT signaling pathway	ko04151	Col6a6	Collagen type Ⅵ alpha 6 chain	0.52
		Nr4a1	Nuclear receptor subfamily 4, group A, member 1	0.41
		Sgk1	Serum/glucocorticoid regulated kinase 1	0.51
		Gng7	G protein subunit gamma 7	1.78
ECM-receptor interaction	ko04512	Col6a6	Collagen type Ⅵ alpha 6 chain	0.52
Complement and coagulation cascades	ko04610	-	-	-
Renin-angiotensin system	ko04614	Thop1	Thimet oligopeptidase 1	0.66
		Klk1c2	Kallikrein 1-related peptidase C2	1.78
TNF signaling pathway	ko04668	-	-	-
Protein digestion and absorption	ko04974	Slc7a7	Solute carrier family 7 member 7	0.60
		Col6a6	Collagen type Ⅵ alpha 6 chain	0.52
Metabolism of xenobiotics by cytochrome P450	ko00980	Ugt2b35	UDP glucuronosyltransferase 2 family, polypeptide B35	1.92
Prolactin signaling pathway	ko04917	Cish	Cytokine inducible SH2-containing protein	0.45

Pathway	Pathway ID	Gene	Description	Fold change
PI3K-AKT signaling pathway	ko04151	Col6a6	Collagen type Ⅵ alpha 6 chain	0.52
		Nr4a1	Nuclear receptor subfamily 4, group A, member 1	0.41
		Sgk1	Serum/glucocorticoid regulated kinase 1	0.51
		Gng7	G protein subunit gamma 7	1.78
ECM-receptor interaction	ko04512	Col6a6	Collagen type Ⅵ alpha 6 chain	0.52
Complement and coagulation cascades	ko04610	-	-	-
Renin-angiotensin system	ko04614	Thop1	Thimet oligopeptidase 1	0.66
		Klk1c2	Kallikrein 1-related peptidase C2	1.78
TNF signaling pathway	ko04668	-	-	-
Protein digestion and absorption	ko04974	Slc7a7	Solute carrier family 7 member 7	0.60
		Col6a6	Collagen type Ⅵ alpha 6 chain	0.52
Metabolism of xenobiotics by cytochrome P450	ko00980	Ugt2b35	UDP glucuronosyltransferase 2 family, polypeptide B35	1.92
Prolactin signaling pathway	ko04917	Cish	Cytokine inducible SH2-containing protein	0.45

基于转录组测序技术探讨黄芪水提物干预多柔比星肾病大鼠的分子机制

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贺升升 ¹^,² , 李爱平 ¹^,^* , 张王宁 ¹ , 秦雪梅 ¹^,^*

药学学报 | 研究论文 2019,54(8): 1439-1448

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药学学报 | 研究论文 2019, 54(8): 1439-1448

基于转录组测序技术探讨黄芪水提物干预多柔比星肾病大鼠的分子机制

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贺升升¹^,², 李爱平¹^,^*, 张王宁¹, 秦雪梅¹^,^*

作者信息

1.山西大学中医药现代研究中心, 山西太原 030006

2.山西大学化学化工学院, 山西太原 030006

通讯作者:

*李爱平, Tel:86-351-7011501, E-mail:qinxm@sxu.edu.cn;

秦雪梅, Tel:86-351-7018379, E-mail:aipingli@sxu.edu.cn

Exploring the molecular mechanism of aqueous extract from Huang qi on doxorubicin induced nephropathy in rats using transcriptome sequencing technique

Sheng-sheng HE¹^,², Ai-ping LI¹^,^*, Wang-ning ZHANG¹, Xue-mei QIN¹^,^*

Affiliations

1. Modern Research Center for Traditional Chinese Medicine, Shanxi University, Taiyuan 030006, China

2. College of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006, China

出版时间: 2019-08-12 doi: 10.16438/j.0513-4870.2019-0225

文章导航

摘要

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为了研究黄芪水提物对多柔比星肾病大鼠基因表达谱的影响，探讨黄芪水提物干预多柔比星肾病大鼠的分子机制。采用转录组测序技术检测对照组、模型组和黄芪水提物组大鼠肾组织的基因表达谱，通过STEM趋势分析软件筛选趋势表达的差异表达基因（differentially expressed genes，DEGs），并针对DEGs进行GO功能富集与KEGG通路分析，用实时荧光定量PCR（RT-qPCR）对基因的表达量进行验证。实验过程中对动物的处置符合动物实验伦理标准。结果显示，与对照组相比，模型组共筛选DEGs 432个；与模型组相比，黄芪水提物组共筛选DEGs811个。KEGG通路分析与荧光定量PCR结果指示，PI3K-AKT通路（Col6a6、Nr4a1、Sgk1、Gng7）与脂质代谢相关基因（Cpt1b、Pcsk9、Abca1、Ascm5）是黄芪水提物治疗多柔比星肾病、发挥肾脏保护作用的关键通路与基因。总之，黄芪水提物干预多柔比星肾病大鼠的分子机制与凋亡相关基因和脂质代谢相关基因密切相关，此结果为后续黄芪治疗多柔比星肾病的关键基因验证和作用机制研究提供了研究基础。

关键词

黄芪 / 水提物 / 多柔比星肾病 / 转录组学 / 分子机制

Abstract

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We studied the effect of aqueous extract from Huang qi on gene expression profile of doxorubicin induced nephropathy in rats, and explored the molecular mechanism of the intervention. The gene expression profiles of control group, model group and aqueous extract from Huang qi group were detected by using transcriptome sequencing technique. The differentially expressed genes (DEGs) were screened by STEM trend analysis software. GO function enrichment and KEGG pathway analysis were performed for DEGs, and the gene expression level was verified by real-time fluorescence quantitative PCR (RT-qPCR). The results showed that, compared with the control group, 432 DEGs were obtained in doxorubicin nephropathy model group; compared with the model group, 811 DEGs were obtained due to aqueous extract of Huang qi. The results of GO function enrichment and KEGG enrichment analysis indicated that PI3K-AKT pathway (Col6a6, Nr4a1, Sgk1, Gng7) and lipid metabolism-related genes (Cpt1b, Pcsk9, Abca1, Ascm5) were the key pathways and genes in the treatment of doxorubicin induced nephropathy by aqueous extract from Huang qi, which played a protective role in kidney. In conclusion, the molecular mechanism of aqueous extract from Huang qi in protection against doxorubicin induced nephropathy rats is closely related to apoptosis-related genes and lipid metabolism-related genes, suggesting for the need of follow-up study for key gene validation and mechanism of action of aqueous extract from Huang qi for prevention of doxorubicin induced nephropathy.

Key words

Huang qi / aqueous extract / doxorubicin nephropathy / transcriptomics / molecular mechanism

引用本文

贺升升, 李爱平, 张王宁, 秦雪梅. 基于转录组测序技术探讨黄芪水提物干预多柔比星肾病大鼠的分子机制. 药学学报, 2019 , 54 (8) : 1439 -1448 . DOI: 10.16438/j.0513-4870.2019-0225

Sheng-sheng HE, Ai-ping LI, Wang-ning ZHANG, Xue-mei QIN. Exploring the molecular mechanism of aqueous extract from Huang qi on doxorubicin induced nephropathy in rats using transcriptome sequencing technique[J]. Acta Pharmaceutica Sinica, 2019 , 54 (8) : 1439 -1448 . DOI: 10.16438/j.0513-4870.2019-0225

正文

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肾病综合征(nephrotic syndrome, NS)是儿童和成人常见的肾小球疾病, 其主要特征表现为高蛋白尿, 常伴有低蛋白血症、高脂血症及水肿。多柔比星肾病[adriamycin (doxorubicin) nephropathy, AN]是公认的能够模拟人体肾脏疾病的一种药理学实验模型, 其方法是通过使用多柔比星干预实验动物, 使其表现出与NS相似的病理特征和临床表现, 其机制可能是多柔比星通过诱导肾脏活性氧的生成, 引起肾小球足细胞基底膜结构和功能损害, 介导足细胞损伤和蛋白尿的发生^[1]。目前, 对AN的研究内容主要涉及足细胞损伤^[2]、氧化应激^[3]、肠道菌群变化^[4]、脂质代谢紊乱^[5]、炎症^[6]与纤维化^[7]等方面, 采用细胞生物学、微生物组学、基因组学和代谢组学等多种方法进行研究。

黄芪又名“黄耆”, 为豆科植物蒙古黄芪或膜荚黄芪的干燥根^[8], 始载于《神农本草经》, 具有补气固表、利水消肿、托毒排脓和生肌等功效^[9]。中医认为肾病综合征的病因病机为虚实夹杂、肾虚为本, 肾阴与肾阳的机能失衡是导致其水肿发生的主要原因^[10], 而黄芪的升提、固表、摄精、补气和利尿的作用与肾病综合征的本虚密切相关, 是中医临床治疗肾病综合征的常用药物^[11]。

黄芪治疗NS的药效学研究表明, 其有效成分多以黄酮类^[12]和皂苷类^[13]为主。已报道, 黄芪甲苷^[14]可通过上调足细胞结构蛋白nephrin和podocin的表达来保护AN大鼠, 降低尿蛋白水平。Zhang等^[15]通过核磁代谢组学结合网络药理学的方法研究黄芪总黄酮对AN大鼠的保护作用, 结果表明黄芪总黄酮可通过调节柠檬酸循环、丙酮酸代谢和肾素-血管紧张素系统保护AN大鼠肾脏过滤功能, 减轻蛋白尿的排泄。本课题组前期以黄芪为君药的防己黄芪汤作为研究对象, 发现防己黄芪汤干预AN的利水消肿作用的过程中, 防己黄芪汤全方与缺黄芪阴性方干预AN大鼠的药效差别显著, 结果显示缺黄芪的阴性方药效明显减弱, 而单独给予AN大鼠黄芪水提物后发现肌酐、胆固醇和白蛋白等传统药效指标均有不同程度的回调, 进一步确证黄芪水提物治疗AN大鼠药效确切, 然而其发挥药效的分子机制尚不明确。

转录组测序技术能够从整体水平研究基因功能及基因结构, 揭示特定生物学过程及疾病发生过程中的分子机制, 已广泛应用于研究中药材的生物合成途径和药效作用机制^{[16, 17]}。本研究采用转录组测序技术, 考察黄芪水提物对AN大鼠肾组织全基因表达谱的影响, 通过分析差异表达基因(differentially expressed genes, DEGs)的表达趋势和富集的GO功能和KEGG信号通路的变化, 荧光定量PCR检测趋势表达DEGs的表达量, 阐释黄芪水提物治疗AN大鼠的分子机制, 以期为黄芪在肾病综合征的临床应用提供实验药理学的研究基础, 为后续黄芪治疗肾病综合征的关键基因验证和调控分子机制研究提供基础资料。

材料与方法

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动物与仪器 选用SPF级雄性大鼠30只, 体质量(210 ± 10) g, 鼠龄6周, 北京维通利华实验动物技术有限公司提供, 动物许可SCXK (京) 2016-0011。动物饲养室保持相对温度(23.0 ± 1.5) ℃, 相对湿度(45 ± 15)%, 日光照12 h, 自由觅食饮水。实验过程中对动物的处置符合动物实验伦理标准。NanoDrop 2000超微量分光光度计(美国Thermo公司); 2100生物分析仪(美国Agilent公司); DQ-103/104台式方形中药切片机(温岭市林大机械有限公司); JEM-1011透射电子显微镜(日本日立公司); ABI Stepone Plus实时荧光定量PCR仪(美国应用生物系统公司); PCR仪(美国BIO-RAD公司); 生化自动分析仪(芬兰Thermo Scientific公司)。

药物与试剂 黄芪购自山西浑源县泽清茂业公司, 经山西大学中医药现代研究中心秦雪梅教授鉴定为蒙古黄芪Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao.的干燥根。Trizol试剂(日本Takara公司); RNA6000 Nano kit (美国Agilent公司); DEPC处理水(北京索莱宝公司); 氯仿、异丙醇、无水乙醇均属于分析级(天津市大茂化学试剂厂); 盐酸多柔比星(每瓶20 mg, 批号: 622B021, 北京索莱宝公司); 白蛋白(ALB)、血清尿素氮(BUN)、血清肌酐(Scr)、总胆固醇(TCHO)、三酰甘油(TG)、总蛋白(TP)试剂盒(上海复星长征医学科学有限公司)。

黄芪水提物的制备 采用课题组前期研究的制备方法, 黄芪经切片机切片后, 浸泡4 h, 加10倍量的水回流提取2 h, 滤过, 滤渣加8倍量的水回流提取2 h, 滤过, 合并取上清, 浓缩至生药质量浓度1 g·mL^-1, 置于-20 ℃冰箱备用。基于HPLC-UV-ELSD联用技术构建的黄芪水提物指纹图谱^[18], 对其进行质量控制, 以保证质量稳定可控。

动物分组、造模、给药 30只大鼠称体质量, 随机分为3组, 分别为对照组(control group, C)、多柔比星肾病模型组(model group, M)和黄芪水提物组(Huang qi group, HQ), 每组各10只。大鼠适应1周, 体质量约300 g后, M组与HQ组大鼠分2次尾静脉注射(iv)多柔比星进行造模。造模方案为实验第1天给予多柔比星3.5 mg·kg^-1, 第8天给予多柔比星1 mg·kg^-1, 复制AN模型; C组大鼠两次分别给予等体积生理盐水。黄芪水提物给药剂量依据本实验室前期研究^[18], 灌胃给予1.5 g·kg^-1·d^-1, 给药体积为10 mL·kg^-1, 于实验第15天开始进行药物干预, 用药周期为5周。

血清生化指标测定与肾组织病理检查 实验结束后, 动物禁食12 h, 腹腔注射10%水合氯醛麻醉, 股动脉取血, 收集所有大鼠的肾组织。使用生化自动分析仪分析血清ALB、TP、TG、TCHO、BUN、Scr。切取左肾部分肾皮质, 经10%中性福尔马林固定, 蒸馏水冲洗固定液, 乙醇梯度脱水后, 对组织进行二甲苯透明, 浸蜡后包埋; 包埋后的组织切3~5 μm薄片, 经60 ℃烤片后进行苏木精-伊红(hematoxylin-eosin, HE)染色, 透明, 封片后进行光镜检查。切取右肾部分肾皮质, 制成1 mm³的小块2~5枚, 置于2.5%戊二醛中, 4 ℃固定2 h, 0.1 mol·L^-1磷酸缓冲液浸洗3次, 1%四氧化锇固定液中4 ℃固定2 h, 0.1 mol·L^-1磷酸缓冲液浸洗3次, 乙醇梯度洗脱, 过氧丙烷置换10 min, 包埋后作超薄切片50~70 nm, 醋酸铀、柠檬酸铅染后, 在透射式电子显微镜(transmission electron microscope, TEM)下观察。

转录组测序 各组分别选取3只大鼠肾脏组织, Trizol试剂对样本进行总RNA的提取, 并采用RNA专用琼脂糖凝胶电泳对其浓度及纯度进行检测。通过Oligo (dT)磁珠富集总RNA中带有polyA结构的mRNA, 采用离子打断的方式, 将RNA打断到200~300 bp片段。随后通过随机引物和逆转录酶的作用合成cDNA第一链, 并以第一链cDNA为模板进行第二链cDNA的合成, 建立链特异性文库。使用安捷伦2100生物分析仪对文库进行质检, 采用第二代测序技术, 基于Illumina HiSeq测序平台, 对文库进行双末端测序, 得到FASTQ格式的原始数据(raw data)。使用Cutadapt^[19]软件对原始数据进行过滤, 然后使用TopHat2^[20]软件将过滤后得到的高质量序列(clean data)的Reads比对到参考基因组上。

转录组测序数据分析 使用HTSeq^[21]统计每组样本比对到每一个基因上的Read数, 作为基因的原始表达量, 然后采用FPKM^[22] (fragments per kb per million Reads)对表达量进行标准化。采用DESeq对基因表达进行差异分析, 筛选差异表达基因条件:表达差异倍数|log₂(fold change)| > 1.5, P-value < 0.05。采用R语言ggplots2软件包绘制差异表达基因的火山图, 使用韦恩图统计DEGs个数以及各组差异分析之间的共有基因数量。为进一步筛选显著变化的基因群, 采用STEM^[23]软件将DEGs进行趋势分析。将所有基因映射到Gene Ontology数据库(http://geneontology.org/)各个Term, 计算每个Term的DEGs数目, 以整个基因组为背景, 采用超几何分布计算DEGs显著富集的GO条目。将DEGs注释到KEGG数据库中, 进而确定差异表达基因主要参与的代谢途径和信号通路。

实时荧光定量PCR验证 如上述转录组测序的方法, 各组重新选取3只大鼠肾组织进行Trizol法提取RNA。选取转录组数据中有趋势表达的DEGs进行荧光定量PCR验证, GADPH作为管家基因。引物采用Primer3^[24] (http://primer3.ut.ee/)在线软件设计, 由生工生物工程(上海)股份有限公司合成(表 1)。根据定量试剂盒说明书操作, 每个样本重复测定3次, 采用SYBR green法在ABI Stepone Plus实时荧光定量PCR仪完成测定。PCR反应条件: 95 ℃×15 s, 60 ℃×15 s, 72 ℃×15 s, 40个循环。采用${{2}^{-\Delta \Delta {{C}_{t}}}}$方法计算趋势表达的DGEs mRNA的相对表达量, 其中C_t值为循环阈值, 相对表达量=2^-ΔΔC_t; △△C_t=(C_t_{分析组目标基因}-C_t_{分析组内标基因GADPH})-(C_t_{对照组目标基因}-C_t_{对照组GADPH}_内标基因), 计算各组相对于对照组的表达量。

统计学方法 使用统计学软件GraphPad Prism 6.01进行统计分析, 实验结果采用x ± s表示, 通过t检验比较组间差异。P < 0.05表示具有统计学意义。

结果与讨论

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1 药效指标观察

Scr和BUN水平反映肾功能的重要指标, TP和ALB水平反映肾病大鼠蛋白状况, TCHO和TG反映机体脂质代谢情况。从表 2可见, 与C组相比, M组血清BUN、TG和TCHO水平升高, 给予黄芪水提物干预后各指标均有所回调, 且具有显著性差异(P <0.05); M组血清ALB和TP水平降低, 给予黄芪水提物干预后各指标均回调(P <0.05, P <0.01);而血清Scr水平无显著性变化。综上, 从血清生化测定结果可知, AN模型复制成功, 给予黄芪水提物干预后, 各指标发生不同程度回调, 表明黄芪对多柔比星肾病有确切疗效。

给药5周后, 对C组、M组和HQ组肾组织进行HE染色和TEM观察来评价黄芪水提物对AN大鼠的治疗效果(图 1)。HE染色结果表明, 与C组比较, M组肾小管上皮细胞萎缩, 管腔变大。给予黄芪水提物后, HQ组比M组肾小管上皮细胞管腔间隙缩小。TEM扫描结果表明, 与C相比, M组肾小球足细胞足突融合, 部分脱落, 基底膜增厚, 内皮细胞轮廓模糊。给药后, HQ组比M组足突融合减轻, 仅局部融合, 有完整的足突存在, 内皮细胞轮廓清晰。综上, 从肾组织的病理分析结果可知, AN模型复制成功, 黄芪水提物可明显改善AN大鼠肾小球足细胞的损伤。

2 差异表达基因筛选

采用P值与log₂(fold change)来筛选DEGs。由图 2A可知, 与C组相比, 从M组中共筛选出432个DEGs, 其中266个上调, 166个下调。由图 2B可见, 与M组相比, 从HQ组共筛选出811个DEGs, 其中546个上调, 265个下调。由图 2C可见, 123个DEGs在给药前后均有变化。

3 差异表达基因趋势分析

趋势分析是分析基因的表达变化规律和特征, 将相同变化特征的基因集中在一种变化趋势中, 从而找到实验变化过程中最具代表性基因群的一种方法。为了筛选具有显著变化的基因, 采用STEM软件将C组与M组比较、M组与HQ组比较筛选出的所有DEGs进行趋势分析, 结果如图 3。DEGs分为8个趋势, 其中, 趋势Profile B和Profile E是作者最感兴趣的表达变化趋势。结果如表 3, 共有52个基因, 其中35个基因在M组中表达上调, 给予黄芪水提物后基因表达下调; 而17个基因在M组中表达下调, 给予黄芪水提物后基因表达上调。因此, 这些趋势表达的DEGs可能是黄芪水提物干预AN大鼠发挥药效作用的关键基因。

4 差异表达基因的GO功能富集与KEGG通路分析

将筛选所得的DEGs分别从生物学过程(biological process)、分子功能(molecular function)、细胞成分(cellular component)三个层面进行GO功能富集, 并采用KEGG通路分析研究DEGs可能参与调控的相关信号通路。由图 4可见, M组与C组相比, 432个DEGs可显著注释到120条途径, 其中85条生物学过程, 主要涉及一元羧酸代谢过程(monocarboxylic acid metabolic process)、氧酸代谢过程(oxoacid metabolic process)、不饱和脂肪酸代谢过程(unsaturated fatty acid metabolic process)、脂质代谢过程(lipid metabolic process)和凋亡过程的调节(regulation of apoptotic process)等; 15条分子功能, 主要涉及细胞外基质(extracellular matrix)、膜区(membrane region)、次级跨膜转运蛋白活性(secondary active transmembrane transporter activity)、细胞外间隙(extracellular space)等; 其余20条途径为细胞成分。这些结果提示, 多柔比星可能通过诱导大鼠体内不饱和脂肪酸与脂质代谢异常, 参与足细胞凋亡过程, 进而介导蛋白尿的发生。已有研究表明^{[25, 26]}, AN的发生总是伴随着大鼠体内脂质代谢的异常。此外, 蛋白尿发生的根本原因是足细胞骨架解体和足细胞的脱落与凋亡引起裂孔隔膜间隙增大, 基底膜结构的破坏, 最终导致蛋白随尿液从肾小球滤过^[27], 多柔比星诱导的脂质代谢异常可能作为足细胞损伤和蛋白尿发生的关键因素。

KEGG通路富集分析指出, DEGs可显著富集到29条信号通路, 包括PPAR、不饱和脂肪酸的生物合成、脂肪酸代谢、PI3K-AKT和肾素-血管紧张素系统等, 这些通路可能作为AN大鼠足细胞损伤和蛋白尿发生的机制。已报道, 转录因子过氧化物酶体增生物激活受体γ (PPARγ)的表达与肾组织炎症反应、足细胞损伤密切相关^[28]。多柔比星可介导足细胞中PI3K的p110γ亚型蛋白上调, nephrin蛋白表达下调, 最终导致细胞骨架的解体和细胞凋亡^[29]。此外, 多柔比星可诱导肾脏组织自由基的形成, 自由基可通过产生氧化脂质(包括不饱和脂肪酸)而对细胞质膜造成损害, 促进炎症反应的进程^[30]。

由图 5可见, HQ组与M组相比, 811个DEGs可显著注释到467条途径, 其中373条生物学过程, 主要涉及免疫系统过程的调节(regulation of immune system process)、细胞增殖调节(regulation of cell proliferation)、凋亡过程的调节(regulation of apoptotic process)和细胞对脂质的反应(cellular response to lipid)等; 38条分子功能, 主要涉及肌动蛋白细胞骨架(actin cytoskeleton)、膜筏(membrane raft)、蛋白质细胞外基质(proteinaceous extracellular matrix)和细胞连接(cell junction)等; 其余56条途径为细胞成分。KEGG通路富集分析指出, DEGs可显著富集到41条信号通路, 包括补体和凝血级联、PI3K-AKT、肿瘤坏死因子(tumor necrosis factor, TNF)、细胞外基质(extracellular matrix, ECM)等。这些结果提示, 黄芪水提物可能通过调节免疫功能、脂质代谢过程和细胞增殖与凋亡保护肌动蛋白骨架的完整性, 增强足细胞与基底膜的黏附。已有文献^[31]报道, 足细胞损伤是蛋白尿产生和肾小球硬化的关键, 足突-基底膜黏附分子功能受损是足细胞从基底膜剥离和脱落的重要因素, Gui^[32]研究表明, 黄芪水提物对体外培养的多柔比星刺激下的足细胞损伤有明显保护作用, 其机制可能与上调黏附分子α-dystroglycan、α3 integrin蛋白及mRNA表达、改善足细胞与基底膜之间黏附功能有关。

据文献^[33]报道, AN的发生与脂质代谢失调和血脂异常有关。在趋势表达的DEGs中, Abca1、Acsm5、Pcsk9和Cpt1b的功能与脂质代谢密切相关。当AN发生时, Pcsk9升高、脂肪酶活性降低, Pcsk9可通过降低低密度脂蛋白受体(LDLR)的表达而升高血浆胆固醇水平, 导致严重高胆固醇血症^[34]。肉毒碱棕榈酰基转移酶1B (Cpt1b)能够促进长链脂肪酸向线粒体的转运, 为β-氧化提供底物, 增加线粒体肉毒碱的水平^{[35, 36]}。Zhang等^[37]研究表明, 肉毒碱可作为早期诊断和反映肾病综合征进程的潜在生物标志物, 其含量随着AN的肾脏损伤程度加重而减少。Gao等^[38]使用微阵列技术分析了中药益气消颗粒对慢性肾小球肾炎大鼠基因表达的影响, 结果显示, 与模型组相比, Ascm5表达显著提高, 给药后Ascm5表达下降。综上, 脂质代谢相关基因(包含Abca1、Acsm5、Pcsk9、Cpt1b)可能是黄芪水提物治疗AN大鼠的关键基因。

通过对M组与C组比较、HQ组与M组比较, 筛选出的DEGs显著富集的通路, 取交集得PI3K-AKT、ECM受体相互作用、补体和凝血级联、肾素-血管紧张素系统、TNF、催乳素信号通路、细胞色素P450对异种生物的代谢、蛋白质消化吸收为HQ干预AN的信号通路。由表 4可知, 共同参与的信号通路中, 趋势表达的DEGs多分布在PI3K-AKT信号通路上, 这提示PI3K-AKT信号通路(包含Col6a6、Nr4a1、Sgk1、Gng7)可能作为黄芪水提物干预AN大鼠的关键通路和药效靶点。研究表明, 在线粒体膜上, 核受体Nr4a1通过与Bcl-2相互作用, 介导Bcl-2构象改变, 使其从抗凋亡蛋白转变为促凋亡蛋白, 促进细胞凋亡^[39]。黄芪甲苷可通过抑制PTEN/AKT/mTOR减缓肾组织的损伤^[40]。

5 实时荧光定量PCR验证测序结果

为了验证转录组测序数据的准确性, 将上述8个与PI3K-AKT通路和脂质代谢相关的趋势表达DEGs进行荧光定量PCR验证。8个基因分别是Col6a6、Nr4a1、Sgk1、Gng7、Abca1、Acsm5、Pcsk9和Cpt1b, 结果见图 6。这8个基因的相对定量值与转录组测序数据趋势一致, 说明转录组测序数据可靠且重复性好。黄芪水提物可通过调节PI3K-AKT通路和脂质代谢相关基因来改善AN大鼠肾脏足细胞的凋亡和脂质代谢紊乱。

结论

收起

本文以AN大鼠为研究对象, 通过对C组、M组和HQ组大鼠的肾组织进行HE染色和TEM观察来评价黄芪水提物对AN大鼠的干预效果。通过分析转录组测序数据, 对DEGs进行趋势分析, 共筛选出52个黄芪水提物发挥药效作用的关键基因。通过将DEGs进行GO富集分析与KEGG通路分析, 结果显示, PI3K-AKT信号通路(Col6a6、Nr4a1、Sgk1、Gng7)、脂质代谢过程中相关基因(Cpt1b、Pcsk9、Abca1、Ascm5)与黄芪水提物干预AN的药效机制密切相关, RT-qPCR检测其基因表达量与转录组测序数据结果一致。综上, 可推测黄芪水提物可能通过作用于脂质代谢相关基因和PI3K-AKT信号通路干预AN大鼠肾组织的脂质代谢与凋亡过程, 这也可能是其治疗AN、发挥肾脏保护作用的分子机制。后期研究可通过靶标脂质组学技术探讨肾组织脂质类成分的差异, 通过分子生物学技术从蛋白层面检测PI3K-γ、AKT等蛋白的磷酸化与非磷酸化蛋白的表达, 进一步确证黄芪水提物发挥药效的作用机制。

基金

收起

国家自然科学基金资助项目(31570346)

参考文献

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2019年第54卷第8期

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doi: 10.16438/j.0513-4870.2019-0225

接收时间：2019-04-01
首发时间：2026-01-26
出版时间：2019-08-12

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收稿日期：2019-04-01
修回日期：2019-06-04

基金

国家自然科学基金资助项目(31570346)

作者信息

1.山西大学中医药现代研究中心, 山西太原 030006

2.山西大学化学化工学院, 山西太原 030006

通讯作者:

*李爱平, Tel:86-351-7011501, E-mail:qinxm@sxu.edu.cn;

秦雪梅, Tel:86-351-7018379, E-mail:aipingli@sxu.edu.cn

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Gene symbol	Gene bank	Primer sequence
Cpt1b	ENSRNOG00000010438	CATGTATCGCCGCAAACTGG	CCTGGGATGCGTGTAGTGTT
Pcsk9	ENSRNOG00000006280	AGGGCCAGAGAAGCAATGTC	GCACAGCCTGTCCTCTCATT
Abca1	ENSRNOG00000018126	CTGAAGCCACCACAACGTTG	AGAAACATCACCTCCTGCCG
Ascm5	ENSRNOG00000031211	GTGAAAGCAGCCTGGACTCT	TGTACAAGCCGCCGGAAGAG
Gng7	ENSRNOG00000019857	CAGAGAGGGCCAGGACTTTG	TCCCGGTCCCCAGAATGTAT
Col6a6	ENSRNOG00000023007	GACCAGCTGCTCAGGGAAAT	ACTGCTAAAGAACGTGGCGA
Sgk1	ENSRNOG00000011815	GCAGTGACGAGCATCCAGAT	ACGGCTCTGACTGACAACTG
Nr4a1	ENSRNOG00000007607	TGCCAAGTACATCTGCCTGG	GGCTGCTTGGGTTTTGAAGG
Gapdh	ENSRNOG00000018630	AAAGGGTCATCATCTCCGCC	AGTGATGGCATGGACTGTGG

Gene symbol

Gene bank

Primer sequence

Forward primer

Reverse primer

Cpt1b

ENSRNOG00000010438

CATGTATCGCCGCAAACTGG

CCTGGGATGCGTGTAGTGTT

Pcsk9

ENSRNOG00000006280

AGGGCCAGAGAAGCAATGTC

GCACAGCCTGTCCTCTCATT

Abca1

ENSRNOG00000018126

CTGAAGCCACCACAACGTTG

AGAAACATCACCTCCTGCCG

Ascm5

ENSRNOG00000031211

GTGAAAGCAGCCTGGACTCT

TGTACAAGCCGCCGGAAGAG

Gng7

ENSRNOG00000019857

CAGAGAGGGCCAGGACTTTG

TCCCGGTCCCCAGAATGTAT

Col6a6

ENSRNOG00000023007

GACCAGCTGCTCAGGGAAAT

ACTGCTAAAGAACGTGGCGA

Sgk1

ENSRNOG00000011815

GCAGTGACGAGCATCCAGAT

ACGGCTCTGACTGACAACTG

Nr4a1

ENSRNOG00000007607

TGCCAAGTACATCTGCCTGG

GGCTGCTTGGGTTTTGAAGG

Gapdh

ENSRNOG00000018630

AAAGGGTCATCATCTCCGCC

AGTGATGGCATGGACTGTGG

Group	ALB/g·L^-1	BUN/mmol·L^-1	Scr/μmol·L^-1	TG/mmol·L^-1	TP/g·L^-1	TCHO/mmol·L^-1
C	33.06 ±1.73	5.06 ± 0.91	46.09 ± 11.32	0.36 ± 0.052	54.46 ± 1.79	1.42 ± 0.32
M	30.36 ± 2.19^*	5.69 ± 1.10^*	46.95 ± 9.43	0.512 ± 0.17^*	52.26 ± 2.90^**	2.49 ± 0.55^**
HQ	31.96 ± 0.97^#	5.07 ± 0.91^#	34.06 ± 2.85^##	0.37 ± 0.089^#	55.46 ± 2.02^##	1.66 ± 0.38^#

Group

ALB/g·L^-1

BUN/mmol·L^-1

Scr/μmol·L^-1

TG/mmol·L^-1

TP/g·L^-1

TCHO/mmol·L^-1

33.06 ±1.73

5.06 ± 0.91

46.09 ± 11.32

0.36 ± 0.052

54.46 ± 1.79

1.42 ± 0.32

30.36 ± 2.19^*

5.69 ± 1.10^*

46.95 ± 9.43

0.512 ± 0.17^*

52.26 ± 2.90^**

2.49 ± 0.55^**

31.96 ± 0.97^#

5.07 ± 0.91^#

34.06 ± 2.85^##

0.37 ± 0.089^#

55.46 ± 2.02^##

1.66 ± 0.38^#

Gene bank	Gene symbol	P-value	Fold change	Description	Trend
ENSRNOG00000032508	Acot5	0.047	0.423 797	Acyl-CoA thioesterase 5	↑↓
ENSRNOG00000005780	Adamts13	0.047	0.271 354	ADAM metallopeptidase with thrombospondin type 1 motif, 13	↑↓
ENSRNOG00000029844	Akr1c2	0.031	0.520 374	Aldo-keto reductase family 1, member C2	↑↓
ENSRNOG00000007980	Anxa13	0.004	0.479 046	Annexin A13	↑↓
ENSRNOG00000006365	Asb15	0.001	0.393 735	Ankyrin repeat and SOCS box containing 15	↑↓
ENSRNOG00000020685	Atp12a	0.042	0.547 411	ATPase H⁺/K⁺ transporting non-gastric alpha 2 subunit	↑↓
ENSRNOG00000002381	Bmp3	0.001	0.382 219	Bone morphogenetic protein 3	↑↓
ENSRNOG00000010438	Cpt1b	0.016	0.543 593	Carnitine palmitoyltransferase 1B	↑↓
ENSRNOG00000006873	Dnase1	0.000 07	0.408 402	Deoxyribonuclease 1	↑↓
ENSRNOG00000013770	Folh1	0.021	0.513 944	Folate hydrolase 1	↑↓
ENSRNOG00000011599	Gldc	0.001	0.450 592	Glycine decarboxylase CRA_a	↑↓
ENSRNOG00000003616	Grem2	0.041	0.364 554	Gremlin 2, DAN family BMP antagonist	↑↓
ENSRNOG00000029237	Klk1c2	0.001	0.306 455	Kallikrein 1-related peptidase C2	↑↓
ENSRNOG00000056076	Mif	0.001	0	Macrophage migration inhibitory factor	↑↓
ENSRNOG00000034078	Mxi1	0.006	0.537 201	MAX interactor 1, dimerization protein	↑↓
ENSRNOG00000007607	Nr4a1	0.004	0.414 562	Nuclear receptor subfamily 4, group A, member 1	↑↓
ENSRNOG00000051837	Nwd2	0.001	0.034 365	NACHT and WD repeat domain containing 2	↑↓
ENSRNOG00000006280	Pcsk9	0.001	0.040 151	Proprotein convertase subtilisin/kexin type 9	↑↓
ENSRNOG00000007387	Per1	0.012	0.541 879	Period circadian clock 1	↑↓
ENSRNOG00000014761	Rasd2	0.008	0.459 304	RASD family, member 2	↑↓
ENSRNOG00000029651	Rdh16	0.001	0.439 728	Retinol dehydrogenase 16 (all-trans)	↑↓
ENSRNOG00000014524	S1pr3	0.011	0.460 415	Sphingosine-1-phosphate receptor 3	↑↓
ENSRNOG00000011815	Shisa3	0.001	0.230 381	Shisa family member 3	↑↓
ENSRNOG00000025670	Sgk1	0.002	0.506 214	Serum/glucocorticoid regulated kinase 1	↑↓
ENSRNOG00000017072	Slc16a14	0.021	0.583 361	Solute carrier family 16, member 14	↑↓
ENSRNOG00000018131	Slc16a4	0.001	0.463 205	Solute carrier family 16, member 4	↑↓
ENSRNOG00000014338	Slc25a25	0.002	0.504 666	Solute carrier family 25, member 25	↑↓
ENSRNOG00000007591	Slc5a8	0.007	0.541 283	Solute carrier family 5, member 8	↑↓
ENSRNOG00000006367	Slc45a3	0.037	0.586 863	Solute carrier family 45, member 3	↑↓
ENSRNOG00000033830	Slc7a12	0.004	0.514 671	Solute carrier family 7, member 12	↑↓
ENSRNOG00000036984	Slco1a1	0.003	0.518 432	Solute carrier organic anion transporter family, member 1a1	↑↓
ENSRNOG00000021726	Tlr3	0.046	0.611 419	Toll-like receptor 3	↑↓
ENSRNOG00000039464	Tmem229a	0.016	0.438 692	Transmembrane protein 229A	↑↓
ENSRNOG00000019486	Trpv1	0.023	0.483 102	Transient receptor potential cation, subfamily Ⅴ, member 1	↑↓
ENSRNOG00000003832	Vash2	0.001	0.153 739	Vasohibin 2	↑↓
ENSRNOG00000018126	Abca1	0.034	1.803 634	ATP binding cassette subfamily A, member 1	↓↑
ENSRNOG00000031211	Acsm5	0.001	3.260 038	Acyl-CoA synthetase medium-chain family member 5	↓↑
ENSRNOG00000006460	Amdhd2	0.037	1.783 825	Amidohydrolase domain containing 2	↓↑
ENSRNOG00000029543	Cish	0.021	1.879 914	Cytokine inducible SH2-containing protein	↓↑
ENSRNOG00000023007	Col6a6	0.001	2.460 307	Collagen type Ⅵ alpha 6 chain	↓↑
ENSRNOG00000008534	Dusp15	0.043	2.175 131	Dual specificity phosphatase 15	↓↑
ENSRNOG00000009660	Enpp6	0.011	1.909 883	Ectonucleotide pyrophosphatase/phosphodiesterase 6	↓↑
ENSRNOG00000001806	Fetub	0.001	4.472 346	Fetuin B	↓↑
ENSRNOG00000000168	Gatm	0.001	2.314 874	Glycine amidinotransferase	↓↑
ENSRNOG00000019857	Gng7	0.045	1.784 171	Acyl-CoA thioesterase 5	↓↑
ENSRNOG00000014233	Krt19	0.009	2.704 678	ADAM metallopeptidase with thrombospondin type 1 motif, 13	↓↑
ENSRNOG00000006857	Ndnf	0.003	7.001 253	Aldo-keto reductase family 1, member C2	↓↑
ENSRNOG00000015353	Prss12	0.007	2.502 539	Annexin A13	↓↑
ENSRNOG00000014861	Rnf183	0.001	3.599 888	Ankyrin repeat and SOCS box containing 15	↓↑
ENSRNOG00000057620	Slc6a8	0.042	1.584 699	ATPase H⁺/K⁺ transporting non-gastric alpha 2 subunit	↓↑
ENSRNOG00000010296	Slc7a7	0.001	3.960 724	Bone morphogenetic protein 3	↓↑
ENSRNOG00000019924	Thop1	0.048	1.757 756	Carnitine palmitoyltransferase 1B	↓↑

Gene bank

Gene symbol

P-value

Fold change

Description

Trend

ENSRNOG00000032508

Acot5

0.047

0.423 797

Acyl-CoA thioesterase 5

↑↓

ENSRNOG00000005780

Adamts13

0.047

0.271 354

ADAM metallopeptidase with thrombospondin type 1 motif, 13

↑↓

ENSRNOG00000029844

Akr1c2

0.031

0.520 374

Aldo-keto reductase family 1, member C2

↑↓

ENSRNOG00000007980

Anxa13

0.004

0.479 046

Annexin A13

↑↓

ENSRNOG00000006365

Asb15

0.001

0.393 735

Ankyrin repeat and SOCS box containing 15

↑↓

ENSRNOG00000020685

Atp12a

0.042

0.547 411

ATPase H⁺/K⁺ transporting non-gastric alpha 2 subunit

↑↓

ENSRNOG00000002381

Bmp3

0.001

0.382 219

Bone morphogenetic protein 3

↑↓

ENSRNOG00000010438

Cpt1b

0.016

0.543 593

Carnitine palmitoyltransferase 1B

↑↓

ENSRNOG00000006873

Dnase1

0.000 07

0.408 402

Deoxyribonuclease 1

↑↓

ENSRNOG00000013770

Folh1

0.021

0.513 944

Folate hydrolase 1

↑↓

ENSRNOG00000011599

Gldc

0.001

0.450 592

Glycine decarboxylase CRA_a

↑↓

ENSRNOG00000003616

Grem2

0.041

0.364 554

Gremlin 2, DAN family BMP antagonist

↑↓

ENSRNOG00000029237

Klk1c2

0.001

0.306 455

Kallikrein 1-related peptidase C2

↑↓

ENSRNOG00000056076

Mif

0.001

Macrophage migration inhibitory factor

↑↓

ENSRNOG00000034078

Mxi1

0.006

0.537 201

MAX interactor 1, dimerization protein

↑↓

ENSRNOG00000007607

Nr4a1

0.004

0.414 562

Nuclear receptor subfamily 4, group A, member 1

↑↓

ENSRNOG00000051837

Nwd2

0.001

0.034 365

NACHT and WD repeat domain containing 2

↑↓

ENSRNOG00000006280

Pcsk9

0.001

0.040 151

Proprotein convertase subtilisin/kexin type 9

↑↓

ENSRNOG00000007387

Per1

0.012

0.541 879

Period circadian clock 1

↑↓

ENSRNOG00000014761

Rasd2

0.008

0.459 304

RASD family, member 2

↑↓

ENSRNOG00000029651

Rdh16

0.001

0.439 728

Retinol dehydrogenase 16 (all-trans)

↑↓

ENSRNOG00000014524

S1pr3

0.011

0.460 415

Sphingosine-1-phosphate receptor 3

↑↓

ENSRNOG00000011815

Shisa3

0.001

0.230 381

Shisa family member 3

↑↓

ENSRNOG00000025670

Sgk1

0.002

0.506 214

Serum/glucocorticoid regulated kinase 1

↑↓

ENSRNOG00000017072

Slc16a14

0.021

0.583 361

Solute carrier family 16, member 14

↑↓

ENSRNOG00000018131

Slc16a4

0.001

0.463 205

Solute carrier family 16, member 4

↑↓

ENSRNOG00000014338

Slc25a25

0.002

0.504 666

Solute carrier family 25, member 25

↑↓

ENSRNOG00000007591

Slc5a8

0.007

0.541 283

Solute carrier family 5, member 8

↑↓

ENSRNOG00000006367

Slc45a3

0.037

0.586 863

Solute carrier family 45, member 3

↑↓

ENSRNOG00000033830

Slc7a12

0.004

0.514 671

Solute carrier family 7, member 12

↑↓

ENSRNOG00000036984

Slco1a1

0.003

0.518 432

Solute carrier organic anion transporter family, member 1a1

↑↓

ENSRNOG00000021726

Tlr3

0.046

0.611 419

Toll-like receptor 3

↑↓

ENSRNOG00000039464

Tmem229a

0.016

0.438 692

Transmembrane protein 229A

↑↓

ENSRNOG00000019486

Trpv1

0.023

0.483 102

Transient receptor potential cation, subfamily Ⅴ, member 1

↑↓

ENSRNOG00000003832

Vash2

0.001

0.153 739

Vasohibin 2

↑↓

ENSRNOG00000018126

Abca1

0.034

1.803 634

ATP binding cassette subfamily A, member 1

↓↑

ENSRNOG00000031211

Acsm5

0.001

3.260 038

Acyl-CoA synthetase medium-chain family member 5

↓↑

ENSRNOG00000006460

Amdhd2

0.037

1.783 825

Amidohydrolase domain containing 2

↓↑

ENSRNOG00000029543

Cish

0.021

1.879 914

Cytokine inducible SH2-containing protein

↓↑

ENSRNOG00000023007

Col6a6

0.001

2.460 307

Collagen type Ⅵ alpha 6 chain

↓↑

ENSRNOG00000008534

Dusp15

0.043

2.175 131

Dual specificity phosphatase 15

↓↑

ENSRNOG00000009660

Enpp6

0.011

1.909 883

Ectonucleotide pyrophosphatase/phosphodiesterase 6

↓↑

ENSRNOG00000001806

Fetub

0.001

4.472 346

Fetuin B

↓↑

ENSRNOG00000000168

Gatm

0.001

2.314 874

Glycine amidinotransferase

↓↑

ENSRNOG00000019857

Gng7

0.045

1.784 171

Acyl-CoA thioesterase 5

↓↑

ENSRNOG00000014233

Krt19

0.009

2.704 678

ADAM metallopeptidase with thrombospondin type 1 motif, 13

↓↑

ENSRNOG00000006857

Ndnf

0.003

7.001 253

Aldo-keto reductase family 1, member C2

↓↑

ENSRNOG00000015353

Prss12

0.007

2.502 539

Annexin A13

↓↑

ENSRNOG00000014861

Rnf183

0.001

3.599 888

Ankyrin repeat and SOCS box containing 15

↓↑

ENSRNOG00000057620

Slc6a8

0.042

1.584 699

ATPase H⁺/K⁺ transporting non-gastric alpha 2 subunit

↓↑

ENSRNOG00000010296

Slc7a7

0.001

3.960 724

Bone morphogenetic protein 3

↓↑

ENSRNOG00000019924

Thop1

0.048

1.757 756

Carnitine palmitoyltransferase 1B

↓↑

Pathway	Pathway ID	Gene	Description	Fold change
PI3K-AKT signaling pathway	ko04151	Col6a6	Collagen type Ⅵ alpha 6 chain	0.52
		Nr4a1	Nuclear receptor subfamily 4, group A, member 1	0.41
		Sgk1	Serum/glucocorticoid regulated kinase 1	0.51
		Gng7	G protein subunit gamma 7	1.78
ECM-receptor interaction	ko04512	Col6a6	Collagen type Ⅵ alpha 6 chain	0.52
Complement and coagulation cascades	ko04610	-	-	-
Renin-angiotensin system	ko04614	Thop1	Thimet oligopeptidase 1	0.66
		Klk1c2	Kallikrein 1-related peptidase C2	1.78
TNF signaling pathway	ko04668	-	-	-
Protein digestion and absorption	ko04974	Slc7a7	Solute carrier family 7 member 7	0.60
		Col6a6	Collagen type Ⅵ alpha 6 chain	0.52
Metabolism of xenobiotics by cytochrome P450	ko00980	Ugt2b35	UDP glucuronosyltransferase 2 family, polypeptide B35	1.92
Prolactin signaling pathway	ko04917	Cish	Cytokine inducible SH2-containing protein	0.45

Pathway

Pathway ID

Gene

Description

Fold change

PI3K-AKT signaling pathway

ko04151

Col6a6

Collagen type Ⅵ alpha 6 chain

0.52

Nr4a1

Nuclear receptor subfamily 4, group A, member 1

0.41

Sgk1

Serum/glucocorticoid regulated kinase 1

0.51

Gng7

G protein subunit gamma 7

1.78

ECM-receptor interaction

ko04512

Col6a6

Collagen type Ⅵ alpha 6 chain

0.52

Complement and coagulation cascades

ko04610

Renin-angiotensin system

ko04614

Thop1

Thimet oligopeptidase 1

0.66

Klk1c2

Kallikrein 1-related peptidase C2

1.78

TNF signaling pathway

ko04668

Protein digestion and absorption

ko04974

Slc7a7

Solute carrier family 7 member 7

0.60

Col6a6

Collagen type Ⅵ alpha 6 chain

0.52

Metabolism of xenobiotics by cytochrome P450

ko00980

Ugt2b35

UDP glucuronosyltransferase 2 family, polypeptide B35

1.92

Prolactin signaling pathway

ko04917

Cish

Cytokine inducible SH2-containing protein

0.45