Exploring the mechanism of Carbonized Typhae Pollen in treating blood stasis syndrome through metabolic profiling: the synergistic effect of hemostasis without blood stasis

Ethnopharmacological relevance

Removing blood stasis and stopping bleeding traditional Chinese medicines (RBSB-TCM) formed a unique class of TCM, characterized by vasodilating, removing stasis and hemostatic effects. Carbonized Typhae Pollen (CTP), derived from Typhae Pollen (TP) through carbonization, has emerged as a particularly valuable therapeutic agent. It has been widely used in clinical practice to treat hemorrhagic disorders caused by blood stasis syndrome (BSS). However, the potential mechanism for CTP to achieve the dual synergistic effect of promoting blood flow and hemostasis remains unclear.

Aim of the study

From the standpoint of metabolite profiles, this study attempts to investigate the fundamental mechanism of CTP in the elimination of blood stasis and the cessation of bleeding.

Materials and methods

First, chemical constituents, absorbed constituents and metabolites in rats following oral administration of CTP were identified by ultra-high performance liquid chromatography coupled with the quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) method combined with MetabolitePilot 2.0.4 software. Subsequently, the pharmacological effects of CTP were systematically investigated using rat models with BSS and zebrafish with cerebral hemorrhage. Specifically, the impact on coagulation function and histopathology in rats, as well as the effect on cerebral hemorrhage in zebrafish, were thoroughly evaluated. Untargeted metabolomics based on rat plasma was applied to analyze the metabolic profile changes, revealing the potential action mechanism. The underlying mechanism was furtherly confirmed by gut microbiome analysis and systemic molecular biology experiments.

Results

34 prototype chemicals and 71 metabolites from the liver, heart, spleen, lung, kidney, small intestine, uterus, and serum were found. CTP improved the abnormal coagulation system, promoted blood circulation, and reduced pathological damage caused by BSS. Plasma metabolomics revealed that BSS significantly altered bile acid (BA) metabolism and arachidonic acid (AA) metabolism. Gut microbiome analysis and fecal microbiota transplantation (FMT) experiments further demonstrated that CTP modulated the gut microbiota. This modulation promoted BA production and activated endothelial nitric oxide synthase (eNOS), leading to increased nitric oxide (NO) levels. These changes contributed to the therapeutic effect of CTP in removing blood stasis. Systemic molecular biology experiments showed that CTP activated key components of the AA metabolic pathway. It promoted PLCγ1 phosphorylation, increased intracellular Ca²⁺ levels, and upregulated COX-2 expression. In addition, CTP enhanced the production of AA-related metabolites, including 6-keto-prostaglandin F_1α (6-keto-PGF_1α), prostaglandin E₂ (PGE₂), and thromboxane B₂ (TXB₂). It also increased the transcription of AA metabolism-related genes, such as PLCγ1, PTGS2a, PTGS2b, PTGIS, PTGES, TXBAS, and vWF.

Conclusions

CTP could promote the generation of AA metabolites through PLCγ1/Ca²⁺/COX-2 to stop bleeding, while also enhancing eNOS activity and NO synthesis through gut microbiota-bile acid axis to remove blood stasis. These two effect were balanced to achieve hemostasis without blood stasis.