Mechanisms of Dangua Fang (丹瓜方) in multi-target and multi-method regulation of glycolipid metabolism based on phosphoproteomics

doi:10.19852/j.cnki.jtcm.20230908.001

Abstract

Abstract:

OBJECTIVE: To explore the mechanism of Dangua Fang (丹瓜方, DGR) in multi-target and multi-method regulation of glycolipid metabolism based on phosphoproteomics.

METHODS: Sprague-Dawley rats with normal glucose levels were randomly divided into three groups, including a conventional diet control group (Group A), high-fat-high-sugar diet model group (Group B), and DGR group (Group C, high-fat-high-sugar diet containing 20.5 g DGR). After 10 weeks of intervention, the fasting blood glucose (FBG), 2 h blood glucose [PBG; using the oral glucose tolerance test (OGTT)], hemoglobin A1c (HbA1c), plasma total cholesterol (TC), and triglycerides (TG) were tested, and the livers of rats were removed to calculate the liver index. Then, hepatic portal TG were tested using the Gross permanent optimization-participatiory action planning enzymatic method and phosphoproteomics was performed using liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis followed by database search and bioinformatics analysis. Finally, cell experiments were used to verify the results of phosphoproteomics. Phosphorylated mitogen-activated protein kinase kinase kinase kinase 4 (MAP4k4) and phosphorylated adducin 1 (ADD1) were detected using western blotting.

RESULTS: DGR effectively reduced PBG, TG, and the liver index (P < 0.05), and significantly decreased HbA1c, TC, and hepatic portal TG (P < 0.01), showed significant hematoxylin and eosin (HE) staining, red oil O staining, and Masson staining of liver tissue. The total spectrum was 805 334, matched spectrum was 260 471, accounting for accounting 32.3%, peptides were 19 995, modified peptides were 14 671, identified proteins were 4601, quantifiable proteins were 4417, identified sites were 15 749, and quantified sites were 14659. Based on the threshold of expression fold change ( > 1.2), DGR up-regulated the modification of 228 phosphorylation sites involving 204 corresponding function proteins, and down-regulated the modification of 358 phosphorylation sites involving 358 corresponding function proteins, which included correcting 75 phosphorylation sites involving 64 corresponding function proteins relating to glycolipid metabolism. Therefore, DGR improved biological tissue processes, including information storage and processing, cellular processes and signaling, and metabolism. The metabolic functions regulated by DGR mainly include energy production and conversion, carbohydrate transport and metabolism, lipid transport and metabolism, inorganic ion transport and metabolism, secondary metabolite biosynthesis, transport, and catabolism. In vitro phosphorylation validation based on cell experiments showed that the change trends in the phosphorylation level of MAP4k4 and ADD1 were consistent with that of previous phosphoproteomics studies.

CONCLUSION: DGR extensively corrects the modification of phosphorylation sites to improve corresponding glycolipid metabolism-related protein expression in rats with glycolipid metabolism disorders, thereby regulating glycolipid metabolism through a multi-target and multi-method process.

Key words: phosphoproteomics, glycolipid metabolism, therapeutic mechanism, compound formula, Dangua Fang

HENG Xianpei, WANG Zhita, LI Liang, YANG Liuqing, HUANG Suping, JIN Lang, HE Weidong. Mechanisms of Dangua Fang (丹瓜方) in multi-target and multi-method regulation of glycolipid metabolism based on phosphoproteomics[J]. Journal of Traditional Chinese Medicine, 2024, 44(2): 334-344.

Figures/Tables 7

Table 1 Comparison of glycolipid metabolism index between the groups ($\bar{x}±s$)

Group	n	FBG (mmol/L)	PBG (mmol/L)	HbA1c (%)	TG (mmol/L)	Hepatic portal TG (mmol/L)	TC (mmol/L)	Liver index (%)
A	6	6.05±0.30	6.58±0.43	6.05±0.35	1.07±0.22	0.22±0.06	2.35±0.32	2.39±0.20
B	6	6.26±0.43	8.00±0.80^a	6.48±0.30^c	1.80±0.20^a	0.40±0.14^a	5.99±0.68^a	4.37±0.34^a
C	6	6.06±0.56	7.38±0.19^b	5.87±0.31^d	1.49±0.19^b	0.19±0.13^d	5.06±0.41^d	3.98±0.29^b

Figure 1 Functional classification and subcellular structure location A, B: GO functional classification; C, D: Distribution of the subcellular structure of the corresponding proteins of the differential phosphorylation modification sites. A, C: group B vs group A; B, D: group C vs group B. Rats in group A (n = 7) were fed conventional chow. Rats in groups B (n = 7) and C (n = 7) were fed high-fat-high-sugar diet lasting for 4 weeks. Then, DGR liquid was administered according to 20.5 g·kg-1·d-1 to rats in group C and the same amount of clean water was administered to rats in group B until the end of the experiment. DGR: Dangua Fang; GO: Gene Ontology.

Table 2 Differentially modified modification sites (modified proteins) summary (filtered with threshold value of expression fold change)

Site comparison	Regulated type	Fold change >1.2	Fold change >1.3	Fold change >1.5	Fold change >2
B vs A	up-regulated	1171 (777)	1105 (745)	896 (607)	487 (332)
B vs A	down-regulated	788 (570)	728 (525)	536 (390)	245 (196)
C vs B	up-regulated	228 (204)	200 (179)	145 (130)	70 (63)
C vs B	down-regulated	432 (358)	358 (302)	180 (164)	35 (32)

Figure 2 Analysis of GO function enrichments involving in the corresponding proteins of the differential phosphorylation modification site regulated by DGR (group C vs group B) A, B: biological processes; C, D: molecular function; G: cellular components. A, D: down-regulated; B, E: up-regulated; C, F, G: the directed acyclic graph. The circle in figures represent the GO classification where the differentially modified proteins are located. Red represents the highly significant (P < 0.01) enriched GO classification of the differentially modified protein, yellow represents the significantly enriched GO classification of the differentially modified protein (P < 0.05), and blue represents the non-significantly enriched GO category. The line with arrows represents the upper and lower levels of GO classification, and the size of the circle represents the degree of enrichment (fold enrichment). Rats in group A (n = 7) were fed conventional chow. Rats in groups B (n = 7) and C (n = 7) were fed high-fat-high-sugar diet lasting for 4 weeks. Then, DGR liquid was administered according to 20.5 g·kg-1·d-1 to rats in group C and the same amount of clean water was administered to rats in group B until the end of the experiment. DGR: Dangua Fang; GO: Gene Ontology.

Figure 3 Corrected pathological phosphorylation modification sites and corresponding amino acids and their proteins (C vs B) A: proteins with modification sites that were all down-regulated; B: proteins with modification sites that were partially down-regulated; C: proteins with modification sites that were all up-regulated; D: proteins with modification sites that were partially up-regulated. Rats in group A (n = 7) were fed conventional chow. Rats in groups B (n = 7) and C (n = 7) were fed high-fat-high-sugar diet lasting for 4 weeks. Then, DGR liquid was administered according to 20.5 g·kg-1·d-1 to rats in group C and the same amount of clean water was administered to rats in group B until the end of the experiment. DGR: Dangua Fang.

Table 3 Quantity contrast of the corresponding proteins of the differential phosphorylation modification sites involving glycolipid metabolism in the GO function enrichment analysis

COG/KOG category	Category description	No. of proteins of B vs A		No. of proteins of C vs B
COG/KOG category	Category description	Up	Down	Up	Down
Information storage and processing	[J] Translation, ribosomal structure, and biogenesis	13	11	4	6
	[A] RNA processing and modification	11	13	6	11
	[K] Transcription	26	19	5	16
	[L] Replication, recombination, and repair	7	1		1
	[B] Chromatin structure and dynamics	11	2		4
Cellular processes and signaling	[D] Cell cycle control, cell division, and chromosome partitioning	15	9	2	3
	[V] Defense mechanisms	6	3	1
	[Y] Nuclear structure	1	4		1
	[T] Signal transduction mechanisms	105	36	11	21
	[N] Cell motility	2	1		2
	[Z] Cytoskeleton	29	8	3	5
	[W] Extracellular structures	3	1	1
	[U] Intracellular trafficking, secretion, and vesicular transport	23	13	3	7
	[O]Posttranslational modification, protein turnover, and chaperones	18	10	11	6
Metabolism	[C] Energy production and conversion	4	8	1	3
	[G] Carbohydrate transport and metabolism	4	9	4
	[E] Amino acid transport and metabolism	2	17	1
	[F] Nucleotide transport and metabolism	4	2	1
	[H] Coenzyme transport and metabolism		1
	[I] Lipid transport and metabolism	14	14	2	3
	[P] Inorganic ion transport and metabolism	12	5	1	1
	[Q] Secondary metabolites biosynthesis, transport, and catabolism	1	5	2
Poorly characterized	[R] General function prediction only	55	38	14	12
Poorly characterized	[S] Function unknown	19	14	5	6

Figure 4 Phosphorylation levels of p-GCN2 and p-SREBP1 in cell experiment detected using western blot A, C: western blot; western blots in A are control, 5%DGR, model, 10%DGR. in proper order and in C are control, model, 5%DGR, 10%DGR. B, D: quantitative comparison of western blot detection results. Control: conventional culture without intervention; Model: without drug intervention after modelling; 5%DGR: 5% medium-dose medicated serum + 10% blank serum after modelling; 10%DGR: 10% medium-dose medicated serum + 5 % blank serum. DGR: Dangua Fang; p-GCN2: phospho-general control non-derepressible 2 (n = 4); p-SREBP 1: phospho- sterol-regulatory element binding protein 1 (n = 6). Compared with control group: aP < 0.05; compared with model group: bP < 0.05, cP < 0.01.

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[1]	HENG Xianpei, WANG Zhita, YANG Liuqing, LI Liang, HUANG Suping. Dangua Fang (丹瓜方) regulating tricarboxylic acid cycle and respiratory chain and its mechanism in diabetic rats [J]. Journal of Traditional Chinese Medicine, 2023, 43(6): 1150-1159.
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