Network pharmacology combined with in vivo experiments to explore the molecular mechanism of Jiawei Erzhi pill (加味二至丸) protects against atherosclerosis by inhibiting ferroptosis

doi:10.19852/j.cnki.jtcm.2025.06.012

Abstract

Abstract:

OBJECTIVE: To elucidate the possible mechanism of Jiawei Erzhi pill (加味二至丸, JWEZP) in the treatment of atherosclerosis (AS).

METHODS: The chemical constituents of JWEZP were identified using ultra-performance liquid chromatography-mass spectrometry. A high-fat diet (HFD) was used to establish AS models in ApoE^-/- mice. The ApoE^-/- mice were randomly divided into the Model (normal saline), Simvastatin (normal saline), Low-dose JWEZP, Medium-dose JWEZP and High-dose JWEZP groups (n = 15), and C57BL/6 mice on a normal diet were used as the control group. Mice were treated with JWEZP at different doses (3.9, 7.8, 15.6 g·kg?¹·d?¹) or with simvastatin (2.6 mg·kg?¹·d?¹) for ten weeks. The inhibitory effects of JWEZP on AS were assessed by measuring serum lipid levels and changes in atherosclerotic plaques, lipid peroxidation and ferroptosis. Common targets and key regulatory pathways of JWEZP-mediated inhibition of ferroptosis were predicted using network pharmacology and verified using real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting.

RESULTS: We identified 46 active compounds in JWEZP. Mice in the JWEZP group had lower body weights and serum cholesterol levels compared to HFD mice. The results of Hematoxylin-Eosin and Oil Red O staining showed that JWEZP alleviated AS. Masson staining showed that JWEZP improved the stability of atherosclerotic plaques. In addition, JWEZP-treated mice had lower levels of reactive oxygen species (ROS) in thoracic aortic tissue according to ROS fluorescence staining. The ELISA results showed that JWEZP decreased the levels of iron, lipid peroxide, malondialdehyde and nicotinamide adenine dinucleotide phosphate and increased the levels of glutathione (GSH) and GSH-PX in the thoracic aortic tissues of mice. The expression of glutathione peroxidase 4 in the thoracic aorta of mice in the JWEZP group was upregulated in the results of the immunofluorescence assay. Network pharmacology results indicated that the action mechanisms of JWEZP-mediated inhibition of ferroptosis were closely related to the p53, mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (Akt) signaling pathways. RT-qPCR and Western blotting results demonstrated that JWEZP inhibited the p53 and MAPK pathways, and activated the PI3K/Akt pathway to regulate ferroptosis.

CONCLUSION: JWEZP improved AS by inhibiting ferroptosis. The study provides a scientific basis for further research and validation of JWEZP as a potential therapeutic for AS.

Key words: atherosclerosis, ferroptosis, network pharmacology, Jiawei Erzhi pill

MA Guiping, CHEN Ran, LI Junlong, SUN Le, HU Shiping, ZHANG Yiyi, HONG Chuangxiong. Network pharmacology combined with in vivo experiments to explore the molecular mechanism of Jiawei Erzhi pill (加味二至丸) protects against atherosclerosis by inhibiting ferroptosis[J]. Journal of Traditional Chinese Medicine, 2025, 45(6): 1330-1341.

Figures/Tables 5

Table 1 Effects of JWEZP on blood lipids of mice ($\bar{x}$ ± s, mmol/L)

Group	n	TC	TG	HDL-C	LDL-C
CON	10	3.523±0.113^a	2.518±0.297	2.841±0.063^a	0.363±0.022^a
MOD	10	48.706±2.770^b	3.685±0.30	1.583±0.115^b	35.021±1.109^b
SIM	10	37.045±3.721^ab	2.961±0.932	1.801±0.067^bc	26.905±4.955^ab
L-JWEZP	10	43.981±2.871^b	3.293±1.437	1.771±0.191^b	34.682±2.277^b
M-JWEZP	10	43.735±1.765^b	2.910±0.403	1.998±0.236^ab	30.428±3.337^ab
H-JWEZP	10	40.393±2.883^bc	3.556±0.958	1.930±0.305^ab	29.086±1.896^ab

Figure 1 JWEZP improves AS in ApoE-/- mice A: body weight change trend in mice from week 12 to week 22; B: body weight of mice at week 12; C: body weight of mice at week 22; D: Oil-red O staining of aorta; E: HE staining of the aortic sinus (magnification at × 400; scale bar, 50 μm. F: Masson staining of the aortic sinus (magnification at × 400; scale bar, 50 μm); G: percentages of plaque lesions in the aorta; H: atherosclerotic plaque areas in the aortic sinus; I: collagen fiber positive areas in the aortic sinus; D1, E1, F1: CON group; D2, E2, F2: MOD group; D3, E3, F3: SIM group; D4, E4, F4: L-JWEZP group; D5, E5, F5: M-JWEZP group; D6, E6, F6: H-JWEZP group. CON: treated only with physiological saline; MOD: treated only with physiological saline; SIM: treated with simvastatin by gavage (2.6 mg·kg?1·d?1); L-JWEZP: treated with low-dose Jiawei Erzhi pill by gavage (3.9 g·kg?1·d?1); M-JWEZP: treated with medium-dose Jiawei Erzhi pill by gavage (7.8 g·kg?1·d?1); H-JWEZP: treated with high-dose Jiawei Erzhi pill by gavage (15.6 g·kg?1·d?1). CON: control group; MOD: model group; SIM: simvastatin group; L-JWEZP: low-dose Jiawei Erzhi pill; M-JWEZP: medium-dose Jiawei Erzhi pill; H-JWEZP: high-dose Jiawei Erzhi pill. The results are expressed as mean ± standard deviation (n = 3), and the groups were compared using one-way analysis of variance. aP < 0.01, compared with model group; bP < 0.01, compared with control group; cP < 0.05, compared with model group.

Figure 2 JWEZP ameliorates ferroptosis in arterial tissues of mice A: content of GSH in the aortas of mice; B: content of LPO in the aortas of mice; C: content of MDA in the aortas of mice; D: content of NADPH in the aortas of mice; E: content of GSH-PX in the aortas of mice; F: content of iron in the aortas of mice; G: expression of ROS in thoracic aortic tissues; H: expression of GPX4 in thoracic aortic tissues were evaluated by immunofluorescence. G1, H1, I1, J1: CON group; G2, H2, I2, J2: MOD group; G3, H3, I3, J3: SIM group; G4, H4, I4, J4: L-JWEZP group; G5, H5, I5, J5: M-JWEZP group; G6, H6, I6, J6: H-JWEZP group. CON: treated only with physiological saline; MOD: treated only with physiological saline; SIM: treated with simvastatin by gavage (2.6 mg·kg?1·d?1); L-JWEZP: treated with low-dose Jiawei Erzhi pill by gavage (3.9 g·kg?1·d?1); M-JWEZP: treated with medium-dose Jiawei Erzhi pill by gavage (7.8 g·kg?1·d?1); H-JWEZP: treated with high-dose Jiawei Erzhi pill by gavage (15.6 g·kg?1·d?1). CON: control group; MOD: model group; SIM: simvastatin group; L-JWEZP: low-dose Jiawei Erzhi pill; M-JWEZP: medium-dose Jiawei Erzhi pill; H-JWEZP: high-dose Jiawei Erzhi pill; GSH: glutathione; LPO: lipid peroxide; MDA: malondialdehyde; NADPH: nicotinamide adenine dinucleotide phosphate; ROS: reactive oxygen species; DAPI: 4',6-Diamidino-2-Phenylindole; GPX4: glutathione peroxidase 4. The results are expressed as mean ± standard deviation (n = 3), and the groups were compared using one-way analysis of variance. aP < 0.01, compared with model group; bP < 0.01, compared with control group; cP < 0.05, compared with model group.

Figure 3 Target-pathway network for JWEZP in ferroptosis A: venn diagram of JWEZP and ferroptosis targets; B: protein-protein interaction network of JWEZP and ferroptosis; C: results of KEGG enrichment analysis. JWEZP: Jiawei Erzhi pill; KEGG: Kyoto Encyclopedia of Genes and Genomes.

Figure 4 JWEZP inhibits ferroptosis via p53, MAPK and PI3K/Akt signaling pathways against AS A: protein expression of p53 signaling pathway; B: protein expression of p53; C: protein expression of COX2; D: protein expression of FTH1; E: protein expression of SLC7A11; F: protein expression of GPX4; G: mRNA expression of p53; H: mRNA expression of PTGS2; I: mRNA expression of FTH1; J: mRNA expression of SLC7A11; K: mRNA expression of GPX4; L: protein expression of MAPK and PI3K/Akt signaling pathways; M: protein expression of p-JNK/JNK; N: protein expression of p-PI3K/PI3K; O: protein expression of p-Akt/Akt. CON: treated only with physiological saline; MOD: treated only with physiological saline; SIM: treated with simvastatin by gavage (2.6 mg·kg?1·d?1); L-JWEZP: treated with low-dose Jiawei Erzhi pill by gavage (3.9 g·kg?1·d?1); M-JWEZP: treated with medium-dose Jiawei Erzhi pill by gavage (7.8 g·kg?1·d?1); H-JWEZP: treated with high-dose Jiawei Erzhi pill by gavage (15.6 g·kg?1·d?1). CON: control group; MOD: model group; SIM: simvastatin group; L-JWEZP: low-dose Jiawei Erzhi pill; M-JWEZP: medium-dose Jiawei Erzhi pill; H-JWEZP: high-dose Jiawei Erzhi pill; COX2: cyclooxygenase-2; FTH1: ferritin heavy chain 1; SLC7A11: solute carrier family 7 member 11; GPX4: glutathione peroxidase 4; JNK: c-Jun N-terminal kinase; PI3K: phosphatidylinositol 3-kinase; Akt: protein Kinase B; MAPK: mitogen-activated protein kinase. The results are expressed as mean ± standard deviation (n = 3), and the groups were compared using one-way analysis of variance. aP < 0.01, compared with model group; bP < 0.01, compared with control group; cP < 0.05, compared with model group.

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