Modified Huanjingjian Prevents Chemotherapy-Induced Alopecia by Inhibiting Genomic DNA Methylation of the Wnt Signaling Pathway in Mice

Abstract

Aim: Cyclophosphamide (CTX), a cornerstone in breast cancer combination chemotherapy, frequently induces adverse effects including myelosuppression, gastrointestinal disturbances, hepatic impairment, and alopecia. Chemotherapy-induced alopecia severely impacts patients' quality of life and psychological well-being. Modified Huanjingjian (MHJJ), a traditional Chinese herbal formula, demonstrates clinical efficacy in alleviating chemotherapy-related side effects, yet its mechanisms against CTX-induced alopecia remain uncharacterized. And our main aim was to explore the efficacy and the mechanism of MHJJ in mice.

Methods: UPLC-QE-Orbitrap-MS characterized MHJJ's chemical composition. A CTX-induced alopecia murine model was established. Systemic toxicity was evaluated through body weight monitoring, automated biochemical analysis (ALT/AST levels), and hematological profiling (WBC/PLT counts). Hair follicle histopathology was assessed via H&E staining. IHC and IF staining quantified proliferation markers and hair follicle stem cell (HFSC) biomarkers. Reduced representation bisulfite sequencing (RRBS) was used to map DNA methylation patterns. Wnt pathway dynamics were analyzed through qRT-PCR and IF staining.

Results: We identified 110 bioactive compounds in MHJJ. MHJJ intervention attenuated alopecia severity, restored follicular architecture, and increased follicular density compared to CTX monotherapy (p<0.05). HFSC proliferation markers (Ki67/CD34) showed significant upregulation, while apoptosis markers (Caspase-3) were suppressed. RRBS revealed MHJJ-mediated hypomethylation in differentially methylated regions, with gene body methylation constituting 60% of total methylation changes. Methylation-modulated genes predominantly localized to Wnt signaling pathways: MHJJ enhanced Wnt3/Wnt10a expression while suppressing Cer1/Axin1. Corresponding methylation reductions at promoter and gene body regions were confirmed at mRNA and protein levels.

Conclusion: MHJJ mitigates CTX-induced alopecia through epigenetic regulation of HFSCs, specifically via DNA hypomethylation-mediated activation of Wnt3/Wnt10a and suppression of Cer1/Axin1. This mechanism promotes follicular regeneration by restoring Wnt signaling homeostasis, positioning MHJJ as a promising adjuvant for chemotherapy-induced alopecia management.

Keywords: DNA methylation; Wnt signaling pathway; chemotherapy-induced alopecia; hair follicle; traditional Chinese medicine.

Graphical abstract

Figure 1

Main components of MHJJ were identified by UPLC-QE-Orbitrap-MS. (A) Components were detected in the positive ion mode. (B) Components were detected in the negative ion mode. (C) Number of active components were identified (spectral similarity scores > 0.9). (D) Proportion of the identified components, which are previously identified as characteristic chemicals of several TCMs used in the MHJJ formula. (E) Based on the chemical structures, the main chemicals were classified into certain categories.

Figure 2

Prevention of CTX-induced alopecia by MHJJ. (A) Treatment and observation schedule of mice. (B) Alopecia on the back of mice in each group on day 28. (C and D) Hair regrowth on the back of mice in each group on day 49. (n = 5). Data were presented as mean ± SD. * P < 0.05, ** (P < 0.01).

Figure 3

Effects of MHJJ on HFSCs and cell proliferative gene Ki67 in histological skin sections (magnification, 200×; scale bar, 50 µm). (A) MHJJ could repair damaged HFSCs in CTX-treated mice. (B) Ki67 expressed higher in the dorsal skin of mice after MHJJ treatment (n = 5). Significant differences between the CTX and MHJJ groups were denoted by * (P < 0.05), ** (P < 0.01), and *** (P < 0.001). The yellow arrows indicated the positive staining of Ki67.

Figure 4

MHJJ promoted the proliferation of HFSCs. (A) Representative images of IF-stained HFSCs markers LGR5, CD49f, SOX9, S100A4, CK15, and FZD10 in HFs of dorsal skin tissues (magnification, 200×; scale bar, 50 µm). The yellow arrows indicated the positive staining of HFSCs markers, respectively. (B) IF staining positive area ratio (n = 5 per group). Significant differences between the CTX and MHJJ groups were denoted by ^ns (not significant), * (P < 0.05), ** (P < 0.01), *** (P < 0.001).

Figure 5

Regulation of differential DNA methylation by MHJJ analyzed by RRBS. (A) The Circos plot showed the distribution of methylation levels on the chromosome scale. The outermost circle was the label of the chromosome karyotype, and the five heatmap bars from the outside to the inside in order represented GC content (green), gene density (red), CG methylation level (purple), CHG methylation level (Orange), and CHH methylation level (light blue). Darker colors indicated higher methylation levels. (B) The motifs of methylation sites were identified. (C) The distribution of methylation levels of cytosine sites was identified. (D) The methylation levels of motifs with different methylation sites in genomic functional elements in each group of samples were compared. (E) The Circos plot illustrated significant differences in DMRs among the groups. (F) The lengths of total DMRs and the lengths of more frequently methylated DMRs. (G) The DMR methylation levels in each group. (H) Distribution of differential methylation levels of DMRs of genomic regions in each group. (I) The Cluster heatmap showed the methylation levels of DMR in each group.

Figure 6

MHJJ regulates methylation modification of Wnt signaling pathway genes affecting the expression of key proteins. (A) IF images of co-expression of the HFSCs marker LGR5 and four target genes of the Wnt pathway, Wnt3, Wnt10a, Axin1 and Cer1, in hair follicles of dorsal skin tissue (magnification, 200×; scale bar, 50 μm), and the yellow arrows indicated the positive staining of the HFSCs marker and the Wnt pathway genes, respectively; (B) IF staining positive area ratio (n = 5). Significant differences between CTX and MHJJ groups are indicated by ^ns (not significant), * (P < 0.05), ** (P < 0.01) and *** (P < 0.001).

Figure 7

MHJJ regulates proliferation and apoptosis mRNA levels by reducing methylation modification of Wnt signaling pathway genes. (A) mRNA expression of proliferation-related markers in HFSCs. (B) mRNA expression of cell apoptosis markers. (C) mRNA expression of DNA methylesterase and demethylase. (D) The left Venn diagram suggested the signal transduction pathways involved in differentially methylated genes in each group, and the right one showed the Wnt pathway genes in which promoter and gene body regions were methylated. (E) IGV analysis showed that the key node genes in the Wnt pathway were sequenced, indicating gene methylation was related to the effect of medium dose of MHJJ on treating chemotherapy-induced alopecia in the CTX-induced model. (F) The mRNA expression levels of Cer1, Axin1, Wnt3 and Wnt10a were detected by qRT-PCR in the skin tissues of each group. (n = 5). Significant differences were denoted by ^ns (not significant), * (P < 0.05), ** (P < 0.01) and *** (P < 0.001).