A network pharmacology approach to discover active compounds and action mechanisms of San-Cao Granule for treatment of liver fibrosis

Figures & data

Figure 1 The whole framework of this study based on an integrative strategy of network pharmacology and experimental verification.

Abbreviations: SCG, San-Cao Granule; TAA, thioacetamide; ELISA, enzyme-linked immunosorbent assay; HE, hematoxylin-eosin; TTD, Therapeutic Target Database; TDT, TCM Database@Taiwan; HIT, Herbal Ingredients’ Targets Database; OMIM, Online Mendelian Inheritance in Man; PPI, protein-protein interaction.

Figure 2 The main network of compound–target–disease of SCG.

Notes: The red triangles represent active chemical constituents of SCG, the blue dots represent the indirect targets for drugs, the yellow dots represent the targets of the specific disease of liver fibrosis, the yellow squares represent the common targets of herbs and liver fibrosis, and the purple dots represent the interactional proteins with the targets of liver fibrosis and drugs.
Abbreviation: SCG, San-Cao Granule.

Table 1 The targets and its relevant topological parameters and corresponding active compounds of SCG screened by network pharmacology

Direct target	Target protein	Degree	Closeness centrality	Betweeness centrality	Compound
P05067	Amyloid β A4 protein	12	0.9167	0.9818	Cryptotanshinone
P60709	Actin, cytoplasmic 1	9	0.5313	0.7794	Danshensu
P01137	Transforming growth factor β-1	8	0.4473	0.4265	Danshensu
P09038	Fibroblast growth factor 2	6	0.65	0.641	Ursolic acid
P11802	Cyclin-dependent kinase 4	11	0.2577	0.0729	Glycyrrhetinic acid
P31749	RAC-α serine/threonine-protein kinase	13	0.2673	0.061	Salvianolic acid A
P03372	Estrogen receptor	21	0.2407	0.0906	Formononetin
O15392	Baculoviral IAP repeat-containing protein 5	8	0.1717	0.0363	Ursolic acid
Q07817	Bcl-2-like protein 1	4	0.205	0.0054	Ursolic acid
P07550	β-2 adrenergic receptor	3	1	1	11,12,13-trinorguai-6-ene-4β,10β-diol
P22303	Acetylcholinesterase	1	0.5	0	Ursolic acid
P04141	Granulocyte-macrophage colony-stimulating factor	3	1	1	Glycyrrhizic acid
P01008	Antithrombin-III	3	1	0	Cryptotanshinone
P62736	Actin, aortic smooth muscle	0	0	0	Salvianolic acid B
Q9UKK3	Poly [ADP-ribose] polymerase 4	0	0	0	TanshinoneII A
P09917	Arachidonate 5-lipoxygenase	0	0	0	Alizarin

Abbreviation: SCG, San-Cao Granule.

Figure 3 Potential pathways annotation of putative targets.

Notes: The bar map shows the top 22 pathways of putative targets with SCG compounds. X-axis represents the percentage of targets in the total pathways. Y-axis represents the names of putative pathways.
Abbreviations: TGF, transforming growth factor; MAPK, mitogen-activated protein kinase; SCG, San-Cao Granule; NOD, nucleotide-binding oligomerization domain.

Table 2 The 15 identified components in SCG

Number	Identified components	Molecular formula	Attribution
1	Rubimaillin	C17H16O4	Rubia cordifolia
2	Purpurin	C14H8O5	Rubia cordifolia
3	Gentiopicroside	C16H20O9	Gentiana macrophylla
4	Tanshinone IIA	C19H18O3	Salvia miltiorrhiza
5	Salvianolic acid B	C36H30O16	Salvia miltiorrhiza
6	Danshensu	C9H10O5	Salvia miltiorrhiza
7	Glycyrrhizic acid	C42H62O16	Glycyrrhiza uralensis
8	Glycyrrhetinic acid	C30H46O4	Glycyrrhiza uralensis
9	Alizarin	C14H8O4	Rubia cordifolia
10	Cryptotanshinone	C19H20O3	Salvia miltiorrhiza
11	Ferulic acid	C10H10O4	Forsythia suspense
12	Formononetin	C16H12O4	Glycyrrhiza uralensis
13	Orientin	C21H20O11	Siegesbeckia orientalis
14	Sweroside	C16H22O9	Gentiana macrophylla
15	Swertamarin	C16H22O10	Gentiana macrophylla

Abbreviation: SCG, San-Cao Granule.

Figure 4 The serum levels of ALT, AST, ALB, TB, HA, LN, and CIV of experimental groups.

Notes: (A): ALT, AST, ALB, TB; (B): HA, LN, CIV. Data are expressed as the mean ± SD. (SCG-H: TAA + SCG [3.6 g/kg]; SCG-M: TAA + SCG [1.8 g/kg]; SCG-L: TAA + SCG [0.9 g/kg]). *P<0.01 compared with normal group, **P<0.01 compared with model group, ^#P<0.05 compared with model group.
Abbreviations: SCG, San-Cao Granule; UDCA, ursodeoxycholic acid; TAA, thioacetamide; ALT, alanine transaminase; AST, aspartate aminotransferase; ALB, albumin; TB, total bilirubin; HA, hyaluronic acid; LN, laminim; CIV, IV collagen.

Figure 5 The effect of SCG on the expressions of α-SMA and CTGF.

Notes: (A) The expression of α-SMA detected by immunohistochemistry staining (a: Normal; b: Model; c: UDCA; d: SCG-H; e: SCG-M; f: SCG-L); (B, C) the expression of α-SMA and CTGF detected by Western blot; (D) the level of Hyp in liver tissue; (E) the percentage of collagen content detected by image software through liver tissue stained by Masson (100×); (F) the integrated optical density (IOD) value of α-SMA. (SCG-H: TAA + SCG [3.6 g/kg]; SCG-M: TAA + SCG [1.8 g/kg]; SCG-L: TAA + SCG [0.9 g/kg]). *P<0.01 compared with normal group, **P<0.01 compared with model group.
Abbreviations: SCG, San-Cao Granule; UDCA, ursodeoxycholic acid; TAA, thioacetamide; SMA, smooth muscle actin; CTGF, connective tissue growth factor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Hyp, hydroxyproline.

Figure 6 The effect of SCG on the pathological features of liver tissues of experimental rats stained with HE (A) and Masson (B). (SCG-H: TAA + SCG [3.6 g/kg]; SCG-M: TAA + SCG [1.8 g/kg]; SCG-L: TAA + SCG [0.9 g/kg]).

Abbreviations: SCG, San-Cao Granule; UDCA, ursodeoxycholic acid; TAA, thioacetamide; HE, hematoxylin-eosin.

Table 3 The degree of hepatic fibrosis in rats

Groups	n	0	I	II	III	IV	Staging scores (mean ± SD)
Normal	10	10	0	0	0	0	0
Model	10	0	0	0	7	3	3.3±0.48*
UDCA	10	0	1	4	5	4	2.4±0.69**
TAA + SCG (3.6 g/kg)	10	0	8	2	0	0	1.2±0.42**
TAA + SCG (1.8 g/kg)	10	0	5	4	1	0	1.6±0.68**
TAA + SCG (0.9 g/kg)	10	0	2	5	3	0	2.1±0.73**

Notes:

* P<0.01 compared with normal group,

** P<0.01 compared with model group.

Abbreviations: SCG, San-Cao Granule; UDCA, ursodeoxycholic acid; TAA, thioacetamide; SD, standard deviation.

Figure 7 The effect of SCG on the expressions and semiquantitative analysis of TGF-β1, p-Smad3, Smad3, and Smad7 detected by Western blot.

Notes: (SCG-H: TAA + SCG [3.6 g/kg]; SCG-M: TAA + SCG [1.8 g/kg]; SCG-L: TAA + SCG [0.9 g/kg]). *P<0.01 compared with normal group, **P<0.01 compared with model group. (A) Western blot analysis for TGF-β1, p-Smad3, Smad3, and Smad7; Densitometry analysis of TGF-β1 (B), Smad7 (C), and p-Smad3/Smad3 (D) on Western blot.
Abbreviations: SCG, San-Cao Granule; TAA, thioacetamide; TGF-β1, transforming growth factor β1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.