ObjectiveTo elucidate the molecular mechanisms underlying the vascular irritation reaction induced by Huachanshu injection （HCSI） and screen the related substances.MethodsFirstly， the model of vascular irritation reaction induced by HCSI and its components was established in rabbits. Sixteen clean-grade male white rabbits were randomly allocated into six groups： blank （2 rabbits， normal saline）， HCSI （2 rabbits， 0.16 L·L^-1）， polypeptide （3 rabbits，0.232 g·L^-1）， nucleic acid （3 rabbits，0.232 g·L^-1）， tryptamine （3 rabbits，0.022 4 g·L^-1）， and toxic ligand （3 rabbits，0.016 8 g·L^-1）. The rabbits were administrated with corresponding agents once a day for three consecutive days. Visual observation， pathological examination， and scoring were performed 24 h after the last administration. The model of increased vascular permeability was established in mice. According to the body weight， 78 ICR mice were assigned into 13 groups （n=6）： blank （normal saline）， positive drug （histamine， 20 mg·kg^-1）， high- and low-dose （12.8，3.2 mg·kg^-1） HCSI， high-， medium-， and low-dose （3， 1.5， and 0.75 mg·kg^-1） 5-hydroxytryptamine （5-HT）， high-， medium-， and low-dose （3， 1.5， and 0.75 mg·kg^-1） bufotenine （BUF）， and high-， medium-， and low-dose （3， 1.5， and 0.75 mg·kg^-1， respectively） bufotenidine （BTD）. The ear blue staining was observed within 30 min after administration. Evans blue （EB） exudation from the mouse ear tissue was detected by spectrophotometry （a microplate reader）. The serum levels of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） were determined by enzyme-linked immunosorbent assay. Western blot was employed to determine the protein levels of p38 mitogen-activated protein kinase （p38 MAPK）， p-p38 MAPK， inhibitor of nuclear factor-κB （IκB）， p-IκB， IκB kinase （IKK）， and glyceraldehyde-3-phosphate dehydrogenase （GAPDH） in the lung tissue of mice. Digital quantitative polymerase chain reaction（Digital PCR） was employed to detemine the mRNA levels of nuclear factor-κB （NF-κB） and p-NF-κB p65 in the ear tissue and the lung tissue.ResultsThe rabbit model induced by HCSI showed obvious vascular irritation reaction， which was mild in the tryptamine group and not observed in the nucleic acid group， peptide group， or toxic ligand group. This result suggested that tryptamine may be the main contributor of the vascular irritation reaction induced by HCSI. High-dose HCSI， 5-HT， BUF， and high-dose BTD increased the ear blue staining reaction rate， ear blue staining score， and EB exudation compared with the blank group （P<0.05， P<0.01）， and the degree of reaction was positively correlated with the dose. High-dose HCSI and high-dose BUF increased the content of TNF-α and IL-6 （P<0.01） and up-regulated the protein levels of p-p38 MAPK/p38 MAPK， p-IκB/IκB， and IKK （P<0.05， P<0.01） and the mRNA levels of NF-κB and p-p65 （P<0.05， P<0.01）.ConclusionThe p38 MAPK signaling pathway and NF-κB activation are involved in the vascular hyperpermeability and vascular irritation reaction induced by HCSI and BUF in mice. Indole derivatives such as BUF may be key contributors to the vascular irritation reaction induced by HCSI.

Huachansu injection;vascular irritation reaction;vascular hyperpermeability;buftenine;p38 mitogen-activated protein kinase （MAPK） signaling pathway