Network pharmacology and experimental validation to reveal the pharmacological mechanisms of Sini decoction (四逆汤) against renal fibrosis

doi:10.19852/j.cnki.jtcm.20230630.003

Abstract

Abstract:

OBJECTIVE: To investigate the mechanism by which Sini decoction (四逆汤, SND) improves renal fibrosis (Rf) in rats based on transforming growth factor β1/Smad (TGF-β1/Smad) signaling pathway.

METHODS: Network pharmacology was applied to obtain potentially involved signaling pathways in SND's improving effects on Rf. The targets of SND drug components and the targets of Rf were obtained by searching databases, such as the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCSMP) and GeenCard. The intersection targets of two searches were obtained and underwent signaling pathway analysis using a Venn diagram. Then experimental pharmacology was utilized to prove and investigate the effects of SND on target proteins in the TGF-β1/Smad signaling pathway. The Rf rat model was established by unilateral ureteral occlusion (UUO). The expression levels of transforming growth factor, matrix metalloproteinase-9 (MMP-9), matrix metal protease-2 (MMP-2), connective tissue growth factor (CTGF), and tissue inhibitor of metalloproteinase-1 (TIMP-1) were determined by Masson staining of rat renal tissue, and immunohistochemical methods. The expression levels of Smad3, Smad2, and Smad7 in renal tissue were determined by Western blotting (WB). The mechanism of the improving effects of SND on Rf was investigated based on TGF-β1/Smad signaling pathway.

RESULTS: A total of 12 drug components of Fuzi (Radix Aconiti Lateralis Preparata), 5 drug components of Ganjiang (Rhizoma Zingiber), and 9 drug components of Gancao (Radix Glycy et Rhizoma) were obtained from the database search, and 207 shared targets were found. A total of 1063 Rf targets were found in the database search. According to the Venn diagram, in total, 96 intersection targets were found in two database searches. The metabolic pathways involved included TGF-β signaling pathway, phosphatidylinositol-3-kinase/serine-threonine protein kinase signaling (PI3K/Akt) pathway, and hypoxia-inducible factor-1 (HIF-1) signaling pathway. Masson staining analysis showed that compared with the model group, the renal interstitial collagen deposition levels in the SSN and SND groups were significantly lower (P < 0.05). Immunohistochemical analysis, compared with the control group, the positive cell area expression levels of MMP-9/TIMP-1 and MMP-2/TIMP-1 in the kidney tissue of the model group were significantly decreased (P < 0.05, P < 0.01), and the positive cell area expression levels of CTGF and TGF-β1 were significantly increased (P < 0.01). Compared with the model group, the positive cell area expression levels of MMP-9/TIMP-1 and MMP-2/TIMP-1 in the kidney tissue of the SSN and SND groups were significantly increased (P < 0.05, P < 0.01), and the positive cell area expression levels of CTGF and TGF-β1 in the kidney tissue were significantly decreased (P < 0.05, P < 0.01). WB results showed that the SSN group and the SND group could reduce the expression of Smad2 and Smad3 (P < 0.05) and increase the expression of Smad7 (P < 0.05).

Key words: renal fibrosis, network pharmacology, transforming growth factor beta1, smad proteins, Sini decoction

WANG Yan, DENG Fanying, LIU Shiqi, WANG Yingli. Network pharmacology and experimental validation to reveal the pharmacological mechanisms of Sini decoction (四逆汤) against renal fibrosis[J]. Journal of Traditional Chinese Medicine, 2024, 44(2): 362-372.

Figures/Tables 7

Table 1 Temperature and wildlife count in the three areas covered by the study

Herb	Number of drug components found in TCMSP	Number of drug components in Chinese Pharmacopoeia	Targets of drug components
Fuzi (Radix Aconiti Lateralis Preparata)	7	5	138
Ganjiang (Rhizoma Zingiber)	4	1	134
Gancao (Radix Glycy Et Rhizoma)	7	2	69

Table 2 Key targets of SND acting on renal tissue (top 30)

Target	Name	Degree	Betweenness centrality	Closeness centrality
EGFR	Epidermal growth factor receptor	98	0.103	0.669
HSP90AA1	Heat shock protein 90 alpha family class a member 1	96	0.117	0.650
JUN	Jun proto-oncogene, ap-1 transcription factor subunit	88	0.061	0.628
MAPK3	Mitogen-activated protein kinase 3	84	0.045	0.628
STAT3	Signal transducer and activator of transcription 3	84	0.038	0.616
ESR1	Estrogen receptor 1	78	0.058	0.616
PPARG	Peroxisome proliferator activated receptor gamma	76	0.080	0.608
MTOR	Mechanistic target of rapamycin kinase	76	0.018	0.596
PTGS2	Prostaglandin-endoperoxide synthase 2	72	0.059	0.600
MDM2	MDM2 proto-oncogene	70	0.025	0.596
BCL2L1	BCL2 like 1	68	0.019	0.581
PIK3CA	Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha	68	0.018	0.578
MMP9	Matrix metallopeptidase 9	58	0.017	0.560
KDR	Kinase insert domain receptor	54	0.022	0.560
JAK2	Janus kinase 2	54	0.008	0.544
AR	Androgen receptor	52	0.010	0.544
NR3C1	Nuclear receptor subfamily 3 group c member 1	52	0.056	0.567
GSK3B	Glycogen synthase kinase 3 beta	52	0.012	0.554
IGF1R	Insulin like growth factor 1 receptor	50	0.004	0.547
MAP2K1	Mitogen-activated protein kinase 1	48	0.004	0.538
APP	Amyloid beta precursor protein	46	0.052	0.554
MMP2	Matrix metallopeptidase 2	44	0.004	0.531
CDK2	Cyclin dependent kinase 2	44	0.003	0.520
PIK3CB	Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit beta	40	0.005	0.482
COMT	Catechol-o-methyltransferase	38	0.026	0.482
CYP19A1	Cytochrome p450 family 19 subfamily a member 1	38	0.021	0.531
PARP1	Poly(ADP-Ribose) polymerase 1	36	0.001	0.503
TERT	Telomerase reverse transcriptase	34	0.001	0.505
JAK1	Janus kinase 1	34	0.001	0.487
ESR2	Estrogen receptor 2	34	0.022	0.528

Figure 1 GO function enrichment and KEGG signaling pathway analysis results of SND in the treatment of Rf A: GO function enrichment; B: KEGG signaling pathway. SND: Sini decoction; Rf: renal fibrosis. ErbB: epidermal growth factor receptor; HIF-1: hypoxiainduciblefactor-1; RAC1/pak1/p38/MMP2: ras-related C3 botulinum toxin substrate 1/p21-activated kinases 1/p38 mi‐togen-activated protein kinase; FoxO: forkhead box protein O; cAMP: cyclic adenosine monophosphate; TGF:transforming growth factor; PI3K-Akt: phosphatidylinositol-3-kinase/serine-threonine protein kinase; MAPK: mitogen-activated protein kinase.

Figure 2 Molecular docking diagram of chemical composition to target A: 6-Gingerol vs Smad7; B: 6-Gingerol vs Smad2; C: 6-Gingerol vs TGF-β1; D: Hypaconitine vs Smad3; E: Aconitine vs Smad3; F: Mesaconitine vs TGF-β1. TGF-β1: transforming growth factor β1.

Table 3 Molecular docking results of the active ingredient and the target

Active compounds	Combined energy (kcal/mol)
Active compounds	Smad2	Smad3	Smad7	TGF-β1
6-Gingerol	－7.9	－7.5	－8.9	－7.8
Hypaconitine	－6.9	－7.2	－6.7	－6.9
Aconitine	－7.2	－7.4	－5.9	－6.3
Mesaconitine	－7.0	－7.2	－6.4	－7.2

Figure 3 Images of renal tissue of Rf rats, masson staining and IHC stained TGF-β1, MMP-2, MMP-9, CTGF, and TIMP-1 granules (× 400) A: masson staining; B: expression of MMP-2; C: expression of MMP-9; D: expression of CTGF; E: expression of TIMP-1; F: expression of TGF-β1. Control: the unilateral ureter was separated without ligation (A1, B1, C1, D1, E1, F1); model: the unilateral ureter was separated and ligation (A2, B2, C2, D2, E2, F2); SSN: the unilateral ureter was separated and ligation, than gavage Shenshuaining 0.432 g·kg-1·d-1 2 weeks (A3, B3, C3, D3, E3, F3); SND: the unilateral ureter was separated and ligation, than gavage Sini decoction 0.585 g·kg-1·d-1 2 weeks (A4, B4, C4, D4, E4, F4). Rf: renal fibrosis; MMP-2: matrix metal protease-2; MMP-9: matrix metalloproteinase-9; CTGF: connective tissue growth factor; TIMP-1: tissue inhibitor of metalloproteinase-1; TGF-β1: transforming growth factor β1.

Figure 4 Effect of SND on TGF-β1/Smad pathway A: the expression of MMP-2/TIMP-1 (A1), MMP-9/TIMP-1 (A2), TGF-β1 (A3), and CTGF (A4) in renaltissue of rats with Rf (n = 6); B: expression levels of Smad2 (B1), Smad3 (B2) and Smad7 (B3) in renal tissue of four groups (n = 3). Control: the unilateral ureter was separated without ligation; model: the unilateral ureter was separated and ligation; SSN: the unilateral ureter was separated and ligation, than gavage Shenshuaining 0.432 g·kg-1·d-1 2 weeks; SND: the unilateral ureter was separated and ligation, than gavage Sini decoction 0.585 g·kg-1·d-1 2 weeks. MMP-2: matrix metal protease-2; MMP-9: matrix metalloproteinase-9; CTGF: connective tissue growth factor; TIMP-1: tissue inhibitor of metalloproteinase-1; TGF-β1: transforming growth factor β1. Data are expressed as the mean ± standard deviation; t-test, aP < 0.01, bP < 0.05 as compared to the model.

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