Wenjing decoction exerts analgesic effects on cold coagulation and stasis PD through BDNF/ TRKB/CREB pathway

Wenjing decoction (WJD) is a traditional Chinese medicine formula (from "Fu Ren Da Quan Liang Fang") used in the clinical treatment of primary dysmenorrhea (PD) that regulates endocrine levels, reduces inflammatory factor levels, improves microcirculation, regulates neurotransmitter levels, alleviates uterine smooth muscle spasms, and relieves pain. However, its potential mechanism of action is not yet clear.

The aim of this study was to explore the role of pain sensitization in the occurrence of PD, clarify the mechanism by which WJD exerts analgesic effects in PD model rats with cold coagulation and blood stasis (CCBS) syndrome, and provide experimental evidence for the clinical application of WJD.

Ultrahigh-performance liquid chromatography‒mass spectrometry (UHPLC‒MS/MS) technology was used to identify the main compounds in traditional Chinese medicine compound preparations. Eight-week-old female Sprague-Dawley (SD) rats were randomly divided into a control group, a model group, an ibuprofen group, and low-, medium-, and high-dose WJD groups (WJD-L, WJD-M, WJD-H), with 8 rats in each group. A PD rat model of CCBS syndrome was established using oestradiol, benzoate and oxytocin treatment combined with an ice-water bath. The intervention effect of different doses of WJD in PD model rats was evaluated by measuring the CCBS symptom score, uterine surface microcirculation blood flow and temperature, serum prostaglandins and vasoactive substances, pain behaviour, and uterine histomorphology of the rats. After the dose screening experiment, the WJD-M group was renamed the WJD group, and subsequent experiments were conducted at this dose of WJD. Using RNA-seq technology, we explored the pathogenesis of PD with CCBS syndrome, as well as the targets and mechanisms of action of WJD. By evaluating pain response factors in the serum, the mRNA and protein expression levels of peripheral pain sensitization markers in uterine tissue were determined. The response of the central pain sensitization pathway, BDNF/TrkB/CREB, was investigated to explore the analgesic mechanism of WJD in PD model rats with CCBS syndrome.

Thirty-three compounds were identified in WJD. Moreover, the use of WJD can effectively alleviate CCBS syndrome and pain behaviour in PD model rats, improve microcirculation in the uterus, alleviate inflammatory damage to uterine tissue, and reduce the levels of pain response factors in the serum. The RNA-seq results revealed that the expression of genes related to hypothalamic neuroendocrine regulation, neural signal transduction, and immune regulation increased in the PD with CCBS syndrome model, whereas the use of WJD downregulated the expression of the Trpv1, Bdnf, and Pgf genes and the expression of genes related to neuropeptide receptor binding, ion channel activity, and other signalling pathways. RT‒qPCR, IHC, and WB revealed that the mRNA and protein expression levels of BDNF, TRPV1, and PTGFR were increased in the uterine tissue of the model group, and the key factors of the BDNF/TRKB/CREB pathway and the expression of CD11b and c-FOS were increased in the hypothalamic tissue. After the administration of WJD, the expression of these genes was downregulated.

Central and peripheral pain sensitization are involved in PD with CCBS syndrome. The use of WJD reduced the phosphorylation of CREB mediated by BDNF in the hypothalamus and the expression of the key regulatory factor TRKB inhibited the activation of central microglia, alleviating peripheral pain in uterine tissues and exerting analgesic effects in rat models of PD with CCBS syndrome. The use of WJD can inhibit the activation of central microglia and central neurons by reducing the phosphorylation of CREB and the expression of TrkB mediated by BDNF in the hypothalamus to reduce the peripheral pain sensitization effect on uterine tissue and has an analgesic effect in a rat model of PD with CCBS syndrome.Through RNA seq and molecular experiments, it was elucidated that Wenjing Decoction inhibits central and peripheral pain sensitization by regulating the BDNF/TrkB/CREB pathway, providing a scientific basis for its TCM treatment of primary dysmenorrhea(PD).