Experimental Verification of Erchen Decoction Plus Huiyanzhuyu Decoction in the Treatment of Laryngeal Squamous Cell Carcinoma Based on Network Pharmacology

Abstract

Background: The prescription of Chinese herbal medicine (CHM) consists of multiple herbs that exhibit synergistic effects due to the presence of multiple components targeting various pathways. In clinical practice, the combination of Erchen decoction and Huiyanzhuyu decoction (EHD) has shown promising outcomes in treating patients with laryngeal squamous cell carcinoma (LSCC). However, the underlying mechanism by which EHD exerts its therapeutic effects in LSCC remains unknown.

Methods: Online databases were utilized for the analysis and prediction of the active constituents, targets, and key pathways associated with EHD in the treatment of LSCC. The protein-protein interaction (PPI) network of common targets was constructed and visualized using Cytoscape 3.8.1 software. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to investigate the functional roles of core targets within the PPI network. Protein clustering was conducted utilizing the MCODE plug-in. The obtained results highlight the principal targets and pathways involved. Subsequently, clinical samples were collected to validate alterations in the levels of these main targets through Western blotting (WB) and immunohistochemistry (IHC). Furthermore, both in vivo and in vitro experiments were conducted to investigate the therapeutic effects of EHD on healing LSCC and elucidate its underlying mechanism. Additionally, to ensure experimental reliability and reproducibility, quality control measures utilizing HPLC were implemented for EHD herbal medicine.

Results: The retrieval and analysis of databases in EHD medicine and LSCC disease yielded a total of 116 overlapping targets. The MCODE plug-in methods were utilized to acquire 8 distinct protein clusters through protein clustering. The findings indicated that both the first and second clusters exhibited a size greater than 6 scores, with key genes PI3K and ErbB occupying central positions, while the third and fourth clusters were associated with proteins in the PI3K, STAT3, and Foxo pathways. GO functional analysis reported that these targets had associations mainly with the pathway of p53 mediated DNA damage and negative regulation of cell cycle in terms of biological function; the death-induced signaling complex in terms of cell function; transcription factor binding and protein kinase activity in terms of molecular function. The KEGG enrichment analysis demonstrated that these targets were correlated with several signaling pathways, including PI3K-Akt, FoxO, and ErbB2 signaling pathway. On one hand, we observed higher levels of key genes such as P-STAT3, P-PDK1, P-Akt, PI3K, and ErbB2 in LSCC tumor tissues compared to adjacent tissues. Conversely, FOXO3a expression was lower in LSCC tumor tissues. On the other hand, the key genes mentioned above were also highly expressed in both LSCC xenograft nude mice tumors and LSCC cell lines, while FOXO3a was underexpressed. In LSCC xenograft nude mice models, EHD treatment resulted in downregulation of P-STAT3, P-PDK1, PI3K, P-AKT, and ErbB2 protein levels but upregulated FOXO3a protein level. EHD also affected the levels of P-STAT3, P-PDK1, PI3K, P-AKT, FOXO3a, and ErbB2 proteins in vitro: it inhibited P-STAT3, P-AKT, and ErbB2, while promoting FOXO3a; however, it had no effect on PDK1 protein. In addition, HPLC identified twelve compounds accounting for more than 30% within EHD. The findings from this study can serve as valuable guidance for future experimental investigations.

Conclusion: The possible mechanism of EHD medicine action on LSCC disease is speculated to be closely associated with the ErbB2/PI3K/AKT/FOXO3a signaling pathway.

Keywords: Chinese herbal medicine; ErbB2/PI3K/AKT/Foxo3a signaling pathway; Erchen decoction plus Huiyanzhuyu decoction; laryngeal squamous cell carcinoma; phlegm coagulation and blood stasis syndrome.

Figure 1.

Network analysis of targets. (A) PPI of EHD key targets, (B) PPI of key targets in LSCC, and (C) Venn diagram of medicine targets and disease targets.

Figure 2.

The 116 targets’ protein clustering. Screened hub targets in Cytoscape with MCODE. (A and B) MCODE score ≥ 6; (C-E) MCODE score = 4. (F) MCODE score = 3.33. (G and H) MCODE score = 3.

Figure 3.

GO enrichment analysis and KEGG signaling pathways analysis of 116 common targets. (A) Cellar component analysis, biological process analysis, molecular functions analysis, (B) KEGG signaling pathways analysis. EHD medicines-common targets- signaling pathways interaction diagram.

Figure 4.

Network of EHD medicines- common targets-signaling pathways. Red, yellow, green oval represent EHD medicines, common targets, and signaling pathways, respectively, and black lines indicate mutual relationships. The nodes in the network represent the medicines, signaling pathways and targets. The connections of the edges represent interactions and relationships between medicines and targets and between targets and the signaling pathways.

Figure 5.

Protein expression levels in tumor specimens and corresponding normal tumor margin tissues from LSCC patients. (A and B) Western blot analysis: P-STAT3, P-PDK1, P-AKT, PI3K, FOXO3a, and ErbB2 proteins expression level in clinical laryngeal cancer samples (Tu, Tumor) and safe margins (Adj, Adjacent), respectively. (P < .05 or P < .001 comparison of two groups). (C) Proteins in the tumor tissues samples from the different groups detected by immunohistochemistry.

Figure 6.

The expression of P-STAT3, P-PDK1, P-AKT, PI3K, FOXO3a, and ErbB2 proteins in LSCC xenografts nude mice was suppressed by EHD. (A and B) Western blot analysis: P-STAT3, P-PDK1, P-AKT, PI3K, FOXO3a, and ErbB2 proteins expression level in different groups. (**P < .05 compared to the control group; ***P < .01 compared to the control group; ****P < .001 compared to the control group). (C) Proteins in the tumor tissues samples from the different groups detected by immunohistochemistry.

Figure 7.

(A) The expression of P-STAT3, PDK1, AKT, PI3K, FOXO3a, and ErbB2 proteins in LSCC cells was inhibited by EHD. (b) Western blot analysis: P-STAT3, P-PDK1, P-AKT, PI3K, FOXO3a, and ErbB2 proteins expression level in different groups. *P < .05 compared to the control group. ****P < .001 compared to the control group.

Figure 8.

Chinese herb quality control of EHD. The brown corresponds to wavelength 270 nm. The pink corresponds to wavelength 280 nm. The green corresponds to wavelength 230 nm. The red corresponds to wavelength 210 nm. The blue corresponds to wavelength 254 nm. The numbers refer to the main components identified by the control sample.