Tongnao Decoction Facilitates Angiogenesis via the VEGFA/ PI3K/AKT/JUNB Signaling Axis to Promote Stroke Recovery
Wanhui Peng, Fei Ma, Yan Liu, Ziyan Cai, Yongxing Deng, Shufan Xu, Zhaoyao Chen, Qinghua Feng, Liqin Luan, Wenlei Li, Yuan Zhu, Minghua WuIntroduction:
Acute ischemic stroke (AIS) is a leading cause of global disability and mortality, with treatments limited by narrow time windows. Tongnao Decoction (TND), a traditional Chinese medicine formula, has shown neuroprotective potential. This study investigates TND’s mechanism in promoting post-stroke recovery.
materials and methods:
Materials and Methods Preparation of TND. TND is formulated with the following components: Processed Ramulus Uncariae Cum Uncis (Batch No. 20231225; Bozhou Xintai Pharmaceutical Co., Ltd., China); Rhizoma arisaematis (Batch No. 20231013; Bozhou Xintai Pharmaceutical Co., Ltd., China); Ligusticum chuanxiong (Batch No. 20230922; Tongren Tongde Pharmaceutical Co., Ltd., China); Rhizoma Gastrodiae (Batch No. 20231102; Tongren Tongde Pharmaceutical Co., Ltd., China); Rhizoma Anemones altaicae (Batch No. 20231218; Tongren Tongde Pharmaceutical Co., Ltd., China); Pheretima (Batch No. 202330901; Tongren Tongde Pharmaceutical Co., Ltd., China); Hirudo (Batch No. 20230219; Ma'anshan Jingquan Pharmaceutical Co., Ltd., China); and Radix et Rhizoma Rhodiolae Crenulatae (Batch No. 20231125; Bozhou Xiehecheng Co., Ltd., China). Comprehensive details regarding the herbal constituents and medicinal origins of TND are presented in Table 1. The preparation of decoction pieces in TCM involves extraction and concentration methods, succeeded by lyophilization, with the objective of efficiently retaining the bioactive constituents.This careful process results in a powdered form that exhibits high stability.For a comprehensive description of the preparation methodology, please refer to our earlier study(Wang et al., 2020a). UHPLC-QTOF-MS analysis The extraction of TND metabolites was conducted following the methodology outlined(Han et al., 2024).The sample analysis was conducted utilizing an Agilent 1290 ultra-high performance liquid chromatography (UHPLC) system, which was fitted with a Waters ACQUITY UHPLC BEH C18 column (1.7 μm, dimensions of 2.1 × 100 mm). The flow rate of the samples was consistently regulated at 0.4 mL/min, with an injection volume set at 5 μL. The mobile phase consisted of two components: phase A, which was a solution of 0.1% formic acid in water, and phase B, which comprised 0.1% formic acid in acetonitrile.The multi-step linear elution gradient procedure was carried out in the following manner: during the initial 0 to 3.5 minutes,a composition of 95
Methods:
Chemical analysis of TND using UHPLC-QTOF-MS identified 20 bioactive compounds. A photothrombosis-induced mouse stroke model was established and confirmed by TTC staining and MRI.
Results:
Behavioral tests showed TND significantly improved motor function, an effect reduced by the VEGFR2 inhibitor SKLB1002.
discussion:
Discussion In this study, we utilized UHPLC-QTOF-MS to identify several key bioactive components(L. Zhang et al., 2021), Additionally, we demonstrated through in vivo and in vitro experiments that TND promotes angiogenesis via the VEGFA/PI3K/AKT pathway. TND was developed based on the therapeutic principles of National Master of Chinese Medicine Professor Zhou Zhong ying and Academician of the Chinese Academy of Engineering Professor Wu Yiling. It is characterized by its dual actions of “clearing heat and resolving phlegm, as well as breaking blood stasis and promoting regeneration”. Various herbal compounds and formulations have been found to enhance vascular regeneration through several molecular pathways, including VEGFA signaling, PI3K/AKT activation, and the regulation of angiogenic transcription factors(R. Zhang et al., 2025; Zhou et al., 2024). For instance, compounds such as ginsenoside Rg1 and tetramethylpyrazine have exhibited pro-angiogenic effects in both laboratory and living organism experimental models of cerebral ischemia(Xu et al., 2023). The complex nature and diverse target interactions of TCM allow it to influence complex pathological processes, such as inflammation, oxidative stress, and endothelial dysfunction, thus creating a conducive microenvironment for angiogenesis(Fan et al., 2025). The VEGFA/PI3K/AKT signaling axis is a key regulator of angiogenesis, neuroprotection, and cell survival after ischemic stroke(Shi et al., 2019). Downstream transcription factors such as JUNB and CFOS contribute significantly to endothelial cell proliferation, migration, and vascular maturation(Wake et al., 2025; Wang et al., 2024). PI3K/AKT activation promotes endothelial nitric oxide synthase (eNOS) expression and inhibits apoptosis, thereby supporting vascular integrity and neovascularization(He et al., 2024)JUNB, an AP-1 component, further regulates VEGFA expression and facilitates vascular remodeling under ischemia(Xu et al., 2020).In the PT mouse model, TND improved motor function and preserved neuronal structure. It also counteracted stroke-induced suppression of neuroprotective genes via PI3K/AKT activation(Hou et al., 2018). In order to validate the specificity of this pathway, we employed the inhibition of VEGFR2(El-Hanboshy et al., 2021). The incomplete reversal observed with SKLB1002 suggests that there remains some activity within the pathway, which aligns with the multi-target mechanisms characteristic of TCM(Abdelsaid et al., 2017), and highlights the crucial importance of VEGFA-PI3K signaling in the process of angiogenesis following a stroke(Pang et al., 2024; W. Zhang et al., 2021).In vitro, TND enhanced angiogenic functions, an effect dependent on PI3K signaling(Wang et al., 2020b). SKLB1002 co-treatment reduced TND-induced migration and tube formation, accompanied by suppressed expression of VEGFA, JUNB, and CFOS(Abdurahman et al., 2022). Despite the promising findings, several limitations should be considered. The absence of pharmacokinetic data on the bioactive compounds in TND limits the ability to optimize dosing strategies effectively. Future research should aim to track the biodistribution of specific compounds and investigate the synergistic interactions among the eight herbal components of TND. Additionally, while the PT model allows for reproducible infarcts, its thrombo-inflammatory pathophysiology may not fully capture the complexity and variability of human stroke cases(Wiersma and Winship, 2018). Consequently, validating the results obtained from embolic models is crucial, along with executing long-term recovery investigations that surpass a duration of 30 days. This approach will facilitate a deeper comprehension of the therapeutic potential of TND within clinical environments.
Discussion:
In human cerebral microvascular endothelial cells (HCMECs), TND (1375 μg/mL) enhanced viability by 53.3%, proliferation by 1.5-fold, migration by 73%, and tube formation by 1.3-fold. Western blot analysis revealed that TND upregulates p-PI3K/PI3K, p-AKT/AKT, and JUNB, which was reversed by SKLB1002.
Conclusion:
In conclusion, TND facilitates functional recovery after AIS by promoting angiogenesis via the VEGFA/PI3K/AKT/JUNB pathway, supporting its therapeutic potential for ischemic stroke.