[Mechanism of Shenmai Injection in inducing immediate hypersensitivity by activating RhoA/ROCK signaling pathway]

This study aimed to elucidate the mechanism of Shenmai Injection(SMI) in inducing immediate hypersensitivity reaction by activating the Ras homolog gene family member A(RhoA)/Rho-associated coiled-coil forming protein kinase(ROCK) signaling pathway. In the in vivo experiment, ICR mice were randomly divided into a negative control group, SMI groups of low-dose(7.5 mL·kg~(-1)), medium-dose(15 mL·kg~(-1)), and high-dose(30 mL·kg~(-1)), as well as a positive control group. The effect of SMI on vascular leakage was detected by mouse auricular blue staining, and histopathological changes in the lung tissue were observed. In the in vitro experiment, the effect of SMI on the vascular endothelial barrier and cytoskeleton was evaluated by endothelial monolayer permeability assays and phalloidin staining. Western blot was employed to measure changes in the key protein expressions of the RhoA/ROCK signaling pathway, including phospho-myosin light chain 2(p-MLC2), myosin light chain 2(MLC2), phospho-myosin phosphatase target subunit 1(p-MYPT1), myosin phosphatase target subunit 1(MYPT1), guanosine triphosphate-RhoA(GTP-RhoA), and total RhoA, in endothelial cells as well as in mouse ear and lung tissues after SMI administration. To verify the role of the RhoA/ROCK signaling pathway in SMI-induced immediate hypersensitivity reactions, mice were pretreated with the ROCK inhibitor fasudil to observe the vascular hyperpermeability induced by SMI. The results showed that SMI caused a dose-dependent increase in ear vascular permeability in ICR mice, with the 30 mL·kg~(-1) dose group exhibiting marked ear vascular extravasation and slight alveolar wall thickening, while resulting in concentration-dependent endothelial barrier function decrease and cytoskeleton changes. Mechanistically, SMI could significantly upregulate GTP-RhoA/RhoA, p-MLC2/MLC2, and p-MYPT1/MYPT1 levels in vascular endothelial cells, as well as in mouse ear and lung tissues. Additionally, the ROCK inhibitor fasudil could significantly reduce the SMI-induced vascular hyperpermeability, indicating the key role of the RhoA/ROCK signaling pathway in SMI-induced hypersensitivity reactions. The results suggest that the risk of SMl-induced immediate hypersensitivity reactions may be associated with excessively high doses or rapid administration, and that the RhoA/ROCK signaling pathway plays an important role in this response.