Efficacy of Ganshuang granules (肝爽颗粒) on non-alcoholic fatty liver and underlying mechanism: a network pharmacology and experimental verification

doi:10.19852/j.cnki.jtcm.20231215.001

Abstract

Abstract:

OBJECTIVE: To investigate the potential pharmacological mechanisms of Ganshuang granules (肝爽颗粒, GSG) in treating non-alcoholic fatty liver (NAFLD).

METHODS: All the active components and targets of GSG were retrieved from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform. Protein-Protein interaction network, Kyoto Encyclopedia of Genes and Genomes and Gene Ontology function annotation of common targets were analyzed to predict the mechanisms of action of GSG in the treatment of NAFLD. Then, the mouse models of NAFLD were constructed in a diet-induced manner and treated with GSG. The levels of interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α) and phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway-related proteins in the liver of mice in each group were measured by enzyme linked immunosorbent assay and Western blot, respectively.

RESULTS: Network pharmacology revealed a total of 159 potential targets of GSG for the treatment of NAFLD. Functional enrichment analysis indicated that the PI3K/AKT signaling pathway may be involved during GSG treatment of NAFLD. Further experiments showed that the significantly decreased alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total cholesterol, triglyceride and low-density lipoprotein cholesterol levels in NAFLD model mice serum after GSG treatment, as well as the expression levels of IL-6 and TNF-α in the liver. Furthermore, drug intervention increased the protein expression levels of phosphorylated-PI3K (P-PI3K) and P-AKT in the liver of the model group mice, and decreased the protein expression level of sterol regulatory element-binding protein 1.

CONCLUSION: We found that GSG is effective in treating NAFLD and the potential therapeutic targets may be involved in PI3K/AKT signaling pathway.

Key words: fatty liver, alcoholic, network pharmacology, models, animal, phosphatidylinositol 3-kinase, proto-oncogene proteins c-akt, signal transduction, Ganshuang granules

ZHI Guoguo, SHAO Bingjie, ZHENG Tianyan, JI Shaoxiu, LI Jingwei, DANG Yanni, LIU Feng, WANG Dong. Efficacy of Ganshuang granules (肝爽颗粒) on non-alcoholic fatty liver and underlying mechanism: a network pharmacology and experimental verification[J]. Journal of Traditional Chinese Medicine, 2024, 44(1): 122-130.

Figures/Tables 7

Figure 1 PPI core target The smaller the degree, the lighter the color and the smaller the circle. PPI: protein-protein interaction; TNF: tumor necrosis factor; IL-6: interleukin 6; PIK3R1: phosphoinositide-3-kinase regulatory subunit 1; VEGFA: vascular endothelial growth factor A; CXCL8: C-X-C motif chemokine ligand 8; APP: amyloid precursor protein; STAT3: signal transducer and activator of transcription 3; Akt: protein kinase B; TP53: tumor protein p53.

Figure 2 Compound-target-pathway network diagram Green: circular nodes represent the active ingredients of GSG; purple: inverted triangle nodes represent signaling pathways; orange: rectangular nodes represent core goals. hsa04933: advanced glycation endproduct (AGE)-the receptor of advanced glycation endproduct (RAGE) signaling pathway in diabetic complications; hsa04657: IL-17 signaling pathway; hsa04066: hypoxia-inducible factor (HIF)-1 signaling pathway; hsa04920: adipocytokine signaling pathway; hsa04668: TNF signaling pathway; hsa04152: adenosine 5‘-monophosphate (AMP)-activated protein kinase (AMPK) signaling pathway; hsa04620: toll-like receptor signaling pathway; hsa04625: C-type lectin receptor signaling pathway; hsa04068: forkhead box O (FoxO) signaling pathway; hsa04151: PI3K-Akt signaling pathway; hsa04660: T cell receptor signaling pathway; hsa04910: insulin signaling pathway; hsa04630: Janus kinase (JAK)-STAT signaling pathway; hsa04010: mitogen-activated protein kinase (MAPK) signaling pathway; hsa04064: NF-kappa B signaling pathway; hsa04211: Longevity regulating pathway; hsa04621: NOD-like receptor signaling pathway; hsa04062: chemokine signaling pathway; hsa04115: tumor suppressor gene (p53) signaling pathway; hsa04370: VEGF signaling pathway; MOL007088: cryptotanshinone; MOL004635: saikosaponin A; MOL000422: kaempferol; MOL000098: quercetin; MOL000006: luteolin; MOL004328: naringenin; MOL007154: tanshinone IIA; MOL005828: nobiletin; MOL000472: emodin; MOL002268: rhein; MOL007074: salvianolic acid B; MOL005812: naringin; MOL005814: tangeretin; MOL001924: paeoniflorin; MOL000358: beta-sitosterol; MOL000354: isorhamnetin; MOL000043: atractylenolide; MOL013289: polydatin.

Table 1 Changes in indicators related to liver injury and lipid metabolism were reflected in the serum of each group ($\bar{x}±s$)

Item	Control (n = 6)	Model (n = 6)	GSG (n = 6)
ALT (U/L)	72.55±33.26	152.65±8.67^a	104.04±46.48^b
AST (U/L)	166.98±19.03	350.22±58.19^a	269.08±50.45^b
ALP (U/L)	102.10±6.23	138.03±24.10^a	103.56±23.21^b
TC (mmol/L)	3.62±0.57	5.07±0.44^a	3.58±0.89^b
TG (mmol/L)	0.99±0.21	2.04±0.29^a	0.75±0.17^b
LDL (mmol/L)	0.26±0.04	0.32±0.04^a	0.26±0.06^b

Figure 3 Results of liver pathological sections in each group by HE staining (× 20) A: control group; B: model group (× 20); C: GSG group (× 20). Control group: treated with normal diet and physiological saline; Model group: treated with high-fat diet and physiological saline; GSG group: treated with HFD and GSG of 0.1 mL/10 g. HE: hematoxylin and eosin; HFD: high-fat diet; GSG: Ganshuang granules.

Figure 4 Expression of lipids in the liver of each group by oil red staining (× 20) A: control group; B: model group; C: GSG group. Control group: treated with normal diet and physiological saline; Model group: treated with high-fat diet and physiological saline; GSG group: treated with HFD and GSG of 0.1 mL/10 g. HFD: high-fat diet; GSG: Ganshuang granules.

Table 2 Changes in mouse LW, BW, and LW/BW in each group ($\bar{x}±s$)

Item	Control (n = 6)	Model (n = 6)	GSG (n = 6)
LW (g)	0.943±0.050	1.970±0.084^a	1.598±0.151^b
BW (g)	27.517±1.848	31.183±2.882^a	29.180±1.778^b
LW/BW	0.034±0.001	0.063±0.004^a	0.055±0.005^b

Table 3 Expression of proteins in P-PI3K, P-AKT, SREBP-1, IL-6 and TNF-α in liver tissues changed ($\bar{x}±s$)

Item	Control (n = 4)	Model (n = 4)	GSG (n = 4)
IL-6 (pg/mg)	181.912±27.074	299.723±85.682^a	206.379±32.358^b
TNF-α (pg/mg)	11.700±2.369	34.001±7.657^a	14.192±3.424^b
SREBP-1/GAPDH	0.431±0.081	0.847±0.032^a	0.643±0.020^b
P-PI3K/GAPDH	0.317±0.050	0.221±0.044^a	0.365±0.051^b
P-AKT/GAPDH	0.894±0.160	0.542±0.121^a	0.849±0.038^b

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