Tianma-Chuanxiong synergically inhibit glioma by regulating the miR-101/COX-2/AKT signal

Ethnopharmacological relevance
Tianma (Gastrodiae Rhizoma, TM) and Chuanxiong (Chuanxiong Rhizoma, CX) are well-established medicinal and food homologous substances in Traditional Chinese Medicine (TCM). The two herbs also hold important places in ethnic medical systems such as Tibetan and Miao medicine. According to TCM theory, TM and CX are commonly used in combination (TMCX) to enhance their respective therapeutic effects and alleviate neurological symptoms associated with glioma, such as headache, dizziness, and seizures. However, the potential synergistic anti-glioma activity of TMCX and the underlying molecular mechanisms remain to be elucidated. Although the traditional Chinese medicine pair Tianma and Chuanxiong (TMCX) demonstrates potential in alleviating glioma-related symptoms, its synergistic anti-glioma efficacy and underlying mechanisms remain unexplored.

Aim of the study
This study aims to investigate the synergistic therapeutic efficacy of TMCX against glioma and elucidate its molecular mechanisms.

Materials and methods
Subcutaneous and orthotopic glioma mouse models were established to evaluate the anti-glioma effects of TMCX. Animals received TM, CX, or TMCX extracts via oral gavage. The anti-glioma effects were assessed using bioluminescence imaging, immunohistochemistry (IHC), H&E staining, and Western blotting. The miRNA-seq and mRNA-seq analyses were conducted on orthotopic glioma tissues to explore possible mechanism of TMCX. The major active components of TMCX was analyzed by using UPLC-MS/MS. Furthermore, the anti-glioma effect of Gastrodin (the major active of TM), Chuanxiong oil (the major active fraction of CX) and Gastrodin-Chuanxiong oil combination (GC) were examined on U87 cell line, and their impact on the miR-101/COX-2/AKT signaling pathway was evaluated by PCR and Western blot. The crucial role of miR-101 in mediating the synergistic anti-glioma effect of GC were validated on miR-101-knocked down U87 cells.

Results
TMCX significantly reduced the tumor burden, decreased Ki67 and downregulated Bcl-2, MMP-2, and MMP-9 in glioma-bearing mice. In the orthotopic mouse model, TMCX demonstrated superior efficacy compared to TM or CX alone, as evidenced by notably smaller tumor sizes, lower Ki67, and decreased levels of Bcl-2, MMP-2 and MMP-9. Transcriptomic analysis of miRNAs and mRNAs identified that TMCX inhibits pro-cancer miR-101/COX-2 and PI3K-AKT signaling. Specifically, TMCX increased miR-101 expression while decreased COX-2 and p-AKT/AKT, which is consolidated by qRT-PCR and Western blotting. UPLC-MS/MS analysis revealed Gastrodin and Chuanxiong oil (GC) as the main active components/fraction. In vitro, GC combinational treatment synergistically suppressed U87 cell proliferation and migration more effectively than when used individually. Notably, GC elevated miR-101, while decreased COX-2, Bcl-2, MMP-2, MMP-9, and p-AKT/AKT. Lentiviral knockdown of miR-101 in U87 cells abolished the anti-glioma effects of GC and reversed its suppression of COX-2, p-AKT/AKT, Bcl-2 and MMP-2, establishing miR-101 as the pivotal mediator.

Conclusion
TMCX exerts a synergistic anti-glioma effect by regulating the miR-101/COX-2/AKT signaling pathway, primarily mediated through its active components Gastrodin and Chuanxiong oil.