Shen-Ying-Yang-Zhen formula promotes angiogenesis around hair follicles, alleviates oxidative stress, and inhibits hair follicle apoptosis through the VEGF/Akt/Caspase-9 signaling axis

Abstract

Background: Androgenic alopecia (AGA) represents the most predominant form of hair loss in clinical practice, yet currently approved pharmacological options (e.g., finasteride and minoxidil) face limitations in safety and long-term efficacy. Shen-Ying-Yang-Zhen formula (SYF) is a classical prescription in traditional Chinese medicine to AGA; nevertheless, its specific mechanisms of action in addressing AGA remain to be further explored and elucidated. In our study, we for the first time propose that SYF uniquely reshapes the perifollicular microenvironment through dual regulation of oxidative stress and angiogenesis, thus providing a potential medication for AGA.

Methods: In this study, the chemical constituents of SYF were identified using UPLC-MS. An AGA mouse model was established by induction with testosterone propionate (TP), and a human dermal papilla cell (HDPC) model was induced by dihydrotestosterone (DHT). The mechanism underlying SYF treatment for AGA was examined by merging network pharmacology with RNA sequencing, and the findings were corroborated through flow cytometry, multiple immunofluorescences, Western blotting, RT-qPCR, and ELISA techniques.

Results: In the AGA mouse model, histological analysis revealed that oral administration of SYF significantly increased skin thickness and hair follicle density (2.8-fold vs. TP group, p < 0.001). Immunofluorescence staining further confirmed that SYF treatment promoted a 2.9-fold increase in p-S6-positive hair follicles-indicative of anagen-phase induction (p < 0.001). Network pharmacology and RNA-seq analyses showed that SYF treatment was associated with cellular oxidative stress and the angiogenic microenvironment around AGA hair follicles, which may be related to targets such as VEGF and Akt Additionally, expression of Ki67, SRY-box transcription factor 9, phospho-S6 ribosomal protein (p-S6), and Nrf2 was enhanced in the skin of the SYF-treated mice, whereas reactive oxygen species expression was decreased. RT-qPCR analysis revealed downregulation of follicle degradation-related factors DKK1, IL-6, and TGFβ1, along with decreased Caspase-9 expression and upregulated key targets VEGF and Akt in the VEGF/Akt/Caspase-9 signaling axis. In the DHT-HDPC model, SYF promoted the proliferation of HDPCs and elevated the Bcl-2/Bax ratio, which in turn suppressed apoptosis of HDPCs in vitro.

Conclusion: SYF shows promise as a therapeutic agent for prevention of hair loss by improving oxidative stress and angiogenesis in the area surrounding hair follicles in AGA. It reduces apoptosis in DHT-treated HDPCs by regulating the VEGF/Akt/Caspase-9 signaling axis. This multi-target, systems-level approach highlights SYF's potential as a novel therapeutic agent for AGA.

Keywords: Androgenic alopecia; Angiogenesis; Apoptosis; Oxidative stress; Shen-Ying-Yang-Zhen formula; Traditional Chinese medicine.