Potential Targets of Baicalin
15 potential targets were screened from the Swiss Target Prediction databases (Table 1). These targets are possibly involved in improving the metabolic function of Baicalin.
Table 1
Potential targets of Baicalin
ID | Gene | Target |
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P15121 | AKR1B1 | Aldose reductase |
P30542 | ADORA1 | Adenosine A1 receptor |
P01375 | TNF | TNF-alpha |
P60568 | IL2 | Interleukin-2 |
P47989 | XDH | Xanthine dehydrogenase |
P35354 | PTGS2 / COX-2 | Prostaglandin G/H synthase 2 / Cyclooxygenase-2 |
P51812 | RPS6KA3 | Ribosomal protein S6 kinase alpha 3 |
P00533 | EGFR | Epidermal growth factor receptor |
P22303 | ACHE | Acetylcholinesterase |
P16083 | NQO2 | Quinone reductase 2 |
Q9GZQ4 | NMUR2 | Neuromedin-U receptor 2 |
P08913 | ADRA2A | Alpha-2a adrenergic receptor |
P18825 | ADRA2C | Adrenergic receptor alpha-2 |
Q9NPH5 | NOX4 | NADPH oxidase 4 |
P05091 | ALDH2 | Aldehyde dehydrogenase |
Disease-associated Targets
Used “Myocardial fibrosis” as keywords to retrieve all targets from the GeneCard, OMIM, and TTD databases, then combined the target genes and removed repetitions of 4399 disease targets of MF were found.
Analysis of PPI Network and Key Targets of Baicalin in Improving MF
15 potential targets of Baicalin and 4399 targets of MF were inputted into the online visualization platform to construct a Venn diagram and identify thirteen key target genes (Fig. 2). 13 target genes were then entered into the String database to create a PPI network (Fig. 3A), including 13 target proteins and 24 target protein interaction lines, with an average node degree of 3.69 and an average local clustering coefficient of 0.641. The constructed PPI network was imported into Cytoscape3.9 software for visualization processing, with one nonessential target being removed. In descending order of degree value, the key targets of Baicalin for improving MF are TNF-α, PTGS2, EGFR, AKR1B1, XDH, IL2, NOX4, ACHE, ADORA1, ALDH2, ADRA2A, and ADRA2C (Fig. 3B).
GO and KEGG Pathway Enrichment Analyses
We introduced the 13 key targets into the David database (P<0.05) for GO enrichment analysis and found that the key targets of Baicalin were mainly enriched within 15 biological processes (BPs), 6 cellular components (CCs), and 7 molecular functions (MFs). The results indicated that the BPs of the key targets of Baicalin in improving MF were mainly related to negative regulation of apoptotic process, positive regulation of MAP kinase activity, and negative regulation of lipid catabolic process, etc.. The analysis of CCs revealed that the identified targets were primarily associated with the extracellular space, integral component of plasma membrane, and basolateral plasma membrane. Additionally, electron carrier activity, protein homodimerization activity, and alpha2-adrenergic receptor activity were all found to be related to the MFs. KEGG pathway analysis (P<0.05) identified 6 pathways: C-type lectin receptor signaling pathway, Yersinia infection, Alcoholic liver disease, cGMP-PKG signaling pathway, Human cytomegalovirus infection, and Coronavirus disease - COVID-19. This suggests that Baicalin may potentially impact these pathways in treating MF. The bioinformatics platform (https://www.bioinformatics.com.cn/) conducted a visualized analysis of the 10 target genes identified in the GO and KEGG pathway enrichment analysis based on P-value (Fig. 4–5). The target proteins involved in the first C-type lectin receptor signaling pathway (Fig. 6), including TNF-α, PTGS2, and IL2, were all located downstream and were hypothesized to be the core targets of baicalin's efficacy in improving MF.
Molecular Docking
The molecular docking analysis revealed that Baicalin exhibits strong binding affinity with TNF-α, PTGS2 and IL2, as indicated by the respective binding energies of -7.3, -8.9 and − 8.3 kJ/mol, all of which are lower than − 5 kJ/mol. The results showed that Baicalin forms hydrogen bonds with specific amino acid residues of TNF-α (SER-38, LYS-39, ASN-48, GLU-56 and SER-57), PTGS2 (SER-49, ASP-133, GLY-135, VAL-155, ASP-157 and GLN-327) and IL2 (SER-38, LYS-39, NAS-48, GLU-56, SER57) (Fig. 7).
Effects of Baicalin on ECGs in Rats
The ECG waveforms of rats in the normal group displayed typical characteristics. In contrast, the model group showed a reduction in the S-T segment of the electrocardiogram, low T wave amplitude, and the presence of atrial flutter. However, both the captopril group and the low-, middle- and high-dose baicalin groups exhibited restoration of S-T segment and T wave amplitude in the electrocardiogram, along with a decrease in atrial flutter (Fig. 8).
Effects of Baicalin on HWI and LVWI in Rats
Compared to the control group, both HWI and LVWI showed significant increases in the model group, captopril group, and low-, middle-, and high-dose baicalin groups (P < 0.05). HWI was significantly decreased in the captopril group and middle/high dose baicalin groups compared to the model group (P < 0.05), while LVWI was significantly decreased in the captopril group and all baicalin dose groups compared to the model group (P < 0.05). Furthermore, HWI of the low-dose baicalin group was significantly increased compared to the captopril group (P < 0.05), and LVWI of all baicalin dose groups was significantly increased compared to both the captopril and low-dose baicalin groups (P < 0.05); Additionally, HWI in middle/high dose baicalin groups was significantly increased compared to the low-dose baicalin group (P < 0.05) (Fig. 9).
Effects of Baicalin on Myocardium CK, LDH and NT-proBNP levels in Rats
Compared to the normal group, the levels of CK and LDH in the myocardium were significantly elevated in the model group, captopril group, and low-, middle-, and high dose baicalin groups (P < 0.05). Additionally, NT-proBNP levels in the myocardium were significantly increased in the model group and low-, middle-, and high dose baicalin groups (P < 0.05). Furthermore, compared to the model group, levels of CK, LDH and NT-proBNP in myocardial tissue of rats were significantly reduced in the captopril group as well as low-, middle- and high dose baicalin groups (P < 0.05). Lastly, compared to the captopril group, levels of CK, LDH and NT-proBNP in myocardium were significantly increased in low-, middle-, and high dose baicalin groups (P < 0.05), while compared with low-dose Baicalein, the levels of CK, LDH and NT-Pro BNP in the myocardial tissue of rats were significantly decreased in the middle- and high-dose groups (P < 0.05) (Fig. 10).
Effects of Baicalin on Myocardium Col Ⅰ and Col Ⅲ levels in Rats
The levels of Col Ⅰ and Col Ⅲ in myocardial tissue were significantly increased in the model group, captopril group, and low-, middle-, and high dose baicalin groups compared to the normal group (P < 0.05). In addition, the levels of Col Ⅰ and Col Ⅲ in myocardium were significantly decreased in the captopril group and low-, middle-, and high dose baicalin groups compared to the model group (P < 0.05). Furthermore, the levels of Col Ⅰ and Col Ⅲ in myocardium were significantly increased in low- and middle dose baicalin groups compared to the captopril group (P < 0.05), while they were significantly decreased in middle- and high dose baicalin groups compared to low dose baicalin group (P < 0.05) (Fig. 11).
Effects of Baicalin on Myocardium tissue pathology in Rats
The myocardial structure in the normal group appeared to be intact, with clear transverse lines and no evidence of fibrous tissue hyperplasia or inflammatory cell infiltration. In contrast, the model group exhibited myocardial damage, necrosis, hyperplasia of fibrous tissue filling the necrotic space, and inflammatory cell infiltration in the myocardium interstitium. However, in the captopril group and low-, middle- and high-dose baicalin groups, the myocardial structure was orderly with reduced fibrous tissue hyperplasia and no signs of necrosis or inflammatory cell infiltration. Masson staining revealed minimal collagen deposition in the myocardium of the normal group (stained blue), while a large amount was observed in the model group. The degree of collagen deposition was improved to varying degrees in rats from captopril group and low-, middle- and high-dose baicalin groups compared to those from both normal and model groups. Additionally, compared to the model group alone, there was a significant decrease (P < 0.05) in Masson-staining positive area proportion within each low-, middle-, and high-dose baicalin groups (Fig. 12).
Effects of Baicalin on Myocardium TNF-α, PTGS2 and IL2 Protein Expression Levels in Rats
The levels of TNF-α and PTGS2 in the myocardium were significantly elevated in the model group, captopril group, and low, medium, and high dose baicalin groups compared to the normal group (P < 0.05). Conversely, the levels of IL2 in the myocardium were significantly reduced in the model group and low dose baicalin group (P < 0.05). In comparison to the model group, both TNF-α and PTGS2 levels in the myocardium were significantly decreased in the captopril group and low, medium, and high dose baicalin groups (P < 0.05), while IL2 levels were significantly increased (P < 0.05). Furthermore, when compared to the captopril group, TNF-α levels in the myocardium were notably increased in both low and medium dose baicalin groups; PTGS2 levels were also notably increased across all three baicalin dosage groups (P < 0.05), with a significant decrease observed for IL2 levels specifically within the low dose baicalin group (P < 0.05). Finally, when comparing with the low dose baicalin group alone,TNF-α levels within rat myocardium from both medium and high-dose groups showed a significant decrease (P < 0.05), whereas IL2 was found to be markedly increased (P < 0.05) (Fig. 13).