In acupuncture practice, the most important step is to confirm the location of a sensitized acupoint which reflects a diagnosis and can be stimulated with a specialized needle to treat the disease. Abnormal symptoms such as hyperalgesia or allodynia at the sensitized acupoints in patients with visceral disorders are considered to be in relation with referred pain and neurogenic inflammation. Yet, limited study has investigated the cutaneous neurochemical changes of the sensitized acuponits. The resent study developed an animal model of gastric mucosal injury (GMI) by HCl administered into the stomach of the rats. Evans Blue (EB) dye was applied by injection of tail vein after mucosal damage to observe the neurogenic plasma extravasation dots in the skin of the rats. The EB dots extravagated in the skin were compared with locations of acupoints. Immnohistochemistry analysis was used to detect the expression of calcitonin gene-related peptide (CGRP)- or substance P (SP)-labeled nerve fibers, histamine (HA)-, serotonin (5-HT)-, and tryptase-labeled cells in EB dots. Images were recorded and analyzed by Confocal imaging system and Olympus Image Processing Software. The results showed that GMI resulted in neurogenic plasma extravasation in the skin of the acupoints over the back and abdomen, which mostly occurred in the T9-11 dermatomere. The EB extravasation dots appeared after GMI and disappeared gradually during the natural self-recovery of the gastric mucosa. More SP and CGRP positive nerve fibers were distributed in EB dots than that in regions beside EB dots and in the control, mostly distributed in the nerve fibers around both the vessels and root of hair follicle. Mast cells also aggregated and degranulated to release algogenic substances of 5-HT and HA around the vessels in areas of the EB dots. Our results indicates that the mechanism of EB extravasation in the skin of the acupoints induced by GMI are closely related to neurogenic inflammation, and that the high expression of local allergic substances and nociceptive neuropeptides in the local skin including SP, CGRP, HA, 5-HT, and mast cell tryptase may be the underlying mechanism of the acupoint sensitization.

Type 2 diabetes mellitus has become a prevalent metabolic disease worldwide. Current synthetic PPARγ (Peroxisome proliferator-activated receptor γ)agonists are associated with adverse effects, highlighting the need to explore natural compounds with potential PPARγ agonistic activity. PPARγ is a vital therapeutic target for type 2 diabetes. To facilitate the discovery of natural PPARγ agonists, this study integrated graph neural network (GNN) prediction, molecular docking, ADMET evaluation, in vitro activity assessment, and molecular dynamics simulations for multistage virtual screening. Three GNN models, including GCN, GAT, and MPNN, were constructed and systematically compared, with GAT showing the best overall classification performance. The GAT model was subsequently applied to screen 4,112 natural products from the ZINC database, yielding 291 candidates for molecular docking. Following docking and ADMET evaluation, five compounds were prioritized for experimental assessment. Among them, glycyrrhisoflavone exhibited significant concentration-dependent PPARγ agonistic activity, with an EC ₅₀ of 9.98 μM, whereas the other four candidates showed weak activity. Molecular docking revealed that glycyrrhisoflavone adopted a binding mode similar to that of rosiglitazone, while molecular dynamics simulations further supported the stability of the glycyrrhisoflavone–PPARγ complex. These findings demonstrate the utility of GNN-assisted multistage virtual screening for prioritizing natural PPARγ agonist candidates and identify glycyrrhisoflavone as a candidate for further investigation.