Yixin-Fumai granules modulate autophagy through the PI3K/AKT/FOXO pathway and lead to amelioration of aging mice with sick sinus syndrome
Authors: Jin Lianzi , Hou Ping 
Published: July 2024

Abstract
Objective
By employing network pharmacology alongside molecular docking techniques, we can delve into the intricate workings of Yixin-Fumai granules (YXFMs) and their impact on sick sinus syndrome (SSS) within wrinkles mice. Specifically, we aim to understand how YXFMs enhance autophagy through the PI3K/AKT/FOXO path.

Methods
The active ingredients and medicinal uses of Ginseng, ligusticum wallichii, Ophiopogon, Schisandra, salvia, and astragalus were compiled using the BATMAN-TCM database. We also used Genecards, OMIM, and Disgenet files to identify the disease goals. A hierarchical diagram of “disease-drug-key targets” was generated using the Cytoscape programs. In addition, we established a target protein interaction (PPI) network using the STRING database. Then, the Cluster Profiler R package was used to conduct GO functional enrichment evaluation and KEGG pathway enrichment analyses of the targets. Based on the PPI system, we chose the top communicating targets and substances over molecular docking. In vivo studies were performed to validate these selections further. The mouse model was induced to study the damaged sinoatrial node (SAN) in mice with lower heart rates due to age-related changes. Electrocardiogram and Masson staining assessments were performed to obtain the results. The transmission electron microscope was used to assess the autophagy level of SAN cells. Western blot was employed to analyze the impact of YXFMs on protein expression in the PI3K/AKT/FOXO signaling process throughout SSS therapy in aging mice.

Results
One hundred forty-two active ingredients, 1858 targets, 1226 disease targets, and 266 intersection targets were obtained. The key targets of the PPI network encompassed TP53, AKT1, CTNNB1, INS, and TNF, among others. According to GO functional analysis, the mechanism underlying YXFMs in SSS treatment may primarily be associated with the control of ion transport across membranes, cardiac contraction, regulation of blood circulation, and other biological processes. Based on the results of KEGG pathway enrichment analysis, it was determined that they were mainly enriched in multiple pathways of signaling such as the PI3K-Akt signaling route, MAPK signaling process, AGE-RAGE signaling path, FOXO signaling path, HIF-1 signaling process, and several other paths. Molecular docking demonstrated that five compounds had excellent binding to the key candidate target proteins AKT1 and INS. Through the in vivo studies, we noticed notable effects when administering YXFMs. These effects included the suppression of aging-induced SSS, a decrease in the R-R interval, a rise in heart rate, a reduction in fibrosis, a boost in the autophagy process level, and a spike in the levels of expression of key protein molecules in the PI3K/AKT/FOXO signaling path.

Conclusion
This research has made preliminary predictions about the potential of YXFMs in treating SSS. It suggests that YXFMs may have the ability to target key proteins and critical paths associated with the condition. Further testing has been conducted to discover new findings and evidence of ideas for tackling SSS triggered by aging.