Potential targets of Yixin-Fumai granules in the treatment of SSS

BAT-TCM platforms screened a hundred and forty-two different YXFMs that might be active, and 1858 targets were obtained after predicting and eliminating duplicates.

Based on the search keyword “Sick Sinus Syndrome”, the data were searched in Genecards, OMIM, and Disgenet databases, and 1226 targets of cerebral ischemia were obtained following retrieving and removing duplicates.

The active ingredient targets of YXFMs and disease targets were introduced into the Venny analysis platform to obtain the intersection targets. There are 1,858 gene medications, 1,266 genetic disorders, and 266 genes that interact with both types of pharmaceuticals (Fig. 1). In addition, the primary compounds in YXFMs are identified, and a hierarchical model called “drug-active ingredients-target” was created using the Cytoscape3.7.2 program. This model consists of 262 nodes and 4276 edges (Fig. 2). The findings reveal that the initial eight substances identified in the study are Miltionone I, Neocryptotanshinone II, Tanshiquinone B, Dihydrokaranone, Nootkatone, (E)-9-Isopropyl-6-Methyl-5, 9-dodecene-2-1, and 13-Methyl Pentadecanoic Acid.

Fig. 1

The junction points between YXFMs and SSS vector representation

Fig. 2

“Disease-drug-component-target” network diagram

The target protein interaction network was obtained by querying the STRING database with the common target data of medications and illnesses. An interaction score of at least 0.4 was required. The PPI data was loaded into Cytoscape 3.7.2 to build a PPI network [9] (Fig. 3). The level value, which indicates the significance of a node in a network, increases as the graph becomes brighter and more prominent. Using R software, PPI data were imported to know the number of connected nodes of core genes, and the bar chart of the top 30 core genes were drawn, which included TP53, AKT1, CTNNB1, INS, TNF, ALB, IL6, MYC, BCL2, and IL1B etc. (Fig. 4).

Fig. 3

YXFMs’ PPI network for SSS treatment

Fig. 4Bioinformatics analysis based on target genes

To identify possible targets, we used the R program plus the underlying database “org. Hs.eg.db” to catalog genes. Subsequently, a Bioconductor performed GO functional enrichment analysis on these potential targets. Each category was ranked according to significance, and bar graphs represented the top 10 enrichment entries (Fig. 5). The analysis revealed that YXFMs played a role in regulating ion transmembrane transport, heart shrinkage, blood flow, cation channel complex, ion channel complex, channel activity, ion-gated channel action, and more. The line length suggests the amount of the genes enriched in GO, and the color reflects the importance of enrichment.

Fig. 5

GO enrichment analysis of YXFMs

The gene ID of desired targets was obtained using R software and the associated database “org.Hs.eg.db”. Besides, Bioconductor was utilized to perform KEGG function enrichment analysis of these potential targets. According to their significance, 20 enrichment items were rated in the top spot, and a bubble plot was created to display them (Fig. 6). The findings show that YXFMs primarily target specific signaling pathways in treating SSS, including the PI3K-Akt, MAPK, AGE-RAGE, FOXO, HIF-1, and Thyroid hormone signaling pathways. The active parts of YXFMs could target many signaling pathways in their effort to cure SSS. One essential mechanism that regulates the whole network is the PI3K/Akt/FOXO signaling path; this suggests that YXFMs may influence the SSS development process via this pathway. On the autophagy-enriched pathway, we further screened to obtain the autophagy mechanism of the INS-PI3K-Akt-FOXO-SIRT1 signaling pathway (Fig. 7).

Fig. 6

KEGG pathway enrichment analysis of YXFMs

Fig. 7

FOXO pathway map is the most significantly enriched pathway

Molecular docking

Combining the component-target network relationship and core target screening results, a variety of components in YXFMs, including Adenosine, Neocryptotanshinone Ii, Miltionone I, (E) -9-isopl-6-methyl-5, 9-decadiene-2-one, and Adenosine Triphosphates were found to be strongly associated with the core targets AKT1 and INS, and A total of five compounds were chosen for molecular docking experiments using their corresponding targets. Besides, the minimum binding energy ≤-5.0 kJ·mol-1 shows excellent docking impact of the drug molecule and the protein. The findings showed that these chemicals could dock with AKT1 and INS with stable energies (Table 1). The 3D molecular docking findings were generated using the PyMOL program [12] (Fig. 8).

Table 1 The chemical formulas and results of molecular dockingFig. 8

Complex-INS/AKT1 molecular docking shown in three dimensions

YXFMs improved SAN function and increased heart rate in SSS mice

To begin the animal investigation, the SAN function in mice was assessed using Masson staining and an electrocardiogram (resting heart rate and R-R interval). The ECG showed that YXFMs mice had a considerable improvement in heart rate compared to (Control group) CON mice, but the heart rate of SSS animals fell considerably with age (Fig. 9). The Masson staining showed that the CON group had much less SAN fibrosis than the SSS group and that the YXFMs group had even more SAN fibrosis than the SSS group (Fig. 10). Finally, YXFMs significantly decreased age-related SAN fibrosis while simultaneously improving SAN function.

Fig. 9

YXFMs enhanced SAN function and led to an increase in heart rate in SSS mice. A Heart rates at rest for each mouse group (125 ms/div) CON, SSS, YXFMs groups, respectively from up to down. B A statistical evaluation of the heart rates of the mice in each group. C Statistical evaluation of the R-R intervals of the ECG in every mouse group. **Compared to SSS, **P < 0.01. The control group is negative. SSS stands for sick sinus syndrome model group. Yixin-Fumai granule therapy group (YXFMs)

Fig. 10

The SAN region of CON, SSS, and YXFMs groups was stained with Masson’s trichrome

YXFMs increased autophagy in SAN of SSS mice

Transmission electron microscopy showed that as SSS mice aged, the amount of autophagy and the amount of autophagosomes in their SANs were reduced. Nonetheless, the level of autophagy in the YXFMs group was significantly improved, and lysosomes (Ly), autophagolysosomes (ASS), and autophagosomes (AP) were visible (Fig. 11). Finally, in aging-induced SSS, YXFMs considerably enhanced SAN function and raised SAN autophagy levels.

Fig. 11

The SAN region of CON, SSS, and YXFMs groups was observed via electron microscopy. Five micrometers and two micrometers serve as the scale bars

YXFMs increased the level of autophagy protein in SAN of SSS mice

Autophagy indicator molecules include Beclin-1, with a p62, LC3-II, Atg8, and Atg12. In order to assess the impact of YXFMs upon autophagy, Western blot analysis was used to identify the extent of expression of proteins for Beclin-1, p62, LC3-II, Atg8, and Atg12. A decrease in the protein expression amounts of Beclin-1, LC3-II, Atg8, and Atg12 proteins, as well as an increase in the gene expression level of p62 protein within the SSS category compared to the CON category, suggest the fact that the SSS category impeded the autophagic function of SAN. While p62 protein production was lowered in senile SSS mice treated with YXFMs, Beclin-1, LC3-II, Atg8, and Atg12 protein expression levels were substantially elevated. There were significantly significant differences in protein expression across the groups (Fig. 12), suggesting that YXFMs may increase the autophagic activities of the SANin-aged mice.

Fig. 12

Autophagy in the SAN region of CON, SSS, and YXFMs groups measured by protein assay. A The expression of Beclin-1, p62, LC3-II, Atg8, and Atg12 in CON, SSS, and YXFMs groups was measured by Western blotting; B The findings of the Western blotting were quantitatively analyzed. P < 0.05 in comparison to not using SSS. CON: Control group, The SSS model group represents sick sinus syndrome. YXFMs: Group treated with Yixin-Fumai granules

Interactions between YXFMs and the PI3K/AKT/FOXO signals

In the second part of the animal experiment, the SAN of each group was analyzed using western blotting to determine the expression of INS, INSR, PI3K, p-PI3K, AKT, p-AKT, FOXO1, Ace-FOXO1, and SIRT1. In addition, the amount of INS, INSR, p-AKT/AKT, and p-PI3K/PI3K showed an essential rise in SSS mice. This suggests that the PI3K/AKT route, responsible for activating various cellular processes, became active in SSS mice, leading to the phosphorylation of PI3K/AKT. However, the gene expression levels of INS, INSR, p-AKT, and p-PI3K significantly decreased in SSS mice treated with YXFMs. In the SSS group, there was an important rise in the levels of ACE-FOXO1/FOXO1 and a reduction in the levels of SIRT1 compared to the CON category. In the YXFMs group, a notable enhancement was noticed in the gene expression of Ace-FOXO1/FoxO1 compared to the SSS organization. On the other hand, the AKT phosphorylation agonist SC79 had a counteractive influence on the impact of YXFMs in this context. The differences in the expression of proteins between the categories were statistically significant (Fig. 13).

Fig. 13

Test of the SAN region PI3K/AKT/FOXO signaling pathway in CON, SSS, YXFMs SC79 groups. A The expression of INS, INSR, PI3K, p-PI3K, AKT, p-AKT, FOXO1, Ace-FOXO1, and SIRT1 was detected by Western blotting; B Quantitative analysis of the Western blotting findings was performed. #P < 0.05 in comparison to SSS. Compared to YXFMs, #P<0.05. Downside: the control group. SSS-model group: sick sinus syndrome. YXFMs (Yixin-Fumai granules): group that received therapy.SC79: female mice given YXFMs in addition to SC79 agonist for phosphorylation of AKT (SC79)

Thus, YXFMs may increase autophagy efficiency by blocking PI3K/AKT signaling cascade phosphorylation, decreasing FOXO1 acetylation. However, the impact of YXFMs was counteracted through the AKT phosphorylation agonist SC79.