Multiple components in Astragalus Injection target CCL2/FOS/PPARG/CXCL8 to suppress cervical cancer progression

doi:10.12122/j.issn.1673-4254.2026.09.19

Abstract

Abstract:

Objective To predict the functional targets and molecular mechanisms of Astragalus Injection for suppressing cervical cancer. Methods The overlapping targets between Astragalus Injection and cervical cancer were used to construct a protein-protein interaction network. GO functional annotation and KEGG pathway enrichment analysis were performed, and molecular docking study was conducted to evaluate the binding capacity between drug active components and the core targets. SiHa cells were treated with Astragalus Injection and the changes in cell proliferation, migration and invasion were examined. The expressions of the core targets (FOS, PPARG, CXCL8, and CCL2), key molecules in the IL-17 pathway (NF-κB p65, p-p65, STAT3, and p-STAT3), Bax, Bcl-2, caspase-3, and cleaved caspase-3 were detected in the treated cells. The changes in microfilament, mitochondrial membrane potential, cell apoptosis and membrane permeability were observed using Coomassie brilliant blue, phalloidin, JC-10 and AO/EB staining. In a mouse model bearing U14 cervical cancer xenograft, the effect of Astragalus Injection on tumor growth was observed, tumor histopathology was examined using HE staining, and CCL2, PPARG and FOS expressions were analyzed with immunohistochemistry. Results Five major active components of Astragalus Injection were found to stably bind to 4 core targets (CCL2, CXCL8, FOS, and PPARG) with docking binding energies all below -5 kJ/mol. Astragalus Injection significantly inhibited the proliferation, migration and invasion of SiHa cells, disrupted cytoskeletal microfilament structure, and induced cell apoptosis, causing also significantly lowered protein levels of FOS, CXCL8, CCL2, PPARG, Bcl-2, p-p65 and p-STAT3 and increased expressions of Bax and cleaved caspase-3. In the tumor-bearing mice, Astragalus Injection significantly suppressed xenograft growth, induced tumor tissue necrosis, and reduced the expressions of CCL2, PPARG and FOS. Conclusion Astragalus Injection inhibits cervical cancer progression by downregulating multiple target proteins, damaging microfilament structure, weakening tumor cell motility and promoting apoptosis.

Key words: Astragalus injection, cervical cancer, network pharmacology, microfilament, cell apoptosis

Yuanyuan MA, Yue LIU, Ning GAO, Yumeng HUO, Meini CHEN, Jiawen LI, Jiahao LIU, Manqiu ZHANG, Bingbing YAN, Nuo XU, Jumei ZHAO. Multiple components in Astragalus Injection target CCL2/FOS/PPARG/CXCL8 to suppress cervical cancer progression[J]. Journal of Southern Medical University, 2026, 46(9): 2206-2221.

Figures/Tables 13

Fig.1 Astragalus Injection inhibits the proliferation of cervical cancer cells. A: CCK-8 assay for assessing proliferation of SiHa cells treated with Astragalus Injection for 0, 24, and 48 h. B: SiHa cell viability after intervention with different concentrations of Astragalus Injection for 24 and 48 h. C: Colony formation assay for assessing the effect of Astragalus Injection on proliferation of SiHa cells. D: Quantitative analysis of cell colony formation (n=3). ***P<0.001 vs NC.

Fig.2 Astragalus Injection inhibits migration and invasion of cervical cancer cells. A, B: Scratch assay for assessing the effect of Astragalus Injection on migration of cervical cancer cells (Scale bar=50 μm; n=3). C, D: Transwell assay for assessing migratory ability of SiHa cells (Scale bar=20 μm; n=3). E, F: Transwell assay for evaluating invasive ability of SiHa cells (n=3). *P<0.05, ***P<0.001 vs NC.

Tab.1 Information of active components of Astragalus Injection

Mol ID	Molecule name	OB (%)	DL
MOL000211	Mairin	55.38	0.78
MOL000239	Jaranol	50.83	0.29
MOL000296	hederagenin	36.91	0.75
MOL000033	(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-[(2R,5S)-5-propan-2-yloctan-2-yl]-2,3,4,7, 8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol	36.23	0.78
MOL000354	isorhamnetin	49.6	0.31
MOL000371	3,9-di-O-methylnissolin	53.74	0.48
MOL000374	5'-hydroxyiso-muronulatol-2',5'-di-O-glucoside	41.72	0.69
MOL000378	7-O-methylisomucronulatol	74.69	0.3
MOL000379	9,10-dimethoxypterocarpan-3-O-β-D-glucoside	36.74	0.92
MOL000380	(6aR,11aR)-9,10-dimethoxy-6a,11a-dihydro-6H-benzofurano[3,2-c]chromen-3-ol	64.26	0.42
MOL000387	Bifendate	31.1	0.67
MOL000392	formononetin	69.67	0.21
MOL000398	isoflavanone	109.99	0.3
MOL000417	Calycosin	47.75	0.24
MOL000422	kaempferol	41.88	0.24
MOL000433	FA	68.96	0.71
MOL000438	(3R)-3-(2-hydroxy-3,4-dimethoxyphenyl)chroman-7-ol	67.67	0.26
MOL000439	isomucronulatol-7,2'-di-O-glucosiole	49.28	0.62
MOL000442	1,7-Dihydroxy-3,9-dimethoxy pterocarpene	39.05	0.48
MOL000098	quercetin	46.43	0.28

Fig.3 Target screening of Astragalus Injection-cervical cancer interaction. A: Venn diagram of potential targets in cervical cancer. B: Venn diagram of shared targets between astragalus injection and cervical cancer. C: Protein-protein interaction (PPI) network map. D: Subnetwork analysis using MCODE plugin. E: Core targets of Astragalus Injection-cervical cancer interaction.

Fig.4 "Traditional Chinese Medicine-Bioactive Compounds -Targets - Diseases" Network Diagram.

Tab.2 Top 5 active components of Astragalus Injection by degree value

Mol ID	Molecule name	Degree value
MOL000098	quercetin	125
MOL000422	kaempferol	44
MOL000392	formononetin	27
MOL000354	isorhamnetin	25
MOL000378	7-O-methylisomucronulatol	25

Fig.5 Enrichment analysis of biological process (A), cellular component (B), molecular function (C) and KEGG enrichment analysis (D).

Fig.6 Molecular docking of active components with target proteins. A: Molecular docking binding energy results. B-F: Molecular docking interactions between the active components and CXCL8, CCL2, PPARG, and FOS, respectively.

Fig.7 Effect of Astragalus Injection on key pathways and target protein expression in SiHa cells. A, C: Western blotting for assessing alterations in the expression levels of key pathway and target proteins across the experimental groups, with GAPDH serving as the internal reference protein. B, D: Quantitative grayscale analysis of protein expression levels (n=3). *P<0.05, **P<0.01 vs NC.

Fig.8 Astragalus Injection disrupts microfilament structure of SiHa cells. A: Morphology of cervical cancer SiHa cells after Astragalus Injection intervention (Coomassie Brilliant Blue staining, Scale bar=10 μm); B: Phalloidin staining of cervical cancer SiHa cells before and after treatment with Astragalus injection (Scale bar=10 μm).

Fig.9 Effectof Astragalus Injection on mitochondrial membrane potential in SiHa Cells. A: JC-10 staining showing normal mitochondrial membrane potential in the control group, exhibiting red fluorescence. In contrast, the Astragalus Injection treatment group showed decreased mitochondrial membrane potential with enhanced green fluorescence (Scale bar=50 μm). B: JC-10 quantitative statistical analysis (n=3). ****P<0.0001 vs NC.

Fig.10 Astragalus Injection promotes apoptosis in SiHa cells. A: Astragalus Injection induces apoptosis via the mitochondrial pathway, as assessed by AO/EB staining.The nuclei in the control group exhibited uniform green fluorescence; The treated cells displayed characteristic apoptotic features, including chromatin condensation (bright green) and nuclear fragmentation (orange-red) (Scale bar=20 μm). B: AO/EB quantitative statistical analysis (n=3). ****P<0.0001 vs NC; C: Western blotting of expression levels of apoptosis-related protein BAX, BCL2, caspase-3, and cleaved caspase-3, with GAPDH serving as the internal control protein. D: Quantitative grayscale analysis of protein expression levels (n=3). ***P< 0.001, ****P<0.0001 vs NC.

Fig.11 Inhibitory effect of Astragalus injection on cervical cancer xenograft growth in mice. A: Macroscopic observation of subcutaneous tumor-bearing mice in the control group and the Astragalus injection treatment group. B: Tumor volume growth curve (n=4). **P<0.01 vs NC. C: Organ observation of mice in the control group and the Astragalus injection group From top to bottom: heart, liver, spleen, lung, kidney. D: Dissected tumors at the end of treatment. E: Tumor tissue HE staining (Scale bar=10 μm). F: Tumor tissue immunohistochemical staining for CCL2, FOS, and PPARG (Scale bar=5 μm). G: Quantitative analysis of immunohistochemistry results. ****P<0.0001 vs NC.

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